Part:BBa_K4634010

PD-L1 nanobody
PD-L1 nanobody is a camelid single-domain antibody fragment blocking PD-L1 (programmed cell death–ligand 1). It acts as an immune check point inhibitor, which have revolutionized the paradigm of cancer immunotherapy treatments. Unlike antibodies with a molecular size of ~150 kDa, nanobodies are typically ~15 kDa and lack an Fc region that requires glycosylation by mammalian cells, allowing them to be recombinantly produced in bacteria. Nanobodies provide multiple advantages, including their small size, which allows increased diffusion within the TME, and more rapid clearance from the bloodstream through glomerular filtration, thereby reducing off-target effects.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 247
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 247
  Illegal NotI site found at 200
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 247
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 247
  Illegal AgeI site found at 25
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Functional Verification

We designed experiments to verify if PD-L1 can be expressed and secreted by E.coli.

We transformed plasmid pET-28a-OmpA-PD-L1 nano (Figure 1)into BL21. OmpA-PD-L1 nanobody expression was induced by 1mM IPTG. The induced cultures were centrifuged, and the supernatant was collected and filtered. SDS-PAGE and silver staining was performed to detect OmpA-PD-L1 nanobody. When OmpA and IPTG presented simultaneously, we found a clear band at the position of 15kDa on the silvered stained gel (lane 2). There was also a band when only IPTG presented (lane 4), but it appeared lighter. (Figure 2)These results indicated that OmpA-PD-L1 nanobody was successfully secreted by E.coli. The band on lane 4 might be due to the rupture of E.coli in the present of IPTG.

Figure 1. Plasimd profile of pET-28a-OmpA-PD-L1 nanobody.

Figure 2. Silver stained SDS-PAGE gel.

Conclusion

The above results show that PD-L1 nanobody can be successfully expressed and secreted by E.coli.

References

[1] Gurbatri CR, Lia I, Vincent R, Coker C, Castro S, Treuting PM, Hinchliffe TE, Arpaia N, Danino T. Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci Transl Med. 2020 Feb 12;12(530):eaax0876. doi: 10.1126/scitranslmed.aax0876. PMID: 32051224; PMCID: PMC7685004.