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Team:NCTU Formosatest2
From 2014.igem.org
To help doctors with innovated methods to judge whether to use monoclonal antibody targeted drugs more directly ,we redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin® anti-VEGF)[1], Cetuximab (Erbitux® anti-EGFR)[2] and Trastuzumab (Herceptin® anti-HER2)[3] into recombinant antibodies : scFv (single chain variable fragment). For detecting the specific molecules ("targeted molecules"), which are specific to these scFv of targeted drugs, scFv are displayed on the surfaces of E.coli by using a transmembrane fusion protein, Lpp-OmpA[5] respectively; that is one APOllO E.Cotector displays one kind of targeted drugs of scFv each time. Take an example, while the anti-EGFR scFv (originate from Cetuximab) is displayed on the surface of E.coli, we can detect EGFR by this APOllO E.Cotector. Furthermore, as APOllO E.Cotector expressed fluorescence protein at the same time, the specific molecules ("targeted molecules") that correspond to those scFv of targeted drugs can be identified individually in direct by those different colors APOllO E.Cotector. In a simple word, because of the APOllO E.Cotector that display scFv of targeted drugs can we identify the corresponding specific molecules ("targeted molecules") ,then achieve to offer a prescription of monoclonal antibody targeted drug in direct .
Figure 1. Concept of APOllO E.Cotecter:Displaying scFv of antibody drugs on the surface of E.coli
Figure 2. Concept of our Application: Cell Staining
scFv is a fusion protein of the variable regions of the heavy chains(VH) and light chains (VL) of antibody connected by a flexible linker peptide. scFv still reserve completely functional antigen-binding fragment and specificity of the original immunoglobulin; that is , the property of specificity of antibody to antigen has being maintained. Moreover, scFv is only 20 percent the size as antibody [4], therefore it will not cause stress to E.coli for displaying it
In clinical situation, doctors may stain cancer tissue slides by specific antibody to judge whether to use targeted drugs therapy. As we utilizing those fluorescence APOllO E.Cotectors, which respectively display scFv of targeted drugs, such as anti-EGFR scFv , anti-VEGF scFv and anti-HER2 scFv to stain the cancer cells, they may specify each of the targeted molecules –EGFR,VEGFR or HER2 on the cell directly in same time . Therefore, doctors may obtain results to define whether these monoclonal antibody targeted drugs is proper to use for the patient or not. That is, by this application, we will offer a multimarker diagnosis for doctors to judge whether to use combination targeted drugs
1.The design of scFv of targeted drugs
2.The usage of Cell Staining
Reference
[1] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112
[2] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002
[3] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072
[4] Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626261/
[5] 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
While imaging tests and tissue biopsies are the most common methods for diagnosing cancer, serum tests can also help doctors identify the cancer by the usage of certain indicators, the antigens. Via measuring the levels of antigens, our APOllO E.Cotector Plus can help doctors to suggest the diagnosis as the reference for prescription of target therapy[1].
In our APOllO E.Cotector Plus design, we engineered the E.coli to display dual display system. One of the system displayed scFv by the transmembrane protein Lpp-OmpA. Meanwhile, the other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip, the material commonly used in biosensors[2].
By processing our E.Cotector Plus, with gold binding polypeptide connecting to the gold chip, we simplified the procedures of the self-assemble modification (SAM), the technique to immobilize antibodies on the gold surface[3]. We skipped the procedure of the gold surface decoration with sulfur bond and omitted the carbon chains attaching to antibody, which may occasionally block the binding sites of scFv,
further increasing the efficiency of antibody-antigen interactions.
Combined with the idea of the biosensor, we deemed our E.cotectorplus played the role of biological recognition part and the gold chip acted as the transducer part.
Therefore, our E.cotectorplus, which is able to attach on gold chip, can be regarded as the platform for precise physicochemical nanoscale detectors, such as Quartz crystal microbalance, Surface plasmon resonance Spectroscopy, Dual-polarization interferometry, ellipsometry, etcetera.
With our powerful APOllO E.Cotector Plus , we solved the time-consuming and sophisticated process of gold surface modification. What's more, by coupling our E.Cotector Plus to precise measurement instruments, we can achieve a huge boost to provide more sensitive and specific scFv detecting techniques options,
and give more reliable diagnosis for doctors to apply monoclonal antibody target drugs.
Figure 3. APOllO E.Cotector Plus contains dual display system.
One system displayed the transmembrane protein Lpp-OmpA fused with the sequence of scFv, which can bind with targeted antigens.
The other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip.
Figure 4. (a) When targeted antigens in patients' serum attach to scFv on APOllO E.Cotector Plus, the signal change of mass on the gold surface will be caught by the machines, such as Quartz crystal microbalance (QCM), Surface plasmon resonance Spectroscopy (SPR).Therefore, we can quantize and evaluate the level of binding antigens, and give the more accurate diagnosis for doctors.
(b)A biosensor is an analytical device, which can be divided into three elements including the sensitive biological element, the transducer and the detector with data evaluation device.
The gold binding polypeptide, abbreviated as GBP, is the three-repeated of following 14 aminoacid sequences: [MHGKTQATSGTIQS], which was developed in an E. coli cell-surface display system[4]. According to the paper, the binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)[5]. The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding. It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface[6].
The gold is the best choice for our biosensor substrate because of its advantages of stability to external environment, the excellent capability of transducing electronic signals, the sensitive physicochemical properties and, most important of all, the specific interaction with gold binding polypeptide.
Reference
[1]Blood Tests and Biomarkers http://www.asbestos.com/mesothelioma/blood-test.php
[2]Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface Tae Jung Park1,2, Shun Zheng1,2, Yeon Jae Kang2 & Sang Yup Lee1,2,3, Oxford University press, FEMS Microbiology Letters (2009)
[3]Biosensor surface chemistry for oriented protein immobilization and biochip patterning Linköping Studies in Science and Technology Licentiate Thesis No. 1573 (2013)
[4] Molecular characterization of a prokaryotic polypeptide sequence that catalyzes Au crystal formation, John L. Kulp III,a Mehmet Sarikayab and John Spencer Evans, Journal of Materials Chemistry(2004)
[5] Adsorption of genetically engineered proteins studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Part A: data acquisition and principal component analysis (PCA), Noriaki Suzuki,1 Lara Gamble,2 Candan Tamerler,3 Mehmet Sarikaya,1 David G. Castner2,4 (2007)
[6] Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)
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 the 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.
- 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. Want to see more, please see Achievements page.
E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins:
E.Cotectors Plus detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
NCTU_Formosa APOllO
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