Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors.

Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies.

BACKGROUND: The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. RESULTS: We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). CONCLUSIONS: The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma patient-derived xenografts.

Monoclonal antibodies specific for biomarkers expressed on the surface of uveal melanoma (UM) cells would simplify the immune capture and genomic characterization of heterogeneous tumor cells originated from patient-derived xenografts (PDXs). Antibodies against four independent tumor antigens were isolated by panning a nanobody synthetic library. Such antibodies enabled flow cytometry-based sorting of distinct cell subpopulations from UM PDXs and to analyze their genomic features. The complexity and specificity of the biochemical and genomic biomarker combinations mirrored the UM tumor polyclonality. The data showed that MUC18 is highly and universally displayed on the surface of UM cells with different genetic background and consequently represents a reliable pan-biomarker for their identification and purification. In contrast, the other three biomarkers were detected in very variable combinations in UM PDX cells. The availability of the identified nanobodies will be instrumental in developing clone-specific drug evaluation and rational clinical strategies based on accurate genomic profiling.