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Objective: CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cellmalignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developinga bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTaggedanti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells.Materials and Methods: In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vectorand expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the elutedprotein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed inE. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher andSpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nbwas tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometryResults: SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio ofSpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flowcytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is nobinding to the CD22- K562 control cells.Conclusion: The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficialexpression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

Nanobody (Nb) is derived from the variable domain of heavy-chain antibody (HCAb), naturally displaying notable properties like nano-scale size, exceptional stability, high specificity, low immunogenicity, and cryptic epitope accessibility. These features contribute to its great therapeutic potential as a valuable research tool across diverse diseases, especially autoimmune diseases (AIDs). Caplacizumab (Cablivi®) is the first nanobody drug approved for treating acquired thrombotic thrombocytopenic purpura (aTTP). This review summarizes the biomolecular structure, usage of Nb as a foundation of recombinant constructs, and biochemical properties of nanobodies. As attractive therapeutic candidates, many clinical trials of Nbs have been conducted, elucidating potential therapeutic strategies for AIDs. Therefore, the preclinical development and application of Nbs in AIDs are emphasized throughout this review.

Abstract Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.

Single-domain antibodies (sdAbs), initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs, have emerged as a promising alternative to conventional therapeutic antibodies. These sdAbs have many superior physicochemical and pharmacological properties, including small size, good solubility and thermostability, easier accessible epitopes, and strong tissue penetration. However, the inherent challenges associated with the animal origin of sdAbs limit their clinical use. In recent years, various innovative humanization technologies, including complementarity-determining region (CDR) grafting or complete engineering of fully human sdAbs, have been developed to mitigate potential immunogenicity issues and enhance their compatibility. This review provides a comprehensive exploration of sdAbs, emphasizing their distinctive features and the progress in humanization methodologies. In addition, we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment, such as sdAb–drug conjugates, multispecific sdAbs, sdAb-based delivery systems, and sdAb-based cell therapy.

Objective: CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cell malignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developing a bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTagged anti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells. Materials and Methods: In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vector and expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the eluted protein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed in E. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher and SpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nb was tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometry Results: SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio of SpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flow cytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is no binding to the CD22- K562 control cells. Conclusion: The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficial expression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22 in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs). [ABSTRACT FROM AUTHOR]

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications. [ABSTRACT FROM AUTHOR]

Purpose: The cluster of differentiation (CD70) is a potential biomarker of clear cell renal cell carcinoma (ccRCC). This study aims to develop CD70-targeted immuno-positron emission tomography/computed tomography (immunoPET/CT) imaging tracers and explore the diagnostic value in preclinical studies and the potential value in detecting metastases in ccRCC patients. Methods: Four novel CD70-specific single-domain antibodies (sdAbs) were produced and labelled with 18F by the aluminium fluoride restrained complexing agent (AlF-RESCA) method to develop radiotracers. The visualisation properties of the tracers were evaluated in a subcutaneous ccRCC patient–derived xenograft (PDX) model. In a registered prospective clinical trial (NCT06148220), six patients with pathologically confirmed RCC were included and underwent immunoPET/CT examination exploiting one of the developed tracers (i.e., [18F]RCCB6). Results: We engineered four sdAbs (His-tagged RCCB3 and RCCB6, His-tag-free RB3 and RB6) specifically targeting recombinant human CD70 without cross-reactivity to murine CD70. ImmunoPET/CT imaging with [18F]RCCB3 and [18F]RCCB6 demonstrated a high tumour-to-background ratio in a subcutaneous ccRCC PDX model, with the latter showing better diagnostic potential supported by higher tumour uptake and lower bone accumulation. In comparison, [18F]RB6, developed by sequence optimisation, has significantly lower kidney accumulation than that of [18F]RCCB6. In a pilot translational study, [18F]RCCB6 immunoPET/CT displayed ccRCC metastases in multiple patients and demonstrated improved imaging contrast and diagnostic value than 18F-FDG PET/CT in a patient with ccRCC. Conclusion: The work successfully developed a series of CD70-targeted immunoPET/CT imaging tracers. Of them, [18F]RCCB6 clearly and specifically identified inoculated ccRCCs in preclinical studies. Clinical translation of [18F]RCCB6 suggests potential for identifying recurrence and/or metastasis in ccRCC patients. [ABSTRACT FROM AUTHOR]

