Screening and identification of single-chain fragment variable against the receptor-binding domain of spike protein of SARS-CoV-2 / 中国生物制品学杂志

@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.

Cytokine release assessment:a good de-risk approach to bi-specific T-cell engagers in non-clinical development / 中国药理学与毒理学杂志

Bi-specific T-cell engagers (BiTEs) show great clinical outcomes for anti-cancer purposes. However, potential cytokine release syndrome (CRS) is notorious to all BiTEs. The mechanism underlying CRS is still not fully known, even though such toxicities are considered to be cytokine release related. Assessment of CRS is a key to non-clinical de-risk programs for BiTEs therapeutic development. In the present review, possible mechanisms are discussed, especially factors contributing to CRS develop?ment. T cell activation may be just an initiation of the CRS cascade, and other cell types can greatly contribute to CRS, such as a chain reaction triggered by downstream B-cells, monocytes, and endothe?lium cells. A non-clinical de-risk program can be designed based on these components in the CRS cascade. Combination of in vitro cytokine release assay, and in vivo mouse and non-human primates studies should be reliable enough to predict and mitigate CRS risk in the clinics. Further more, a good de-risk program should be able to provide ranking for candidates for further development and provide enough confidence to select a first-in-human dose.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Screening and identification of human anti-C3d single chain antibodies / 国际药学研究杂志

Objective: To screen human single chain antibodies against human C3d from phage-displayed single-chain variable fragment(scFv)library, and analyze their binding activities. Methods: The phage display library was exposed to 3-round selections in immunotubes coated with recombinant C3d at a decreasing concentration range and progressively stringent washing conditions. The positive clones were identified by ELISA, followed with sequencing to determine the specific genes which were then cloned intoex-pression vectors. The single chain antibodies were expressed in the FreeStyleTM 293-F system and harvested by affinity purification. The binding activities with recombinant C3d were determined by using Bio-Layer Interferometry technology. Results: These experiments resulted in three novel single chain antibodies(that is, A1, A3 and B6)against C3d with high affinity ranging from 22.7 to 171 pmol/ L. Conclusion: The high affinity human single chain antibodies against human C3d were obtained.

Combination of a single-chain variable fragment JZC00 with 2-deoxyglucose inhibited tumor growth in murine models / 中国药科大学学报

@#This study aimed to investigate the antitumor efficacy of a single-chain variable fragment JZC00 combined with 2-deoxyglucose(2-DG)on murine non-small lung cancer cell and breast cancer cell models. JZC00 was expressed by E. coli and identified using SDS-PAGE and Western blot. The combination inhibited the proliferation of LLC and 4T1 cells. The concentration of glucose and lactic acid in the medium were determined by glucose and lactate kit, respectively, then calculated the tumor cell glucose uptake inhibition rate and lactate release inhibition rate. In vivo, the tumor volume and tumor weight were analyzed after 15-day treatment. The results showed that the molecular weight of JZC00 expressed was correct, and it could inhibit the proliferation of tumor cells in vitro. JZC00 and 2-DG could inhibit the glycolysis of tumor cells, respectively, and JZC00 combined with 2-DG could inhibit glycolysis synergistically. When hypoxic microenvironment was induced in vitro, the inhibition of glycolysis by JZC00 treatment decreased. However, it was reversed with the addition of 2-DG. The in vivo models the combination showed a significantly improved tumor suppressive effect compared with JZC00 treated group, suggesting that 2-DG could improve the anti-tumor effect of anti-angiogenic antibodies and its combination has the potentialial value in the treatment of solid tumors.

Preparation of fusion protein of PD-L1 single-chain antibody and verification of its antitumor activity / 中国免疫学杂志

Objective: Using yeast surface presentation technology, secreted anti-PD-L1 single-chain antibody fragment ( sc Fv), then purify the sc Fv that specifically binds PD-L1 antigen. The sc Fv antibody gene sequence was synthesized based on the single chain antibody gene sequence. We express this sc Fv-mFc protein by using p Fuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of lung adenocarcinoma cells ( A549). Methods: Recombinant plasmid p Fuse-scFv was constructed by gene engineering. The recombinant plasmid p Fuse-scFv was transfected into 293 F ( human embryonic kidney cells) and cultured in serum-free Pro293 a-CDM for 72 hours, then the fusion protein was collected, and use the Rapid Protein Liquid Phase Separation and Purification System to purify the sc Fv-mFc fusion protein. Then the fusion protein and the tumor cells were detected by immunohistochemistry; the affinity of fusion protein and tumor cells was analyzed by flow cytometry; ADCC was used to determine the proliferation of tumor cells in vitro. The nude mice inoculated with lung adenocarcinoma cells, and use the fusion protein to verify its anti-tumor effect in vivo. Results: sc Fv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells; immunohistochemistry and flow cytometry showed that the fusion protein was highly expressed with the surface of PD-L1 protein;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro; the results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor. At the dose of 5 mg/kg, The tumor volume growth rate decreased from 14. 90％ to3. 72％, the two independent samples t test P＜0. 05, the difference was statistically significant. Conclusion: The fusion protein containing single chain antibody was successfully prepared, which had good binding ability to A549 cells and inhibited the proliferation of tumor cells in vitro and in vivo, and provided the laboratory basis for the development of targeted anti-tumor drugs.

Effect of midkine on expression of non-coding RNA in ovarian cancer / 临床检验杂志

Objective@#To explore the correlation of non-coding RNA and the tumor-associated antigen midkine (MK) in SKOV3cells and the clinical significance for diagnosis of ovarian cancer. @*Methods@#The Agilent′s gene chips (miRNAs chip and lncRNAs chip) were used to analyze the differential expression of miRNAs and lncRNAs in both MK-overexpressing SKOV3-MK cells and the control SKOV3-Con cells to screen the potential biomarkers in ovarian cancer. The clinical significance of midkine in the serum and tissues samples was analyzed for the patients with ovarian cancer by quantitative PCR combined with clinical data. @*Results@#Compared with control SKOV3-con cells, MK overexpression significantly promoted the expressions of 11 miRNAs and 7 lncRNAs in SKOV3 cells (P＜0.01, ratio＞3 fold), reduced the expressions of 8 miRNAs and 13 lncRNAs (P＜0.01, ratio＜0.3). Results of qPCR showed that the expression level of miR489 was significantly lower in ovarian cancer tissues than that of the contralateral normal ovarian tissues, while HOTAIR was significantly elevated (P＜0.05). The expression level of HOTAIR in the serum of ovarian cancer patients was significantly higher than that in healthy controls group with same age (0.036±0.024 vs 0.019±0.020, P=0.002). ROC curve analysis of HOTAIR showed that the specificity was 66.7%, the sensitivity was 75.6% and the AUC value was 0.749 as a marker for serum detection of ovarian cancer when the cutoff value was 0.017 6. @*Conclusion@#Long-chain non-coding RNA HOTAIR may be served as a potential biomarker in serum of ovarian cancer patients.

Optimización del algoritmo diagnóstico de anticuerpos antinucleares / Optimization of the diagnostic algorithm of antinuclear antibodies

Introducción: La prueba de anticuerpos antinucleares es una poderosa herramienta en el diagnóstico de las enfermedades reumáticas. Los anticuerpos antinucleares se determinan en el laboratorio por un algoritmo o secuencia que se inicia con prueba de cribado y sigue con la identificación de las especificidades antinucleares más comunes. Pero, ¿cómo interpretar los resultados discordantes entre los dos niveles de estudio de anticuerpos antinucleares? Objetivo: Determinar las especificidades antinucleares menos frecuentes en pacientes positivos de cribado de ANA y negativos de las especificidades más comunes. Métodos: Estudio prospectivo de 88 pacientes consecutivos remitidos para la detección rutinaria de ANA con resultado positivo de cribado por ensayo inmuno-adsorbente ligado a enzima (ELISA) pero negativo de anticuerpos anti-ADN de doble cadena (dc, IgG) y anti-antígenos nucleares extraíbles comunes (ENAc). Las muestras séricas correspondientes fueron evaluadas por inmunofluorescencia indirecta sobre células de carcinoma epidermoide laríngeo humano (IFI-HEp-2) y por ELISA para la detección individual de ANA específicos. Resultados: La prueba de ANA por IFI/HEp-2 resultó positiva en 56/88 (63,6 por ciento) y las especificidades antinucleares se detectaron en 57/88 (64,8 por ciento) muestras, en el orden decreciente de Anti-Nucs: 16/88 (18,2 por ciento); anti-centrómero (CENP-B): 15/88 (17,0 por ciento); -histona: 15/88 (17 por ciento); -PM/Scl: 13/88 (14,8 por ciento); -ADNsc: 11/88 (12,5 por ciento) y -ENAc individuales: 8/88 (9,1 por ciento). La sensibilidad de la IFI-HEp-2 para las especificidades antinucleares fue de 0,83 (IC95 por ciento: 0,72-0,93). De los pacientes negativos de subserología (26/31), 83,9 por ciento no tenían antecedentes de enfermedad reumática asociada a ANA. Conclusiones: La mayoría de los pacientes con resultados discordantes entre el primer y segundo nivel de ANA fueron positivos de especificidades antinucleares menos comunes, pero de reconocido valor diagnóstico(AU)

Introduction: The antinuclear antibody test is a powerful tool for diagnosing rheumatic diseases. Antinuclear antibodies are determined in the laboratory by an algorithm or sequence that starts with a screening test and continues with the identification of the most common antinuclear specificities. But how to interpret the discordant results between the two levels of study of antinuclear antibodies? Objective: To determine the less frequent antinuclear specificities in positive patients of ANA screening and negative of the most common specificities. Methods: A prospective study was done on 88 consecutive patients referred for the routine ANA screening with a positive result of screening by enzyme-linked immunosorbent assay (ELISA) but negative for anti-double-stranded DNA (dc, IgG) and common extractable anti-nuclear antigens (ENAc). The corresponding serum samples were evaluated by indirect immunofluorescence on human laryngeal epidermoid carcinoma cells (IFI-HEp-2) and by ELISA for the individual detection of specific ANA. Results: The ANA test by IFI / HEp-2 was positive in 56/88 (63.6 percent) and the antinuclear specificities were detected in 57/88 (64.8 percent) samples, in decreasing Anti-Nucs order: 16/88 (18.2 percent); anti-centromere (CENP-B): 15/88 (17.0 percent); -histona: 15/88 (17 percent); -PM / Scl: 13/88 (14.8 percent); -ADNsc: 11/88 (12.5 percent) and -ENAc individual: 8/88 (9.1 percent). The sensitivity of IFI-HEp-2 for antinuclear specificities was 0.83 (95 percent CI: 0.72-0.93). No history of rheumatic disease associated with ANA was read in (26/31) 83.9 percent patients with negative subserology. Conclusions: The majority of patients with discordant results between the first and second level of ANA were positive of less common antinuclear specificities, but of recognized diagnostic value(AU)

Desenvolvimento de novos vetores para a produção de bibliotecas de anticorpos pelo sistema do phage display / Development of a antibody display library system targeted against vascular growth factor

Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display

Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system

Agonistic anti-CD40 single-chain antibody enhances anti-tumor effect of mouse NK cells / 中华微生物学和免疫学杂志

