Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity.
Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.
Chowdhury
P S
PS
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Viner
J L
JL
Beers
R
R
Pastan
I
I
D000818:Animals; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D004247:DNA / Q000276:immunology*; D005098:Exotoxins; D005260:Female; D058851:GPI-Linked Proteins; D015723:Gene Library; D006801:Humans; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D000922:Immunotoxins / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D000090204:Mesothelin; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D005098:Exotoxins; D058851:GPI-Linked Proteins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D011994:Recombinant Proteins; D004247:DNA; D000090204:Mesothelin