f BASIC-TRANSLATIONAL REVIEW
At the Bedside: Adoptive cell therapy for melanoma— clinical development Jeffrey S. Weber1 Moffitt Cancer Center and the Donald A. Adam Comprehensive Melanoma Research Center, Tampa, Florida, USA RECEIVED MAY 21, 2013; REVISED JANUARY 5, 2014; ACCEPTED MARCH 13, 2014. DOI: 10.1189/jlb.0513293
‹ SEE CORRESPONDING ARTICLE ON PAGE 867
ABSTRACT Adoptive cell therapy for melanoma, particularly using TIL, consists of a complex and difficult set of procedures, although it has a strong preclinical background and justification and has been pursued clinically by one small group of investigators over the last 20 years. More recent developments and a better understanding of the molecular basis of the anti-tumor immune response have led to the conduct of clinical trials that use lymphoid depletion with chemotherapy and/or TBI to exploit the favorable immune milieu of homeostatic lymphoid reconstitution during transfer of effector T cells. Improved ways of propagating T cells ex vivo have also simplified and shortened the cell-growth process. Current TIL trials have now been expanded beyond the initial center where it was developed, reproducing excellent objective response rates of 40 –50% in previously treated melanoma patients and more importantly, demonstrating that a significant proportion of patients will be alive and free of disease 3–5 years after treatment, raising the possibility that those patients may be cured of their disease. Newer methods for growing the infiltrating T cells using immune-checkpoint antibodies or other agents to condition the tumor before harvest and improved technology to simplify the complex and often cumbersome cell-growth process suggest that this technology may be able to be disseminated to a wide selection of cancer centers and may be a candidate for testing in a randomized Phase III trial to show definitively its benefit in patients with metastatic melanoma. In the accompanying review, the preclinical work that supports the idea of adoptive cell therapy with TIL and expands the concept in promising new ways will be explored. J. Leukoc. Biol. 95: 875– 882; 2014.
INITIAL DEMONSTRATION OF SUCCESSFUL ADOPTIVE CELL THERAPY: LAK CELLS WITH IL-2 The earliest adoptive cell therapy trials in the 1980s were conducted with LAK cells administered with high-dose IL-2 [1– 4]. LAK cells were autologous peripheral blood monocytes from a leukapheresis blood specimen that were incubated with IL-2 in media in large plastic plates and harvested after 72 h of activation to induce nonclass I-restricted lytic activity. The LAK trials resulted in significant response rates in patients with previously treated melanoma and established that IL-2 was a treatment modality for melanoma that could induce long-term survival in the modest proportion (15%) of patients who had CR and PR. The toxicity of the combination regimen was severe, predominantly as a result of the use of high-dose IL-2, resulting in a capillary leak syndrome that in some cases, was associated with noncardiogenic pulmonary edema, tachyarrhythmias, and renal failure. Initial trials in small numbers of patients showed response rates in melanoma for LAK therapy of ⬎20%, with 23 responses of 106 patients treated in one study. A subsequent, randomized trial of LAK cells, plus high-dose IL-2 compared with high-dose IL-2 alone in 181 cancer patients who had failed all standard therapy, included 54 melanoma patients. The data showed a trend toward improved survival for patients with melanoma who received IL-2 plus LAK cells compared with those who received IL-2 alone (at 24 months, 32% vs. 15% OS with a two-sided P⫽0.064). None of 26 patients with melanoma who received IL-2 alone was alive at 5 years of follow-up; five of 28 who received IL-2 plus LAK cells were alive, and three continued in CR beyond 5 years. These borderline data diminished enthusiasm for the use of LAK cells, as new murine preclinical data showed that antigen-specific T cells grown from tumors might be more therapeutically active. LAK cell therapy was the first adoptive cell treatment pursued in melanoma that resulted in tumor regression. The use of LAK cells with IL-2 was ultimately shown to be no better than high-dose IL-2 alone, however.
Abbreviations: CR⫽complete response(s), irRC⫽immune-related response criteria, LAK⫽lymphokine-activated killer, NCI⫽National Cancer Institute, NMA⫽nonmyeloablative, ORR⫽overall response rate, OS⫽overall survival, PR⫽partial response(s), REP⫽rapid expansion protocol, TBI⫽total body irradiation, TIL(s)⫽tumor-infiltrating lymphocytes
0741-5400/14/0095-875 © Society for Leukocyte Biology
1. Correspondence: Moffitt Cancer Center, 12902 Magnolia Dr., SRB-2, Tampa, FL 33612, USA. E-mail: [email protected]
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Important questions: what was the real difference between the LAK cell phenotype and T cell phenotype? What is their relation to NK cells and whether innate immune cells can ultimately be used for successful adoptive cell therapy? Does the concept of LAK cells need to be revisited as a result of greater understanding of innate immune effector features/phenotypes?
INITIAL EXPERIENCE WITH TIL TRIALS FROM 1988: TIL WITH HIGH-DOSE IL-2 The evolution of murine data, detailed in the accompanying review, paved the way for the initiation of clinical trials of adoptive therapy using T cells expanded from tumor digests administered with high-dose IL-2. Much if not all of this early technology was developed at the Surgery Branch of the NCI (Bethesda, MD, USA), and it was there in 1988 that the first patient was successfully treated with TIL [5]. All of the lessons from murine experiments were incorporated into the initial clinical trials, including the use of high doses of IL-2 in vitro to propagate the T cells, enzymatic digestion to generate the single-cell suspension, and the frequent transfer under sterile conditions of expanded T cells from 24-well plates to larger plates to gas-permeable bags. In the initial trial, 20 patients were treated with 11 responses seen and an ORR of 55%. Most were PR and of short duration, with a median OS of only ⬃12 months. No long-term survival data were available. A number of observations were made in that trial that have been seen consistently in subsequent trials of adoptive T cell therapy. Tumor regression occurred in some cases very quickly, and patients who had progressed through multiple lines of therapy could respond to treatment with TIL and IL-2. It appeared that CD8 predominant cultures were more successful than CD4 predominant cultures, but some patients with CD4 TIL could still show a response. Targeting studies did not show that the TIL could preferentially target tumors, and the longterm survival (greater than a few weeks) of the circulating TIL was very low [6 – 8]. Several variations on the idea of i.v. administration occurred, including the intra-arterial administration of TIL to patients with large, regionally advanced tumors, but that maneuver did not appear to benefit patients. TIL were found to be therapeutically active in melanoma but induced responses of short duration and did not traffic to tumor.
Important questions: what was the important clinical measure with TIL: is a high anti-tumor ORR important, or is long-term OS the key to therapeutic success? Or is duration of response the critical parameter to measure with TIL?
RECOGNITION OF CLINICAL AND TIL-RELATED FACTORS ASSOCIATED WITH POSITIVE OUTCOME As the clinical experience with adoptive transfer of TIL with high-dose IL-2 grew, sufficient patients were treated so that factors associated with clinical benefit, defined by overall response, could be assessed [9 –16]. Response rates remained at 876 Journal of Leukocyte Biology
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40 –50% in patients who had been treated previously with high-dose IL-2, with most responses not long-lasting. The number of CD8 T cells infused, the anti-tumor reactivity of the cells administered, and the rapidity of the kinetics with which the cells grew were all factors that were associated with an antitumor response. Interestingly, administration of predominant CD4 T cells was also shown to result in clinical responses, and those cells were often class II-restricted and capable of recognizing tumor cells by release of cytokine [17]. Nonetheless, median OS was only 12 months, and as in prior trials, follow-up was short, with little long-term survival data available. The number of TIL infused and the rapidity with which they expanded seemed to be associated with clinical response. The tumor reactivity of the successful TIL indicated that testing for tumor recognition might be a strategy to select the most effective TIL. This functional assay, however, did not allow a definition of the phenotype of the cells that were most likely to be beneficial for patients, which to this day, is a critical unanswered question in the adoptive cell-therapy field.
