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+ | <br> | ||
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<p> | <p> | ||
− | + | Taking <b>breast cancer</b> which has the highest incidence rate in the world as an opponent, we need to be very careful in dealing with every step of implementing our project into reality. | |
+ | In the real world, we believe that thousands of researchers and drug companies are also worried about the plight of breast cancer treatment and come up with countless ideas. | ||
+ | However, if we want to really <b>improve the survival status</b> of breast cancer patients, we need to prove our project is valuable enough to <b>contribute to society</b>. | ||
+ | <br> | ||
+ | In this page, we extend the content about <b>Proof of Concept</b> on the basis of the project implementation plan, and explain that each part of our project is running in a relevant | ||
+ | context. | ||
</p> | </p> | ||
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+ | <div class="section Cloning" id="technical_feasibility"> | ||
<div class="container1"> | <div class="container1"> | ||
− | <h2 | + | <h2>Technical Feasibility</h2> |
− | < | + | <h3>Interaction of Two Recombinant Proteins</h3> |
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<p> | <p> | ||
− | + | Using the interaction system of Fc-ZZ as our linker, we linked the mamC expressed in <i>E.coli</i> BL21 and scFv expressed in <i>E.coli</i> SHuffle® as a compound BioBrick, indicated that we | |
− | + | can immobilized scFv on the surface of magnetosomes produced by <i>Magnetospirillum gryphiswaldense</i>. | |
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− | <img src="https://static.igem.org/mediawiki/2020/ | + | <img src="https://static.igem.org/mediawiki/2020/thumb/f/fc/T--ZJU-China--Proof_Of_Concept_fig1.jpg/800px-T--ZJU-China--Proof_Of_Concept_fig1.jpg"></img> |
− | <h6> | + | <h6>Fig1. Western-blot results of immunoprecipitation between mamC-ZZ and scFv-Fc.</h6> |
− | + | ||
</div> | </div> | ||
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− | < | + | <br> |
− | + | <p>All the protein concentrations from each block were determined by UV spectrophotometry. Total protein obtained mamC-ZZ was <b>24.8mg/mL</b>, purified mamC was <b>0.3mg/mL</b>, purified scFv was | |
− | < | + | <b>0.2mg/mL</b>. In industry, it is not difficult to achieve that level of protein concentration, which proves that our products are easy to be manufactured.</p> |
− | </ | + | <h3>Effectiveness of scFv</h3> |
− | <br> | + | <p>In order to prove that scFv-Fc fusion protein can specifically target HER2 positive breast cancer cells, we have demonstrated the specificity and effectiveness of this targeting by flow cytometry.</p> |
+ | <br><br> | ||
<div class="imgbox"> | <div class="imgbox"> | ||
− | < | + | <img src="https://static.igem.org/mediawiki/2020/c/ce/T--ZJU-China--Results_fig10_newone.jpg" alt=""> |
− | + | <h6>Fig2. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.</h6> | |
− | + | ||
</div> | </div> | ||
+ | <p>Obviously, the high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence (about 10 times) than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Fig2).</p> | ||
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− | < | + | <h3>Modeling Results</h3> |
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− | + | We simulated the process of the two recombinant proteins from the internal expression of bacteria to the interaction and binding in vitro, and simulated the whole process of | |
− | + | our final product diffusion in tumors and binding with HER2 as well. | |
− | + | <br> | |
− | + | Through all those modelling results delivered in <a href="https://2020.igem.org/Team:ZJU-China/Model" class="outerlink">Model</a>, we have a general understanding of the | |
− | + | production of two recombinant in <i>E.coli</i> and <i>Magnetospirillum</i>, and the speed and | |
− | + | proportion of the Fc-ZZ interaction system in vitro, and the dispersion and binding of scFv modified magnetosomes in tumor. These data provide us a possible range of contrast | |
− | < | + | agent injection dose in future clinical trials. |
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− | + | <h3>Further Approach of Technical Feasibility</h3> | |
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<p> | <p> | ||
− | + | After we obtain the engineered magnetosomes and equip them with scFv, we will carry out in vitro experiments on cytotoxicity and contrast effect of MagHER2some. When all | |
+ | indexes show a good trend, we will carry out in vivo experiments on mice and follow-up clinical trials through legal application according to the requirements of China's laws. | ||
+ | You can learn more from <b>the Clinical Trial</b> section in <a href="https://2020.igem.org/Team:ZJU-China/Implementation" class="outerlink">Implementation</a>. | ||
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+ | <div class="section about" id="commercial_feasibility"> | ||
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− | < | + | <h2>Commercial Feasibility</h2> |
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− | + | <h3>Market Investigation</h3> | |
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<p> | <p> | ||
− | + | As a team targeted to develop a novel contrast agent with a innovative design, we conducted a comprehensive investigation with patients, medical specialists in breast cancer, | |
+ | radiologist, researchers in pharmocochemistry, company researchers, etc. You can learn more from the <a href="https://2020.igem.org/Team:ZJU-China/Human_Practices" class="outerlink">Human Practice</a> section. | ||
+ | <br> | ||
+ | We also developed industry analysis from various aspects, you can learn more from the <a href="https://2020.igem.org/Team:ZJU-China/Implementation" class="outerlink">Implementation</a> section. | ||
+ | <br> | ||
+ | We gradually figured out that there was a lack of targeted contrast agents in the market. Targeted contrast agents such as MagHER2some have dual economic and social value, | ||
+ | because they can reduce the proportion of empirical judgement in the analysis of MRI results, so as to achieve a more accurate prognosis treatment of breast cancer. As a | ||
+ | result, the treatment of breast cancer patients will be improved, and much more people will free from the torture of the disease. | ||
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+ | <h3>Advantage Demonstration</h3> | ||
<p> | <p> | ||
− | + | Compared with gadolinium based contrast agent (GBCA), MagHER2some goes beyond it from the following three aspects: | |
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− | <img src="https://static.igem.org/mediawiki/2020/ | + | <img src="https://static.igem.org/mediawiki/2020/thumb/6/6a/T--ZJU-China--Proof_Of_Concept_table1.jpg/800px-T--ZJU-China--Proof_Of_Concept_table1.jpg" alt=""> |
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− | <p> | + | <p>As the table showed above, MagHER2some is an innovative and superior contrast agent compared with traditional products. Based on what we have done during iGEM, we can foresee that MagHER2some will be a highly specific, non-toxic and environmentally friendly product to enhance the MRI contrast.</p> |
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− | </p> | + | <h3>Commercial Strategy</h3> |
+ | <p>We have developed a business plan based on our experiments and investigation results. The contents of business plan are a prototype that demonstrate the method that we implement Proof of Concept activities at the early stage of commercialization. You can learn more from the <a href="https://2020.igem.org/Team:ZJU-China/Implementation" class="outerlink">Implementation</a> section.</p> | ||
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<div class="top-arrow"> | <div class="top-arrow"> |
Revision as of 14:44, 27 October 2020
Proof Of Concept
Taking breast cancer which has the highest incidence rate in the world as an opponent, we need to be very careful in dealing with every step of implementing our project into reality.
In the real world, we believe that thousands of researchers and drug companies are also worried about the plight of breast cancer treatment and come up with countless ideas.
However, if we want to really improve the survival status of breast cancer patients, we need to prove our project is valuable enough to contribute to society.
In this page, we extend the content about Proof of Concept on the basis of the project implementation plan, and explain that each part of our project is running in a relevant
context.
Technical Feasibility
Interaction of Two Recombinant Proteins
Using the interaction system of Fc-ZZ as our linker, we linked the mamC expressed in E.coli BL21 and scFv expressed in E.coli SHuffle® as a compound BioBrick, indicated that we can immobilized scFv on the surface of magnetosomes produced by Magnetospirillum gryphiswaldense.
Fig1. Western-blot results of immunoprecipitation between mamC-ZZ and scFv-Fc.
All the protein concentrations from each block were determined by UV spectrophotometry. Total protein obtained mamC-ZZ was 24.8mg/mL, purified mamC was 0.3mg/mL, purified scFv was 0.2mg/mL. In industry, it is not difficult to achieve that level of protein concentration, which proves that our products are easy to be manufactured.
Effectiveness of scFv
In order to prove that scFv-Fc fusion protein can specifically target HER2 positive breast cancer cells, we have demonstrated the specificity and effectiveness of this targeting by flow cytometry.
Fig2. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.
Obviously, the high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence (about 10 times) than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Fig2).
Modeling Results
We simulated the process of the two recombinant proteins from the internal expression of bacteria to the interaction and binding in vitro, and simulated the whole process of
our final product diffusion in tumors and binding with HER2 as well.
Through all those modelling results delivered in Model, we have a general understanding of the
production of two recombinant in E.coli and Magnetospirillum, and the speed and
proportion of the Fc-ZZ interaction system in vitro, and the dispersion and binding of scFv modified magnetosomes in tumor. These data provide us a possible range of contrast
agent injection dose in future clinical trials.
Further Approach of Technical Feasibility
After we obtain the engineered magnetosomes and equip them with scFv, we will carry out in vitro experiments on cytotoxicity and contrast effect of MagHER2some. When all indexes show a good trend, we will carry out in vivo experiments on mice and follow-up clinical trials through legal application according to the requirements of China's laws. You can learn more from the Clinical Trial section in Implementation.
Commercial Feasibility
Market Investigation
As a team targeted to develop a novel contrast agent with a innovative design, we conducted a comprehensive investigation with patients, medical specialists in breast cancer,
radiologist, researchers in pharmocochemistry, company researchers, etc. You can learn more from the Human Practice section.
We also developed industry analysis from various aspects, you can learn more from the Implementation section.
We gradually figured out that there was a lack of targeted contrast agents in the market. Targeted contrast agents such as MagHER2some have dual economic and social value,
because they can reduce the proportion of empirical judgement in the analysis of MRI results, so as to achieve a more accurate prognosis treatment of breast cancer. As a
result, the treatment of breast cancer patients will be improved, and much more people will free from the torture of the disease.
Advantage Demonstration
Compared with gadolinium based contrast agent (GBCA), MagHER2some goes beyond it from the following three aspects:
As the table showed above, MagHER2some is an innovative and superior contrast agent compared with traditional products. Based on what we have done during iGEM, we can foresee that MagHER2some will be a highly specific, non-toxic and environmentally friendly product to enhance the MRI contrast.
Commercial Strategy
We have developed a business plan based on our experiments and investigation results. The contents of business plan are a prototype that demonstrate the method that we implement Proof of Concept activities at the early stage of commercialization. You can learn more from the Implementation section.