The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. AU transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of >99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosinecontaining non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.

We have shown previously that high-affinity receptors for interleukin-13 (IL-13Ralpha2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13Ralpha2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13Ralpha2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13Ralpha2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13Ralpha2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13Ralpha2(scFv)-PE38 was 200 microg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13Ralpha2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE). This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE. To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted. We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL. From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines. This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55. To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-basepair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.

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