Important question: Are response rates to TIL-adoptive therapy at 40–50% as a result of pretherapy-intrinsic tumor factors, or does “tolerance”, as a result of IL-2-induced effects, need to be overcome?
USE OF SELECTION FOR GROWTH OF ANTIGEN-SPECIFIC TIL The key difference between the way TIL were grown in early adoptive transfer trials in the 1980s and 1990s and the newer trials of the late 1990s and early 2000s was the use of methods for rapid selection of TIL early in their proliferation for antigen specificity and a protocol to nonspecifically expand that population to large numbers, called a “REP”. In vitro experiments were performed to show that multiple fragments of the tumor could be plated in plastic 24-well plates, and the T cells that migrated out of the fragments could be expanded to large numbers over a several-week period by passaging them in 24-well plates. The resulting cells were easily tested for antigen specificity by incubating a small aliquot with autologous or cultured HLA-matched tumor targets and assessing release of IFN-␥ in the supernatant by an ELISA assay. In this manner, multiple cultures could be chosen for tumor specificity and pooled and then expanded in the presence of allogeneic feeder cells, IL-2, and the anti-CD3 antibody in the REP before adoptive transfer. The antigen specificity achieved by selection and large numbers achieved during the REP were important for the subsequent success of TIL protocols. Selection of TIL for tumor specificity, and the development of rapid expansion protocols were key innovations to facilitate more widespread clinical use of TIL and increase the likelihood of benefit from this complex treatment. Key unanswered questions were whether newer assays that were phenotypic and not just functional could be developed to be able to define clinically active TIL selectively and whether that knowledge could allow selective growth and expansion of only the most effective TIL.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
ADOPTION OF LYMPHOID-DEPLETION PROTOCOLS BEFORE TIL TRANSFER
TABLE 1. List of Past and Current Adoptive Cell Therapy Trials for Melanoma
The realization that homeostatic lymphoid proliferation that occurred after lymphoid depletion in rodent models resulted in increased immune “space” that favored the outgrowth and proliferation of adoptively transferred effector cells with increased anti-tumor activity led to the adoption of a lymphoiddepletion protocol for preparation before TIL transfer [18 – 25]. The 43 patients in the first series of Stage IV melanoma patients who received NMA chemotherapy before TIL and one cycle of high-dose IL-2 had an ORR rate of 49%. Five of those patients (12%) had a CR and were alive and progression-free at times ranging from 6.9 years to 8.6 years. The 16 PR all developed progressive disease, with 15 of the 16 patients progressing 2–36 months after treatment and only one long-term responder who did not progress until 7 years after treatment. There was no association between clinical response and sex, age, HLA type, metastatic stage, or numbers of TIL cells administered for patients in this NMA-only TIL trial. Clinical responders did differ from nonresponders, tolerating fewer doses of postinfusion IL-2. This study was important in that it revealed for the first time that patients could have long-term survival free of progression after TIL therapy. This regimen in various forms has been repeated at several other institutions recently, including Moffitt Cancer Center (Tampa, FL, USA) and MD Anderson Cancer Center (Houston, TX, USA). Investigators at MD Anderson have reported on 31 patients with metastatic melanoma, differing from NCI, in that anti-tumor reactivity was tested at the pre-REP stage but was not used as an absolute criterion to select patients for therapy [16]. Patients without tumor reactivity using in vitro assays were also treated. Clinical responses were not associated with in vitro anti-tumor reactivity pre-REP. Patients were administered two cycles of high-dose IL-2, one immediately after TIL infusion and the second 21 days later. A new approach to determining clinical response using irRC, felt to be more relevant to immunotherapy, was used in that trial. The MD Anderson investigators achieved an irRC ORR of 48% with a CR rate of 6.5%, with the longest follow-up at 36 months and no long-term survival data available. Progression-free survival of 12 months or more was observed for nine of 15 (60%) responding patients. The total number of TIL infused, the proportion of CD8⫹ T cells, a more differentiated effector phenotype of the CD8⫹ TIL, and a higher frequency of CD8⫹ T cells coexpressing the negative costimulation molecule B- and T-lymphocyte attenuator were parameters that were associated with clinical response. At the Moffitt Cancer Center, 13 patients were treated with TIL, selected for antigen reactivity, similar to the NCI protocols, to which are added the fastest growing cultures, in spite of selection [26]. In that trial, 31% had a PR, and 15% had a CR; these results are similar to the NCI data of 37% PR and 12% CR. The longest duration of follow-up of the Moffitt patients is 15 months, so that long-term survival data are unavailable. Investigators at Uppsala Medical Center (Sweden) obtained tumor material by ultrasound-guided core needle biopsy or surgery and were able to isolate from 0.5 to 30 billion TIL from 23 of 24 patients, 11 of which had core biopsies
• NCI-Surgery Branch Past: TIL with chemotherapy (NMA) ⫾ TBI Current: Phase II randomized trial of TIL/NMA versus TIL/NMA ⫹ TBI • MD Anderson Cancer Center Past: TIL with NMA without selection Current: TIL with NMA with DC vaccination • Moffitt Cancer Center Past: TIL with NMA with selection Current: Ipilimumab before TIL harvest and then TIL/NMA ⫹ ipilimumab • Sheba Medical Center, Israel Past: Young TIL with NMA Current: Rapidly expanded, young TIL with NMA • Herlev Medical Center, Denmark Past: Young TIL with NMA with low-dose IL-2 Current: Young TIL with NMA with intermediate-dose IL-2 • Uppsala Medical Center, Sweden Past: Selected TIL grown from core biopsies with low-dose IL-2
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for the TIL harvest. Selected and expanded TIL were used for treatment in combination with daily low-dose, s.c. IL-2 after prior lympho-depleting chemotherapy. One objective of the clinical responses was seen in one patient of 13 treated with TIL obtained from surgery and in four patients of 11 treated with TIL from core biopsies. The low numbers of TIL infused, low doses of IL-2, or the small sample of tumor for cell growth and expansion from the 11 patients who had core biopsies may account for the low response rate [27]. These trials and others are summarized in Table 1. Lymphoid depletion was the key to allowing TIL to persist in the recipient circulation and mediate improved clinical activity. It appeared to have many beneficial effects: it augmented IL-7 and IL-15 levels, created lymphoid space, and decreased endogenous T regulatory cells. Which of these effects is paramount is a critical, unanswered question in the field of adoptive cell therapy. The role of high-dose IL-2 or whether any IL-2 is required post-transfer is also important.
Important questions: How does cell volume in blood monitors get altered after adoptive therapy, and how does this affect homeostatic mechanisms of proliferation? Do IL-15 and IL-7 levels as a “cytokine sink” affect the efficacy of adoptive cell therapy and how? Is IL-2 required for in vivo expansion and persistence of adoptively transferred TIL?
