Difference between revisions of "Team:NCTU Formosa/Results"

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<div class="title">Overview</div>
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Single chain variable fragment as probe
 
+
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<main class="cd-content">
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<div class="cd-container">
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<img src="https://static.igem.org/mediawiki/2015/1/16/2015_NCTU_Formosa_%E5%B0%8F%E6%A8%99.png"; width=300vw;height=200vh;>
+
 
+
<p>To prove that our E.Coli have successfully displayed scFv of Bevacixumab and Cetuxicumab respectively and the high specificity of our E.Cotector,
+
  we designed cell staining experiment. The mainly material that we needed are fluorescent E.Coli with scFv and without scFv, the cancer cells that expressed or displayed the specific antigens for staining used.
+
  
  <br>
+
<div class="content">
 
+
    Single chain variable fragment (scFv) Abs are one of the <font color=#b51c48> recombinant antibody(rAb)</font> fragments, which are popular therapeutic alternatives to full length of monoclonal Abs. Compared to generating whole Abs from animal cell culture, scFv are smaller and can be expressed rapidly, economically and in large quantities in a bacterial host, such as<font color=#b51c48> E. coli</font>. A scFv <font color=#b51c48>possesses the complete antigen binding site</font>, which contains the variable heavy (VH) and variable light domain of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide linker. A scFv is capable of binding its target antigens with an affinity similar to that of the parent mAb. Due to containing the specific antigen binding unit, scFv fragments show tremendous versatility and importance in<font color=#b51c48> human therapeutics and diagnostics</font>. [1] In addition, scFv fragments can be envisaged to be applied in the non-pharmaceutical sector, such as in the food, cosmetic or environmental industries. The unique and highly specific antigen-binding ability might, for example, be exploited to block specific enzymes (e.g. enzymes that cause food spoilage), bacteria (e.g. in toothpaste or mouthwashes) or to detect environmental factors present in very low concentrations (as biosensors).[2]
    Introduction of cell lines that we used: <br>
+
<br>
+
        1. MCF7 cell line: <br>
+
          MCF7 is a kind of epithelial cell of human breast cancer cells. There are overexpressed epithermal growth factor receptor (EGFR) on the cell surfaces.<br>
+
        <br>
+
        2. MDA-MB-231 cell line: <br>
+
          MDA-MB-231 is also a kind of epithelial cell that can produce vascular epithermal growth factor (VEGF) that will trigger the abnormal growth of vascular, angiogenesis.
+
           
+
+
<br>
+
        Before undergoing cell staining, we would cultivate MCF7 and MDA-MB-231 each in a 6-well plate. Besides, we would also cultivate our anti-EGFR E.Cotector, anti-VEGF E.Cotector and green fluorescent E.Coli as control
+
in experiment for 18 hours to make sure that the concentration of E.Coli are good enough for undergoing cell staining. Before cell staining, we had to add triton into some wells to permeabilize the cell membrane of MDA-MB-231.  
+
The reason to do so is that VEGF is growth factor that is produced and released by cancer cells and not receptors that are displayed on those cell surfaces.
+
        After injecting E.Coli into the wells, we had shaken the plates in darkness for 1hour. After staining for 1 hour, we will wash away unbind E.Coli with PBS solution for a few times before observing the staining result under fluorescent microscope.
+
        In our hypothesis, if we abled to observe anti-VEGF E.Cotector penetrate into the MDA-MB-231 cells and bind specifically with VEGF inside the cells, our anti-VEGF E.Cotector can be said to be perform well and successfully.
+
In the other hand, when our anti-EGFR E.Cotector can bind with EGFR which are displayed on the MCF7 cell surfaces by staining on the surfaces of MCF7 cells.  
+
  
