| Grant number: | 23/09167-7 |
| Support Opportunities: | Regular Research Grants |
| Start date: | November 01, 2023 |
| End date: | October 31, 2025 |
| Field of knowledge: | Biological Sciences - Biochemistry - Chemistry of Macromolecules |
| Principal Investigator: | Vitor Marcelo Silveira Bueno Brandão de Oliveira |
| Grantee: | Vitor Marcelo Silveira Bueno Brandão de Oliveira |
| Host Institution: | Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil |
| Associated researchers: | Fábio Dupart Nascimento ; Jair Ribeiro Chagas ; Marcelo Yudi Icimoto |
Abstract
We intend to use two specific cell surface tumor biomarkers, the prostate specific membrane antigen (PSMA) and the carcinoembryonic antigen (CEA), as targets for directing fluorescent probes, antitumor drugs, or therapeutic bacteria. For this, two previously described and characterized nanobodies will be used for targeting these cell surface biomarkers: the anti-PSMA nanobody called NB7 and the anti-CEA nanobody called NbCEA5. Two nanobody guided antitumor methodologies will be tested and compared:1 - Nanobodies carrying antitumor drugs. A p-azido-phenylalanine residue (p-azido-Phe or pAzF) will be bioincorporated in a specific position of these nanobodies, during expression in E. coli cells. The presence of this azide group will allow the subsequent conjugation of these nanobodies with fluorescent probes (Cy5) or antitumor drugs (monomethyl Auristatin E MMAE) by click-chemistry. Cleavable and non-cleavable spacers will be used for comparison regarding cytotoxicity in tumor cells.2 - Nanobodies targeting therapeutic bacteria. Therapeutic (antitumor) bacteria will be prepared and transformed to express and expose these nanobodies on their surfaces, in such a way that the therapeutic bacteria are guided to positive PSMA or CEA tumor cells. This exposure of the nanobodies will be done through fusion with a protein from the outer membrane of E. coli called intimin. In addition, for the programming of antitumor bacteria, we will use plasmid vectors with a circuit of genes activated by a minimum concentration of a quorum sensing signal (N-acylhomoserine lactone, AHL), which is only reached when there is a minimum population of bacteria growing in the same environment. And this circuit of genes, when activated, makes the bacteria produce an antitumor (therapeutic) protein, and a protein that causes the lysis of the bacterial cell, in such a way that the therapeutic protein is released into the neighborhood by the synchronized lysis of a minimum population of bacterial cells.For a comparative scale of effectiveness, toxicity and specificity, we will treat positive (PSMA+ or CEA+) and negative (PSMA- or CEA-) cultured cells with: 1 - Anti-biomarker antibodies (PSMA or CEA) unconjugated, conjugated with groups fluorescent (Cy5) or conjugated with antitumor drugs; 2 - Bacteria programmed for synchronized lysis and 3 - Bacteria programmed for synchronized lysis driven by anti-biomarker antibodies (PSMA or CEA). Prostate cancer and colorectal cancer model cells will be used.At the same time, through collaborations, we intend to develop new antibody fragments against PSMA and CEA, and in the future, against other tumor cell biomarkers and/or bacterial biofilms, to extend and apply the developed technologies. (AU)
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