Single-domain antibodies (sdAbs), initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs, have emerged as a promising alternative to conventional therapeutic antibodies. These sdAbs have many superior physicochemical and pharmacological properties, including small size, good solubility and thermostability, easier accessible epitopes, and strong tissue penetration. However, the inherent challenges associated with the animal origin of sdAbs limit their clinical use. In recent years, various innovative humanization technologies, including complementarity-determining region (CDR) grafting or complete engineering of fully human sdAbs, have been developed to mitigate potential immunogenicity issues and enhance their compatibility. This review provides a comprehensive exploration of sdAbs, emphasizing their distinctive features and the progress in humanization methodologies. In addition, we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment, such as sdAb–drug conjugates, multispecific sdAbs, sdAb-based delivery systems, and sdAb-based cell therapy. This review provides a comprehensive exploration of sdAbs, emphasizing the progress in humanization methodologies and their distinctive applications in solid tumor treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

Single‐domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross‐inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications. [ABSTRACT FROM AUTHOR]

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine‐quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody‐based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single‐domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small‐Angle X‐ray Scattering. Lentiviral transduction of S10 into HepG2‐NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next‐generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is classified as an urgent antibiotic resistance threat by the Centers for Disease Control and Prevention (CDC). C. difficile infection (CDI) is mainly caused by the C. difficile exotoxin TcdB, which invades host cells via receptor-mediated endocytosis. However, many natural variants of TcdB have been identified including some from the hypervirulent strains, which pose significant challenges for developing effective CDI therapies. Here, we review the recent research progress on the molecular mechanisms by which TcdB recognizes Frizzed proteins (FZDs) and chondroitin sulfate proteoglycan 4 (CSPG4) as two major host receptors. We suggest that the receptor-binding sites and several previously identified neutralizing epitopes on TcdB are ideal targets for the development of broad-spectrum inhibitors to protect against diverse TcdB variants.

Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens (e.g., norovirus) in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab (VHH, nanobody) against norovirus on the cell surface of Lactobacillus, a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall-anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection.

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics. [ABSTRACT FROM AUTHOR]

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti- CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders. Methods We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39. Results Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC50

AbstractHelicobacter pylori (H. pylori) is recognized as a pathogen associated with several gastrointestinal diseases. The current treatments exhibit numerous drawbacks, including antibiotic resistance. H. pylori can adhere to and colonize the gastric mucosa through H. pylori adhesin A (HpaA), and antibodies against HpaA may be an effective therapeutic approach. The variable domain of immunoglobulin new antigen receptor (VNAR) is a novel type of single-domain antibody with a small size, good stability, and easy manufacturability. This study isolated VNARs against HpaA from an immune shark VNAR phage display library. The VNARs can bind both recombinant and native HpaA proteins. The VNARs, 2A2 and 3D6, showed high binding affinities to HpaA with different epitopes. Furthermore, homodimeric bivalent VNARs, biNb-2A2 and biNb-3D6, were constructed to enhance the binding affinity. The biNb-2A2 and biNb-3D6 had excellent stability at gastrointestinal pH conditions. Finally, a sandwich ELISA assay was developed to quantify the HpaA protein using BiNb-2A2 as the capture antibody and BiNb-3D6 as the detection antibody. This study provides a potential foundation for novel alternative approaches to treatment or diagnostics applications of H. pylori infection.

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. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders. Methods: We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39. Results: Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC50 < 100nM). We finally found that sdAb39 (EC50 ~ 4nM) can compete with ACE2 for binding to RBD. Conclusion: Overall, this two-stage strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD sdAb with potential therapeutic activity, and this two-stage strategy can potentially be used for rapid discovery of sdAbs against other targets. [ABSTRACT FROM AUTHOR]

Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nMaffinity dependent on the species.G13 affinity was difficult to determine by biolayer interferometry due to lowand heterogeneous signals. G13 andG6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species. [ABSTRACT FROM AUTHOR]

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization. [ABSTRACT FROM AUTHOR]

In the past decade, various single-domain antibodies from llamas, also known as VHH or nanobody, have been discovered with applications in tumor imaging and cancer therapy. However, the potential application of anti-HER2 VHHs as a diagnostic tool suitable for ELISA, flow cytometry, cell imaging, bispecific antibody engineering, and immunohistochemistry has not been fully elucidated. To investigate this potential, HER2 antigen was expressed in HEK293 F cells, purified, and used to immunize llama. Using phage display, anti-HER2 VHHs with high affinity and specificity were isolated, sequenced, and constructed with a Histag and c-Myc tag. The constructed anti-HER2 VHHs were then expressed in E. coli, purified, and evaluated for their use in ELISA, flow cytometry, cell imaging, and immunohistochemistry. The affinities of the anti-HER2 VHHs toward the HER2 antigen were determined using biolayer interferometry. Furthermore, the binding sites of the anti-HER2 VHHs were evaluated by epitope mapping and in silico modeling and docking. Here, we report the sequence of an anti-HER2 VHH with high affinity (sub-nanomolar), specificity, and selectivity. This VHH binds to the same epitope as trastuzumab and can be utilized to generate bispecific antibodies or used as a diagnostic tool to differentiate HER2+ from HER2- antigens on plates, cells, and tissues. This discovery has broad applications in biochemical, biological, and medical sciences. [ABSTRACT FROM AUTHOR]

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

Dandan Wu,1,&ast; Junxiao Cong,1,&ast; Jiali Wei,1 Jing Hu,1 Wenhao Sun,1 Wei Ran,2 Chenghui Liao,1 Housheng Zheng,1 Liang Ye1 1Department of Immunology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, People’s Republic of China; 2State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Liang Ye, Department of Immunology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, 518055, People’s Republic of China, Tel +86-0755-26631420, Fax +86-0755-86671943, Email liangyeszu@163.comBackground: The emergence of the coronavirus disease 2019 (COVID-19) pandemic and the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) requires the continuous development of safe, effective, and affordable prevention and therapeutics. Nanobodies have demonstrated antiviral activity against a variety of viruses, providing a new candidate for the prevention and treatment of SARS-CoV-2 and its variants.Methods: SARS-CoV-2 glycoprotein spike 1 subunit (S1) was selected as the target antigen for nanobody screening of a naïve phage display library. We obtained a nanobody, named Nb-H6, and then determined its affinity, inhibition, and stability by ELISA, Competitive ELISA, and Biolayer Interferometry (BLI). Infection assays of authentic and pseudotyped SARS-CoV-2 were performed to evaluate the neutralization of Nb-H6. The structure and mechanism of action were investigated by AlphaFold, docking, and residue mutation assays.Results: We isolated and characterized a nanobody, Nb-H6, which exhibits a broad affinity for S1 and the receptor binding domain (RBD) of SARS-CoV-2, or Alpha (B.1.1.7), Delta (B.1.617.2), Lambda (C.37), and Omicron (BA.2 and BA.5), and blocks receptor angiotensin-converting enzyme 2 (ACE2) binding. Moreover, Nb-H6 can retain its binding capability after pH or thermal treatment and effectively neutralize both pseudotyped and authentic SARS-CoV-2, as well as VOC Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.2 and BA.5) pseudoviruses. We also confirmed that Nb-H6 binds two distinct amino acid residues of the RBD, preventing SARS-CoV-2 from interacting with the host receptor.Conclusion: Our study highlights a novel nanobody, Nb-H6, that may be useful therapeutically in SARS-CoV-2 and VOC outbreaks and pandemics. These findings also provide a molecular foundation for further studies into how nanobodies neutralize SARS-CoV-2 and variants and imply potential therapeutic targets for the treatment of COVID-19.Keywords: COVID-19, antiviral medication, single-domain antibody, neutralizing antibody, bio-screening

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.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nM affinity dependent on the species. G13 affinity was difficult to determine by biolayer interferometry due to low and heterogeneous signals. G13 and G6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species.

Antibody maturation is the central function of the adaptive immune response. This process is driven by the repetitive selection of mutations that increase the affinity toward antigens. We hypothesized that a precise observation of this process by high-throughput sequencing along the time course of immunization will enable us to predict the antibodies reacting to the immunized antigen without any additional in vitro screening. An alpaca was immunized with IgG fragments using multiple antigen injections, and the antibody repertoire development was traced via high-throughput sequencing periodically for months. The sequences were processed into clusters, and the antibodies in the 16 most abundant clusters were generated to determine whether the clusters included antigen-binding antibodies. The sequences of most antigen-responsive clusters resembled those of germline cells in the early stages. These sequences were observed to accumulate significant mutations and also showed a continuous sequence turnover throughout the experimental period. The foregoing characteristics gave us >80% successful prediction of clusters composed of antigen-responding VHHs against IgG fragment. Furthermore, when the prediction method was applied to the data from other alpaca immunized with epidermal growth factor receptor, the success rate exceeded 80% as well, confirming the general applicability of the prediction method. Superior to previous studies, we identified the immune-responsive but very rare clusters or sequences from the immunized alpaca without any empirical screening data.

ABSTRACTHuman metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract disease in children and adults. The hMPV fusion protein F is a trimeric class I fusion protein that is initially synthesized as a precursor (F0) and requires proteolytic activation by a host cell protease to generate the metastable, fusion-competent prefusion conformation of F. hMPV F is considered the main target of the neutralizing antibody response against hMPV infection. We isolated single-domain antibodies (sdAbs) directed against hMPV F that potently neutralize hMPV A and B strains. One of these sdAbs, sdHMPV16, specifically bound to cleaved and uncleaved prefusion F. Co-crystal structure analysis revealed that sdHMPV16 binds to a site located at the trimer interface of prefusion F. Moreover, prophylactic treatment with a sdHMPV16-Fc fusion protein reduced viral titers in the lungs of hMPV-infected cotton rats. In summary, sdHMPV16 broadly neutralizes hMPV, can be turned into a candidate biologic that restricts hMPV replication in an in vivo model, and, unexpectedly, binds to an unconventional epitope at the prefusion F trimer interface.IMPORTANCEHuman metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or “breathing” on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.

The shark-derived single-domain antibody VNAR (variable domain of new antigen receptor) has many advantageous features that make the VNAR suitable for improving current monoclonal antibody therapy deficiencies or disease diagnosis methods. In order to discover more VNARs, it is necessary to improve the efficiency of the isolation process. This research aims to enhance the VNAR discovery platform by dual displaying the semi-synthetic VNAR library and green fluorescent protein tag on the yeast surface. The GFP tag can be used to determine the degree of VNAR expression. The diversity of the semi-synthetic VNAR library constructed in this study is verified to be 1.97 × 109 by next-generation sequencing (NGS). We conveniently screened VNARs against the feline neonatal Fc receptor or feline infectious peritonitis virus nucleocapsid protein by sequential MACS and FACS. To find more diverse VNARs, we analyzed the NGS data of VNAR CDR3 genes before and after biopanning. By comparing the frequency change of each sequence, we found that the amplification factor of sequences was increased by biopanning. Four VNAR candidates selected by the high-frequency and high-amplification factor criteria showed an antigen-binding ability. The results demonstrate that biopanning from a yeast surface displaying a semi-synthetic VNAR library followed by the NGS assay can generate antigen binders rapidly without the need for shark rearing and long-term immunization. [ABSTRACT FROM AUTHOR]

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFRoverexpressing spontaneous tumors. [ABSTRACT FROM AUTHOR]

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with KD of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans. [ABSTRACT FROM AUTHOR]

