Tim4 enables large peritoneal macrophages to cross-present tumor antigens at early stages of tumorigenesis.

Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.

Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma.

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients.

GD2 CAR T cells against human glioblastoma.

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF ß1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors.

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells.

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.

MSC-Delivered Soluble TRAIL and Paclitaxel as Novel Combinatory Treatment for Pancreatic Adenocarcinoma.

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. Results: PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.

Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

Role of Capsid Anchor in the Morphogenesis of Zika Virus.

The flavivirus capsid protein (C) is separated from the downstream premembrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the endoplasmic reticulum (ER). To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of Zika virus was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression in cis with respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of heterologous, but not homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.IMPORTANCE The capsid anchor (Ca) is a single-pass transmembrane domain at the C terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further examine the role of Ca in Zika virus life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

DNA-immunisation with dengue virus E protein domains I/II, but not domain III, enhances Zika, West Nile and Yellow Fever virus infection.

Dengue virus (DENV), the causative agent of dengue disease, is among the most important mosquito-borne pathogens worldwide. DENV is composed of four closely related serotypes and belongs to the Flaviviridae family alongside other important arthropod-borne viral pathogens such as Zika virus (ZIKV), West Nile virus (WNV) and Yellow Fever virus (YFV). After infection, the antibody response is mostly directed to the viral E glycoprotein which is composed of three structural domains named DI, DII and DIII that share variable degrees of homology among different viruses. Recent evidence supports a close serological interaction between ZIKV and DENV. The possibility of worse clinical outcomes as a consequence of antibody-dependent enhancement of infection (ADE) due to cross-reactive antibodies with poor neutralisation activity is a matter of concern. We tested polyclonal sera from groups of female Balb/C mice vaccinated with DNA constructs expressing DI/DII, DIII or the whole sE from different DENV serotypes and compared their activity in terms of cross-reactivity, neutralisation of virus infection and ADE. Our results indicate that the polyclonal antibody responses against the whole sE protein are highly cross-reactive with strong ADE and poor neutralisation activities due to DI/DII immunodominance. Conversely, anti-DIII polyclonal antibodies are type-specific, with no ADE towards ZIKV, WNV and YFV, and strong neutralisation activity restricted only to DENV.

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells.

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.

Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through α5ß1 Integrin.

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5ß1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5ß1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5ß1 modulates the expression/activity of another integrin, α2ß1, involved in the cell adhesion to collagen I. These results suggest that α5ß1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2ß1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5ß1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.

New tags for recombinant protein detection and O-glycosylation reporters.

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation.

CD4 and BST-2/tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules.

CD4 and BST-2/Tetherin are cellular membrane proteins targeted to degradation by the HIV-1 protein Vpu. In both cases proteasomal degradation following recruitment into the ERAD pathway has been described. CD4 is a type I transmembrane glycoprotein, with four extracellular immunoglobulin-like domains containing three intrachain disulfide bridges. BST-2/Tetherin is an atypical type II transmembrane glycoprotein with an N-terminal transmembrane domain and a C-terminal glycophosphatidylinositol anchor, which dimerizes through three interchain bridges. We investigated spontaneous and Vpu-induced retro-translocation of CD4 and BST-2/Tetherin using our novel biotinylation technique in living cells to determine ER-to-cytosol retro-translocation of proteins. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and then becomes deglycosylated and reduced to monomers. These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation.

Selective targeting of proteins within secretory pathway for endoplasmic reticulum-associated degradation.

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.

A vector design that allows fast and convenient production of differently tagged proteins.

Recombinant-tagged proteins have a widespread use in experimental research as well as in clinical diagnostic and therapeutic approaches. Well-stocked sets of differently tagged variants of a same protein would be of great help. However, the construction of differently tagging vectors is a demanding task since cloning procedures need several tailored DNA inserts. In this study, we describe a novel vector system that allows a cost- and time-effective production of differently tagged variants of a same protein by using the same DNA fragment and a set of vectors each carrying a different tag. The design of these expression vectors is based on an intronic region that becomes functional upon cloning the insert sequence, splicing of which attaches a certain tag to the protein termini. This strategy allows for the cloning of the fragment that codes for the protein of interest, without any further modification, into different vectors, previously built and ready-to-use, each carrying a tag that will be joined to the protein. Proof of principle for our expression system, presented here, is shown through the production of a functional anti-GD2 Fab fragment tagged with biotin or polyhistidine, or a combination of both, followed by the demonstration of the functional competencies of both the protein and the tags.

Efficient detection of proteins retro-translocated from the ER to the cytosol by in vivo biotinylation.

Retro-translocation from the ER to the cytosol of proteins within the secretory pathway takes place on misfolded molecules that are targeted for degradation by the cytosolically located 26S proteasome complex. Retro-translocation occurs also for other proteins (such as calreticulin) that, despite being synthesized and transported to the ER, are in part dislocated to the cytosol. We have taken advantage of the E. coli derived biotin-ligase (BirA) expressed in the cytosol of mammalian cells to specifically biotin-label in vivo proteins within the secretory pathway that undergo retro-translocation. We validated the method using four different proteins that are known to undergo retro-translocation upon different conditions: the human trans-membrane protein MHC class-I α chain (MHC-Iα), the Null Hong Kong mutant of the secretory α1 anti-trypsin (NHK-α1AT), the immunoglobulin heavy chain (HC) and the ER chaperone calreticulin (Crt). We observed specific mono-biotinylation of cytosolically dislocated molecules, resulting in a novel, reliable way of determining the extent of retro-translocation.

CD57+ T lymphocytes and functional immune deficiency.

CD57(+) expression in T lymphocytes has been recognized for decades as a marker of in vitro replicative senescence. In recent years, accumulating evidences have pointed on the utility of this marker to measure functional immune deficiency in patients with autoimmune disease, infectious diseases, and cancers. We review here the relevant literature and implications in clinical settings.