Thirdborough, S. M. | bioRxiv Science

bioRxiv2026

Bispecific Antibody Architecture and TNFRSF Target Selection Determine CD8+ T Cell Differentiation and Anti-tumour Immunity

Tumour necrosis factor receptor superfamily (TNFRSF)-targeting bispecific antibodies (bsAb) enable tumour-localised T cell co-stimulation, yet how antibody architecture and receptor choice govern activity remains unclear. Here we define how bsAb format and TNFRSF target selection shape CD8+ T cell responses and anti-tumour immunity. Using B7-H3 as a tumour-associated antigen, we show that dual-bivalent 2x2 bsAb elicit maximal agonistic activity, whereas the corresponding monovalent 1x1 format is least active. A 2x1 format retaining TNFRSF bivalency but monovalent B7-H3 binding preserves substantial activity, identifying co-stimulatory receptor bivalency as a key determinant of efficacy. Across TNFRSF targets, 4-1BB drives the strongest cytotoxic CD8+ T cell differentiation and anti-tumour response. This superiority is conserved in human T cells and reproduced by cognate ligands, indicating that the observed functional hierarchy reflects receptor-intrinsic biology. Mechanistically, efficacy requires tumour-associated B7-H3 and T cell-dependent 4-1BB signalling. Together, these findings establish general principles linking antibody architecture and receptor biology to co-stimulatory bsAb efficacy and provide a framework for rational design leading to optimal therapeutics.

immunology↗

bioRxiv2020

Activation of the myeloid checkpoint inhibitor LILRB3 (ILT5) elicits profound immununomodulation

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppresive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

immunology↗