Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects.

The evolving development landscape of biotherapeutics and their growing complexity from simple antibodies into bi- and multi-specific molecules necessitates sophisticated discovery and engineering platforms. This review focuses on mammalian display technology as a potential solution to the pressing challenges in biotherapeutic development. We provide a comparative analysis with established methodologies, highlighting key aspects of mammalian display technology, including genetic engineering, construction of display libraries, and its pivotal role in hit selection and/or developability engineering.

Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies.

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target.

Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes.

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance.

discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom.

The monocled cobra () is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments.

Advances in antibody phage display technology.

Phage display technology can be used for the discovery of antibodies for research, diagnostic, and therapeutic purposes. In this review, we present and discuss key parameters that can be optimized when performing phage display selection campaigns, including the use of different antibody formats and advanced strategies for antigen presentation, such as immobilization, liposomes, nanodiscs, virus-like particles, and whole cells. Furthermore, we provide insights into selection strategies that can be used for the discovery of antibodies with complex binding requirements, such as targeting a specific epitope, cross-reactivity, or pH-dependent binding.

Cross-Reactive SARS-CoV-2 Neutralizing Antibodies From Deep Mining of Early Patient Responses.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response.

Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation.

In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver.

Beyond affinity: selection of antibody variants with optimal biophysical properties and reduced immunogenicity from mammalian display libraries.

The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode of therapeutic action. As more protein therapeutics pass through the development pipeline; however, it is clear that non-optimal biophysical properties can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems can lead to delay or failure in traversing the development process.

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing.

The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization.

Targeting early changes in the synovial microenvironment: a new class of immunomodulatory therapy?

Objectives: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA).

Methods: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display.

Publisher Correction: In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

In the original version of this Article, the sixth sentence of the first paragraph of the Introduction incorrectly read 'Particularly, elapid antivenoms often have an unbalanced antibody content with relatively low amounts of antibodies against small neurotoxic venom components that have low immunogenicity, which often leads to low immune cgqtns in production animals'. The correct version states 'responses' instead of 'cgqtns'. This has been corrected in both the PDF and HTML versions of the Article.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals.

Basics of Antibody Phage Display Technology.

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments.

Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo.

Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1.

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity.

Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo.

The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions.

The INNs and outs of antibody nonproprietary names.

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g.

Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2).

Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp).

Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display.

Identification of optimal protein binders through the use of large genetically encoded display libraries.

The use of large genetically encoded binder libraries in co-operation with display technologies has matured over the past 25 years, and is now one of the primary methods used for selection of protein binders. Display technology has proven to be a robust and versatile method for generating binders to almost any antigen of interest. The evolution of this technology beyond antibody phage display has opened up new aspects for the concept of designer biologics.

Temporal and embryonic lineage-dependent regulation of human vascular SMC development by NOTCH3.

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes.

Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody.