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.

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.

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.

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.

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).

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).

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.

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.

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.

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.

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.

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.

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.

Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through α5β1 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 α5β1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver.

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).

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.

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.

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.

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.

Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models.

Background: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.

Gangliosides, Ab1 and Ab2 antibodies IV. Dominance of VH domain in the induction of anti-idiotypic antibodies by gene gun immunization.

The heavy chain of anti-N-glycolyl-ganglioside P3 mAb plays the main role in its binding properties. At least one hybrid idiotype consisting on the P3 VH and an unrelated VL domain retains antigen recognition. Moreover, the unusual immunogenic properties of P3 idiotype could be modified by single mutations of H-CDR residues.

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner.

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA.

An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts.

To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The epsilonSIP molecule consisted of a single-chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the epsilon-CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the epsilonSIP molecule was flanked by the leader peptide from the original murine antibody at its N-terminus and an endoplasmic reticulum retention signal (HDEL) at its C-terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum.

Recombinant dimeric small immunoproteins neutralize transmissible gastroenteritis virus infectivity efficiently in vitro and confer passive immunity in vivo.

Small immunoproteins (SIPs) are single-chain molecules comprising the variable regions of an antibody assembled in a single polypeptide (scFv) and joined to the immunoglobulin heavy-chain dimerizing domain. To investigate the potential of these molecules to provide protection against enteric infections when supplied orally, SIPs were generated against Transmissible gastroenteritis virus (TGEV), a highly pathogenic porcine virus. Different variants of TGEV-specific SIPs were created, of epsilon and alpha isotypes, by exploiting the dimerizing domains epsilonCH4 and alphaCH3 of human and swine origin.