Development of single-chain Fv of antibody to DNA as intracellular delivery vehicle

Application of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in nuclei as well as cytoplasm of L929 cells. Overall, the G2-6 single-chain variable fragment might be a candidate vehicle which could be used to deliver specific genes or biomolecules for therapy or diagnosis into the cytoplasm or cell nucleus.     

Symmetry of binding sites of a mouse IgA myeloma protein (MOPC 315)

We have investigated the mechanism of monovalency of the 7S subunit of a mouse IgA myeloma protein (MOPC 315) against a large antigen. This subunit, although it clearly can bind two molecules of a small hapten, fails to precipitate or hemagglutinate the relevant multivalent antigen. In an equilibrium Farr assay, we have shown that the subunit has only one valence for a univalent 40,000 molecular weight antigen (dinitrophenyl-dextran). We have investigated how various levels of affinity labeling quantitatively affect (a) the valence observed in the equilibrium Farr assay against a large antigen, and (b) the binding of the MOPC 315 to an insoluble antigenic matrix. Our results indicate that the Fab regions of the 7S subunit are arranged symmetrically and that the inactivity of one of them toward a large antigen is probably due to steric hindrance caused by the antigen bound to the adjacent site.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Rapid Purification of a New Humanized Single-chain Fv Antibody／Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

Overexpression of the proto-oncogene c-erbB-2, neuor HER2 has been shown in many human tumor cells,especially in breast cancer cells [1–5], which is thought tobe important in human carcinogenesis. c-erbB-2 geneencodes a 185 kD transmembrane glycoprotein …     

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V_L). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K _a) for hu/muCC49 and muCC49 constructs were 1.1 × 10⁶ M⁻¹ and 1.4 × 10⁶ M⁻¹ respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Received: 29 December 1999 / Accepted: 4 February 2000     

Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy

Field pea (Pisum sativum L.) appears well suited for the production of high-value molecules such as recombinant antibodies, with well-established agricultural practices world-wide and seeds that are easily stored and distributed. In order to evaluate the suitability of this grain legume for the production of biologically active antibodies, we transformed peas with a cDNA encoding the single-chain Fv fragment scFvT84.66. This scFv is derived from the monoclonal antibody T84.66, which recognises the well-characterised tumour-associated carcinoembryonic antigen. The antibody is useful for in vitro immunodiagnosis and in vivo imaging of human cancers. We expressed scFvT84.66 cDNA under the control of the seed-specific legumin A promoter. We targeted the antibody to the endoplasmic reticulum for better stability and high accumulation. Transgenic plants produced up to 9 g per gram fresh weight of functional scFvT84.66 in their seeds. The transgene was stably inherited and expressed in the progeny, and the antibody remained active after storage in dried transgenic seeds for two months at room temperature. Our results demonstrate the suitability of grain legume seeds to produce biologically active recombinant antibodies, and the utility of field pea seeds as production vehicles for recombinant pharmaceutical macromolecules.     

羊朊毒体单抗结合表位分析

通过分段表达PrP核心片段和人工合成多肽,分析5株羊朊毒体单抗结合表位。分段表达PrP核心片段,通过PCR方法扩增目的片段,经酶切、连接后,将目的片段插入质粒pET32a,在大肠杆菌BL21中表达。将表达的系列融合蛋白与单抗进行免疫转印试验,根据反应情况确定单抗结合的大致部位,在此基础上设计合成多条针对性多肽,用ELISA方法进一步确定3株单抗的结合部位;通过与6段融合蛋白反应证明5株单抗的结合部位分别为:2H3在199aa～213aa之间,4C6、5F11和7F11在139aa～168aa之间,7F1在214aa～227aa之间,与3段人工合成多肽进行ELISA反应进一步得到4C6、5F11和7F11抗原结合表位在149aa～158aa之间;本研究确定了5株单抗在PrP分子上的结合部位,为羊痒病和牛海绵状脑病的检测、发病机制的研究奠定了基础。     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.     

