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Background Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds. Results Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity. Conclusions The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host. Electronic supplementary material The online version of this article (10.1186/s12858-018-0101-0) contains supplementary material, which is available to authorized users.

Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start-up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient-specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial-scale production. We discuss the processes that are required for scale-out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.

The aim of this study was to examine the effect of two of the most commonly used viral vectors, that is, retrovirus and adenovirus, on the antigen presentation of dendritic cells (DCs). DCs were generated from CD34(+) hematopoietic precursors and CD14(+) monocytes of the same prostate cancer patients. Adenoviral transduction of monocyte-derived DCs (MO-DCs) resulted in upregulation of CD80, CD86, and CD83 expression. Adenovirus-transduced MO-DCs were also more potent stimulators of allogeneic lymphocytes, produced increased amounts of the cytokines tumor necrosis factor alpha and interleukin 12 p70, and exhibited increased expression of NF-kappaB and antiapoptotic molecules Bcl-X(L) and Bcl-2. Enhanced expression of the antiapoptotic molecules correlated with increased resistance of adenovirus-transduced MO-DCs to spontaneous as well as Fas-mediated cell death. In contrast to the adenoviral construct, no significant transduction of MO-DCs with the retrovirus could be obtained. Transduction of CD34(+) cell-derived DCs with the retrovirus or the adenovirus did not significantly alter expression of the costimulatory molecules or cytokines studied. At lower stimulation ratios, CD34(+) cell-derived DCs transduced with retrovirus were less potent in their ability to stimulate allogeneic lymphocytes in comparison with nontransduced DCs. Our results indicate that adenoviral vectors may be more suitable for gene delivery to DCs for immunotherapy.

Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.

The purpose of this study was to determine the safety and antitumor activity of IFN-gamma retroviral vector in patients with advanced melanoma. Seventeen patients (9 single courses, 8 multiple courses) received a total of 363 intratumor injections of IFN-gamma retroviral vector (1 x 10(7) PFU/ml administered at 0.3, 0.5, and 1.0 ml per cohort). No grade III/IV adverse events were attributed to study medication. Replication-competent retrovirus was not detected in any of the 17 patients by polymerase chain reaction studies. Eight patients showed elevated anti-tumor antibody responses in comparison with baseline by ELISA. One of nine patients treated with a single course had an optimal response of stable disease, compared with eight of eight multiple-injected patients. Median survival of single-injected patients was 150 days, and patients who received multiple injections have still not achieved median survival duration, with four of eight still living (p = 0.0462, Wilcoxon; p = 0.0273, log rank). We conclude that intratumor injection of IFN-gamma is safe and well tolerated. Evidence of antitumor activity is suggested in patients with advanced malignancy that received multiple injections.

Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.

The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.

Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells., Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity., Author Summary Graft-versus-host disease (GVHD) is the most serious complication of bone marrow transplants between individuals (so-called allogenic transplants). The class of suppressor immune cells called regulatory T cells (Tregs) inhibit GVHD by dampening the effects of donor immune cells in the grafted tissue. The cellular and molecular mechanisms involved in this process have not been fully characterized, particularly for human cells. In this study, we report that human Tregs, which we generated from precursor cells ex vivo, express high levels of a cell surface protein called PD-L1 (programmed death ligand-1) that is known to mediate immune suppression. Coculture of these Tregs with allogeneic antigen-presenting cells (APCs), which are known to initiate GVHD, increased, in turn, the amount of PD-L1 on the APCs. The Treg-conditioned APCs were then less able than unconditioned APCs to provoke GVHD in a mouse model of the condition, preventing the death of the animals after transplantation. We found that an antibody against PD-L1 blocked the immunosuppressive effects of Tregs or Treg-conditioned APCs, indicating that this protein is an important part of the molecular mechanism. These findings are potentially important for attempts to modulate immune responses in disease by transplanting T cells into patients.

Background: Interferon-γ (IFN-γ) gene/retroviral vector cell vaccinations have generated protective responses from unmodified tumor cell challenges as well as a regression of established tumors in animal models. The purpose of this trial was to determine the feasibility and safety of a direct intratumoral injection of IFN-γ retroviral vector in advanced melanoma patients. Methods: This was a phase I study, in which 13 patients received a single daily injection of a retroviral vector with the IFN-γ gene for 5 consecutive days (1.5 × 108 colony-forming units total dose); patients subsequently underwent resection of the injected lesion to confirm DNA transduction in situ. Results: No toxicity related to the injected vector was observed. Replication competent retrovirus was not observed in any prepared samples (n = 65). IFN-γ expression was confirmed in 3 of 10 harvested tumor samples; one was equivocal, and DNA transduction was unable to be confirmed by enzyme-linked immunospot assay in six samples. Conclusions: An injection of IFN-γ gene/retroviral vector is well tolerated. DNA transduction was demonstrated in human subjects, confirming the feasibility of the direct injection approach for the gene therapy of solid tumors. Further trials to determine optimal schedule and potential efficacy are indicated.

The generation of a lasting systemic immune response is a primary goal for cancer immunotherapy. Here we examine the ability of high-titer IFN-gamma retroviral vector injected into an accessible tumor to generate significant antitumor responses at a distal untreated site. CT26 or B16F10 murine tumors were inoculated subcutaneously to form solid tumors in BALB/c or C57BL/6 mice. Seven to 10 days postinoculation, high-titer IFN-gamma retroviral vector was directly injected into the subcutaneous tumor nodule, and optimal dose and course of therapy were determined. As a model for disseminated disease, mice were inoculated intravenously with CT26 cells to form pulmonary lesions, at the same time as the subcutaneous injections. Regression of subcutaneous tumor correlated with a systemic response at the distal lung metastases in the IFN-gamma-treated group (p < 0.0005). Splenocytes from mice with completely regressed tumors had a twofold increase in percent specific cytotoxicity in a standard CTL assay as compared with nonresponding mice. CD8+ T cells were shown to be essential for the regional and systemic antitumor response, as determined by in vivo cell depletion experiments. These data demonstrate that IFN-gamma retroviral vector gene therapy delivered intralesionally can generate significant inhibition of pulmonary tumor formation distal to the treatment site. The data from these preclinical studies suggest the potential clinical value of retroviral vector-mediated cytokine gene therapy for systemic cancer.

A total of 17 patients with metastatic melanoma were treated with intratumoral interferon-gamma (IFN-gamma) retroviral vector in a phase I clinical trial. A cycle of treatment consisted of five daily injections every 2 weeks. Patients were divided into two treatment arms that involved a single course (one cycle) of treatment (group I; n = 9) and multiple cycles (six cycles) of treatment (group II; n = 8). Patients received intratumoral injections of IFN-gamma (10(7) plaque-forming units/mL administered at 0.3, 0.5, and 1.0 mL per cohort of patients). All patients receiving multiple injections either maintained stable disease (n = 5) or achieved a partial or complete response (n = 3) of the injected lesion, whereas in patients receiving a single cycle of treatment, only one of nine patients had a response. Patients were assessed for immunoglobulin G antibody (Ab) responses to the melanoma-associated antigens (MAA) tyrosinase, gp100, TRP-2, and MAGE-A1 by affinity enzyme-linked immunosorbent assay. Anti-MAGE-A1 and tyrosinase Ab were significantly elevated from baseline (day 0) to week 16 during treatment (P = .005; P = .002, respectively) in patients who received multiple injections. Patients undergoing treatment who had a clinical response (stable disease or better) also had significantly more elevated Ab responses to a greater number of MAA (P = .0004). The induction of systemic Ab responses to multiple MAA also correlated with systemic clinical responses. These studies suggest that multiple anti-MAA Ab responses are associated with clinical responses to IFN-gamma retroviral treatment and may be used as surrogate response markers.