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Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Ne-(o-azidobenzyloxycarbonyl)- l -lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Carcinoembryonic antigen (CEA) is a classic tumor‐specific antigen that is overexpressed in several cancers, including gastric cancer. Although some anti‐CEA antibodies have been tested, to the best of our knowledge, there are currently no clinically approved anti‐CEA antibody therapies. Because of this, we have created the novel anti‐CEA antibody, 15‐1‐32, which exhibits stronger binding to membrane‐bound CEA on cancer cells than existing anti‐CEA antibodies. 15‐1‐32 also shows poor affinity for soluble CEA; thus, the binding activity of 15‐1‐32 to membrane‐bound CEA is not influenced by soluble CEA. In addition, we constructed a 15‐1‐32‐monomethyl auristatin E conjugate (15‐1‐32‐vcMMAE) to improve the therapeutic efficacy of 15‐1‐32. 15‐1‐32‐vcMMAE showed enhanced antitumor activity against gastric cancer cell lines. Unlike with existing anti‐CEA antibody therapies, antitumor activity of 15‐1‐32‐vcMMAE was retained in the presence of high concentrations of soluble CEA.

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C{sub 2}H{sub 2}-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.

Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5′-(G/T)GGGCGG(G/A)(G/A)(C/T)-3′ (GC box). In order to determine whether the three-zinc finger peptide Sp1(530–623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5′-GGGTGGGCGTGGC-3′ (s-GC box), which is flanked by a novel conserved guanine triplet on the 5′-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530–623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5′-GGGGCGGGGC-3′ (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530–623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530–623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline–copper complex (OP-Cu), binding of Sp1(530–623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530–623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.

The unique linker sequence of the native nine zinc finger transcription factor IIIA (TFIIIA) appears to significantly affect its novel DNA recognition mode. An artificial new nine zinc finger peptide Sp1ZF9T has been created by connecting three units of the three zinc finger domains of Sp1 with the TFIIIA-type linker. The DNA-binding characteristics of Sp1ZF9T were evaluated by the gel mobility shift, DNase I footprinting, and methylation interference assays, and compared with those of the previous Sp1ZF9 with a Kruppel-type linker. The gel mobility shift assays revealed that Sp1ZF9T forms two complex species, a short-lived species (B-2) and a long-lived species (B-1), with GCIII DNA (5′-GGG GCG GGG GGG GCG GGG GGG GCG GGGCC-3′). The B-2 complex dissociated into the free peptide and DNA, whereas the B-1 complex was stable even after 72 h. The DNase I footprinting and methylation interference results indicated that 3′- and central portions of GCIII DNA are recognized by Sp1ZF9T in the B-1 complex. The present DNA binding mode of Sp1ZF9T is evidently different from that of Sp1ZF9. Namely, fingers 1–5 participate in the DNA contact of Sp1ZF9T, and fingers 1–9 in that of Sp1ZF9. Therefore, the linker sequence among the zinc finger domains has a significant effect on the specific DNA recognition by the multi-zinc finger proteins.

To understand the DNA recognition mechanism of zinc finger motifs of transcription factor Sp1, we have determined the solution structure of DNA-binding domain of the Sp1 by solution NMR techniques. The DNA-binding domain of Sp1 consists of three Cys(2)His(2)-type zinc finger motifs. They have typical betabetaalpha zinc finger folds and relatively random orientations. From DNA-binding analysis performed by NMR and comparison between structures determined here and previously reported structures of other zinc fingers, it was assumed that DNA recognition modes of fingers 2 and 3 would be similar to those of fingers of Zif268, in which each finger recognizes four base pairs strictly by using residues at positions -1, 2, 3, and 6 of the recognition helix. On the contrary, finger 1 can use only two residues for DNA recognition, Lys550 and His553 at positions -1 and 3 of the helix, and has more relaxed sequence and site specificity than other Cys(2)His(2) zinc fingers. It is proposed that this relaxed property of finger 1 allows transcription factor Sp1 to bind various DNA sequences with high affinity.