An epidemiological survey for the causes of a high incidence of primary liver cancer (PLC) in Haimen city, Jian-Su province and Fusui county, Guangxi province in China, found a close correlation between the incidence of PLC and the drinking of pond and ditch water. With an aim to clarify whether microcystins (MC), a hepatotoxic peptide produced by water bloom algae, contaminate the drinking water in the endemic areas of PLC in China, a highly sensitive enzyme-linked immunosorbent assay with a detection limit of 50 pg/ml, was introduced to monitor the MC. Three trials to survey the drinking water were carried out in 1993-1994. Samples, 1135 in total, were collected from different sources such as: ponds, ditches, rivers, shallow wells and deep wells in Haimen city. The first survey in September 1993 found that three out of 14 ditch water specimens were positive for MC, with a range of 90-460 pg/ml. Several toxic algae such as Oscillatoria agardhii were present in some of the ditches. In the second trial, samples were collected from five ponds/ditches, two rivers, two shallow wells and two deep wells monthly for the whole year of 1994. These data showed that MC was highest in June to September, with a range of 62-296 pg/ml. A third trial on the 989 different water samples collected from the different types of water sources in July 1994 revealed that 17% of the pond/ditch water, 32% of the river water, and 4% of the shallow-well water were positive for MC, with averages of 101, 160 and 68 pg/ml respectively. No MC was detected in deep well water. A similar survey on 26 drinking water samples in Fusui, Guangxi province, demonstrated a high contamination frequency of MC in the water of ponds/ditches and rivers but no MC in shallow and deep wells. These data support a hypothesis that the blue-green algal toxin MC in the drinking water of ponds/ditches and rivers, or both, is one of the risk factors for the high incidence of PLC in China. Based on previous findings on the epidemiology of PLC and the present results from the mass screening of MC in the drinking water, an advisory level of MC in drinking water was proposed to below 0.01 microg/l. The combined effect of a potent hepatocarcinogen AFB1 and an intermittent intake of MC in drinking water in the summer season was discussed as an etiology of PLC.

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-H in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNase cells showed that the former encoded the 28-kDa protein and the latter encoded the 40-and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28-and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.

Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of PtdIns(3,4,5)P3 are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds PtdIns(3,4,5)P3. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind PtdIns(3,4,5)P3 blocked membrane ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses PtdIns(3,4,5)P3-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired membrane ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22-8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.antibody engineering ͉ BL22 ͉ HA22 ͉ immunotherapy ͉ Pseudomonas exotoxin A I mmunotoxins (ITs) are hybrid proteins that are composed of a cancer-specific antibody attached to a bacterial or plant toxin (1). Initially ITs were made by chemically coupling toxins to whole antibodies. Now they are made using a combination of antibody and protein engineering (2, 3). ITs kill cells by binding to a cell surface protein, being internalized by endocytosis and eventually reaching the cytosol, where they arrest protein synthesis by inactivating EF2 or ribosomes (4, 5). Our laboratory has developed recombinant immunotoxins (RITs) in which the Fv portion of an antibody is directly fused to a 38-kDa portion of the bacterial toxin Pseudomonas exotoxin A (PE). Three RITs are currently in clinical trials and all three have shown anti-tumor activity in phase 1 trials. LMB-2 [anti-Tac-(Fv)-PE38] targets CD25 expressed on many T cell malignancies and some B cell malignancies (6). BL22 [anti-CD22-(Fv)-PE38] targets CD22 expressed on most B cell malignancies (7), and SS1P antimesothelin-(Fv)-PE38 targets the mesothelin antigen expressed on mesotheliomas and on ovarian, lung, pancreatic, and gastric cancers (8). Because these ITs contain a portion of a bacterial protein, they can induce the formation of neutralizing antibodies, hindering their efficacy. In patients with B-and T-cell malignancies the formation of neutralizing antibodies is infrequent because of the immune-suppressed state of patients with these malignancies (6, 7). However, in patients with solid tumors treated with SS1P and other ITs, antibody formation was very frequently detected 21 days after the first treatment cycle, preventing readministration of the IT (9).Previous studies have shown that the formation of antibodies to foreign proteins can be prevented by coupling the protein to high-mol...

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.

. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of antiTac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor ␣ subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then sitespecifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37°C in mouse serum, a 5-to 8-fold increase in plasma half-lives in mice, and a 3-to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.Pseudomonas exotoxin ͉ cancer therapy ͉ immunotherapy ͉ pharmacokinetics ͉ liver toxicity

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

IL‐6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte‐colony stimulating factor (G‐CSF) receptor and the cytoplasmic domain of gp130, a signal‐transducing subunit of the IL‐6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant‐negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL‐6‐induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.