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Sci Adv
2025 Nov 07;1145:eaea3737. doi: 10.1126/sciadv.aea3737.
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Xenopus IgX informs engineering strategies of IgM and IgG hexamers.
Zhang R, Ji C, Li S, Li N, Gao N, Xiao J.
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Polymeric immunoglobulins are essential components of the immune system in jawed vertebrates. While mammalian immunoglobulin M (IgM) typically forms a pentamer linked by the joining chain (J-chain), Xenopus laevis IgX can assemble into a J-chain-independent polymer. Here, we present the cryo-electron microscopy (cryo-EM) structure of IgX, revealing its hexameric configuration. By incorporating the IgX tailpiece into human IgM, we achieved efficient IgM hexamer formation. Truncating IgM's natural tailpiece to a range of 11 to 16 residues also substantially enhanced hexamerization efficiency. Furthermore, introducing a shortened IgM tailpiece to IgG resulted in effective IgG hexamer formation. We further show that the engineered IgM and IgG hexamers targeting CD20 demonstrated robust complement-dependent cytotoxicity (CDC) against several B lymphoma cells. In addition, the IgG-Fc hexamer functioned as a decoy, attenuating CDC in cell cultures. These findings deepen our understanding of polymeric immunoglobulin evolution and introduce innovative strategies for the development of IgM- and IgG-based biologics.
Fig. 1. Xenopus Fcχ can form a stable hexamer.(A) 2D cryo-EM analyses showed that both Fcχ and FcχCχ3-Cχ4-χtp form hexamers. (B) Cryo-EM reconstruction of the FcχCχ3-Cχ4-χtp hexamer at a 3.29-Å resolution, shown in two orientations. The EM density map is superimposed on the structural model. The χtp regions were not clearly resolved because of the blurred densities in this region, as indicated by a dashed circle. (C) Side-by-side comparisons of the Fcχ hexamer (blue), human Fcμ-J pentamer (Fcμ in teal and J-chain in orange), and teleost Fcμ tetramer (cyan). (D) Side-by-side comparisons of the Fc-Fc interactions in the Fcχ hexamer, human Fcμ-J pentamer, and teleost Fcμ tetramer. The interface area, the interchain disulfide bonds, and the FG loops of Cμ4 or Cχ4 domains are highlighted in pink, yellow, and red, respectively.
Fig. 2. The Fcμ-χtp chimera forms a hexamer.(A) Sequence alignment of the tailpiece regions of Xenopus IgX and IgM, teleost IgM (from Oncorhynchus mykiss), and human IgM. The tailpiece regions are highlighted. (B) SV-AUC analysis of Fcμ-μtp18 suggested that the monomer, tetramer, pentamer, and hexamer are all present in solution. AU, absorbance units. (C) 2D cryo-EM analyses suggested that the polymers of Fcμ-μtp18 consist of a mixture of tetramer, pentamer, and hexamer. (D) SV-AUC analysis indicated that the Fcμ-χtp polymer is uniformly hexameric in contrast to Fcμ-μtp18. (E) 2D cryo-EM analyses of the Fcμ-χtp chimera. (F) Cryo-EM reconstruction of the Fcμ-χtp hexamer at a 3.29-Å resolution. The EM density map is superimposed on the structural model.
Fig. 3. Human Fcμ variants with shorter tailpieces can form hexamers.(A) SV-AUC analysis suggested that besides a monomer, Fcμ-μtp11 is predominantly present as a hexamer in solution. (B) Cryo-EM reconstruction of the Fcμ-μtp11 hexamer at a 3.17-Å resolution. The EM density map is superimposed on the structural model. (C) 2D cryo-EM analyses of Fcμ-μtp variants with different tailpiece lengths. (D) Structural comparison between the Fcμ-μtp11 hexamer and the Fcμ-J pentamer focusing on the Cμ4 regions. The Fcμ molecules in the Fcμ-μtp11 hexamer are represented in green, while those in the Fcμ-J pentamer are shown in white. The J-chain is highlighted in orange.
Fig. 4. hFcγ-μtp11 hexamer.(A) Sequences of Fcμ, Fcγ, and the engineered Fcγ-μtp11 chimera at the C-terminal regions. (B) SEC analysis suggested that Fcγ-μtp11 is mainly present as a hexamer in solution. The positions of several molecular weight standards running on SEC are shown with purple arrows. UV, ultraviolet. (C) Cryo-EM reconstruction of the Fcγ-μtp11 hexamer at a 2.75-Å resolution. (D) Structural overlay of the Fcγ-μtp11 hexamer and the IgG1-b12 hexamer within the crystal lattice [PDB (Protein Data Bank) ID: 1HZH]. The Fcγ-μtp11 hexamer is shown in purple, while the IgG1-b12 hexamer is depicted in light cyan. (E) Fc-Fc interactions in the Fcγ hexamer. The interface area in one of Fcγ protomers is highlighted in pink. Important regions in the Cγ3 domains that are involved in the interactions between two adjacent Fcγ molecules, including the C-strand and the FG loop and G-strand, are highlighted in red and yellow.
Fig. 5. IgM and IgG hexamers with μtp11 display enhanced CDC activities.(A) CDC activities of the engineered RTX-IgG and RTX-IgM antibodies were assessed using OCI-Ly10 cells in the presence of human complement. Data were plotted as the means ± SD. n = 3 biological replicates. Source data are provided in the Supplementary Materials. (B) CDC activities of the engineered RTX-IgG and RTX-IgM antibodies toward the Daudi cells. To facilitate the assay, the Daudi cells, and also the Raji cells in (C), were first cultured for several days in the RPMI-1640 medium supplemented with 10% heat-inactivated FBS. Data were plotted as the means ± SD. n = 3 biological replicates. (C) CDC activities of the engineered RTX-IgG and RTX-IgM antibodies toward the Raji cells. Data were plotted as the means ± SD. n = 3 biological replicates.
Fig. 6. The engineered IgG-Fc hexamer with μtp11 blocks RTX-IgG–mediated CDC.(A) The IgG-Fc hexamer with μtp11 blocks RTX-IgG–mediated CDC in the Daudi cells. The IgM-Fc hexamers with μtp11 and IgG-Fc monomers exhibit no inhibitory effect. Data were plotted as the means ± SD. n = 3 biological replicates. Source data are provided in the Supplementary Materials. (B) The IgG-Fc hexamer with μtp11 blocks RTX-IgG–mediated CDC in the Raji cells. Data were plotted as the means ± SD. n = 3 biological replicates. (C) Enzyme-linked immunosorbent assay measurement compares C4d generation in human serum. The background (BKG) C4d level in serum is indicated with a dashed line. Ab, antibody. Data were plotted as the means ± SD. n = 3 biological replicates.
