Part:BBa_K4839000
The scfv of tumor GPC3
Glypican-3 (GPC3) is a glycophosphatidylinositol (GPI)-anchored cell surface heparan sulfate proteoglycan that is expressed during early development. The overexpression of GPC3 is associated with hepatocellular carcinoma (HCC).
In our design, we use Huh7 cells as our invitro model of hepatocellular carcinoma. Huh7 cells exhibit several key characteristics associated with HCC, including the expression of tumor markers such as glypican-3 (GPC3).
To enable our artificially designed macrophages to target the GPC3 receptor, we searched for a series of highly effective GPC3 antibodies in the literatures. In our design, we wish to assemble the GPC3 antibody to our SNIPR protein (See design). In order to minimize the protein size and enhance expression efficiency as much as possible, we wish to find a Single-Chain Fragment Variable (scfv) protein instead of full-length antibody.
Zhang and Mitchell generated a series of high affinity anti-GPC3 mouse monoclonal antibodies (YP7, YP8, YP9, YP9.1) by immunizing mice with a C-terminal peptide derived from human GPC3 isoform 2. They initially tested one of these antibodies (YP7) and found that it had very specific binding towards HCC tumor cells in patient tissues and inhibited the growth of a hepatoblastoma xenograft tumor in nude mice. (Figure 1.)
Figure1. The binding efficiency of different antibodies
To humanize anti-GPC3 antibodies for potential clinical development, the YP7 and YP9.1 murine Fvs were selected because of their high affinity and cytotoxicity, (Figure2 and Figure3.)
Figure2. The protein sequence of YP7 and humanized YP7 (hYP7)
Figure3. The binding efficiency of different antibodies
Based on the existing work of Zhang and Mitchell, we are able to utilize this high affinity anti-GPC3 scfv in our design. We fused the variable heavy chain (VH) and variable light chain (VL) of hYP7 with GGGGS linker (Figure4). The combination of VH and VL domain of hYP7, which also called anti-GPC3 scfv will work together to target the GPC3 receptor.
In our design, we fuse the anti-GPC3 scfv to our SNIPR receptor (Figure5) for targeting GPC3-positive cancer cell (Huh7 or THP-1). Our experiment shows that the anti-GPC3 scfv can specifically target GPC3-positive cell and mediate downstream signal transduction.
Figure4. Structure of anti-GPC3 scfv
Figure5. Structure of anti-GPC3 SNIPR
[1] Zhu I , Liu R , Wittsten A H ,et al.Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells[J]. 2021.DOI:10.1101/2021.05.21.445218.
[2] Zhang Y F , Ho M .Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma[J].Scientific Reports, 2016, 6:33878.DOI:10.1038/srep33878.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 159
Illegal PstI site found at 241 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 159
Illegal PstI site found at 241 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 159
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 159
Illegal PstI site found at 241 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 159
Illegal PstI site found at 241 - 1000COMPATIBLE WITH RFC[1000]
| None |