Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen.

Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.

Crystal structure of streptavidin mutant with low immunogenicity.

We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein Sci., 22, 213-221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin.

Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin.

The defining property of core streptavidin (cSA) is not only its high binding affinity for biotin but also its pronounced thermal and chemical stability. Although potential applications of these properties including therapeutic methods have prompted much biological research, the high immunogenicity of this bacterial protein is a key obstacle to its clinical use. To this end, we have successfully constructed hypoimmunogenic cSA muteins in a previous report. However, the effects of these mutations on the physicochemical properties of muteins were still unclear. These mutations retained the similar electrostatic charges to those of wild-type (WT) cSA, and functional moieties with similar hydrogen bond pattern. Herein, we performed isothermal titration calorimetry, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to gain insight into the physicochemical properties and functions of these modified versions of cSA. The results indicated that the hypoimmunogenic muteins retained the biotin-binding function and the tetramer structure of WT cSA. In addition, we discuss the potential mechanisms underlying the success of these mutations in achieving both immune evasion and retention of function; these mechanisms might be incorporated into a new strategy for constructing hypoimmunogenic proteins.

Synthesis, characterization, and binding property of isoelectronic analogues of nucleobases, B(6)-substituted 5-aza-6-borauracils and -thymines.

As isoelectronic BN-containing analogues of 6-substituted uracil and thymine, a series of B(6)-substituted 5-aza-6-borauracils (U(BN)s) and -thymines (T(BN)s) were synthesized and fully characterized. The crystallographic and spectroscopic analyses of the analogues revealed that the framework and hydrogen-bonding pattern of T(BN)s were similar to those of the original nucleobase, thymine.