At the bench: adoptive cell therapy for melanoma.

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At the Bench: Adoptive cell therapy for melanoma Walter J. Urba1 Earle A. Chiles Research Institute, Providence Cancer Center, Portland, Oregon, USA RECEIVED MAY 28, 2013; REVISED JANUARY 22, 2014; ACCEPTED FEBRUARY 20, 2014. DOI: 10.1189/jlb.0513301

‹ SEE CORRESPONDING ARTICLE ON PAGE 875

ABSTRACT The cellular and molecular principles that furnish the foundation for ACT of melanoma and their implications for further clinical research are reviewed. The parallel advances in basic immunology, preclinical animal studies, and clinical trials over the last two decades have been integrated successfully with improvements in technology to produce an effective ACT strategy for patients with melanoma. From the initial observation that tumors could be treated effectively by the transfer of immune cells to current strategies using preconditioning with myeloablative therapy before adoptive transfer of native or genetically altered T cells, the role of preclinical animal models is discussed. The importance of the pmel transgenic mouse model in the determination of the mechanisms of lymphodepletion, the ongoing work to identify the optimal T cells for adoptive immunotherapy, and the early impact of the emerging discipline of synthetic biology are highlighted. The clinical consequences of the research described herein are reviewed in the companion manuscript. J. Leukoc. Biol. 95: 867– 874; 2014.

EXPERIMENTS DEMONSTRATING THE EFFICACY OF ACT IN ANIMAL MODELS The recent history of ACT provides an excellent example of the application of the fundamental principles of translational research to clinical medicine. The parallel advances in basic immunology, preclinical animal studies, and clinical trials over the last two decades have been integrated successfully, with improvements in technology, particularly synthetic biology, to produce an effective therapeutic strategy that is accessible to more patients with melanoma each year. The first attempts to use immune cells for the ACT of cancer predated the identification of T cells and lymphocyte subsets [1]. During the 1960s and 1970s, multiple inves-

Abbreviations: aAPC⫽artificial APC, ACT⫽adoptive cellular therapy, CAR⫽chimeric antigen receptor, CD62L⫽L-selectin, CRT⫽calreticulin, D⫽diversity, GD2/GD3⫽gangliosides, HMGB1⫽high-mobility group box 1 protein, HSC⫽hematopoietic stem cell, iCAR⫽inhibitory chimeric antigen receptor, J⫽joining, LAK⫽lymphokine-activated killer, MAGE-A3⫽melanomaassociated antigen 3, MART-1⫽melanoma antigen recognized by T cell 1, MTD⫽maximum tolerated dose, pmel⫽premelanosomal protein or gp100, scFv⫽single-chain variable fragment, TCM⫽central memory T cell, TEM⫽effector memory T cell, TIL⫽tumor-infiltrating lymphocyte, Treg⫽regulatory T cell, V⫽variable

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tigators showed that established tumors in rodents could be treated effectively by the transfer of immune cells [2]. A significant advance was the development of techniques to produce large numbers of anti-tumor immune cells via in vitro sensitization. Cheever et al. [3] first showed that the combination of in vitro-sensitized T cells and chemotherapy was effective against the Friend murine leukemia virus-induced erythroleukemia lymphoma. ACT expanded dramatically when it became possible to grow T lymphocytes in culture. Growth depended on an activity found in peripheral blood or spleen cell culture supernatants that kept T cells alive and proliferating [4]. Growth promotion was a result of the presence of IL-2, which was eventually cloned and arguably led to a revolution in cancer immunotherapy. Once sufficient IL-2 was available, investigators were able to demonstrate that IL-2 alone could inhibit the growth of micrometastases from a variety of immunogenic and nonimmunogenic murine sarcomas, melanomas, and adenocarcinomas [5]. There was a direct relationship between the dose of IL-2 administered and its therapeutic benefit. The best antitumor effects were obtained when mice were treated at the MTD, although there was a very low toxic:therapeutic ratio in mice. These preclinical results formed the basis of the philosophy for the development of IL-2 as therapy for melanoma, wherein each patient is treated to his or her individual MTD—a philosophy that remains in effect to this day. Most of the side effects observed in patients treated with IL-2, particularly those related to lymphocytic infiltration of visceral organs and capillary leak, were foreshadowed in the murine models. IL-2: IL-2 is a member of the common ␥-chain cytokine family that is necessary for the growth, proliferation, and differentiation of T cells. It also causes NK cells to exhibit nonantigen-specific, MHC-restricted tumor cell cytotoxicity.

IL-2 AND THE DEVELOPMENT OF LAK CELL IMMUNOTHERAPY IL-2 had no direct antitumor effects in vitro; however, murine and human lymphocytes cultured in IL-2 lysed fresh tumor

1. Correspondence: Earle A. Chiles Research Institute, Providence Cancer Center, 4805 N.E. Glisan St., Ste. 2N35, Portland, OR 97213, USA. E-mail: [email protected]

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cells, but not normal cells [6]. These killer cells were predominantly activated NK cells and called LAK cells, which could be distinguished from cytotoxic T cells by their phenotype and their ability to kill tumor cells in a nonantigen-specific, nonMHC-restricted manner. LAK cells could be produced easily from murine spleen cells (or peripheral blood from patients) by short-term in vitro culture in IL-2. When given i.v., in combination with IL-2, they eliminated liver and lung metastases and could cure mice with disseminated tumor. Combined LAK cell and IL-2 therapy proved to be superior to IL-2 alone in animal models, and a direct relationship between efficacy and the dose of IL-2 and the number of LAK cells administered was observed [7]. Multiple clinical trials, discussed in the companion manuscript, were initiated to test high-dose IL-2 and LAK cells in patients with melanoma. Substantial evidence of antitumor activity was observed in patients with metastatic melanoma and renal cell cancer. However, as response rates after IL-2 and LAK cell therapy were not superior to IL-2 alone, this cellular approach was abandoned, and high-dose IL-2 became a standard treatment for these patients, producing a low rate (⬃5%) of durable complete remissions. LAK cells: LAK cells are IL-2-activated NK cells that can cure mice of established tumors when administered with IL-2. Clinical trials in patients showed that LAK cells did not add significantly to the antitumor effects of IL-2.

ADOPTIVE TRANSFER OF ANTIGENSPECIFIC T CELLS IN MICE The other major cell population activated by IL-2 in vitro was tumor-reactive T cells. Murine models had demonstrated the efficacy of the adoptive transfer of antigen-specific T cells obtained from repetitively immunized syngeneic mice. As repetitive immunization of cancer patients was not feasible, other sources of antigen-specific T cells were identified. Cheever and Chen [8] showed that therapy with cultured tumor-specific T cells, generated from the circulation, could cure otherwise fatal disseminated leukemias in mice, and investigators from the Surgery Branch of the National Cancer Institute demonstrated that TILs obtained from transplantable murine tumors also contained tumor-reactive T cells. Following in vitro culture, the adoptive transfer of murine TILs with IL-2 eradicated established micrometastases from sarcomas and carcinomas [9]. TILs were produced in vitro by culturing minced tumor in an enzyme digest (DNAase, collagenase, hyaluronidase), filtering the debris and then incubating the lymphocytes in high doses of IL-2 (1000 U/ml). During culture, the lymphocytes expanded, and tumor cells were eliminated. As adoptive therapy, TILs were 50 –100 times more potent on a per-cell basis than LAK cells and IL-2. Larger, established macrometastases of the same tumor types could be treated effectively if high doses of cyclophosphamide were administered in conjunction with TILs and IL-2, a setting in which LAK cells were ineffective [9]. This established a role for preconditioning of hosts before ACT. TILs comprised CD8 and CD4 T cells in varying proportions with multiple phenotypes. In contrast to the broad MHC-unrestricted, nonantigen specificity of LAK 868 Journal of Leukocyte Biology


TABLE 1. Possible Mechanisms by Which Lymphodepletion Enhances the Efficacy of ACT Elimination of immunosuppressive cells 䡠 Tregs (CD4⫹ C25⫹ Forkhead box P3⫹) 䡠 Myeloid suppressor cells (CD11c⫹ Gr1⫹) Minimizing cellular cytokine sinks 䡠 Homeostatic proliferation 䡠 Decreased competition for IL-7, IL-15 Improved APC function and availability

cells, TILs demonstrated antigen specificity for the tumor from which they were derived and recognized tumor cells in a MHC-restricted fashion. TILs: TILs are antigen-specific, MHC-restricted T cells obtained from tumors that after in vitro culture in IL-2, are 50 –100 times more effective than LAK cells against metastases in tumor models.

These findings marked the departure point for ACT for patients with melanoma; subsequent preclinical and clinical studies moved from the study of nonspecific cellular immunity to antigen-specific T cells. The early preclinical work defined principles of ACT that still hold today. T Cell therapy can be exquisitely specific; it is quantitative (the more T cells given, the better the anti-tumor effects); efficacy depends on the persistence of tumor-specific T cells (peptide stimulation or treatment with growth factors, e.g., IL-2, can help accomplish this); and immunosuppressants (lymphodepletion) help maintain increased numbers of functional donor T cells. Peripheral blood and tumor-infiltrating, antigen-specific T cells have been used in clinical trials. ACT with TILs has garnered more attention, but a group at the University of Washington used in vitro-sensitized peripheral blood T cells to treat patients with melanoma and observed some impressive responses (see companion review by Weber).

PRECLINICAL JUSTIFICATION FOR LYMPHODEPLETION The key discovery that depletion of immune cells before ACT significantly improved the anti-tumor efficacy of adoptively transferred CD8 T cells was made in animal models years ago [3, 10]. However, the mechanisms by which lymphodepletion improved the efficacy of ACT remained largely unknown until recent work by a number of investigators identified multiple factors that contributed to its salutary effects. Initially attributed to making “space” for the transferred T cells, later experiments showed that T cell homeostatic proliferation, which occurred following the induction of lymphopenia by radiation, chemotherapy, or transfer of T cells to RAG-deficient mice, was posited as the mechanism of enhanced antitumor immunity in a variety of preclinical models [11, 12]. Subsequent experiments have identified multiple possible explanations [13] that will be discussed further (Table 1).

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Homeostatic proliferation: T cells have the capacity to undergo extensive proliferation spontaneously after transfer into immunodeficient hosts. A normal mechanism to maintain the peripheral pool of T cells at a normal size, homeostatic proliferation requires cytokines (IL-7 and IL-15) and interaction with peptide—MHC complexes.

Although the mechanism(s) responsible for the antitumor activity were not elucidated completely, empiric observations from preclinical models were adopted and translated to the clinic, where lymphodepletion was included in the preparative regimen for ACT. The exact composition of lymphodepletion, which improved response rates, has evolved in parallel with new data from preclinical models. The trend has been to induce more profound lymphodepletion (chemotherapy and radiation)—a strategy strongly supported by findings from the pmel-1 transgenic mouse model [14]. RAG-deficient mice: The complex of enzymes that performs somatic recombinations of V, D, and J segments is called the V(D)J recombinase. The lymphoid-specific components of the recombinase are encoded by two RAGs: RAG1 and RAG2. Lymphocyte development in mice, in which either of the RAG genes has been knocked out, is blocked at the gene-rearrangement stage. These mice make few B and T cells and are termed SCID mice.

PMEL-1 MURINE MODEL FOR ADOPTIVE IMMUNOTHERAPY Overwijk et al. [14] developed a valuable transgenic mouse model that has served to guide the design of clinical trials and to define many of the current principles of adoptive immunotherapy. pmel-1 is a TCR transgenic mouse strain, in which all of the transgenic T cells recognize the melanoma antigen pmel-1 (murine gp100). pmel-1 transgenic mice inoculated with gp100⫹ B16 melanoma tumor cells developed tumors at the same rate as nontransgenic littermates, indicating that the mere presence of naive or activated anti-tumor T cells alone was not sufficient to cause regression of subcutaneous tumor. These tolerant cells could be activated to a functional antitumor state by immunization with an altered peptide ligand, in this case, a heteroclitic human gp100 peptide. The pmel model allowed investigators to develop therapeutic strategies that led to the destruction of large vascular tumors comprising poorly immunogenic B16 melanoma cells. This led to a series of experiments that improved our understanding of the mechanisms of a strategy for tumor eradication, which could then be deployed in clinical trials. The model had three required elements: (1) adoptive transfer of tumor-specific CD8⫹T cells; (2) immunization with an altered peptide ligand; and (3) administration of a T cell growth factor (IL-2).

MECHANISMS BY WHICH LYMPHOID DEPLETION AUGMENTS ACT EFFICACY The pmel-1 model was used to explore the immune-enhancing mechanisms of lymphodepletion. Upon examination of the role of CD4⫹ T cells in successful ACT with CD8⫹ T cells, Antony et al. [15] paradoxically found that elimination of

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Adoptive cell therapy for melanoma

CD4⫹ T cells enhanced the efficacy of ACT. Enhancement was attributed to removal of CD4⫹ CD25⫹ Tregs that inhibited the antitumor response. CD4⫹ CD25⫺ Th cells contributed to antitumor immunity, but they could be replaced by administration of exogenous IL-2. Thus, removal of Tregs was established as one mechanism by which lymphodepletion enhanced ACT. Depletion of Tregs is not the entire story, however. Gattinoni et al. [16] have shown that even in the genetically determined absence of Tregs, inclusion of a nonmyeloablative radiotherapeutic regimen augmented ACT with gp100-specific CD8 T cells. They further demonstrated that the improvement in efficacy was not merely a product of an increased number of tumor-reactive T cells, as might be predicted if homeostatic proliferation were responsible, but that upon exposure to the lymphopenic environment, there were qualitative changes in the transferred T cells themselves. The transferred cells exhibited enhanced production of IFN-␥, IL-2, GM-CSF, TNF-␣, and MIP-1␣ in response to gp100. The enhanced functionality and improved antitumor effects were related to exposure to the ␥-chain cytokines IL-7 and IL-15, which are known to be key regulators of homeostatic CD8⫹ T cell expansion. Further support for the important role of IL-15 and IL-7 comes from experiments in IL-15 knockout mice, in which the enhancement in efficacy was partially abrogated and from IL-7 and IL-15 double-knockout mice, in which the efficacy of ACT was impaired dramatically by the absence of both cytokines. Removal of endogenous cells (e.g., nontumor-specific T cells and NK cells) that could serve as cytokine sinks for IL-7 and IL-15 also enhanced tumor responses. For example, depletion of NK cells in RAG-deficient mice by antibody treatment enhanced ACT, whereas ACT, in mice that lacked B cells, T cells, and NK cells, was not improved by lymphodepleting radiotherapy. Thus, removal of endogenous leukocytes, which appear to compete with transferred T cells for supportive cytokines, is another mechanism by which lymphodepletion enhances the efficacy of ACT. Lymphodepleting irradiation or chemotherapy can also facilitate antigen presentation by causing tumor cell death and enhancing the function of the APCs themselves [17, 18]. Irradiation and chemotherapy can produce an inflammatory environment that results in an immunogenic cell death. The release of damage-associated molecular patterns, such as HMGB1 and ATP, and translocation of CRT to the cell surface could enhance antigen uptake by DCs via the “eat me” signal from CRT and triggering of the TLR4 by HMGB1. This would enhance cross-presentation to CD4 and CD8 cells. Radiation has also been shown to facilitate the recruitment of activated T cells to the tumor, up-regulate expression of adhesion molecules, and induce production of T cell chemokines, all of which can enhance antitumor effects. Cross-presentation: Some APCs can form peptide:MHC class I complexes from extracellular sources [e.g., viruses, phagocytized dying (tumor) cells], which can be presented to T cells in a process called cross-presentation.

Tumor-infiltrating myeloid cells, including macrophages and myeloid-derived suppressor cells, can also suppress tumor immunity; this is accomplished via production of NO, reactive Volume 95, June 2014


oxygen species, and the expression of iNOS, IDO, and/or arginase [19]. The potential importance of these cells is illustrated by the fact that inhibition of CSF-1R signaling decreased the number of tumor-infiltrating macrophages and led to enhanced pmel T cell function and superior efficacy against metastatic melanoma [20]. Nonmyeloablative lymphodepletion, combined with ACT, exhibited antitumor activity in mice and humans; however, there was still substantial room for improvement in clinical activity in patients with melanoma. Wrzesinski et al. [21] investigated whether increasing the intensity of lymphodepletion would improve the antitumor effects of ACT. With the use of the pmel-1 model, the intensity of lymphodepletion was increased to a degree that necessitated rescue with murine HSCs. Under these circumstances, antigen-preactivated pmel-1 CD8 T cells were more effective when transferred to mice that received a myeloablative regimen (9 Gy radiation) with stem cell rescue than mice that received a nonmyeloablative dose of 5 Gy without stem cells. Mice receiving myeloablative therapy and stem cells had persistently higher levels of tumor-reactive T cells. The increase in tumor-reactive T cells was not solely related to enhanced lymphodepletion and the consequent reduction in host regulatory elements and cytokine sinks. It appeared that the addition of lin⫺/c-kit⫹ HSCs drove T cell proliferation by increasing production of IL-7 and IL-15. An additional benefit of the more pronounced lymphodepletion and addition of HSCs in the pmel-1 model was the elimination of the requirement for antigen stimulation in vivo to achieve eradication of large established tumors, i.e., both preactivated and naive pmel-1 CD8⫹ T cells proliferated and reached their full antitumor potential in the myeloablated host that received stem cells. This advance was translated quickly to the clinic, where Dudley et al. [22] showed that a preparative regimen of total body irradiation plus CD34⫹ HSCs, followed by ACT with TIL and IL-2, increased the response rates further in patients with melanoma. Confirming the results of the mouse model, they reported that serum levels of IL-7 and IL-15 were elevated and that response rates correlated with telomere length in the transferred cells. Clinical questions: What extent of lymphodepletion is necessary to achieve optimal clinical results? Which modality or combination of modalities is most effective?

Telomere: Telomere is a region at the end of each chromatid of repetitive nucleotide sequences that protects the end of the chromosome from degradation. Telomeres are consumed during cell division and replenished by telomerase. Telomere length is correlated with the survival and replicative capacity of T cells.

SUBPOPULATIONS OF T CELLS THAT MEDIATE SUCCESSFUL ACT Substantial progress has been made in our understanding of the properties of various subpopulations of T cells and how their state of differentiation relates to their ability to protect against viral challenge [23] or mediate tumor regres870 Journal of Leukocyte Biology


sion [24]. With the use of the pmel-1 model, Gattinoni et al. [24] demonstrated that the differentiation state of the transgenic CD8 T cells affected the efficacy of ACT. T cells at progressive stages of differentiation (naive, early effector, intermediate effector, effector) were generated in vitro and transferred to tumor-bearing mice after lymphodepleting therapy, and IL-2 was administered. The ability of cells to mediate tumor regression correlated inversely with their state of differentiation. Fully differentiated effector cells, the population used most frequently in clinical trials, were the least effective, and naive cells were the most effective. Naive pmel-1 cells expressed CD62L, CCR7, and CD27; they produced low levels of IFN-␥ and high levels of IL-2 and exhibited little cytotoxicity in vitro. At the other end of the spectrum, effector cells had little expression of CD62L, CCR7, and CD27 and high levels of granzyme B and were cytotoxic in vitro. The differentiation state was inversely related to the proliferative capacity of T cells; less differentiated cells proliferate more and lead to a substantial increase in the total number of tumor-reactive T cells in vivo after transfer. The following five populations of CD8⫹ T cells have emerged as candidates for adoptive immunotherapy: naive cells, T memory stem cells, TCMs, TEMs, and terminally differentiated effector T cells [25]. A detailed description of the biology of these cells is beyond the scope of this review. Instead, the focus will be on the relevance of each of these subsets, which although referred to as discrete populations, probably represent a continuum of differentiation and maturation, to effective ACT for melanoma. The preclinical data support the hypothesis that less-differentiated T cells will be the most effective population for ACT-based immunotherapy [26] and that clinical strategies should avoid infusion of cells with senescent and exhausted phenotypes. Clinical question: What particular T cell lineage would be the best subpopulation for use in ACT?

There has been substantial interest in the central memory population, those T cells that express CD62L and CCR7 (proteins that promote migration into lymph nodes) and proliferate rapidly upon re-exposure to antigen. Additional research has suggested that even less differentiated cells, e.g., naïve cells or T stem cells, may be even more effective in ACT [26, 27]. This principle has been tested in nonhuman primates, where Berger et al. [28] showed that sorted CMV-specific TCM, but not TEM, persisted long-term in vivo, reacquired phenotypic and functional properties of memory T cells, and occupied memory T cell niches. The same group showed that after transfer to immunodeficient mice, human-derived CMV-specific TCM, when compared with TEM, were less prone to apoptosis and established a reservoir of functional T cells that persisted in the host [29]. This suggested that superior engraftment fitness of TCM-derived human CD8⫹ effector cells made them a preferred cell type for ACT. These findings pose a challenge to successful translation to the clinic because of the difficulty in isolating, purifying (although it is not certain that

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isolation will be necessary), and preparing these subpopulations for administration to patients. TCM: TCMs have the most potent anti-tumor effects in ACT, and TEMs are the least potent. T stem cell memory cells may be the most promising for treatment.

Butler et al. [30, 31] developed a strategy to increase the persistence of adoptively transferred CD8 T cells without lymphodepletion by creating a human cell-based aAPC, which can stimulate antigen-specific T cells to acquire TCM and TEM phenotypes. The aAPCs were K562 cells transduced to express HLA-A0201, CD80, and CD83. After pulsing with melanoma peptides, these aAPCs produced large numbers of CTLs after in vitro culture of peripheral CD8⫹ T cells with IL-2 and IL15. With the use of this strategy, MART-1-specific T cells were generated from the peripheral blood of nine patients with melanoma. Immune monitoring after adoptive transfer showed that CTLs with a memory phenotype and function were present in vivo. The cells persisted in vivo long-term, trafficked to tumor sites, and were associated with tumor regression. One patient achieved a near-complete tumor regression that lasted for more than 25 months. These results were achieved without lymphodepletion, vaccination, or administration of IL-2 after adoptive transfer of the in vitro-generated T cells. Among five patients whose tumors did not respond to treatment with ACT, salvage therapy with ipilimumab showed clinical benefit in three with augmentation of the MART-1-specific memory response. These results illustrate the opportunity to combine ACT with other immunotherapies, particularly checkpoint inhibitors and immunostimulatory antibodies. Clinical question: Will there be a role for checkpoint inhibitors and immunostimulatory antibodies in combination with ACT?

Other strategies have been used to isolate human TCM for adoptive transfer in patients with melanoma [32]. Taking advantage of the observation that TCM exhibit high relative ratios of IL-2 and IFN-␥ mRNA production following antigen stimulation, high-throughput methods were developed to isolate these high ratio cells and grow them in vitro for adoptive transfer. Effector cells obtained from precursors exhibiting high (TCM) or low (TEM) indices for the ratio of IL-2/IFN-␥ production exhibited similar cytolytic function but differed substantially in their proliferative ability (high index⬎low index) and expression of cell survival/antiapoptosis genes (higher expression in high-index cells). These factors may play an important role in governing the fate in vivo of the respective T cell clones. Five patients with melanoma were treated with T cell clones derived from peripheral blood high IL-2:IFN index precursors, stimulated with a gp100 peptide. T cells were infused and IL-2 administered after a lymphodepleting-preparative regimen. The T cells were expanded in vitro and at the time of adoptive transfer, comprised predominantly fully differentiated effector T cells, as measured by phenotype and function. The evidence for efficacy was very modest—a finding that has been reported previously for T cell clones (see companion review by Weber). However, there was evidence of (1) in vivo trafficking of effector cells, (2) targeting of gp100⫹ melanocytes documented by

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biopsy of skin rashes, (3) in vivo persistence, and (4) self-renewal of T cells following adoptive transfer. The authors concluded that despite the extensive in vitro expansion and the subsequent in vivo proliferation, there was an intrinsic difference in the subset of TEM derived from TCM that allows them to engraft, persist long-term, and repopulate the TCM pool after adoptive cell transfer. The disappointing clinical results may be related to the fact that only a single gp100 epitope was targeted. The major focus of ACT of cancer has been in vitro-activated, tumor-reactive CD8⫹ CTLs. CD4 T cells alone have been shown to exhibit antitumor activity. Transgenic CD4⫹ T cells, specific for a peptide from the endogenous melanocyte differentiation antigen tyrosinase-related protein 1, can expand and differentiate following adoptive transfer and cause regression of large, established tumors without in vitro manipulation [33, 34]. These models included lymphodepletion and demonstrated that the mechanism of tumor rejection was class II-restricted, antigen-specific killing by cytotoxic CD4 T cells. CD4 T cell expansion did not require exogenous cytokines, and antitumor effects were improved by the addition of antiCTLA-4. Clinical question: How effective will CD4⫹ T cells be for ACT, and will they be effective in the absence of exogenous cytokines?

TCR ENGINEERING There are many limitations to ACT with TILs. Among those are the requirement for surgery, the inability to obtain tumorreactive T cells from patients with cancers other than melanoma, and the low affinity of the resultant tumor-reactive T cells, which for the most part, recognized self-antigens. To overcome these problems, approaches to engineer T cells genetically to express specific receptors that target tumors have been developed. TCRs with high-affinity and antigen specificity have been isolated from patients or mice and cloned into a retrovirus for transduction of autologous T cells [7]. Most of this work has been performed with human cells, and the clinical work related to melanoma is discussed in the companion manuscript. Significant antitumor activity has been observed in melanoma patients treated with anti-MART-1, anti-gp100 [35], or anti-MAGE-A3 [36] TCR gene-modified cells and in colon cancer patients receiving T cells engineered to target carcinoembryonic antigen [37]. However, in all three trials, substantial side-effects were observed, as normal tissue was inadvertently targeted. Skin, eye, and ear toxicity, secondary to melanocyte destruction, was observed after administration of high-avidity T cells to melanoma patients, and severe transient colitis was observed in patients with colorectal cancer. The TCR can be modified by site-directed mutagenesis to increase the functional avidity of T cells. Devised to enhance antitumor activity, this strategy can also lead to unexpected toxicity, as was evidenced by the induction of fatal cardiovascular toxicity in two patients with melanoma, who received T cells engineered to express an affinity-enhanced TCR against MAGE-A3 [38, 39]. There has always been the theoretical conVolume 95, June 2014


cern that the engineered ␣- and ␤-chains could combine with the endogenous TCR chains and create a new, unpredictable binding capacity that could produce toxicity. That appeared not to be the case in this trial, where despite extensive preclinical investigations that failed to detect off-target antigen recognition, the authors found that the altered TCR recognized a cross-reactive peptide from the muscle protein titin. This crossreactivity was not detected during preclinical testing against normal cardiac tissue, as it could only be detected on beating cardiomyocytes generated in culture from induced pluripotent stem cells. These studies highlight the destructive power of small numbers of highly avid genetically engineered T cells and the serious and unpredictable off-target and organ-specific toxicities that can be seen. They also highlight the need for better preclinical methods to define the specificity of engineered TCRs. Clinical question: What are the optimal methods to detect potentially dangerous cross-reactivity with self-tissues before genetically engineered T cells can be released for treatment of patients?

CARs CARs are synthetic proteins that comprise a scFv from an antibody gene (for tumor targeting) fused with a transmembrane domain (usually from CD4 or CD8) and an intracellular signaling molecule (some combination of the ␨-chain, CD28, 4-1BB or OX40). CAR-transfected T cells have the advantages of being able to target any extracellular moiety that can be recognized by an antibody and being free of any requirement for MHC restriction. Furthermore, the affinity of CARs is several orders of magnitude higher than TCRs. However, CARs contain foreign sequences that can be immunogenic and cannot recognize intracellular proteins derived from mutated genes, which may be the most important targets in certain cancers (e.g., melanoma and lung cancer). This strategy has moved to the clinic rapidly, and excellent responses have been observed following treatment of patients with refractory B cell malignancies using CAR T cells that recognize CD19 [40]. Patients have experienced cytokine storm and exhibited signs of tumor lysis syndrome days after cell infusion. Patients also have ongoing B cell aplasia, presumably maintained by persistent engineered T cells. Following these initial successes, a major question for the field is whether this strategy will be effective against solid tumors. There is a concern that CAR-based therapies could cause on-target/offtumor toxicity via recognition of healthy cells sharing the target antigen. The potential for CAR T cells against solid tumors was shown in 1995 by Hwu et al. [41]. A chimeric receptor gene from the MOv18 mAb, which recognized the folate-binding receptor expressed by human ovarian carcinomas, was inserted into mouse TIL; these CAR-expressing T cells improved the survival of nude mice with human tumor xenografts. Preclinical models for CARs, directed against carcinoembryonic antigen, human epidermal growth factor receptor 2/neu, and mesothelin, have demonstrated significant antitumor activity [42, 43]. 872 Journal of Leukocyte Biology


Investigators have developed CARs that redirect T cells to recognize melanoma cell lines and tumor cells. Theses CAR T cells proliferate, produce cytokines, and exhibit cytotoxicity [44 – 46]. The CAR T cells have targeted gangliosides GD2 [44] and GD3 [45, 46] and the high molecular weight melanoma-associated antigen 46]. The GD2- and GD3-specific CAR T cells exert antitumor effects in human tumor xenografts, and the GD2-reactive CARs have been given safely to patients with neuroblastoma. Although there is an ongoing trial for patients with solid tumors (including melanoma) of CAR T cells that target vascular endothelial growth factor receptor (2NCT01218867), there has yet to be a clinical trial of CARmodified T cells that target a melanoma-specific antigen. A major concern, of course, is safety, particularly after the sideeffects observed in patients treated with TCR-transfected T cells targeting MART-1 and MAGE antigens. A significant safety measure would be the development of strategies that can control the fate of the adoptively transferred cells. Multiple innovative strategies have been used to date. These include: (1) insertion of a suicide gene, such as herpes simpex virus thymidine kinase [47], (2) tagging the TCR with sequences (e.g., myc) that can be recognized by an antibody that could deplete CAR⫹ cells [48], (3) including an inducible T cell safety switch, which when triggered, activates caspase 9, leading to rapid death of cells expressing this construct [49], and (4) production of CAR T cells using CAR mRNA, which is expressed only for days, rather than using viral transfer that leads to integration of the construct and persistence of CAR T cells with attendant difficulty controlling toxicity should it occur [50]. As mRNA CAR T cells have a short half-life, toxicity should be easier to manage, and patients can be treated with serial infusions [5]; another novel approach to divert off-target immunotherapy responses is the production of an antigenspecific iCAR, which includes a CTLA-4 or programmed cell death protein 1 inhibitory domain linked to a scFv that targets a different antigen than the one expressed by the antitumor CAR T cell [51]. Thus, tumor cells, expressing just the target antigen, would be killed by the CAR T cell, whereas a normal cell that expressed both the target antigen plus the antigen recognized by the iCAR would be spared. This will be challenging to test clinically but is illustrative of the creativity that has been unleashed by the opportunities presented by synthetic biology. Clinical questions: What will be the optimal configuration of a CAR T cell? What is the best configuration of signaling domains to optimize proliferation and survival? What will be the best strategies to control the fate of adoptively transferred T cells?

The ability, through the application of synthetic biology and genetic engineering, to create new T cells with enhanced function is a major advance [52]. The possible combinations of scFv, signaling domains, and subpopulations of T cell recipients to be studied should keep investigators busy for years to come. A detailed description of this emerging area is beyond the scope of this review, but the interested reader is invited to consult a recent review [52].

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TABLE 2. Summary of Questions for Future Clinical-Translational Research (1) What extent of lymphodepletion is necessary to achieve optimal clinical results? Which modality or combination of modalities is most effective? (2) What particular T cell lineage would be the best subpopulation for use in ACT? (3) Will there be a role for checkpoint inhibitors and immunostimulatory antibodies in combination with ACT? (4) How effective will CD4⫹ T cells be for ACT, and will they be effective in the absence of exogenous cytokines? (5) What are the optimal methods to detect potentially dangerous cross-reactivity with self-tissues before genetically engineered T cells can be released for treatment of patients? (6) What will be the optimal configuration of a CAR T cell? What is the best configuration of signaling domains to optimize proliferation and survival? What will be the best strategies to control the fate of adoptively transferred T cells?

12. 13.

14.

15.

16.

17.

SUMMARY Adoptive T cell therapy is effective in patients with metastatic melanoma. The evolution of this effective immunotherapy is a story about the close ties between the bench and the bedside. The value of the preclinical model in designing effective clinical strategies and in investigating clinical findings is beyond doubt. There are, however, limitations to the animal models, particularly in the prediction of some of the toxicity that has been observed, wherein the best model may be a well-designed clinical trial. A number of questions remain (Table 2), but ACT should play an increasing role in the management of patients with melanoma and other solid tumors.

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KEY WORDS: murine 䡠 transgenic 䡠 lymphoid depletion 䡠 cytokines

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At the bedside: adoptive cell therapy for melanoma-clinical development.

Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells.

Adoptive cell therapy for sarcoma.

Adoptive T-Cell Therapy for Solid Tumors.

Adoptive T-cell therapy for Leukemia.

Adoptive T cell therapy for the treatment of viral infections.

Adoptive T-cell therapy for fungal infections in haematology patients.

Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy.

Reassessing target antigens for adoptive T-cell therapy.

CT halo sign as an imaging marker for response to adoptive cell therapy in metastatic melanoma with pulmonary metastases.

Stem cell therapy for bronchopulmonary dysplasia: bench to bedside translation.

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics.

Tracking the T-cell repertoire after adoptive therapy.

Erratum: Tracking the T-cell repertoire after adoptive therapy.

The coming of age of adoptive T-cell therapy for viral infection after stem cell transplantation.

Research highlights: cell separation at the bench and beyond.

Adoptive cell therapies for glioblastoma.

Is there a future for adoptive cell transfer in melanoma patients?

Metastasectomy Following Immunotherapy with Adoptive Cell Transfer for Patients with Advanced Melanoma.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

Corrigendum to "Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma".

Virus-specific T-cell banks for 'off the shelf' adoptive therapy of refractory infections.

Risky business: target choice in adoptive cell therapy.

Clinical application of adoptive T cell therapy in solid tumors.