Klervi Even-Desrumeaux

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes

UNLABELLED Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Masked Selection: A Straightforward and Flexible Approach for the Selection of Binders Against Specific Epitopes and Differentially Expressed Proteins by Phage Display

Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers.

State of the Art in Tumor Antigen and Biomarker Discovery

Our knowledge of tumor immunology has resulted in multiple approaches for the treatment of cancer. However, a gap between research of new tumors markers and development of immunotherapy has been established and very few markers exist that can be used for treatment. The challenge is now to discover new targets for active and passive immunotherapy. This review aims at describing recent advances in biomarkers and tumor antigen discovery in terms of antigen nature and localization, and is highlighting the most recent approaches used for their discovery including “omics” technology.

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates.

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

Phage display and selections on cells.

Traditional methods of phage display panning bind purified antigen to plates or other solid phases to which libraries are then applied. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. We describe here a procedure of a panning strategy on cell surface receptors including a depletion step. We explain every step of the protocol: production of phage library, depletion and selection, elution, screening by ELISA, and analysis of soluble antibodies by ELISA and flow cytometry. Finally, several possible variants of the protocol are explained in Subheading 4.

Affinity Determination of Biotinylated Antibodies by Flow Cytometry

Affinity determination is a crucial step of an antibody characterization. Here, we describe a method for antibody affinity determination by flow cytometry, relying on the unique affinity of biotin for streptavidin for easy and efficient antibody labeling. Several labeling approaches are described and discussed in this chapter, including chemical and enzymatic (in vivo and in vitro) biotinylation. Finally, a procedure for K (D) determination by flow cytometry is precisely described.