Epitope Identification of an mGlu5 Receptor Nanobody Using Physics-Based Molecular Modeling and Deep Learning Techniques.

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development.

Improved antibody pharmacokinetics by disruption of contiguous positive surface potential and charge reduction using alternate human framework.

Optimal pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) are essential to achieve the desired pharmacological benefits in patients. To accomplish this, we followed an approach comprising structure-based mAb charge engineering in conjunction with the use of relevant preclinical models to screen and select humanized candidates with PK suitable for clinical development. Murine mAb targeting TDP-43, ACI-5891, was humanized on a framework (VH1-3/VK2-30) selected based on the highest sequence homology.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galβ1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response.

A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers.

There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations.

Systematic functional identification of cancer multi-drug resistance genes.

Background: Drug resistance is a major obstacle in cancer therapy. To elucidate the genetic factors that regulate sensitivity to anti-cancer drugs, we performed CRISPR-Cas9 knockout screens for resistance to a spectrum of drugs.

Results: In addition to known drug targets and resistance mechanisms, this study revealed novel insights into drug mechanisms of action, including cellular transporters, drug target effectors, and genes involved in target-relevant pathways.

Human Antibody Bispecifics through Phage Display Selection.

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.

Expression of IgG Monoclonals with Engineered Immune Effector Functions.

The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites.

Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood.

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).

Transient expression of human antibodies in mammalian cells.

Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells.

Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation.

Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways.

In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody (99m)Tc-D10 for visualizing small tumour lesions with volumes below 100 mm(3) by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody (99m)Tc-D10 of a size as small as 15.

Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers.

The epidermal growth factor receptor (EGFR) is a cell-surface receptor with a single transmembrane domain and tyrosine kinase activity carried by the intracellular domain. This receptor is one of the four members of the ErbB family including ErbB2, ErbB3, and ErbB4. Ligand binding, like EGF binding, induces a conformational rearrangement of the receptor and induces a homo/hetero dimerization essentially with ErbB family receptors that leads to the phosphorylation of the kinase domain, triggering a signaling cascade.

Force measurements of TCR/pMHC recognition at T cell surface.

The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed.