Construction And Therapeutic Analysis Of Recombinant Immunotoxins For Treating Pancreatic Cancer

Pancreatic cancer,one of the devastating malignancies,is the seventh leading cause of cancer-related death worldwide with a 5-year overall survival rate of only 9%.Traditional treatment methods for pancreatic cancer include surgical resection,radiotherapy,and chemotherapy.Currently,there are no effective treatments in clinical practice and it’s difficult to overcome drug resistance and recurrence in pancreatic cancer therapy.Besides,immunotherapy,such as immune checkpoint blockades,has not shown significant efficacy in the clinical setting possibly due to the low immunogenicity of pancreatic cancer.Therefore,novel strategies are urgently needed to further improve survival of patients with pancreatic cancer.Recombinant immunotoxin（RIT）,a recombinant protein composed of the variable region of an antibody fused to a bacterial exotoxin（Pseudomonas exotoxin A,PE）,is a potent cytotoxin for cancer therapy.It combines specificity of the antibody with high efficiency of the toxin.The mechanism of action employed by PE-derived immunotoxins is protein synthesis inhibition and cell apoptosis induction,which is different from any other anti-cancer therapeutics used in the clinic.Thus,immunotoxins can be treated on pancreatic cancer patients that are resistant to chemotherapy.In present study,antibodies with high affinity and specificity against two different pancreatic-tumor-associated antigens,mesothelin and glypican-1,were isolated and fused to a truncated PE（called LR）with lower immunogenicity to generate LR-based immunotoxins.Anti-tumor efficacy has been further evaluated in vivo and in vitro.1.Preparation and evaluation of anti-GPC1 immunotoxinsGlypican-1（GPC1）,a cell surface glycoprotein,is encoded by human Gpc1 gene and contains558 amino acids with a predicted molecular weight of 62 k Da.GPC1 is a member of the glypican family,composed of a core protein and three heparan sulfate（HS）chains and attached to the cell surface by glycosylphosphatidylinositol（GPI）anchor.Overexpression of GPC1 in pancreatic cancer promotes the proliferation and migration of pancreatic tumor cells,and the high expression is associated with the poor prognosis of pancreatic cancer patients.GPC1 can be a potential biomarker for clinical diagnosis.Anti-GPC1 immunotoxins has not been reported yet.The present study is aimed to investigate whether GPC1 can be a target for immunotoxin treatment in pancreatic cancer.Camel single domain antibody D4,human heavy chain single domain antibody h VH and shark single domain antibody A1 as well as mouse monoclonal antibody HM2 were isolated by phage display technology and hybridoma technology,respectively.Immunotoxins D4-LR,h VH-LR,A1-LR and HM2-LR were successfully prepared after vector construction,fermentation,inclusion body preparation,denaturation,refolding,anion exchange chromatography as well as size exclusion chromatography purification.Among all,D4-LR and HM2-LR,with good purity and high yield,showed high affinity on antigen and great killing capacity on GPC1 overexpressing tumor cells H8/A431 as well as 2B9/KLM1.D4-LR was further engineered into bivalent immunotoxins,D4-AAA-D4-LR and D4-GGS-D4-LR.When compared with D4-LR,the bivalent immunotoxins showed 70-fold higher affinity and exhibited better cell killing activity on native pancreatic cancer cells T3M4 and KLM1.The anti-tumor efficacy of anti-GPC1 immunotoxins were further evaluated in xenograft mice model.Anti-GPC1 immunotoxins HM2-LR,D4-LR and D4-AAA-D4-LR were observed to significantly inhibit tumor growth and remarkedly extend survival rate in both H8/A431 and T3M4 xenograft mouse models.2.Preparation and evaluation of anti-MSLN immunotoxinsMesothelin（MSLN）is another cell surface glycoprotein with a predicted molecular weight of about 40 k Da and attached to the cell surface by GPI anchor.MSLN is a tumor specific marker and confined to expressed on normal mesothelial cells of the pleura,pericardium,and peritoneum.Overexpression of MSLN has been observed in multiple solid tumors,including pancreatic cancer,and is related to the poor prognosis.The abnormal expression of MSLN promotes cancer cell proliferation,invasion,metastasis as well as anti-apoptotic ability.Two immunotoxins（SS1P and LMB-100）targeting MSLN are undergoing clinical trials.This study is aimed to isolate camel single domain antibodies（also known as"nanobodies"）and construct nanobody-based immunotoxins with smaller molecular weight and better tissue permeability as compared with the immunotoxins currently used in clinic.The properties and cytotoxicity of the anti-MSLN nanobody based immunotoxins were characterized and evaluated further.Four MSLN-targeted nanobodies,A101,G8,G11 and A6,were isolated via phage display technology.The corresponding immunotoxins were produced.A101-LR was identified to be the one with good affinity,good thermal stability and the best anti-tumor activity by ELISA,thermal stability analysis,ADP-ribosylation assay as well as WST assay.A101-LR was engineered to be a bivalent one,A101-AAA-A101-LR,for better cell proliferation inhibition ability.A101-AAA-A101-LR had 10-fold higher affinity as compared to A101-LR.It also showed significant enhanced cytotoxicity on MSLN overexpression tumor cells H9/A431（18-fold）,native pancreatic cancer cells T3M4（3-fold）and KLM1（8-fold）.3.Preparation and evaluation of bispecific immunotoxins targeting GPC1/MSLNWe proposed that bispecific immunotoxins simultaneously targeting GPC1 and MSLN may have better synergistic anti-tumor efficacy when we found those two antigens were co-expressed on pancreatic cancer cells.D4 and A101 nanobodies were connected by G₉S₈ linker and fused to LR to produce bispecific immunotoxins,D4/A101-LR and A101/D4-LR.ELISA,flow cytometry and Octet results indicated that the bispecific ones not only retained their ability to bind each antigen,but also could bind GPC1 and MSLN simultaneously.The bispecific immunotoxins significantly inhibited the growth of double-positive cells,2G2/A431,with a very low IC₅₀ of 0.09n M.They also showed enhanced proliferation inhibition activity on pancreatic cancer cells T3M4and KLM1,as compared to the monospecific ones D4-LR or A101-LR.To sum up,this project is aimed to construct therapeutic immunotoxins for pancreatic cancer therapy.The purified immunotoxins targeting GPC1 or MSLN with high purity and good specificity could efficiently inhibit the proliferation of pancreatic cancer cells.Besides,the engineered bivalent immunotoxins showed increased affinity and enhanced anti-tumor activity in vitro.Bispecific immunotoxins,capable of binding GPC1 and MSLN,exhibited increased cytotoxicity on double positive pancreatic tumor cells.More importantly,anti-GPC1immunotoxins significantly inhibited tumor growth and remarkably extended survival rate in xenograft mice models.Immunotoxins targeting GPC1 or MSLN are worthy of more preclinical research to further evaluate their efficacy in pancreatic cancer therapy.