Cancer Immunol Immunother (2014) 63:1061–1071 DOI 10.1007/s00262-014-1575-2
ORIGINAL ARTICLE
A phase I clinical trial combining dendritic cell vaccination with adoptive T cell transfer in patients with stage IV melanoma Isabel Poschke · Tanja Lövgren · Lars Adamson · Maria Nyström · Emilia Andersson · Johan Hansson · Roger Tell · Giuseppe V. Masucci · Rolf Kiessling
Received: 27 March 2014 / Accepted: 18 June 2014 / Published online: 4 July 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Adoptive transfer of in vitro-expanded tumorinfiltrating lymphocytes (TIL) has shown great clinical benefit in patients with malignant melanoma. TIL therapy itself has little side effects, but conditioning chemo- or radiotherapy and postinfusion interleukin 2 (IL-2) injections are associated with severe adverse advents. We reasoned that combining TIL infusion with dendritic cell (DC) vaccination could circumvent the need for conditioning and IL-2 support and thus represent a milder treatment approach. Eight patients with stage IV melanoma were enrolled in the MAT01 study, consisting of vaccination with autologous tumor-lysate-loaded DC, followed by TIL infusion. Six of eight patients were treated according to protocol, while one patient received only TIL and one only DC. Treatments were well tolerated with a single grade 3 adverse event. The small study size precludes analysis of clinical responses, though interestingly one patient showed a complete remission and two had stable disease. Analysis of the infusion products revealed that mature DC were generated in all cases. TIL after expansion were CD3+ T cells, dominated
Giuseppe V. Masucci and Rolf Kiessling have contributed equally to this study. Electronic supplementary material The online version of this article (doi:10.1007/s00262-014-1575-2) contains supplementary material, which is available to authorized users. I. Poschke · T. Lövgren · L. Adamson · M. Nyström · E. Andersson · J. Hansson · R. Tell · G. V. Masucci · R. Kiessling Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden I. Poschke (*) German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany e-mail:
[email protected]
by effector memory CD8+ cytotoxic T cells. Analysis of the T cell receptor repertoire revealed presence of highly dominant clones in most infusion products, and many of these could be detected in the circulation for weeks after T cell transfer. Here, we report the first combination of DC vaccination and TIL infusion in malignant melanoma. This combined treatment was safe and feasible, though after evaluating both clinical and immunological parameters, we expect that administration of lymphodepleting chemotherapy and IL-2 will likely increase treatment efficacy. Keywords Melanoma · DC vaccine · Adoptive T cell transfer · Tumor-infiltrating lymphocytes · Combination (immuno)therapy Abbreviations ACT Adoptive T cell transfer AJCC American Joint Committee on Cancer CDR3 Complimentarity determining region 3 Ct Threshold cycle value CR Complete response CT Computer tomography CYP Cyclophosphamide DC Dendritic cells DTH Delayed type hypersensitivity GM-CSF Granulocyte macrophage colony-stimulating factor IL Interleukin MDSC Myeloid-derived suppressor cells MRI Magnetic resonance imaging LN Lymph node OS Overall survival PBMC Peripheral blood mononuclear cells PD Progressive disease PET Positron emission tomography
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PR Partial response RESIST Response evaluation criteria in solid tumors SD Stable disease SOP Standard operating procedure TCR T cell receptor TIL Tumor-infiltrating lymphocytes TNF Tumor necrosis factor TNM Tumor node metastasis Tregs Regulatory T cells SC Subcutaneous WHO/ECOG World Health Organization/Eastern Cooperative Oncology Group
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hypersensitivity (DTH) responses as well as some benefit for the patients [16–20]. We reasoned that a combination of DC vaccination using monocyte-derived DC loaded with autologous tumor lysate and ACT using in vitro-expanded TIL might potentiate the effect of the DC vaccine and reduce the need for conditioning and in vivo IL-2 support for the T cells. We report here “MAT01,” the first clinical trial in patients with advanced metastatic melanoma making use of such a combined approach, and demonstrate that it is feasible and safe in this patient population.
Materials and methods Introduction
Patients
Adoptive T cell transfer (ACT) has recently emerged as a powerful therapeutic option for solid [1–3] as well as liquid cancers [4–6]. T cell-based therapies include the infusion of in vitro-expanded tumor-infiltrating lymphocytes (TILs) [1–3, 7] or genetically modified T cells carrying receptors specific for tumor-associated antigens [6, 8, 9]. Since TIL therapy was first explored around 20 years ago, it has become increasingly clear that “conditioning” prior to T cell infusion is crucial for therapeutic success. Conditioning, either non-myeloablative, lymphodepleting chemotherapy or total body irradiation, seems to act in several ways: (1) making “space” for the infused T cells, thus triggering their expansion by homeostatic proliferation, (2) creating an environment where they have exclusive access to cytokines and antigen-presenting cells, while (3) suppressive cell populations, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC), are absent [10]. Some evidence suggests that increased intensity conditioning is correlated with superior treatment responses [11]. On the other hand, such conditioning is accompanied by considerable toxicity and might not be applicable to a vast majority of patients with advanced cancer. The same is true for infusion of high-dose interleukin 2 (IL-2), which is used to support T cell proliferation in vivo [12]. As IL-2 infusion is associated with significant side effects, regimens using no or lower doses of IL-2 need to be explored and such studies are currently ongoing in our lab and elsewhere [13]. Being professional antigen-presenting cells, dendritic cells (DC) were expected to be potent inducers of tumorspecific immune responses in a vaccine setting. DC can be generated in vitro from monocytes with relative ease and flexibly loaded with many different types of antigens. Unfortunately, DC vaccination trials have rarely resulted in striking clinical responses [14, 15], though a great number of trials report induction of T cell and delayed type
This trial was approved by the local ethical committee, as well as the Swedish medical authorities (EU-nr 2008000694-38). All patients’ treatment adhered to the guidelines defined by the Declaration of Helsinki, and patients were enrolled after written informed consent. All patients enrolled had advanced metastatic melanoma verified by imaging within 30 days before treatment initiation, ambulatory performance status 0–2, were aged between 18 and 74 and had a life expectancy >3 month. Patients had not received chemo- or radiotherapy in the 4 weeks preceding start of treatment and no other biotherapy 8 weeks preceding treatment. Exclusion criteria included a significant history of cardiovascular or respiratory disease, autoimmune disease (except vitiligo), allogeneic organ transplantation, infection with blood-borne pathogens, prior or concurrent additional malignancies, CNS metastases, immunodeficiency, as well as other serious illnesses or pregnancy. Patient characteristics are detailed in Table 1.
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Treatment schedule After patient enrollment and clinical verification of disease status, surgery to obtain tumor tissue was performed. This tissue was used to expand TIL and to generate tumor lysate. After surgery, patients underwent leukapheresis to obtain monocytes for DC generation. As soon as either successful TIL expansion or DC vaccine production was confirmed, the patients received a conditioning of orally administered cyclophosphamide (CYP, 50 mg/m2) daily for 7 days. One day after the last CYP dose, DC vaccination was initiated. At least three DC vaccinations were scheduled at weekly intervals. Two weeks after the last DC vaccination, T cell infusion commenced. If the T cell product was not ready by this time, additional DC vaccination could be administered to bridge the time until the T cell product was produced and released for infusion. Depending on the cell number
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Table 1 Patient characteristics Patient no.
Age
Sex
Stage at treatment start AJCC
TNM
WHO/ECOG performance status
Sites of disease SC
LN
Lung
X X X X
X
X
001 002 003 004 005 006 007
55 75 35 36 43 59 75
F M F F F F M
IV IV IV IV IV IV IV
M1b M1c M1c M1c M1b M1c M1b
0 0 0 0 0 0 0
X
008
70
F
IV
M1c
0
X
Liver
Other visceral
Bone
Brain
X X X X
X
X X
X X X
X
X
X
X
X
X
X
AJCC American Joint Committee on Cancer, TNM tumor node metastasis, WHO/ECOG World Health Organization/Eastern Cooperative Oncology Group, SC subcutaneous, LN lymph node
a
b
Fig. 1 MAT01 treatment regimen. a Interventions and blood samples (black arrows) in the MAT01 phase I clinical trial. *Cyclophosphamide (50 mg/m2 perorally daily for 7 days). b Overview of treatments administered to patient p001–p008
obtained, patients received 1–3 T cell infusions at weekly intervals. Figure 1a presents an overview of the study protocol, including the time points where blood samples for immunomonitoring were obtained. Details of the different treatment steps are provided in the following paragraphs.
Surgery All patients underwent surgery to remove a skin or lymph node metastasis for generation of tumor lysate and TIL. Surgery was performed under local anesthesia.
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During surgery, a piece of tumor was collected in a sterile container. Generation of tumor lysate A piece of the tumor (approximately 1 cm3) was minced, and a lysate was generated by 6 freeze–thaw cycles in liquid nitrogen and a 56 °C water bath. The final lysate was irradiated at 60 Gy, filtered and aliquoted. Protein content of the lysate was determined using a nano-drop spectrophotometer (Thermo Fisher Scientific, Wilmington, USA), and sterility and endotoxin (Limulustest) tests of the lysate were performed by the Department of Clinical Microbiology/Pharmacology at Karolinska University Hospital. Generation of DC vaccine For vaccine production, DC were generated from bloodderived monocytes. Monocytes were isolated from leukapheresis products by elutriation [counter-flow centrifugation, ELUTRA, (CaridianBCT, Lakewood, USA)]. The average monocyte purity of the starting product was 83 % (range 72–90 %). Monocytes were cultured in CellGro medium at 2 million cells/mL in VueLife cell culture bags in presence of 100 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) and 20 ng/mL IL-4. On day 2 of culture, one volume of fresh cytokine containing medium was added. On day 5, immature DC (iDC) were loaded with 10–30 μg tumor lysate per million cells, adjusted to 2 million cells/mL in medium supplemented with IL-4 and GMCSF as above, and maturation of iDC was induced by addition of 20 ng/mL tumor necrosis factor (TNF)-α. On day 7, mature DC (mDC) were harvested, counted, characterized and frozen in aliquots of 17.5 million cells. All culture media, cytokines and cell culture bags were GMP-grade and purchased from CellGenix (Freiburg, Germany). On the day(s) of DC vaccination, one ampule of frozen DC was rapidly thawed in a 37 °C water bath, counted and the viability was assessed. Cells were resuspended in 0.2 mL 0.15 M NaCl and administered intra-dermally using an insulin syringe (Mircro-Fine, BD Medical, San Diego, USA).
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of the medium volume in each well was replaced with fresh medium supplemented with plasma and IL-2. After 3–4 days of culture, lymphocytes could usually be seen emerging from the tumor piece. Cultures were monitored regularly and split before reaching confluence. After approximately 2 weeks, expanding wells were pooled and counted. To generate large T cell numbers, TIL were cultured for approximately 14 days in presence of autologous, irradiated feeders (40 Gy, TIL:feeder ratio 1:50–1:80) with 300 IU/mL IL-2 and 30 ng/mL anti-CD3 antibody (OKT3, Cilag, Zug, Switzerland). Cells were split or medium exchanged as necessary. Cultures were initiated in standing T75 flasks, but transferred to VueLife cell culture bags 1 week before harvest. At the end of culture, cells were harvested, pooled, characterized, counted and frozen. On the day(s) of ACT, T cells were rapidly thawed in a 37 °C water bath, counted and administered intra-venously in 50 mL 0.15 M NaCl solution (Braun, Melsungen, Germany) over a period of 15 min. Immunomonitoring In addition to leukapheresis and blood collection for isolation of plasma, 100 mL blood was collected before DC vaccination, before each T cell infusion and 4 weeks after the cell administration. Peripheral blood mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque (GE, Uppsala, Sweden), characterized by flow cytometry, and remaining cells were frozen until further analysis. Antibodies used for flow cytometry are listed in supplementary Table 1. Extra-cellular and intra-cellular staining were performed as described previously [21, 22]. Melanoma-specific CD8 T cells were detected in the infusion product and peripheral blood by staining with a panel of MHC-multimers (Melanoma collection 1 dextramers, Immudex, Copenhagen, Denmark) for the tumor-associated antigens MART-1, NY-ESO-1, MAGE-A3, Tyrosinase, gp100 or MAGE-A1, according to the manufacturer’s instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences, San Jose USA) and analyzed using FlowJo software (Treestar, Ashland, USA). T cell receptor repertoire determination
