Engineering of E. coli bacteria for targeting human and murine epithelial tumor cells expressing HER2 and PD-L1 markers and their application in the colonization of mouse bladder tumors in vivo.

• Autores: Eva Pico Sánchez
• Directores de la Tesis: Luis Ángel Fernández Herrero (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2020
• Idioma: inglés
• Tribunal Calificador de la Tesis: José María Almendral del Río (presid.), Mar Valés Gómez (secret.), Molly Ingersoll (voc.), Jerome Bonnet (voc.), Juan Ignacio Aguiló Anento (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias agrarias
    • Fitopatología
      • Bacterias
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Anaerobic bacteria have been investigated as therapeutic agents in cancer treatments due to their natural capacity to colonize and grow preferentially in the hypoxic and immunosuppressed tumor microenvironment, that is found in solid tumors. Other bacterial features are also advantageous in cancer therapies, including their natural cytotoxicity, potent immune stimulation, and active motility that enable them to reach tumor regions non-accessible to therapeutics by passive diffusion. These beneficial features of bacteria can be enhanced by bacterial engineering using synthetic biology. One important aspect is the targeting of bacteria towards tumor cells, which could reduce systemic side effects to patients as well as increase specific toxicity against tumor cells. In this regard, our laboratory developed the so-called synthetic adhesins (SAs) that enable to precisely program the adhesion properties of E. coli bacteria to specific antigens present on abiotic and biotic surfaces, including the surface of tumor cells. SAs are chimeric fusion proteins based on the N-terminal domain of Intimin from enterohemorrhagic E. coli (EHEC), which anchors in the bacterial outer membrane, and a camelid single domain antibody fragment (nanobody, VHH), which provides binding specificity being displayed on E. coli surface.

    In this work, we have engineered E. coli strains with SAs binding human HER2 and mouse PD-L1 (mPD-L1), two tumor cell surface proteins frequently upregulated in cancers from different origins (i.e., breast, bladder, colon, melanoma). We have selected nanobodies against these tumor associated antigens from available VHH sequences against HER2 and from an immune library constructed after dromedary immunization with the ectodomain of mPD-L1. We demonstrated the functional surface display expression of the SAs in E. coli upon chromosomal integration and that the modified bacteria attach specifically to human and mouse tumor cell lines expressing these tumor antigens. Using an orthotopic syngeneic murine bladder cancer model, based on the implantation of MB49luc cells in the bladder of C57BL/6 mice, we demonstrated the ability of the engineered E. coli bacteria binding mPD-L1 to colonize these bladder tumors after their intravesical transurethral administration. Importantly, unlike an uropathogenic E. coli (UPEC) strain, the engineered bacteria were not able to colonize the bladders of healthy animals. At high bacterial doses (107 CFU), tumor colonization was observed irrespective of the specificity of the SA expressed by the engineered strain. However, with low doses (i.e., 105 -106 CFU), only the engineered strain binding mPD-L1 was found in large tumors and tumors derived from MB49 cells treated with interferon-g, which upregulate mPD-L1 levels. These data demonstrate in immunocompetent mice the specific colonization of bladder tumors by the engineered E. coli strain, opening its future development as a chassis for the expression of therapeutic proteins against bladder cancer.