In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT

Abstract

Nanobodies are a novel type of immunoglobulinlike, antigenbinding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. Methods: We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and 99m Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. Results: The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEAtransfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with 99m Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected 99m Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. Conclusion: This study shows the potency of antigenbinding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.

Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies

“…scFvs often show reduced affinity/ specificity compared with their parental Ab and their linking to other chains may further reduce their affinity or that of the Ab to which they are linked (32). ScFv, like nanobodies, may also show higher immunogenicity (55). Our format in contrast maintains the full structure of Fab, helping with affinity and a decrease in immunogenicity.…”

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

“…The general humanized nanobody scaffold h-NbBcII10 FGLA referenced published research papers. 14,20 construction of immunotoxins…”

“…Due to their small molecular weight, 13 rapid tissue penetration, 14 high solubility and stability, 15 high specificity of antigen binding, and ability for large amounts to be synthesized, 16 they are often used in cancer treatment. Including the structural recombinant immunotoxins, 17 the therapeutic fusion proteins, they are are formed from the fusion of the antibody part that can specifically bind to tumor cell surface antigens and the part with cytotoxic activity.…”

Single-domain antibody fragments derived from heavy-chain antibodies: a review

“…In contrast to radiolabelled monoclonal antibodies, small sdAb fragments show rapid antigen targeting and fast clearance from blood resulting in a contrast-enhanced imaging signal, reduced accumulation of labelled fragments in liver and lower radiation burden. The main limitation of sdAb-based imaging probes, however, is their high non-specific uptake in kidney and bladder (Vaneycken et al 2010(Vaneycken et al , 2011b). An innovative approach based on sdAb fragments fused to fluorescent proteins and expressed in living cells as "chromobodies" offers the possibility to trace intracellular antigens and to modulate protein function in living cells .…”