Culture of tumor‑infiltrating lymphocytes A piece of the tumor (approximately 0.5 cm3) was minced into 1 mm3 pieces. Each piece was placed in one well of a 24-well plate containing 1 mL/well CellGro medium supplemented with 2 % autologous plasma (generated by centrifugation and heat-inactivation of plasma collected on the day of surgery) and 6,000 IU/mL IL-2 (Proleukin, Novartis, Basel, Switzerland). On day 1 of culture, half
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The final TIL products were subjected to high-throughput deep-sequencing of the TCRB gene complementarity determining region 3 (CDR3) (ImmunoSeq; Adaptive biotechnologies, Seattle, USA) to identify high-frequency T cell clones. Based on this analysis, primers for the 3–5 most dominant T cell clone TCRB gene CDR3 regions were designed (CyberGene, Stockholm, Sweden). Genomic DNA from TIL infusion products and peripheral blood
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mononuclear cells (PBMC) (5–10 million cells for each sample) was prepared using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, USA). The concentration was measured on a NanoDrop ND 1000 (Thermo Fischer Scientific, Waltham, USA). Quantitative PCRs were set up using 100 ng genomic DNA as template, 0.4 µM forward and reverse primer and 1× SYBR green PCR master mix (Applied Biosystems, Foster City, USA) in a total volume of 10 µL. In addition to CDR3 TCRB-specific primers, primers for the hemoglobin beta-unit gene (HBB) and the tumor protein 53 (TP53) gene were used as references to normalize for differences in DNA content between samples. Reactions were run on an Applied Biosystems 7900HT FAST instrument using a cycling protocol of 50 °C for 2 min, 95 °C for 10 min and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Specificity of the PCR was confirmed by performing a melting curve. The threshold cycle (Ct) value was determined using the SDS software (Applied Biosystems). Frequencies of cells expressing the dominant CDR3 regions were calculated assuming 100 % PCR efficiencies and a diploid expression of the reference genes, by the formula 200 × 2(mean Ct for HBB, TP53 − Ct for dominant CDR3).
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prevented generation of a DC vaccine. As TIL were available for this patient, she received three ACT without prior vaccination. P007 was the only individual where no TIL could be expanded from the tumor explant cultures. Consequently, this patient received only DC vaccinations but not ACT. Since additional aliquots of DC vaccine were available and the patient had stable disease after three vaccinations, an additional three DC vaccines were administered off protocol. Such maintenance vaccination was also applied to patient p001 until disease progression. As p001 had a low number of TIL at the end of culture, she also received a second infusion of peripheral blood-derived T cells that had been re-stimulated three times with the DC vaccine. Figure 1b shows a schematic representation of the treatments administered to each individual patient. On average, patients received 2.6 DC vaccines and 2.5 T cell infusions, containing a mean total of 47 × 106 DC and 1.2 × 109 T cells, respectively. Overall, the administration of DC vaccine after conditioning and followed by ACT was feasible in the majority of patients and failed only once due to lack of expandable TIL (p007), while the second failure (p006) could be attributed to aseptic difficulties related to surgery.
Clinical monitoring Infusion products Clinical examinations were carried out at least 1–3 weeks before start of treatment, the day of the first DC vaccine injection, the day of each adoptive T cell transfer and 4 weeks after the last adoptive T cell transfer injection. Radiologic evaluations (CT, PET-CT or MRI) were performed during the 3 weeks preceding treatment initiation and again 4 weeks after the last ACT. Treatment response was evaluated by objective methods following the “Response Evaluation Criteria In Solid Tumors” (RECIST) guidelines version 1.1.
