Design And Application Of Novel Immunotoxins With Low Non-specific Toxicity

An immunotoxin is a fusion protein which contains a targeting moiety linked to a toxin moiety.Wherein,the targeting moiety is usually a biological active molecule such as an antibody,an antibody fragment or a growth factor with targeting ability.The targeting moiety specifically recognizes antigens on the cell surface thus brings the toxin moiety specifically to the diseased cells.The toxins,usually come from microorganisms,plants,insects,and vertebrates,generally have a powerful cell killing effect,which can be exerted after being internalized into the targeted cells.Pseudomonas aeruginosa exotoxin(PE)and diphtheria toxin(DT)are two widely studied bacterial toxins,and the corresponding immunotoxins made from PE and DT,Lumoxiti and Ontak,have been approved for treatment of hairy cell leukemia and cutaneous T cell lymphoma in clinical.However,they may be even widely used if some improvements were made,for example,immunogenicity,low permeability,and non-specific toxicity.The non-specific toxicity of the immunotoxin mainly comes from two sides.The non-specific binding of the toxin molecule to the endothelial cells,which damages the endothelial cells and leads to the occurrence of vascular leakage syndrome.The other question is the targeting part non-specifically binds normal cells that express the tumor-associated antigens.Due to the heterogeneity of the toxin itself and the rare presence of tumor-specific antigens,the non-specific toxicity of immunotoxins is often difficult to be avoided.In order to reduce the non-specific toxicity of immunotoxins,we designed new types of immunotoxins to modify the toxin and targeting parts of conventional immunotoxins.To design the novel two-fragment immunotoxin,the immunotoxin scFvPE38,which targeted HER2/erbB2,was divided into two non-toxic segments and each fused with the split intein.Intein is a self-processing domain capable of self-splicing and linking flanked proteins(exteins)with natural covalent bonds.Activities of the two-fragment immunotoxins were were studied separately.To make sure whether the activity was recovered on the target cell surface,cytotoxicity of the two-fragment immunotoxins and the reconstituted immunotoxin was detected.Results of cell viability assay showed that the two-fragment immunotoxins lost its original activity and did not produce cytotoxicity even with high concentrations.The intact immunotoxin was reconstituted on the targeted tumor cells surface where the trans-splicing reaction occurred.The biological activity of the immunotoxin was restored,which led to binding and killing of the tumor cells with high antigen expression.The two-fragment immunotoxins could undergo an intein-mediated protein trans-splicing reaction under reducing conditions to produce an intact immunotoxin,and restore the cytotoxicity of the immunotoxin,which have the potential to reduce non-specific toxicity of immunotoxin.To design the novel masked immunotoxin M-Fab-PE24,a masking peptide was linked to the N-terminal of the Fab light chain.The masking peptide could only be excised by the tumor-specific proteases to expose the antigen binding site of Fab,which would restore the antigen binding affinity.Then the protease activated immunotoxins specially killed the tumor cells,with reduced non-specific toxicity to the normal cells.The antigen-binding affinity and biological activity of the masked immunotoxin were verified both in vitro and in vivo.The results of in vitro studies showed that the antigen binding affinity and cytotoxicity of the masked immunotoxin were significantly decreased,but the activity could be restored after protease digestion.The tumor inhibition effect of immunotoxins was verified on xenograft mouse models.The masked immunotoxins M-Fab-PE24 had obvious antitumor effect and lower liver damage compared with Fab-PE24,which means the masked immunotoxins reduced non-specific cytotoxicity and had better safety profile.Novel immunotoxins with low non-specific toxicity were designed and studied to demonstrate that it is a generic method of reducing the non-specific toxicity,while bioactivity of specific cytotoxicity were recovered in tumor environment.The method can be possibly employed in any combination of different antibodies and toxins without limiting to the source of the toxin and the specific antigen expression.The research has contributed potentially to widespread use of immunotoxins in clinical.The design of new immunotoxins with lower non-specific toxicity is innovative and had high application value.