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Enhancement of the Stability of Single Chain Fv Molecules with the Amino Acid Substitutions Predicted by High-Performance Computer
by Tyuji Hoshino, Keisuke Matsuo Graduate School of Pharmaceutical Sciences, Chiba University, Japan
Jun Komano Department of Clinical Laboratory, Nagoya Medical Center, Japan
Fumiko Maeda & Masataka Takekoshi Department of Molecular Life Science, Tokai University School of Medicine, Japan


A single chain variable fragment (scFv) is a kind of fusion protein keeping the function of immunoglobulin, i.e., high affinity and high specificity to an antigen. ScFv usually consists of variable regions of immunoglobulin, composed of heavy (H) and light (L) chains, and a short linker connecting the regions. Because mass-weight of scFv is smaller than that of antigen-binding fragment (Fab), the possibility of the entry of engineered scFv molecules into cells has been widely examined [1, 2]. The clinical application of scFv is one of the potent and key technologies in the field of biomolecule-based therapy [3, 4].

Because the number of amino acid residues composing scFv is about half of that of Fab, the binding force between H- and L-chains of scFv is weaker compared to that of Fab. The weak H-L binding of scFv sometimes causes the instability of the protein. The structural instability leads to the misfolding of scFv, which results in the decrease of affinity with antigen and also the lowering of expression level. Accordingly, enhancing the structural stability of scFv is beneficial for the broad use of scFv.

The cell surface glycoprotein CD4 plays an important role in the cellular signal transduction and human immunodeficiency virus type 1 (HIV-1) infection in T lymphocytes. Anti-CD4 monoclonal antibody is known to suppress the replication of HIV-1 and also have a high potential for therapeutic applications. Takekoshi et al. identified an anti-CD4 monoclonal antibody labelled as HO538-213 [5], using the oligoclonal library that consisted of anti-CD4 antibody-producing cells of peripheral lymphocytes from a healthy individual [6]. The therapeutic potential of HO538-213 was already suggested by a cell-based assay.

Exemplifying a scFv molecule derived from HO538-213, we carried out a designed protein modification. By introducing amino mutations at the interface between H- and L-chains of the scFv, we tried to enhance the structural stability of scFv. The mutations to be introduced into the protein were determined by molecular calculation with high performance computer. The scFv mutants were expressed in E. coli in a Glutathione S-transferase (GST)-conjugated form and purified by GST-affinity chromatography. The activity of the purified scFv was confirmed by enzyme-linked immunosorbent assay (ELISA) and isothermal titration calorimetry (ITC).

II. Method

II-1 Computational prediction of effectual amino mutations

Since no crystallographic study was performed for HO538-213, the details in molecule structure is unknown for the protein, particularly for the loop structures of the variable regions. Hence, a model for HO538-213 scFv was built by homology modeling (Figure 1(a)) with referring a crystal structure of a variable fragment for lysozyme; PDB code: 1C08. One of the approaches to stabilize the scFv will be enforcing the H-L contact. The residues positioned at the interface were extracted by measuring the minimum distances of the respective residues from their opposite chain. Totally 32 residues, 16 of H-chain and 16 of L-chain, were selected for computational analysis for virtual mutation. Each of the 32 residues was converted to another kind of amino residue other than Cys, that is, 18 kinds of mutations were possible for each residue. Accordingly, 32 × 18 = 576 derivative models were generated from the original HO538-213 scFv (Figure 1(c) & (d)).

The binding force between H- and L-chains for every derivative model was calculated by in-house software, Orientation. The computation was carried out using the FX-10 super-computer system at the University of Tokyo. In FX-10 system, 192 calculations can be executed in parallel in a normal run. Therefore, all the computation was finished within a week.

II-2 Expression and purification of scFv

A DNA coding for the scFv was cloned into pGEX-6p-2 expression vector. The E. coli strain, Origami B, transformed with a pGEX-6p-2 vector containing scFv gene was cultured in 300 mL LB medium. The protein was expressed at 20 °C for 48 hours after induction at an OD600 value of 0.8~1.0 with 0.15 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Bacterial cells collected with centrifugation was re-suspended and disrupted by French-press. After removing unnecessary disrupted fragments from lysate by centrifugation, the expressed protein was obtained from the supernatant. The protein was purified by a glutathione sepharose column with a step-wise rise of the concentration of reduced glutathione. The eluted protein fraction was dialyzed overnight and then GST-tag was cleaved by HRV 3C protease. The cleaved protein was again purified by the glutathione sepharose column to remove the cleaved GST-tag, the remaining uncleaved protein, and HRV 3C protease.