Results Administered treatments A total of eight patients were enrolled in this clinical trial. All had advanced melanoma, metastatic to sites including skin, lymph nodes, liver, lung and bone (Table 1). Of the eight patients included in the trial, six were treated according to protocol and received at least three DC vaccinations, with exception of p001 who received only two injections, followed by at least two T cell administrations. Details of the infused cellular products are summarized in Table 2. The skin metastasis used for generation of tumor lysate for p006 did not pass sterility testing and thus
As mentioned above, DC vaccines could be produced for 7/8 patients. The standard operating procedure (SOP) used for DC generation has, with minor modifications, already been applied in a number of clinical trials [23, 24]. This 7-day protocol resulted in high numbers of mature antigen-loaded DC in all cases. On average, 10 vaccine doses per patient were produced (range 6–14), and the average yield of DC relative to cultured monocytes was 23 %. The viability of the product was high both at harvest and in the thawed vaccine before injection (mean ± SD: 91 ± 6 and 86 ± 15.4 %, respectively). On day 5, iDC was potently able to engulf antigens as shown by FITC-Dextran assays (data not shown). On day 7, the final DC product clearly exhibited a mature phenotype characterized by high expression of CD83, DC-Sign and HLA-DR (Fig. 2a, b). Additional information on the functional characteristics of DC generated in parallel to the vaccine products has been published elsewhere [25]. TIL could be expanded from 7/8 patients of patients, though expansion varied from 53-fold to 650-fold. The viability of the T cell product was good at harvest (89 ± 8 %) and after thawing for infusion (92 ± 6.3 %). The average duration of TIL culture was 31 days (range 24–38). At the day of cell harvest, cultures were predominantly T cells (88 ± 9 % CD3+ cells), with a strong skewing toward the CD8+ subpopulation (Fig. 2c, d), with the exception of the
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Table 2 Overview of infused cellular products, side effects and responses Toxicityb
Clinical responsec
OSe (months)
PD
11†
CRd PD
>50 10†
PD PD PD
7† 14† 10†
No TIL growth from None 24 cultures
SD
20†
None
SD
10† Median PD = 10 Median SD + CR = 20
Patient no.
No. of DC vaca
No. of ACT
Total no. Total no. of of injected DC infused T cells (×106)a (×108)
Comment
001
2
2
72
1.1 + 1 (ACT-B)
1× fresh TIL, 1× Local reaction, vaccine stim T cells vitiligo
002 003
3 4
3 3
57 36
18.9 13.4
004 005 006
3 3 –
3 3 3
42 38 –
4.5 20.6 16.8
007
3
–
44
–
008 Mean
3 2.6
3 2.5
43 47.4
18.3 11.7
Range
0–4
0–3
0–72
1.1–20.6
No DC due to contamination of tumor lysate
Local reaction Local reaction, myalgia Local reaction Skin rash, itching None
PD: 7–14 SD + CR: 10–50
CR complete response, PR partial response, SD stable disease, PD progressive disease, OS overall survival †
Deceased
a
According to protocol (including maintenance vaccination to allow time for T cell preparation)
b Patients experienced only transient, reversible adverse reactions. One patient (#003) experienced grade 3 toxicity (injection site pain) according to the National Cancer Institute Common Toxicity Criteria (NCICTC). No other grade 3 or 4 toxicities were noted c
Best overall response measured by RECIST
d
The CR could be established clearly after a series of liver examinations (MRT, PET, CT and Ultrasounds). The liver metastases were detected at the beginning of treatment, and 2 years later, only liquid filled cystic formations of unchanged size remain. (Cytological verification could not be performed). No new lesions have been detected since the start of the treatment e
As of February 2014
vaccine-stimulated peripheral blood T cells of p001, which contained 72 % CD4+ T cells. No living tumor cells could be detected in the final T cell products (data not shown). The expanded T cells were predominantly CD45RO+ memory T cells with low CCR7 expression, suggesting that the majority were of the effector memory subtype (Fig. 2e– h). The T cell activation marker CD69 was expressed on 62 % of CD8+ T cells and 50 % of CD4+ T cells (Fig. 2i). At 2.7 % (±2.4) of CD4+ and 0.5 % (±0.7) of total cells, Tregs did not constitute a dominant T cell subset, which was reassuring with regard to the long exposure to high doses of IL-2 (Fig. 2j). In the HLA-A2 positive patients, T cell specificities in the final T cell product were interrogated using a panel of MHC class I multimers loaded with common melanoma antigens (gp100, MAGE-A1, MAGE-A3, MART-1, NYESO-1, Tyrosinase). The most commonly detected T cell response was directed against MART-1 (5/8 patients), though it was a minor response representing a maximum
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of 0.78 % of CD8+ T cells in patient p008 (see supplementary Figure 1). While many patients exhibited several multimer reactive T cell populations, none of these known reactivities could be detected in the infusion product of p004. As the multimer-positive populations were in the range of 0.01–0.8 % of CD8+ T cells (maximum 0.2 % of CD3), it was difficult to perform phenotypical comparisons between multimer-positive and multimer-negative cells, though multimer-positive cells appeared to be exclusively effector memory T cells (CD45RA-CCR7-). Clinical observations The combination of DC vaccination and ACT could be safely administered to all patients (Table 2). P003 experienced grade 3 toxicity in the form of injection site pain after the second injection. No other grade 3 or 4 adverse events were observed. Five patients experienced grade 1–2 adverse events including injection site reactions, myalgia, itching, skin rash or vitiligo.
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a
b
c
g
d
e
f
i
j
h
Fig. 2 Properties of the DC vaccine product and the T cell infusion product. DC vaccine product: a raw data of p004 (open histograms show isotype controls) and b pooled data of all 7 patients that received DC vaccine illustrate the phenotype of the mature and antigen-loaded DC on day 7 of culture. Each symbol denotes one patient. T cell infusion product: frequency of c CD3+ T cells, CD19+ B cells and CD3-CD56+ NK cells in total cells; d CD8+ and CD4+
T cells in CD3+ cells; e CD8+ and f CD4+ CD45RA+ (naïve) and CD45RO+ (memory) T cells subsets; g CCR7+ (naïve or central memory) T cells in the CD4 and CD8 subset and h the gating used to analyze naïve and memory subsets; i CD69+ activated T cells in CD4+ and CD8+ subsets and j regulatory T cells (Tregs) in the infusion products of all patients receiving adoptive T cell transfer
The size of the study does not allow conclusions about the clinical efficacy of the treatment. However, it is interesting to report that p002 experienced a complete response that is ongoing at 50 months post-treatment. Seven patients progressed during/after treatment though two patients achieved stable disease for 2 and 10 month, respectively, measured from the first evaluation 4 weeks after the last vaccination.
differentiation, activation status, presence of Tregs and cells with phenotypes associated with MDSC. While T cell activation and differentiation seemed to increase following DC vaccination, there were no significant phenotypic changes in any of the investigated markers/cell subsets associated either with DC vaccination or with adoptive T cell transfer (data not shown). Further, no decrease in Treg frequencies could be observed as a result of cyclophosphamide conditioning (data not shown). While several patients had multimer responses in the blood (mainly against MART-1), we did not observe changes in their frequency after adoptive T cell transfer (not shown), as might be expected based on the low frequencies of multimer-positive cells in the infusion products (Supplementary Figure 1).
