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. IC 50 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.