Decoupling Individual Host Response and Immune Cell Engager Cytotoxic Potency.

Immune cell engagers are molecular agents, usually antibody-based constructs, engineered to recruit immune cells against cancer cells and kill them. They are versatile and powerful tools for cancer immunotherapy. Despite the multiplication of engagers tested and accepted in the clinic, how molecular and cellular parameters influence their actions is poorly understood.

Rapid nanobody-based imaging of mesothelin expressing malignancies compatible with blocking therapeutic antibodies.

Introduction: Mesothelin (MSLN) is overexpressed in a wide variety of cancers with few therapeutic options and has recently emerged as an attractive target for cancer therapy, with a large number of approaches currently under preclinical and clinical investigation. In this respect, developing mesothelin specific tracers as molecular companion tools for predicting patient eligibility, monitoring then response to mesothelin-targeting therapies, and tracking the evolution of the disease or for real-time visualisation of tumours during surgery is of growing importance.

Methods: We generated by phage display a nanobody (Nb S1) and used enzymatic approaches were used to site-directed conjugate Nb S1 with either ATTO 647N fluorochrome or NODAGA chelator for fluorescence and positron emission tomography imaging (PET) respectively.

Anti-NKG2D single domain-based antibodies for the modulation of anti-tumor immune response.

The natural killer group 2 member D (NKG2D) receptor is a C-type lectin-like activating receptor mainly expressed by cytotoxic immune cells including NK, CD8 T, γδ T and NKT cells and in some pathological conditions by a subset of CD4 T cells. It binds a variety of ligands (NKG2DL) whose expressions is finely regulated by stress-related conditions. The NKG2DL/NKG2D axis plays a central and complex role in the regulation of immune responses against diverse cellular threats such as oncogene-mediated transformations or infections.

A Bispecific Antibody-Based Approach for Targeting Mesothelin in Triple Negative Breast Cancer.

Triple negative breast cancers (TNBC) remain a major medical challenge due to poor prognosis and limited treatment options. Mesothelin is a glycosyl-phosphatidyl inositol-linked membrane protein with restricted normal expression and high level expression in a large proportion of TNBC, thus qualifying as an attractive target. Its overexpression in breast tumors has been recently correlated with a decreased disease-free survival and an increase of distant metastases.

Nanobody-CD16 Catch Bond Reveals NK Cell Mechanosensitivity.

Antibodies are key tools in biomedical research and medicine. Their binding properties are classically measured in solution and characterized by an affinity. However, in physiological conditions, antibodies can bridge an immune effector cell and an antigen-presenting cell, implying that mechanical forces may apply to the bonds.

Preclinical Evaluation of Mesothelin-Specific Ligands for SPECT Imaging of Triple-Negative Breast Cancer.

Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several types of cancer, including triple-negative breast cancer not responding to trastuzumab or hormone-based therapies. Mesothelin-targeting therapies are currently being developed. However, the identification of patients potentially eligible for such a therapeutic strategy remains challenging.

Taking up Cancer Immunotherapy Challenges: Bispecific Antibodies, the Path Forward?

As evidenced by the recent approvals of Removab (EU, Trion Pharma) in 2009 and of Blincyto (US, Amgen) in 2014, the high potential of bispecific antibodies in the field of immuno-oncology is eliciting a renewed interest from pharmaceutical companies. Supported by rapid advances in antibody engineering and the development of several technological platforms such as Triomab or bispecific T cell engagers (BiTEs), the "bispecifics" market has increased significantly over the past decade and may occupy a pivotal space in the future. Over 30 bispecific molecules are currently in different stages of clinical trials and more than 70 in preclinical phase.

Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle-Based Biomedical Applications.

Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems.

A FcγRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy.

Trastuzumab is established as treatment of HER2high metastatic breast cancers but many limitations impair its efficacy. Here, we report the design of a Fab-like bispecific antibody (HER2bsFab) that displays a moderate affinity for HER2 and a unique, specific and high affinity for FcγRIII. In vitro characterization showed that ADCC was the major mechanism of action of HER2bsFab as no significant HER2-driven effect was observed.

Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif.

Therapeutic antibodies for the treatment of pancreatic cancer.

