Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Abstract

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. 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. 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. 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). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

“…E. coli BL21(DE3) strains transformed with expression plasmids (pETh-CRT3-LASP-PAS200, pETh-CRT4-LASP-PAS200, or pETh-Fn3(DGR)-LASP-PAS200) were used to purify PASylated CRT-targeting L-ASNases (CRT3LP, CRT4LP, or #DGRLP) as described 49 . E. coli BL21(DE3) strains transformed with expression plasmids pETh-CRT3-Rluc8, pETh-CRT4-Rluc8, or pETh-Fn3(DGR)-Rluc8 were used to purify Renilla luciferase variant 8 (Rluc8)-conjugated monobodies (CRT3-Rluc8, CRT4-Rluc8, or #DGR-Rluc8) as described 48 . The conjugated proteins were expressed in the above bacterial strains and purified using His GraviTrap columns (GE Healthcare, USA) and PD-10 desalting column (GE Healthcare, USA), as previously described 49 .…”

“…In a previous study, we developed CRT-targeting monobodies (CRT3 and CRT4) based on the human fibronectin domain III (FN3) scaffold 48 . These monobodies specifically target ecto-CRT in solid tumors of mice treated with ICD-inducing chemotherapy.…”

Trial watch: chemotherapy-induced immunogenic cell death in oncology

“…Finally, optimizing the detection of ICD in response to chemotherapy has also been investigated further since the last Trial Watch publication 235 . Zhang et al (Chonnam National University Medical School, Hwasun, Korea) engineered calreticulin-targeting monobodies to detect ICD more accurately 236 . Via this method, they were able to detect surface expression in multiple cancer cell lines and in mice treated with ICD inducers.…”

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma

“…Strategic immunotherapies that involved CALRmediated activation of immune responses to treating multiple cancers for have been documented [26,31,32]; nonetheless, whether CALR plays functional role in radiotherapy is yet to be delineated. In present study, we explored whether CALR could be impacted by irradiation or mediate cancer malignancies of NB.…”

“…Similar to various HSPs, endoplasmic reticulum chaperone CALR is known for its ability in activating anticancer immunity by immunogenic cell death (ICD) [22], and expression levels of CALR have been utilized for novel therapeutic strategies via elevation of anticancer immunity in various cancer types including colorectal carcinoma, nonsmall-cell lung carcinoma, acute myeloid leukemia, and ovarian cancer [23][24][25]. The successful development of anti-CALR antibody for detection of ICD has greatly facilitated early assessments of therapeutic efficacies [26]. In present study, we thus investigated the underrated role of CALR in NB by unprecedentedly uncovering CALR-mediated regulation of radioresistance and cancer stemness.…”