title: Designing the furin-cleavable linker in recombinant immunotoxins based on Pseudomonas exotoxin A.
abstract: Recombinant immunotoxins (RITs) are fusion proteins that join antibodies to protein toxins for targeted cell killing. RITs armed with Pseudomonas exotoxin A (PE) are undergoing clinical trials for the treatment of cancer. The current design of PE-based RITs joins an antibody fragment to the catalytic domain of PE using a polypeptide linker that is cleaved by the protease furin. Intracellular cleavage of native PE by furin is required for cytotoxicity, yet the PE cleavage site has been shown to be a poor furin substrate. Here we describe the rational design of more efficiently cleaved furin linkers in PE-based RITs, and experiments evaluating their effects on cleavage and cytotoxicity. We found that changes to the furin site could greatly influence both cleavage and cytotoxicity, but the two parameters were not directly correlated. Furthermore, the effects of alterations to the furin linker were not universal. Identical mutations in the anti-CD22 RIT HA22-LR often displayed different cytotoxicity from mutations in the anti-mesothelin RIT SS1-LR/GGS, underscoring the prominent role of the target site in their intoxication pathways. Combining several beneficial mutations in HA22-LR resulted in a variant (HA22-LR/FUR) with a remarkably enhanced cleavage rate and improved cytotoxicity against five B cell lines and similar or enhanced cytotoxicity in five out of six hairy cell leukemia patient samples. This result informs the design of protease-sensitive linkers and suggests that HA22-LR/FUR may be a candidate for further preclinical development.
authors.affiliation: †Department of Biological Sciences, Jess and Mildred Fisher College of Science and Mathematics, Towson University, Towson, Maryland 21252, United States.|‡Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: John E
authors.initials: JE
authors.lastname: Weldon
authors.affiliation: ‡Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Skarzynski
authors.affiliation: †Department of Biological Sciences, Jess and Mildred Fisher College of Science and Mathematics, Towson University, Towson, Maryland 21252, United States.
authors.forename: Jamy A
authors.initials: JA
authors.lastname: Therres
authors.affiliation: †Department of Biological Sciences, Jess and Mildred Fisher College of Science and Mathematics, Towson University, Towson, Maryland 21252, United States.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Ostovitz
authors.affiliation: ‡Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
authors.affiliation: ‡Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kreitman
authors.affiliation: ‡Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D045683:Furin / Q000378:metabolism*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D007938:Leukemia / Q000188:drug therapy; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D037521:Virulence Factors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D045683:Furin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A