Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents. Methods The naive phage nanobody library was constructed and its diversity was analyzed. Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target, and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing. Results The capacity of the constructed naive phage nanobody library was 3.89×10⁹ CFU/mL, and the insertion of effective fragments was higher than 85%. Based on this library, seven anti-human CD22 nanobodies were screened, and the amino acid sequence comparison results showed that the overall similarity was 70.34%, and all of them were hydrophilic proteins. The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22, and the main forces were hydrophobic interaction and hydrogen bonding. Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22, successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.
Camelus/immunology* ; Single-Domain Antibodies/chemistry* ; Peptide Library ; Humans ; Animals ; Sialic Acid Binding Ig-like Lectin 2/genetics* ; Amino Acid Sequence ; Molecular Docking Simulation

Antibodies currently comprise the predominant treatment modality for a variety of diseases; therefore, optimizing their properties rapidly and efficiently is an indispensable step in antibody-based drug development. Inspired by the great success of artificial intelligence-based algorithms, especially deep learning-based methods in the field of biology, various computational methods have been introduced into antibody optimization to reduce costs and increase the success rate of lead candidate generation and optimization. Herein, we briefly review recent progress in deep learning-based antibody optimization, focusing on the available datasets and algorithm input data types that are crucial for constructing appropriate deep learning models. Furthermore, we discuss the current challenges and potential solutions for the future development of general-purpose deep learning algorithms in antibody optimization.
Deep Learning ; Humans ; Antibodies/chemistry* ; Algorithms ; Artificial Intelligence ; Drug Development

With advances of drug design and preparation technology, the development of long-acting drugs has become an important research focus in precision medicine and chronic disease management. These drugs are designed to improve the patients' compliance and quality of life by achieving prolonged maintenance of an effective drug concentration in the body with a reduced dosing frequency. Small molecule drugs, monoclonal antibodies and nucleic acid drugs all have their own difficulties in achieving long actions, which can be especially challenging for the latter two because of their structural complexity. This review provides an overview of the strategies for designing long-acting small molecule drugs, monoclonal antibodies, and nucleic acid drugs.
Humans ; Drug Design ; Antibodies, Monoclonal/chemistry* ; Nucleic Acids ; Precision Medicine ; Delayed-Action Preparations

Bio-mineralization has emerged as a promising strategy to enhance vaccine immunogenicity. This study optimized the calcium phosphate (CaP) mineralization process of foot-and-mouth disease virus-like particles (FMD VLPs) to achieve high mineralization efficiency and scalability. Key parameters, including concentrations of Ca²⁺, HPO₄^2-, NaCl, and VLPs, as well as stirring speed, were systematically optimized. Stability of the scaled-up reaction system and immunogenicity of the mineralized vaccine were evaluated. Optimal conditions [25.50 mmol/L Ca(NO₃)₂, 15 mmol/L Na₂HPO₄, 300 mmol/L NaCl, 0.75 mg/mL VLPs, and 1 500 r/min] yielded CaP-mineralized VLPs (VLPs-CaP) with high mineralization efficiency, uniform morphology, and a favorable particle size. Scaling up the reaction by 25 folds maintained consistent mineralization efficiency and particle characteristics. Immunization in mice demonstrated that VLPs-CaP induced higher titers of specific antibodies and neutralizing antibodies than unmineralized VLPs (P < 0.05). Higher IgG2a/IgG1 ratio and enhanced IFN-γ secretion (P < 0.05) further indicated robust cellular immune responses. We establish a stable and scalable protocol for VLPs-CaP, providing a theoretical and technical foundation for developing high-efficacy VLPs-CaP vaccines.
Vaccines, Virus-Like Particle/immunology* ; Immunogenicity, Vaccine ; Calcium Phosphates/chemistry* ; Foot-and-Mouth Disease Virus ; Biomineralization ; Particle Size ; Animals ; Mice ; Antibodies, Neutralizing/blood* ; Antibodies, Viral/blood* ; Immunity, Cellular

As the need for antibody production rises, there is an urgent need to lower the costs and enhance the efficiency of the separation process. Currently, the chromatographic media used for antibody separation and purification often focus on individual properties of antibodies, such as affinity, hydrophobicity, and charge, leading to issues like low purification efficiency or inadequate adsorption capacity. To address this, an electrostatically coupled polypeptide affinity medium (FD7-3, 5-diaminobenzoic acid n-sepharose, FD7-DA-Sepharose) was developed for rapid purification of antibodies from cell culture supernatant. This medium utilized 3, 5-diaminobenzoic acid as a spacer to attach the heptapeptide-affinity ligand (FYEILHD, FD7) to agarose microspheres. Antibodies could be adsorbed through charge interactions with the carboxyl functional group of the FD7-DA-Sepharose spacer, while FD7 enhanced electrostatic coupling and affinity adsorption through synergistic effects, significantly increasing the adsorption capacity while maintaining the affinity and specificity. The influences of pH and ionic strength on adsorption capacity were investigated with human immunoglobulin as a model protein. The static adsorption capacity (Q_m) of FD7-DA-Sepharose in the solution of pH 6.0 reached 67.73 mg/mL, representing a 52.68% increase compared with that (44.36 mg/mL) of the commercial Protein A affinity medium. Furthermore, the elution conditions for FD7-DA- Sepharose were mild (20 mmol/L PB, 0.5 mol/L NaCl, pH 6.0), in contrast to the harsh acidic elution (pH 2.7-3.6) typically associated with Protein A, which can damage antibody integrity. The FD7-DA-Sepharose medium was then employed to purify antibodies from cell culture supernatant, achieving the yield of 94.8% and the purity of 98.4%. The secondary structure of the purified antibody was determined by circular dichroism spectroscopy. The results demonstrated that FD7-DA-Sepharose enabled efficient purification of antibodies from cell culture supernatant, which provided a cost-effective solution (approximately one-third the price of commercial Protein A affinity medium) with gentle elution conditions that preserve the natural conformation of antibodies. This approach paves a novel, economical, and efficient way for the separation and purification of antibodies from cell culture supernatant.
Chromatography, Affinity/methods* ; Static Electricity ; Humans ; Sepharose/analogs & derivatives* ; Peptides/chemistry* ; Adsorption ; Antibodies/isolation & purification*

Nanobodies (Nbs), the unique single-domain antibodies discovered in the species of Camelidae and sharks, are also known as the variable domain of the heavy chain of heavy-chain antibody (VHH). They offer strong antigen targeting and binding capabilities and overcome the drawbacks such as large size, low stability, high immunogenicity, and slow clearance of conventional antibodies. Nbs can be boosted by bioconjugation with toxins, enzymes, radioactive nucleotides, fluorophores, and other functional groups, demonstrating potential applications in the diagnosis and treatment of human and animal diseases. This article introduces the structures and characteristics of Nbs, the construction and screening of Nb libraries, and the strategies for affinity maturation and then reviews the current applications of Nbs in diagnosis and treatment, providing a reference for the development of diagnostic reagents and clinical therapies for infectious diseases.
Single-Domain Antibodies/chemistry* ; Animals ; Humans ; Camelidae/immunology*

This study developed ferritin-based nanoparticles carrying the African swine fever virus (ASFV) p30 protein and evaluated their immunogenicity, aiming to provide an experimental basis for the research on nanoparticle vaccines against ASFV. Initially, the gene sequences encoding the p30 protein and SpyTag were fused and inserted into the pCold-I vector to create the pCold-p30 plasmid. The gene sequences encoding SpyCatcher and ferritin were fused and then inserted into the pET-28a(+) vector to produce the pET-F-np plasmid. Both plasmids were expressed in Escherichia coli upon induction. Subsequently, the affinity chromatography-purified p30 protein was conjugated with ferritin in vitro, and the p30-ferritin (F-p30) nanoparticles were purified by size-exclusion chromatography. The morphology and structural integrity of F-p30 nanoparticles were examined by a particle size analyzer and transmission electron microscopy. Mice were immunized with F-p30 nanoparticles, and the humoral and cellular immune responses were assessed. The results showed that F-p30 nanoparticles were successfully prepared, with the particle size of approximately 20 nm. F-p30 nanoparticles were efficiently internalized by bone marrow-derived dendritic cells (BMDCs) cells in vitro. Compared with the p30 protein alone, F-p30 nanoparticles induced elevated levels of specific antibodies and cytokines in mice and stimulated the proliferation of follicular helper T cell (T_FH) and germinal center B cell (GCB) in lymph nodes as well as CD4⁺ and CD8⁺ T cells in the spleen. In conclusion, we successfully prepared F-p30 nanoparticles which significantly enhanced the immunogenicity of p30 protein, giving insights into the development of vaccines against ASFV.
Animals ; Nanoparticles/chemistry* ; Mice ; African Swine Fever Virus/genetics* ; Ferritins/chemistry* ; Swine ; Viral Vaccines/genetics* ; African Swine Fever/immunology* ; Mice, Inbred BALB C ; Viral Proteins/genetics* ; Escherichia coli/metabolism* ; Dendritic Cells/immunology* ; Immunogenicity, Vaccine ; Antibodies, Viral/blood* ; Female ; Capsid Proteins/genetics*

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
Cricetinae ; Animals ; Antibodies, Monoclonal/metabolism* ; Caprylates/chemistry* ; Cell Culture Techniques ; Chromatography, Affinity ; CHO Cells ; Cricetulus ; Chemical Precipitation

This study was to develop a new method for detecting circulating tumor cells (CTCs) with high sensitivity and specificity, therefore to detect the colorectal cancer as early as possible for improving the detection rate of the disease. To this end, we prepared some micro-column structure microchips modified with graphite oxide-streptavidin (GO-SA) on the surface of microchips, further coupled with a broad-spectrum primary antibody (antibody1, Ab₁), anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody to capture CTCs. Besides, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were coupled with colorectal cancer related antibody as specific antibody 2 (Ab₂) to prepare complex. The sandwich structure consisting of Ab₁-CTCs-Ab₂ was constructed by the microchip for capturing CTCs. And the electrochemical workstation was used to detect and verify its high sensitivity and specificity. Results showed that the combination of immunosensor and micro-nano technology has greatly improved the detection sensitivity and specificity of the immunosensor. And we also verified the feasibility of the immunosensor for clinical blood sample detection, and successfully recognitized detection and quantization of CTCs in peripheral blood of colorectal cancer patients by this immunosensor. In conclusion, the super sandwich immunosensor based on micro-nano technology provides a new way for the detection of CTCs, which has potential application value in clinical diagnosis and real-time monitoring of disease.
Humans ; Nanotubes, Carbon/chemistry* ; Neoplastic Cells, Circulating/pathology* ; Biosensing Techniques ; Immunoassay/methods* ; Antibodies ; Colorectal Neoplasms/diagnosis* ; Electrochemical Techniques/methods* ; Gold/chemistry*

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive and stable biomarker for evaluating DNA oxidative damage. A rapid and sensitive colloidal gold immunochromatographic strip was developed for 8-OHdG detection by a competitive method. The sample pad (glass cellulose film), bonding pad (glass cellulose film), nitrocellulose film and absorbent pad were pasted on the polyvinyl chloride (PVC) base plate to construct the test strip. Colloidal gold (AuNPs) was prepared by the reduction of chloroauric acid with sodium citrate. 8-OHdG antibody (Ab) was coated on the outer layer of AuNPs to form Ab@AuNPs as a probe. Bovine serum albumin (BSA) and 8-OHdG were conjugated with carbodiimide hydrochloride to prepare an artificial antigen, which was used as the coating antigen of detection line. Goat anti mouse polyclonal antibody IgG was used as the coating antibody of control line. The experimental parameters were optimized including the type of nitrocellulose membrane, the formula of loading solution, and the spraying amount of gold labeled antibody. The results showed that the appropriate nitrocellulose membrane was CN 95. The optimal loading solution included BSA (1%), Tween-20 (3%), sucrose (3%) and NaCl (0.9%). The optimal spraying amount of gold labeled antibody was 4 μL. 8-OHdG can be detected by the strip under visible light, and the level of 8-OHdG in urine can be preliminarily determined by comparing the color intensity of T line and C line. The 8-OHdG concentration in urine was further calculated by the gray value of T line and the threshold of detection was 2.55 μg/L. This colloidal gold immunochromatographic strip is simple, rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.
8-Hydroxy-2'-Deoxyguanosine ; Animals ; Antibodies, Monoclonal ; Gold ; Gold Colloid/chemistry* ; Metal Nanoparticles ; Mice ; Sensitivity and Specificity