Antibodies represent a relatively mature detection means and serve as therapeutic drug carriers in the clinical diagnosis and treatment of cancer—among which monoclonal antibodies (mAbs) currently occupy a dominant position. However, the emergence and development of small-molecule monodomain antibodies are inevitable due to the many limitations of mAbs, such as their large size, complex structure, and sensitivity to extreme temperature, and tumor microenvironments. Thus, since first discovered in Chondroid fish in 1995, IgNAR has become an alternative therapeutic strategy through which to replace monoclonal antibodies, thus entailing that this novel type of immunoglobulin has received wide attention with respect to clinical diagnoses and tumor therapies. The variable new antigen receptor (VNAR) of IgNAR provides an advantage for the development of new antitumor drugs due to its small size, high stability, high affinity, as well as other structural and functional characteristics. In that respect, a better understanding of the unique characteristics and therapeutic potential of IgNAR/VNAR in clinical and anti-tumor treatment is needed. This article reviews the advantages of its unique biochemical conditions and molecular structure for clinical diagnoses and novel anti-tumor drugs. At the same time, the main advantages of the existing conjugated drugs, which are based on single-domain antibodies, are introduced here, thereby providing new ideas and methods for the development of clinical diagnoses and anti-tumor therapies in the future. [ABSTRACT FROM AUTHOR]

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or “VHHs”), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library’s diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library’s vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

Arming oncolytic viruses with transgenes encoding immunomodulators improves their therapeutic efficacy by enhancing and/or sustaining the innate and adaptive anti-tumoral immune responses. We report here the isolation, selection, and vectorization of a blocking anti-human PDL1 single-domain antibody (sdAb) isolated from PDL1-immunized alpacas. Several formats of this sdAb were vectorized into the vaccinia virus (VV) and evaluated for their programmed cell death protein 1 (PD1)/PD1 ligand (PDL1) blocking activity in the culture medium of tumor cells infected in vitro. In those conditions, VV-encoded homodimeric sdAb generated superior PDL1 blocking activity compared to a benchmark virus encoding full-length avelumab. The sdAb was further used to design simple, secreted, and small tumor necrosis factor superfamily (TNFSF) fusions with the ability to engage their cognate receptors (TNFRSF) only in the presence of PDL1-positive cells. Finally, PDL1-independent alternatives of TNFRSF agonists were also constructed by fusing different variants of surfactant protein-D (SP-D) oligomerization domains with TNFSF ectodomains. An optimal SP-D–CD40L fusion with an SP-D collagen domain reduced by 80% was identified by screening with a transfection/infection method where poxvirus transfer plasmids and vaccinia virus were successively introduced into the same cell. However, once vectorized in VV, this construct had a much lower CD40 agonist activity compared to the SP-D–CD40L construct, which is completely devoid of the collagen domain that was finally selected. This latest result highlights the importance of working with recombinant viruses early in the payload selection process. Altogether, these results bring several complementary solutions to arm oncolytic vectors with powerful immunomodulators to improve their immune-based anti-tumoral activity.

Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient’s quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer.Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration.Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies.Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases.

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. [ABSTRACT FROM AUTHOR]

Antibodies are the largest class of biotherapeutics. However, in recent years, single-domain antibodies have gained traction due to their smaller size and comparable binding affinity. Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, singledomain antibodies lack a light chain and so have just three CDR loops. Given this inherent structural difference, it is important to understand whether Abs and sdAbs are distinguishable in how they engage a binding partner and thus, whether they are suited to different types of epitopes. In this study, we use non-redundant sequence and structural datasets to compare the paratopes, epitopes and antigen interactions of Abs and sdAbs. We demonstrate that even though sdAbs have smaller paratopes, they target epitopes of equal size to those targeted by Abs. To achieve this, the paratopes of sdAbs contribute more interactions per residue than the paratopes of Abs. Additionally, we find that conserved framework residues are of increased importance in the paratopes of sdAbs, suggesting that they include non-specific interactions to achieve comparable affinity. Furthermore, the epitopes of sdAbs are only marginally less accessible than those of Abs: we posit that this may be explained by differences in the orientation and compaction of sdAb and Ab CDR-H3 loops. Overall, our results have important implications for the engineering and humanization of sdAbs, as well as the selection of the best modality for targeting a particular epitope. [ABSTRACT FROM AUTHOR]

Simple Summary: The overlapping presence of free-ranging long-tailed macaques (Macaca fascicularis) and humans in tropical and southeast Asian countries, for example, Thailand and Indonesia, has resulted in instances of physical harm, vandalism, and potential transmission of zoonotic diseases. The development of contraception in the free-ranging long-tailed macaques is aimed at maintaining testosterone production to keep the social hierarchy and dominance behavior. Using phage-display library technology, this study developed a novel form of contraceptive for the long-tailed macaques, consisting of small antibody fragments called single-chain variable fragments (scFvs) against the extracellular domain of the follicular-stimulating hormone receptor (anti-FSHr). The purity, size, and binding efficiency of the anti-FSHr scFv were characterized. Then, the study evaluated the effects of intratesticular administration of the anti-FSHr scFv on testicular function and serum testosterone levels in three adult macaques. The results indicated that the anti-FSHr scFv reduced sperm output without altering testosterone levels. This study provides promising insights into the development of new methods of contraception for long-tailed macaques. FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (KD) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 106 cells) to day 56 (1.6 × 106 cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. Key points: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells [ABSTRACT FROM AUTHOR]

Single-domain antibodies, or VHH, nanobodies, are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. Single-domain antibodies have potential for use in sensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use of immobilized antibodies on a sensing substrate. Amine coupling was used to immobilize the single-domain antibodies on the substrate through a robust covalent bond. First, for two model single-domain antibodies with lysines at four highly conserved positions (K48, K72, K84, and K95), we mutated the lysines to alanine and measured the binding activity of the mutants (the percentage of immobilized antibodies that can bind antigen) using surface plasmon resonance. The two model single-domain antibodies tended to have higher binding activities when K72, which is close to the antigen binding site, was mutated. Adding a Lys-tag to the C-terminus of single-domain antibodies also increased the binding activity. We also mutated the lysine for another model single-domain antibodies with the lysine in a different position than the four residues mentioned above and measured the binding activity. Thus, single-domain antibodies immobilized in an orientation accessible to the antigen tended to have a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy of single-domain antibodies with high binding activity included mutating the lysine at or near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine. It is noteworthy that mutating K72 close to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition, and immobilization at the N-terminus close to the antigen binding site did not have such a negative effect on the binding activity compared to immobilization at the K72. • Model sdAbs mutants were immobilized by amine coupling and binding activities were measured by surface plasmon resonance. • Mutation of K72, one of the highly conserved lysines in sdAbs, increased binding activity after immobilization unless it destabilized the structure. • Mutation of K72 was more effective than N-terminal capping or addition of a Lys-tag to the C-terminus in designing sdAbs with high binding activity. • Mutating a residue away from the antigen binding site to lysine increased the binding activity of sdAbs. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 is the etiological agent of the COVID‐19 pandemic. Antibody‐based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS‐CoV‐2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID‐19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR) antibodies. VNARs are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNARs that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS‐CoV‐2 virus. Several binders, including S2A9, showed cross‐reactivity against S2 subunits from other β coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS‐CoV‐2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single‐domain antibodies against emerging viral pathogens. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.

Despite advances in immunotherapy for the treatment of cancers, not all patients can benefit from programmed cell death ligand 1 (PD-L1) immune checkpoint blockade therapy. Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level; hence, accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects. Therefore, based on the high affinity antibody Nb109, a new site-specifically radiolabeled tracer, 68Ga-NODA-cysteine, aspartic acid, and valine (CDV)-Nb109, was designed and synthesized to accurately monitor PD-L1 expression. The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95% and radiochemical purity of 97%. It showed high affinity for PD-L1 with a dissociation constant of 12.34 ± 1.65 nM. Both the cell uptake assay and positron emission tomography (PET) imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells. Meanwhile, dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression, allowing timely interventional immunotherapy. In conclusion, this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.