II-3 Measurement of scFv activity

The activity of the purified scFv was checked by enzyme-linked immunosorbent assay (ELISA) using recombinant human CD4 as an antigen. Since HA-tag was linked at the C-terminal side of the scFv, the scFv binding to CD4 was detected by anti-HA secondary antibody conjugated with horseradish peroxidase (HRP) enzyme. The amount of the secondary antibody bound to scFv was monitored by tetramethylbenzidine (TMB) ELISA substrate.

The binding affinity of the scFv was also confirmed by ITC measurement. A 14-mer peptide, TYIAEVEDQKEEVQ, which corresponds to the residues 106-119 of CD4 D1 domain, was synthesized and used for antigen. The purified scFv was contained in the sample cell in a concentration of 8.1 μM and the solution containing 90 μM peptide was titrated from a syringe.

III. Results & Discussion
III-1 Potent amino acid mutations

The computational binding forces of the H-L contact for 576 derivative models were compared to that of the original scFv model. The comparison indicated that 92 models of 576 showed higher affinity than the original one. In particular, the binding affinity was greatly increased in 15 models. The amino mutations in these 15 models were H:V37W, H:G44F, H:G44K, H:G44R, H:G44Y, H:C102F, H:C102Q, H:C102T, H:C102W, H:C102Y, L:A43D, L:A43K, L:A43N, L:A43R, L:A43S in Kabat numbering. Accordingly, 4 residues, H:V37, H:G44, H:C102 (this residue is located at the last of complementarity determining region (CDR) H3 in HO538-213), L:A43, were predicted to be potent residues to be converted (Figure 1(b)). Multiple mutations such as H:V37W & H:G44K resulted in a further increase of the H-L binding force in calculation.

III-2 Expression level of scFv

Based on the calculation results, we introduced several amino acid mutations in the HO538-213 scFv molecule. Because H:V37, H:G44, H:C102, and L:A43 were predicted to be potent targets, one amino mutation was chosen for each of the 4 residues. Hence, the conversions of H:V37W, H:G44K, H:C102T, and L:A43R were selected for the mutations in experimental examination. Totally 5 different mutants were expressed in E. coli and purified in the same protocol as the original. Table 1 shows the final yield of the purified scFv mutants. The expression level of the original HO538-213 scFv is quite low. In contrast, the expression level was increased due to the introduction of amino mutations. A multiple mutations of H:V37W & H:G44K & H:C102T markedly promoted the final yield of the purified scFv. A comparison in yield of the purified scFv among the mutants suggests that H:V37W mutation is effective for enhancing the expression while L:A43R seems to hamper the enhancement.

III-3 Activity Measurement

The activity of the purified scFv mutant was checked by ELISA measurement. A scFv molecule including a single amino mutation, H:V37W, was used in the measurement. TMB is a substrate of the chemical reaction caused by the HRP activity coupled to the secondary antibody. The absorbance at 450nm, which is the peak wave-length of TMB reaction product, indicates the quantity of scFv molecules bound to the antigen. The absorbance value was almost proportional to the amount of scFv applied on the plate. Therefore, the scFv mutant with H:V37W was confirmed to be active in terms of the binding ability to CD4.

The activity of the scFv H:V37W mutant was also examined by ITC measurement. The binding of the scFv mutant with 14-mer peptide extracted from CD4 D1 domain was monitored. The ITC measurement also suggested the scFv capability of binding to the antigen peptide. From the ITC curve, the binding was found to be entropic-driven and the association constant Ka was estimated to be 2 × 105.

IV. Conclusion

The conversion from Fab to scFv sometimes causes the instability of protein molecules, which often leads to a decrease in activity of the antibody. The effective amino acid mutations at the interface between H- and L-chains of scFv were predicted by parallel computer calculations to strengthen the H-L contact. The introduction of the predicted mutations greatly promoted the expression level of scFv while maintaining the activity of the antibody.

About the Author

Tyuji Hoshino is the Associated Professor at Chiba University. He received his Ph.D. from Waseda University. After three years of Research Associate at Waseda University, he was appointed as a Faculty member of Graduate School of Pharmaceutical Sciences, Chiba University.



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