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 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.

Figure 1

Phage displaying SS scFv bind specifically to mesothelin (amino acids 291–606) coated ELISA wells in a dose-dependent manner. Phage were exposed to wells coated with mesothelin, p55, BSA, and streptavidin or to uncoated wells. Bound phage was detected as described in Materials and Methods.

Figure 2

Epitopes for SS scFv and K1 are different. Mesothelin-coated wells were incubated with various dilutions of SS scFv or K1 scFv phage in the presence or absence of mAb K1 at 1 μg/ml. Bound phage was detected as mentioned in Materials and Methods.

Figure 3

Binding of SS scFv phage to mesothelin positive and negative cells as detected by immunofluorescence. (A and B) NIH 3T3 K20 cells. (C and D) NIH 3T3 cells. A and C are phase contrast, and B and D are immunofluorescence pictures. All cells were exposed to SS scFv phage as described in Materials and Methods. Binding of anti-Tac phage to both the cell lines was tested and found to be negative (data not shown).

Figure 4

Stability of SS scFv-PE38 at 37°C. SS scFv-PE38 (10 μg/ml) was incubated at 37°C for up to 40.5 hr, and then its cytotoxic activity was measured. The % of initial activity remaining after various periods of incubation is shown.

Figure 5

Anti-tumor effect of SS scFv-PE38 in nude mice. Groups of five animals were injected with 1.5 × 10⁶ A431 K5 cells on day 0. On day 5, tumors reached a size of 50 mm³. Animals were treated intravenously on days 5, 7, and 9 with 2.6 μg (⧫) or 4.3 μg (□) of SS scFv-PE38 in DPBS containing 0.2% human serum albumin. Control groups received either the carrier alone (○) or 3 μg anti-Tac(scFv)-PE38 (•). No death or toxicity was observed at these doses.