Chain Variable Fragment Antibody Research Articles

Machine learning modeling for solubility prediction of recombinant antibody fragment in four different E. coli strains

The solubility of proteins is usually a necessity for their functioning. Recently an emergence of machine learning approaches as trained alternatives to statistical models has been evidenced for empirical modeling and optimization. Here, soluble production of anti-EpCAM extracellular domain (EpEx) single chain variable fragment (scFv) antibody was modeled and optimized as a function of four literature based numerical factors (post-induction temperature, post-induction time, cell density of induction time, and inducer concentration) and one categorical variable using artificial neural network (ANN) and response surface methodology (RSM). Models were established by the CCD experimental data derived from 232 separate experiments. The concentration of soluble scFv reached 112.4 mg/L at the optimum condition and strain (induction at cell density 0.6 with 0.4 mM IPTG for 24 h at 23 °C in Origami). The predicted value obtained by ANN for the response (106.1 mg/L) was closer to the experimental result than that obtained by RSM (97.9 mg/L), which again confirmed a higher accuracy of ANN model. To the author’s knowledge this is the first report on comparison of ANN and RSM in statistical optimization of fermentation conditions of E.coli for the soluble production of recombinant scFv.

Brolucizumab for the treatment of diabetic macular edema.

To review the available data supporting the use of brolucizumab in the treatment of diabetic macular edema (DME). Brolucizumab is a humanized single- chain variable antibody fragment (scFv), the smallest functional subunit of an antibody approved for intravitreal use. Three phase III studies demonstrate that at 52 weeks, brolucizumab has statistically superior anatomical outcomes of reducing retinal thickness (54.0-57.5% of brolucizumab treated eyes achieved central subfield thickness <280 μm compared to 40.1 - 41.4% of aflibercept treated eyes) and retinal fluid (present in 54.2-60.3% of brolucizumab treated eyes compared to 72.9-78.2% of aflibercept treated eyes). Brolucizumab also demonstrated a prolonged durability up to 16 weeks, thus reducing treatment burden. The visual outcomes appear noninferior to current anti-VEGF agents with an increased risk for intraocular inflammatory events (0.3-4.7% compared to 0.6-1.7%). Results from recent phase III trials showing the efficacy and safety of brolucizumab presents an additional therapeutic option in the DME treatment landscape. It can reduce treatment burden in DME with increased inter-treatment intervals while conferring efficacy in both functional and anatomical outcomes. Caution should be taken regarding the risks of intraocular inflammation, retinal vasculitis, and retinal vascular occlusion.

Haemagglutinin antigen selectively targeted to chicken CD83 overcomes interference from maternally derived antibodies in chickens

Maternally derived antibodies (MDAs) are important for protecting chickens against pathogens in the neonatal stage however, they often interfere with vaccine performance. Here, we investigated the effects of MDAs on a targeted antigen delivery vaccine (TADV), which is developed by conjugating H9 subtype avian influenza virus haemagglutinin (HA) antigen to single chain fragment variable (scFv) antibodies specific for the chicken antigen presenting cell receptor CD83. Groups of 1-day-old chickens carrying high levels of MDAs (MDA++) and 14-day old chickens carrying medium levels of MDAs (MDA+) were immunised with TADV (rH9HA-CD83 scFv), untargeted rH9HA or inactivated H9N2 vaccines. Immunogenicity in these vaccinated chickens was compared using haemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISA). The results showed that the TADV (rH9HA-CD83 scFv) induced significantly higher levels of H9HA-specific antibody titres compared to the untargeted rH9HA and inactivated H9N2 vaccines in MDA++ and MDA+ chickens. Overall, the data demonstrates immune responses induced by TADV are not affected by the MDA in chickens.

Construction and evaluation of an antibody phage display library targeting heparan sulfate

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a ‘single pot’ phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

Abstract 2781: "Wheelz": A novel engineered human antibody for possible CAR T-cell therapy

Abstract The purpose of this study is to present a novel engineered human antibody specific to thymidine kinase 1 (TK1). Recent developments in chimeric antigen receptor (CAR) T-cell therapeutic approaches have rocketed into the spotlight in the field of cancer immunotherapy. One of the limitations to CAR T-cell therapy is the need for novel targets on cancer cells. The upregulation of serum TK1 has been shown to be an important biomarker in cancer detection and prognosis. Furthermore, we have previously reported the cell surface expression of TK1 on multiple cancer cell lines and in clinical samples. This data merits an investigation of the use of TK1 as a target for CAR T-cells. We have created an engineered human antibody against TK1. We present “Wheelz,” a single chain fragment variable monoclonal antibody with a constant region (scFv-Fc) to potentially target cancer cells expressing surface TK1. Our engineered antibody was isolated from a yeast display library (donated by the Wittrup lab at MIT) using magnetic sorting and flow cytometry to find the top 0.1% of TK1-binding single chain fragments. We fused the resulting scFv region to a constant region (Fc) fragment through restriction digestion and ligation. We then transformed the construct with a secretion vector into yeast and induced the transformed yeast to produce the antibody, which we called “Wheelz.” We purified the antibody using His-tag. In order to confirm specificity to TK1, we used dot blotting and saw specific binding to purified TK1. We performed a western blot using chemiluminescence imaging. This indicated that the engineered human scFv-Fc binds to TK1 present in normal serum, cancer serum, and cancer cell extract in TK1's monomeric, dimeric, and tetrameric forms and did not bind to any other human cellular proteins. In the past, anti-TK1 scFv antibodies of murine origin were used in CAR experiments with some cytotoxic effect. Because “Wheelz” is an engineered human antibody, it could improve previous models by minimizing immune rejection in the patient. Future research will explore the cytotoxic-inducing effects of “Wheelz” before inserting the antibody into a CAR. We will also explore the specificity and cytotoxicity of eight other scFv regions isolated from yeast display that preliminarily appear to be specific for TK1. The specific binding of the engineered scFv-Fc antibody to cell-surface biomarker TK1 makes it a promising approach in cancer immunotherapy. “Wheelz” has the potential to expand the repertoire of tumor targets in CAR T-cell therapy. Citation Format: Kiara V. Whitley, Edwin J. Velazquez, Kelsey B. Bennion, Brianne M. Kingery, Scott K. Weber, Kim L. O'Neill. "Wheelz": A novel engineered human antibody for possible CAR T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2781.

Abstract 5765: Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody

Abstract Introduction: Biomarkers for early detection, disease stratification as well as treatment strategies are a major area of focus in prostate cancer (CaP) research. The gene fusion involving ERG and the TMPRSS2 gene promoter contributes to expression of ERG in CaP, serving as a biomarker for CaP. This led to the development of monoclonal or polyclonal antibodies against ERG protein for diagnostic or therapeutic purposes. As the use of intact monoclonal antibody is limited due to large size and low cell penetration, we hypothesized that single chain fragment variable (scFv) and single domain antibody (sdAb) known as minibodies will be valuable as therapeutic agents targeting ERG protein. Towards this goal, the present study addresses the following: i) Characterization of variable heavy (VH) and variable light (VL) chain sequences and generation of expression vectors; ii) Assessing the specificity of binding of scFv and sdAb to ERG protein; iii) Functional evaluation of scFv and sdAb in cellsMethods: We generated VH and VL encoding sequences using RNA derived from hybridoma cells secreting anti-ERG monoclonal antibody designated 9FY. scFv-9FY and sdAb-9FY fragments represent scFv and sdAb , respectively were cloned in an eukaryotic and prokaryotic expression vectors. The proteins (scFv and sdAb) were analyzed by western blot and ELISA. The binding of scFv and sdAb to ERG protein in live cells was evaluated by bimolecular fluorescence complementation (BiFC) assay. Biological effects of scFv and sdAb were assessed in HEK293, VCaP and LNCaP cells. Results: Analysis was carried out to determine the primary DNA sequence of scFv fragments. Using the predicted amino acid sequence, we identified CDR1, CDR2 and CDR3 regions. scFv expressed in prokaryotic vector was purified and used for the evaluation of immunoreactivity to ERG by ELISA. The affinity of scFv and sdAb to ERG was demonstrated through their binding to ERG protein in HEK293 cells by using BiFC assay. This involved the generation of chimeric proteins in which scFv or sdAb coding sequences were fused in-frame to the Venus N- or C-terminal segment separated by a flexible linker of 10 amino acids. Minibodies scFv and sdAb, upon introduction into VCaP cells through transfection, showed cytotoxic effects similar to the observations noted with siRNA against ERG.Conclusions: Here we showed that scFv and sdAb minibodies exhibit high affinity for ERG protein. This property enables them to disrupt the functions mediated by ERG in cells. Given the prevalence of ERG overexpression in prostate cancer, the novel agents based on scFv and sdAb have the potential to interfere with the effect or functions of ERG and are likely to be beneficial in the treatment of CaP. Source of Funding: Center for Prostate Disease Research, USU Grant HU0001-10-2-0002, NCI/EDRN Grant ACN12011-001-0, and the NCI Grant R01CA162383. Citation Format: Shyh-Han Tan, Anshu Rastogi, Sreedatta Banerjee, Annie Bagga, Charles Xavier, Ahmed Mohamed, Denise Young, Gyorgy Petrovics, Albert Dobi, Isabell A. Sesterhenn, Jacob Kagan, Sudhir Srivastava, David G. David, Inger L. Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5765.

Functional and binding characterization of a single chain Fv antibody to abscisic acid and conjugated abscisic acid

ABSTRACTIn the present research, we studied the binding characterization of a single chain fragment variable (scFv) antibody S12B8 with abscisic acid (ABA) and conjugated ABA by competitive enzyme-linked immunosorbent assay (ELISA) and in silico molecular docking. The nucleotide sequences of variable light (VL) and variable heavy (VH) were obtained from hybridoma cells (F12B8), which secrete monoclonal antibodies recognizing ABA and conjugated ABA. And then the genes of VL and VH were constructed to a scFvS12B8 with a nucleotide sequence of a flexible linker (G4S)3 by splicing overlap extension PCR (SOE-PCR). The fragment of S12B8 was cloned into pMAL-c2x with a maltose binding protein tag and expressed in Escherichia coli with a calculated molecular mass of 68 kDa. The results of ELISA showed dose-dependent inhibition of the purified recombinant protein S12B8 by ABA, (ABA-ME) and ABA glucose ester tetra acetyl (ABAGE tetra acetyl). The detection limits (10% inhibition) of ABA, ABA-ME and ABAGE tetra acetyl were 19.71, 8.06 and 3.95 μg/ml, respectively. On the other hand, we investigated the interactions between S12B8 and ligands by homology modeling using RosettaAntibody and molecular docking by Discovery studio 2.5 (DS 2.5). The binding energy of ABA, ABA-ME and ABAGE when fused to the S12B8 antibody was −37.1, −69.6 and −112.0 kcal/mol, respectively, which agreed well with our competitive ELSIA results.

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

Generation and immune-characterization of single chain fragment variable (scFv) antibody recognize breast cancer cells line (MCF-7)

Meeting abstracts Phage display technologies were used to produce single-chain antibodies (scFv) against breast cancer cell line (MCF-7). The mouse B cell hybridoma line C3A8 was the starting material, which generates a monoclonal antibody against breast cancer cells (MCF-7). The best candidate

Abstract 2674: Preparation and characterization of scFv antibody against tissue factor (TF) for the delivery of an MRI contrast agent.

Abstract Background: There have been many clinical reports of the increased risk of thrombosis in cancer patients. Tumor cells and tumor vascular endothelial cells express tissue factor (TF) at high levels. This clinical evidence and various biochemical data suggest that TF is involved in the activation of cancer-related blood coagulation. We hypothesized that anti-TF antibody could be a useful tool for drug delivery to solid tumors. Antibodies of various sizes to suit the purpose could be used to deliver a payload to cancers. Here, we prepared an anti-TF single chain variable fragment (scFv) antibody as a potential delivery tool for diagnostic agents. Method: Anti-TF IgG (clone name, 1157) was obtained from a hybridoma that produced IgG with high affinity for TF antigen. Products of the VL and VH genes of 1157 were obtained from the hybridoma cDNA and were constructed in both orientations, i.e. VH-linker-VL and VL-linker-VH. The scFv was produced in Escherichia coli and was purified by using a conventional method. The affinities of anti-TF IgG and scFv for TF protein were analyzed with flow cytometry (FCM). These antibodies were labeled with Alexa647 and given intravenously to mice bearing chemically induced cutaneous tumors with marked similarity to human solid cancers generally in terms of growth rate and stroma. Biodistribution of the antibodies was analyzed with an OV110 in vivo imaging system. The ratio of the concentration of each antibody in the tumor to that in the surrounding normal tissue area (T/N ratio) was calculated. Results and Discussion: In vitro, the scFv gene VH-linker-VL was expressed as inclusion bodies in E. coli, whereas the scFv gene VL-linker-VH was expressed in the cytoplasm. scFv gene construction with VL-linker-VH was therefore more promising than that with VH-linker-VL, because scFv could be obtained from the former without the need to refold the scFv protein. FCM analysis revealed that the anti-TF scFv could bind to TF protein on the membrane of several cancer cell lines. In vivo, the T/N ratios of scFv in the mice 1, 3, and 6 h after injection were 1.02, 1.46, and 1.90, respectively. In contrast, those of the IgG were 1.02, 1.01, and 1.23, respectively. Because a higher T/N ratio can result in clearer contrast of the tumor, the scFv may be more useful than IgG as a molecular imaging tool in the 6 h after injection. Conclusion and Future Plans: Anti-TF scFv accumulated selectively in tumor tissue for 6 h after iv injection. Our next step will be to quantify the ratio of the concentration of scFv in the tumor to that in the plasma (T/P ratio) by using radioisotopes. The scFv may be a useful tool for the delivery of MRI contrast agents. Citation Format: Ryuta Sato, Ryou Tsumura, Toshifumi Obonai, Satoshi Muneoka, Kouhei Tsumoto, Yoshikatsu Koga, Masahiro Yasunaga, Yasuhiro Matsumura. Preparation and characterization of scFv antibody against tissue factor (TF) for the delivery of an MRI contrast agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2674. doi:10.1158/1538-7445.AM2013-2674

Molecular Breeding of Solasodine Glycoside for Solanum khasianum using Anti-Solamargine Single-Chain Fv Gene

A recombinant single chain fragment variable (scFv) antibody was constructed from hybridoma cell lines expressing anti-solamargine (As) monoclonal antibody (MAb). The characteristics of the As-scFv protein, expressed in both recombinant Escherichia coli and hairy root, were almost same with those of the original MAb. Up to 220 ng recombinant As-scFv was produced per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The solasodine glycosides concentration was 2.3 fold higher in the transgenic, than in the wild-type hairy root, reflecting from the soluble As-scFv level. The recombinant S. khasianum plant regenerated from recombinant hairy roots contained 2.3 fold concentration of solasodine glycoside, compared to the regenerated plant from wild-type hairy root. These results suggested that the scFv antibody expressed in transgenic plant controlled the antigen level. This methodology is the first success for molecular breeding of secondary metabolites, without understanding of biosynthetic enzyme.

EFFICIENT RESISTANCE TO POTATO VIRUS Y INFECTION CONFERRED BY CYTOSOLIC EXPRESSION OF ANTI-VIRAL PROTEASE SINGLE-CHAIN VARIABLE FRAGMENT ANTIBODY IN TRANSGENIC POTATO PLANTS

The production of transgenic potato plants that express anti-Potato virus Y (PVY) single chain variable fragment (scFv) antibodies directed against a viral protease is reported. Complementary DNA encoding the recombinant anti-PVY NIa protease antibody was transferred to potato plants (cvs Belle de Fontenay, Claustar and Nicola) and inserted downstream of the Tobacco mosaic virus (TMV) O leader sequence in order to increase translation efficiency. Protein expression in transgenic plants was verified by Western blot and its subcellular localization by immunocytochemical analyses. The intracellular accumulation of scFv in protoplasts from transgenic potato leaves was assessed. Transgenic potato plants were transferred to a greenhouse and mechanically inoculated with PVYO. Transgenic potato plants expressing anti-PVY protease scFv antibodies in the cytosol led to complete resistance against the target virus.

Baculovirus display of single chain antibody (scFv) using a novel signal peptide

BackgroundCells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.ResultsTwo versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.ConclusionExpression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.

Abstract 3365: Identification of target antigens on breast cancer stem cells using phage-developed scFv antibodies: A Proteomic Approach

Abstract The term ‘cancer stem cell’ has only recently emerged, and is defined as a cancer cell that has the ability to self-renew and give rise to another malignant stem cell, as well as differentiate to give rise to the phenotypically diverse non-tumorigenic cancer cells. The ‘cancer stem cell’ hypothesis emerged from the fact that tumors contain a heterogeneous mixture of cells and that not all the cells within a tumor contribute to tumor growth. Using phage display technology, our previous studies have identified two single chain variable fragment (scFv) antibodies (clones 207, 208) that were found to specifically bind human SUM159 breast cancer stem cells (BCSC). We have also shown in a murine xenograft tumor model prepared from the implanted SUM159 cells that these scFv antibodies predominantly bind to the cells located at the periphery of the tumors. Taking in light of these discoveries, the next logical question to be investigated was, “What cellular-antigens are these scFvs binding to?” In order to answer this in the present investigation, we used proteomic approaches based on a small scale immunoprecipitation, followed by purification and mass-spectrometry-based identification of the binding target antigens. A pure population of BCSC was acquired by flow cytometry, using ALDEFLUOR®-based sorting of the SUM159 cell line. Cell lysates of the sorted BCSC were prepared and the target antigens were immunoprecipitated with scFv-207 and scFv-208 antibodies using a solid-state immunoprecipitation assay. The immunoprecipitated targets were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The target antigens were purified and mass-spectrometric techniques (LQT-MS/ESI mass-fingerprinting) were employed to compare the proteomic data to protein databases. The details of the recovery of peptide-based target antigens and their identification using the proteomic approach as outlined above, will be presented and discussed. The identification of BCSC-specific targets will be a crucial step in designing and developing better and more specific antibody-based therapies against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3365.