Makabe, K. | Search bioRxiv

bioRxiv2024

Structural basis for bispecific antibody design: arrangement of domain linkage produces activity enhancement

A bispecific antibody (BsAb) is a protein genetically engineered from two different antibodies, allowing simultaneous binding to two kinds of antigen to bring them into close proximity. BsAbs have been developed as anti-cancer drugs that accumulate lymphocytes onto cancer cells by bridging antigens present on each. Ex3 is a bispecific diabody composed of the two fused variable regions (Fvs) of an anti-epidermal growth factor receptor (EGFR) antibody and an anti-CD3 antibody with potent cancer cytotoxic activity. In Ex3, the LH-type, in which the variable regions of the light chain (VLs) are located at the N-terminus of those of the heavy chain (VHs), exerted 1000-fold greater anticancer activity than the HL-type, in which the VHs are located at the N-terminus of the VLs. This effect (termed activity enhancement), in which the activity is greatly enhanced by domain rearrangement, has been reported not only for Ex3 but also for several other BsAbs. However, the molecular details of this activity enhancement have yet to be elucidated. In this study, we determined the cryo-EM structures of Ex3 LH- and HL-types in complex with CD3 and EGFR. Structural comparison of the LH- and HL-types showed that rearrangement of the domain linkage produces drastic structural differences in the overall shape of these complexes, and dynamics attributed to the flexibility between the two Fvs. These findings provide valuable insights into the molecular mechanism for the activity enhancement of BsAbs. This study will be a stepping stone towards establishing a design foundation for BsAb development.

biochemistry↗

bioRxiv2022

intra-single cell sequencing (iSCseq) spotlights transcriptomic and epigenetic heterogeneity inside multinucleated osteoclast

Single-cell RNA-seq (scRNA-seq) has clarified cellular heterogeneity within cell populations. However, scRNA-seq and spatial transcriptomics cannot capture the dynamic transcriptomic changes inside living cells. To decode subcellular gene expression, we developed intra-single cell sequencing (iSCseq), a novel approach that combines confocal imaging, repeatedly picking up cellular components inside living cells, and next-generation sequencing (intra single-cell RNA-seq; iSCseq). iSCseq illustrated the subcellular heterogeneity of gene expression. iSCseq revealed not only multiple differentiation stages embedded in the same cell, but also physical cytoskeletal connections, physiological activity of mitochondria, and intracellular calcium, as confirmed by transcriptomic evidence. Inclusive iSCseq with in vivo scRNA-seq datasets identified new osteoclast subsets in physiological and pathological bones. Network analysis with centrality provided insights into the connection between subcellular components, and clearly divided differentiation and fusion processes in multinucleation. The iSCseq approach has the potential to enhance cell biology at subcellular resolution and identify new therapeutic targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/506360v4_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@115c1d8org.highwire.dtl.DTLVardef@1695ad2org.highwire.dtl.DTLVardef@1c9d257org.highwire.dtl.DTLVardef@1107ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefintra-single cell sequencing (iSCseq) enhances single-cell technology by combining live cell imaging, subcellular sampling from living cells and sequencing, offering deeper insights into cell functions and pathology at subcellular resolution through inclusive analysis with scRNA-seq and advanced centrality-focused network analysis. HighlightsO_LIintra-single cell sequencing (iSCseq) clarifies subcellular heterogeneity C_LIO_LIiSCseq connects morphological and physiological features with transcriptome C_LIO_LIInclusive iSCseq unveils osteoclast subsets in physiological and pathological bones C_LIO_LILinkage at subcellular resolution reveals key players in characteristic fusion C_LI

genomics↗