Antibodies are key molecules in the human immune defense system. On November 6, 2025, a research team led by Professor Junyu Xiao and Professor Ning Gao from Peking University published a study entitled "Xenopus IgX informs engineering strategies of IgM and IgG hexamers" in Science Advances. This study, in tracing the evolution of antibody molecules, drew inspiration from IgX antibodies from the African frog and developed a strategy suitable for constructing human IgM and IgG hexamers, providing new insights for the design of related macromolecular drugs.

IgM antibodies are the most evolutionarily ancient and are widely found in jawed vertebrates. In mammals, IgM assembles into a stable pentamer by binding to the J chain through the "tailpiece (tp)" region, which consists of 18 amino acids at the C-terminus of its heavy chain (1). The J-chain imparts unique features to the IgM pentamer and enhances its interactions with various receptors and binding partners such as pIgR, FcμR, and CD5L (1–4). Without the J-chain, mammalian IgMs can form multiple polymeric structures, including hexamers, pentamers, and tetramers. In addition to binding to and neutralizing pathogens in a multivalent manner, IgM can also efficiently activate the complement system and synergistically eliminate microbial pathogens and abnormal cells (5, 6). Notably, the IgM hexamer is significantly more effective in activating the complement system than the pentamer, likely due to the hexameric structure of the C1q adaptor (7–10).

Fig. 1. Xenopus Fcχ can form a stable hexamer.

IgX in Xenopus laevis represent a unique branch of antibody evolution. This study first discovered that IgX can autonomously assemble into stable hexamers independent of the J chain (Fig. 1A). Unlike IgM tp (μtp), IgX tp (χtp) contains only 11 amino acids (Fig. 1B); removing this region disrupts its ability to form hexamers, indicating that χtp is crucial for IgX hexamer formation. Using cryo-electron microscopy (cryo-EM), the structure of the IgX-Fc hexamer was resolved. In the EM density map obtained using C1 symmetry calculations, the resolution of the χtp region was low; however, after processing the complex particles using a C6 symmetry expansion method, the β-sheet conformation of the χtp region was revealed.

To further investigate the ability of χtp to mediate hexamer formation, this study replaced the μtp of human IgM with the χtp of IgX. The results showed that the obtained chimera (Fcμ-χtp) could efficiently assemble into a uniform hexamer even without the J chain. Based on this result, it is speculated that the length of tp may be the key to regulating hexamer formation. Subsequently, by systematically shortening the μtp of human IgM, it was found that uniform hexamer assembly could be achieved with μtp lengths between 11 and 16 amino acids (Fig. 2, A and B). From a molecular mechanism perspective, μtp lengths of less than 11 amino acids impair the integrity of the β-sheet, thereby disrupting the stability of the IgM hexamer; while μtp lengths of more than 16 amino acids hinder hexamer formation due to steric hindrance. Specifically, Cys575, the second-to-last molecule, and Tyr576, the terminal molecule, participate in the interaction between IgM-Fc and the J chain, which is more conducive to the assembly of IgM-J pentamers, but will affect the formation of hexamers.

Fig. 2. The engineered IgM-Fc and IgG-Fc can form hexamers.

Notably, introducing a truncated μtp (μtp₁₁) from IgM into the C-terminus of human IgG also yielded stable IgG hexamers (Fig. 2C). In functional validation, this study designed IgM and IgG hexamers targeting CD20 and tested their cytotoxic effects in various B lymphoma cell lines, including OCI-Ly10, Daudi, and Raji. The results showed that these engineered hexamer antibodies exhibited significantly enhanced complement-dependent cytotoxicity (CDC) at the cellular level (Fig. 3A). Furthermore, the engineered IgG-Fc hexamer can also act as a "molecular decoy," inhibiting overactivated complement responses at the cellular level (Fig. 3B).

Fig. 3. The functional characterization of the engineered antibodies.

In summary, this study reveals the structural principles of a natural hexameric antibody from a molecular evolution perspective and develops a universal strategy to efficiently induce the formation of IgM or IgG hexamers by chimeric χtp or truncated μtp sequences. Engineered IgM-Fc hexamers are also expected to serve as the molecular scaffold to showcase other biologically active proteins, thereby expanding their application potential. This work deepens our understanding of the evolution of polyimmunoglobulins and provides a platform technology for the development of novel biologics.

Dr. Ruixue Zhang (Ph.D. graduate from the PKU Center for Life Sciences program), a postdoctoral fellow at the School of Life Sciences/BIOPIC of Peking University, and Dr. Chenggong Ji, a former postdoctoral fellow at Peking University, are co-first authors of this paper. Professor Junyu Xiao (Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Gene Function and Modulation Research, Peking University School of Life Sciences/BIOPIC, Changping Laboratory), Professor Ning Gao (Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Changping Laboratory), and Dr. Chenggong Ji are co-corresponding authors. Doctoral student Shuhan Li and Dr. Ningning Li from the School of Life Sciences at Peking University made significant contributions to this work. This research received support from the National Natural Science Foundation of China, the National Key Research and Development Program of China, the Peking-Tsinghua Center for Life Sciences, the Qidong-SLS Innovation Fund at Peking University, and the Frontier Innovation Fund of Peking University Chengdu Academy for Advanced Interdisciplinary Biotechnologies. Data collection and processing were strongly supported by the cryo-EM platform of Peking University and the Changping Laboratory.

Original link：https://doi.org/10.1126/sciadv.aea3737

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