Single-chain Fv Antibody Fragment Research Articles

Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization.

Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies’ initial properties after storage, without any drop in affinity for any of the tested antibody clones.

Anti-EGFR Single-Chain Fv Antibody Fragment Displayed on the Surface of Ferritin H-Chain Protein Nanoparticle for Asthma Therapy.

Epidermal growth factor receptor (EGFR)-dependent signaling contributes to the pathophysiology of asthma. However, these findings have not been translated into a clinical application. We recently generated ferritin H-chain protein (FTH1)-based nanoparticles with an anti-EGFR single-chain Fv (anti-EGFR scFv) on the surface of FTH1, namely, anti-EGFR scFv-FTH1/FTH1 nanoparticles. In the present study, we found that these nanoparticles could specifically bind to EGFR-expressing cells, leading to downregulation of EGFR and mucin 5AC (MUC5AC) protein expression and growth suppression of House Dust Mite (HDM)-stimulated human bronchial epithelial 16HBE and lipopolysaccharides (LPS)-activated murine macrophage-like RAW264.7 cells. In vivo, intraperitoneal administration of anti-EGFR scFv-FTH1/FTH1 nanoparticles, but not FTH1 nanoparticles, alleviated the major pathological symptoms including airway hyperresponsiveness, airway inflammation, goblet cell hyperplasia, mucus hyperproduction, and increased release of Th2 cytokines in an allergen ovalbumin (OVA)-induced asthma mouse model. Importantly, during the dosing period these nanoparticles were safe for both heathy and asthmatic mice, and more effective in controlling airway inflammation than cetuximab, an EGFR monoclonal antibody. Altogether, our studies provide insights into the control of airway inflammation for treatment of asthma by targeting EGFR. The similar strategy can be used to fabricate scFv-based recombinant protein nanoparticles for other clinical applications.

Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering.

Depending on the linker length between the V and the V domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin

ABSTRACTGranulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of intratumoral injection of recombinant granulysin using in vivo models of breast cancer and multiple myeloma. In the present work we have developed a granulysin gene fusion to the anti-carcinoembryonic antigen (CEA/CEACAM5) single chain Fv antibody fragment MFE23. Both granulysin and the granulysin-based immunotoxin were expressed in Pichia pastoris. The immunotoxin specifically recognized CEA, purified or expressed on the cell surface. Moreover, the bioactivity of the immunotoxin against several CEA+ cell lines was higher than that of granulysin alone. Granulysin and the immunotoxin were tested as a treatment in in vivo xenograft models in athymic mice. When injected intratumorally, both granulysin and the immunotoxin were able to inhibit tumor growth. Furthermore, systemic administration of the immunotoxin demonstrated a decrease in tumor growth in a CEA+ tumor-bearing mouse model, whereas granulysin did not exhibit a therapeutic effect. This is the first granulysin-based immunotoxin and the present work constitutes the proof of concept of its therapeutic potential.

Cancer cell killing by target antigen engagement with engineered complementary intracellular antibody single domains fused to pro-caspase3

Many tumour causing proteins, such as those expressed after chromosomal translocations or from point mutations, are intracellular and are not enzymes per se amenable to conventional drug targeting. We previously demonstrated an approach (Antibody-antigen Interaction Dependent Apoptosis (AIDA)) whereby a single anti-β-galactosidase intracellular single chain Fv antibody fragment, fused to inactive procaspase-3, induced auto-activation of caspase-3 after binding to the tetrameric β-galactosidase protein. We now demonstrate that co-expressing an anti-RAS heavy chain single VH domain, that binds to mutant RAS several thousand times more strongly than to wild type RAS, with a complementary light chain VL domain, caused programmed cell death (PCD) in mutant RAS expressing cells when each variable region is fused to procaspase-3. The effect requires binding of both anti-RAS variable region fragments and is RAS-specific, producing a tri-molecular complex that auto-activates the caspase pathway leading to cell death. AIDA can be generally applicable for any target protein inside cells by involving appropriate pairs of antigen-specific intracellular antibodies.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli.

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.

Single-step colony assay with autoinduction of scFv expression for the screening of antibody libraries.

We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG.

