The non-pathogenic protozoon Leishmania tarentolae interferes with the activation of NLRP3 inflammasome in human cells: new perspectives in the control of inflammation.

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.

Chimeric Antigen Receptor T Cell with an Inducible Caspase 9 Suicide Gene Eradicates Uveal Melanoma Liver Metastases via B7-H3 Targeting

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic disease for which there are no effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T cell-based immunotherapy targeting B7-H3. EXPERIMENTAL DESIGN: B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, metastatic UM patient-derived organotypic tumor spheroids (PDOTS), and in immunodeficient and humanized murine models. RESULTS: B7-H3 is expressed at high levels on >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable anti-tumor response, even upon tumor re-challenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody. CONCLUSIONS: These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.

A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

Interplay between B7-H3 and HLA class I in the clinical course of pancreatic ductal adenocarcinoma.

Human leukocyte antigen (HLA) class I defects are associated with cancer progression. However, their prognostic significance is controversial and may be modulated by immune checkpoints. Here, we investigated whether the checkpoint B7-H3 modulates the relationship between HLA class I and pancreatic ductal adenocarcinoma (PDAC) prognosis. PDAC tumors were analyzed for the expression of B7-H3, HLA class I, HLA class II molecules, and for the presence of tumor-infiltrating immune cells. We observed defective HLA class I and HLA class II expressions in 75% and 59% of PDAC samples, respectively. HLA class I and B7-H3 expression were positively related at mRNA and protein level, potentially because of shared regulation by RELA, a sub-unit of NF-kB. High B7-H3 expression and low CD8+ T cell density were indicators of poor survival, while HLA class I was not. Defective HLA class I expression was associated with unfavorable survival only in patients with low B7-H3 expression. Favorable survival was observed only when HLA class I expression was high and B7-H3 expression low. Our results provide the rationale for targeting B7-H3 in patients with PDAC tumors displaying high HLA class I levels.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Implications of High Tumor Burden on Chimeric Antigen Receptor T-Cell Immunotherapy: A Review.

Importance: Chimeric antigen receptor (CAR) T-cell therapy has redefined the therapeutic landscape of several hematologic malignant tumors. Despite its clinical efficacy, many patients with cancer experience nonresponse to CAR T-cell treatment, disease relapse within months, or severe adverse events. Furthermore, CAR T-cell therapy has demonstrated minimal to no clinical efficacy in the treatment of solid tumors in clinical trials. Observations: A complex interplay between high tumor burden and the systemic and local tumor microenvironment on clinical outcomes of CAR T-cell therapy is emerging from preclinical and clinical data. The hallmarks of advanced cancers-namely, inflammation and immune dysregulation-sustain cancer progression. They negatively affect the production, expansion, antitumor activity, and persistence of CAR T-cell products. Understanding of CAR T-cell therapy, mechanisms underlying its failure, and adverse events under conditions of high tumor burden is critical for realizing the full potential of this novel treatment approach. Conclusions and Relevance: This review focuses on linking the efficacy and safety of CAR T-cell therapy with tumor burden. Its limitations relative to high tumor burden, systemic inflammation, and immune dysregulation are discussed. Emerging clinical approaches to overcome these obstacles and more effectively incorporate this therapeutic strategy into the treatment paradigm of patients with solid malignant tumors are also described.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Stressed target cancer cells drive nongenetic reprogramming of CAR T cells and solid tumor microenvironment.

The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquire early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogram and reverse the immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells of healthy donors or metastatic female breast cancer patients, induce robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a promising therapy for solid tumors.

Rectal Administration of Leishmania Cells Elicits a Specific, Th1-Associated IgG2a Response in Mice: New Perspectives for Mucosal Vaccination against Leishmaniasis, after the Repurposing of a Study on an Anti-Viral Vaccine Candidate.

The mucosal immune system plays a pivotal role in the control of infections, as it represents the first line of defense against most pathogens, from respiratory viruses to intestinal parasites. Mucosal vaccination is thus regarded as a promising strategy to protect animals, including humans, from infections that are acquired by ingestion, inhalation or through the urogenital system. In addition, antigens delivered at the mucosal level can also elicit systemic immune responses. Therefore, mucosal vaccination is potentially effective also against systemic infections acquired through non-mucosal routes, for example, through the bite of hematophagous insects, as in the case of leishmaniasis, a widespread disease that affects humans and dogs. Here, we explored the potential of antigen rectal administration for the generation of anti-Leishmania immunity. Mice were immunized through rectal administration of whole cells of the model parasite Leishmania tarentolae (using a clone engineered to express the spike protein of the SARS-CoV-2 virus generated in a previous study). A specific anti-Leishmania IgG antibody response was detected. In addition, the recorded IgG2a/IgG1 ratio was higher than that of animals injected subcutaneously; therefore, suggesting a shift to a Th1-biased immune response. Considering the importance of a Th1 polarization as a protective response against Leishmania infections, we suggest that further investigation should be focused on the development of novel types of vaccines against these parasites based on rectal immunization.

B7-H3-targeted CAR T cell activity is enhanced by radiotherapy in solid cancers.

Adoptive cell therapy utilizing T cells genetically modified to express a chimeric antigen receptor (CAR) has demonstrated promising clinical results in hematological malignancies. However, solid cancers have not seen a similar success due to multiple obstacles. Investigating these escape mechanisms and designing strategies to counteract such limitations is crucial and timely. Growing evidence in the literature supports the hypothesis that radiotherapy has the potential to enhance the susceptibility of solid tumors to CAR T cell therapy, by overcoming mechanisms of resistance. Radiation treatment can increase the susceptibility of different types of solid cancers (TNBC, HNSCC, PDAC) to B7-H3 CAR T cell-mediated eradication. Multiple mechanisms, including reduced cancer cell proliferation, upregulation of the targeted antigen, modulation of apoptotic molecules may contribute to this signal. The information in the literature and the results we describesupport the ability of radiotherapy to improve the efficacy of CAR T cell therapy in solid tumors.

Intracellular persistence of Leishmania tarentolae in primary canine macrophage cells.

Leishmania tarentolae is a non-pathogenic species first isolated from geckoes in the Mediterranean basin. The finding that dogs test positive against both Leishmania infantum and L. tarentolae raises questions regarding the ability of the latter species to persist and adapt to new hosts. This study aimed to evaluate in vitro the capability of L. tarentolae to colonize, survive and persist in canine primary monocyte-derived mononuclear cells. Monocytes were isolated from dog whole blood samples and placed in 24-well plates for differentiation into macrophages and for incubation with L. tarentolae field-isolated strains (RI-325 and SF-178) and laboratory (LEM-124) strain; the parasite burden was assessed at different time points post-infection. The L. infantum laboratory strain (MON-1) was used as control. Infection parameters were evaluated by microscopy, counting the number of amastigotes/200 infected cells, and by duplex real-time PCR from supernatants and detached cells. Similar to L. infantum, L. tarentolae strains developed into round-shaped amastigote-like forms, with higher infection rates detected at 4 h followed by an overall decrease until 48 h. RI-325 presented also a higher infection rate at 72 h. Data showed that L. tarentolae strains infect and persist inside in vitro primary canine mononuclear cells, opening new perspectives for further laboratory studies.

Efficacy of mucosal vaccination using a protozoan parasite as a vehicle for antigen delivery: IgG and neutralizing response after rectal administration of LeCoVax-2, a candidate vaccine against COVID-19.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation.

Leishmania tarentolae as an Antigen Delivery Platform: Dendritic Cell Maturation after Infection with a Clone Engineered to Express the SARS-CoV-2 Spike Protein.

BACKGROUND: Protozoa of the genus Leishmania are characterized by their capacity to target macrophages and Dendritic Cells (DCs). These microorganisms could thus be exploited for the delivery of antigens to immune cells. Leishmania tarentolae is regarded as a non-pathogenic species; it was previously used as a biofactory for protein production and has been considered as a candidate vaccine or as an antigen delivery platform. However, results on the type of immune polarization determined by L. tarentolae are still inconclusive. METHODS: DCs were derived from human monocytes and exposed to live L. tarentolae, using both the non-engineered P10 strain, and the same strain engineered for expression of the spike protein from SARS-CoV-2. We then determined: (i) parasite internalization in the DCs; and (ii) the capacity of the assayed strains to activate DCs and the type of immune polarization. RESULTS: Protozoan parasites from both strains were effectively engulfed by DCs, which displayed a full pattern of maturation, in terms of MHC class II and costimulatory molecule expression. In addition, after parasite infection, a limited release of Th1 cytokines was observed. CONCLUSIONS: Our results indicate that L. tarentolae could be used as a vehicle for antigen delivery to DCs and to induce the maturation of these cells. The limited cytokine release suggests L. tarentolae as a neutral vaccine vehicle that could be administered in association with appropriate immune-modulating molecules.

Recruitment, Infiltration, and Cytotoxicity of HLA-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models.

Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.

CAR T Cell-Based Immunotherapy for the Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in adults. Current treatment options typically consist of surgery followed by chemotherapy or more frequently radiotherapy, however, median patient survival remains at just over 1 year. Therefore, the need for novel curative therapies for GBM is vital. Characterization of GBM cells has contributed to identify several molecules as targets for immunotherapy-based treatments such as EGFR/EGFRvIII, IL13Rα2, B7-H3, and CSPG4. Cytotoxic T lymphocytes collected from a patient can be genetically modified to express a chimeric antigen receptor (CAR) specific for an identified tumor antigen (TA). These CAR T cells can then be re-administered to the patient to identify and eliminate cancer cells. The impressive clinical responses to TA-specific CAR T cell-based therapies in patients with hematological malignancies have generated a lot of interest in the application of this strategy with solid tumors including GBM. Several clinical trials are evaluating TA-specific CAR T cells to treat GBM. Unfortunately, the efficacy of CAR T cells against solid tumors has been limited due to several factors. These include the immunosuppressive tumor microenvironment, inadequate trafficking and infiltration of CAR T cells and their lack of persistence and activity. In particular, GBM has specific limitations to overcome including acquired resistance to therapy, limited diffusion across the blood brain barrier and risks of central nervous system toxicity. Here we review current CAR T cell-based approaches for the treatment of GBM and summarize the mechanisms being explored in pre-clinical, as well as clinical studies to improve their anti-tumor activity.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Betulinic acid is a PPARγ antagonist that improves glucose uptake, promotes osteogenesis and inhibits adipogenesis.

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

Cytokine-Induced Killer Cells Kill Chemo-surviving Melanoma Cancer Stem Cells.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4 Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP- counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277-88. ©2016 AACR.

Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis.

This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.

Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.

Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 µg/well, T = 37 °C) was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP KM = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP KM = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.