Cognate Antibodies Research Articles

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

In biofilms, bacteria that possess a negatively charged surface are embedded within a matrix of polymers consisting mainly of negatively charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively charged substance, for example, a basic protein, exists within biofilms to neutralize charge–charge repulsions and act as a ‘glue’ attaching negatively charged bacteria to negatively charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly abundant nucleoid-associated histone-like protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, for example, (a) availability in association with e-DNA; (b) multivalent DNA binding; (c) non–sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU–cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and noncanonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical–chemical, microscopy-based, and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS, that this facilitates DNA–DNA, DNA–LPS, and LPS–LPS interactions, and that this facilitates bacterial clumping and attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (nonshaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide, and move away from each other, in opposition to the accreting influence of HUs).

The Allosteric Effect in Antibody-Antigen Recognition.

We studied the molecular details of the recognition of antigens by the variable domain of their cognate antibodies in as well as those elicited by the constant domains, which do not directly interact with antigens. Such effects are difficult to study experimentally; however, molecular dynamics simulations and subsequent residue interaction network analysis provide insight into the allosteric communication between the antigen-binding CDR region and the constant domain. We performed MD simulations of the complex of Fab and prion-associated peptide in the apo and bound forms and follow the conformational changes in the antibody and cross-talk between its subunits and with antigens. These protocols could be generally applied for studies of other antigens-antibody recognition systems.

RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets.

Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes. C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement. Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250μg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies. Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.

The Discovery of the Hepatitis D Virus: Three Princes of Serendip and the Recognition of Autoantibodies to Liver-Kidney Microsomes.

The Discovery of the Hepatitis D Virus: Three Princes of Serendip and the Recognition of Autoantibodies to Liver-Kidney Microsomes.

Comparison and in-silico modeling of monoclonal antibodies recognizing the immuno-dominant Ana o 3 cashew nut allergen

Food allergies negatively affect quality of life and can be a significant medical burden. Allergic reactions to cashew nuts can often be severe and are rarely outgrown. The Ana o 3 protein is an immuno-dominant cashew allergen. Ana o 3 is a small 13kDa protein composed of 2 subunits held together by disulfide bonds that can be disrupted by treatment with reducing agents. The stability and solubility properties of Ana o 3 make it an excellent target for detection methods and tools. Several monoclonal antibodies targeted towards Ana o 3 were generated and 4 of them were characterized by immunoblot and ELISA. Selected Ana o 3 cognate antibody genes were sequenced, and in-silico modeling was used for epitope prediction. All 4 of the monoclonal antibodies recognized both recombinant and native Ana o 3. The calculated KD for each of the antibodies for native Ana o 3 is as follows, 2H5 - 21.7 nM, 5B7F8 - 9.2 pM, 6B9C1 - 0.465 nM, and 19C9A2 - 0.365 nM. The 2H5 antibody was specific for cashew nut extract, while the 6B9C1 and 19C9A2 antibodies also recognized pistachio extract, and the 5B7F8 antibody also recognized pecan extract. The 6B9C1 and 19C9A2 antibodies likely recognized conformational epitopes as binding by these antibodies was disrupted after treatment of Ana o 3 with a reducing agent. In contrast, recognition of Ana o 3 by the 2H5 and 5B7F8 antibodies was increased after reduction of Ana o 3, suggesting linear epitopes. The amino acids predicted to be important for interaction with Ana o 3 epitopes are unique for each of the 4 antibodies. The 5B7F8 antibody is likely the best candidate for continued study and assay development for the creation of cashew allergen detection or diagnostic devices. This collection of antibodies represents useful reagents for the study and characterization of the Ana o 3 cashew nut allergen using cell based and immunoassay methods.

Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands.

The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for drug discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (enzyme-linked immunosorbent assay, or "ELISA"), to small molecule ligands, using fluorescein conjugates and antifluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 μM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.

Identification of a novel anti‑heat shock cognate 71kDa protein antibody in patients with Kawasaki disease.

Kawasaki disease (KD) is an idiopathic form of acute systemic vasculitis, which clinically mimics febrile diseases. Although it has been hypothesized that immune system malfunction is associated with KD, its etiology remains unclear. The aim of the present study was to identify a KD-associated antibody. Immunoproteomic methods were used to identify KD-associated antigens that could be recognized in the sera of patients with KD. HeLa cells were used as an antigen source and KD sera were used as probe antibodies to determine the binding of the antibodies using an indirect immunofluorescence assay. Western blotting was performed to identify KD-associated antigens in HeLa whole cell lysates. Eight out of 12 serum samples obtained from patients with KD demonstrated immunoreactive bands at ~70 kDa, which was later determined to be heat shock cognate 71 kDa protein (HSP7C) by mass spectrometry. The diagnostic value of serum anti-HSP7C antibodies for KD was assessed using ELISA. Using a cut-off value of 0.267, anti-HSP7C antibodies were observed to be present in the sera of 60.00% (30/50) of patients with KD, in 21.05% (8/38) of non-KD febrile controls, and in 5.26% (2/38) of healthy controls. High serum levels of anti-HSP7C antibodies were detected in the peripheral circulation of patients with KD. To the best of our knowledge, the present study is the first to observe the high expression levels of anti-HSP7C antibodies in patients with KD. Therefore, anti-HSP7C antibodies may be used as a diagnostic marker to detect KD.

Structural basis of polyethylene glycol recognition by antibody

BackgroundPolyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated.MethodsThe Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC).ResultsA common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment.ConclusionsThe differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

Use of DosR Dormancy Antigens from Mycobacterium tuberculosis for Serodiagnosis of Active and Latent Tuberculosis.

As more than two billion people possibly have a latent tuberculosis (LTB) infection, early LTB diagnosis is crucial for the efficient control and elimination of tuberculosis (TB). The aim of this study is to detect the serum antibody responses to dormancy-related DosR regulon antigens of Mycobacterium tuberculosis for the diagnosis of active and latent TB infections. A membrane array with 25 latency antigens detected by silver-enhanced gold nanoparticles was used to determine the corresponding cognate antibody levels in clinical serum samples from healthy controls, TB patients, and individuals with LTB. The array is sized to fit into a 24-well ELISA plate and follows an ELISA-like experimental procedure without expensive instrumentation. Linear discriminant analysis (LDA) of the resulting antibody profiling data set identified a panel of nine DosR antigens with significant discriminatory capability among different subjects with ≥90% sensitivity, specificity, and overall accuracy. Furthermore, the high predictive performance validated by an independent test sample set reflects the robustness and reliability of the LDA classification model. Our current data demonstrate that the nine DosR antigen combination associated with the proposed membrane array platform is a clinically feasible approach for distinguishing different TB infection statuses. The proposed methodology in this study could be further developed for multiple disease serodiagnoses with high sensitivity and specificity.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF).

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long α-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

A symmetric geometry of transmembrane domains inside the B cell antigen receptor complex

B lymphocytes have the ability to sense thousands of structurally different antigens and produce cognate antibodies against these molecules. For this they carry on their surface multiple copies of the B cell antigen receptor (BCR) comprising the membrane-bound Ig (mIg) molecule and the Igα/Igβ heterodimer functioning as antigen binding and signal transducing components, respectively. The mIg is a symmetric complex of 2 identical membrane-bound heavy chains (mHC) and 2 identical light chains. How the symmetric mIg molecule is asymmetrically associated with only one Igα/Igβ heterodimer has been a puzzle. Here we describe that Igα and Igβ both carry on one side of their α-helical transmembrane domain a conserved amino acid motif. By a mutational analysis in combination with a BCR rebuilding approach, we show that this motif is required for the retention of unassembled Igα or Igβ molecules inside the endoplasmic reticulum and the binding of the Igα/Igβ heterodimer to the mIg molecule. We suggest that the BCR forms within the lipid bilayer of the membrane a symmetric Igα-mHC:mHC-Igβ complex that is stabilized by an aromatic proline-tyrosine interaction. Outside the membrane this symmetry is broken by the disulfide-bridged dimerization of the extracellular Ig domains of Igα and Igβ. However, symmetry of the receptor can be regained by a dimerization of 2 BCR complexes as suggested by the dissociation activation model.

HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions

The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (Kd), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the Kd of antigen-antibody interactions in solution. The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the Kd values of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent Kd values. This HiBiT-qIP assay showed a considerable variation in the Kd values among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.

Structural differences between the ectodomains of murine and human CD98hc.

The CD98 heavy chain (CD98hc) constitutes both a promising cell surface target for the treatment of cancers and a transcytosis receptor potentially useful for the brain delivery of therapeutics. However, pharmacokinetic studies and safety assessment of cognate antibodies or nonimmunoglobulin binding proteins in rodents is hampered by cross-species variability of both amino acid sequence and glycosylation pattern. Here, we report the crystal structure of the murine CD98hc extracellular domain and a comprehensive comparison with its human ortholog, revealing only one conserved surface patch that is neither shielded by glycosylation nor by the cell membrane with an accessible surface area typical for an antibody epitope. Our results imply the necessity of a surrogate approach for CD98hc-specific binding proteins with predictive power for clinical investigations.

Antibody Specific B-Cell Epitope Predictions: Leveraging Information From Antibody-Antigen Protein Complexes.

B-cells can neutralize pathogenic molecules by targeting them with extreme specificity using receptors secreted or expressed on their surface (antibodies). This is achieved via molecular interactions between the paratope (i.e., the antibody residues involved in the binding) and the interacting region (epitope) of its target molecule (antigen). Discerning the rules that define this specificity would have profound implications for our understanding of humoral immunogenicity and its applications. The aim of this work is to produce improved, antibody-specific epitope predictions by exploiting features derived from the antigens and their cognate antibodies structures, and combining them using statistical and machine learning algorithms. We have identified several geometric and physicochemical features that are correlated in interacting paratopes and epitopes, used them to develop a Monte Carlo algorithm to generate putative epitopes-paratope pairs, and train a machine-learning model to score them. We show that, by including the structural and physicochemical properties of the paratope, we improve the prediction of the target of a given B-cell receptor. Moreover, we demonstrate a gain in predictive power both in terms of identifying the cognate antigen target for a given antibody and the antibody target for a given antigen, exceeding the results of other available tools.

