Domain Of Heavy-chain Antibodies Research Articles

CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs.

Redirecting the recognition specificity of T lymphocytes to designated tumour cell surface antigens by transferring chimeric antigen receptor (CAR) genes is becoming an effective strategy to combat cancer. Today, CAR T-cell therapy has proven successful in the treatment of haematological malignancies and the first CD19 CAR T-cell products has already entered the market. This success is expanding CAR design for broader malignancies including solid tumours. Nevertheless, CARs such as those built on antigen-specific single chain antibody variable fragment (scFv) may induce some adverse effects. Here, we briefly review CAR T-cell bioengineering and discuss selected important initiatives for improved T-cell reprogramming, function and safety. In this respect, we further elaborate on unconventional CARs structured on single variable domain of heavy chain (VHH) antibodies (single-domain antibodies) as an alternative to scFv, because of their interesting immunological and physicochemical characteristics and unique structure, which shows a high degree of homology with human VH3 gene family.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A.

Variable domain of heavy chain antibody (nanobody, Nb) derived from camelids is an efficient reagent in monitoring environmental contaminants. Oriented conjugates of Nbs and bacterial magnetic particles (BMPs) provide new tools for the high-throughput immunoassay techniques. An anti-tetrabromobisphenol-A (TBBPA) Nb genetically integrated with an extra cysteine residue at the C terminus was immobilized onto BMPs enclosed within the protein membrane, using a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithiol) propionate, to form a solid BMP-Nb complex. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of BMP-Nb and T5-horseradish peroxidase was developed for the analysis of TBBPA, with a total assay time of 30min and a half-maximum signal inhibition concentration (IC50) of 1.04ng/mL in PBS (pH10, 10% methanol and 0.137 moL/L NaCl). This assay can even be performed in 100% methanol, with an IC50 value of 44.3ng/mL. This assay showed quantitative recoveries of TBBPA from spiked canal water (114-124%) and sediment (109-113%) samples at 1.0-10ng/mL (or ng/g (dw)). TBBPA residues determined by this assay in real canal water samples were below the limit of detection (LOD) and in real sediments were between <LOD and 23.4ng/g (dw). The BMP-Nb-based ELISA shows promising application in environmental monitoring. Graphical abstract ᅟ.

Generation and characterization of an anti-delta like ligand-4 Nanobody to induce non-productive angiogenesis

Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it. In this work, a Nanobody library against human recombinant DLL4 was developed. After panning, the periplasmic-extract (PE) of individual colonies were screened through ELISA. The interactions between Nanobody and DLL4 were assessed using immunohistochemistry and FACS. The functional assessment was carried out via tube formation assay. We selected a Nanobody (3Nb3) with a high binding signal to DLL4, associated with a binding affinity of 3.6 nM. It was demonstrated that 3Nb3 binds to native DLL4 on the surface of MKN cells and gastric carcinoma tissue, and also inhibits the maturation of capillary-like structures in HUVECs. The results were indicative of the potential of Nanobody for DLL4 identification and can broaden the scope for development of cancer diagnosis and treatment techniques.

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.

Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Nanobody-targeted photodynamic therapy for oncology

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 339 for photodynamic therapy in cancer and a potential alternative treatment to conventional chemotherapeutics, such as cisplatin. http://dx.doi.org/10.1016/j.pdpdt.2015.07.057 Synthesis of a new bacteriochlorin derivative with long-wavelength absorption: A potential dye for use as a photosensitizer Francisco F. de Assis, Timothy J. Brocksom, Kleber T. de Oliveira Department of Chemistry, Federal University of Sao Carlos, Brazil Bacteriochlorins are very attractive candidates for use in photodynamic therapy (PDT) due to their important photophysical properties and absorption bands at the infrared region. These compounds canbeobtained fromnatural sources, suchas cyanobacteria and are denominated bacteriochlorophylls. However, the syntheses of these structures are very laborious and represent a challenge from the synthetic point of view. In our research, we have prepared a new bacteriochlorin compound, which presents a strong absorption band around 800nm. This structure was obtained by the Sonogashira cross-coupling between a tetrabromo bacteriochlorin, prepared by Lindsey’smethodology and phenylacetylene. Currently, we are evaluating the photophysical properties of this new compound as a photosensitizer for use in PDT treatment. http://dx.doi.org/10.1016/j.pdpdt.2015.07.058 Nanobody-targeted photodynamic therapy for oncology Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, The Netherlands Monoclonal antibodies have been employed to target photosensitizers (PS) and improve tumor selectivity in photodynamic therapy (PDT). However, antibody-PS conjugates have a long blood half-life and a relatively poor tumor penetration, which result in long photosensitivity in patients and limited therapeutic efficacy. In an attempt to target PS specifically and homogenously to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). Nanobodies are the variable domain of heavy-chain antibodies existent in camelids and have a great potential in cancer imaging and therapy. The selected PS is a silicon-phthalocyanine derivative that is more hydrophilic than common PS and is suitable for nearinfrared imaging. The fluorescent nanobody-PS conjugates allow the distinction of cell lineswith different expression levels of EGFR. These conjugates specifically induce cell death of EGFR overexpressing cells, while PS alone or the nanobody-PS conjugates in absence of light induce no toxicity. Delivery of PS using internalizing biparatopic nanobody-PS conjugates results in even more pronounced toxicities. Initial in vivo studies point towards a potent therapy, enabling the combination with molecular imaging, which can have a significant impact in the field of targeted PDT. http://dx.doi.org/10.1016/j.pdpdt.2015.07.059 Ru(II) complexes as potent photosensitizers in Photodynamic Therapy

