Construction,expression And Purification Of Anti-AGR2/PD-1 And Anti-AGR2/CTLA-4 Bispecific Antibodies

Immune checkpoint inhibitors(ICIs),which target cytotoxic T lymphocyte antigen-4(CTLA-4)and programmed cell death protein-1/ligand-1(PD-1/PD-L1),have already transformed the treatment landscape of many cancers types.However,broad immune activation associated with ICIs often causes toxic side effects against various tissue and damage vital organs.These toxic effects caused by over activation of T cells lead to auto immune-related adverse events(ir AEs),thus preventing patients from the full efficacy of cancer immunotherapeutics effect(CITE).To increase the CITE while bringing the ir AEs under control,we hypothesized that a second antibody targeting tumor microenvironment(TME)will guide the powerful ICI antibodies to enrich in solid tumor using the bispecifc antibody(Bs Ab)constructs,thus reducing non-specific activation of immune system.AGR2(Human Anterior Gradient Homolog-2)is a member of protein disulfide isomerase(PDI)family anchored on endothelium reticulum(ER)critical in proper protein folding inside cells but also secreted as a paracrine signal promoting cell growth.AGR2 is overexpressed in many types of cancer and detected at high levels in extracellular space,the interstitial fluids,of solid tumor.The intracellular form of AGR2 is critical in protein folding while secreted AGR2 function as a paracrine signal promoting tumor microenvironment(TME)formation.Our laboratory developed a monoclonal antibody against AGR2,18A4.Both 18A4 and its humanized form 18A4 Hu,can inhibit angiogenesis,xenograft tumor growth and lung metastasis,and has no adverse effect on mouse weight and various organs.We found that our antibody against AGR2 was specifically located at tumor site in A549 and H460 xenograft tumor models.Based on the therapeutic and targeting properties of 18A4 Hu,we designed anti-AGR2/PD-1 and anti-AGR2/CTLA-4 bispecific antibodies combinations to guide these ICIs towards tumors.The backbone 18A4 Hu plasmid was optimized,and the synthesized sc Fv fragments of the variable regions of commercial anti-PD-1(Pembrolizumab)or anti-CTLA-4(Ipilimumab)were inserted into 18A4 Hu in two Ig G appended formats of Bs Abs.HEK293 T cells were used for eukaryotic expression and the secreted Bs Abs were collected and purified through Protein G affinity columns.Western blot and SDS-PAGE results showed that the constructed Bs Abs were expressed successfully.Prokaryotic expressed GST-PD-1,GST-CTLA-4 and his-AGR2-Ds Red recombinant proteins were used to confirm their binding activities.The preliminary co-culture experiments showed that the in vitro bioactivity of anti-AGR2/PD-1 Bs Abs.This study successfully constructed bispecific antibodies targeting tumor microenvironment signal AGR2 and immune checkpoints PD-1 or CTLA-4,confirmed their binding activity,and tested the in vitro activity in preliminary in vitro cell experiments.This study helps to overcome the barrier of ICIs’ toxic effects,and the further study on the anti-tumor effects and mechanisms of anti-AGR2/PD-1 and anti-AGR2/CTLA-4 bispecific antibodies will promote more effective and novel therapeutic antibody development.