Targeting the Tetraspanins with Monoclonal Antibodies in Oncology: Focus on Tspan8/Co-029.

Tetraspanins are exposed at the surface of cellular membranes, which allows for the fixation of cognate antibodies. Developing specific antibodies in conjunction with genetic data would largely contribute to deciphering their biological behavior. In this short review, we summarize the main functions of Tspan8/Co-029 and its role in the biology of tumor cells. Based on data collected from recently reported studies, the possibilities of using antibodies to target Tspan8 in immunotherapy or radioimmunotherapy approaches are also discussed.

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) for targeted antibody blocking.

Targeted antibody blocking enables characterization of binding sites on immunoglobulin G (IgG), and can efficiently eliminate harmful antibodies from organisms. In this report, we present a novel peptide-denoted as a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF)-for targeted blocking of antibodies. Synthesis of DCAF was achieved by native chemical ligation, and the molecule consists of three functional parts: a specific antigenic peptide, a linker and the Fc-III mimetic peptide, which has a high affinity toward the Fc region of IgG molecules. We demonstrate that DCAF binds the cognate antibody with high selectivity by simultaneously binding to the Fab and Fc regions of IgG. Animal experiments revealed that DCAF molecules diminish the antibody-dependent enhancement effect in a dengue virus infection model, and rescue the acetylcholine receptor by inhibiting the complement cascade in a myasthenia gravis model. These results suggest that DCAFs could have utility in the development of new therapeutics against harmful antibodies.

Identification of B cell epitopes enhanced by protein unfolding and aggregation

Aggregation of therapeutic proteins is a key factor in the generation of unwanted immunogenicity, and can result in reduced serum half-life, neutralization of function and adverse health effects. There is currently little information regarding how aggregates interact with B-cell receptors or cognate antibodies at the protein sequence level, or whether non-native, aggregate-induced epitopes predominate in these interactions. Using an antibody fragment (single chain antibody variable fragment; scFv) that forms aggregates readily at low temperature, anti-scFv IgG antibody responses were generated by intraperitoneal injection of BALB/c strain mice with monomer or aggregate preparations. Aggregate-specific immunosignatures were identified by oligo-peptide microarray fine epitope mapping, using overlapping 15mer peptides based on the linear sequence of scFv, printed onto glass slides. IgG antibodies from mice immunized with aggregated scFv preferentially recognized a patch of overlapping peptides. This region mapped to a β-strand located at the interface between the VH and VL domains. Molecular dynamics simulations indicated that the VL domain is less stable than the VH domain, suggesting the interface region between the two domains becomes exposed during partial unfolding of the scFv during aggregate formation. These data are consistent with the hypothesis that epitopes from partially unfolded states are revealed, or are more fully exposed, in the aggregated state, and that this can augment the IgG antibody response. This observation offers the theoretical possibility that epitopes preferentially associated with aggregates can be identified from the anti-drug antibody serum IgG response which may, in turn, lead to better methods for detection of anti-drug antibody responses, and improved design of therapeutic proteins to control immunogenicity.

Glutazumab, a novel long-lasting GLP-1/anti-GLP-1R antibody fusion protein, exerts anti-diabetic effects through targeting dual receptor binding sites

AimsGlucagon like-peptide-1 (GLP-1)-based drugs have been proposed as mono- or combined therapy for type 2 diabetes mellitus. Thus we characterized a novel antibody fusion protein engineered by linking the human GLP-1 derivative to a humanized GLP-1 receptor (GLP-1R) antibody via a peptide linker. Materials and methodsGlutazumab was characterized by receptor binding and reporter activation assays, and its specificity was investigated with the aid of the cognate receptor antagonist exendin (9-39) and antibody Ab1. Pharmacokinetics was evaluated in Sprague-Dawley (SD) rats and cynomolgus monkeys, and pharmacodynamics was assessed in normal ICR and spontaneous type 2 diabetic KKAy mice. Hypoglycemic effects were evaluated after acute administration and glucose metabolism and β-cell function were assessed with repeated administrations. Dulaglutide was a positive control in all experiments. ResultsGlutazumab significantly bound and activated GLP-1R, but the receptor antagonist exendin (9-39) did not inhibit the activation except when combined with Ab1. Single injection of glutazumab reduced the blood glucose in ICR mice and KKAy mice, and the half-lives in SD rats and cynomolgus monkeys were 18 h and 33.6 h. Repeated injections of glutazumab controlled glycemic fluctuations and improved β-cell function in KKAy mice. ConclusionsAs a novel GLP-1R agonist, glutazumab may be a potential treatment for T2DM.

In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia.

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. Herein, we investigated the relationship between these two processes in normal and leukemic B cells. We found that leukemic B cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B cells. Furthermore, ligation of either surface CD79B, a B-cell receptor component required for downstream signaling, or surface Immunoglobulin M (IgM) by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to the dissociation of these two molecules in leukemic cells. A comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node- and peripheral blood-derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton’s tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization.