Variable Heavy Chain Research Articles

Monitoring Phage Biopanning by Next-Generation Sequencing.

Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of the variable heavy chain gene (VH) and variable light chain gene (VL). The method is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection by phage biopanning.Next-Generation Sequencing (NGS) coupled with bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library.Here, we describe a method that demonstrates how NGS analysis enhances phage biopanning of complex antibody libraries as well as facilitates the antibody discovery process.

Single Domain Antibodies as New Biomarker Detectors.

Biomarkers are defined as indicators of biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers have been widely used for early detection, prediction of response after treatment, and for monitoring the progression of diseases. Antibodies represent promising tools for recognition of biomarkers, and are widely deployed as analytical tools in clinical settings. For immunodiagnostics, antibodies are now exploited as binders for antigens of interest across a range of platforms. More recently, the discovery of antibody surface display and combinatorial chemistry techniques has allowed the exploration of new binders from a range of animals, for instance variable domains of new antigen receptors (VNAR) from shark and variable heavy chain domains (VHH) or nanobodies from camelids. These single domain antibodies (sdAbs) have some advantages over conventional murine immunoglobulin owing to the lack of a light chain, making them the smallest natural biomarker binders thus far identified. In this review, we will discuss several biomarkers used as a means to validate diseases progress. The potential functionality of modern singe domain antigen binders derived from phylogenetically early animals as new biomarker detectors for current diagnostic and research platforms development will be described.

Peptides of the variable IgG domain as potential biomarker candidates in primary open-angle glaucoma (POAG).

Autoantibody profiling has gained increasing interest in the research field of glaucoma promising the detection of highly specific and sensitive marker candidates for future diagnostic purposes. Recent studies demonstrated that immune responses are characterized by the expression of congruent or similar complementarity determining regions (CDR) in different individuals and could be used as molecular targets in biomarker discovery. Main objective of this study was to characterize glaucoma-specific peptides from the variable region of sera-derived immunoglobulins using liquid chromatography--mass spectrometry (LC-MS)-based quantitative proteomics. IgG was purified from sera of 13 primary open-angle glaucoma patients (POAG) and 15 controls (CTRL) and subsequently digested into Fab and Fc by papain. Fab was further purified, tryptic digested and measured by LC-MS/MS. Discovery proteomics revealed in total 75 peptides of the variable IgG domain showing significant glaucoma-related level changes (P < 0.05; log2 fold change ≥ 0.5): 6 peptides were high abundant in POAG sera, whereas 69 peptides were low abundant in comparison to CTRL group. Via accurate inclusion mass screening strategy 28 IgG V domain peptides were further validated showing significantly decreased expression levels in POAG sera. Amongst others 5 CDR1, 2 CDR2 and 1 CDR3 sequences. In addition, we observed significant shifts in the variable heavy chain family distribution and disturbed κ/λ ratios in POAG patients in contrast to CTRL. These findings strongly indicate that glaucoma is accompanied by systemic effects on antibody production and B cell maturation possibly offering new prospects for future diagnostic or therapy purposes.

Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex.

The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn’s disease, rheumatoid arthritis, and psoriasis.

Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses.

Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103–105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

Aberrant expression of CD10 and BCL6 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by distinctive histological and molecular features. Aberrant expression of BCL6 and CD10 has been reported occasionally, but the biological features of such cases are largely unknown. This study aimed to define the epidemiological, histological and cytogenetic characteristics of BCL6 and CD10-positive MCLs, also investigating possible biological features. A total of 165 cases of cyclin D1 and t(11;14)(q13;q34)-positive MCLs were studied for CD10 and BCL6 immunohistochemical expression, which was documented in 26 of 165 (15.8%) cases (BCL6 17 of 165; CD10 11 of 165; BCL6 and CD10 co-expression two of 165). CD10-positivity was significantly more frequent in females (63.3%; P < 0.01). Either expression correlated significantly with higher mean proliferation index and higher prevalence of MUM1 positivity (P < 0.05). Fluorescence in-situ hybridization (FISH) for BCL6 (3q27) gene derangements was performed on the BCL6- and CD10-positive cases and 98 matched controls: amplifications were documented more frequently in BCL6-positive than -negative cases (50.0% versus 19.4% of cases) (P < 0.05). The mutational status of the variable immunoglobulin heavy chain genes (IGVH) was investigated by Sanger sequencing: five of the six successfully tested cases (83.3%) showed no somatic hypermutations. Aberrant CD10 and BCL6 expression defines a subset of MCLs with higher mean Ki-67 index and higher prevalence of MUM1 expression. BCL6 protein positivity correlates with cytogenetic aberrations involving the BCL6 gene. Although examined successfully in few cases, the high prevalence of unmutated IGVH genes also points at a pregerminal cell origin for these phenotypically aberrant cases.

Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

Aging has a strong impact on the activity of the immune system, enhancing susceptibility to pathogens and provoking a predominant pre-inflammatory status, whereas dampening responses to vaccines in humans and mice. Here, we demonstrate a loss of marginal zone B lymphocytes (MZ, CD19+CD45R+CD21++CD23lo) and a decrease of naive B cells (CD19+IgD+), whereas there is an enhancement of a CD19+CD45Rlo innate-like B cell population (B1REL) and the so-called aged B cell compartment (ABC, CD45R+CD21loCD23loCD5−CD11b−) in aged senescence-accelerated (SAMP8) mice but not in aged senescence-resistant (SAMR1) mice. These changes in aged SAMP8 mice were associated with lower IgG isotype levels, displaying low variable gene usage repertoires of the immunoglobulin heavy chain (VH) diversity, with a diminution on IgG1-memory B cells (CD11b−Gr1−CD138−IgM−IgD−CD19+CD38+IgG1+), an increase in T follicular helper (TFH, CD4+CXCR5+PD1+) cell numbers, and an altered MOMA-1 (metallophilic macrophages) band in primary follicles. LPS-mediated IgG1 responses were impaired in the B1REL and ABC cell compartments, both in vitro and in vivo. These data demonstrate the prominent changes to different B cell populations and in structural follicle organization that occur upon aging in SAMP8 mice. These novel results raise new questions regarding the importance of the cellular distribution in the B cell layers, and their effector functions needed to mount a coordinated and effective humoral response.

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: VH22 and VH92 in the variable heavy chain (VH) and VL23, VL87 and VL88 in the variable light chain (VL). The influence of two unusual contiguous Cys at positions VL87 and VL88 was studied by considering the wild type fragment and mutant variants: VL-C88S, VL-C87S, VL-C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that VL-C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the VL-C87 by a usual residue at this position like Tyr caused distant structural changes at the VH region that confers a higher mobility to the VH-CDR2 and VH-CDR3 loops improving the scFv binding to the antigen.

Application of nanobody in cancer treatment

Camelidae can produce a unique antibody that lacks light chain called variable heavy chain domain, also known as nanobodies. This antibody contains only one variable region, with high affinity, high stability, strong tissue penetration, efficient expression. Besides, their toxicity and immunogenicity are both low to be used for both therapeutic and diagnostic applications, as well as research tools. In this review, we discuss how nanobody has been explored as therapeutics in oncology, and provide ideas for the further development of nanobody.

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview.

Single domain antibodies (sdAbs) are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (Tms) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their Tm, making them stable until exposed to extreme temperatures. SdAbs derived from the variable heavy chains of camelid and shark heavy chain-only antibodies are termed VHH and VNAR, respectively, and generally exhibit some ability to refold and bind antigen after heat denaturation. This ability to refold varies from 0 to 100% and is a property dependent on both intrinsic factors of the sdAb and extrinsic conditions such as the sample buffer ionic strength, pH, and sdAb concentration. SdAbs have also been engineered to increase their solubility and refolding ability, which enable them to function even after exposure to temperatures that exceed their melting point. In addition, efforts to improve their stability at extreme pH and in the presence of chemical denaturants or proteases have been undertaken. Multiple routes have been employed to engineer sdAbs with these enhanced stabilities. The methods utilized to achieve these goals include grafting complementarity-determining regions onto stable frameworks, introduction of non-canonical disulfide bonds, random mutagenesis combined with stringent selection, point mutations such as inclusion of negative charges, and genetic fusions. Increases of up to 20°C have been realized, pushing the Tm of some sdAbs to over 90°C. Herein, we present an overview of the work done to stabilize sdAbs derived from camelids and sharks. Utilizing these various strategies sdAbs have been stabilized without significantly compromising their affinity, thereby providing superior reagents for detection, diagnostic, and therapeutic applications.

Humanization of chimeric anti-CD20 antibody by logical and bioinformatics approach with retention of biological activity.

To develop a fully bioactive humanized antibody from the chimeric rituximab for potential clinical applications using a relatively simpler and faster logical and bioinformatics approach. From bioinformatics data, mismatched mouse amino acids in variable light and heavy chain amphipathic regions were identified and substituted with those common to human antibody framework. Appropriate synthetic DNA sequences inserted into vectors were transfected into HEK293 cells to produce the humanized antibody. Humanized antibodies showed specific binding to CD20 and greater cytotoxicity to cancer WIL2-NS cell proliferation than rituximab in vitro. A humanized version of rituximab with potential to be developed into a biobetter for treatment of B-cell disorders has been successfully generated using a logical and bioinformatics approach.

Self-reactive VH4-34-expressing IgG B cells recognize commensal bacteria.

The germline immunoglobulin (Ig) variable heavy chain 4-34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34-expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34-expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34-encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34-encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

CNS Aquaporin-4-specific B cells connect with multiple B-cell compartments in neuromyelitis optica spectrum disorder.

ObjectivesNeuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory disorder of the central nervous system (CNS) targeted against aquaporin‐4 (AQP4). The origin and trafficking of AQP4‐specific B cells in NMOSD remains unknown.MethodsPeripheral (n = 7) and splenic B cells (n = 1) recovered from seven NMOSD patients were sorted into plasmablasts, naïve, memory, and CD27‐IgD‐ double negative (DN) B cells, and variable heavy chain (VH) transcriptome sequences were generated by deep sequencing. Peripheral blood (PB) VH repertoires were compared to the same patient's single‐cell cerebrospinal fluid (CSF) plasmablast (PB) VH transcriptome, CSF immunoglobulin (Ig) proteome, and serum Ig proteome. Recombinant antibodies were generated from paired CSF heavy‐ and light chains and tested for AQP4 reactivity.ResultsApproximately 9% of the CSF VH sequences aligned with PB memory B cells, DN B cells, and plasmablast VH sequences. AQP4‐specific VH sequences were observed in each peripheral B‐cell compartment. Lineage analysis of clonally related VH sequences indicates that CSF AQP4‐specific B cells are closely related to an expanded population of DN B cells that may undergo antigen‐specific B‐cell maturation within the CNS. CSF and serum Ig proteomes overlapped with the VH sequences from each B‐cell compartment; the majority of matches occurring between the PB VH sequences and serum Ig proteome.InterpretationDuring an acute NMOSD relapse, a dynamic exchange of B cells occurs between the periphery and CNS with AQP4‐specific CSF B cells emerging from postgerminal center memory B cells and plasmablasts. Expansion of the PB DN B‐cell compartment may be a potential biomarker of NMOSD activity.

Variable Heavy Chain Domain Derived from a Cell-Penetrating Anti-DNA Monoclonal Antibody for the Intracellular Delivery of Biomolecules

ABSTRACTTargeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (VH) and light (VL) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides VH-JUN-HIS and VL-FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of VH and VL. The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency.

Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

Pronounced effect of hapten binding on thermal stability of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain.

Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64-66°C,while that of Gly95-type was 47-48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.

Dinutuximab in the treatment of neuroblastoma

ABSTRACTIntroduction: Neuroblastoma is the cancer of the sympathetic nervous system. Nearly half of the patients with neuroblastoma present with high-risk features and have poor outcome despite aggressive therapy. The high expression of GD2 – a cell surface disialoganglioside – in neuroblastoma and its restricted distribution in normal tissues make anti-GD2 monoclonal antibodies suitable for immunotherapy.Areas covered: Dinutuximab is an anti-GD2 monoclonal antibody which is a chimeric construct composed of the variable region heavy and light chain genes of the murine mAb14.G2a and the human constant region genes for heavy chain IgG1 and light chain kappa. Dinutuximab is a unique orphan drug, that was approved by the FDA on 3/10/2015 for use in high-risk neuroblastoma patients who achieve at least a partial response to prior first-line multiagent, multimodality therapy in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), aldesleukin (interleukin-2 [IL-2]), and isotretinoin. The author examines the published pharmacokinetic, safety and efficacy data from the Phase I/II/III studies.Expert opinion: Although dinutuximab has significant side-effects such as neuropathic pain, hypersensitivity reactions, fever, hypotension and capillary leak, its use when combined with GM-CSF, IL-2 and isotretinoin has led to improvements in event-free survival (EFS) and overall survival (OS) in high-risk neuroblastoma patients who have responded to the standard front-line multiagent, multimodality treatment.

Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia

A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced in vitro T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne–azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.