Research On HIL2-anti-hCCR4/anti-hCCR4-hIL2 Bispecific Immunotoxin

Objective: Human CD25 and CCR4 are highly expressed on cutaneous T cell lymphoma(CTCL).Our preliminary research showed that the Ontak?-like human hIL2 fusion toxin(hIL2 IT)can inhibit CTCL by targeting CD25,and human CCR4 immunotoxin can inhibit CTCL by targeting CCR4.Due to the limited affinity of single target,those two kind of inmmunotoxin/ fusion toxin’s efficacy can’t be enhanced completely by targeting the same cell line together.Therefore,we constructed a new hIL2-anti-h CCR4 bispecific immunotoxin,to improve the function of inhibiting the CD25~+ CCR4~+ CTCL tumor cells.Method:(1)Based on DT-bivalent-CCR4 and DT-bivalent-hIL2,we construct the expression vector of hIL2-anti-h CCR4/anti-h CCR4-hIL2,transfer it to Pichia pastoris system and express it using methanol.For the purification of the bispecific immunotoxin,we use Ni SepharoseTM 6 Fast Flow and POROS 50 HQ Strong Anion Exchange Resin.For the identification of it,we use SDS-PAGE and Western Blot.(2)We use different concentration of the immunotoxin to target the CD25~+CCR4~+ T HUT 102/6TG cell line,use cell viability assay to analyze the in vitro inhibition function of the immunotoxin,and use flow cytometry to analyze the binding affinity.(3)We transfer CD25~+ CCR4~+ CTCL tumor cells into NSG mouse by intravenous injection to make CD25~+ CCR4~+ tumor bearing mouse model.The immunotoxin was IP injected daily for 10 continuously days.We test the in vivo function of this bispecific immunotoxin by observing the survival days.We also do pathological anatomy and microscopic examination to check the pathological changes of the liver。Result:(1)Rresults of SDS-PAGE and Western Blot shows that the bispecific immunotoxin was expressed successfully.(2)The result of cell viability assay shows that the bispecific immunotoxin can dose dependently inhibit CD25~+ CCR4~+ CTCL tumor cells.(3)The results of binding assay shows that the binding efficacy of hIL2-anti-h CCR4 IT and anti-h CCR4-hIL2 IT is higher than hIL2 IT and CCR4 IT.(4)The result of survival assay shows that bispecific immunotoxin can significantly prolong the lives of tumor bearing mice.(5)The result of pathological anatomy and microscopic examination shows that Both bispecific immunotoxins inhibited CD25~+CCR4~+ tumor cells more strongly than CCR4 immunotoxins and hIL2 fusion toxins alone.Conlusion: 1.The bispecific immunotoxin was successfully expressed in Pichia pastoris.After purification,7 mg of hIL2-anti-h CCR4 IT and 7 mg of anti-h CCR4-hIL2 IT were obtained in 1 L of culture medium;2.hIL2 IT,CCR4 IT,hIL2-anti-h CCR4 IT and anti-h CCR4-hIL2 IT have a killing effect on CD25~+CCR4~+ T cell line HUT 102/6TG.hIL2-anti-h CCR4 IT/hIL2-anti-h CCR4 IT’s in vivo efficacy is stronger than hIL2 IT and CCR4 IT...