Summary Nanobodies are the products of an intriguing invention in the evolution of immunoglobulins. This invention can be traced back approximately 45 million years to the common ancestor of extant ...dromedaries, camels, llamas, and alpacas. Next to conventional heterotetrameric H2L2 antibodies, these camelids produce homodimeric nanobody‐based heavy chain antibodies, composed of shortened heavy chains that a lack the CH1 domain. Nanobodies against human target antigens are derived from immunized animals and/or synthetic nanobody libraries. As a robust, highly soluble, single immunoglobulin domain, a nanobody can easily be fused to another protein, for example to another nanobody and/or the hinge and constant domains of other immunoglobulins. Nanobody‐derived heavy chain antibodies hold promise as a new form of immunotherapeutics.
ATP, NAD
+
, and nucleic acids are abundant purines that, in addition to having critical intracellular functions, have evolved extracellular roles as danger signals released in response to cell ...lysis, apoptosis, degranulation, or membrane pore formation. In general ATP and NAD
+
have excitatory and adenosine has anti-inflammatory effects on immune cells. This review focuses on recent advances in our understanding of purine release mechanisms, ectoenzymes that metabolize purines (CD38, CD39, CD73, ENPP1, and ENPP2 autotaxin), and signaling by key P2 purinergic receptors (P2X7, P2Y2, and P2Y12). In addition to metabolizing ATP or NAD
+
, some purinergic ectoenzymes metabolize other inflammatory modulators, notably lysophosphatidic acid and cyclic GMP-AMP (cGAMP). Also discussed are extracellular signaling effects of NAD
+
mediated by ADP-ribosylation, and epigenetic effects of intracellular adenosine mediated by modification of
S
-adenosylmethionine-dependent DNA methylation.
Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells
is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of ...a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused
a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration
. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors
. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.
Microglia survey the brain microenvironment for signals of injury or infection and are essential for the initiation and resolution of pathogen‐ or tissue damage‐induced inflammation. Understanding ...the mechanism of microglia responses during pathology is hence vital to promote regenerative responses. Here, we analyzed the role of purinergic receptor P2X4 (P2X4R) in microglia/macrophages during autoimmune inflammation. Blockade of P2X4R signaling exacerbated clinical signs in the experimental autoimmune encephalomyelitis (EAE) model and also favored microglia activation to a pro‐inflammatory phenotype and inhibited myelin phagocytosis. Moreover, P2X4R blockade in microglia halted oligodendrocyte differentiation in vitro and remyelination after lysolecithin‐induced demyelination. Conversely, potentiation of P2X4R signaling by the allosteric modulator ivermectin (IVM) favored a switch in microglia to an anti‐inflammatory phenotype, potentiated myelin phagocytosis, promoted the remyelination response, and ameliorated clinical signs of EAE. Our results provide evidence that P2X4Rs modulate microglia/macrophage inflammatory responses and identify IVM as a potential candidate among currently used drugs to promote the repair of myelin damage.
Synopsis
Innate immune cells contribute to axonal damage and demyelination in multiple sclerosis (MS) but they are also pivotal in promoting repair responses. Modulating microglia/macrophage P2X4R activation determines clinical outcome in the experimental autoimmune encephalomyelitis (EAE) model of MS.
Expression of P2x4r and the two transcription factors controlling its expression, Irf8 and Irf5, was increased in EAE.
EAE clinical symptoms were exacerbated by P2X4R blockage and ameliorated by P2X4R potentiation with the allosteric modulator ivermectin.
P2X4R blockage does not interfere with the immune priming or with blood‐brain‐barrier permeability during the acute phase of EAE.
P2X4R activation favors a switch of microglia/macrophage to an anti‐inflammatory phenotype and increases BDNF release, that promotes oligodendrocyte differentiation.
P2X4R activation increases myelin phagocytosis and degradation at lysosomes, thus indirectly promoting remyelinating responses.
Innate immune cells contribute to axonal damage and demyelination in multiple sclerosis (MS) but they are also pivotal in promoting repair responses. Modulating microglia/macrophage P2X4R activation determines clinical outcome in the experimental autoimmune encephalomyelitis (EAE) model of MS.
COVID‐19, caused by SARS‐CoV‐2, has emerged as a global pandemic. While immune responses of the adaptive immune system have been in the focus of research, the role of NK cells in COVID‐19 remains ...less well understood. Here, we characterized NK cell‐mediated SARS‐CoV‐2 antibody‐dependent cellular cytotoxicity (ADCC) against SARS‐CoV‐2 spike‐1 (S1) and nucleocapsid (NC) protein. Serum samples from SARS‐CoV‐2 resolvers induced significant CD107a‐expression by NK cells in response to S1 and NC, while serum samples from SARS‐CoV‐2‐negative individuals did not. Furthermore, serum samples from individuals that received the BNT162b2 vaccine induced strong CD107a expression by NK cells that increased with the second vaccination and was significantly higher than observed in infected individuals. As expected, vaccine‐induced responses were only directed against S1 and not against NC protein. S1‐specific CD107a responses by NK cells were significantly correlated to NK cell‐mediated killing of S1‐expressing cells. Interestingly, screening of serum samples collected prior to the COVID‐19 pandemic identified two individuals with cross‐reactive antibodies against SARS‐CoV‐2 S1, which also induced degranulation of NK cells. Taken together, these data demonstrate that antibodies induced by SARS‐CoV‐2 infection and anti‐SARS‐CoV‐2 vaccines can trigger significant NK cell‐mediated ADCC activity, and identify some cross‐reactive ADCC‐activity against SARS‐CoV‐2 by endemic coronavirus‐specific antibodies.
Antibodies induced by SARS‐CoV‐2 infection or vaccination mediate NK cell activation, resulting in the release of cytotoxic granules, referred to as ADCC. Highest levels of ADCC were observed when using plasma from BNT162b2‐vaccinated individuals. (Created with BioRender.com)
The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more ...than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors
. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.
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.
Extracellular ATP activates the P2X7 receptor, leading to inflammasome activation and release of pro‐inflammatory cytokines in monocytes. However, a detailed analysis of P2X7 receptor expression and ...function in the human T cell compartment has not been reported. Here, we used a P2X7‐specific nanobody to assess cell membrane expression and function of P2X7 on peripheral T lymphocyte subsets. The results show that innate‐like T cells, which effectively react to innate stimuli by secreting high amounts of pro‐inflammatory cytokines, have the highest expression of P2X7 in the human T cell compartment. Using Tγδ cells as example for an innate‐like lymphocyte population, we demonstrate that these cells are more sensitive to P2X7 receptor activation than conventional T cells, affecting fundamental cellular mechanisms like calcium signaling and ATP‐induced cell death. The increased susceptibility of innate‐like T cells to P2X7‐mediated cell death provides a mechanism to control their homeostasis under inflammatory conditions. Understanding the expression and function of P2X7 on human immune cells is essential to assume the benefits and consequences of newly developed P2X7‐based therapeutic approaches.
Innate‐like T cells are ready‐to‐go effectors and have the highest expression of the ATP receptor P2X7 in the human T cell compartment. P2X7 expression renders these cells susceptible to ATP‐mediated cell death, which may constitute a mechanism for controlling immune pathology in an ATP‐rich, inflammatory environment.
Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3
T cells isolated from the ...peripheral blood (
= 20), malignant ascites (
= 16), and tumor tissue (
= 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8
T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1
CD8
T cell population was detected, which differed from PD-1
CD8
T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8
T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (
= 14) and malignant ascites (
= 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24
EpCAM
tumor cells showed a higher frequency of CD39
or CD73
cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8
T cells.
ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety ...from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.
Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 - 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.
The pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.
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