Chemokine receptor-specific antibodies in cancer immunotherapy: achievements and challenges.

The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications.

Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein.

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones, polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein, respectively, were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence, immunoelectron microscopy and cell surface protein labeling. In addition, measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed.

Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods.

OBJECTIVES: In the past, one of the major problems in gluten analysis has been the unavailability of an efficient, universal, extraction procedure of gliadins - the alcohol-soluble proteins of gluten - from both heat processed and unprocessed products. This study was designed to develop a universal, extraction procedure capable of extracting the totality of gliadins from both unprocessed and heat processed foods for coeliac patients. METHODS: A simple quantitative extraction solution containing 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride ('cocktail'), was developed to extract gliadins from heated foods. RESULTS: The diluted reducing and disaggregating agents reaching the micro plate at low concentration do not affect the ELISA system based on the R5 monoclonal antibody. The recovery of gliadins extracted by the cocktail from spiked samples was nearly complete, with an average mean value of 95.5%, which is clearly superior to 44.4% obtained with conventional 60% aqueous ethanol. The cocktail always yielded either slightly similar or higher values than 60% aqueous ethanol depending on the type of foods: 1.1-fold in unheated foods, 1.4-fold in wheat starches and 3.0-fold in heated foods. False positives or negatives were never observed using the cocktail solution. CONCLUSION: We present a general complete gliadin extraction procedure based on reducing and disaggregating agents for both heated and unheated foods as a crucial tool for gliadin analysis. The new extraction solution is used for corresponding proteins from rye (secalins) and barley (hordeins). The cocktail was employed as the extraction method in the international ring trial evaluation of sandwich R5-ELISA as proposed by the Codex Alimentarius and organized by the Working Group on Prolamin Analysis and Toxicity.

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol.

OBJECTIVES: There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. METHODS: Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested. RESULTS: R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods. CONCLUSION: We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.