Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

Phage display of disulfide-stabilized Fv fragments.

Phage display of single-chain Fvs (scFvs) is a powerful tool to enrich and isolate specific antibody fragments from large pools (libraries) of Fv coding genes. However, many scFvs and scFv fusion proteins are unstable, not only as soluble proteins but also on the surface of phage. This limits and biases the recovery of specific Fv phage from display libraries to relatively stable scFvs. Also, the peptide linker in scFvs can diminish antigen binding of scFvs and scFv-fusion proteins. Disulfide-stabilized Fvs (dsFv) which have the VH-VL heterodimer stabilized by an interchain disulfide bond connecting framework regions in VH and VL rather than a peptide linker are more stable than scFvs and in some instances show better binding. To analyze whether these advantages can be utilized in a phage display system and to prove the feasibility of dsFv display, we constructed phage for dsFv display of the anti-Tac antibody and a dsFv-phage library. We find that dsFv phage can specifically bind antigen although the titer of dsFv phage in supernatants appears to be reduced compared to scFv phage. But this reduction in titer does not hamper the isolation of dsFv phages from large pools (libraries) as demonstrated by 'panning' of anti-Tac scFv and dsFv phages on living leukemia cells in suspension. In addition, dsFv phage are more stable than scFv phage. Therefore, display of dsFvs on phage is a useful alternative and addition to scFv-phage display.

Primate antibody response to immunotoxin: serological and computer-aided analysis of epitopes on a truncated form of Pseudomonas exotoxin.

NLysPE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) in which domain Ia (amino acids 1 to 252) and part of domain Ib (365 to 380) are deleted and an 11-amino-acid N-terminal peptide is added. LMB-1 is an immunotoxin in which the monoclonal antibody B3 is coupled to NLysPE38 near its N terminus. LMB-7 is a single-chain immunotoxin in which the Fv fragment of B3 is fused to PE38. To identify the antigenic regions of PE38, 12 polyclonal serum samples from monkeys immunized with the immunotoxins LMB-1 (six monkeys) and LMB-7 (six monkeys) were tested for their reactivity to a panel of 120 synthetic, overlapping peptides representing the amino acid sequence of NLysPE38. The antibody responses to peptides were similar among the 12 serum specimens, identifying several major immunodominant B-cell epitopes. Predominant reactivity was seen in six locations: amino acids 272 to 287, 341 to 359, 504 to 516, 540 to 564, and 573 to 591 and the C-terminal amino acids 591 to 613. The sera did not react with approximately 75% of the peptides. Furthermore, a computer-aided analysis was done to predict the immunologically relevant areas and revealed the same antigenic regions defined by serum reactivity to peptides. Competition enzyme-linked immunosorbent assays and neutralization assays were performed with domain II, III, or III plus Ib of PE38 and confirmed the immunodominance of domain III. To analyze the role of specific amino acids in antibody binding, individual amino acids of PE38 with large accessible surface areas were altered by site-directed mutagenesis. These results also show that the predicted areas of immunogenicity agree with the reactivity of the anti-PE38 antibodies to peptides and to the mutants of PE.

Role of de novo protein synthesis in target cells recognized by cytotoxic T lymphocytes specific for vesicular stomatitis virus.

The requirements for viral and host protein synthesis in the generation of target antigens for cytotoxic T lymphocytes (CTL) was evaluated by using vesicular stomatitis virus (VSV) inactivated by UV irradiation (UV-VSV). EL4 target cells incubated with UV-VSV were recognized and lysed by anti-VSV CTL, indicating that de novo synthesis of viral proteins was not required for the generation of antigens recognized by antiviral CTL. Anti-VSV CTL from H-2b mice primarily recognize determinants derived from the VSV N protein bound to the class I major histocompatibility complex (MHC) antigen H-2Kb. Comparison of a cloned CTL line representing this specificity and a heterogeneous population of anti-VSV CTL showed that determinants other than that recognized by the cloned CTL were generated more efficiently from UV-VSV. By using vaccinia virus recombinants that express deletion fragments of the N protein, it was shown that these additional determinants were probably derived from VSV proteins other than the N protein. The protein synthesis inhibitor emetine was used to determine whether newly synthesized host proteins were required for antigen generation. The addition of emetine to target cells prior to or at the time of the addition of UV-VSV inhibited lysis by anti-VSV CTL. This inhibition could be due to depletion of newly synthesized MHC molecules from intracellular membranes. This hypothesis was supported by using brefeldin A to delay membrane protein transport in target cells during the time of incubation with emetine and UV-VSV, which resulted in partial reversal of the effect of emetine. These results suggest that newly synthesized class I MHC molecules are required for the generation of antigens recognized by anti-VSV CTL.