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Antibody-immunotoxin Conjugate Using FcBP-mediated Photoconjugation to Treat Cancer
Kwang-Hyeok Kim1, Jisoo Park2, Jin-Ock Kim1
1College of Pharmacy, Ajou University, Suwon, Republic of Korea.
Background/Aim:
Cytotoxic payload conjugation to antibodies efficiently suppresses tumors and contributes to the improvement of cancer survival. In our previous study, c-Kit targeting antibody-drug conjugate (2G4-DM1) with DM1, a microtubule inhibitor, efficiently suppressed tumor growth. However, slow-growing c-Kit-positive tumors, such as GIST-48, did not efficiently respond to DM1. In this study, we aimed to treat tumors using 2G4 immunotoxin with Pseudomonas exotoxin A (PE) as a payload.
Materials And Methods:
Modified FcBP-PE24 containing p-benzoyl-L-phenylalanine, unnatural amino acid, was expressed in E. coli and purified. Then, photoconjugation of 2G4 antibody and FcBP-PE24 at 365 nm was carried out and 2G4 immunotoxin was purified using anion exchange chromatography. In vitro cytotoxicity of 2G4 immunotoxins was assessed in HMC-1.2, GIST-48, and MDA-MB-453 cells. Then, in vivo efficacy analysis was performed using C.B-17 SCID mice.
Results:
2G4 immunotoxin efficiently induced cytotoxicity in 2G4-DM1-resistant HMC-1.2 and GIST-48 cells by inhibiting protein synthesis but not in c-Kit-negative MDA-MB-453 cells. The results showed ~200-fold or more increase in cytotoxicity against c-Kit-positive cells compared to IC50 of 2G4-DM1. In addition, 2G4 immunotoxin suppressed tumor growth in the in vivo xenograft mouse model.
Conclusion:
2G4 immunotoxins could be an alternative therapeutic strategy for microtubule inhibitor- resistant cancer cells.
Insights
This study developed a novel 2G4 immunotoxin using Pseudomonas exotoxin A (PE) to overcome resistance to microtubule inhibitors like DM1 in c-Kit-positive cancers. The new immunotoxin demonstrated significantly enhanced cytotoxicity and tumor suppression in preclinical models.
Area of Science:
- Oncology
- Immunotherapy
- Bioconjugation Chemistry
Background:
- Antibody-drug conjugates (ADCs) improve cancer survival by delivering cytotoxic payloads.
- Previous 2G4-DM1 (microtubule inhibitor) showed limited efficacy against slow-growing c-Kit-positive tumors (e.g., GIST-48).
- Resistance to microtubule inhibitors necessitates alternative therapeutic payloads.
Purpose of the Study:
- To develop and evaluate a 2G4 immunotoxin utilizing Pseudomonas exotoxin A (PE) as a payload.
- To assess the efficacy of the 2G4 immunotoxin against c-Kit-positive and DM1-resistant cancer cells.
- To investigate the therapeutic potential of 2G4 immunotoxins in preclinical cancer models.
Main Methods:
- Engineered a modified FcBP-PE24 payload incorporating an unnatural amino acid.
- Utilized photoconjugation (365 nm) to link the 2G4 antibody with the FcBP-PE24 payload.
- Purified the 2G4 immunotoxin via anion exchange chromatography.
- Assessed in vitro cytotoxicity in HMC-1.2, GIST-48, and MDA-MB-453 cell lines.
- Evaluated in vivo efficacy in a xenograft mouse model.
Main Results:
- 2G4 immunotoxin exhibited potent cytotoxicity against c-Kit-positive, DM1-resistant cells (HMC-1.2, GIST-48) by inhibiting protein synthesis.
- Demonstrated over a 200-fold increase in cytotoxicity compared to 2G4-DM1 in c-Kit-positive cells.
- Showed no significant cytotoxicity in c-Kit-negative MDA-MB-453 cells.
- Successfully suppressed tumor growth in vivo.
Conclusions:
- 2G4 immunotoxins represent a promising alternative therapeutic strategy.
- This approach is effective against cancer cells resistant to microtubule inhibitors.
- Further development of immunotoxins with novel payloads warrants investigation.
