The assembly of single domain antibodies into bispecific decavalent molecules

Bispecific antibodies present unique opportunities in terms of new applications for engineered antibodies. However, designing ideal bispecific antibodies remains a challenge. Here we describe a novel bispecific antibody model in which five single domain antibodies (sdAbs) are fused via a linker sequence to the N-terminus of the verotoxin B (VTB) subunit, a pentamerization domain, and five sdAbs are fused via a linker sequence to the VTB C-terminus. Fifteen such decavalent bispecific molecules, termed decabodies, were constructed and characterized for the purpose of identifying an optimal decabody design. One of the fifteen molecules existed in a non-aggregated decavalent form. In conjunction with the isolation of sdAbs with the desired specificities from non-immune phage display libraries, the decabody strategy provides a means of generating high avidity bispecific antibody reagents, with good physical properties, relatively quickly.     

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture,Studied in Weight-Bearing and Non–Weight-Bearing Human Bone

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non–weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > −1.0) to osteoporotic (T-score ≤ −2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and proteoglycans in cancer emerged at both sites, whereas fatty acid metabolism and focal adhesion were only identified in iliac bone. Lasso analysis and cross-validations identified sets of nine bone ncRNAs correlating strongly with adjusted total hip BMD in both femoral and iliac bone. Twenty-eight iliac ncRNAs were associated with risk of fracture (FDR < 0.1). The small nucleolar RNAs, RNU44 and RNU48, have a function in stabilization of ribosomal RNAs (rRNAs), and their association with fracture and BMD suggest that aberrant processing of rRNAs may be involved in development of osteoporosis. Cis-eQTL (expressed quantitative trait loci) analysis of the iliac bone biopsies identified two loci associated with microRNAs (miRNAs), one previously identified in a heel-BMD genomewide association study (GWAS). In this comprehensive investigation of the skeletal genetic background in postmenopausal women, we identified functional bone ncRNAs associated to fracture and BMD, representing distinct subsets in WB and NWB skeletal sites. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.