Detailed Abstract
[E-poster - Basic Research (Basic Research)]
[EP 097] Engineering calreticulin-targeting monobodies to detect immunogenic cell death in cancer chemotherapy
ZHANG YING*1
1 Department Of Nuclear Medicine, Chonnam National University Medical School, REPUBLIC OF KOREA
Background : Cancer cell surface?exposed calreticulin (ecto-CRT) is the primitive form of signal during immunogenic cell death (ICD). It is a well-known candidate to allow “eat-me” signal from dying cells, which further contributes to their perception in directing the immune system. Various forms of anticancer agents and ionizing radiation can facilitate the ICD via ecto-CRT exposure. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. 93
Methods : To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment.
Results : Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein?engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine).
Conclusions : Taken together, our data clearly demonstrate the functional properties of engineered CRT-targeting monobodies to detect ICD during cancer chemotherapy. This strategy of engineering novel monobodies using peptides may simplify the process required to generate high-affinity biomolecules for inaccessible or challenging targets.
Methods : To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment.
Results : Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein?engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine).
Conclusions : Taken together, our data clearly demonstrate the functional properties of engineered CRT-targeting monobodies to detect ICD during cancer chemotherapy. This strategy of engineering novel monobodies using peptides may simplify the process required to generate high-affinity biomolecules for inaccessible or challenging targets.
SESSION
E-poster
E-Session 03/03 ~ 03/05 ALL DAY