Full-length Antibodies Research Articles

Comparison of C-type Nanoantibody Produced in Different Expression Systems Implying Potential Clinical Applications.

In the pharmaceutical industry, the Chinese hamster ovary cell, a type of mammalian cell, is extensively employed for the production of conventional full-length monoclonal antibodies. Nanobody is one of the most attractive directions for the development of next-generation antibody drugs. However, a suitable expression system for its manufacture has not yet been comprehensively evaluated. Previously, we proposed that the immunoglobulin constant CH2 domain could be a promising scaffold for developing C-type nanoantibodies (C-Nabs) as candidate therapeutics. Here, we used an antiviral C-Nab, which we identified previously (under review), as a model for investigation. We expressed C-Nabs without a tag in different systems, including a bacterium (C-Nabbac), yeast (C-Nabyeast), and mammalian cell (C-Nabmam). After purification, the binding and neutralizing activities of C-Nabs from different expression systems are similar. Their secondary structures are rich in β-strand. The melting temperatures of C-Nabbac (71.5 °C) and C-Nabmam (70.2 °C) are similar, which are slightly higher than that of C-Nabyeast (65.6 °C), while C-Nabyeast and C-Nabmam are more resistant to urea-induced unfolding than C-Nabbac. C-Nabyeast and C-Nabmam demonstrate higher resistance to aggregation compared to C-Nabbac. C-Nabyeast exhibits greater resistance to enzyme digestion compared to C-Nabbac and C-Nabmam. Notably, when administered via intraperitoneal injection in mice, C-Nabyeast shows superior pharmacokinetics. Overall, after comparing C-Nab proteins from various expression systems, we determined that yeast is the most suitable host for producing C-Nabs. This finding is beneficial for the production of nanobodies as potential drug candidates.

Machine Learning Models for Predicting Monoclonal Antibody Biophysical Properties from Molecular Dynamics Simulations and Deep Learning-Based Surface Descriptors.

Monoclonal antibodies (mAbs) have found extensive applications and development in treating various diseases. From the pharmaceutical industry's perspective, the journey from the design and development of mAbs to clinical testing and large-scale production is a highly time-consuming and resource-intensive process. During the research and development phase, assessing and optimizing the developability of mAbs is of paramount importance to ensure their success as candidates for therapeutic drugs. The critical factors influencing mAb development are their biophysical properties, such as aggregation propensity, solubility, and viscosity. This study utilized a data set comprising 12 biophysical properties of 137 antibodies from a previous study (Proc Natl Acad Sci USA. 114(5):944-949, 2017). We employed full-length antibody molecular dynamics simulations and machine learning techniques to predict experimental data for these 12 biophysical properties. Additionally, we utilized a newly developed deep learning model called DeepSP, which directly predicts the dynamical and structural properties of spatial aggregation propensity and spatial charge map in different antibody regions from sequences. Our research findings indicate that the machine learning models we developed outperform previous methods in predicting most biophysical properties. Furthermore, the DeepSP model yields similar predictive results compared to molecular dynamic simulations while significantly reducing computational time. The code and parameters are freely available at https://github.com/Lailabcode/AbDev. Also, the webapp, AbDev, for 12 biophysical properties prediction has been developed and provided at https://devpred.onrender.com/AbDev.

Pretargeted alpha therapy in MUC16-positive high-grade serous ovarian cancer

BackgroundPeritoneal metastasis with micrometastatic cell clusters is a common feature of advanced ovarian cancer. Targeted alpha therapy (TAT) is an attractive approach for treating micrometastatic diseases as alpha particles release enormous amounts of energy within a short distance. A pretargeting approach — leveraging the inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and trans-cyclooctene (TCO) — can minimize off-target toxicity related to TAT, often associated with full-length antibodies. We hypothesized that a pretargeting strategy could effectively treat high-grade serous (HGS) ovarian tumors while minimizing toxicity. MethodsWe utilized the humanized antibody, AR9.6, labeled with actinium-225 (225Ac). AR9.6 targets fully glycosylated and hypoglycosylated isoforms of MUC16. For biodistribution and radioimmunotherapy studies, AR9.6-TCO was injected into OVCAR3-bearing mice 72 h before administering [225Ac]Ac-mcp-PEG8-Tz, e.g. using a 1,2,4,5-tetrazine conjugated to the macropa chelator via a polyethylene glycol (PEG) linker. ResultsBiodistribution data revealed that the pretargeting approach achieved substantial tumor uptake. Cerenkov luminescence imaging confirmed successful in vivo pretargeting during TAT studies. Compared to the control groups, TAT with AR9.6-TCO and [225Ac]Ac-mcp-PEG8-Tz significantly suppressed tumor growth and improved overall survival in OVCAR3 tumor-bearing mice. Renal and ovarian pathology compatible with toxicity was observed in mice in addition to transient hematologic toxicity. ConclusionWe confirmed that pretargeting with AR9.6-TCO and [225Ac]Ac-mcp-PEG8-Tz has durable antitumor effects in high MUC16-expressing tumors. These findings demonstrate great potential for using pretargeting in combination with TAT for the treatment of ovarian cancer. ClassificationBiological Sciences; Applied Biological Sciences.

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli.

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the C H 2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N- linked glycans within the QYNST sequon of the IgG C H 2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native C H 2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N -glycan at the QYNST sequon of the E. coli -derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

Immuno-PET Imaging of EGFR with 64Cu-NOTA Panitumumab in Subcutaneous and Metastatic Nonsmall Cell Lung Cancer Xenografts.

Objective: About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [64Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [18F]FLT PET. Immuno-PET with [64Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [64Cu]CuCl2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [64Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein (n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein (n = 3). PET imaging revealed high uptake of [64Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV)mean reached 4.70 ± 0.42 and 5.37 ± 0.40 (n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 (n = 4; p < 0.001). In the metastatic model, the following SUVmean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 (n = 4) to 3.67 ± 0.33% ID/g (n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 (n = 4) to 8.35 ± 1.30% ID/g (n = 5; p < 0.001), respectively. Conclusions: [64Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

Recent Advances and Future Perspectives in Radiolabeled Antibody Fragments for Breast Cancer Molecular Imaging

Breast Cancer (BC) is the leading cause of cancer-related deaths in women and the most common cancer worldwide. It is classified based on its anatomical origin, the presence of Human Epidermal Growth Factor Receptor 2 (HER-2), and the presence of Estrogen Receptor (ER) and/or Progesterone Receptor (PR). Around 20% of breast cancers are HER-2 positive. While biopsy-based diagnoses are valuable in clinical settings, they have limitations in terms of sampling and interpretation. However, laboratory tests such as Immunohistochemistry (IHC) or Fluorescence In Situ Hybridization (FISH) are also limited, including being time-consuming, expensive, and requiring specialized equipment. Ongoing research and technological advancements aim to address the challenges associated with biopsy-based diagnoses and laboratory tests to develop more accurate and efficient methods for assessing HER-2 status. To this end, various radioactively labeled proteins and small compounds, such as single-chain variable Fragments (scFv), F(ab')2, affibody, and nanobody, have been developed to target HER-2 using molecular array techniques. These smaller targeted compounds offer improved image quality, shorter circulating half-life, and reduced immunogenicity compared to their larger counterparts. This is due to their better biodistribution, clearance, and stability. This study investigates the current understanding and ongoing efforts in utilizing antibody fragments for molecular imaging. The specific objectives were to evaluate the advantages of antibody fragments over full-length antibodies regarding biodistribution, clearance, and stability. Additionally, this study aims to assess the current knowledge and ongoing research in utilizing antibody fragments for molecular imaging.

Rational design of environmentally responsive antibodies with pH-sensing synthetic amino acids

Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-l-tyrosine, 3-cyano-l-tyrosine, and 3, 5-halogenated-l-tyrosine, each with near neutral pKa, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions. Cell-based assays showed that these modifications achieved up to 140-fold tighter binding to antigens and several-fold tighter cytotoxicity to antigen-expressing cell at pH 6.0 than pH 7.4. The pH-dependent binding effect was retained in full-length antibodies. In silico structural analyses revealed electrostatic repulsion at neutral pH between antigens and antibodies or inside the antibody as the underlying mechanisms of the acid preference, and this finding increases the designability of pH-dependent antigen binding. The development of antibodies responsive to the microenvironments of diseased tissues will allow more disease-related antigens to be targeted in treatments, because of the reduced cross-reactivity toward healthy tissues.

Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide.

Efficient functionalization of peptides and proteins has widespread applications in chemical biology and drug discovery. However, the chemoselective and site-selective modification of proteins remains a daunting task. Herein, a highly efficient chemo-, regio-, and stereoselective hydrosulfuration of ynamide was identified as an efficient method for the precise modification of peptides and proteins by uniquely targeting the thiol group of cysteine (Cys) residues. This novel method could be facilely operated in aqueous buffer and was fully compatible with a wide range of proteins, including small model proteins and large full-length antibodies, without compromising their integrity and functions. Importantly, this reaction provides the Z-isomer of the corresponding conjugates exclusively with superior stability, offering a precise approach to peptide and protein therapeutics. The potential application of this method in peptide and protein chemical biology was further exemplified by Cys-bioconjugation with a variety of ynamide-bearing functional molecules such as small molecule drugs, fluorescent/affinity tags, and PEG polymers. It also proved efficient in redox proteomic analysis through Cys-alkenylation. Overall, this study provides a novel bioorthogonal tool for Cys-specific functionalization, which will find broad applications in the synthesis of peptide/protein conjugates.

An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies.

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

Immunoassay Analysis Using Full-Length and Phage Antibodies for Detection of Antibiotics: A Review of the Literature

Immunoassay Analysis Using Full-Length and Phage Antibodies for Detection of Antibiotics: A Review of the Literature

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3–4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies.

Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70°C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

Increasing the Dye Payload of Cetuximab-IRDye800CW Enables Photodynamic Therapy.

Cetuximab (Cet)-IRDye800CW, among other antibody-IRDye800CW conjugates, is a potentially effective tool for delineating tumor margins during fluorescence image-guided surgery (IGS). However, residual disease often leads to recurrence. Photodynamic therapy (PDT) following IGS is proposed as an approach to eliminate residual disease but suffers from a lack of molecular specificity for cancer cells. Antibody-targeted PDT offers a potential solution for this specificity problem. In this study, we show, for the first time, that Cet-IRDye800CW is capable of antibody-targeted PDT in vitro when the payload of dye molecules is increased from 2 (clinical version) to 11 per antibody. Cet-IRDye800CW (1:11) produces singlet oxygen, hydroxyl radicals, and peroxynitrite upon activation with 810 nm light. In vitro assays on FaDu head and neck cancer cells confirm that Cet-IRDye800CW (1:11) maintains cancer cell binding specificity and is capable of inducing up to ∼90% phototoxicity in FaDu cancer cells. The phototoxicity of Cet-IRDye800CW conjugates using 810 nm light follows a dye payload-dependent trend. Cet-IRDye800CW (1:11) is also found to be more phototoxic to FaDu cancer cells and less toxic in the dark than the approved chromophore indocyanine green, which can also act as a PDT agent. We propose that antibody-targeted PDT using high-payload Cet-IRDye800CW (1:11) could hold potential for eliminating residual disease postoperatively when using sustained illumination devices, such as fiber optic patches and implantable surgical bed balloon applicators. This approach could also potentially be applicable to a wide variety of resectable cancers that are amenable to IGS-PDT, using their respective approved full-length antibodies as a template for high-payload IRDye800CW conjugation.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus-1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. Additionally, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared to TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared to TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles.

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic β-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

A novel sustainable immunoassay for sensitive detection of atrazine based on the anti-idiotypic nanobody and recombinant full-length antibody

Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC50) of 1.66 ng mL−1 for ATR and a linear range of 0.35 − 8.73 ng mL−1. The cross-reactivity of the assay to ametryn was 64.24 %, whereas that to terbutylazine was 38.20 %. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40 − 105.36 % for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Off-label use of intravitreal bevacizumab: A global conundrum.

Bevacizumab is a monoclonal, humanized, full-length antibody targeting vascular endothelial growth factor(VEGF-A), known for its anti-angiogenic properties. The off-label use of bevacizumab has stirred legal, financial, industrial, and ethical complexities. With its potential to treat diverse ocular conditions, this commentary delves into the multifaceted dimensions of bevacizumab's off-label utilization, encompassing clinical trials, regulatory frameworks, safety considerations, comparative effectiveness, and economic implications.