Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncated pseudomonas exotoxin.
To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. A431-CD30 cells were injected s.c. on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v. Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated. Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies. Published 2001 Wiley-Liss, Inc.
Rozemuller
H
H
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Chowdhury
P S
PS
Pastan
I
I
Kreitman
R J
RJ
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D019496:Cancer Vaccines; D002460:Cell Line; D003062:Codon; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005098:Exotoxins / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D017730:Ki-1 Antigen / Q000737:chemistry* / Q000276:immunology*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008954:Models, Biological; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D019496:Cancer Vaccines; D003062:Codon; D004247:DNA; D018076:DNA, Complementary; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D017730:Ki-1 Antigen; D019151:Peptide Library; D012313:RNA; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A