Nano-seahorse T-cell-recruiting antibody assembly for cancer therapy

Structural biology describes hierarchical structure of proteins: functional modules of domains, fragments, and subunits, are clustered as building blocks to create fine machinery molecules with autonomic systematical... [ view full abstract ]

Structural biology describes hierarchical structure of proteins: functional modules of domains, fragments, and subunits, are clustered as building blocks to create fine machinery molecules with autonomic systematical functions. Antibody is a protein which has been fragmented and rearranged to form recombinant proteins with non-native structure and function, because antibody is a typical module protein that is composed of structurally and functionally independent fragments. In this study, we propose a new structural format of high cytotoxic bispecific antibody assembly recruiting to cancer and lymphocyte cells. Small bispecific T-cell–recruiting antibodies have the potential of low-cost bacterial expression and contributes to low immunogenicity and high penetration into the tumor mass; so that, several bispecific structures are proposed, and some compact antibodies, such as tandem single-chain Fv, have been used in clinical trials. However, most of the compacted structures are monovalency which lead to weaker affinity for target-displaying cells than full-length IgG antibodies. Here, we constructed a compact bispecific and bivalent antibody from antigen-binding modules of single variable domain of the heavy chain of a heavy chain camel antibody (VHH) and single chain Fv (scFv) with two variable domains of heavy chain and light chain joined via a flexible polypeptide linker. The single domain format of VHH is an appropriate building block for generating fusion proteins and the scFv has the potential of intermolecular interaction that induces dimerization: the VHH domains with affinity for the epidermal growth factor receptor (EGFR) overexpressed on cancer cells were fused to the self-dimerized scFv recruiting to CD3 receptors on T-cells, resulting in the formation of bispecific and bivalent antibodies (BiBian) (Fig.1). The VHH-fused scFv fragments were spontaneously assembled to BiBian forms with a unique seahorse conformation in bacterial expression, and affinity increment for both target cells by bispecific and bivalent design of BiBian caused a drastic enhancement of cytotoxicity against tumor spheroids in vitro and in vivo (Fig2, 3). We show a promising sea-horse-shaped high cytotoxic antibody assembly formed from small antibody modules expressed in bacterial expression.