Complete and long-lasting clinical responses in immune checkpoint inhibitor-resistant, metastasized melanoma treated with adoptive T cell transfer combined with DC vaccination.

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses. This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI. An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols. A second cohort (first-in-man, 5 patients) received TIL combined with autologous tumor lysate-loaded DC vaccination. All patients received cyclophosphamide/fludarabine preconditioning prior to, and intravenous (i.v.) IL-2 after, TIL transfer. The DC vaccine was given as five intradermal injections after TIL and IL-2 administration. [18F]-FDG PET/CT radiology was performed to evaluate clinical response, according to RECIST 1.1 (on the CT part). Immunological monitoring was performed by flow cytometry and T-cell receptor (TCR) sequencing. In the safety/optimization cohort, all patients had a mixed response or stable disease, but none durable. In the combination cohort, two patients experienced complete responses (CR) that are still ongoing (>36 and >18 months, respectively). In addition, two patients had partial responses (PR), one still ongoing (>42 months) with only a small bone-lesion remaining, and one of short duration (<4 months). One patient died early during treatment and did not receive DC. Long-lasting persistency of the injected TILs was demonstrated in blood. In summary, we report clinical responses by TIL therapy combined with DC vaccination in 4 out of 4 treated MM patients who previously failed ICI.

Human serum albumin as a clinically accepted cell carrier solution for skin regenerative application.

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

Transplantation of tissue-engineered cell sheets for stricture prevention after endoscopic submucosal dissection of the oesophagus.

BACKGROUND AND OBJECTIVE: Endoscopic mucosal dissection (ESD) is a treatment option for oesophagus tumours localized to the mucosa enabling en bloc removal of large lesions. The resulting larger mucosal defects have resulted in an increase in the occurrence of post-treatment strictures. Transplantation of autologous cell sheets, cultured from oral mucosa, has been shown to prevent post-ESD strictures. The aim of the study was to assess the efficacy and safety of cell sheet transplantation after oesophageal ESD in a Western patient population where reflux-associated pre-malignant and malignant conditions predominate. METHODS: Patients with Barrett's oesophagus associated high-grade dysplasia or early adenocarcinoma where ESD entailed a resection >3 cm in length and ≥75% of the circumference were eligible for treatment under hospital exemption. Cell sheets were cultured from buccal mucosa according to Good Manufacturing Practice and were endoscopically applied to the post-ESD defect directly after resection. Patients were followed with weekly endoscopy examinations, including confocal laser microscopy, for a total of four weeks. RESULTS: Five patients were treated. ESD was extensive with resections being circumferential in three patients and 9-10 cm in length in two. The number of transplanted cell sheets ranged from two to six. Three patients developed strictures requiring two to five dilatation sessions. CONCLUSIONS: Cell sheet transplantation shows to be safe and feasible in a Western population. Results suggest that transplantation has a protective effect on the mucosal defect after ESD, decreasing both the risk for and extent of stricture formation.

Fedbatch design for periplasmic product retention in Escherichia coli.

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h(-1) where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control.

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.

Solubility and proteolysis of the Zb-MalE and Zb-MalE31 proteins during overproduction in Escherichia coli.

From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation. Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure. It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (< or = 5 microM), this formation is almost abolished while at a comparatively high concentration (> or = 300 microM) 50% of the total product accumulated was in the form of inclusion bodies.

Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli.

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P(malK) promoter; and the cytoplasmic part of the production was compared with production from the P(lacUV5) promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P(malK) promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.