Team:Fatih-Medical/Sherlocoli/Design

We need an effective tumor specific antibody to discriminate tumor cells in the media.
CD225 is an antibody which has the high binding affinity to the epithelial cell adhesion molecules (EpCAMs) which are widely present on the cell membranes of various tumor cells such as some types of breast cancer or colorectal cancer subtypes. We aim to use this ability as a recognition signal inducer with help of transmembrane proteins using as bases. CD225 has three different binding domains on EpCAM antigens, which poses a great opportunity to carry out several binding styles to induce the first signal reaction.

Our bacteria should easily present these antibodies on their cell wall.
OmpA is a transmembrane protein which is generally located at outer membrane of gram negative bacteria such as E.coli. It is widely used as protein presenting membrane “docks” both in the outer or inner media of bacterial cell. With the help of binding fragments which are attached both of the sides of OmpA; the desired protein can be presented in the desired location. In our design, we intend to add our antibody into the location of outer media of OmpA with aim of using it as antibody presenting site. (Figure 1)

Figure 1

The recognition system should be only activated in the course of antibody-antigen reaction.
To establish such system, we designed a two antibody mediated transfection system based on TEV protease N and C terminal fragments. As it was mentioned above, EpCAM has several binding sites for CD225 antibodies. This allows us to use this antibody to enhance different bindings in the case using only this type of antibody. Thus, we planned to assemble our antibodies with OmpAs which are attached with N terminus and C terminus of TEV protease separately. (Figure 2) So, in the course of binding different sites, TEV protease fragments would come together and get fixed physically which is called transfection. (Figure 3) It is planned to use this newly fixed TEV protease to induce signal amplification.

Figure 2

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Figure 3

The system must activate a reporter system in the bacterial cytoplasm.
After the generation of fully functional TEV protease, our pre-synthesized anchor-inducer proteins (Figure 4 and 5) which are built up in the cytoplasm would be cleaved in order to release the inducer free. (Figure 6) Therefore, the freeing inducer can activate the first sender device which is present in the plasmid of our tumor recognizing bacterium to start a signal amplification reaction between the colonies of E.coli in the tumor cell including media. (Figure 7) It is also planned alternatively to add an anchor-reporter system to visualize the first recognition with help of the freeing reporter protein (in this case, fluorescent proteins or characteristic enzymes) in the case of the tumor cell binding. Figure 4

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Figure 5

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Figure 6

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Figure 7