Unveiling inverted D genes and D-D fusions in human antibody repertoires unlocks novel antibody diversity.

Antibodies, essential components of adaptive immunity, derive their remarkable diversity primarily from V(D)J gene rearrangements, particularly within the heavy chain complementarity-determining region 3 (CDR-H3) where D genes play a major role. Traditionally, D genes were thought to recombine only in the forward direction, despite having identical recombination signal sequences (12 base pair spacers) at both ends. This observation led us to question whether these symmetrical sequences might enable bidirectional recombination.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire.

A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery.

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing.

Landscape of Non-canonical Cysteines in Human V Repertoire Revealed by Immunogenetic Analysis.

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (Vs) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions.

Tumors attenuating the mitochondrial activity in T cells escape from PD-1 blockade therapy.

PD-1 blockade therapy has revolutionized cancer treatments. However, a substantial population of patients is unresponsive. To rescue unresponsive patients, the mechanism of unresponsiveness to PD-1 blockade therapy must be elucidated.

Retraction: A highly concise and practical route to clavaminols, sphinganine and (+)-spisulosine indium mediated allylation of α-hydrazino aldehyde and a theoretical insight into the stereochemical aspects of the reaction.

[This retracts the article DOI: 10.1039/C3RA43048K.].

Recombinant human B cell repertoires enable screening for rare, specific, and natively paired antibodies.

The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes.

PPAR-Induced Fatty Acid Oxidation in T Cells Increases the Number of Tumor-Reactive CD8 T Cells and Facilitates Anti-PD-1 Therapy.

Although PD-1 blockade cancer immunotherapy has shown potential for a wide range of patients with cancer, its efficacy is limited, in part, due to the loss of effector cytotoxic T lymphocytes (CTLs) via terminal differentiation-induced apoptosis. We previously demonstrated that mitochondrial activation, by the agonists of peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC-1α)/transcription factor complexes, had synergistic effects with a PD-1-blocking monoclonal antibody in a mouse tumor model. In the current study, we examined the molecular mechanism of the synergistic effects of bezafibrate, an agonist of PGC-1α/ PPAR complexes, which enhanced the tumoricidal effects of PD-1 blockade.

Development and analytical performance of a new ARCHITECT automated dipeptidyl peptidase-4 immunoassay.

Background: Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations.

Methods: We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E.

Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity.

Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor.

Development of a new ARCHITECT automated periostin immunoassay.

Background: Periostin is being investigated as a potential biomarker for T-helper-2 (Th2)-driven asthma or eosinophilic inflammation and may help to identify patients more likely to benefit from interleukin-13-targeted treatments. We report the development and analytic performance of the investigational use only ARCHITECT Periostin Immunoassay, a new automated assay developed to detect serum periostin concentrations.

Methods: We assessed assay performance in terms of precision, sensitivity, linearity, interference from classical immunoassay interferents and representatives of common asthma medications, specimen handling, and isoform reactivity.

A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings.

Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays.

S100A9 induced inflammatory responses are mediated by distinct damage associated molecular patterns (DAMP) receptors in vitro and in vivo.

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors.

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA.

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor.

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells.

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment.

Tailor-made antibody therapeutics.

Therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry. There are currently 18 monoclonal antibodies in the market that have been approved by the FDA and over 150 in clinical developments. Driven by innovation and technological developments, scientists have gone beyond the traditional antibody molecules.