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD)

The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of “human Abs” based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.     

Development and characterization of a recombinant chicken single-chain Fv antibody detecting <Emphasis Type="Italic">Eimeria acervulina</Emphasis> sporozoite antigen

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V_H and V_L genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l^–1. The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V_L genes suggested that the gene conversion with V pseudogenes might contribute to the diversification of V_L genes in chickens.     

Actin interacts with CCT via discrete binding sites: a binding transition-release model for CCT-mediated actin folding

The chaperones prefoldin and the cytosolic chaperonin CCT-containing TCP-1 (CCT) guide the cytoskeletal protein actin to its native conformation. Performing an alanine scan of actin, we identified discrete recognition determinants for CCT interaction. Interestingly, one of these is similar and functional in the non-homologous protein Cdc20, suggesting that some of the binding information in the CCT target proteins is shared. The information in actin for recognition by CCT and for folding is different, as all but one of the mutants in the recognition determinants are folding-competent. In addition, some other actin mutants remain CCT-arrested and are not released in a native conformation, whereas others do fold but remain bound to CAP. Kinetic experiments provide evidence that CCT-mediated folding of non-native actin occurs in at least two steps, in which initially the recognition determinant 245-249 contacts CCT and the other determinants interact at later stages. Actin mutants that are CCT-arrested demonstrate that some regions neighbouring the recognition determinants are involved in modulating the correct folding transitions of actin on CCT, or its release from this chaperonin. Further, we found that the ATP binding of actin is not a prerequisite for its release, and we suggest that CAP may be involved in charging the nucleotide. Based on the kinetics of CCT binding and folding of actin and actin mutants, we propose a multi-step recognition-transition-release model. This also implies that the currently accepted notion of CCT-mediated actin folding is probably more complex.     

From fluorescence polarization to Quenchbody: Recent progress in fluorescent reagentless biosensors based on antibody and other binding proteins

Recently, antibody-based fluorescent biosensors are receiving considerable attention as a suitable biomolecule for diagnostics, namely, homogeneous immunoassay and also as an imaging probe. To date, several strategies for “reagentless biosensors” based on antibodies and natural and engineered binding proteins have been described. In this review, several approaches are introduced including a recently described fluorescent antibody-based biosensor Quenchbody, which works on the principle of fluorescence quenching of attached dye and its antigen-dependent release. The merits and possible demerits of each approach are discussed. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

Engineering a single-chain Fv antibody to alpha v beta 6 integrin using the specificity-determining loop of a foot-and-mouth disease virus

The αvβ6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-β1 and transforming growth factor-β3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O₁ British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for αvβ6, and we recently reported that a peptide derived from VP1 exhibited αvβ6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to αvβ6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to αvβ6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with αvβ6 but not with α5β1, αvβ3, αvβ5, αvβ8 or CEA. B6-2 was internalized into αvβ6-expressing cells and inhibited αvβ6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of αvβ6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block αvβ6-mediated cancer cell invasion or to deliver and internalize toxins specifically to αvβ6-expressing tumors.     

The ligand binding site of the synaptosomal choline transporter: A provisional model based on inhibition studies

A topographic model of the ligand binding site of the choline transporter was deduced from inhibition studies with the help of CPK molecular models. It is posited that there are two identical or closely similar hydrophilic anionic sites separated from each other by an hinged, essentially planar but conformationally flexible cationic hydrophobic domain. Subsequently to attachment of external choline to either one of the anionic sites, both sites cooperate in enveloping the ligand by a Venus fly-trap mechanism. This leads to rapid configurational changes by which the closed-liganded form of the transporter opens up to the interior to release the bound choline. Intracellular K⁺, a ligand for the choline-binding site, is proposed to be countertransported by a reversal of the above mechanism.Special issue dedicated to Dr. Morris H. Aprison.     

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3x10(-7) M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.     

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)