Immunological properties of a single-chain fragment of the anti-idiotypic antibody ACA125

Vaccination with anti-idiotypic antibodies has been described as a promising concept for treatment of several malignant diseases. The murine monoclonal anti-idiotypic antibody ACA125 imitates a specific epitope of the tumor-associated antigen CA125 expressed by 80% of ovarian carcinomas. In the first clinical trial it could be shown that mAb ACA125 is able to elicit anti-anti-idiotypic antibodies (Ab3) with anti-CA125 specificity in patients with advanced ovarian cancer. In order to improve the capabilities of anti-idiotype vaccines we generated a genetically engineered single-chain fragment (scFv) ACA125 composed of heavy- and light-chain variable regions connected by a flexible linker. The antigenicity of scFv ACA125 was demonstrated by immunizing rats i.p. with scFv or complete mAb in complete/incomplete Freund's adjuvants (CFA/IFA) or precipitated by aluminium hydroxide. Negative control groups included applications of irrelevant mouse IgG or adjuvants alone. Anti-anti-idiotypic antibodies (Ab3) directed against the mAb ACA125 as well as specific anti-CA125 antibodies (Ab1′) could be detected in all animals treated with scFv in CFA/IFA. Nevertheless, antibody titers were lower than when the complete mAb ACA125 was used. Suprisingly, an increase of specificity could not be observed in scFv-immunized animals, which had been expected because of the lack of heavy- and light-chain constant regions that could raise rather unspecific anti-isotypic and anti-allotypic rat anti-(mouse Ig) antibodies (RAMA). In contrast, the RAMA responses detected in these rats were even stronger than those following immunization with complete mAb ACA125. In conclusion, the anti-idiotypic scFv ACA125 alone cannot improve the immunogenic features of the corresponding mAb, but provides a useful tool for the further development of genetic vaccines. Received: 20 January 2000 / Accepted: 24 April 2000     

Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody

Here, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations.     

Variable region primary structures of a high affinity anti-fluorescein immunoglobulin M cryoglobulin exhibiting oxazolone cross-reactivity

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 mouse following hyperimmunization with fluorescein (Fl)-conjugated keyhole limpet hemocyanin, demonstrated a high affinity for Fl (Ka = 2.9 x 10(10) M-1) and cryoprecipitation that was abrogated upon Fl binding to the antibody-combining site. V region sequences of 18-2-3 were determined by Edman degradation and nucleotide sequence analysis. The VH region of 18-2-3 was encoded by a gene VHI(B) of the Q52 VH family with 96% homology to anti-oxazolone antibody NQ7.5.3 but utilized a larger D region (DQ52 plus N region). The V kappa region of 18-2-3 was encoded by a gene V kappa IV with an amino acid sequence 97% homologous to that of anti-oxazolone antibody NQ11.1.18. Although monoclonal anti-Fl antibodies 18-2-3 and 4-4-20 possessed similar binding affinities and quenched bound fluorescein to the same extent (Qmax greater than 96%), they utilized different VH, D, V kappa, and J kappa genes, but the same JH gene segment (JH4). Solid-phase analyses showed that 18-2-3 was not idiotypically related to 4-4-20 and 9-40, prototypic anti-Fl antibodies. Fine specificity binding patterns of Fl analogues by 18-2-3 IgM and IgMs were distinct from other anti-Fl antibodies. Monoclonal antibody 18-2-3 bound phenyloxazolone bovine serum albumin with a lower affinity than for Fl-bovine serum albumin. The first hypervariable region of the 18-2-3 light chain showed homology to human cryoglobulins. This is the first variable region sequence of a murine IgM which self-aggregates at low temperature.     

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of single-chain antibody affinity with temperature-responsive elastin-like polypeptide linkers

Single-chain antibodies are genetically engineered constructs composed of a VH and VL domain of an antibody linked by a flexible peptide linker, commonly (GGGGS)3. We asked whether replacement of this flexible linker with peptides known to undergo environmentally induced structural transitions could lead to antibodies with controlled binding and release characteristics. To this end, we genetically modified and produced a series of anti-fluorescein single-chain antibodies with the general linker sequence (VPGXG)n, where n is 1.2 to 3 and X is Val or His, to evaluate the effects of linker length and composition. Our results indicate that single-chain antibodies containing elastin-like polypeptide linkers have equilibrium affinity (KD) comparable to wild-type (GGGGS)3 at room temperature but altered binding kinetics and faster ligand release as the temperature is raised. These results are consistent with the increased molecular order and contraction that elastin-like polypeptides are known to undergo with increased temperature. Modulation of antibody affinity using stimulus-responsive linkers may have applications in biosensors, drug delivery, and bioseparations.     

Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.     

Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active site structures than mAb 4-4-20.     

Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

Background

Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques.

Results

The sequences encoding the variable regions of heavy (V_H) and light (V_L) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)₃ and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab).

Conclusion

This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.     

Engineering and structural characterization of a linear polyubiquitin-specific antibody

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.     

Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system

Monoclonal antibody A21 reacts specifically with the extracellular domain of p185c-erbB-2 oncoprotein, a member of the epidermal growth factor receptor family. In a previous study, we constructed a single-chain chimeric antibody, assembled using an A21 single-chain Fv antibody and a human IgG1 Fc fragment. In this study, we expressed this chimeric antibody using a CHO-GS system, and developed a simple and efficient method for its purification. After only one step using affinity purification, the recovery rate and purity of the antibody attained was 60 and 91%, respectively. After a second step, using reverse phase HPLC purification, the purity was above 99%. The high purity of the recombinant antibody allowed us to identify a number of its intrinsic molecular properties, including antigen binding activity, measurement of affinity constant, N-terminal sequencing, and mass spectrometer analysis. These results further augment the potential of this recombinant antibody to be a drug candidate for cancer therapy.     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Thermodynamic and structural characterization of an antibody gel

Although extensively studied, protein–protein interactions remain highly elusive and are of increasing interest in drug development. We show the assembly of a monoclonal antibody, using multivalent carboxylate ions, into highly-ordered structures. While the presence and function of similar structures in vivo are not known, the results may present a possible unexplored area of antibody structure-function relationships. Using a variety of tools (e.g., mechanical rheology, electron microscopy, isothermal calorimetry, Fourier transform infrared spectroscopy), we characterized the physical, biochemical, and thermodynamic properties of these structures and found that citrate may interact directly with the amino acid residue histidine, after which the individual protein units assemble into a filamentous network gel exhibiting high elasticity and interfilament interactions. Citrate interacts exothermically with the monoclonal antibody with an association constant that is highly dependent on solution pH and temperature. Secondary structure analysis also reveals involvement of hydrophobic and aromatic residues.     

In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody

 The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of ³²P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate. Received: 20 March 1997 / Accepted: 5 February 1998     

Selective depletion and enrichment of alloreactive cytolytic effector lymphocytes using anti-fluorescein affinity columns

Peritoneal exudates from BALB/c mice rejecting C57BL ascites lymphoma EL4 are a rich source of cytolytic effector lymphocytes (CL); however, these preparations are still contaminated even after removal of adherent cells with other mononuclear cells which do not appear to be cytolytic. The relationship of the cytolytic and “non-cytolytic” cells to graft rejection in vivo is not completely understood. We have used anti-fluorescein (α-FL) columns to separate sensitized lymphoid populations into fractions enriched or depleted in cytolytic activity. EL4 were directly labeled with fluorescein isothiocyanate (FL-EL4), centrifuged with CL and the mixture was applied to a column of horse α-FL antibody conjugated to Sepharose 4B. FL-EL4 and lymphocytes bound to them were retained on the column, while non-bound lymphocytes were collected in a medium wash (passed cells). CL bound to FL-EL4 were then eluted with EDTA (eluted cells). Cytolytic activity of the two fractions was compared to that of the unfractionated population in an in vitro⁵¹Cr release assay. Passed cells were consistently depleted in cytolytic activity compared to unfractionated cells or manipulation controls reaching 100% depletion in some experiments. Enrichment of cytolytic activity in eluted populations was frequently but not invariably observed. Rate of cytolysis was used as a measure of cytolytic activity in fractionated populations. Specificity of binding was investigated in reciprocal experiments using CS7BL/6J effectors raised against BALB/c lymphoma RL♂ 1. Viability of recovered cells was high and the procedure was rapid, efficient and versatile. In contrast to monolayer cellular immunoabsorbents, contamination of fractions with absorbing cells was consistently less than 5%. Both enriched and depleted populations are available for further study of surface markers and function.     

Monoclonal antibody with high affinity for 1,25-dihydroxycholecalciferol

We have developed a monoclonal antibody capable of detecting 1 pg/ml of 1,25-dihydroxycholecalciferol. At a dilution of 1:80,000 of ascitic fluid this antibody has an apparent K_D of 3.3 × 10^?11ML^?1. The immunogen used was a vitamin D analogue, calcitroic acid [1α, 3 β-dihydroxy-9, 10 seco-24-nor 5,7,10 (19) cholatriene-23-oic acid], conjugated to bovine serum albumin. Although this antibody is extremely sensitive, it also recognizes other important vitamin D₃ metabolites.     

Immunological and structural characterization of sarafotoxin/endothelin family of peptides

A highly specific and sensitive radioimmunoassay (RIA) was developed for the potent vasoconstrictor peptides, sarafotoxin-b and human endothelin. The antigenic determinants of the antibodies employed in studies with these assays were found to be localized within the amino acid sequence at positions 4-7. This was confirmed by CNBr cleavage of the methionyl residue at position 6 in the sarafotoxin and at position 7 in the endothelin. The chemically characterized modified peptides showed very low cross reactivity in the RIAs. On the other hand, the binding properties as well as the ability to induce phosphoinositide hydrolysis were very similar in the modified and native peptides, indicating that despite cleavage of the peptide bond the biologically active conformation responsible for either binding or phosphoinositide hydrolysis is retained, probably because of the disulfide bonds. Thus, structural alteration might be a valuable means of curtailing some of the various activities induced by the sarafotoxin/endothelin family of peptides.     

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).     

Preparation and characterization of a single-chain calcineurin-calmodulin complex

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.     

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K_D) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K_D = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.