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

Abstract

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°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.

Novel systemic therapy against malignant pleural mesothelioma

“…The mouse-human chimeric IgG1k monoclonal antibody amatuximab has a binding affinity of 1.5 nM for human mesothelin (137). Two phase I clinical trials identified a maximum tolerated dose of 200 mg/m 2 ; however, no responses were seen in patients with MPM who received this agent (138,139).…”

An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

“…In contrast with published antimesothelin antibodies (10,(32)(33)(34)(35), our mouse monoclonals were raised to unfolded, nonglycosylated mesothelin extracellular domain (with and without MPF). Hybridoma supernatants were screened by ELISA, followed by FACS and IHC analysis of cells stably transfected with mesothelin to select those capable of detecting native glycosylated and formalin-fixed mature mesothelin, respectively (data not shown).…”

The Role of Inflammation in Development and Therapy of Malignant Mesothelioma

“…The primary outcome is progression free survival (Clinicaltrials.gov identifier: NCT 00738582). SS1P is a recombinant immunotoxin made up of an antimesothelin Fv fragment linked to a truncated Pseudomonas exotoxin (Chowdhury and Viner, 1998). A single center, phase I clinical trial evaluating dose escalation of SS1P with concurrent administration of cisplatin and pemetrexed in unresectable epithelioid MPM patients is underway (Clinicaltrials.gov identifier: NCT01445392).…”