The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor-targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G 4 S) 3 linker, generating FcA22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor-targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor-targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs.

Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.

The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors death receptor (DR) 4 and DR5 are attractive targets to develop the receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell death-inducing activity. Here, we report a novel agonistic mAb, AY4, raised against human DR4 in mice. ELISA analysis revealed that AY4 specifically bound to DR4 without competition with TRAIL for the binding. Despite distinct binding regions of AY4 on DR4 from those of TRAIL, AY4 as a single agent induced caspase-dependent apoptotic cell death of several tumor types through the extrinsic and/or intrinsic pathways without substantial cytotoxicity to normal human hepatocytes. Further, the AY4-sensitive cells followed the same cell death characteristics classified as type I and type II cells by the response to TRAIL, suggesting that the cell death profiles in responses to DR4 and/or DR5 stimulation are determined by the downstream signaling of the receptor rather than the kind of receptor. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which have been reported to be resistant to other anti-DR4 agonistic mAbs, most likely due to the unique epitope property of AY4. In vivo administration of AY4 significantly inhibited tumor growth of human non-small cell lung carcinoma preestablished in athymic nude mice. Conclusively, our results provide further insight into the DR4-mediated cell death signaling and potential use of AY4 mAb as an anticancer therapeutic agent, particularly for