Pancreatic cancer is a devastating disease with the worst mortality rate and an overall 5-year survival rate lower than 5%. In the U.S.

Modification of pancreatic lipase properties by directed molecular evolution.

Cystic fibrosis is associated with pancreatic insufficiency and acidic intraluminal conditions that limit the action of pancreatic enzyme replacement therapy, especially that of lipase. Directed evolution combined with rational design was used in the aim of improving the performances of the human pancreatic lipase at acidic pH. We set up a method for screening thousands of lipase variants for activity at low pH.

Computational study of colipase interaction with lipid droplets and bile salt micelles.

Colipase is a key element in the lipase-catalyzed hydrolysis of dietary lipids. Although devoid of enzymatic activity, colipase promotes the pancreatic lipase activity in physiological intestinal conditions by anchoring the enzyme at the surface of lipid droplets. Analysis of structures of NMR colipase models and simulations of their interactions with various lipid aggregates, lipid droplet, and bile salt micelle, were carried out to determine and to map the lipid binding sites on colipase.

Exploring the active site cavity of human pancreatic lipase.

Within the scope of improving the efficiency of pancreatic enzyme replacement therapy in cystic fibrosis, the feasibility of shifting the pH-activity profile of pancreatic lipase toward acidic values was investigated by site specific mutagenesis in different regions of the catalytic cavity. We have shown that introducing a negative charge close to the catalytic histidine induced a shift of the pH optimum toward acidic values but strongly reduced the lipase activity. On the other hand, a negative charge in the entrance of the catalytic cleft gives rise to a lipase with improved properties and twice more active than the native enzyme at acidic pH.

Structure and orientation of pancreatic colipase in a lipid environment: PM-IRRAS and Brewster angle microscopy studies.

Colipase is a key element in lipase-catalyzed dietary lipids hydrolysis. Although devoid of enzymatic activity, colipase promotes pancreatic lipase activity in the physiological intestinal conditions by anchoring the enzyme on the surface of lipid droplets. Polarization modulation infrared reflection absorption spectroscopy combined with Brewster angle microscopy studies was performed on colipase alone and in various lipid environments to obtain a global view of both conformation and orientation and to assess lipid perturbations.

Role of the lid hydrophobicity pattern in pancreatic lipase activity.

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear.

Crystallization of a proteolyzed form of the horse pancreatic lipase-related protein 2: structural basis for the specific detergent requirement.

Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties.

Inhibitory effect of the pancreatic lipase C-terminal domain on intestinal lipolysis in rats fed a high-fat diet: chronic study.

Objective: Previous in vitro experiments, as well as acute assays in rat showed that the C-terminal domain (CT-domain) of porcine pancreatic lipase behaves as a potent specific noncovalent inhibitor of pancreatic lipase. Nevertheless, the potential use of the CT-domain as a therapeutic tool against obesity in humans requires further investigation and would be best achieved using the human CT-domain. In the present study, we investigated the inhibitory effects of the recombinant human CT-domain, in vivo, upon chronic administration to rats fed a high-fat diet.

Activation of horse PLRP2 by bile salts does not require colipase.

Although structurally similar to pancreatic lipase (PL), the key enzyme of intestinal fat digestion, pancreatic lipase-related protein type 2 (PLRP2) differs from PL in certain functional properties. Notably, PLRP2 has a broader substrate specificity than PL, and unlike that of PL, its activity is not restored by colipase in the presence of bile salts. In the studies presented here, the activation mechanism of horse PLRP2 was studied through active site-directed inhibition experiments, and the results demonstrate fundamental differences with that of PL.

High expression in adult horse of PLRP2 displaying a low phospholipase activity.

The physiological role of the two lipase-related proteins, PLRP1 and PLRP2, still remains obscure although some propositions have been made concerning PLRP2. In this paper, we report the presence of high amounts of PLRP2 in adult horse pancreas whereas no PLRP1 could be detected. As well, a non-parallel expression of PLRP2 and PLRP1 is observed in adult cat and dog, since no PLRP2 could be detected in these two species.

The lipase C-terminal domain. A novel unusual inhibitor of pancreatic lipase activity.

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme.

Interactions of bile salt micelles and colipase studied through intermolecular nOes.

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy.