Camelid Heavy-chain Antibodies Research Articles

Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling.

Detection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

Immunogenicity and humanization of single-domain antibodies.

Single-domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy-chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid VH Hs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed VH :VL interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5-specific VH H TAS266 and the TNFR1-specific VH GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid VH Hs are nonimmunogenic while some fully human VH s elicit ADAs. The presence of preexisting ADAs directed against the exposed C-termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low-level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for VH H and VNAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability.

Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies.

The antibody immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the antibody repertoire. Here, we developed a proteomic strategy to survey, at an unprecedented scale, the landscape of antigen-engaged, circulating camelid heavy-chain antibodies, whose minimal binding fragments are called VHH antibodies or nanobodies. The sensitivity and robustness of this approach were validated with three antigens spanning orders of magnitude in immune responses; thousands of distinct, high-affinity nanobody families were reliably identified and quantified. Using high-throughput structural modeling, cross-linking mass spectrometry, mutagenesis, and deep learning, we mapped and analyzed the epitopes of >100,000 antigen-nanobody complexes. Our results revealed a surprising diversity of ultrahigh-affinity camelid nanobodies for specific antigen binding on various dominant epitope clusters. Nanobodies utilize both shape and charge complementarity to enable highly selective antigen binding. Interestingly, we found that nanobody-antigen binding can mimic conserved intracellular protein-protein interactions. A record of this paper's Transparent Peer Review process is included in the Supplemental information.

Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate.

"Quenchbody (Q-body)" is a quench-based fluorescent biosensor labeled with a fluorescent dye near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules quickly by simply mixing with a sample. However, the development of Q-bodies using VHH-nanobodies derived from camelid heavy-chain antibodies has not been reported despite their favorable characteristics. Here, we report a "mini Q-body" that can detect the chemotherapy agent methotrexate (MTX) by using anti-MTX nanobody. Three kinds of constructs each encoding an N-terminal Cys-tag and anti-MTX VHH gene with a different length of linker (GGGS)n (n = 0, 2, and 4) between them were prepared followed by the expression in Escherichia coli and labeling with several dye maleimides. When the fluorescence intensities in the presence of varied MTX concentrations were measured, TAMRA-labeled nanobodies showed a higher response than ATTO520- or R6G-labeled ones. Especially, TAMRA C6-labeled mini Q-body with no linker showed the highest response of ∼6-fold and a low detection limit of 0.56 nM. When each Trp residue in the mini Q-body was mutated to address the quenching mechanism, the major role of Trp34 at CDR1 in quenching was revealed. Furthermore, the mini Q-body could detect MTX in 50% human serum with a low detection limit of 1.72 nM, showing its applicability to therapeutic drug monitoring. This study is expected to become the basis of the construction of highly responsive mini Q-bodies for sensitive detection of many molecules from small haptens to larger proteins, which will lead to broader applications such as point-of-care tests.

Phase 1 Study of CART-Ddbcma, a CAR-T Therapy Utilizing a Novel Synthetic Binding Domain for the Treatment of Subjects with Relapsed and Refractory Multiple Myeloma

Phase 1 Study of CART-Ddbcma, a CAR-T Therapy Utilizing a Novel Synthetic Binding Domain for the Treatment of Subjects with Relapsed and Refractory Multiple Myeloma

Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.

Development and production of nanobodies specifically against green fluorescence protein.

Variable domains of heavy chains of camelid heavy-chain antibodies (VHHs) are known as nanobodies. Nanobodies are approximately 15kDa in size with high affinity to their antigens. They can be easily manipulated and produced in microorganisms. In this study, an alpaca was immunized with purified green fluorescence protein (GFP) and a VHH library from lymphocytes of the immunized alpaca was constructed with a capacity of 6.7 × 107. The library was biopanned against GFP by phage display technique and four unique DNA sequences coding for anti-GFP nanobodies were identified by enzyme-linked immunosorbent assay, named a12, e6, d5, and b9. The four DNA sequences were then cloned into pADL-10b-6×His or pBAD24-Flag-6×His for expression in bacteria. Purified A12, E6, D5, and B9 were demonstrated to bind GFP specifically both in vitro by enzyme-linked immunosorbent assay and native-PAGE analysis and in vivo by immunofluorescence and immunoprecipitation. Taken together, our results demonstrate that anti-GFP nanobodies are successfully selected from the immune library, are produced in bacteria, and are available for basic research.Key Points• Four different GFP binders were successfully obtained from an immune VHH library.• The four GFP binders were successfully purified from bacteria. • Purified GFP binders can bind GFP both in vitro and in vivo and are ready for use in basic research.

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Nanobodies as versatile tools: A focus on targeted tumor therapy, tumor imaging and diagnostics.

Monoclonal antibodies and vaccines have widely been studied for the immunotherapy of cancer, though their large size appears to limit their functionality in solid tumors, in large part due to unique properties of tumor microenvironment. Smaller formats of antibodies have been developed to throw such restrictions. These small format antibodies include antigen binding fragments, single-chain variable fragments, single variable domain of camelid antibody (so-called nanobody (Nb) or VHH). Since their serendipitous discovery, nanobodies have been studies at length in the fields of research, diagnostics and therapy. These antigen binding fragments, originating from camelid heavy-chain antibodies, possess unusual hallmarks in terms of (small) size, stability, solubility and specificity, hence allowing cost-effective production and sometimes out performing monoclonal antibodies. In addition, these small camelid heavy-chain antibodies are highly adaptable tools for cancer research as they enable specific modulation of targets, enzymatic and non-enzymatic proteins alike. Molecular imaging studies benefit from the rapid, homogeneous tumor accumulation of nanobodies and their fast blood clearance, permitting previously unattainable fast tumor visualization. Moreover, they are endowed with considerable therapeutic potential as inhibitors of receptor-ligand pairs and deliverers of drugs or drug-loaded nanoparticles towards tumors. In this review, we shed light on the current status of nanobodies in diagnosis and imaging of tumor and exploiting nanobodies revert immunosuppressive events, modulation of immune checkpoints, and as deliverers of drugs for targeted tumor therapy.

Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF.The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%.We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.

The novel llama-human chimeric antibody has potent effect in lowering LDL-c levels in hPCSK9 transgenic rats

BackgroundThe advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)–inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost-effective application of camelid anti-PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti-PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half-life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub-type 4 (IgG4 Fc) was incorporated to form a novel llama-human chimeric molecule (VHH-hFc).ResultsThe PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague–Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH-hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama-human chimeric VHH-Fc protein had a similar reduction (~ 40%) of the LDL-c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity.ConclusionsDue to the high yield and low cost of Pichia pastoris, lipid-lowering effect and strong stability, the llama-human chimeric antibody (VHH-Fc) offers a potent therapeutic candidate for the control of the serum lipid level.

Abstract 4774: Engineering an oncolytic adenovirus to target CEA in colorectal cancer

Abstract Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most commonly diagnosed cancer overall. Screening methods can identify precancerous polyps before they progress, leading to five-year survival rates of 92%. However, if diagnosed in later stages the survival rates decrease to 12%. While treatment options are available for advanced stages, they remain inadequate to affect overall survival. Thus, new therapies are needed to improve treatment outcomes. Oncolytic virotherapy is a unique therapeutic approach in which a virus is genetically modified to target cancer cells while sparing normal tissue. Oncolytic virotherapy takes advantage of the lytic properties of the viral life cycle. These viruses are used replicate inside cancer cells, lyse them and spread to adjacent cells throughout the tumor. The adenovirus is a well-characterized vector that is easily manipulated and produced and is capable of infecting a wide range of cells. However, the endogenous receptor for adenovirus, the Coxsackie and adenovirus receptor (CXADR), is often downregulated in cancers, thus decreasing the ability to selectively infect cancer cells. In this study, an adenovirus was genetically engineered to present the single variable domain (VHH) derived from a camelid heavy chain antibody B2. The B2 antibody recognizes the human carcinoembryonic antigen (CEA), which is often overexpressed in a variety of cancers, including colorectal cancer. Here, the wild-type fiber and knob domain of the adenovirus fiber gene was replaced with a modified T4 fibritin fiber linked to the B2 VHH. To provide for visual tracking of adenovirus infection and biodistribution, a red fluorescent protein (RFP) marker gene was also fused to the capsid protein IX (pIX). This virus was rescued and amplified in the HEK293 F28 packaging cell line with a final round of amplification in HEK293 cells. The virus was purified by CsCl ultracentrifugation and dialysis. The resulting viral stock was titered and characterized for the presence of the modified fiber and knob. Subsequently, the vector was modified to express the human sodium-iodide symporter (NIS) transgene, to allow for noninvasive imaging of the adenovirus uptake and replication as well as radiotherapy using 131I. Further experiments will be completed to confirm viral selectivity for CEA and subsequent lysis of colorectal cancer cells. The results of this study will advance targeting oncolytic adenoviruses to metastatic colorectal cancer. Note: This abstract was not presented at the meeting. Citation Format: Jessica C. Schaumburg. Engineering an oncolytic adenovirus to target CEA in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4774.

Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy‐chain antibody domain

The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health. VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4ng mL-1 and a limit of detection (LOD) of 0.3ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively. The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Targeted Nanobody-Based Molecular Tracers for Nuclear Imaging and Image-Guided Surgery.

Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific background limits their applicability. Nanobodies, unique antibody-binding fragments derived from camelid heavy-chain antibodies, have excellent properties for molecular imaging as they are able to specifically find their target early after injection, with little to no nonspecific background. Nanobody-based tracers using either nuclear or fluorescent labels have been heavily investigated preclinically and are currently making their way into the clinic. In this review, we will discuss different important factors in nanobody-tracer design, as well as the current state of the art regarding their application for nuclear and fluorescent imaging purposes. Furthermore, we will discuss how nanobodies can also be exploited for molecular therapy applications such as targeted radionuclide therapy and photodynamic therapy.

Anti-MET VHH Pool Overcomes MET-Targeted Cancer Therapeutic Resistance.

Receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) play crucial roles in many human malignancies. Numerous drugs have been developed against kinase center of MET or HGF-mediated activation; however, the outcomes in patients are not so promising. Increasing evidence show that MET has kinase-independent effects on tumorigenesis and dissemination, which explains the low efficacy in kinase inhibition-based strategy. VHH is the recombinant variable region of Camelid heavy-chain antibody. As a nanoscale antigen-binding unit, VHH has become an appealing drug candidate in cancer therapy. In our study, we choose a novel strategy to construct an anti-MET VHH pool against the whole ecto-domain of MET. Comparing to monoclonal antibody or single VHH, the anti-MET VHH pool strongly promotes MET degradation through Clathrin-dependent endo-lysosomal pathway. Thus, the anti-MET VHH pool not only blocks kinase activity of MET, but also reduces protein level of MET. As a consequence, anti-MET VHH pool dramatically suppresses cancer cell proliferation, viability, and colony formation in vitro, and inhibits tumorigenesis and growth in mice. Taken together, VHH pool-based strategy greatly improves MET-targeted therapeutic effects on cancer.

Nanobody as a new generation of functional proteins

Nanobody (Nb) or VHH is the smallest binding domain of camelid heavy chain antibody (HcAb). Light chains of HcAb naturally removed and because of some evolutionary changes, Nbs have unique properties rather than conventional antibodies. The size of Nb is about one-tenth (0.1) of whole antibodies and this size improved some problems of four chains antibodies such as high yield of expression in prokaryotic systems and penetration to tissues. Some other characterizes of Nb like close homology to human VH, high stability in the extended range of pH and temperature, the capability to the identification of unusual epitope is very attractive for research and development of new Nb candidates for diagnosis, research, and therapeutic applications. Discovery of Nb almost coincided with advancement in phage display technology that was used along with Hybridoma technology in monoclonal antibody development. Currently, many of research groups focused on high-quality Nbs development against different targets especially in cancers and fortunately there are many of Nbs in clinical trial stages for use in extended ranges of diseases such as cancers, autoimmune and inflammatory diseases and infectious diseases. Recently two of them were approved for clinical use. Big companies like Ablynx and Merck have been invested in this field and in future, further drugs base Nbs were approved in different areas of health science. In this review, we focused on production, features, and clinical application of Nbs and will be noted to Nbs in clinical trials.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen, and to provide experimental basis for subsequent functional studies. Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times. The peripheral blood was collected and the lymphocytes were isolated. Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH), and to construct a phage display library. The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA). The PD-1 antigen, which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml), was coated in an ELISA plate, and the phage display library was subjected to 3 rounds of affinity selection. Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA. According to the results of DNA sequencing, three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector, and transformed into E. coli BL21(DE3) competent cells, and then induced by isopropyl-β-D-thiogalactoside. The recombinant VHH antibody protein was purified by nickel column affinity chromatography, and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA. Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc, high titer specific antibody was stimulated, and the immune serum titer reached 1∶32 000. A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes. After 3 rounds of affinity selection, 46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA. Among them, three clones of VHH-B7, VHH-H5 and VHH-H12 had higher repeats, indicating that significant enrichment was obtained. The results of Western Blot and ELISA showed that the purified B7, H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity. Their affinity constants were 1.19×1011 and 1.63×1011, 1.59×1011 L/mol, respectively. Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library, which can bind to the PD-1 antigen with high affinity. This study can provide an experimental basis for subsequent functional studies. Key words: PD-1; Nano antibodies; Heavy chain antibody variable region; Identification; Phage display

The structural basis of nanobody unfolding reversibility and thermoresistance

Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.

Combinatorial Design of a Nanobody that Specifically Targets Structured RNAs

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.