Objective To obtain a mouse CD40-specific single-chain antibody (scFv) with high purity and to investigate its in vivo and in vitro agonistic activity on natural killer(NK) cells against tumor. Methods Agonistic anti-mouse CD40 scFv with high purity was obtained by genetic engineering. Mouse dendritic cells (DCs) were treated with different strategies including anti-CD40 monoclonal antibody (McAb),anti-CD40 scFv and negative control. Expression of IL-12 in the supernatants of DC cultures was measured by ELISA. Then DCs and NK cells were co-cultured to obtain activated NK cells,which were co-cultured with T6-17 cells. WST-8 was used to test the cytotoxic effects of NK cells on T6-17 cells. A mouse tumor model was established by injecting BALB/c nude mice with T6-17 cells. Anti-CD40 scFv was injected into tumor to evaluate its inhibitory effect on tumor growth. Levels of IL-12 and IFN-γ in the tumor microen-vironment were detected by ELISA. Changes in the percentages of tumor-infiltrating NK cells and the expres-sion of NKG2D protein(natural-killer group 2,member D) were detected by flow cytometry and immunohis-tochemistry,respectively. Results The recombinant plasmid CD40 scFv-pET28a was confirmed to be con-structed correctly. Results of SDS-page and His-tag Western blot revealed that anti-CD40 scFv could be ex-pressed successfully with a relative molecular mass of 27×103. The level of IL-12 in the supernatant of DC culture of anti-CD40 scFv group was (555.86 ±40.48) pg/ml, which was significantly higher than that of negative control group (P<0.05). The killing ability of NK cells in anti-CD40 scFv group was (72.23 ± 3.99)%,which was significantly higher than that in negative control group (P<0.05). Anti-CD40 scFv significantly inhibited the tumor growth in BALB/c nude mice as compared with negative control group(P<0.05). The levels of IL-12 and IFN-γ in tumor microenvironment of anti-CD40 scFv group were(188.801± 32.718) pg/ml and(121.428±30.994) pg/ml,respectively,which were significantly higher than those in normal saline(NS) group(P<0.05). The positive rate of NKG2D protein and the percentage of CD3-DX5+cells in anti-CD40 scFv group were respectively(8.18±2.01)% and(19.15±2.24)%,which were signifi-cantly higher than those in NS group (P<0.05). Conclusion Agonistic anti-mouse CD40 scFv could en-hance the anti-tumor ability of NK cells by activating DCs.

Expression and functional detection of recombinant immunotoxins anti-B7-H4-scFv/PE38KDEL / 中国免疫学杂志

Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody (scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography (Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease di-gestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.

Application of a single-chain fragment variable (scFv) antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

Background: Hydatid disease is a serious parasitic disease threatening public health. Because of its rarity in non-endemic coastal areas, determining the nature and origin of a chronic, enlarged liver cystic mass is challenging in these regions. Under these circumstances, physicians need a confirmatory diagnostic tool beyond immunological and radiological examinations. This study investigated a novel human single-chain fragment variable (scFv) antibody for the confirmative diagnosis of 18 atypical hydatid disease cases in non-endemic coastal areas. Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P b 0.05). The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

Expression, purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Imaging atherosclerosis model rabbits with 99Tcm-single chain antibody fragment against vascular cell adhesion molecule-1 / 中华核医学与分子影像杂志

Objective To investigate the imaging performance and feasibility of 99Tcm labeled scFv against VCAM-1(99Tcm-scFv-VCAM-1) on atherosclerosis model rabbits.Methods HYNIC was used as a chelator for 99Tcm labeling.The labeling efficiency and radiochemical purity of 99Tcm-scFv-VCAM-1 were measured by instant thin layer chromatography after PD-10 purification.New Zealand white rabbits were employed for establishing atherosclerotic animal models by endothelia immunity injury and high fat diet, and plaques at aorta lesions were examined by HE staining.Model rabbits were sacrificed after administration of 99Tcm-scFv-VCAM-1 at 1 or 2 h respectively, and tissue samples were measured with gamma counter and weighted to obtain in vivo biodistribution data.Planar imaging was performed 1 and 2 h after the injection of 99Tcm-scFv-VCAM-1 to investigate radioactivity of abdominal aorta.After imaging study, atherosclerosis plaque and VCAM-1 expression at aortas were confirmed by the immunohistochemistry (IHC) study.Two-sample t test was used to analyze data.Results 99Tcm-scFv-VCAM-1 was successfully synthesized.Its labeling efficiency was 75%-83%, radiochemistry purity was (98.54±1.03)% and specific activity was 216 MBq/nmol.Atherosclerosis plaque was confirmed at the aortas of experimental rabbits by HE staining, while no plaque was observed in controls.Biodistribution data indicated that the tracer was cleared mainly through the kidneys.Planar imaging showed that the tracer uptake in abdominal aorta of model rabbits was higher than that of control rabbits, the T/B ratios at 2 h of the model group and control group were statistically different (3.68±0.73 vs 2.42±0.39;t=2.950, P<0.05;n=5).Atherosclerosis plaque and high level of VCAM-1 expression were observed at aortas of model rabbits by IHC study.Conclusions It is feasible and effective to detect vulnerable plaques using 99Tcm-scFv-VCAM-1.It may provide a promising way for early diagnosis and accurate evaluation of atherosclerosis.

Radioimmunoimaging of 131I Labeled Human scFv Against Anaplastic Thyroid Carcinoma in Tumor Bearing Nude Mice / 中国医学影像学杂志

Purpose To study the radioactive purity and activity of 131I labeled human single chain variable fragments antibodies (scFv) against anaplastic thyroid carcinoma (ATC),and to explore its distribution and radioimmunoimaging characteristics in tumor bearing nude mice model in vivo so as to provide a new method for anaplastic thyroid carcinoma diagnosis and treatment.Materials and Methods The nude mice model bearing human anaplastic thyroid carcinoma was constructed.The chloramine T method was used to label scFv with 131I and the Sephadex G25M was used for purification of labeled scFv.Labeling rate was determined by trichloroacetic acid method;radiochemical purity,room temperature stability and serum stability were examined using paper chromatography.131I-scFv was injected via tail vein in mice,and the distribution of 131I-scFv in body tissues and organs was analyzed at 12,24,48,72 h after injection.Static SPECT imaging was performed at 12,24,48,72 h after injection to observe the intratumoral accumulation of radioactivity.The SPECT/CT image fusion was performed when the tumor tissues were clearly visible.Results 131I-scFv was purified,and the labeling rate was 91.64％;the radiochemical purity was (93.3 ±0.3)％.The radiochemical purity of 131l-scFv placed at room temperature and the serum for 1,6,12,24 h were all ＞90％.The radioactive distribution of 131I-scFv in tumor,liver,kidney,intestine and blood was high.SPECT imaging showed 131I-scFv was selectively concentrated in tumor tissue;the target/non-target ratio was the highest at 48 h,and the imaging was most satisfactory.Conclusion 131I-scFv can be successfully prepared.SPECT imaging of 131I-scFv in nude mice model is satisfactory,which lays the foundation for further research in ATC diagnosis and treatment.

Screening and characterization of human anti-GPC3 single chain Fv antibody fragment selected by phage display / 药学学报

The aim of the experiments is to screen human single-chain variable fragment (scFv) targeting glypican 3 from phage display library, and analyze its biological activity. After several rounds of panning, the binders with high affinity were obtained through phage ELISA and IMGT analysis. The desired scFv gene was then ligated with pET-22b vector yielding recombinant plasmids, which was then introduced into E. coli Rosetta (DE3). Soluble scFv protein was expressed and further purified using Ni2+ affinity chromatography. The purified proteins were identified by SDS-PAGE and Western blot. Subsequently, the affinity and cell based binding activity were measured using Surface Plasmon Resonance (SPR) and flow cytometry assay, separately. Four enriched sequences with relatively high binding affinity were found (1F7, 1D7, 1D4 and 1B10). We also found that 1F7 scFv showed better targeting ability and higher affinity. The scFv could pave the way for new immunotherapies, such as bispecific anitbody, antibody-drug conjugate and chimeric antigen receptor T-cell immunotherapy cell, etc.

Establishing a genebank for phage single-chain antibody using silicotic rats / 中国职业医学

OBJECTIVE: To establish a genebank for phage single-chain antibody for further screening the specificity of single chain fragment variable( Sc Fv) in lung tissue of silicosis rats by phage display technology. METHODS: Twenty-four specific pathogen free male SD rats were used to construct silicosis model by one-time bronchial perfusion with 1. 0 m L of silicon dioxide suspension( mass concentration,100 g / L). We took periphery blood from 6 rats 3,6,9 and 12 weeks respectively after establishing the model. The peripheral lymphocytes were mixed,and total RNA was extracted using Trizol,and c DNA was synthesized by reverse transcription. The degenerated primers were used to amplify the variable region of heavy chain( VH) gene and variable region of light chain( VL) gene by polymerase chain reaction( PCR).Then VH and VL genes were assembled to form Sc Fv by T4 DNA linker. The cloning recombinant of Sc Fv and plasmid of PCANTAB-5e were transformed into competence E. coli TG1 by calcium chloride. The Sc Fv genebank of silicosis model was constructed by M13K07 helper phage superinfection. There were 10 bacterial colonies for plasmid restriction dualenzyme digestion randomly selected for confirmation. RESULTS: Agarose gel electrophoresis showed that there were two bands of obvious 28 S and 18 S in total RNA of periphery blood lymphocytes of silicosis rats. The total RNA was intact. The size of VH gene fragment was about 400 bp,the size of VL gene fragment was about 350 bp and recombinant Sc Fv gene fragment length was about 750 bp. The helper phage was amplified and placed with double-deck agar plate and observed limpid plaque with the size of a rice grain. The phage titer was 1. 35 × 10~(16) pfu / L. The recombinant plasmids were transformed into E. coli TG1 and total bacterial count was 8. 0 × 10~9 cfu / L in resistant plate. The positive cloned plasmid PCR gel electrophoresis and double enzyme results showed a positive inserting rate of 90. 0%. The capacity of phage single-chain antibody genebank of experimental silicosis was 7. 2 × 10~9 cfu / L. CONCLUSION: The silicosis rat model with phage Sc Fv gnebank could be successfully established,and its capacity and diversity provide support for the follow-up screening.

Combination of scFv-AFP with doxorubicin inhibits ceII proIiferation in hepato-ceIIuIar carcinoma ceII Huh7 / 中国药科大学学报

This study was to investigate the inhibitory effects of single-chain variable fragment of alpha fetopro-tein (scFv-AFP)in combination with doxorubicin on the proliferation of human hepatocellular carcinoma cell lines Huh7.Huh7 cells were treated with different concentration of scFv-AFP or doxorubcin alone or their combi-nation.The inhibitory effects were detected by MTT assay,and cycle arrest and apoptosis of Huh7 cells were ana-lyzed by flow cytometry in different groups using PI and Annexin V /PI-staining respectively.Results showed that scFv-AFP,doxorubicin alone or in combinations dose-dependently inhibited the proliferation of Huh7,and a syn-ergistic effect was observed in their combined action.The combination treatment resulted in significantly higher apoptosis than those in other groups (P <0.05).scFv-AFP (40 μg/mL)markedly blocked the Huh7 cell pro-gression by arresting the cells in the G0 /G1 phase,and the percentage of cells in S phase decreased dramatically (P <0.05);and scFv-AFP combined with doxorubicin blocked the Huh7 cell progression by arresting the cells in G2 /Mphase (P <0.01).

Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.

Desarrollo de una vacuna mejorada de la subunidad del virus de la diarrea viral bovina basada en la glicoproteína E2 fusionada con un anticuerpo de cadena única que se dirige a las células presentadoras de antígenos / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos