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Hindawi Publishing Corporation Journal of Skin Cancer Volume 2011, Article ID 574695, 6 pages doi:10.1155/2011/574695 Research Article Characterization of Ex Vivo Expanded Tumor Inﬁltrating Lymphocytes from Patients with Malignant Melanoma for Clinical Application 1 2 2 1 Niels Junker, PerthorStraten, Mads Hald Andersen, and Inge Marie Svane Center for Cancer Immune Therapy (CCIT), Department of Oncology, University Hospital Herlev, Herlev Ringvej 75, 2730 Herlev, Denmark Center for Cancer Immune Therapy (CCIT), Department of Hematology, University Hospital Herlev, Herlev Ringvej 75, 2730 Herlev, Denmark Correspondence should be addressed to Mads Hald Andersen, firstname.lastname@example.org Received 4 February 2011; Accepted 16 April 2011 Academic Editor: Giuseppe Palmieri Copyright © 2011 Niels Junker et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clinical trials of adoptive transfer of autologous tumor inﬁltrating lymphocytes (TILs) to patients with advanced malignant melanoma have shown remarkable results with objective clinical responses in 50% of the treated patients. In order to initiate a clinical trial in melanoma, we have established a method for expanding TILs to clinical relevant quantities in two steps with in 8 weeks. Further characterization of expanded TILs revealed an oligoclonal composition of T-cells with an eﬀector memory like phenotype. When autologous tumor was available, TILs showed speciﬁc activity in all patients tested. TIL cultures contained speciﬁcity towards tumor cells as well as peptides derived from tumor-associated antigens (TAAs) during expansion procedures. 1. Introduction In our study, we have analysed TIL characteristics from 17 melanoma patients, whereof ﬁve have undergone lym- The incidence of malignant melanoma is increasing world- phodepletion and TIL-based ACT with low-dose IL-2. wide, and upon dissemination has a very poor prognosis . Only two systemic treatments are approved for dissem- 2. Materials and Methods inated disease and encompass IL-2 based immunotherapy (16% response rate and 6% complete responses) and 2.1. Patients. Patients referred to surgery for primary or dacarbazine (6%–15% response rate with no improved sur- recurrent stage III-IV malignant melanoma were eligible for vival) . However, results from clinical trials of TIL- the study. The study protocol was approved by the local based immunotherapy conducted at two centres has shown ethics committee, and all patients were included after signing 50% response rates in patients with advanced disease, and informed consent. Tumor material from the patients was responses were long lasting [4, 5]. TILs were reported to obtained from the surgically removed tumour within 30 be dominated by CD8 T-cells and mediate speciﬁc killing minutes after surgery. of autologous tumor in most patients . Information on TAA-derived peptide speciﬁcities in TIL has mainly shown the occasional large frequency of MART-1 and gp100 speciﬁc 2.2. TIL Bulk Cultures and Rapid Expansion. The TIL cultur- T-cell populations. On the other hand, results on the clon- ing method was adapted from Dudley et al.  constituting otypic and phenotypic composition has been scarce; one a two-step expansion process: (I) initiating bulk cultures publication has revealed a mixed clonal content of TIL by and (II) rapid expansion of selected bulk cultures with a FACS analysis , and two recent studies report surface proliferative potential. Following surgical removal of tumor markers identical to memory like eﬀector T-cells from a lim- tissue from patients with MM the tumour sample were cut ited patient material [8, 9]. into 1-2 mm fragments. Fragments were subsequently placed 2 Journal of Skin Cancer individually in 24-well culture plates (Nunc, Denmark) and 2.8. T-Cell Receptor (TCR) Clonotype Mapping by Denaturing maintained in 2 mL of culture medium (CM) containing Gradient Gel Electrophoresis (DGGE). RNA was extracted RPMI1640 (Invitrogen), penicillin, streptomycin, fungizone using the NucleoSpin RNA II (Macherey-Nagel, Germany). (Bristol-Myers Squibb), 10% human serum (Sigma) and cDNA synthesis and quantitation of cDNA in each sample 7300 or 6000 IU/mL IL-2 (Aldesleukin, Novartis). Each frag- was carried out as previously described . ment initiated an individual TIL culture which was main- For TCR clonotype mapping, cDNA was ampliﬁed using tained separately during subsequent expansion and activa- a primer panel covering the 24 BV region families of tion. the TCR. Resulting PCR products are suited for DGGE Bulk cultures were selected for further expansion accord- [12, 13]. Ampliﬁcations were carried out in a total volume ing to a rapid expansion protocol (REP). TIL were cocul- of 45 μL containing 1xPCR buﬀer (50mM KCl, 20mM Tris tured with irradiated (40 Gy) allogeneic PBMCs serving as pH 8.4, 2.0 mM MgCl , 0.2 mM cresol red, 12% sucrose, feeder cells in a ratio of 1 : 200 in a 1 : 1 mixture of CM 0.005% (wt/v) BSA (Boehringer-Mannheim, Mannheim, and AIM-V (Invitrogen) initially with 10% HS, and con- Germany)), 2.5 pmol of each primer, 40 mM dNTPs (Phar- taining 30 ng/mL OKT-3 (Cilag AG, Suisse) and 7300 or macia LKB, Uppsala, Sweden) and 1.25 units of AmpliTaq 6000 IU/mL IL-2 (Aldesleukin, Novartis) in upright T-ﬂasks. polymerase (Perkin Elmer Cetus Corporation, Emeryville, REPs for preclinical purposes generally were initiated from Calif, USA). Parameters and conditions used for ampliﬁca- 5 6 ◦ ◦ ◦ 1 × 10 TIL per ﬂask, while 1 × 10 TIL were used per ﬂask tion were 94 Cfor 30sec.,60 C for 60 sec., and 72 Cfor in clinical scale REPs. On day 5, half of the medium 60 sec., as described, in [11, 12]. was replaced with fresh medium containing AIM-V, CM For DGGE 10 μL aliquots were loaded onto a denaturing with 10% HS and 7300 IU/mL IL-2. From there on, the gradient gel containing 6% polyacrylamide and a gradient TIL concentration were maintained at 1 × 10 cells/mL by of urea and formamide ranging from 20% to 80%. Gels were adding AIM-V supplemented with Fungizone and 7300 run at 160 V for 4.5 h in 1x TAE buﬀer kept at a constant tem- or 6000 IU/mL IL-2. Half of the patients TIL where cul- perature of 56 C. After electrophoresis, the gel was stained tured in 7300 IU/ml IL-2, while the other half received with SYBR Green I (Molecular Probes, Oregon, USA) and 6000 IU/mL IL-2 during culturing. visualized using the FLA-3000 ﬂuorescence detection system (FUJI ﬁlm, Science Imaging Scandinavia, Sweden). 2.3. Viability. Cell counting and viability testing were per- formed by microscopy. Cells were stained with trypan blue 2.9. Elispot INFγ Measurement. Antitumor activity was followed by counting of live and dead cells in a haemocy- assessed with Elispot INFγ quantiﬁcation as described tometer. previously . In brief, nitrocellulose bottomed 96 well plates (Multiscreen MAIP N45; Millipore, Denmark) were 2.4. Sterility Tests. Bulk and REP cultures were intermittently coated with INFγ capturing antibody (1-DIK; Mabtech, sampled for microbiological testing of fungal and bacterial Sweden) and further washed and blocked with RPMI 1640. A contamination. maximum of 1× 10 eﬀector cells per well were either added alone when stimulated by peptides, or in coculture with target cells (1 × 10 cells per well) consisting of autologous 2.5. Peptides. We used the following HLA-A2 restricted tumor cells. After a four-hour or overnight incubation pe- peptides: SUR1M2 (LMLGEFLKL), HTERT P540 (ILAK- riod, the medium was discarded and wells washed followed FLHWL), Cyclin B1 204 (ILIDWLVQV), MART-1 27–35 by application of secondary biotinylated antibody (7-B6-1- (AAGIGILTV ), and NY-ESO 1 157–165 (SLLMWITQC). Biotin; Mabtech). The plates were incubated for one hour, further washed, and avidin-enzyme (Streptavidin; Mabtech) 2.6. Cell Lines. Autologous tumor cell lines were established conjugate, were added to each well followed by one-hour in- from tumor fragments by outgrowth in 24 well or 6 well cubation at room temperature. Succeedingly, the wells were plates (Nunc) in medium consisting of RPMI1640 (Invit- washed and the enzyme substrate NBT/BCIP (nitro blue rogen), penicillin, streptomycin, fungizone, 10% fetal calf tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; Mab- serum (Invitrogen), and SoluCortef (Pﬁzer). tech) were added into each well. The plates incubated at Tumor cells were cryopreserved in 90% FCS and 10% room temperature for 2 to 10 minutes, while emerging pur- DMSO (Hospital Pharmacy, RegionH, Copenhagen, Den- ◦ ple spots developed. The reaction was terminated with tap mark) and stored at −140 C. water. Spots were counted with the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers) and the frequency of tumor 2.7. Flow Cytometry. Phenotyping were conducted using a speciﬁc TIL couldbe calculatedfrom the numbers of spot FACS-Aria with Diva software (from BD) and ﬂuorescence forming cells. The assays were preferably done in triplets or conjugated monoclonal antibodies (mAb) against CD3 in duplicates in case of low cell numbers. APC-Cy7, CD4 APC, CD8 PerCP, CD25 PE, CD27 PE, CD45RA FITC, CD45RO PE, CD56 PE (all from BD), CCR7 FITC (R&D systems), CD16 FITC (Dako), CD28 2.10. Cr Release Assay. Astandard Cr -release assay was FITC (Immunotech), CD62Ligand PE (BD Pharmingen), used to quantify the speciﬁc cytotoxic ability of selected TIL 3 51 and CD57 FITC (BD Pharmingen) along with corresponding cultures. In brief, 5×10 Cr -labeled tumor cells (duplicates isotypes. or triplicates) were cocultured with TIL (maximum E : T Journal of Skin Cancer 3 ratio of 100 : 1 and titrated) in RPMI containing 10% FCS for a 4 hour incubation period. Thereafter, Cr -release was measured and percentage of tumor lysis calculated as (#count − Min count)/(Max count −Min count) × 100%. 2.11. Statistical Analysis. We utilized Graphpad Prism sta- tistical software to analyse for statistical diﬀerences, using a paired two-tailed t test. P values <.05 were considered signiﬁcant. 3. Results 3.1. Patients. Tumor material were obtained from 17 patients 2weeks with either locally advanced or advanced disease from me- Figure 1: REP fold-expansion. During two weeks 41 TIL cultures tastasis localized either in lymph nodes (majority of spec- (represented by single diamonds) reached a mean 1400 expansion imens) or subcutaneously. A minimum requirement of fold. 1cm of tumor was needed to ensure suﬃcient material for TIL expansion. The mean age was 62 years with an equal gender distribution. 12 of the patients had only been while NK cells (CD16/56 )(Figure 2) were consistently treated surgically prior to inclusion, while ﬁve patients who absent. In bulk cultures, we observed a heterogeneous CD4 were included in our recent established clinical pilot trial and CD8 T-cell distribution among cultures inter- and had previously received IL-2 and/or DC vaccination based intraindividually. There was, however, an overall skewing immunotherapies. Patients showed the following distribu- towards a CD8 (mean = 74% ± 24%, range 30%–94%) tion of HLA-A types: one HLA-A1+, two HLA-A1/A3+, one T-cell predominance in relation to CD4 T-cells (mean = HLA-A3+, two HLA-A3/A11+, one HLA-A11+, one HLA- 19.5% ± 23.5%, range 1%–64%). Next, we investigated the A3+/A2+, four HLA-A2+, one HLA-A2/24+, and four non- occurrence of surface markers identifying T-cell memory HLA-A1/A2/A3/A11/A24. subsets, or alternatively, a diﬀerentiation path of eﬀector + + cells, in the overall CD3 population, and among CD4 and 3.2. TIL Expansion Kinetics. Lymphocytes migrated out from CD8 T-cell subsets in comparison to TIL after two weeks of the fragments within two-to-ﬁve days and expanded into a REP (Figure 2). Overall, there was a distinct predominance + Lo/− conﬂuent layer before splitting the wells. Each well initiated of CD45RO and CCR-7 T-cell populations before and an individual bulk culture and were kept separated from after REP identifying the cells as T eﬀector memory like. other cultures. TIL bulk cultures expanded to at least 5 × TIL were