Team:NCTU Formosa/Project
Contents
Background
Nowadays, the top cancers include lung cancer, colorectal cancer, breast cancer, melanoma and thyroid cancer. According to NCI, in 2015, the estimated number of new cases of these cancers are 724,410. The estimated death cases of these cancers are 260,360. And the number is still growing. It might sound really hopeless .Actually, all of these cancers could be treated by targeted drug in proper condition.
Targeted drugs therapy, such as monoclonal targeted drugs, block the growth and spread of cancer by interfering with specific molecules("molecular targets") that are involved in the growth, progression, and spread of cancer. It often treats cancers with chemotherapy to enhance the effect of cancer therapies. According to the statistics, the usage of targeted drugs therapy does have effective treatment in cancer, decreasing the morality of cancer and increasing the five-year survival rate after treatment. Furthermore, the research also indicated that utilizing different targeted drugs, also known as multi marker, to treat cancers simultaneously attain greater effects than only using one targeted drug. However, targeted drugs therapy must treat patients in the proper condition to reach the best effect of the therapy. What’s worse, the improper usage of targeted drugs therapy would not only waste money and medical resources but also cause the invalid treatment result to patients.
Above all, defining whether to use the targeted drugs might be the crucial point of targeted drugs therapy. Therefore, NCTU_Formosa focuses on creating a multiple marker diagnosis platform for helping doctors to judge whether to use monoclonal antibody (mAb) targeted drugs by innovate methods directly.
Main idea
To offer judgments of using targeted drugs to doctors, in this project our team created a multiple marker diagnosis platform. The Apollo E.cotector was designed to achieve our goal – determining which monoclonal antibody (mAb) target drug to use.
DESIGN
Concept: Displaying single chain variable fragment(scFv) of antibody drugs on the surface of E.coli
scFv (single chain variable fragment)
We redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin®), Cetuximab (Erbitux®) and Trastuzumab (Herceptin®) into a kind of recombinant antibody - scFv, which is a fusion protein of the variable regions of the heavy chains (VH) and light chains (VL) of immunoglobulin connected by a short linker peptide.
Apollo E.cotector
Apollo E.cotector displays these scFv sequences on the surface of E.coli by using the Lpp-OmpA, the transmembrane protein[1]. The scFv antibody fragments, displaying on the surface are anti-VEGF (Bevacizumab), anti-EGFR (Cetuximab) and anti-HER2 (Trastuzumab).
Cell Staining
We applied Apollo E.cotector that displays various scFvs with diverse fluorescence protein to stain cancer cells. With this multiple marker detection, we can determine which combination of targeted drugs is suitable for the patient.
GBP
Summary
This year, our hit project, E.Cotector is to assist the medical practitioners to choose the appropriate targeted drug therapies for various conditions of patients. Before doctors prescribing the targeted drugs for cancer patients, E.Cotector can mark the tumor cells or test the antigens in serum by part of monoclonal antibodies (scFv) which is a kind of targeted drug directly binding with antigens. APOllO organization provided an advanced method in selecting personalized therapy for every particular patient.
- E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins:
- Simultaneously marked multiple kinds of overexpressed unique antigens on the cells.
- Amplified the signal by E. coli expressing fluorescence proteins.
- An innovative indicator to combine synthetic biology and numerous precision measurement technology.
- Achieve the extraordinary degree of precision in detecting concentration of antigens in the serum.
- Enhance the process yield in immobilization of antibodies on the medium gold surface.
E.Cotectors detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
Want to see more, please see Achievements page.
Biobrick Design
Lpp-OmpA-scFv
To display the antibody outside the E.coli outer membrane, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference [1], We chose the first 9 amino acids of Lpp to be the signal peptide, and the 46-159 amino acids of OmpA to be the anchor, Lpp-OmpA then fused the single chain variable fragment (scFv) C-terminally. We added a NcoI restriction side between OmpA and scFv so that we can change any scFv DNA sequence just by NcoI restriction enzyme.
By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA-scFv, we were able to display scFv outside the E.coli outer membrane continuously. At the back of this part, we have added fluorescent proteins as the reporters.
In our current work, we chose three targeted drugs, Avastin (Bevacizumab, anti-VEGF)[2], Erbitux (Cetuximab, anti-EGFR)[3] and Herceptin (Trastuzumab, anti-HER2)[4] from Drugbank, selecting their single chain variable fragments (scFv) to use, which is short and it will not give too much stress to E.coli.
At the back of Lpp-OmpA-scFv part, we ligated the weaker ribosome biding site (BBa_B0030), different fluorescent protein and terminator (BBa_J61048) to make it continuously express the fluorescence and the scFv at the same time so that we can apply our E.coli to cell staining. The reason why we used the weak ribosome biding site so that the expression of scFv will not be affected. In addition, by combining these different types of E.coli with different fluorescence, we are able to create a platform which can detect multimarker.
Reference
[1] C Hartmann et al. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response http://www.nature.com/onc/journal/v29/n32/pdf/onc2010195a.pdf
[2] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112
[3] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002
[4] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072
FadL-GBP
Gold binding peptide (GBP) is a kind of peptide which can bind on gold, usually used to immobilize protein on gold surface. The mechanism of how GBP bind the gold is not so understood, but its polar side-chains, such as serine and threonine, seem to interact with gold. We used a 42 amino acids long GBP, which contain three repeated amino acid sequences(MHGKTQATSGTIQS). To display GBP on cell surface, we used Long-chain fatty acid transport protein (FadL) as a transmembrane protein, selecting the first 384 amino acids to link with GBP [1], signal peptide included.
By ligating the constitutive promoter (BBa_J23110) ribosome binding site (BBa_B0034), FadL-GBP and terminator (BBa_J61048), we can continuously display the GBP outside the E.coli outer membrane so that our E.coli can bind on gold chip to apply on many measuring instruments.
Reference
[1] Tae Jung Park et al. (2009) Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface