Part:BBa_K3117030

scFv against CD3 with SpyTag codon optimized for CHOs

BBa_K3117026 is a fusion protein of an anti-CD3 single-chain variable fragment (scFv) and SpyTag (BBa_K3117015).

Usage and Biology

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 it's 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. 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. When the protein passes the membrane, this leader segment is cleaved off.

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).

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.

Characterization and measurement

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.

To assure DNA was correctly inserted during cloning samples were sequenced. Data is depicted in Fig. 1.

Figure 1: Sequencing data of K2b

Fig. 2 shows the harvest after transfection of HEK 293T cells in a western blot. For detection, the His-Tag (BBa_K3117005) in the K2b sequence provides the opportunity to be used as a target for a primary antibody in a western blot. 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.

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

Furthermore the His-Tag (BBa_K3117006) 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.

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

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 might be explained by formation of a trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Figure 4: Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody

References

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.

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.

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.

4. 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.