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.

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.

Neutralization of KIT Oncogenic Signaling in Leukemia with Antibodies Targeting KIT Membrane Proximal Domain 5.

KIT is a cell surface tyrosine kinase receptor whose ligand stem cell factor (SCF) triggers homodimerization and activation of downstream effector pathways involved in cell survival, proliferation, homing, or differentiation. KIT-activating mutations are major oncogenic drivers in subsets of acute myeloid leukemia (AML), in mast cell leukemia, and in gastrointestinal stromal tumors (GIST). The overexpression of SCF and/or wild-type (WT) KIT is also observed in a number of cancers, including 50% of AML and small cell lung cancer. The use of tyrosine kinase inhibitors (TKI) in these pathologies is, however, hampered by initial or acquired resistance following treatment. Using antibody phage display, we obtained two antibodies (2D1 and 3G1) specific for the most membrane proximal extracellular immunoglobulin domain (D5) of KIT, which is implicated in KIT homodimerization. Produced as single chain variable antibody fragments fused to the Fc fragment of a human IgG1, bivalent 2D1-Fc and 3G1-Fc inhibited KIT-dependent growth of leukemic cell lines expressing WT KIT (UT7/Epo) or constitutively active KIT mutants, including the TKI imatinib-resistant KIT D816V mutant (HMC1.2 cell line). In all models, either expressing WT KIT or mutated KIT, 2D1 and 3G1-Fc induced KIT internalization and sustained surface downregulation. However, interestingly, KIT degradation was only observed in leukemic cell lines with oncogenic KIT, a property likely to limit the toxicity of these antibodies in patients. These fully human antibody formats may represent therapeutic tools to target KIT signaling in leukemia or GIST, and to bypass TKI resistance of certain KIT mutants.

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.

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol-resistant human breast cancer cell line MDA-MB-231.

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and ß-tubulins (more specifically the ßIII and ßIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.

Development of human single-chain antibodies to the transferrin receptor that effectively antagonize the growth of leukemias and lymphomas.

The major route of iron uptake by cells occurs through transferrin receptor (TfR)-mediated endocytosis of diferric-charged plasma transferrin (holo-Tf). In this work, we pursued TfR antibodies as potential cancer therapeutics, characterizing human single-chain variable antibody fragments (scFv) specific for the human TfR isolated from a phage display library. We hypothesized that many of these antibodies would function as ligand mimetics because scFvs from the library were selected for binding and internalization into living cells. In support of this hypothesis, the anti-TfR scFvs identified were antagonists of TfR binding to holo-Tf, particularly two of the most potent antibodies, 3TF12 and 3GH7, which blocked the in vitro proliferation of a number of hematopoietic cancer cell lines. We optimized this activity of 3TF12 and 3GH7 by engineering 55-kDa bivalent antibody formats, namely, F12CH and H7CH, which could block cell proliferation with an IC(50) of 0.1 microg/mL. We found that the mechanism of the scFv antibody cytotoxicity was unique compared with cytotoxic anti-TfR monoclonal antibodies that have been described, causing cell surface upregulation of TfR along with the inhibition of holo-Tf cell uptake and induction of cell death. In a nude mouse model of erythroleukemia, administration of F12CH reduced tumor growth. Together, our findings define a new class of fully human anti-TfR antibodies suitable for immunotherapy against tumors whose proliferation relies on high levels of TfR and iron uptake, such as acute lymphoid and myeloid leukemias.

The aberrant localization of oncogenic kit tyrosine kinase receptor mutants is reversed on specific inhibitory treatment.

Kit is a cell surface type III tyrosine kinase (TK) receptor implicated in cell transformation through overexpression or oncogenic mutation. Two categories of Kit mutants displaying mutations either in the juxtamembrane intracellular domain (regulatory mutants) or in the catalytic domain (catalytic mutants) have been described. To explore the effect of Kit oncogenic mutations on its subcellular localization, we constructed enhanced green fluorescent protein (EGFP)-tagged human Kit chimeras harboring mutations either in the regulatory (V560G) or in the catalytic (D816V) domain. When expressed in Chinese hamster ovary cells, EGFP-tagged wild-type Kit was activated on stem cell factor stimulation, whereas both EGFP-tagged Kit mutants displayed a constitutive TK activity. Constitutively activated mutants exhibited a high-mannose-type N-glycosylation pattern and an intracellular localization, suggesting that these mutants induce downstream oncogenic signaling without the need to reach the cell surface. Inhibition of constitutive Kit TK activity with dasatinib induced a complex, mature N-glycosylation pattern identical to unstimulated wild-type Kit and resulted in the redistribution of the mutants to the plasma membrane. This relocalization was clearly correlated to the inhibition of TK activity because imatinib, a specific inhibitor of the V560G mutant, inactive on the catalytic D816V mutant, induced only the relocalization of the V560G mutant. These data show that on TK inhibition, the aberrant localization of Kit mutants can be fully reversed. Kit mutants are then exported and/or stabilized at the cell surface as inactive and fully N-glycosylated isoforms.