Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS.

Antibody libraries came into existence 30 years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an off-the-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful.

Reciprocal interactions between innate immune cells and astrocytes facilitate neuroinflammation and brain metastasis via lipocalin-2.

Brain metastasis still encompass very grim prognosis and therefore understanding the underlying mechanisms is an urgent need toward developing better therapeutic strategies. We uncover the intricate interactions between recruited innate immune cells and resident astrocytes in the brain metastatic niche that facilitate metastasis of melanoma and breast cancer. We show that granulocyte-derived lipocalin-2 (LCN2) induces inflammatory activation of astrocytes, leading to myeloid cell recruitment to the brain.

Epithelial cell-expressed type II IL-4 receptor mediates eosinophilic esophagitis.

Background: Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease, characterized by eosinophil-rich inflammation in the esophagus. The histopathological and clinical features of EoE have been attributed to overproduction of the type 2 cytokines IL-4 and IL-13, which mediate profound alterations in the esophageal epithelium and neutralizing of their shared receptor component (IL-4Rα) with a human antibody drug (dupilumab) demonstrates clinical efficacy. Yet, the relative contribution of IL-4 and IL-13 and whether the type II IL-4 receptor (comprised of the IL-4Rα chain in association with IL-13Rα1) mediates this effect has not been determined.

Antibody-Mediated Inhibition of Insulin-Degrading Enzyme Improves Insulin Activity in a Diabetic Mouse Model.

Diabetes is a metabolic disease that may lead to different life-threatening complications. While insulin constitutes a beneficial treatment, its use may be limited due to increased degradation and an increase in side effects such as weight gain and hypoglycemia. Small molecule inhibitors to insulin-degrading enzyme (IDE) have been previously suggested as a potential treatment for diabetes through their ability to reduce insulin degradation and thus increase insulin activity.

Inhibition of Sema-3A Promotes Cell Migration, Axonal Growth, and Retinal Ganglion Cell Survival.

Purpose: Semaphorin 3A (Sema-3A) is a secreted protein that deflects axons from inappropriate regions and induces neuronal cell death. Intravitreal application of polyclonal antibodies against Sema-3A prevents loss of retinal ganglion cells ensuing from axotomy of optic nerves. This suggested a therapeutic approach for neuroprotection via inhibition of the Sema-3A pathway.

A key role for IL-13 signaling via the type 2 IL-4 receptor in experimental atopic dermatitis.

IL-13 and IL-4 are potent mediators of type 2-associated inflammation such as those found in atopic dermatitis (AD). IL-4 shares overlapping biological functions with IL-13, a finding that is mainly explained by their ability to signal via the type 2 IL-4 receptor (R), which is composed of IL-4Rα in association with IL-13Rα1. Nonetheless, the role of the type 2 IL-4R in AD remains to be clearly defined.

Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells.

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker.

"BIClonals": Production of Bispecific Antibodies in IgG Format in Transiently Transfected Mammalian Cells.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components.

Design Principles for Bispecific IgGs, Opportunities and Pitfalls of Artificial Disulfide Bonds.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format.

A modular platform for targeted RNAi therapeutics.

Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting, their clinical translation has not occurred.

Antibody Isolation From a Human Synthetic Combinatorial and Other Libraries of Single-Chain Antibodies.

Antibody libraries came into existence 25 years ago when the accumulating sequence data of immunoglobulin genes and the advent of the PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. "Synthetic" or "semisynthetic" libraries are from naive-non-immunized source and considered to be a source for many different targets, including self-antigens.

Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining.

Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of V(H) and V(L) genes obtained from lymphocytes of immunized animals. Recently, a different approach to antibody isolation that circumvents library screening and instead relies on the mining of the V(H) and V(L) gene repertoires obtained by high throughput sequencing of cDNAs from bone marrow antibody-secreting cells was reported. Here we compared the antibodies obtained via phage library screening or via repertoire mining of V gene cDNAs obtained from total splenocytes of mice immunized with the hapten trinitrophenyl (TNP) conjugated to carrier proteins.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle.

Isolation of scFvs that inhibit the NS3 protease of hepatitis C virus by a combination of phage display and a bacterial genetic screen.

The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs.

Design of a human synthetic combinatorial library of single-chain antibodies.

Antibody libraries came into existence 15 years ago when the accumulating sequence data of immunoglobulin genes and the advent of the PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. "Synthetic" or "semi-synthetic" libraries are from naïve, non-immunized source and considered to be a source for many different targets, including self-antigens.

An investigation of antistreptococcal antibody responses in guttate psoriasis.

In two-thirds of patients with guttate psoriasis (GP), there is good evidence that the eruption is triggered by a streptococcal throat infection. We attempted to determine if a specific epitope of the bacterial pathogen was associated with the humoral immune response in GP patients. Antibody titres against beta-haemolytic streptococci (BHS) extracts in sera from 14 patients with GP, 10 healthy controls and 10 chronic plaque psoriasis (CPP) patients were determined by ELISA.