Extending Linker Sequences between Antigen-Recognition Modules Provides More Effective Production of Bispecific Nanoantibodies in the Periplasma of E. coli.

Technology of production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. Production of nAb in periplasm of E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and selection of the most promising nAb variants. The situation is more complicated with production of bi- and multivalent derivatives of the initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between the antigen-recognition modules in the cloned expression constructs were developed and applied in order to increase efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of functionality of all the protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of original monomeric nAbs. The use of sufficiently long linkers could presumably be useful for increasing efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding.

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Anti-Idiotypic Nanobodies Mimicking an Epitope of the Needle Protein of the Chlamydial Type III Secretion System for Targeted Immune Stimulation.

The development of new approaches and drugs for effective control of the chronic and complicated forms of urogenital chlamydia caused by Chlamydia trachomatis, which is suspected to be one of the main causes of infertility in both women and men, is an urgent task. We used the technology of single-domain antibody (nanobody) generation both for the production of targeting anti-chlamydia molecules and for the subsequent acquisition of anti-idiotypic nanobodies (ai-Nbs) mimicking the structure of a given epitope of the pathogen (the epitope of the Chlamydial Type III Secretion System Needle Protein). In a mouse model, we have shown that the obtained ai-Nbs are able to induce a narrowly specific humoral immune response in the host, leading to the generation of intrinsic anti-Chlamydia antibodies, potentially therapeutic, specifically recognizing a given antigenic epitope of Chlamydia. The immune sera derived from mice immunized with ai-Nbs are able to suppress chlamydial infection in vitro. We hypothesize that the proposed method of the creation and use of ai-Nbs, which mimic and present to the host immune system exactly the desired region of the antigen, create a fundamentally new universal approach to generating molecular structures as a part of specific vaccine for the targeted induction of immune response, especially useful in cases where it is difficult to prepare an antigen preserving the desired epitope in its native conformation.

Analysis of Transferrin in the Urine of Patients with Bladder Cancer Using Nanobodies.

It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in the previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time relative abundance of the transferrin forms in urine samples from the patients with bladder cancer (BC). We have shown that increase in the concentration of total Tf in the urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to the later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from the BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and holo-Tf were found in the urine samples of some patients with BC. We hypothesized that the holo-Tf fraction in this case could be largely represented by the "secondary complexes" formed by apo-Tf in combination with ions other than Fe3+, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. By using inductively coupled plasma mass spectroscopy (ICP-MS), we obtained first results confirming our hypothesis. Preparation of the holo-Tf in these urine samples was found to be highly enriched in zinc and nickel. Also, relative enrichment in cadmium has been observed in this preparation, but at much lower concentrations. The obtained data indicate that the used nanobody, while recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities for investigation of relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.

Prognostic Value of Serum Transferrin Analysis in Patients with Ovarian Cancer and Cancer-Related Functional Iron Deficiency: A Retrospective Case-Control Study.

(1) Background: There are no reliable and widely available markers of functional iron deficiency (FID) in cancer. The aim of the study was to evaluate the role of transferrin (Tf) as a marker of cancer of the ovary (CrO) and related FID. (2) Methods: The study groups consisted of 118 patients with CrO and 69 control females. Blood serum iron status was determined on a Beckman Coulter AU (USA) analyzer. Tf quantification was performed by immunoturbidimetry. The relative contents of apo- and holo-Tf (iron-free and iron-saturated Tf, respectively) were determined in eight patients and a control female by immunochromatographic analysis based on the use of monoclonal single-domain antibodies (nanobodies). (3) Results: Four groups of patients with different iron statuses were selected according to ferritin and transferrin saturation values: absolute iron deficiency (AID) (n = 42), FID (n = 70), iron overload (n = 4), normal iron status (n = 2). The groups differed significantly in Tf values (p < 0.0001). Lower values of Tf were associated with FID. Furthermore, FID is already found in the initial stages of CrO (26%). Immunosorbents based on nanobodies revealed the accumulation of apo-Tf and the decrease in holo-Tf in patients with CrO. (4) Conclusions: Tf may be a promising tool for diagnosing both CrO and associated FID.

Generation of high affinity ICAM-1-specific nanobodies and evaluation of their suitability for allergy treatment.

The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10-10 M) and had slow off-rates (10-4 s-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

Isolation of nanobodies with potential to reduce patients' IgE binding to Bet v 1.

BACKGROUND: Recent studies showed that a single injection of human monoclonal allergen-specific IgG antibodies significantly reduced allergic symptoms in birch pollen-allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen-specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE-mediated responses. METHODS: A cDNA-VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope-specificity and affinity to Bet v 1. Furthermore, cross-reactivity to Bet v 1-homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen-induced basophil activation were investigated. RESULTS: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross-react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha-helix at the C-terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1-induced basophil activation. CONCLUSION: We identified high-affinity Bet v 1-specific nanobodies that recognize an important IgE epitope and reduce allergen-induced basophil activation revealing the first proof that allergen-specific nanobodies are useful tools for future treatment of pollen allergy.

Fluorescence Polarization Immunoassay of Human Lactoferrin in Milk Using Small Single-Domain Antibodies.

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and antiLF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.

Nanobodies-Useful Tools for Allergy Treatment?

In the last decade single domain antibodies (nanobodies, VHH) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. Currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. According to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. Their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type I allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence.

The structure of myeloid cell-specific TNF inhibitors affects their biological properties.

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VH H domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF-neutralizing properties in vitro and in vivo of MYSTI-2 and MYSTI-3 antibodies were compared with new variants with interchanged VH H domains and different linker sequences. The most effective structure of MYSTI-2 and MYSTI-3 proteins required the Ser/Gly-containing 'superflexible' linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI-3 with the Pro/Gln-containing 'semi-rigid' linker. Our results may contribute toward the development of more effective drug prototypes.

Modulation of bioavailability of proinflammatory cytokines produced by myeloid cells.

In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers.

Properties of Fluorescent Far-Red Anti-TNF Nanobodies.

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

VHH-Based Bispecific Antibodies Targeting Cytokine Production.

Proinflammatory cytokines, such as TNF, IL-6, and IL-1, play pathogenic roles in multiple diseases and are attractive targets for biologic drugs. Because proinflammatory cytokines possess non-redundant protective and immunoregulatory functions, their systemic neutralization carries the potential for unwanted side effects. Therefore, next-generation anti-cytokine therapies would seek to selectively neutralize pathogenic cytokine signaling, leaving normal function intact. Fortunately, the biology of proinflammatory cytokines provides several such opportunities. Here, we discuss various applications of bispecific antibodies targeting cytokines with specific focus on selective TNF neutralization targeted directly to the surface of specific populations of monocytes and macrophages. These bispecific antibodies combine an anti-TNF VHH with VHHs or scFvs directed against abundant surface molecules on myeloid cells and serve to limit the bioavailability of TNF produced by these cells. Such reagents may become prototypes of a novel class of anti-cytokine biologics.

Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.

Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2).

This work continues a series of recently published studies that employ recombinant single-domain antibody (sdAb, or nanobody®) generation technologies to battle viruses by a passive immunization approach. As a proof of principle, we describe a modified technique to efficiently generate protective molecules against a particular strain of influenza virus within a reasonably short period of time. This approach starts with the immunization of a camel (Camelus bactrianus) with the specified antigen-enriched material presented in as natural a form as possible. An avian influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) adapted for mice was used as a model source of antigens for both the immunization and phage display-based selection procedures. To significantly increase activities of initially selected monovalent single-domain antibodies, we propose a new type of sdAb formatting that involves the addition of a special type of coiled-coil sequence, the isoleucine zipper domain (ILZ). Presumably, the ILZ-containing peptides adopt trimeric parallel conformations. After the formatting, the biological activities (virus neutralization) of the initially selected anti-influenza virus (H5N2) sdAbs were significantly increased. Intraperitoneal or intranasal administration of the formatted sdAb at 2h before or 24h after viral challenge specifically protects mice from lethal infection with influenza virus. We hope that the described approach combined with the selection focused on particular conservative epitopes will lead to the generation of sdAb-based molecules protective against a broad spectrum of influenza virus subtypes.

Passive immunization with a recombinant adenovirus expressing an HA (H5)-specific single-domain antibody protects mice from lethal influenza infection.

One effective method for the prevention and treatment of influenza infection is passive immunization. In our study, we examined the feasibility of creating an antibody-based preparation with a prolonged protective effect against influenza virus. Single-domain antibodies (sdAbs) specific for influenza virus hemagglutinin were generated. Experiments in mouse models showed 100% survivability for both intranasal sdAbs administration 24h prior to influenza challenge and 24h after infection. sdAb-gene delivery by an adenoviral vector led to gene expression for up to 14days. Protection by a recombinant adenovirus containing the sdAb gene was observed in cases of administration prior to influenza infection (14d-24h). We also demonstrated that the single administration of a combined preparation containing sdAb DNA and protein expanded the protection time window from 14d prior to 48h after influenza infection. This approach and the application of a broad-spectrum sdAbs will allow the development of efficient drugs for the prevention and treatment of viral infections produced by pandemic virus variants and other infections.