title: A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity.
abstract: Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.
authors.affiliation: Laboratory of Molecular Biology,  Center for Cancer Research,  National Cancer Institute,  National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4264,  USA.
authors.forename: Wenhai
authors.initials: W
authors.lastname: Liu
authors.forename: Masanori
authors.initials: M
authors.lastname: Onda
authors.forename: Changhoon
authors.initials: C
authors.lastname: Kim
authors.forename: Laiman
authors.initials: L
authors.lastname: Xiang
authors.forename: John E
authors.initials: JE
authors.lastname: Weldon
authors.forename: Byungkook
authors.initials: B
authors.lastname: Lee
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D004220:Disulfides / Q000737:chemistry / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D008223:Lymphoma / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D000922:Immunotoxins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
references.citation: Biophys Chem. 2009 Apr;141(1):21-8
references.pmid: 19155118
references.citation: Clin Cancer Res. 2000 Apr;6(4):1476-87
references.pmid: 10778980
references.citation: J Immunother. 2010 Apr;33(3):297-304
references.pmid: 20445350
references.citation: J Biol Chem. 2004 Nov 12;279(46):47939-51
references.pmid: 15347644
references.citation: Blood. 2009 Apr 16;113(16):3792-800
references.pmid: 18988862
references.citation: Anal Biochem. 1979 Feb;93(1):98-102
references.pmid: 434474
references.citation: Methods Mol Biol. 2004;248:503-18
references.pmid: 14970517
references.citation: Biologicals. 2010 Jan;38(1):144-9
references.pmid: 19783458
references.citation: Mol Cell Proteomics. 2004 Jun;3(6):608-14
references.pmid: 15034119
references.citation: J Exp Med. 2006 Sep 4;203(9):2049-55
references.pmid: 16908625
references.citation: N Engl J Med. 2001 Jul 26;345(4):241-7
references.pmid: 11474661
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6902-6
references.pmid: 8692916
references.citation: Clin Cancer Res. 2010 Mar 15;16(6):1894-903
references.pmid: 20215554
references.citation: Clin Cancer Res. 2007 Sep 1;13(17):5144-9
references.pmid: 17785569
references.citation: J Immunol. 2006 Dec 15;177(12):8822-34
references.pmid: 17142785
references.citation: J Clin Oncol. 2010 Apr 10;28(11):1870-7
references.pmid: 20212249
references.citation: Nat Struct Biol. 2002 Aug;9(8):621-7
references.pmid: 12080331
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11311-6
references.pmid: 18678888
references.citation: Biochemistry. 2000 Apr 18;39(15):4207-16
references.pmid: 10757967
references.citation: J Biol Chem. 1988 Aug 25;263(24):11820-5
references.pmid: 2457027
references.citation: J Clin Oncol. 2000 Apr;18(8):1622-36
references.pmid: 10764422
references.citation: Nat Chem Biol. 2009 Nov;5(11):797-807
references.pmid: 19841629
references.citation: Int J Cancer. 1999 Mar 31;81(1):148-55
references.pmid: 10077166
references.citation: Annu Rev Med. 2007;58:221-37
references.pmid: 17059365
references.citation: Nat Rev Cancer. 2006 Jul;6(7):559-65
references.pmid: 16794638
references.citation: Cell. 1987 Jan 16;48(1):129-36
references.pmid: 3098436
references.citation: Trends Biochem Sci. 1990 Jan;15(1):14-7
references.pmid: 2107612
references.citation: J Immunol Methods. 1985 Mar 18;77(2):305-19
references.pmid: 3981007
references.citation: Adv Drug Deliv Rev. 2006 Aug 7;58(5-6):640-56
references.pmid: 16904789
references.citation: J Clin Oncol. 2005 Sep 20;23(27):6719-29
references.pmid: 16061911
references.citation: Anal Biochem. 2006 Jun 15;353(2):266-71
references.pmid: 16647035
references.citation: Leukemia. 1999 Apr;13(4):629-33
references.pmid: 10214872