DNA IMMUNIZATION TO OBTENTION MONOCLONAL ANTIBODIES AGAINST THE  SURFACE PROTEIN HCD73

Aryanny Paula Sousa Ferreira; Priscila Pini Zenatti

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Trabalho

Título do Trabalho

DNA IMMUNIZATION TO OBTENTION MONOCLONAL ANTIBODIES AGAINST THE SURFACE PROTEIN HCD73

Autores

Aryanny Paula Sousa Ferreira
Priscila Pini Zenatti

Modalidade

Apresentação de Pôster

Área temática

Imunologia

Data de Publicação

09/06/2023

País da Publicação

Brasil

Idioma da Publicação

Inglês

Página do Trabalho

https://www.even3.com.br/anais/v-gbmeeting/610437-dna-immunization-to-obtention-monoclonal-antibodies-against-the--surface-protein-hcd73

ISBN

978-85-5722-785-9

Palavras-Chave

Monoclonal antibody, cDNA immunization, Human NT5E, Recombinant protein

Resumo

Introduction: The CD73 (NT5E) protein is an ectonucleotidase involved in the generation of extracellular adenosine and in the promotion of cell growth and survival in normal tissues. This ecto-enzyme is highly expressed in a variety of tumor cells and acts by regulating the function of immune cells, creating an immunosuppressive tumor microenvironment. The upregulation of CD73 is also associated with tumor growth, metastasis, angiogenesis and, consequently, a worse prognosis in several types of cancers, since tumors use several mechanisms of immune evasion mediated by this enzyme. Objective: Considering the therapeutic potential of this protein, this work proposes to obtain anti-hCD73 monoclonal antibodies from DNA immunization. Methodology: Female Balb/c mice were immunized with the pcDNA3.1_NT5E plasmid by two routes: (I) Intramuscular – IM (n = 5) and (II) Intravenous – IV (n = 5). In I, the mice received 100 µg of plasmid (immunogen) diluted in 100 µL of PBS1x in one of the flanks. In II (Hydrodynamic Immunization (HD)) the mice received 10 µg of plasmid diluted in 1 mL of PBS in the first 3 doses and 100 µg in the last 3 doses, via caudal route in ~10 seconds of injection. The serum of the animals were collected at times D0 (pre-immunization), D15 (I immunization), D39 (II immunization), D70 (III immunization), D88 (IV immunization) and D196 (VI immunization) and the anti-hCD73 antibodies production was monitored by Flow-cytometry and ELISA. For cytometry, 200,000 Daoy cells were incubated with 3 µL of each serum (D196), stained with secondary antibody, read in a FACS Fortessa LSR cytometer and analyzed in FlowJo software. For the ELISA, 20 ng of hCD73 recombinant protein was used as substrate, and the sera from times D0, D88 and D196 (50 µL/well; diluted 1:100) were evaluated in triplicate and read in the Sinergy equipment (absorbance of 450 nm ). Data were analyzed using Graphpad Prism software. The animals will still receive a final booster with (1) hNT5E.Fc recombinant protein, intraperitoneally, combined with incomplete Freund's adjuvant, or with (2) 14 million Daoy cells, according to the methodology of Pelletier et al., 2017. Results: Both methodologies led to the production of specific antibodies for hCD73. In cytometry with Daoy cells, a medulloblastoma cell line with physiological expression of CD73, the mean fluorescence intensity (MFI) was 4.61 for negative controls, 131 for IM samples and 62.85 for HD samples. In the ELISA, the mean absorbance was higher for the HD group (OD450 = 1.025) compared to the IM group (OD450 = 0.612). Conclusion: Immunization with DNA is a more economical alternative, as it does not require the production/purchase of the recombinant protein. Cytometry data showed that the antibodies produced by the IM group had a higher affinity for CD73 in the natural conformation. The ELISA results confirm that there was efficient production of antibodies in both groups. Other assays will be realized in order to characterize the diversity and specificity of the repertoire of antibodies generated in each group.

Título do Evento: V GBMeeting
Cidade do Evento: Campinas
Título dos Anais do Evento: Anais do GBMeeting: Encontro Anual da Pós Graduação em Genética e Biologia Molecular da UNICAMP
Nome da Editora: Even3
Meio de Divulgação: Meio Digital

Como citar

FERREIRA, Aryanny Paula Sousa; ZENATTI, Priscila Pini. DNA IMMUNIZATION TO OBTENTION MONOCLONAL ANTIBODIES AGAINST THE SURFACE PROTEIN HCD73.. In: Anais do GBMeeting: Encontro Anual da Pós Graduação em Genética e Biologia Molecular da UNICAMP. Anais...Campinas(SP) Unicamp, 2023. Disponível em: https//www.even3.com.br/anais/v-gbmeeting/610437-DNA-IMMUNIZATION-TO-OBTENTION-MONOCLONAL-ANTIBODIES-AGAINST-THE--SURFACE-PROTEIN-HCD73. Acesso em: 17/04/2025