Domain Of Heavy-chain Antibodies Research Articles

Preparation of interleukin-1β-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice

Objective: To prepare interleukin-1β-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1β-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1β-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1β-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1β (IL-1β) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1β antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 μg/ml and 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

Next generation single-domain antibodies against respiratory zoonotic RNA viruses.

The global impact of zoonotic viral outbreaks underscores the pressing need for innovative antiviral strategies, particularly against respiratory zoonotic RNA viruses. These viruses possess a high potential to trigger future epidemics and pandemics due to their high mutation rate, broad host range and efficient spread through airborne transmission. Recent pandemics caused by coronaviruses and influenza A viruses underscore the importance of developing targeted antiviral strategies. Single-domain antibodies (sdAbs), originating from camelids, also known as nanobodies or VHHs (Variable Heavy domain of Heavy chain antibodies), have emerged as promising tools to combat current and impending zoonotic viral threats. Their unique structure, coupled with attributes like robustness, compact size, and cost-effectiveness, positions them as strong alternatives to traditional monoclonal antibodies. This review describes the pivotal role of sdAbs in combating respiratory zoonotic viruses, with a primary focus on enhancing sdAb antiviral potency through optimization techniques and diverse administration strategies. We discuss both the promises and challenges within this dynamically growing field.

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects.

Food-borne biotoxins from microbes, plants, or animals contaminate unclean, spoiled, and rotten foods, posing significant health risks. Neutralizing such toxins is vital for human health, especially after food poisoning. Nanobodies (Nbs), a type of single-domain antibodies derived from the genetic cloning of a variable domain of heavy chain antibodies (VHHs) in camels, offer unique advantages in toxin neutralization. Their small size, high stability, and precise binding enable effective neutralization. The use of Nbs in neutralizing food-borne biotoxins offers numerous benefits, and their genetic malleability allows tailored optimization for diverse toxins. As nanotechnology continues to evolve and improve, Nbs are poised to become increasingly efficient and safer tools for toxin neutralization, playing a pivotal role in safeguarding human health and environmental safety. This review not only highlights the efficacy of these agents in neutralizing toxins but also proposes innovative solutions to address their current challenges. It lays a solid foundation for their further development in this crucial field and propels their commercial application, thereby contributing significantly to advancements in this domain.

Enhanced sandwich immunoassay based on bivalent nanobody as an efficient immobilization approach for foodborne pathogens detection

BackgroundNanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation. ResultsHere, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM KD, as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 103 CFU mL−1 for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %. Significance and noveltyIn this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads

BackgroundHuman adipose tissue-derived stem cells (ASCs) are widely used in regenerative medicine and tissue engineering. Magnetic-activated cell sorting (MACS) with monoclonal antibodies (mAbs) that bind surface markers of stem cells such as CD105, CD73, and CD90 is currently applied for the enrichment and isolation of ASCs. Alternatives to mAbs are the variable domains of heavy chain antibodies (VHHs), which are naturally produced in camelids. We report the application of an anti-CD105 VHH conjugated to magnetic beads in the isolation of ASCs in vitro. ResultsTwo identical anti-CD105 VHHs (EngVHH17) were screened by phage display from a VHH cDNA library constructed from the PBMCs of an alpaca immunized with a lysate of human bladder cancer cell line (T24 cells). EngVHH17 was cloned in pET22b(+), expressed in Escherichia coli BL21 and purified in Ni-NTA chromatography resulting in a ∼15 kDa VHH antibody. EngVHH17 binds CD105 with high affinity (Kd = 3.9 × 10−10 M). The specific binding of EngVHH17 to CD105 was visualized by fluorescence inmunolabeling of phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 cells using FITC labeled anti-6 × His Tag IgG1 mouse mAb. EngVHH17-magnetic beads selectively sorted human ASCs from osteoblasts in a mixed culture in vitro. The selective recovery of ASC cells using different ASC/Osteoblast ratios was higher than 85%. ConclusionsEngVHH17 coupled to magnetic beads binds CD105 expressed on the cell surface of ASCs and isolates them from osteoblasts in mixed cultures in vitro by application of an external magnetic field. EngVHH17 can be further evaluated for the isolation of MSCs by MACS.How to cite: Gushiken E, Quintana M, Perez L, et al. Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.06.004.

Sensitive immunoassay of Legionella using multivalent conjugates of engineered VHHs.

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia.

Abstract 5577: PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer

Abstract Introduction: Prostate cancer is the third most frequent cause of cancer-related death in men worldwide. Since prostate-specific membrane antigen (PSMA) shows a significant overexpression on prostatic cancer cells, PSMA can be considered a promising target for prostate cancer imaging and treatment. Although chimeric antigen receptor (CAR) T cells have shown great clinical outcomes in hematological malignancies, there is still limited efficacy in solid tumors due to the lack of tumor-specific antigens, low T cell infiltration in the tumors, immune suppressive tumor microenvironment, and on-target off-tumor toxicity in healthy tissues. The antigen recognition module of CAR T cells is usually a single-chain variable fragment (scFv). In contrast, the variable domains of heavy-chain antibodies (VHH) are small, stable, single-domain antibody fragments with high affinity and do not require the supramolecular assembly. Severe fatalities recently halted clinical trials of PSMA CAR T cells, so affinity tuning may be required to avoid CAR T targeting normal tissues. Here, we developed VHH-based CAR T cells targeting PSMA, able to co-express human somatostatin receptor 2 (SSTR2) actioning as a PET reporter for tracking CAR T cell distribution, as an immunotherapeutic strategy for prostate cancer. Methods: Primary human T cells were isolated and transduced with PSMA CAR lentivirus at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. Mouse models of 8505C cell line transduced to overexpress PSMA were utilized to test the efficacy of CAR T cells. Tumor growth was monitored regularly by bioluminescence imaging. PET/CT imaging was performed to monitor T cell localization and biodistribution using ¹⁸F-NOTA-octreotide radiotracer targeting SSTR2 introduced in CAR T cells. Results: By alanine substitution of CDR3 in the VHH, we identified several low affinity candidates. The affinity of the VHH-based PSMA CAR was determined to be 22 nM by a saturation binding assay using monomeric human PSMA protein. PSMA CAR expression in primary human T cells was higher than 90%. Compared to non-treated mice, PSMA targeting CAR T cells led to tumor reduction and improved survival in animal models. Moreover, high levels of T cell infiltration and localization in tumor tissues were found, based on PET/CT imaging. Considering possible treatment-associated toxicities, which led to 2 patient deaths and the halt of one phase 1 trial of PSMA CAR T cells, we will affinity tune VHH-based CAR to improve the safety profile. Thus, additional studies will be performed to test both activity and safety of affinity variants of PSMA CAR T cells. Conclusions: We developed PSMA targeting CAR T cells able to induce potent and specific killing of prostate cancer cells. This strategy demonstrated significant therapeutic efficacy in vivo against animal models. Therefore, CAR T cells targeting PSMA may be a promising treatment strategy for patients with prostate cancer. Citation Format: Juan Gonzalez-Valdivieso, Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Moonsoo M. Jin. PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5577.

Structural insights into the non-inhibitory mechanism of the anti-EGFR EgB4 nanobody

BackgroundThe epidermal growth factor receptor (EGFR) is involved in various developmental processes, and alterations of its extracellular segment are associated with several types of cancers, in particular glioblastoma multiforme (GBM). The EGFR extracellular region is therefore a primary target for therapeutic agents, such as monoclonal antibodies and variable domains of heavy chain antibodies (VHH), also called nanobodies. Nanobodies have been previously shown to bind to EGFR, and to inhibit ligand-mediated EGFR activation.ResultsHere we present the X-ray crystal structures of the EgB4 nanobody, alone (to 1.48 Å resolution) and bound to the full extracellular EGFR-EGF complex in its active conformation (to 6.0 Å resolution). We show that EgB4 binds to a new epitope located on EGFR domains I and II, and we describe the molecular mechanism by which EgB4 plays a non-inhibitory role in EGFR signaling.ConclusionThis work provides the structural basis for the application of EgB4 as a tool for research, for targeted therapy, or as a biomarker to locate EGFR-associated tumors, all without affecting EGFR activation.

Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody

Rapid and quantitative detection of paraquat is crucial because of its high toxicity. Here, we developed an ultrasensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip based on our synthesized variable domain of heavy chain antibody (VHH, also called Nanobody) for paraquat detection. Briefly, the specific immunogen selected from six designed antigens was employed to immunize alpaca, and a high-efficiency capacity of 1.6 × 1013 pfu mL−1 phage display nanobody library was established for biopanning against paraquat. The selected nanobody exhibited high sensitivity (limit of detection (LOD) was 0.0090 ng mL−1 and IC50 was 0.0588 ng mL−1 in buffer) and stability to high temperatures and denaturants. The molecular docking results indicated that the π-π, cation-π, and hydrogen bond interactions between paraquat and the pocket-like structures of complementarity-determining regions (CDRs) in VHH played a critical role in the antibody-paraquat recognition, competition, and affinity processes. The constructed TRFICA recognized paraquat through a quantitative analysis using the strip reader, and showed no cross-reactivity with other herbicides, and a semi-quantitative analysis using the naked eye. Notably, the potential practical applications of the TRFICA evaluated by performing a quantitative analysis of paraquat in food samples (vegetables, fruits, and grain products) and biological samples (blood and urine) showed a recovery rate range between 76.7% and 133.3% with inter-assay coefficient variation lower than 18.5%. The nanobody from phage display libraries was effective for small molecule recognition and detection, and it is a vital tool for immunoassay.

Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics.

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Camelization of a murine single-domain antibody against aflatoxin B1 and its antigen-binding analysis.

Aflatoxin B1 (AFB1), a highly toxic mycotoxin, always contaminated in a variety of agricultural products. Camelid variable domain of heavy chain antibody (VHH) is a noteworthy reagent in immunoassay, owing to its excellent characteristics. Immunization of camelid animals is a straightforward strategy to produce VHHs. In this study, to avoid the dependence on the large animals, the camelized, murine antibody (cVHs) against AFB1 was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB1. A murine anti-AFB1 VH fragment (VH-2E6) was selected for camelization through replacement of conserved hydrophobic residues in framework region 2 (FR2) (cVH-FR2), point mutation at position 103 in the FR4 region (cVH-103), and CDR3-grafted with a high AFB1-affinity VHH (cVH-Nb26). The cVH-Nb26 had a yield of 5 mg/L as refolded protein expressed from Escherichia coli and 10 mg/L expressed from Pichia pastoris. Compared with anti-AFB1 single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB1-binding interactions. Although the AFB1-affinity of cVH-Nb26 cannot meet the application requirement in the present form, our study provides effective strategies for preparation of camelized antibody in vitro, which could be a promising immunoreagent for AFB1 detection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12550-021-00433-z.

Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Neck Cancer Model.

Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT.

Structure of Enterohemorrhagic Escherichia coli O157:H7 Intimin Virulence Factor Bound to Nanobodies

Enterohemorrhagic E. coli O157:H7 (EHEC) is a food and water-born pathogen that presents a significant risk to human health worldwide. EHEC naturally resides in the intestinal tract of cattle as normal flora but can be transmitted to humans when contaminated food is ingested. Once EHEC invades and colonizes the human intestinal tract, it secretes Shiga-like toxins causing serious and potentially fatal gastrointestinal diseases. Unlike most foodborne bacterial diseases, antibiotics are not recommended for treatment of EHEC as antibiotics can induce enterotoxin release, increasing the risk of hemolytic uremic syndrome. There is currently no effective treatment or vaccine against enterohemorrhagic E. coli. EHEC pathogenicity involves intimin, a virulence factor which mediates bacterial colonization of the host GI tract. Due to its critical role in EHEC pathogenesis, intimin has attracted attention as an antibacterial drug target. Inhibiting the interaction of intimin with its cognate bacterial secreted receptor Tir could neutralize bacterial colonization of the gastrointestinal tract. One novel solution for treating EHEC infection comes from a unique class of antibodies known as nanobodies. Nanobodies are the antigen binding domain of heavy chain antibodies produced by the Camelid family. Intimin specific nanobodies were generated by immunizing a llama followed by phage display and selection for high affinity intimin binding nanobodies. The resultant intimin-specific nanobodies neutralized EHEC in vitro. We hypothesize that nanobodies specifically bind the Tir-binding domain of intimin in order to neutralizing EHEC infection. The goal of this research project is to determine the complex crystal structure of the nanobodies bound to intimin. The structure of nanobodies-intimin would show their protein-protein interaction binding site revealing key amino acids involved in binding. These residues could also be involved in the binding of intimin to its natural ligand, Tir. Thus, revealing such residues could form the bases for identifying and developing novel therapeutics and biosensors for treating and preventing EHEC pathogenesis.We show through size exclusion chromatography that nanobodies bind intimin and form stable complexes. We have set up crystal trails of five nanobodies (Int1, Int2, Int3, Int4, and IntN1) complexed to intimin. Two of the five complexed proteins, Int2-intimin and Int3-intimn formed crystals. The crystal condition for the two protein complexes Int2 and Int3 bound to intimin were optimized by varying the concentration of precipitants and pH range. Crystals of the protein complexes were sent to the Canadian Light Source for X-ray diffraction data collection. Diffraction measurements of Int2-intimin and Int3-intimin complexes were collected and processed in space groups P1 and P222, respectively. Currently, we are analyzing the structure by molecular replacement.

Orally Fed Recombinant Lactococcus lactis Displaying Surface Anti-Fimbrial Nanobodies Protects Piglets against Escherichia coli Causing Post-Weaning Diarrhea

Post-weaning diarrhea (PWD) and edema disease (ED), caused by enterotoxigenic and Shiga toxin producing Escherichia coli (ETEC and STEC) strains, are important diseases of newly weaned piglets worldwide. The objective of this study is to develop a passive immunization strategy to protect piglets against PWD and ED using recombinant Lactococcus lactis added to piglet diet at weaning. The Variable Heavy chain domains of Heavy chain antibodies (VHHs) or Nanobodies (Nbs), directed against the fimbrial adhesins FaeG (F4 fimbriae) and FedF (F18 fimbriae) of E. coli were cloned and expressed on the surface of L. lactis. In vitro, the recombinant L. lactis strains agglutinated and inhibited adhesion of cognate F4 or F18 fimbriae expressing E. coli to pig villous preparation. In vivo, the anti-F4 L. lactis protected weaned piglets against a challenge with an F4-positive ETEC strain. Piglets supplemented with oral anti-F4 L. lactis showed reduced fecal E. coli shedding. We concluded that the surface expressed Nanobodies on L. lactis neutralized the adhesins of targeted E. coli and abrogated gut colonization, the first step in disease pathogenesis. As a proof of concept, we demonstrated the potential of passive immunization with recombinant L. lactis as a viable alternative to antibiotic prophylaxis in preventing piglet-post-weaning diarrhea.

A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells.

Objective Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety, which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and cytotoxicity of redirected human T cells. Materials and Methods In this experimental study, we established a second generation novel CAR (VHH-based anti- MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane domain and signalling endodomains of CD28 and CD3+. Next, we constructed lentiviral vectors that contained this CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+T cells. Results The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared with A431 (MUC1-negative) or untransduced T cells. ConclusionOur results suggested that, given the unique properties of VHHs to prevent immunogenic responses and tonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy.

Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells that Secrete Single-Domain Antibody Fragments.

Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.

Phospholipase-Cγ1 Signaling Protein Down-Regulation by Oligoclonal-VHHs based Immuno-Liposome: A Potent Metastasis Deterrent in HER2 Positive Breast Cancer Cells.

Objective The purpose of this study was to develop multivalent antibody constructs via grafting anti-HER2 antibodies, including Herceptin and oligoclonal-variable domain of heavy chain antibodies (VHHs), onto liposome membranes to enhance antibody activity and compare their effect on phospholipase C (PLC) signaling pathway with control. Materials and Methods In this experimental study, SKBR3 and BT-474 cell lines as HER2 positive and MCF10A cell line as normal cell were screened with anti-HER2 antibodies, including constructs of multivalent liposomal antibody developed with Herceptin and anti-HER2 oligoclonal-VHHs. To confirm the accuracy of the study, immunofluorescent assay, migration assay and immuno-liposome binding ability to HER2 were evaluated. Finally, the antibodies effect on PLCγ1 protein level was measured by an immunoassay method (ELISA). Results In the present study, by using multivalent form of antibodies, we were able to significantly inhibit the PLCγ1 protein level. Interestingly, the results of migration assay, used for study the motility of different types of cell, shows correspondingly decreased number of immigrated cells in SKBR3 and BT-474 cell lines. Since MCF10A cells show no overexpression of HER2, as expected, the result did not show any change in PLCγ1 level. Moreover, immunofluorescent assay has confirmed high expression of HER2 in SKBR3 and BT-474 cell lines and low HER2 expression on MCF10A cell line. High binding of immuno-liposome to SKBR3 and BT-474 cells and low binding to MCF10A confirmed that in this study anti-HER2 antibodies have conserved binding ability to HER2 even after conjugation with liposome. ConclusionPLCγ1 protein levels did indeed decrease after treatment with immuno-liposome form of compounds in both two tested cell lines, verifying the inhibition ability of them. Moreover, an elevated antibody activity is associated with liposomes conjugation suggesting that immuno-liposome may be a potential target for enhancing the antibody activity.

Efficient Screening and Design of Variable Domain of Heavy Chain Antibody Ligands Through High Throughput Sequencing for Affinity Chromatography to Purify Fab Fragments.

To design an affinity ligand for purification of antigen-binding fragment (Fab) antibody, variable domain of heavy chain antibody (VHH) phage libraries were constructed from Fab-immunized Alpaca and subjected to biopanning against Fabs. To find the specific binders, we directly applied high-throughput sequencing (HTS) analysis of the VHH sequences in the panned phages on next-generation sequencer. The efficiently enriched sequences were aligned for construction of the phylogenetic tree to be categorized into five groups. VHHs from three major groups were first selected to analyze their properties as an affinity ligand. However, those VHHs were not suitable as an affinity ligand because of lack of resistance against alkaline pH and/or difficulty in acidic elution from the affinity column. So, we further searched the candidates from minor group sequences. Among five, one VHH showed the binding ability but with low affinity against Fabs. Therefore, we improved its affinity-by-affinity maturation through error-prone PCR library techniques. The final designed VHH showed highly alkaline pH resistance and easy acidic elution together with high affinity to Fabs. These results indicate that HTS techniques combined with biopanning and followed by error-prone PCR library techniques is powerful in designing specific binders with desired properties.

A GPCR seen in the active state

Structural Biology G protein–coupled receptors (GPCRs) are exceptionally good targets for drug development. Warne et al. describe four crystal structures of complexes of a GPCR—the β1-adrenergic receptor—in its active state. They used nanobodies (recombinant variable domains of heavy-chain antibodies) and engineered G protein to stabilize the β1-adrenergic receptor bound to a full agonist, two partial agonists, and a weak partial agonist. Comparison of these structures to the inactive state elucidates how agonist binding is altered in the active conformation. Science , this issue p. [775][1] [1]: /lookup/doi/10.1126/science.aau5595