USE OF YOUNG TIL Investigators at Sheba Medical Center (Tel Hashomer, Israel) treated 42 metastatic melanoma patients with “young” TIL, derived from tumors that were enzymatically digested, with no selection for antigen-specific T cells and bulk culture in preREP of 14 –18 days, followed by a REP phase of 14 days [28]. They reported an ORR of 40% in 42 patients with Stage IV melanoma and a 10% rate of CR. The longest follow-up for any patient was 36 months. This approach has also been tried Volume 95, June 2014
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at the NCI, with a similar procedure that minimized the time in culture and eliminated the individualized tumor-reactivity screening step [29, 30]. Young TIL cultures were established successfully from 83% of patients at NCI. Nineteen of 33 patients (58%) treated with CD8⫹-enriched, young TIL and NMA had an objective response, including three CR. Eleven of 23 patients (48%) treated with TIL and 6 Gy total-body irradiation had an objective response, including two CR. At MD Anderson Cancer Center, young TIL were shown to be therapeutically effective by the irRC, as mentioned previously [16]. Investigators in Denmark at Herlev Hospital treated six patients with lympho-depleting chemotherapy, TIL infusion, and 14 days of s.c. low-dose IL-2 injections at 2 million UI/day [31]. The cells were expanded, not by antigen specificity, but were selected for rapid expansion based on proliferative capacity (the fastest-growing cultures) and the highest percentage of CD3⫹, CD45RO⫹, CCR7⫹/⫺, and CD8⫹ phenotypic markers. Two of the six patients had a CR of 10⫹ and 30⫹ months, two were stable, and two progressed. These data, albeit in very small numbers of patients, suggest that a simplification of the TIL growth may be achieved without sacrificing the high response rate seen with this treatment and suggest that the young TIL approach has promise and may make TIL therapy more practical by decreasing the prolonged time required for cell expansion ex vivo. Young TIL that rapidly grew but were unselected were still therapeutically active and had the same phenotype as selected TIL. Unanswered questions included how to accelerate TIL expansion optimally yet retain therapeutic activity.
Important question: What is the phenotype of an effective TIL cell, and what checkpoint proteins are expressed on TIL?
NEWER WAYS TO GROW TIL A number of important questions have arisen as to the practical implications of using adoptive cell therapy with TIL in patients. The process of expanding TIL would best be done using a closed system to ensure sterility, with a minimal number of steps requiring intervention and with a great level of scalability and a low requirement for technician time. As a result of the high cost of medium and the IL-2 used to grow the cells, a decrease in volume and requirement for media would provide a major cost saving. Gas-permeable flasks and the “WAVE” bioreactor have been successfully used to propagate TIL while saving space and minimizing the use of IL-2 in the rapid expansion, which is quite expensive [32, 33]. The GREX100 is a 400-mL capacity flask with a 100-cm2 gas-permeable silicone bottom and has been used to decrease the requirements for media and IL-2 needed for standard growth of TIL using 30 – 60, 3-L bags. The WAVE bioreactor-generated cultures require medium perfusion and replacement, did not have a reduced requirement for volume of media and amount of IL-2, but generated TIL with a higher percentage of CD4⫹ cells and had a less-activated phenotype. This technology does not appear to result in more clinically active TIL. The potential commercialization of TIL will likely depend on the use of 878 Journal of Leukocyte Biology
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a device to minimize the cytokines and media used and provide a closed system to minimize contamination.
TIL CAN HAVE ACTIVITY IN THE BRAIN Of 17 patients who received TIL at the NCI and had untreated brain metastases, seven (41%) achieved a CR in the brain, and six patients achieved a PR [34]. One patient developed a subarachnoid hemorrhage related to a tumor during the period of lympho-depleting, chemotherapy-induced thrombocytopenia and had an uneventful resection of a brain metastasis. These data suggest that patients with small-volume or previously treated brain metastases would be reasonable candidates for adoptive cell therapy with TIL. The ultimate commercialization of TIL and the ability to conduct a registration trial of TIL versus a standard comparator arm critically depend on the ability to manufacture, easily, reliably, and reproducibly, large quantities of cells for adoptive transfer with a sterile and well-defined growth system. Newer technologies for cell growth using closed systems are therefore critical to the more-widespread use of TIL and other adoptive cell therapies.
Important question: By what means can we improve the expansion of TIL to decrease the time to propagate them to adequate quantities and enrich for therapeutically active cells?
ADDITION OF VACCINE APPROACHES TO T CELL-ADOPTIVE TRANSFER The ability of vaccine-induced, adoptively transferred PBMCs to mediate tumor regression after lymphodepletion was assessed using autologous PBMCs from nine gp100-vaccinated patients with metastatic melanoma [35]. Cells were stimulated ex vivo with the gp100:209-217(210M) peptide and transferred in combination with high-dose IL-2 and additional gp100 peptide. Transferred PBMCs contained highly avid, gp100:209-217 peptide-reactive CD8⫹ T cells. One week after transfer, lymphocyte counts peaked with a median of 14.3 ⫻ 103 cells/mL, with 56% of patients experiencing a lymphocytosis. gp100 peptide-specific CD8⫹ T cells persisted at high levels in the blood of all patients and demonstrated significant tumor-specific, IFN-␥ secretion in vitro. Autoimmunity directed against melanocytes was noted in two patients; however, no objective clinical responses were seen. A patient received sequential treatments with autologous TIL that recognized the gp100 melanocyte-differentiation antigen. Although no clinical response was seen when cells were administered alone, a response to therapy was seen with TIL administered together with a highly immunogenic fowlpox vaccine expressing a gp100:209217(210M) epitope [36]. Persistence of the transferred antigen-specific lymphocytes in the peripheral blood was observed only after adoptive cell therapy, plus administration of fowlpox vaccine. Cell proliferation in vitro was stimulated further by an additional vaccine and IL-2. The patient has an ongoing, PR at 10 months after the last treatment.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
Addition of vaccines to amplify TIL responses is a promising approach still early in testing. The lack of availability of an active vaccine in solid tumor oncology is a major stumbling block to improve upon the results with TIL therapy. Important question: How can we pretreat tumors to augment their infiltration with potentially therapeutic TIL for subsequent harvest?
ADDITION OF TBI TO LYMPHOID DEPLETION BEFORE TIL TRANSFER Work with murine models of adoptive cell therapy detailed above suggested that the addition of radiation and other lympho-depleting influences augmented the therapeutic effect of adoptively transferred T cells [21, 22]. To evaluate this phenomenon in patients, TBI was added to NMA chemotherapy with 2 days of cytoxan and 5 days of fludarabine. When 2 or 12 Gy TBI was added to TIL plus IL-2, the response rates were 52% and 72%, respectively, compared with 49% with NMA alone. Responses were seen in all sites of tumors, including the brain. There was one treatmentrelated death in the 93 patients described in one report from the NCI. Host lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic cytokines IL-7 and IL-15. Animal models and clinical experience suggested that more lymphoid depletion with addition of TBI improved results clinically, but these data have not yet been borne out in clinical practice. The major unanswered questions are why the profound lymphoid depletion seems necessary for the success of adoptive cell therapy and whether less-toxic means can substitute.
USE OF CD8 T CELL CLONES FOR ADOPTIVE THERAPY T cell clones were derived from peripheral blood lymphocytes or TIL of patients who had received prior immunization with the melanoma-associated antigen, gpl00. In response to its cognate antigen, each clone used for treatment secreted large amounts of IFN-␥ and GM-CSF, lesser amounts of IL-2 and TNF-␣, and little or no IL-4 and IL-10. Clones also demonstrated recognition of human leukocyte antigen-matched melanomas by cytokine secretion and lysis assays [37]. Twelve patients received two cycles of cells alone; 11 patients received additional cycles of cells and were randomly allocated to receive one of two schedules of IL-2 (125,000 IU/kg s.c. daily for 12 days vs. 720,000 IU/kg i.v. every 8 h for 4 days). An average of 1 ⫻ 1010 cells was transferred/cycle. Peripheral blood samples were analyzed for persistence of transferred cells by TCR-specific PCR. Transferred cells reached a maximum level at 1 h after transfer but declined rapidly to undetectable levels by 2 weeks. One minor response and one mixed response were observed (both in the high-dose IL-2 arm). This report demonstrates the safety and feasibility of cloned T cell
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transfer as a therapy for patients with melanoma. Transfer of different or additional cell types or modulation of the recipient host environment appears to be required, however, for successful therapy [38]. Investigators at the University of Washington (Seattle, WA, USA) performed a trial to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8⫹ T cell clones targeting melanoma antigens, Melan-A and gp100 [39]. Four infusions were given: the first without IL-2 and subsequent infusions with lowdose IL-2 in escalating doses twice daily for the second, third, and fourth infusions, respectively. No serious toxicity was observed in 10 patients treated. The adoptively transferred T cell clones persisted in vivo after low-dose IL-2 was found to localize to tumor sites and induced minor or mixed responses or stable disease in eight of 10 patients for up to 21 months, but no objective responses were observed. In a follow-up trial, patients received two infusions of a single tumor-reactive, antigen-specific CD8 clone, expanded to 1010/M2; the first infusion was given without fludarabine conditioning, and the second infusion was given after fludarabine (25 mg/m2/day⫻5 days) [40]. Ten patients were treated with no serious toxicities. Three of nine evaluable patients experienced a minor response or stable disease for 5.8 –11.0 months. Fludarabine led to a 2.9-fold improvement in the in vivo persistence of transferred CTL clones from a median of 4.5 days to 13.0 days (P⬍0.05). Fludarabine lymphodepletion increased plasma levels of the homeostatic cytokines IL-7 and IL-15. Recently, 11 patients with refractory melanoma received cyclophosphamide as conditioning before the infusion of PBMC-derived, antigen-specific CD8⫹ clones, followed by low-dose or high-dose IL-2. No lifethreatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 weeks, and one of 11 had a complete remission at 36⫹ months. Immunerelated adverse events of skin rash were observed in patients with stable disease or complete remission at 4 weeks or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to skin and lymph nodes. CD8 CTL that persisted long-term acquired phenotypic and/or functional qualities of central memory-type T cells in vivo [41]. CD8 T cell clones were clinically active but required additional IL-2 and did not persist in the host, mediating modest benefit. Persistence of the adoptively transferred cells in the periphery seems key to their therapeutic success, and that didn’t occur with the CD8 clones. Important unanswered questions are whether oligoclonal T cells are important for successful adoptive cell therapy and whether CD4 T cell help is needed.
USE OF CD4 T CELL CLONES FOR ADOPTIVE THERAPY Investigators at the University of Washington, who worked with CD8 T cell clones for adoptive transfer, have described the use of a CD4 clone infused into a patient with refractory melanoma who had not undergone any previous condiVolume 95, June 2014
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TABLE 2. Important Questions to be Answered in the TIL Field What was the real difference between the LAK cell phenotype and T cell phenotype? What is their relation to NK cells and whether innate immune cells can ultimately be used for successful adoptive cell therapy? Does the concept of LAK cells need to be revisited as a result of greater understanding of innate immune effector features/phenotypes? What was the important clinical measure with TIL: is a high anti-tumor ORR important, or is long-term OS the key to therapeutic success? Or is duration of response the critical parameter to measure with TIL? Are response rates to TIL-adoptive therapy at 40–50% as a result of pretherapy-intrinsic tumor factors, or does “tolerance”, as a result of IL-2-induced effects, need to be overcome? How does cell volume in blood monitors get altered after adoptive therapy, and how does this affect homeostatic mechanisms of proliferation? Do IL-15 and IL-7 levels as a “cytokine sink” affect the efficacy of adoptive cell therapy and how? How does the presence or absence of killer cell Ig-like receptors and other inhibitory receptors affect LAK or TIL function? Is IL-2 required for in vivo expansion and persistence of adoptively transferred TIL? What is the phenotype of an effective TIL cell, and what checkpoint proteins are expressed on TIL? By what means can we improve the expansion of TIL to decrease the time to propagate them to adequate quantities and enrich for therapeutically active cells? How can we pretreat tumors to augment their infiltration with potentially therapeutic TIL for subsequent harvest?
tioning to induce lymphopenia or cytokine treatment. CD4 T cells, stimulated with a DPB1*0401-restricted epitope of a peptide derived from NY-ESO-1, were isolated from the peripheral blood, cloned, and expanded for cellular therapy using a REP protocol similar to that used for TIL [42]. The clonal CD4⫹ T cells mediated a durable clinical remission and led to an endogenous T cell epitope spreading against melanoma antigens other than NY-ESO-1. Transferred CD4 clones remained detectable in the patient’s blood for ⬎80 days, during which time, the frequency fluctuated between 0.7% and 3.0% of the total number of mononuclear cells.
TCR GENE-TRANSFER PROTOCOLS The ability to clone two-chain TCR sequences into retroviral and lentiviral vectors had led to gene therapy trials, in which TCR sequences have been transferred to PBMCs [43– 45]. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels ⬎10% of peripheral blood lymphocytes for at least 2 months after the infusion. High, sustained levels of circulating, gene-engineered cells were observed 1 year after infusion in two patients, who both demonstrated objective regression of metastatic melanoma lesions. Transgenic mice were immunized with human melanoma cells, and high-throughput screening of lymphocytes was conducted to generate TCRs highly reactive to mela noma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA⫺ and CD45RO⫹ after ex vivo expansion. After infusion, persisting cells displayed a CD45RA⫹ and CD45RO⫺ phenotype. Gene-engineered cells persisted at high levels in the blood of all patients, 1 month after treatment. Responding patients demonstrated higher ex vivo antitumor T cell reactivity than nonresponders. Tumor regression was seen in 30% and 19% of patients who re880 Journal of Leukocyte Biology
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ceived the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediated cancer regression in humans and targeted rare cognate antigen-containing cells throughout the body. Patients with NY-ESO-1-positive tumors were treated with autologous, TCR-transduced T cells plus high-dose IL-2 to tolerance after preparative chemotherapy to induce lymphoid depletion. Objective clinical responses were observed in five of 11 patients with mela noma-bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted beyond 1 year. Transduced TCR gene therapy approaches are promising but have the hazard of toxicity as a result of potent cross-reactivity with normal tissues, which is a major concern.
NK CELL-ADOPTIVE TRANSFER TRIALS Eight patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred, in vitro-activated, autologous NK cells after the patients received a lympho-depleting NMA chemotherapy regimen [46]. The infused cells exhibited high levels of lytic activity in vitro. There were no clinical responses seen in this trial. The adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least 1 week post-transfer and in some patients, for several months. Persistent NK cells in the circulation expressed significantly lower levels of the activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2. The persistent NK cells mediated antibody-dependent, cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggested to the authors that coupling adoptive NK cell transfer with mAb administration deserved evaluation.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
CONCLUSIONS As the technology for the generation of tumor-specific T cells has advanced, TIL and adoptive T cell therapy have advanced into the mainstream of clinical trials at multiple institutions around the world. Important elements that have improved the clinical results with TIL therapy include antigen-specific selection, rapid expansion, and lymphoid depletion with chemotherapy and/or radiation. The need for accurate, predictive markers for the success of TIL therapy is vital. We look forward to the use of new checkpoint protein inhibitors, novel cytokines, and genetic modification to facilitate the generation of large numbers of tumor-specific effector cells from tumor biopsies or from the peripheral blood that have the proper phenotype and the ability to persist long-term in the circulation and mediate potent anti-tumor effects to increase the number of patients who are cured of melanoma. Success with melanoma-adoptive cell therapy, particularly with TIL, has led to its use in other histologies that are currently undergoing testing (Table 2).
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(2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin. Cancer Res. 17, 4550 –4557. Pilon-Thomas, S., Kuhn, L., Ellwanger, S., Janssen, W., Royster, E., Marzban, S., Kudchadkar, R., Zager, J., Gibney, G., Sondak, V. K., Weber, J., Mulé, J. J., Sarnaik, A. A. (2012) Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J. Immunother. 35, 615–620. Ullenhag, G. J., Sadeghi, A. M., Carlsson, B., Ahlström, H., Mosavi, F., Wagenius, G., Tötterman, T. H. (2012) Adoptive T-cell therapy for malignant melanoma patients with TILs obtained by ultrasound-guided needle biopsy. Cancer Immunol. Immunother. 61, 725–732. Besser, M. J., Shapira-Frommer, R., Treves, A. J., Zippel, D., Itzhaki, O., Hershkovitz, L., Levy, D., Kubi, A., Hovav, E., Chermoshniuk, N., Shalmon, B., Hardan, I., Catane, R., Markel, G., Apter, S., Ben-Nun, A., Kuchuk, I., Shimoni, A., Nagler, A., Schachter, J. (2010) Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin. Cancer Res. 16, 2646 –2655. Tran, K. Q., Zhou, J., Durflinger, K. H., Langhan, M. M., Shelton, T. E., Wunderlich, J. R., Robbins, P. F., Rosenberg, S. A., Dudley,
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KEY WORDS: T cell 䡠 immunotherapy 䡠 IL-2 䡠 lymphoid depletion 䡠 T cell receptor
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At the Bedside: Adoptive cell therapy for melanoma— clinical development Jeffrey S. Weber1 Moffitt Cancer Center and the Donald A. Adam Comprehensive Melanoma Research Center, Tampa, Florida, USA RECEIVED MAY 21, 2013; REVISED JANUARY 5, 2014; ACCEPTED MARCH 13, 2014. DOI: 10.1189/jlb.0513293
‹ SEE CORRESPONDING ARTICLE ON PAGE 867
ABSTRACT Adoptive cell therapy for melanoma, particularly using TIL, consists of a complex and difficult set of procedures, although it has a strong preclinical background and justification and has been pursued clinically by one small group of investigators over the last 20 years. More recent developments and a better understanding of the molecular basis of the anti-tumor immune response have led to the conduct of clinical trials that use lymphoid depletion with chemotherapy and/or TBI to exploit the favorable immune milieu of homeostatic lymphoid reconstitution during transfer of effector T cells. Improved ways of propagating T cells ex vivo have also simplified and shortened the cell-growth process. Current TIL trials have now been expanded beyond the initial center where it was developed, reproducing excellent objective response rates of 40 –50% in previously treated melanoma patients and more importantly, demonstrating that a significant proportion of patients will be alive and free of disease 3–5 years after treatment, raising the possibility that those patients may be cured of their disease. Newer methods for growing the infiltrating T cells using immune-checkpoint antibodies or other agents to condition the tumor before harvest and improved technology to simplify the complex and often cumbersome cell-growth process suggest that this technology may be able to be disseminated to a wide selection of cancer centers and may be a candidate for testing in a randomized Phase III trial to show definitively its benefit in patients with metastatic melanoma. In the accompanying review, the preclinical work that supports the idea of adoptive cell therapy with TIL and expands the concept in promising new ways will be explored. J. Leukoc. Biol. 95: 875– 882; 2014.
INITIAL DEMONSTRATION OF SUCCESSFUL ADOPTIVE CELL THERAPY: LAK CELLS WITH IL-2 The earliest adoptive cell therapy trials in the 1980s were conducted with LAK cells administered with high-dose IL-2 [1– 4]. LAK cells were autologous peripheral blood monocytes from a leukapheresis blood specimen that were incubated with IL-2 in media in large plastic plates and harvested after 72 h of activation to induce nonclass I-restricted lytic activity. The LAK trials resulted in significant response rates in patients with previously treated melanoma and established that IL-2 was a treatment modality for melanoma that could induce long-term survival in the modest proportion (15%) of patients who had CR and PR. The toxicity of the combination regimen was severe, predominantly as a result of the use of high-dose IL-2, resulting in a capillary leak syndrome that in some cases, was associated with noncardiogenic pulmonary edema, tachyarrhythmias, and renal failure. Initial trials in small numbers of patients showed response rates in melanoma for LAK therapy of ⬎20%, with 23 responses of 106 patients treated in one study. A subsequent, randomized trial of LAK cells, plus high-dose IL-2 compared with high-dose IL-2 alone in 181 cancer patients who had failed all standard therapy, included 54 melanoma patients. The data showed a trend toward improved survival for patients with melanoma who received IL-2 plus LAK cells compared with those who received IL-2 alone (at 24 months, 32% vs. 15% OS with a two-sided P⫽0.064). None of 26 patients with melanoma who received IL-2 alone was alive at 5 years of follow-up; five of 28 who received IL-2 plus LAK cells were alive, and three continued in CR beyond 5 years. These borderline data diminished enthusiasm for the use of LAK cells, as new murine preclinical data showed that antigen-specific T cells grown from tumors might be more therapeutically active. LAK cell therapy was the first adoptive cell treatment pursued in melanoma that resulted in tumor regression. The use of LAK cells with IL-2 was ultimately shown to be no better than high-dose IL-2 alone, however.
Abbreviations: CR⫽complete response(s), irRC⫽immune-related response criteria, LAK⫽lymphokine-activated killer, NCI⫽National Cancer Institute, NMA⫽nonmyeloablative, ORR⫽overall response rate, OS⫽overall survival, PR⫽partial response(s), REP⫽rapid expansion protocol, TBI⫽total body irradiation, TIL(s)⫽tumor-infiltrating lymphocytes
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1. Correspondence: Moffitt Cancer Center, 12902 Magnolia Dr., SRB-2, Tampa, FL 33612, USA. E-mail: [email protected]
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Important questions: what was the real difference between the LAK cell phenotype and T cell phenotype? What is their relation to NK cells and whether innate immune cells can ultimately be used for successful adoptive cell therapy? Does the concept of LAK cells need to be revisited as a result of greater understanding of innate immune effector features/phenotypes?
INITIAL EXPERIENCE WITH TIL TRIALS FROM 1988: TIL WITH HIGH-DOSE IL-2 The evolution of murine data, detailed in the accompanying review, paved the way for the initiation of clinical trials of adoptive therapy using T cells expanded from tumor digests administered with high-dose IL-2. Much if not all of this early technology was developed at the Surgery Branch of the NCI (Bethesda, MD, USA), and it was there in 1988 that the first patient was successfully treated with TIL [5]. All of the lessons from murine experiments were incorporated into the initial clinical trials, including the use of high doses of IL-2 in vitro to propagate the T cells, enzymatic digestion to generate the single-cell suspension, and the frequent transfer under sterile conditions of expanded T cells from 24-well plates to larger plates to gas-permeable bags. In the initial trial, 20 patients were treated with 11 responses seen and an ORR of 55%. Most were PR and of short duration, with a median OS of only ⬃12 months. No long-term survival data were available. A number of observations were made in that trial that have been seen consistently in subsequent trials of adoptive T cell therapy. Tumor regression occurred in some cases very quickly, and patients who had progressed through multiple lines of therapy could respond to treatment with TIL and IL-2. It appeared that CD8 predominant cultures were more successful than CD4 predominant cultures, but some patients with CD4 TIL could still show a response. Targeting studies did not show that the TIL could preferentially target tumors, and the longterm survival (greater than a few weeks) of the circulating TIL was very low [6 – 8]. Several variations on the idea of i.v. administration occurred, including the intra-arterial administration of TIL to patients with large, regionally advanced tumors, but that maneuver did not appear to benefit patients. TIL were found to be therapeutically active in melanoma but induced responses of short duration and did not traffic to tumor.
Important questions: what was the important clinical measure with TIL: is a high anti-tumor ORR important, or is long-term OS the key to therapeutic success? Or is duration of response the critical parameter to measure with TIL?
RECOGNITION OF CLINICAL AND TIL-RELATED FACTORS ASSOCIATED WITH POSITIVE OUTCOME As the clinical experience with adoptive transfer of TIL with high-dose IL-2 grew, sufficient patients were treated so that factors associated with clinical benefit, defined by overall response, could be assessed [9 –16]. Response rates remained at 876 Journal of Leukocyte Biology
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40 –50% in patients who had been treated previously with high-dose IL-2, with most responses not long-lasting. The number of CD8 T cells infused, the anti-tumor reactivity of the cells administered, and the rapidity of the kinetics with which the cells grew were all factors that were associated with an antitumor response. Interestingly, administration of predominant CD4 T cells was also shown to result in clinical responses, and those cells were often class II-restricted and capable of recognizing tumor cells by release of cytokine [17]. Nonetheless, median OS was only 12 months, and as in prior trials, follow-up was short, with little long-term survival data available. The number of TIL infused and the rapidity with which they expanded seemed to be associated with clinical response. The tumor reactivity of the successful TIL indicated that testing for tumor recognition might be a strategy to select the most effective TIL. This functional assay, however, did not allow a definition of the phenotype of the cells that were most likely to be beneficial for patients, which to this day, is a critical unanswered question in the adoptive cell-therapy field.
Important question: Are response rates to TIL-adoptive therapy at 40–50% as a result of pretherapy-intrinsic tumor factors, or does “tolerance”, as a result of IL-2-induced effects, need to be overcome?
USE OF SELECTION FOR GROWTH OF ANTIGEN-SPECIFIC TIL The key difference between the way TIL were grown in early adoptive transfer trials in the 1980s and 1990s and the newer trials of the late 1990s and early 2000s was the use of methods for rapid selection of TIL early in their proliferation for antigen specificity and a protocol to nonspecifically expand that population to large numbers, called a “REP”. In vitro experiments were performed to show that multiple fragments of the tumor could be plated in plastic 24-well plates, and the T cells that migrated out of the fragments could be expanded to large numbers over a several-week period by passaging them in 24-well plates. The resulting cells were easily tested for antigen specificity by incubating a small aliquot with autologous or cultured HLA-matched tumor targets and assessing release of IFN-␥ in the supernatant by an ELISA assay. In this manner, multiple cultures could be chosen for tumor specificity and pooled and then expanded in the presence of allogeneic feeder cells, IL-2, and the anti-CD3 antibody in the REP before adoptive transfer. The antigen specificity achieved by selection and large numbers achieved during the REP were important for the subsequent success of TIL protocols. Selection of TIL for tumor specificity, and the development of rapid expansion protocols were key innovations to facilitate more widespread clinical use of TIL and increase the likelihood of benefit from this complex treatment. Key unanswered questions were whether newer assays that were phenotypic and not just functional could be developed to be able to define clinically active TIL selectively and whether that knowledge could allow selective growth and expansion of only the most effective TIL.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
ADOPTION OF LYMPHOID-DEPLETION PROTOCOLS BEFORE TIL TRANSFER
TABLE 1. List of Past and Current Adoptive Cell Therapy Trials for Melanoma
The realization that homeostatic lymphoid proliferation that occurred after lymphoid depletion in rodent models resulted in increased immune “space” that favored the outgrowth and proliferation of adoptively transferred effector cells with increased anti-tumor activity led to the adoption of a lymphoiddepletion protocol for preparation before TIL transfer [18 – 25]. The 43 patients in the first series of Stage IV melanoma patients who received NMA chemotherapy before TIL and one cycle of high-dose IL-2 had an ORR rate of 49%. Five of those patients (12%) had a CR and were alive and progression-free at times ranging from 6.9 years to 8.6 years. The 16 PR all developed progressive disease, with 15 of the 16 patients progressing 2–36 months after treatment and only one long-term responder who did not progress until 7 years after treatment. There was no association between clinical response and sex, age, HLA type, metastatic stage, or numbers of TIL cells administered for patients in this NMA-only TIL trial. Clinical responders did differ from nonresponders, tolerating fewer doses of postinfusion IL-2. This study was important in that it revealed for the first time that patients could have long-term survival free of progression after TIL therapy. This regimen in various forms has been repeated at several other institutions recently, including Moffitt Cancer Center (Tampa, FL, USA) and MD Anderson Cancer Center (Houston, TX, USA). Investigators at MD Anderson have reported on 31 patients with metastatic melanoma, differing from NCI, in that anti-tumor reactivity was tested at the pre-REP stage but was not used as an absolute criterion to select patients for therapy [16]. Patients without tumor reactivity using in vitro assays were also treated. Clinical responses were not associated with in vitro anti-tumor reactivity pre-REP. Patients were administered two cycles of high-dose IL-2, one immediately after TIL infusion and the second 21 days later. A new approach to determining clinical response using irRC, felt to be more relevant to immunotherapy, was used in that trial. The MD Anderson investigators achieved an irRC ORR of 48% with a CR rate of 6.5%, with the longest follow-up at 36 months and no long-term survival data available. Progression-free survival of 12 months or more was observed for nine of 15 (60%) responding patients. The total number of TIL infused, the proportion of CD8⫹ T cells, a more differentiated effector phenotype of the CD8⫹ TIL, and a higher frequency of CD8⫹ T cells coexpressing the negative costimulation molecule B- and T-lymphocyte attenuator were parameters that were associated with clinical response. At the Moffitt Cancer Center, 13 patients were treated with TIL, selected for antigen reactivity, similar to the NCI protocols, to which are added the fastest growing cultures, in spite of selection [26]. In that trial, 31% had a PR, and 15% had a CR; these results are similar to the NCI data of 37% PR and 12% CR. The longest duration of follow-up of the Moffitt patients is 15 months, so that long-term survival data are unavailable. Investigators at Uppsala Medical Center (Sweden) obtained tumor material by ultrasound-guided core needle biopsy or surgery and were able to isolate from 0.5 to 30 billion TIL from 23 of 24 patients, 11 of which had core biopsies
• NCI-Surgery Branch Past: TIL with chemotherapy (NMA) ⫾ TBI Current: Phase II randomized trial of TIL/NMA versus TIL/NMA ⫹ TBI • MD Anderson Cancer Center Past: TIL with NMA without selection Current: TIL with NMA with DC vaccination • Moffitt Cancer Center Past: TIL with NMA with selection Current: Ipilimumab before TIL harvest and then TIL/NMA ⫹ ipilimumab • Sheba Medical Center, Israel Past: Young TIL with NMA Current: Rapidly expanded, young TIL with NMA • Herlev Medical Center, Denmark Past: Young TIL with NMA with low-dose IL-2 Current: Young TIL with NMA with intermediate-dose IL-2 • Uppsala Medical Center, Sweden Past: Selected TIL grown from core biopsies with low-dose IL-2
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for the TIL harvest. Selected and expanded TIL were used for treatment in combination with daily low-dose, s.c. IL-2 after prior lympho-depleting chemotherapy. One objective of the clinical responses was seen in one patient of 13 treated with TIL obtained from surgery and in four patients of 11 treated with TIL from core biopsies. The low numbers of TIL infused, low doses of IL-2, or the small sample of tumor for cell growth and expansion from the 11 patients who had core biopsies may account for the low response rate [27]. These trials and others are summarized in Table 1. Lymphoid depletion was the key to allowing TIL to persist in the recipient circulation and mediate improved clinical activity. It appeared to have many beneficial effects: it augmented IL-7 and IL-15 levels, created lymphoid space, and decreased endogenous T regulatory cells. Which of these effects is paramount is a critical, unanswered question in the field of adoptive cell therapy. The role of high-dose IL-2 or whether any IL-2 is required post-transfer is also important.
Important questions: How does cell volume in blood monitors get altered after adoptive therapy, and how does this affect homeostatic mechanisms of proliferation? Do IL-15 and IL-7 levels as a “cytokine sink” affect the efficacy of adoptive cell therapy and how? Is IL-2 required for in vivo expansion and persistence of adoptively transferred TIL?
USE OF YOUNG TIL Investigators at Sheba Medical Center (Tel Hashomer, Israel) treated 42 metastatic melanoma patients with “young” TIL, derived from tumors that were enzymatically digested, with no selection for antigen-specific T cells and bulk culture in preREP of 14 –18 days, followed by a REP phase of 14 days [28]. They reported an ORR of 40% in 42 patients with Stage IV melanoma and a 10% rate of CR. The longest follow-up for any patient was 36 months. This approach has also been tried Volume 95, June 2014
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at the NCI, with a similar procedure that minimized the time in culture and eliminated the individualized tumor-reactivity screening step [29, 30]. Young TIL cultures were established successfully from 83% of patients at NCI. Nineteen of 33 patients (58%) treated with CD8⫹-enriched, young TIL and NMA had an objective response, including three CR. Eleven of 23 patients (48%) treated with TIL and 6 Gy total-body irradiation had an objective response, including two CR. At MD Anderson Cancer Center, young TIL were shown to be therapeutically effective by the irRC, as mentioned previously [16]. Investigators in Denmark at Herlev Hospital treated six patients with lympho-depleting chemotherapy, TIL infusion, and 14 days of s.c. low-dose IL-2 injections at 2 million UI/day [31]. The cells were expanded, not by antigen specificity, but were selected for rapid expansion based on proliferative capacity (the fastest-growing cultures) and the highest percentage of CD3⫹, CD45RO⫹, CCR7⫹/⫺, and CD8⫹ phenotypic markers. Two of the six patients had a CR of 10⫹ and 30⫹ months, two were stable, and two progressed. These data, albeit in very small numbers of patients, suggest that a simplification of the TIL growth may be achieved without sacrificing the high response rate seen with this treatment and suggest that the young TIL approach has promise and may make TIL therapy more practical by decreasing the prolonged time required for cell expansion ex vivo. Young TIL that rapidly grew but were unselected were still therapeutically active and had the same phenotype as selected TIL. Unanswered questions included how to accelerate TIL expansion optimally yet retain therapeutic activity.
Important question: What is the phenotype of an effective TIL cell, and what checkpoint proteins are expressed on TIL?
NEWER WAYS TO GROW TIL A number of important questions have arisen as to the practical implications of using adoptive cell therapy with TIL in patients. The process of expanding TIL would best be done using a closed system to ensure sterility, with a minimal number of steps requiring intervention and with a great level of scalability and a low requirement for technician time. As a result of the high cost of medium and the IL-2 used to grow the cells, a decrease in volume and requirement for media would provide a major cost saving. Gas-permeable flasks and the “WAVE” bioreactor have been successfully used to propagate TIL while saving space and minimizing the use of IL-2 in the rapid expansion, which is quite expensive [32, 33]. The GREX100 is a 400-mL capacity flask with a 100-cm2 gas-permeable silicone bottom and has been used to decrease the requirements for media and IL-2 needed for standard growth of TIL using 30 – 60, 3-L bags. The WAVE bioreactor-generated cultures require medium perfusion and replacement, did not have a reduced requirement for volume of media and amount of IL-2, but generated TIL with a higher percentage of CD4⫹ cells and had a less-activated phenotype. This technology does not appear to result in more clinically active TIL. The potential commercialization of TIL will likely depend on the use of 878 Journal of Leukocyte Biology
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a device to minimize the cytokines and media used and provide a closed system to minimize contamination.
TIL CAN HAVE ACTIVITY IN THE BRAIN Of 17 patients who received TIL at the NCI and had untreated brain metastases, seven (41%) achieved a CR in the brain, and six patients achieved a PR [34]. One patient developed a subarachnoid hemorrhage related to a tumor during the period of lympho-depleting, chemotherapy-induced thrombocytopenia and had an uneventful resection of a brain metastasis. These data suggest that patients with small-volume or previously treated brain metastases would be reasonable candidates for adoptive cell therapy with TIL. The ultimate commercialization of TIL and the ability to conduct a registration trial of TIL versus a standard comparator arm critically depend on the ability to manufacture, easily, reliably, and reproducibly, large quantities of cells for adoptive transfer with a sterile and well-defined growth system. Newer technologies for cell growth using closed systems are therefore critical to the more-widespread use of TIL and other adoptive cell therapies.
Important question: By what means can we improve the expansion of TIL to decrease the time to propagate them to adequate quantities and enrich for therapeutically active cells?
ADDITION OF VACCINE APPROACHES TO T CELL-ADOPTIVE TRANSFER The ability of vaccine-induced, adoptively transferred PBMCs to mediate tumor regression after lymphodepletion was assessed using autologous PBMCs from nine gp100-vaccinated patients with metastatic melanoma [35]. Cells were stimulated ex vivo with the gp100:209-217(210M) peptide and transferred in combination with high-dose IL-2 and additional gp100 peptide. Transferred PBMCs contained highly avid, gp100:209-217 peptide-reactive CD8⫹ T cells. One week after transfer, lymphocyte counts peaked with a median of 14.3 ⫻ 103 cells/mL, with 56% of patients experiencing a lymphocytosis. gp100 peptide-specific CD8⫹ T cells persisted at high levels in the blood of all patients and demonstrated significant tumor-specific, IFN-␥ secretion in vitro. Autoimmunity directed against melanocytes was noted in two patients; however, no objective clinical responses were seen. A patient received sequential treatments with autologous TIL that recognized the gp100 melanocyte-differentiation antigen. Although no clinical response was seen when cells were administered alone, a response to therapy was seen with TIL administered together with a highly immunogenic fowlpox vaccine expressing a gp100:209217(210M) epitope [36]. Persistence of the transferred antigen-specific lymphocytes in the peripheral blood was observed only after adoptive cell therapy, plus administration of fowlpox vaccine. Cell proliferation in vitro was stimulated further by an additional vaccine and IL-2. The patient has an ongoing, PR at 10 months after the last treatment.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
Addition of vaccines to amplify TIL responses is a promising approach still early in testing. The lack of availability of an active vaccine in solid tumor oncology is a major stumbling block to improve upon the results with TIL therapy. Important question: How can we pretreat tumors to augment their infiltration with potentially therapeutic TIL for subsequent harvest?
ADDITION OF TBI TO LYMPHOID DEPLETION BEFORE TIL TRANSFER Work with murine models of adoptive cell therapy detailed above suggested that the addition of radiation and other lympho-depleting influences augmented the therapeutic effect of adoptively transferred T cells [21, 22]. To evaluate this phenomenon in patients, TBI was added to NMA chemotherapy with 2 days of cytoxan and 5 days of fludarabine. When 2 or 12 Gy TBI was added to TIL plus IL-2, the response rates were 52% and 72%, respectively, compared with 49% with NMA alone. Responses were seen in all sites of tumors, including the brain. There was one treatmentrelated death in the 93 patients described in one report from the NCI. Host lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic cytokines IL-7 and IL-15. Animal models and clinical experience suggested that more lymphoid depletion with addition of TBI improved results clinically, but these data have not yet been borne out in clinical practice. The major unanswered questions are why the profound lymphoid depletion seems necessary for the success of adoptive cell therapy and whether less-toxic means can substitute.
USE OF CD8 T CELL CLONES FOR ADOPTIVE THERAPY T cell clones were derived from peripheral blood lymphocytes or TIL of patients who had received prior immunization with the melanoma-associated antigen, gpl00. In response to its cognate antigen, each clone used for treatment secreted large amounts of IFN-␥ and GM-CSF, lesser amounts of IL-2 and TNF-␣, and little or no IL-4 and IL-10. Clones also demonstrated recognition of human leukocyte antigen-matched melanomas by cytokine secretion and lysis assays [37]. Twelve patients received two cycles of cells alone; 11 patients received additional cycles of cells and were randomly allocated to receive one of two schedules of IL-2 (125,000 IU/kg s.c. daily for 12 days vs. 720,000 IU/kg i.v. every 8 h for 4 days). An average of 1 ⫻ 1010 cells was transferred/cycle. Peripheral blood samples were analyzed for persistence of transferred cells by TCR-specific PCR. Transferred cells reached a maximum level at 1 h after transfer but declined rapidly to undetectable levels by 2 weeks. One minor response and one mixed response were observed (both in the high-dose IL-2 arm). This report demonstrates the safety and feasibility of cloned T cell
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transfer as a therapy for patients with melanoma. Transfer of different or additional cell types or modulation of the recipient host environment appears to be required, however, for successful therapy [38]. Investigators at the University of Washington (Seattle, WA, USA) performed a trial to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8⫹ T cell clones targeting melanoma antigens, Melan-A and gp100 [39]. Four infusions were given: the first without IL-2 and subsequent infusions with lowdose IL-2 in escalating doses twice daily for the second, third, and fourth infusions, respectively. No serious toxicity was observed in 10 patients treated. The adoptively transferred T cell clones persisted in vivo after low-dose IL-2 was found to localize to tumor sites and induced minor or mixed responses or stable disease in eight of 10 patients for up to 21 months, but no objective responses were observed. In a follow-up trial, patients received two infusions of a single tumor-reactive, antigen-specific CD8 clone, expanded to 1010/M2; the first infusion was given without fludarabine conditioning, and the second infusion was given after fludarabine (25 mg/m2/day⫻5 days) [40]. Ten patients were treated with no serious toxicities. Three of nine evaluable patients experienced a minor response or stable disease for 5.8 –11.0 months. Fludarabine led to a 2.9-fold improvement in the in vivo persistence of transferred CTL clones from a median of 4.5 days to 13.0 days (P⬍0.05). Fludarabine lymphodepletion increased plasma levels of the homeostatic cytokines IL-7 and IL-15. Recently, 11 patients with refractory melanoma received cyclophosphamide as conditioning before the infusion of PBMC-derived, antigen-specific CD8⫹ clones, followed by low-dose or high-dose IL-2. No lifethreatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 weeks, and one of 11 had a complete remission at 36⫹ months. Immunerelated adverse events of skin rash were observed in patients with stable disease or complete remission at 4 weeks or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to skin and lymph nodes. CD8 CTL that persisted long-term acquired phenotypic and/or functional qualities of central memory-type T cells in vivo [41]. CD8 T cell clones were clinically active but required additional IL-2 and did not persist in the host, mediating modest benefit. Persistence of the adoptively transferred cells in the periphery seems key to their therapeutic success, and that didn’t occur with the CD8 clones. Important unanswered questions are whether oligoclonal T cells are important for successful adoptive cell therapy and whether CD4 T cell help is needed.
USE OF CD4 T CELL CLONES FOR ADOPTIVE THERAPY Investigators at the University of Washington, who worked with CD8 T cell clones for adoptive transfer, have described the use of a CD4 clone infused into a patient with refractory melanoma who had not undergone any previous condiVolume 95, June 2014
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TABLE 2. Important Questions to be Answered in the TIL Field What was the real difference between the LAK cell phenotype and T cell phenotype? What is their relation to NK cells and whether innate immune cells can ultimately be used for successful adoptive cell therapy? Does the concept of LAK cells need to be revisited as a result of greater understanding of innate immune effector features/phenotypes? What was the important clinical measure with TIL: is a high anti-tumor ORR important, or is long-term OS the key to therapeutic success? Or is duration of response the critical parameter to measure with TIL? Are response rates to TIL-adoptive therapy at 40–50% as a result of pretherapy-intrinsic tumor factors, or does “tolerance”, as a result of IL-2-induced effects, need to be overcome? How does cell volume in blood monitors get altered after adoptive therapy, and how does this affect homeostatic mechanisms of proliferation? Do IL-15 and IL-7 levels as a “cytokine sink” affect the efficacy of adoptive cell therapy and how? How does the presence or absence of killer cell Ig-like receptors and other inhibitory receptors affect LAK or TIL function? Is IL-2 required for in vivo expansion and persistence of adoptively transferred TIL? What is the phenotype of an effective TIL cell, and what checkpoint proteins are expressed on TIL? By what means can we improve the expansion of TIL to decrease the time to propagate them to adequate quantities and enrich for therapeutically active cells? How can we pretreat tumors to augment their infiltration with potentially therapeutic TIL for subsequent harvest?
tioning to induce lymphopenia or cytokine treatment. CD4 T cells, stimulated with a DPB1*0401-restricted epitope of a peptide derived from NY-ESO-1, were isolated from the peripheral blood, cloned, and expanded for cellular therapy using a REP protocol similar to that used for TIL [42]. The clonal CD4⫹ T cells mediated a durable clinical remission and led to an endogenous T cell epitope spreading against melanoma antigens other than NY-ESO-1. Transferred CD4 clones remained detectable in the patient’s blood for ⬎80 days, during which time, the frequency fluctuated between 0.7% and 3.0% of the total number of mononuclear cells.
TCR GENE-TRANSFER PROTOCOLS The ability to clone two-chain TCR sequences into retroviral and lentiviral vectors had led to gene therapy trials, in which TCR sequences have been transferred to PBMCs [43– 45]. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels ⬎10% of peripheral blood lymphocytes for at least 2 months after the infusion. High, sustained levels of circulating, gene-engineered cells were observed 1 year after infusion in two patients, who both demonstrated objective regression of metastatic melanoma lesions. Transgenic mice were immunized with human melanoma cells, and high-throughput screening of lymphocytes was conducted to generate TCRs highly reactive to mela noma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA⫺ and CD45RO⫹ after ex vivo expansion. After infusion, persisting cells displayed a CD45RA⫹ and CD45RO⫺ phenotype. Gene-engineered cells persisted at high levels in the blood of all patients, 1 month after treatment. Responding patients demonstrated higher ex vivo antitumor T cell reactivity than nonresponders. Tumor regression was seen in 30% and 19% of patients who re880 Journal of Leukocyte Biology
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ceived the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediated cancer regression in humans and targeted rare cognate antigen-containing cells throughout the body. Patients with NY-ESO-1-positive tumors were treated with autologous, TCR-transduced T cells plus high-dose IL-2 to tolerance after preparative chemotherapy to induce lymphoid depletion. Objective clinical responses were observed in five of 11 patients with mela noma-bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted beyond 1 year. Transduced TCR gene therapy approaches are promising but have the hazard of toxicity as a result of potent cross-reactivity with normal tissues, which is a major concern.
NK CELL-ADOPTIVE TRANSFER TRIALS Eight patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred, in vitro-activated, autologous NK cells after the patients received a lympho-depleting NMA chemotherapy regimen [46]. The infused cells exhibited high levels of lytic activity in vitro. There were no clinical responses seen in this trial. The adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least 1 week post-transfer and in some patients, for several months. Persistent NK cells in the circulation expressed significantly lower levels of the activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2. The persistent NK cells mediated antibody-dependent, cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggested to the authors that coupling adoptive NK cell transfer with mAb administration deserved evaluation.
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BASIC-TRANSLATIONAL REVIEW Weber Adoptive cell therapy for melanoma
CONCLUSIONS As the technology for the generation of tumor-specific T cells has advanced, TIL and adoptive T cell therapy have advanced into the mainstream of clinical trials at multiple institutions around the world. Important elements that have improved the clinical results with TIL therapy include antigen-specific selection, rapid expansion, and lymphoid depletion with chemotherapy and/or radiation. The need for accurate, predictive markers for the success of TIL therapy is vital. We look forward to the use of new checkpoint protein inhibitors, novel cytokines, and genetic modification to facilitate the generation of large numbers of tumor-specific effector cells from tumor biopsies or from the peripheral blood that have the proper phenotype and the ability to persist long-term in the circulation and mediate potent anti-tumor effects to increase the number of patients who are cured of melanoma. Success with melanoma-adoptive cell therapy, particularly with TIL, has led to its use in other histologies that are currently undergoing testing (Table 2).
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KEY WORDS: T cell 䡠 immunotherapy 䡠 IL-2 䡠 lymphoid depletion 䡠 T cell receptor
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