Immunomonitoring Blood samples for immunomonitoring were taken on the day of leukapheresis, pre- and post-DC vaccination, as well as at each T cell infusion and 4 weeks after the last treatment. Immunomonitoring included T cell subsets,
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Fig. 3 Clonality of the TCR repertoire. Frequency of the top-10 clones (colored) versus all other clones (gray). The number over the gray bar is the total number of unique TCR sequences (clones) detected in each patient
TCR repertoire analysis TIL infusion products from five patients could be analyzed for clonality by deep-sequencing of the TCR-β chain. Each TIL culture contained several thousand unique T cell clones. The top-10 most frequent clones with productive TCRB CDR3 rearrangements accounted for 8–31 % of the total TCRβ repertoire (Fig. 3). Some cultures contained highly dominant clones, such as the TIL of patient 6 with one clone accounting for 28.3 % of the total repertoire. We wondered if these dominant clones could also be seen in the blood after adoptive T cell transfer and designed primers to detect the 3–5 most commonly occurring CDR3 sequences (including also high-frequency, nonproductive rearrangements, likely to originate from T cell clones with a second, productively rearranged TCRB CDR3 allele) of each patient by PCR (Table 3). All primers served to detect their target in DNA preparations of the TIL product with a reasonable overlap between the frequencies calculated from the PCR and the sequencing approach. Although at much lower levels than in the TIL product, 9/20 tested clones from all patients except p004 could be detected in PBMC samples. Notably, 7 of these 9 were not detectable before treatment. This indicates that TIL-derived T cell clones persisted in the peripheral blood and could be detected at least 4 weeks after the last TIL injection in the majority of patients.
Discussion We have performed one of the first clinical trials combining vaccination using autologous tumor lysate-loaded DC with ACT of in vitro-expanded TIL.
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Studies from several mouse models have suggested that the combination of DC vaccination with ACT is superior to single agent treatments [26–30]. Lutz-Nicoladoni et al. [27] as well as Koike et al. [26] showed delayed tumor outgrowth or better survival and tumor regression as a result of this combination treatment in >40 % of animals inoculated with aggressive tumors. Song et al. [29] as well as Tamai et al. [30] had similar findings and showed that combination treatment led to T cell infiltration into the primary tumor, as well as into metastases and induced immunological memory that protected the mice from re-challenge. We show here that such combination treatment is feasible in patients with stage IV malignant melanoma. The regimen was well tolerated with toxicities not exceeding grade 1–2, with the exception of one grade 3 injection site reaction. The small number of treated patients, the variability in the administered treatments and the phase I trial design do not allow us to draw conclusions about the clinical efficacy of the treatment. However, one patient did experience tumor regression during DC vaccination and has a complete clinical response ongoing for 50 month (as of February 2014). Similar to what we have described, Kandalaft et al. [31] reported safety and feasibility of combining DC vaccination with adoptive T cell transfer. In their trial, ovarian cancer patients that achieved at least stable disease after a vaccination with autologous tumor lysate-loaded DC received adoptive transfer of peripheral blood T cells that had been in vitro stimulated with the DC vaccine. This T cell therapy could convert one partial to a complete response and maintain one stable disease, while the third patient progressed after T cell transfer. Clearly, the in vitro generation of two cellular products is logistically challenging, but in most cases both DC and TIL of sufficient quantity and high quality could be generated with relative ease. One advantage of having two products in a protocol is that it minimizes the problem of leaving included patients without treatment. In this trial, the two cases where either TIL or DC were not available, the other cellular product could be given as a single agent. It has been shown that infusion of high TIL number and persistence of infused TIL in vivo correlates with improved treatment response [1, 32, 33]. Cell numbers infused in our trial were mostly lower than those reported by others, both due to the amount of starting material, but also to limited expansion rates of TIL during rapid expansion. Future trials will make use of optimized TIL protocols and GMP-compatible large-scale expansion devices to consistently reach expansion rates >1,000 fold. We chose to provide TIL as repeated infusions to generate a steady stream of assaults on the tumor. It is currently unknown how such an approach compares to a single infusion of a large T cell
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Table 3 Frequencies of dominant T cell clones Patient no.
2
4
5
6
8
Clone no.
% in TIL product
% in blood
Deep-sequencing
PCR
Pre first ACT
Pre second ACT
Pre third ACT
Post third ACT
1 2 3 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3
4.3 3.7 3.4 2.2b 1.8 1.2 1.0 8.6 6.9 3.8 3.6b 1.9 28 11b 7.2 3.9 18b 7.8b 6.3
1.6 1.5 10.7 5.5 0.90 0.15 0.28 5.8 3.4 0.30 3.3 4.0 20 2.1 1.8 1.8 17 4.8 3.1
NDa ND ND ND ND ND ND ND ND ND ND ND 0.040 ND 0.034 ND ND ND ND
ND 0.019 ND ND ND ND ND ND ND ND ND ND – – – – 0.016 0.0026 0.0059
ND ND ND –a – – – ND ND ND ND ND 0.0073 0.0055 0.0092 ND 0.0061 0.0099 0.0049
ND 0.014 ND – – – – ND ND ND 0.0084 ND 0.0093 ND 0.020 ND ND ND ND
4
4.6
5.7
ND
0.013
ND
ND
ND not detected a
No sample available, b nonproductive re-arrangement
dose, though it could resemble the, albeit unspecific, T cell activation achieved by repeated administration of antiCTLA-4 antibodies, which induces clinical responses in a significant proportion of patients [34]. Interestingly, despite low frequencies of T cells reactive to known melanoma antigens, the infusion products contained large clones that we could also detect in the blood of patients after infusion. The majority of these clones could not be detected in the circulation prior to TIL infusion. The tracking of such clones requires sensitive methods, as their frequency within the total pool of blood cells is low, possibly due to simple dilution, but potentially also due to trafficking of infused cells into the tumor, while others might get stuck in the lungs. In retrospect, one can question the sequence of treatments in our trial, starting with DC vaccination with subsequent T cell infusion. This order was based on an originally different trial design similar to that of Kandalaft et al. [31], intending to increase T cell precursor frequencies in the blood by DC vaccinations and using DC vaccinestimulated peripheral blood-derived T cells as the T cell infusion product. When it became obvious that expansion of large T cell numbers using tumor lysate-loaded DC as stimulators was difficult while TIL could be grown from
the majority of patients, the protocol was modified to focus on TIL administration. At this point, the order of treatment administration was maintained, as the period of DC vaccination allowed sufficient time for the expansion of TIL cultures, which were initiated immediately after the surgery required to obtain tumor material for DC loading. However, we now believe that a reverse design is to be preferred. Consequently, we have initiated a new clinical trial where TIL administration is followed by DC vaccination with the intention to support and boost the administered T cells in vivo by exposing them to autologous tumor lysate presented by the DC. As we did observe neither lymphodepletion nor immunomodulation as a consequence of low-dose cyclophosphamide administration in the MAT01 trial described here, we will also administer a more classical conditioning regimen (60 mg/kg/day cyclophosphamide for 2 days followed by 25 mg/m2/day of fludarabine for 5 days) prior to T cell infusion, as lymphodepletion has been shown to be important for therapy success [11, 35], and other Treg-specific targeting regimens have proven inefficient [36]. In vivo T cell expansion will be supported by IL-2 administration. We still think that side effects due to i.v. infusion of high-dose IL-2 are a concern and will therefore make use of a low-dose IL-2 schedule, consisting
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of 1 × 105 units/kg i.v. every 8 h for up to 14 doses, similar to other studies exploring the use of lower IL-2 doses [13]. We reason that absence of high intensity conditioning and high-dose IL-2 can be compensated by the T cell stimulation that will result from the DC vaccination. This trial is currently enrolling patients in a two armed design allowing interrogation of the effect provided by DC vaccination. In conclusion, the combination of DC vaccination and T cell administration can be safely administered in patients with advanced melanoma. Based on our experiences with the MAT01 clinical trial, we have re-assessed our treatment concept and formulated a novel therapeutic protocol, which we expect to demonstrate superior immunological and clinical efficacy. Acknowledgments We would like to thank B. Näsman-Glaser and K. Heimersson for their expert technical assistance. R. Tell is supported by a 50 % research position from the Karolinska Institutet Thematic Network IMTAC. The study was supported by grants to R. Kiessling from the Swedish Cancer Society (120598 Cancerfonden), the Cancer Society of Stockholm (121103 Cancerföreningen, Radiumhemmets Forskningsfonder), Torsten Söderbergs stiftelse, the Swedish Medical Research Council (K2011-66X-15387-07-3 VR), an ALF-Project grant from Stockholm City Council (20110070 ALF Medicin 2012), the Knut and Alice Wallenberg Foundations and a grant from Magnus Bergvalls Foundation to T. Lövgren. Conflict of interest The authors declare that they have no conflict of interest.
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