Difference between revisions of "Part:BBa K3117029"
 
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<partinfo>BBa_K3117029 short</partinfo>
 
<partinfo>BBa_K3117029 short</partinfo>
  
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BBa_K3117026 is a fusion protein of an anti-CD3 single-chain variable fragment (scFv) and SpyTag (BBa_K3117015).
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The composite part <partinfo>BBa_K3117029</partinfo> is a bispecific antibody targeting GPA33 and CD3. It can be used to direct T lymphocytes to colon carcinoma cells expressing GPA33.  
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (<partinfo>BBa_K3117020</partinfo>, <partinfo>BBa_K3117021</partinfo>, <partinfo>BBa_K3117022</partinfo>, <partinfo>BBa_K3117023</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 (BBa_K3117005) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (<partinfo>BBa_K3117006</partinfo>). When the protein passes the membrane, this leader domain is cleaved off.
  
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The sequence contains a C-terminal His-tag for easy purification and detection. Secretion of the protein is ensured by an Igk leader. When the protein passes the membrane, the leader segment is cleaved off. By connecting the variable regions of the heavy and the light chain of an anti-CD3 antibody with a short GGGGS linker (BBa_K3117004), the scFv retains its antigen-binding ability and is much smaller than a conventional antibody. Thus, it is well suited as part of a fusion protein with another effector. The SpyTag attached to the scFv belongs to the SpyTag/SpyCatcher system and is one part of the FbaB protein of Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyCatcher (BBa_K3117016), they bind covalently. This allows our part to be used in a modular manner in combination with other molecules carrying the SpyCatcher.
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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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Our part targets CD3 on T lymphocytes, and thereby activates them. The most prominent effect of such activation is the release of cytotoxic granula into the environment of the cell. T cells play an integral part in the immune system of the body and are e.g. tasked with the identification and destruction of aberrant cells. Anti-CD3 antibodies are therefore a major tool in cancer research and also in cancer therapy (Ellerman, 2019).
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By providing a modular platform, our part can be used in combination with a large variety of effector molecules and other antibodies to further evaluate the therapeutic potential of this application.
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===Characterization and measurement===
 
===Characterization and measurement===
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This compisite part (K1) was synthesized by IDT. Then, it was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.
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The accuracy of the inserted DNA after cloning was confirmed with sequencing. The data is depicted in Fig. 1.
  
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[[File:T--FAU_Erlangen--Bild_Sequencing_K1_Results_Composite_part.png|thumb|center|800px|'''Figure 1''': Sequencing data of K1]]
  
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The construct 2b (K2b) was synthesized by IDT. It was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.
 
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The accuracy of the inserted DNA after cloning was confirmed with sequencing. The data is depicted in Fig. 1.
 
  
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Fig. 2 shows the western blot of the harvest after transfection into HEK 293T cells. For detection, the His-Tag in the K1 sequence (<partinfo>BBa_K3117005</partinfo>) provides the opportunity to be used as a target for a primary antibody in a western blot.
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K1 can be seen at expected height (54 kDa). The presence of K1 in the medium proves the function of the Igk leader (<partinfo>BBa_K3117006</partinfo>), which is directing the protein into the secretory pathway.
  
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Bild Sequencing K2b
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[[File:T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png|thumb|center|200px|'''Figure 2''': Western blot of the harvest after transfection into HEK 293T cells with an anti His-Tag antibody]]
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Figure 1: Sequencing data of the complete K2b
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Fig. 2 shows the harvest after transfection into HEK 293T cells in a western blot. For detection, the His-Tag in the K2b sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot.  
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Furthermore the His-Tag (<partinfo>BBa_K3117005</partinfo>) in K1 allows purification of the protein with HisTrap columns. The western blot after this purification is shown in Fig. 3, showing the remaining presence of the protein after this step.  
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K2b can be seen at expected height (41 kDa). Presence of K2b in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.  
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Bild Ernte
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[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png|thumb|center|150px|'''Figure 3''': Western blot of the constructs after the purification via HisTrap]]
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Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti-His-Tag antibody
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Furthermore the His-Tag (BBa_K3117005) in K2b allows purification of the protein with HisTrap columns. The western blot after this purification is shown in Fig. 3, showing the remaining presence of the protein after this step.  
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===References===
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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.
  
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Bild Aufreinigung
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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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Figure 3: Western blot of the purified protein with HisTrap columns  with an anti-His_tag antibody
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For the SpyTag/SpyCatcher reaction the protein K2b was added to the protein K2a (BBa_K3117026). A height of 83 kDa was expected in the afterwards performed western blot, depicted in Fig 4. A specific band appeared after adding the two products, but this band was running at ~120 kDa. This size could be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).
 
  
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Bild TagCatcher
 
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Figure 4: Western blot after SpyTag/SpyCatcher reaction
 
  
 
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Latest revision as of 23:27, 21 October 2019


scFv bispecific antibody against GPA33 and CD3 codon optimized for CHOs

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 expressing GPA33.


Usage and Biology

The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (BBa_K3117020, BBa_K3117021, BBa_K3117022, BBa_K3117023), 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 is ensured by an Igk leader (BBa_K3117006). When the protein passes the membrane, this leader domain is cleaved off.

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 947
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization and measurement

This compisite part (K1) was synthesized by IDT. Then, it was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection. The accuracy of the inserted DNA after cloning was confirmed with sequencing. The data is depicted in Fig. 1.

Figure 1: Sequencing data of K1


Fig. 2 shows the western blot of the harvest after transfection into HEK 293T cells. For detection, the His-Tag in the K1 sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot. K1 can be seen at expected height (54 kDa). The presence of K1 in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti His-Tag antibody

Furthermore the His-Tag (BBa_K3117005) in K1 allows purification of the protein with HisTrap columns. The western blot after this purification is shown in Fig. 3, showing the remaining presence of the protein after this step.

Figure 3: Western blot of the constructs after the purification via HisTrap

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.