Difference between revisions of "Part:BBa K3117046"
 
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<partinfo>BBa_K3117046 short</partinfo>
 
<partinfo>BBa_K3117046 short</partinfo>
  
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The part <partinfo>BBa_K3117026</partinfo> is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (<partinfo>BBa_K3117016</partinfo>). This part is codon optimized for the expression in E.coli.
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The composite part BBa_K3117029 is a bispecific antibody targeting GPA33 and CD3. It can be used to direct T lymphocytes to colon carcinoma cells by binding CD3 on T-cells and GPA33 on colon carcinoma cells. This part is codon optimized for the expression in E.coli.
  
 
===Usage and Biology===
 
===Usage and Biology===
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By connecting the variable regions of the heavy and light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (<partinfo>BBa_K3117028</partinfo>), the scFv retains it's antigen-binding ability and is much smaller than a conventional antibody. The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacteria Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyTag (<partinfo>BBa_K3117037</partinfo>), they bind covalently (Hatlem et al., 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag. The sequence contains a C-terminal His-Tag (<partinfo>BBa_K3117005</partinfo>) for easy purification and detection. Secretion of the protein into the perisplasm is ensured by the pelB sequence leader (<partinfo>BBa_K3117012</partinfo>). When the protein passes the membrane, the leader segment is cleaved off.  
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The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (<partinfo>BBa_K3117033</partinfo>, <partinfo>BBa_K3117035</partinfo>, <partinfo>BBa_K3117034</partinfo>, <partinfo>BBa_K3117046</partinfo>), which are connected by a flexible (GGGGS)4 linker (<partinfo>BBa_K3117004</partinfo>). The scFvs are formed by the variable regions of the heavy and light chain of the respective antibodies that are connected by either a (GGGGS)4 or (GGGGS)3 linker (<partinfo>BBa_K3117004</partinfo>, <partinfo>BBa_K3117028</partinfo>). The sequence contains a C-terminal His-Tag (<partinfo>BBa_K3117005</partinfo>) for easy purification and detection. Secretion of the protein into the periplasm is ensured by a pelB sequence (<partinfo>BBa_K3117012</partinfo>).
  
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Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.  
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T lymphocytes are part of the adaptive immune system and perform a wide variety of functions. Among these are the identification and the subsequent destruction of aberrant, e.g. cancerous, cells. Antibodies that target the T cell marker CD3 have been shown to be sufficient for activation of the lymphocytes. Which makes them an attractive tool for research and clinic, especially for cancer therapy (Ellerman, 2019). By targeting GPA33, expressed on over 95% of colon cancer cells (Rageul et al., 2009), our part is meant to link T cells with colon cancer cells and simultaneously activate them, so that the cancer cells are killed.  
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3117026 SequenceAndFeatures</partinfo>
 
  
 
===References===
 
===References===
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1. Hatlem, D., Trunk, T., Linke, D., & Leo, J. C. (2019). Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. International journal of molecular sciences, 20(9), 2129.
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1. Ellerman, D. (2019). Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety. Methods, 154, 102-117.
  
 
2. Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., . . . Denis, M. G. (2009). KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines. International journal of cancer, 125(12), 2802-2809.
 
2. Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., . . . Denis, M. G. (2009). KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines. International journal of cancer, 125(12), 2802-2809.
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3. Schoene, C., Fierer, J. O., Bennett, S. P., & Howarth, M. (2014). SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme. Angewandte Chemie International Edition, 53(24), 6101-6104.
 
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<partinfo>BBa_K3117026 parameters</partinfo>
 
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Latest revision as of 22:01, 21 October 2019


scFv bispecific antibody against GPA33 and CD3 codon optimized for E.coli

The composite part BBa_K3117029 is a bispecific antibody targeting GPA33 and CD3. It can be used to direct T lymphocytes to colon carcinoma cells by binding CD3 on T-cells and GPA33 on colon carcinoma cells. This part is codon optimized for the expression in E.coli.

Usage and Biology

The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (BBa_K3117033, BBa_K3117035, BBa_K3117034, BBa_K3117046), which are connected by a flexible (GGGGS)4 linker (BBa_K3117004). The scFvs are formed by the variable regions of the heavy and light chain of the respective antibodies that are connected by either a (GGGGS)4 or (GGGGS)3 linker (BBa_K3117004, BBa_K3117028). The sequence contains a C-terminal His-Tag (BBa_K3117005) for easy purification and detection. Secretion of the protein into the periplasm is ensured by a pelB sequence (BBa_K3117012).

T lymphocytes are part of the adaptive immune system and perform a wide variety of functions. Among these are the identification and the subsequent destruction of aberrant, e.g. cancerous, cells. Antibodies that target the T cell marker CD3 have been shown to be sufficient for activation of the lymphocytes. Which makes them an attractive tool for research and clinic, especially for cancer therapy (Ellerman, 2019). By targeting GPA33, expressed on over 95% of colon cancer cells (Rageul et al., 2009), our part is meant to link T cells with colon cancer cells and simultaneously activate them, so that the cancer cells are killed.


References

1. Ellerman, D. (2019). Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety. Methods, 154, 102-117.

2. Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., . . . Denis, M. G. (2009). KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines. International journal of cancer, 125(12), 2802-2809.