dianas 11(1) > Mendoza-Calvo etal

dianas | Vol 11 Num 1 | marzo 2022 | e202203a07

Establishment of nanobody´s selection tools to identify biomarkers in DIPG.

Noelia Mendoza-Calvo^a, Ruth González-Gómez, Rocío Bayona-Ramón y Cajal, Alberto Jiménez-Schuhmacher

Centro de Investigación Biomédica de Aragón (CIBA).

a. nmendoza@iisaragon.es

VII Congreso de Señalización Celular, SECUAH 2022.
14 a 18 de marzo, 2022. Universidad de Alcalá. Alcalá de Henares, Madrid. España.

Keywords: DIPG; Nanobody; Libraries; Diagnosis; Immuno-PET

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive cancer which mainly affects children between 5 and 7 years old. Tumor develops in the brainstem and its cells growth in a diffuse pattern, spreading and infiltrating through the healthy brainstem tissue, producing the affectation of vital function such as breath and heart rate. Due to its specific growth pattern, it is not possible to make a biopsy of the tumor, and therefore it will not be possible to obtain histological and molecular information. Moreover, imaging diagnosis it is also imprecise in the characterization of this malignancy. All this information is critical to obtain an accurate diagnosis of the patient, apply them the correct treatment and assess their prognosis. To perform an accurate diagnosis and molecular identification of DIPG surface biomarkers, we have generated three nanobody libraries through the immunization of three camelid with two DIPG cell lines and one with a GBM cell line. From these libraries, it is possible to isolate a given nanobody which can recognize in a highly specific way, a previously identified and characterized biomarker in the tumor cell surface. This biomarker must be exposed in the external surface of the plasma membrane. In the laboratory we have set up three different strategies to efficiently isolate a specific nanobody from one of the three libraries. Two are cell-based approaches, this means that an overexpression (OE) and a knock-out (KO) cell lines must be generated. In the first strategy KO and OE cells are attached to a cell culture plate in order to perform several rounds of negative and positive selection, respectively. The second strategy harness the sorter technology, after the negative selection in plate, the library is sorted join to the OE population. The third procedure is based on MACs technology. Obtained Nb can be used to different applications such as nano-CAR construction and immuno-PET diagnosis.