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Background The aim of this study was to evaluate an improved technique for expansion of tumor-infiltrating lymphocytes (TILs) based on the WAVE Bioreactor system with perfusion and tube-welding techniques. Our hypothesis was that the bioreactor would allow for optimized provision of nutrients and removal of spent media while minimizing culture volumes. These refinements might lead to a better quality of expanded cells with lower amounts of exhausted cells compared to static expansions in culture bags. Procedures Tumor-infiltrating lymphocytes from 4 melanoma patients were expanded and compared in parallel using either the WAVE Bioreactor 2/10 System or traditional static culture methods. The parameters viability, final cell number, phenotype and effector function were measured. Results Our results show that the bioreactor system with perfusion is suitable for large-scale expansion of tumor-infiltrating lymphocytes and allows for higher cell densities and absolute cell numbers as compared to static culture conditions. Phenotypic characteristics of TILs were compared pre and post expansion and showed no consistent difference between the two expansion methods. TILs harvested had the phenotype and function corresponding to intermediate to late effector cells. The system allows one technician to operate several bioreactors simultaneously, thereby reducing the labor for one expansion to approximately 1/3 compared to static expansion. Discussion The WAVE Bioreactor system is suitable for large-scale expansion of TILs. Due to constant perfusion of fresh media and removal of spent media much higher cell densities were achieved while the culture volume and the glucose and glutamine levels were kept constant. Expansion of TILs in the bioreactor system represents a labor- and cost-effective method to reach large numbers of T cells for adoptive cell transfer therapy in the clinic. Conclusion The system presented herein offers an effective alternative to large-scale production of cell products for clinical use while meeting requirements of therapeutic cell quantities and qualities of current protocols for treatment of malignant melanoma.

Background . Successful cell therapy relies on the identifi cation and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. Material and methods. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Results and conclusion . Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP � 16 � 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP � 356 � 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF- β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artifi cial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specifi city, as determined by IFN γ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artifi cial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specifi city.

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5 × 10(6) TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.

Autologous or allogeneic transfer of tumor-infiltrating T-lymphocytes is a promising treatment for metastatic cancers, but a major concern is the difficulty in evaluating cell trafficking and distribution in adoptive cell therapy. This study presents a method of tracking transfusion of T-lymphoblasts in a porcine model by (18)F-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG) and positron emission tomography.T-lymphoblasts were labeled with the positron-emitting tracer [(18)F]FDG through incubation. The T-lymphoblasts were administered into the bloodstream, and the distribution was followed by positron emission tomography for 120 min. The cells were administered either intravenously into the internal jugular vein (n=5) or intraarterially into the ascending aorta (n=1). Two of the pigs given intravenous administration were pretreated with low-molecular-weight dextran sulphate.The cellular kinetics and distribution were readily quantifiable for up to 120 min. High (78.6% of the administered cells) heterogeneous pulmonary uptake was found after completed intravenous transfusion. The pulmonary uptake was decreased either by preincubating and coadministrating the T-lymphoblasts with low-molecular-weight dextran sulphate or by administrating them intraarterially.The present work shows the feasibility of quantitatively monitoring and evaluating cell trafficking and distribution following administration of [(18)F]FDG-labeled T-lymphoblasts. The protocol can potentially be transferred to the clinical setting with few modifications.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. We report a pilot study of local adenovector CD40L (AdCD40L) immunogene treatment in 19 cases of canine melanoma (14 oral, 4 cutaneous, and 1 conjunctival). Three patients were World Health Organization stage I, 2 were stage II, 10 stage III, and 4 stage IV. One to 6 intratumoral injections of AdCD40L were given every 7 days, followed by cytoreductive surgery in 9 cases and only immunotherapy in 10 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response included 5 complete responses, 8 partial responses, and 4 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 160 days (range, 20-1141 d), with 3 dogs still alive at submission. Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is in progress.

Malignant melanoma is a common type of solid tumor that causes high cancer-related mortality in young adults of Northern Europe. The incidence of melanoma increases rapidly which renders us a special responsibility to investigate this disease in depth. One recent promising approach to treat malignant melanoma is adoptive cell therapy with tumor-directed autologous T cells. This thesis aims to improve this therapy in four different studies. We first sought to establish a protocol for the assessment of melanoma-specific T-cell cultures in order to screen for optimal specificity and reactivity in a robust, reliable and simple manner. The conclusion was that reactive cells could be found in a majority of patients and could be screened for specificity by stimulation with melanoma cell lines. In the next study, 28 melanoma patients with advanced disease were treated with autologous tumor-infiltrating T cells. Objective responses (18%) including one sustained complete response were observed. This is the first study in cancer patients with autologous T cell transfer combined with low-dose s.c. IL-2 as supportive cytokine. In the following two studies we wanted to improve management and culture conditions of the T cells. When investigating methods for improved handling and preservation of large numbers of T cells, we observed that freeze-thawing of T cells could impair the metabolic activity of the T cells. Another conclusion was that rapid expansion of T cells could lead to loss of antigenic specificity and apoptosis. These adverse effects could be prevented with short time recovery. In order to improve expansion methods, mass expansion of T cells in an automated bioreactor was evaluated. We concluded that the bioreactor is suitable for this task and allows for higher cell densities and absolute cell numbers compared to traditional culturing conditions without influencing cell phenotype or reactivity. Taken together, my current studies present guiding principles and encouragement for the further development of immunotherapies for treatment of patients with malignant melanoma.

Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.