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Selection and Characterization of Specific Nanobody Against Human Immunoglobulin G.

Nanobodies (or VHHs) are the smallest antigen-binding domain of heavy chain antibodies, which naturally occur in camelidae. The small size, monomeric nature, low immunogenicity, high solubility and stability, as well as high affinity to target in nanomolar range, makes nanobodies a promising tool for diagnostic and therapeutic application. In the present study, we developed and identified the nanobody against human IgG from an immune library using phage display technique. For this goal, we performed four rounds of selection procedures on immobilized human IgG through biopanning. A clone named R1Nb was selected and expressed as His-tagged protein, and purified by nickel affinity chromatography. In addition, R1Nb was further characterized for binding specificity and affinity. Results demonstrated that R1Nb was highly specific for human IgG and its constant affinity was about 7.5 nM. Taken together, our achieved results indicate the potential of R1Nb as a promising tool in research and may be used for therapeutic purposes as a fusion part of target-specific nanobody.

Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay.

The emerging technologies on mobile-based diagnosis and bioanalytical detection have enabled powerful laboratory assays such as enzyme-linked immunosorbent assay (ELISA) to be conducted in field-use lab-on-a-chip devices. In this paper, we present a low-cost universal serial bus (USB)-interfaced mobile platform to perform microfluidic ELISA operations in detecting the presence and concentrations of BDE-47 (2,2',4,4'-tetrabromodiphenyl ether), an environmental contaminant found in our food supply with adverse health impact. Our point-of-care diagnostic device utilizes flexible interdigitated carbon black electrodes to convert electric current into a microfluidic pump via gas bubble expansion during electrolytic reaction. The micropump receives power from a mobile phone and transports BDE-47 analytes through the microfluidic device conducting competitive ELISA. Using variable domain of heavy chain antibodies (commonly referred to as single domain antibodies or Nanobodies), the proposed device is sensitive for a BDE-47 concentration range of 10(-3)-10(4 ) μg/l, with a comparable performance to that uses a standard competitive ELISA protocol. It is anticipated that the potential impact in mobile detection of health and environmental contaminants will prove beneficial to our community and low-resource environments.

Heterologous antigen selection of camelid heavy chain single domain antibodies against tetrabromobisphenol A.

Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules.

A novel nanobody against urease activity of Helicobacter pylori

Helicobacter pylori infection is associated with gastritis and in some cases with gastric and duodenal ulcers, and even adenocarcinoma. Antibiotic therapy has significant limitations, such as the high cost and the emergence of antibiotic-resistant strains, generating the need for new treatments. The administration of antibody against H. pylori is a new effective therapeutic strategy. In this study, we successfully developed a single-variable domain of heavy chain antibody against recombinant UreC. A VHH phagemid library was constructed from immune camel heavy chain antibodies. The nanobodies were displayed on M13 phage. Library selection was performed against UreC recombinant protein. A specific single-variable domain of heavy chain antibody against UreC was screened in five rounds of panning. The nanobody with the highest score in the phage ELISA was selected for soluble expression. The nanobody was purified with a nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Affinity, specificity, and urease inhibitory properties of the nanobody were assayed. Here we showed the isolation and purification of a specific nanobody with high affinity against UreC recombinant protein that can inhibit urease activity. The isolated UreC nanobody can specifically detect and bind to UreC and inhibit urease activity. This nanobody could be a novel class of treatment measure against H. pylori infection.

Fluoreszierende Biomarker zum Nachweis nativer Zellstrukturen

Chromobodies are a new class of extremely small, highly stable fluorescent antibodies. The key elements of this technology are the antigen-binding domains of heavy chain antibodies from Camelidae, fused to a fluorescent protein. Chromobodies are efficiently introduced into any cell type as plasmid DNA by means of simple transfection and target and trace native, endogenous proteins in real-time.