further characterized by surface markers 10 cells were considered suﬃciently expanding. This was according to a proposed model of eﬀector CD8 T-cell obtained in 15 out of 17 patients (88%) in 6% to 100% of the diﬀerentiation stages by Gattinoni et al. . Expression of bulk cultures (mean 58%) within 3–5 weeks. We found that the lymphoid homing marker CD62L was signiﬁcantly + + growth rates varied markedly even between cultures from the reduced after REP in the CD3 population and the CD8 same patient, and there was no diﬀerence in success rate of subsets and remained unchanged among the CD4 sub- TIL growth from LN or SC tumor material, nor between the sets. Concerning expression of costimulatory markers, we IL-2 concentrations (data not shown). observed a signiﬁcantly higher expression of CD27 among + + + We next tested the proliferative potential of a range of CD8 bulk TIL compared to CD4 cells, while CD4 cells bulk cultures from 12 of the 15 patients with suﬃciently had sustained higher CD28 expression in bulk cultures and growing TIL. This rapid expansion procedure (REP) involves after REP. Although there was a relatively high percentage the addition of allogeneic feeder cells and a CD3 antibody of CD27/28 double positive cells in a few bulk TIL, they and has shown to increase TIL expansion rates considerably, were downregulated after REP. Finally, there was a signiﬁcant in previously reports in both melanoma and head and increase in the high-aﬃnity IL-2 receptor (CD25) after REP + + neck squamous cell carcinoma. Again, the kinetics could in the CD4 population. In conclusion, the CD8 population + Lo Lo vary between cultures from the same patient; however, the express surface markers (CD45RO , CCR-7 , CD62L , Lo Lo Lo procedure could eﬃciently expand TIL bulk cultures to over CD27 ,CD28 ,and CD57 ) resembling intermediate to a 1000-fold in more than half of the cultures in 2 weeks late-stage eﬀector cells as reported by other groups [8, 9, 16]. (Figure 1). Selected expanded cultures were analyzed for the pres- ence of clonally expanded T cells by RT-PCR/DGGE-based 3.3. Phenotypes and Clonal Composition. TIL were visualized TCR clonotype mapping. Analysis revealed the presence of at in the microscope, showing a blasted morphology related to least 10 diﬀerent T-cell clonotypes in bulk cultures as well as actively dividing lymphocytes laying either as single cells or in rapidly expanded cultures (data not shown). The results in clusters/clones. support our previous ﬁndings in expanded TIL from head In acquisition of cells by ﬂow cytometry, gating of viable and neck cancer patients, that expansion by high-dose IL-2 cells was performed on the basis of the forward and side and CD3 antibody seems to support the continued expansion scatter dot plots. T-cells (CD3 ) predominated the cultures, of bulk T-cell clones. Expansion fold 4 Journal of Skin Cancer CD16/56 100 10 80 8 60 6 20 2 0 0 Bulk REP Bulk REP Bulk REP Bulk REP + + CD4 CD8 DP (a) + + + + CD3 CD3 CD3 CD3 P = .034 100 100 100 P = .014 80 80 80 80 60 60 60 40 40 40 40 20 20 20 0 0 0 0 + + + + + + + + + CCR-7/ CD45RA CD45RO CCR-7 CD62L CD27 CD28 CD27/28 CD57 CD25 CD62L (b) + + + + CD4 CD4 CD4 CD4 P = .034 100 100 P = .005 80 80 60 60 60 40 40 40 40 20 20 20 20 0 0 0 + + + + + + + + + CCR-7/ CD45RA CD45RO CCR-7 CD62L CD27 CD28 CD27/28 CD57 CD25 CD62L (c) + + + + CD8 CD8 CD8 CD8 100 100 100 P = .006 P = .002 P = .002 80 80 80 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 + + + + + + + + + CCR-7/ CD27 CD28 CD27/28 CD57 CD25 CD45RA CD45RO CCR-7 CD62L CD62L (d) Figure 2: Phenotypes. FACS determination of phenotypes of TIL cultures pre- (open circles) and post-REP (closed circles) are represented + + Lo from six patients. The overall T-cell eﬀector memory like phenotype (CD3 CD45R0 CCR-7 ) is preserved after REP with a sustained low expression of CD57 and intermediate CD25 expression. CD28 remains unchanged, while CD62L and CD27 is downregulated, indicating a diﬀerentiation towards a later eﬀector stage. 3.4. Sustained Functional Capacity during Expansion. TIL cer testis antigens (NY-ESO-1) served as known targets, cultures from eight patients were selected to scrutinize the while autologous tumor cell lines presented a panel of un- presence of speciﬁc T-cell populations in bulk cultures and known antigen speciﬁcities. Elispot detection of INFγ release after REP. Peptides derived from over expressed (Telomerase, upon antigenic stimulation revealed a sustained functionality Survivin, and Cyclin B1), diﬀerentiation (MART-1) and can- of TIL after REP. Due to the high sensitivity of the assay, we (%) (%) (%) (%) Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP (%) (%) (%) Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP (%) (%) (%) (%) Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP (%) (%) (%) Bulk Bulk Bulk REP REP REP Bulk Bulk Bulk REP REP REP Journal of Skin Cancer 5 1000 800 0 0 Bulk REP Bulk REP Bulk REP Bulk REP Bulk REP Bulk REP Bulk REP MM 4 MM 10 MM 12 MM 4 MM 9 MM 16 SUR1M2 CB9 204 Tumor MART-1 NY ESO 1 K562 HTERT Daudi (a) (b) 100 100 REPM1 REPM8 80 80 60 60 40 40 20 20 0 0 3:1 11:1 33:1 100 :1 3:1 11 : 1 33 : 1 100 : 1 Tumor 1 K562 Tumor 1 K562 Tumor 2 DAUDI Tumor 2 Daudi (c) Figure 3: Functional capacity. Determination of TAA peptide-speciﬁc populations in TIL pre- and post-REP. Results from Elispot detection of INFγ in three patients exempliﬁes the general tendency of speciﬁcity retention, decline, and/or increase as a consequence of unspeciﬁc stimulation during expansion procedures, (a) Autologous tumor cell lines were available from four patients, and all showed TIL with antitumor activity by measuring INFγ in Elispot. Representative results of TIL from three patients show retained tumor-speciﬁc activity. However, in patient MM + 16 we found a component of unspeciﬁc NK/LAK cell activity, which seemed to decline after REP, (b). Example of preserved lytic capacity of TIL from MM 11 after REP. Both cultures show speciﬁc killing of two established autologous tumor cell lines. Interestingly, Tumor 1 seems more immunogenic than Tumor 2, (c). could follow the presence and loss of low-frequency single- 4. Conclusion peptide-speciﬁc T-cell populations (Figure 3(a), occurring as We were able to establish suﬃciently expanding TIL bulk cul- a consequence of an increase or decrease in cell number of tures in ﬁve weeks from the majority of included melanoma a given speciﬁc cell population, during the unspeciﬁc ex- patients. Further expansion by REP generated a mean ex- pansion procedures provided by IL-2 and anti-CD3. Autol- pansion fold of 1400 in two weeks, ensuring the feasibility to ogous tumor cell lines were available in four patients, and reach clinical relevant quantities for clinical testing. Based on all patients contained TIL showing antitumor activity in earlier studies of T-cell therapy of melanoma patients were Elispot (Figure 3(b) and data not shown). The presence of as low as 1, 3 × 10 infused cells containing 30% MART-1- autologous tumor-speciﬁc T-cell populations was more speciﬁc CD8 T-cells mediated a complete clinical response resistant during REP and showed a sustained functional 9 , we estimate that a minimum of 3 × 10 cells are capacity. Although T-cell-speciﬁc antitumor activity was pre- required to obtain a therapeutic eﬀect. Cell-based analysis dominating in TIL, we observed LAK/NK cell activity in a few revealed an oligoclonal composition of T eﬀector memory cultures (Figure 3(b)) by unspeciﬁc engaging the cell lines cells, predominated by CD8 cells showing an intermediate K562 and Daudi. Finally, we conﬁrmed a sustained tumo- to late stage of diﬀerentiation after REP. TIL retained the ricidal capacity of TIL after REP (Figure 3(c)) indicating that functional capacity measured by INFγ release and lytic TIL expanded to clinical relevant numbers (2400- and 4000- activity against autologous tumor. 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Published: Jun 18, 2011
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