Abstract 4173: Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Survival rates are dismal, at only 8% five year survival. Radiation is one treatment option for PDAC, although historically its efficacy has been controversial. Our research goal is to improve the efficacy of radiation for PDAC patients. An ancillary study of the recent LAP07 trial showed that patients with neutrophilia were more resistant to chemoradiation. Additionally, when patients with systemic inflammation were excluded, one year local control was 80% in the chemoradiation arm compared to 54% in the chemotherapy arm. The EphB4 receptor and ephrinB2 ligand have been shown to play a role in neutrophil mobilization from the bone marrow and infiltration into melanoma tumors. We hypothesize that inhibition of EphB4-ephrinB2 signaling will enhance the efficacy of radiation in PDAC by reducing inflammation by neutrophils. Materials/Methods: To study tumor growth delay, Athymic nude mice were implanted in the flank with a patient derived xenograft (PDX) model of PDAC. To inhibit EphB4-ephrinB2 signaling, mice were administered with 20mg/kg of B11, a human single chain Fv antibody fragment against ephrinB2. Tumors were irradiated with 12Gy and volume was monitored by caliper. For mechanistic studies, C57/BL6 mice were injected in the flank with 10^6 FC1242 cells, which are derived from the pancreata of KPC mice. To inhibit EphB4-ephrinB2 signaling, mice received a hydrodynamic tail vein injection of 20ug/2mL TNYL-RAW-Fc plasmid. TNYL-RAW is a peptide antagonist of EphB4, and it is fused to human Fc. Tumors were irradiated with 12Gy and harvested four days later for flow cytometry analysis. Blood was also collected at various time points and analyzed by blood film white blood cell differential and flow for neutrophils. Plasmid transduction efficiency was confirmed by analyzing plasma samples collected at different time-points on an Fc protein-based ELISA assay. Results: We observed a significant tumor growth delay in PDX implanted mice who received B11 and radiation. An ELISA for human Fc confirmed transduction of the TNYL-RAW-Fc plasmid in FC1242 tumor bearing mice. Flow analysis on FC1242 tumors showed a significant decrease in percentage of infiltrating neutrophils when treated with TNYL-RAW and radiation, and blood film white blood cell differential and flow analysis on blood showed a significant decrease in circulating neutrophils in the same mice. Conclusions: Our findings support the hypothesis that EphB4-ephrinB2 signaling contributes to inflammation by neutrophils in PDAC, and inhibiting this signaling will increase the efficacy of radiation. This data can contribute toward a clinical trial design for combination therapy of EphB4-ephrinB2 inhibition and radiation in patients with pancreatic ductal adenocarcinoma. Citation Format: Shelby Lennon, Shilpa Bhatia, Ayman Oweida, Jaspreet Sharma, Andy Phan, Sanjana Bukkapatnam, Nomin Uyanga, Todd Pitts, Stephen Leong, Karyn Goodman, David Raben, Elena Pasquale, Jorge Martinez-Torrecuadrada, Sana Karam. Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma [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 4173.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours.

Aim of the studyThe arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells.Materials and methodsAs a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized.ResultsWe showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology.ConclusionsWe report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

A sandwich-type ELISA for the detection of Listeria monocytogenes using the well-oriented single chain Fv antibody fragment

Listeria monocytogenes (LM) poses a serious threat to public health. The present study details the development of a sandwich-type enzyme-linked immunosorbent assay (S-ELISA) for the detection of LM using well-oriented single chain Fv antibody fragment (scFv) and a polyclonal antibody. The anti-LM scFv, used as capture antibody, was gene-optimized and expressed with a C-terminal PS-tag in Escherichia coli BL21(DE3) for oriented-immobilization on hydrophilic polystyrene microtiter plates. The anti-LM rabbit polyclonal antibody, used as the detection antibody, was prepared using heat-killed LM cells as an immunogen. The developed S-ELISA showed great specificity to LM and had a detection limit of lg 6.5 CFU/mL, which was better than the control S-ELISA using non-oriented scFv as the capture antibody. When the S-ELISA was applied to pork and pasteurized milk samples spiked with LM, strong responses were observed. In conclusion, the developed S-ELISA shows great potential for the rapid detection of LM.

Linker length effects in the conformation of M3 anti-CEA single chain Fv antibody fragment

Linker length effects in the conformation of M3 anti-CEA single chain Fv antibody fragment

Measuring Protein-Protein Interactions Using Biacore.

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay setup while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.

Targeted heat shock protein 72 for pulmonary cytoprotection.

Heat shock protein 72 (HSP72) is perhaps the most important member of the HSP70 family of proteins, given that it is induced in a wide variety of tissues and cells to combat stress, particularly oxidative stress. Here, we review independent observations of the critical role this protein plays as a pulmonary cytoprotectant and discuss the merits of developing HSP72 as a therapeutic for rapid delivery to cells and tissues after a traumatic event. We also discuss the fusion of HSP72 to a cell-penetrating single-chain Fv antibody fragment derived from mAb 3E10, referred to as Fv-HSP70. This fusion construct has been validated in vivo in a cerebral infarction model and is currently in testing as a clinical therapeutic to treat ischemic events and as a fieldable medical countermeasure to treat inhalation of toxicants caused by terrorist actions or industrial accidents.

Immunoliposomes for Targeted Delivery of an Antifibrotic Drug.

Excessive extracellular matrix formation in organs and tissues arises from an imbalance between the synthesis and degradation of matrix proteins, especially collagen. This condition interferes with proper wound healing and regeneration, and to date, no specific treatment is available. In the present study, we propose a targeted drug delivery system consisting of cell-specific immunoliposomes (ILs) loaded with deferoxamine (DFO) as an antifibrotic drug. ILs were functionalized with polyethylene glycol (PEG) to improve the steric stability and prolong their half-life. In addition, a single-chain Fv (scFv) antibody fragment that specifically targets fibroblast activation protein (FAP) was incorporated. An in vitro fibrosis model was employed to test this construct. This model consisted of highly activated pro-fibrotic fibroblasts with 2- to 6-fold induction of selected fibrosis markers: cell/matrix deposited collagen I, total soluble collagen, and α smooth muscle actin. The activation was accompanied by a significant and cell-specific elevation of FAP expression and activity, thereby confirming that FAP is an adequate target for antifibrotic drug delivery. Purified anti-FAP scFv was shown to bind specifically to these cells without influencing the FAP enzymatic activity. DFO was demonstrated to have a dose-dependent antifibrotic activity as quantified by collagen deposition. Specific binding and intracellular uptake of DiI-labeled ILs into the activated fibroblasts were shown by flow cytometry and microscopy. Finally, DFO-loaded ILs targeted to FAP caused a significant reduction in the collagen deposition, whereas no effect was observed using liposomes that lacked the targeting antibody fragment. These results suggest that the FAP-specific scFv-conjugated liposomes have considerable potential for cell-specific targeting applicable as a therapy for excessive collagen deposition during fibrosis. In general, through liposome encapsulation, bioactive molecules, such as DFO, that have broad effects and poor cell penetration can be converted into cell-specific composites for targeted drug delivery.

A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy

Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli.

Aflatoxin B1 (AFB1) is a toxic secondary metabolic product, which threatens human and animal health. Antibody is a key factor for immunoassay against toxic stuff like AFB1, and single-chain Fv antibody fragment (scFv) has become a popular format of genetically engineered antibody. In this study, four hybridoma cell lines against AFB1 were obtained, and then scFvs 2E6 derived from hybridoma cell line 2E6 were constructed in different V(H)/V(L) orientations. Subsequently, scFvs 2E6 were expressed in E. coli BL21(DE3) mainly in the form of inclusion body. SDS-PAGE, Western blot and ELISA were employed to characterize scFvs 2E6. The results revealed that the yield of inclusion body of scFvs 2E6 in either V(H)/V(L) orientation was similar; however, only the scFv in V(H)-linker-V(L) orientation showed anti-AFB1 bioactivity after refolding. The present study underscores the importance of choosing optimal V(H)/V(L) orientation for scFv construction, and scFv may be favorable for immunoassays in food industry.

Engineered Antibody Therapies Coming of Age for Aging Brains

Engineered Antibody Therapies Coming of Age for Aging Brains

Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells.

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy.