Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function

Two internalizing monovalent single chain antibody fragments (scFv), C6.5 and F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, and their bivalent forms dbC6.5 and F5(scFv')(2), were compared to the growth-inhibiting anti-ErbB2 antibody Herceptin/trastuzumab, in either its bivalent (Her) or monovalent (4D5Fab') form, for their abilities to induce biological responses in the ErbB2-overexpressing breast cancer cells, SkBr-3. Assays compared internalization by receptor-mediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK and PI3K signaling pathways. We found no correlation between ErbB2 epitope affinity or valency on degree of antibody-induced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the monovalent or bivalent forms of the internalizing scFv had any sustained effect on cell growth. Basal levels of MAPK and PI3K signaling in SkBr-3 cells were not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3K signals were noted within 8 h of Her treatment. In summary, antibody-induced ErbB2-mediated endocytosis is not a surrogate marker for resultant biological response, as it shows no correlation with cell cycle, culture proliferation, or intracellular kinase signal induction by internalizing antibodies. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in conjunction with their absence of any growth or signaling impact on ErbB2-overexpressing cells favors their choice as ErbB2 targeting moieties for intracellular delivery of novel cancer therapeutics.     

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.     

Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

Expression of functional single-chain variable domain fragment (scFv) antibody against Metolcarb in Pichia pastoris

A Pichia pastoris system was used to express a single-chain variable fragment (scFv) antibody targeted against Metolcarb. The specific scFv gene was amplified from the phage-display scFv library and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C-scFv, was linearized and transformed into P. pastoris strain X-33. A transformant named X-33-Pp-SMW-12-6, which showed strong expression of antibodies, was isolated, and the culture conditions, including methanol induction concentrations, inoculum densities, and pH, were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of the scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against Metolcarb are comparable to those of scFv antibodies produced in E. coli. Recoveries of Metolcarb demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of Metolcarb residue in environmental and agricultural samples using a competitive inhibition enzyme-linked immunosorbent assay. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against Metolcarb for downstream applications.     

Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the US, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.Key words: anti-IgE, E. coli expression, scFv, antibody engineering, human antibodies, allergy diseases, antibody therapeutics     

Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast

We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single‐chain variable fragment (scFv) antibody library was constructed in a yeast two‐hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin‐8 (hIL8) into the yeast two‐hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error‐prone PCR of the scFv sequence followed by additional rounds of yeast two‐hybrid screening. The scFv antibodies of both primary and affinity‐matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.     

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.     

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus

Severe acute respiratory syndrome (SARS) brought aglobal outbreak in spring of 2003 [1–3], and more andmore attention has been paid on it when a new caseresurfaced in Singapore last September [4]. By the endof May in 2003, WHO reported a cumulative total of 8202infected cases with 725 deaths from 28 countries.Because of the high transmission and morality rate ofSARS, scientists in many countries have made theirefforts in studying SARS coronavirus (SARS-CoV)[5, 6]. Several genomes of…     

A novel single-chain Fv antibody for connective tissue growth factor against the differentiation of fibroblast into myofibroblast

This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.     

Selection of tumor-specific internalizing human antibodies from phage libraries

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.     

Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia

The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off‑target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2‑derived Kasumi‑1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML‑selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA''). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.     

Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli

Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.     

Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support

The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock’n’flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by β-cyclodextrin (β-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365 nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized β-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.     

Cell fusion activities of Hantaan virus envelope glycoproteins

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

Expression and purification of two anti-CD19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells

Antibody-targeted liposomal anticancer drugs combine the specificity of antibodies with large payloads of entrapped drugs. We previously showed that liposomal doxorubicin (DXR) targeted via anti-CD19 monoclonal antibodies (mAb) or their Fab' fragments against the B-cell antigen CD19 led to improved therapeutic effects in murine B-cell lymphoma models relative to non-targeted liposomal DXR. We now are examining the use of anti-CD19 single chain fragments of the antibody variable region (scFv) as a targeting moiety, to test the hypothesis that scFv have advantages over full-sized mAb or Fab' fragments. We expressed two different anti-CD19 scFv constructs, HD37-C and HD37-CCH in E. coli, and purified the scFvs using two different methods. The HD37-CCH construct was selected for coupling studies due to its relative stability and activity in comparison to HD37-C. When coupled to liposomes, the HD37-CCH scFv showed increased binding in vitro to CD19-positive Raji cells, compared to non-targeted liposomes. Cytotoxicity data showed that HD37-CCH scFv-targeted liposomes loaded with DXR were more cytotoxic than non-targeted liposomal DXR. Our results suggest that anti-CD19 scFv constructs should be explored further for their potential in treating B-lymphoid leukemias and lymphomas.     

Toward selection of internalizing antibodies from phage libraries

Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).     

Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.     

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine–HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.