Part:BBa_K3117029

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.