        <br> </p>  
+
</div>
<img src="https://static.igem.org/mediawiki/2015/f/fb/2015_NCTU_Formosa_%E5%B0%8F%E6%A8%991.png"; width=400vw; height=76vh;>
+
<div class="contentitle">Properties and development of targeted drugs</div>
<p>  
+
<div class="content">
        <br>
+
This year, we decided to utilize the scFv as probes to detect cancer markers and aid in the prescription of targeted drugs in cancer treatments.
  1. Cultivate MCF7 cell lines and MDA-MB-231 cell lines in 6-well plates. Cultivate three types of E.Coli: <br><p></p>
+
Targeted drugs therapy utilize compounds that are capable of inhibiting target molecules, the cancer markers which send messages along signaling pathways in cell growth, cell division or cell death. Via specific binding to target molecules, targeted drugs show more accurate attack to cancer cells and less harmful damage to normal tissues. [1] The precision of targeting the cancer cells has enhanced the efficiency of treatment by a large margin. The targeted therapy is a major step forward for many cancers, especially advanced cancers, and physicians and researchers are now focusing on the development of targeted drugs, creating a new era of personalized cancer treatment.[3]Targeted therapy are so-called "personalized medicine" because health care professionals can use clinical test results from a patient to select a specific drug that has a higher likelihood of being effective for that particular person.<br><br>
    the normal green fluorescent E.Coli, the anti-EGFR E.Cotector, the anti-VEGF E.Cotector for 18 hours and 4mL each.<br><p></p>
+
According to the statistics, the usage rate of targeted drug therapy has increased within ten years. In Figure 1, in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over one decade, it is estimated that the usage of targeted drug therapy dramatically increases to<font color=#b51c48> 46%</font>. It indicates targeted drugs therapy is a potential growing field and will become the commonly used therapy in cancer treatments in the near future.
          2. Firstly, remove the cell cultivated solution from the wells. Add in 4% paraformaldehyde to kill and fixed the cells as paraformaldehyde able to prevent the dead cells from floating in the wells. Keep the plates in darkness for 15min.<br>
+
</div>
          3. At the same time, separate the LB broth with bacteria pellets by centrifuge. Wash the pellets with PBS buffer and re-dissolve the pellets with PBS buffer.  <br>
+
<br><br>
  <p></p>
+
<div class="contentitle">Pre-diagnosis of targeted drugs treatment</div>
          4. Remove the paraformaldehyde and wash the wells three times with PBS buffer, the time interval is 10min and shake the plates on shaker.<br><p></p>
+
<div class="content">
          5. Add in triton for permeabilizing the cell membranes and shake in darkness for 10min. Remove triton and repeat the action of washing by using PBS buffer.<br><p></p>
+
To create the new era of tailored targeted drugs, doctors must aim at<font color=#b51c48> appropriate target molecules </font>for patients with particular diseases. In 2014,<font color=#b51c48> the U.S. Food and Drug Administration (FDA) </font>issued a guidance to facilitate the development and review of <font color=#b51c48>diagnostics tests</font>. The diagnostics tests are the steps to identify the abnormal cancer biomarkers. Moreover, the purpose of diagnostics tests are to help medical practitioners <font color=#b51c48>determine which patients could benefit from the certain drugs</font>, conversely, those who should not receive the medication. If the treatment decisions is not optimal, it would not only cause the fatal body damage, but also lead to the waste of time, money and medical resources. FDA encourages the joint of targeted drugs therapies and precise diagnostics tests which are essential for the safe and effective use of targeted drugs.[4]
          6. Add in 500µL of E.Coli in each well. Shake the plates in darkness for 1 hours for cell staining. <br><p></p>
+
</div>
          7. After 1 hour, remove the E.Coli and wash with PBS buffer. Observe the staining result under the fluorescent microscope. <br><p></p>
+
<div class="contentitle">The concept of combination therapy</div>
          </p>  
+
<div class="content">
</div>
+
Although targeted drugs treatments can lead to the dramatic regressions of solid tumors, the responses are often short-lived because resistant cancer cells arise after a period of treatment. The major strategy proposed for overcoming the resistance is <font color=#b51c48>combination therapy</font>. The clinical and preclinical researches further indicated that targeted drug therapy combined with another targeted drug therapy or other types of therapies to treat cancers simultaneously may attain greater effects than using only one therapy. With the concept of combination therapy, we can not only improve the treating effect but also reduce the occurrence of cancer cells resistance toward the targeted drugs as there are less probability that a single mutation will cause cross-resistance to both drugs.[2] </div>
+
<div class="contentitle">APPOllO E.Cotector</div>
</main> <!-- cd-content -->
+
<div class="content">To enhance the <font color=#b51c48>efficiency of diagnosis </font>and provide reference for<font color=#b51c48> proper usage of targeted drugs</font> and <font color=#b51c48>combination therapy</font>, we come up with the idea of detecting multimarker at the same time and this was how our marvelous E.Cotector is borned. This year, NCTU_Formosa commits to creating a multimarker diagnosis platform via scFv as probes for helping physicians to determine and prescribe the usage of targeted drugs in cancer patients, especially the monoclonal-antibody-targeted drugs.</div>
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+
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+
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+
 
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Revision as of 17:27, 8 September 2015

Overview
Single chain variable fragment as probe
Single chain variable fragment (scFv) Abs are one of the recombinant antibody(rAb) fragments, which are popular therapeutic alternatives to full length of monoclonal Abs. Compared to generating whole Abs from animal cell culture, scFv are smaller and can be expressed rapidly, economically and in large quantities in a bacterial host, such as E. coli. A scFv possesses the complete antigen binding site, which contains the variable heavy (VH) and variable light domain of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide linker. A scFv is capable of binding its target antigens with an affinity similar to that of the parent mAb. Due to containing the specific antigen binding unit, scFv fragments show tremendous versatility and importance in human therapeutics and diagnostics. [1] In addition, scFv fragments can be envisaged to be applied in the non-pharmaceutical sector, such as in the food, cosmetic or environmental industries. The unique and highly specific antigen-binding ability might, for example, be exploited to block specific enzymes (e.g. enzymes that cause food spoilage), bacteria (e.g. in toothpaste or mouthwashes) or to detect environmental factors present in very low concentrations (as biosensors).[2]
Properties and development of targeted drugs
This year, we decided to utilize the scFv as probes to detect cancer markers and aid in the prescription of targeted drugs in cancer treatments. Targeted drugs therapy utilize compounds that are capable of inhibiting target molecules, the cancer markers which send messages along signaling pathways in cell growth, cell division or cell death. Via specific binding to target molecules, targeted drugs show more accurate attack to cancer cells and less harmful damage to normal tissues. [1] The precision of targeting the cancer cells has enhanced the efficiency of treatment by a large margin. The targeted therapy is a major step forward for many cancers, especially advanced cancers, and physicians and researchers are now focusing on the development of targeted drugs, creating a new era of personalized cancer treatment.[3]Targeted therapy are so-called "personalized medicine" because health care professionals can use clinical test results from a patient to select a specific drug that has a higher likelihood of being effective for that particular person.

According to the statistics, the usage rate of targeted drug therapy has increased within ten years. In Figure 1, in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over one decade, it is estimated that the usage of targeted drug therapy dramatically increases to 46%. It indicates targeted drugs therapy is a potential growing field and will become the commonly used therapy in cancer treatments in the near future.


Pre-diagnosis of targeted drugs treatment
To create the new era of tailored targeted drugs, doctors must aim at appropriate target molecules for patients with particular diseases. In 2014, the U.S. Food and Drug Administration (FDA) issued a guidance to facilitate the development and review of diagnostics tests. The diagnostics tests are the steps to identify the abnormal cancer biomarkers. Moreover, the purpose of diagnostics tests are to help medical practitioners determine which patients could benefit from the certain drugs, conversely, those who should not receive the medication. If the treatment decisions is not optimal, it would not only cause the fatal body damage, but also lead to the waste of time, money and medical resources. FDA encourages the joint of targeted drugs therapies and precise diagnostics tests which are essential for the safe and effective use of targeted drugs.[4]
The concept of combination therapy
Although targeted drugs treatments can lead to the dramatic regressions of solid tumors, the responses are often short-lived because resistant cancer cells arise after a period of treatment. The major strategy proposed for overcoming the resistance is combination therapy. The clinical and preclinical researches further indicated that targeted drug therapy combined with another targeted drug therapy or other types of therapies to treat cancers simultaneously may attain greater effects than using only one therapy. With the concept of combination therapy, we can not only improve the treating effect but also reduce the occurrence of cancer cells resistance toward the targeted drugs as there are less probability that a single mutation will cause cross-resistance to both drugs.[2]
APPOllO E.Cotector
To enhance the efficiency of diagnosis and provide reference for proper usage of targeted drugs and combination therapy, we come up with the idea of detecting multimarker at the same time and this was how our marvelous E.Cotector is borned. This year, NCTU_Formosa commits to creating a multimarker diagnosis platform via scFv as probes for helping physicians to determine and prescribe the usage of targeted drugs in cancer patients, especially the monoclonal-antibody-targeted drugs.