Welbeck Danquah

Bio: Welbeck Danquah is an academic researcher from University of Hamburg. The author has contributed to research in topics: Single-domain antibody & Antibody. The author has an hindex of 5, co-authored 7 publications receiving 721 citations.

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

Abstract: Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation.

Abstract: Ion channels are desirable therapeutic targets, yet ion channel–directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies—small, single-domain antibody fragments—may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5′-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.

Technical Advance: A new cell preparation strategy that greatly improves the yield of vital and functional Tregs and NKT cells

Abstract: Release of NAD(+) during preparation of murine lymphocytes causes enzymatic ADP-ribosylation of cell-surface proteins on T cells, catalyzed by toxin-related ecto-ADP-ribosyltransferase, ARTC2. ADP-riboslyation activates the cytolytic P2X7 ion channel and affects, in particular, the vitality and function of Tregs and NKT cells. Here, we describe a simple method-injection of an ARTC2-blocking nanobody-to greatly improve Treg and NKT cell vitality and to preserve their function during in vitro assays and in adoptive-transfer experiments. Moreover, we present a method for the sorting of functional, primary NKT cells, based on coexpression of ARTC2 and NK1.1. Our results pave the way for the efficient ex vivo proliferation of Tregs and NKT cells and for new experimental and therapeutic uses of these important regulatory cells.

Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium

Abstract: ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.

Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers.

Abstract: Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.

Nanobodies: Natural Single-Domain Antibodies

Abstract: Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

PEGylation of therapeutic proteins.

Abstract: Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.

Extracellular ATP and P2 purinergic signalling in the tumour microenvironment

Abstract: Modulation of the biochemical composition of the tumour microenvironment is a new frontier of cancer therapy. Several immunosuppressive mechanisms operate in the milieu of most tumours, a condition that makes antitumour immunity ineffective. One of the most potent immunosuppressive factors is adenosine, which is generated in the tumour microenvironment owing to degradation of extracellular ATP. Accruing evidence over the past few years shows that ATP is one of the major biochemical constituents of the tumour microenvironment, where it acts at P2 purinergic receptors expressed on both tumour and host cells. Stimulation of P2 receptors has different effects depending on the extracellular ATP concentration, the P2 receptor subtype engaged and the target cell type. Among P2 receptors, the P2X purinergic receptor 7 (P2X7R) subtype appears to be a main player in host–tumour cell interactions. Preclinical studies in several tumour models have shown that P2X7R targeting is potentially a very effective anticancer treatment, and many pharmaceutical companies have now developed potent and selective small molecule inhibitors of P2X7R. In this Review, we report on the multiple mechanisms by which extracellular ATP shapes the tumour microenvironment and how its stimulation of host and tumour cell P2 receptors contributes to determining tumour fate. In this Review, Di Virgilio et al. describe how extracellular ATP and P2 purinergic signalling can shape the tumour microenvironment to both promote and restrain tumour progression and outline the opportunities to harness nucleotide receptor signalling as an anticancer strategy.