Objective To construct a library of human-derived anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) single-chain variable fragments (scFv) and screen for broad-spectrum neutralizing antibodies to identify candidate molecules for the development of diagnostic and therapeutic agents. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of patients who had recovered from novel coronavirus infection. Total RNA was extracted from these PBMCs and reverse transcribed into cDNA, which was used as a template for constructing a human anti-SARS-CoV-2 scFv library. Phage display technology was used to screen for scFv antibodies specific to the SARS-CoV-2 S protein. Full-length IgG antibodies were synthesized through sequence analysis and human IgG expression, and their binding capacity and neutralizing activity against SARS-CoV-2 were evaluated. Results A human-derived scFv antibody library against SARS-CoV-2 with a capacity of 1.56×10⁷ CFU was successfully constructed. Two specific scFv antibodies were screened from this library and expressed as full-length IgG antibodies (IgG-A10 and IgG-G6). IgG-A10 exhibited strong neutralizing activity against both the original SARS-CoV-2 strain (WT) and the XBB subvariant of the Omicron variant. However, the neutralizing activity of this antibody against the JN.1 sub lineage of the Omicron BA.2.86 variant was moderate. Conclusion This study has successfully constructed a human anti-SARS-CoV-2 scFv antibody library from the peripheral blood of recovered patients, and screened and expressed anti-SARS-CoV-2 IgG antibodies with neutralizing activity, laying a foundation for the prevention, diagnosis, and treatment of SARS-CoV-2 infection.
Humans ; Single-Chain Antibodies/genetics* ; SARS-CoV-2/immunology* ; COVID-19/immunology* ; Immunoglobulin G/genetics* ; Antibodies, Viral/genetics* ; Peptide Library ; Spike Glycoprotein, Coronavirus/immunology* ; Antibodies, Neutralizing/immunology* ; Leukocytes, Mononuclear/immunology* ; Broadly Neutralizing Antibodies/immunology*

To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.
Single-Chain Antibodies/immunology* ; Humans ; Tetraspanin 30/immunology* ; Recombinant Proteins/immunology* ; Pichia/genetics* ; Saccharomycetales/metabolism*

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Animals ; Immunoglobulin Variable Region/genetics* ; Immunoglobulin Fragments/metabolism* ; Single-Chain Antibodies/metabolism* ; Peptide Library ; Mammals/genetics*

Objective To investigate the preventive therapeutic effect and possible mechanism of single chain variable fragments chimeric protein (SD) of ovalbumin epitopes internalizing receptor DEC-205 antibody on food allergy in mice. Methods Mice were randomly divided to five groups (control, PBS, scFv DEC 100 μg, SD 50 μg, SD 100 μg) and treated for 24 hours before OVA administration. After challenge, the serum level of OVA-specific IgE, IgG1, IgG2a and IL-4 were detected by ELISA. Infiltration of eosinophils and mast cells in the jejunum was observed by HE staining and toluidine blue staining respectively. The bone marrow of tibia and femur was isolated and cultured to obtain immature dendritic cells(BMDCs), which were further treated with LPS (10 ng/mL), TSLP (50 ng/mL), scFv DEC protein (1000 ng/mL) and SD protein (10,100,1000)ng/mL for 24 hours, and the IL-10 level of supernatant was assayed by ELISA. Results Compared with PBS group, the number of SD-treated mice with diarrhea was markedly reduced. The difference in rectal temperature and the levels of serum OVA-specific IgE, IgG1, IgG2a and IL-4 decreased significantly after prophylactic administration of SD; The number of eosinophils and mast cells in jejunum also decreased significantly while the IL-10 level in the supernatant of BMDCs increased significantly after SD intervention. Conclusion SD mitigates experimental FA response by fosters the immune tolerance property of dendritic cells.
Mice ; Animals ; Ovalbumin ; Interleukin-10 ; Single-Chain Antibodies/genetics* ; Immunoglobulin E ; Epitopes/therapeutic use* ; Interleukin-4 ; Food Hypersensitivity/prevention & control* ; Immunoglobulin G ; Recombinant Fusion Proteins/genetics* ; Mice, Inbred BALB C ; Disease Models, Animal

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.
Carcinoma, Hepatocellular ; Humans ; Liver Neoplasms ; Neoplastic Stem Cells ; Recombinant Proteins/genetics* ; Single-Chain Antibodies/genetics*

Asthma is a common respiratory disease that affects 300 million of people worldwide, posing a serious health risk and medical burden. Development of new anti-asthmatic drugs and alternative treatment regimens is therefore encouraged. Recent studies have shown that Epidermal Growth Factor Receptor (EGFR) is involved in asthma development. In order to construct nanoparticles targeting EGFR for asthma treatment, a single chain antibody fragment (scFv) against EGFR was genetically engineered and modified at the N-terminal end of the human ferritin H-chain (FTH1) to construct Anti EGFR scFv::FTH1/FTH1 nanoparticles. Transmission electron microscopy showed that the nanoparticles were self-assembled into hollow cage-like structures with the particle size of about 12 nm. Semi-quantitative analysis of the purified nanoparticles by SDS-PAGE revealed the mass ratio of FTH1 to Anti EGFR scFv::FTH1 was 7:3. In House Dust Mite (HDM) driven models, Anti EGFR scFv::FTH1/FTH1 nanoparticles efficiently attenuated several key features of asthma, including goblet cell hyperplasia, mucous metaplasia and subepithelial fibrosis, showing the potential of using ferritin based nanoparticle for asthma treatment.
Asthma/drug therapy* ; Ferritins ; Humans ; Nanoparticles ; Oxidoreductases ; Single-Chain Antibodies/genetics*

Objective: To construct a new CD123- specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. Methods: A hybridoma strain (6E11) capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody (mAb) . The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia (AML) cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. Results: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L) . The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(-) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60%. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(-) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells[ (98.60±1.20) %vs (20.28±6.74) %, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells[ (26.33±3.30) %vs (1.17±0.06) %, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11[IL-2: (92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123(+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. Conclusion: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.
Animals ; Cell Line, Tumor ; Humans ; Interleukin-3 Receptor alpha Subunit ; Leukemia, Myeloid, Acute ; Mice ; Receptors, Chimeric Antigen ; Single-Chain Antibodies

Objective: To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. Methods: Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3(+) T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. Results: Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ(2)=11.73, P=0.008). Conclusion: Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future.
Animals ; Antigens, CD19 ; Cricetinae ; Cytokines ; Humans ; Lentivirus ; Mice ; Single-Chain Antibodies ; T-Lymphocytes

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.
Antibodies ; Bacteriophages ; Chickens ; Clone Cells* ; Cloning, Organism ; Complementarity Determining Regions ; DNA ; Escherichia coli ; Immunoenzyme Techniques ; Mass Screening ; Methods ; Polymerase Chain Reaction ; Prostate-Specific Antigen ; Single-Chain Antibodies