Cloning and characterization of a general amino acid control transcriptional activator from the chestnut blight fungus Cryphonectria parasitica.

We have cloned and characterized a homologue of the Neurospora crassa general amino acid control gene cpc-1 from the chestnut blight fungus Cryphonectria parasitica. The deduced amino acid sequence of C. parasitica CPC1 (cpCPC1) contains regions with significant homology to the transcriptional activation, DNA binding, and dimerization domains previously defined for N. crassa CPC1 (ncCPC1) and the equivalent "b-ZIP" transcription factor from Saccharomyces cerevisiae, GCN4 (scGCN4). Treatment of C. parasitica with low levels of the protein synthesis inhibitor cycloheximide caused cpc-1 transcript levels to undergo a rapid, transient increase similar to that reported for the mammalian b-ZIP transactivators, c-Jun and c-Fos. Northern analysis also revealed that amino acid starvation of C. parasitica elicits an increase in cpc-1 transcript levels. Hypovirus infection did not affect this increase, although transcript accumulation for several amino acid biosynthetic genes was slightly diminished in the hypovirus-containing strain. Recombinant cpCPC1 specifically bound to the consensus DNA binding element (AP-1), 5'-A/GTGACTCAT-3', also located upstream of the C. parasitica cpc-1 coding region. Constitutive transgenic expression of a DNA binding defective cpCPC1 mutant impaired the ability of C. parasitica to adjust to amino acid starvation. Moreover, these transformants showed reduced ability to grow on host chestnut tissue. Our results define a general amino acid control transactivator in a plant pathogenic fungus and suggest that functional modulation of this factor can influence fungal virulence.

Phase I clinical evaluation of a new murine monoclonal antibody (Mu-9) against colon-specific antigen-p for targeting gastrointestinal carcinomas.

BACKGROUND: Colon-specific antigen-p is a tumor-associated antigen present in approximately 60% of colorectal cancers. Preclinical studies have shown that the murine monoclonal antibody Mu-9 has excellent tumor-targeting abilities; therefore, clinical studies were initiated. METHODS: The immunoglobulin G and F(ab')2 were radiolabeled with 131I and administered to 13 and 12 patients, respectively, with advanced gastrointestinal cancer (colon, rectal, and pancreatic) for radioimmunodetection or radioimmunotherapy. RESULTS: Even in patients with highly elevated carcinoembryonic antigen levels, only one patient showed appreciable complexation of the labeled antibody, suggesting the epitope may not be highly expressed in the blood. Fifty percent of 131I-Mu-9 immunoglobulin G was cleared from the blood within 41 +/- 13 hours, while it took only 19 +/- 8 hours for the same amount of 131I-F(ab')2 to be cleared from the blood. Lesion detection in the abdomen, liver, and pelvis was greater than 90% for either the immunoglobulin G or F(ab')2. The dose absorbed by the normal organs, except the kidneys, was two- to threefold less for the F(ab')2 than for the whole immunoglobulin G. The dose to the kidneys was similar for both forms of immunoglobulin. The average tumor dose for 131I-Mu-9 immunoglobulin G was 13.9 +/- 11.0 cGy/mCi, and for 131I-F(ab')2 was 4.9 +/- 2.9. Tumor/red marrow dose ratios for the whole immunoglobulin G were 4.3 +/- 3.0, compared to 3.3 +/- 1.9 for the F(ab')2, suggesting the therapeutic window for the two forms of immunoglobulin may be similar. Eight of nine patients given the whole immunoglobulin G developed highly elevated levels of human anti-mouse antibody, whereas lower values were observed in five of seven patients given the F(ab')2. CONCLUSIONS: These initial results support the need for further evaluation of Mu-9 immunoglobulin G and F(ab')2 for targeting gastrointestinal cancer for radioimmunotherapy.

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.

Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica.

The gene, epn-1, encoding endothiapepsin (Epn), an aspartic protease (AspP) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, Cryphonectria (Endothia) parasitica, was identified and characterized. Inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature Epn [Barkholt, Eur. J. Biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. Of the nine fungal AspP characterized to date, Epn was found to be most closely related to aspergillopepsin and penicillopepsin (52% and 55% identity, respectively), proteases produced by the ascomycetes Aspergillus awamori and Penicillium janthinellum, and least related to proteases produced by the yeasts Candida albicans and Saccharomyces cerevisiae (27% and 26% identity, respectively). Epn production was found to be the same in isogenic virus-free and virus-containing strains, indicating that this AspP is not down-regulated by the presence of a hypovirulence-associated viral double-stranded RNA, as has been reported for several other secreted C. parasitica gene products. Strains containing multiple copies of epn-1 were obtained by transformation with a plasmid vector containing the cloned epn-1. One of these strains was shown to produce seven to ten times more Epn than the parental wild-type strain.

Clinical evaluation of tumor targeting with a high-affinity, anticarcinoembryonic-antigen-specific, murine monoclonal antibody, MN-14.

BACKGROUND: The authors previously reported that an anticarcinoembryonic antigen antibody against a carcinoembryonic antigen (CEA)-specific epitope is preferred for clinical investigations. They developed a second generation, CEA-specific murine monoclonal antibody (MoAb), MN-14 (IMMU-14), that has a tenfold higher affinity. This report summarizes the initial clinical experience with the new MoAb. METHODS: MN-14 immunoglobulin G (IgG) (0.5-6.0 mg) was labeled with radioactive iodine (I131) (5-80 mCi) and injected into 22 patients with cancer. External scintigraphy was used to determine targeting in patients with low and highly elevated plasma CEA. Quantitative external scintigraphy methods were used to determine organ and tumor clearance rates and absorbed radiation doses. Targeting data were correlated with several factors, including MoAb protein dose, plasma CEA, and relative tumor burden. RESULTS: Despite more than 80% complexation with plasma CEA of more than 500 ng/ml, all known tumor sites were disclosed by external scintigraphy. The overall sensitivity of tumor targeting on a lesion basis was 89%. The residence time in the blood was predicted by body weight (P = 0.05) and the log of plasma CEA (P = 0.043). The absorbed dose to the red marrow and total body could be predicted by the body weight of the patient, but no other factor contributed significantly to the clearance rate or absorbed dose to the organs. Individual tumors received an average dose of 9.3 +/- 6.4 cGy/mCi. The absorbed dose to the tumors was negatively correlated to the weight of the tumor, and the percent uptake in the tumor was positively correlated to the estimated total tumor burden. Patients injected with approximately 5 mg of MN-14 IgG were more likely to have anti-mouse antibodies (HAMA) develop than were patients who were injected with less MoAb. CONCLUSIONS: These results suggest that MN-14 targets tumors effectively, even in the presence of elevated circulating CEA. Additional studies are necessary to determine if an advantage for the higher affinity MN-14 MoAb, compared with the lower affinity NP-4 MoAb, can be appreciated clinically.

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.

The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro.

Gene expression by a viral-like double-stranded RNA genetic element associated with reduced virulence (hypovirulence) of the chestnut blight fungus was recently shown to involve an autoproteolytic event which resulted in the release of an encoded protease, designated p29, from a polyprotein during translation. Mutational analysis of p29, described in this report, revealed that residues Cys-162 and His-215 are essential for autocatalytic cleavage. The results were also consistent with previous predictions that cleavage occurs between Gly-248 and Gly-249. Interestingly, p29 bears a striking resemblance to the potyvirus-encoded protease HC-Pro. Both proteases autocatalytically cleave at glycine dipeptides. In addition, there is a significant degree of similarity in the amino acid sequences flanking the essential Cys and His residues of the two proteases and in the spacing of these residues from their respective cleavage sites.

Murine monoclonal antibodies against carcinoembryonic antigen: immunological, pharmacokinetic, and targeting properties in humans.

We have examined three 131I-labeled murine monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA), NP-2, NP-3, and NP-4, after i.v. injection in patients with diverse cancers. Although the MAbs had a similar tumor-targeting ability, several important features were discovered that have led us to the selection of one of these MAbs for further clinical evaluation. We found that it is important to evaluate MAbs with a high immunoreactivity. For example, the MAb NP-2 was used initially in patients with an immunoreactivity between 35 and 50%. Although the tumor-imaging properties of this MAb compared favorably with the affinity-purified, goat anti-CEA antibody that we used previously, further purification of NP-2 to an immunoreactivity greater than 70% uncovered a previously unknown cross-reactivity with human granulocytes. It was also discovered that the MAbs differed in their ability to complex with CEA in the blood. Plasma samples were analyzed by gel filtration at 1 or 24 h after injection. The formation of complexes with circulating CEA was dependent on the CEA:MAb ratio in the blood. NP-3 complexed to a greater degree with CEA than NP-4, but NP-2 did not complex with CEA even at CEA:NP-2 ratios of 55 to 1. NP-3 commonly showed enhanced uptake in the colon by external scintigraphy, and examination of the radioactivity in the stool showed that most of the radioactivity was associated with whole IgG and large-sized fragments of NP-3. We also compared the rate of elimination of radioactivity from the blood for all of the MAbs and compared the clearance of NP-3 to NP-4 at three different ranges of MAb protein doses (less than 1.0 mg, 1 to 5 mg, and 5 to 20 mg). The blood clearance rate for NP-3 was fastest among the other MAbs at protein doses exceeding 1.0 mg. Patients given less than 1.0 mg of NP-4 had a significantly (P less than 0.005) shorter elimination half-life than patients given more than 1.0 mg of NP-4. By virtue of NP-4's good targeting properties in patients and its limited complexation with circulating CEA, it was selected as the MAb of choice for CEA tumor imaging.

Biodistribution and radiation dose estimates for yttrium- and iodine-labeled monoclonal antibody IgG and fragments in nude mice bearing human colonic tumor xenografts.

An anti-carcinoembryonic antigen murine monoclonal antibody designated NP-4, and its F(ab')2 and Fab' fragments, were coupled to the 1/1 mixture of 1-isothiocyanato-benzyl-3-methyl- and 1-methyl-3-isothiocyanato-benzyl-diethylenetriaminepentaacetic acid chelate and labeled with 111In or 88Y. Biodistribution studies in nude mice bearing a human colonic tumor xenograft were performed with these labeled conjugates, and comparisons were made to unconjugated NP-4 IgG and fragments labeled with 131I. Regardless of the labeling method, higher tumor uptake was found with the intact IgG than with the fragments, but due to faster blood clearance, tumor/blood ratios were higher for the fragments than for the IgG. Tumor uptake for the radiometal-labeled NP-4 was generally higher than the 131I-labeled NP-4. Tumor/nontumor ratios for the liver, kidney, and spleen were higher for the 111In- and 88Y-labeled NP-4 IgG than the respective radiometal-labeled fragments, but tumor/nontumor ratios for the 131I-NP-4 fragments were higher than the 131I-NP-4 IgG. Radiometal uptake in the kidney was approximately 8 and 150 times higher than the 131I-NP-4 F(ab')2 and Fab', respectively, and the clearance of radiometal activity in the kidneys was approximately 10 times slower than the radioiodine. Quantitation of 88Y or 111In activity in the femur showed 3-5%/g for the IgG and F(ab')2 and only 1-2%/g for the Fab'. The amount of radioactivity in the femur remained constant over time, and between 60 and 100% of the 88Y activity remained after flushing the core of the femur with saline, whereas 50-70% of the 111In and only 25-30% of the 131I activity remained after washing. Radiation dose estimates derived from these studies suggest that at the maximal tolerated dose 131I-NP-4 IgG would deliver 5.9 times the dose to the tumor as 90Y-labeled NP-4 IgG. 90Y-labeled fragments would not be useful due to higher doses to the kidneys than to the tumor. However, with 131I-labeled IgG and fragments there is greater flexibility to permit tumoricidal doses without excessive toxicity to the normal tissues.

Tumor, red marrow, and organ dosimetry for 131I-labeled anti-carcinoembryonic antigen monoclonal antibody.

Tumor-, red marrow-, and organ-absorbed doses were calculated for patients receiving 131I-labeled monoclonal antibodies against carcinoembryonic antigen for either diagnosis or therapy. Ten patients with confirmed liver tumors who received doses ranging from 10.79 to 200 mCi were evaluated. Urine and blood samples were taken in order to determine total body and red marrow activity, respectively. Anterior and posterior gamma camera images were obtained at multiple times postinjection in order to quantitate activity uptake using the conjugate view counting method for the following organs and regions: lungs, liver, spleen, kidneys, and the liver tumors. In addition, sacral regions of interest were drawn to generate red marrow-absorbed dose estimates for comparison to those obtained by blood sampling. Tumor volumes were obtained from volumetric analysis of the patient's computed tomographic study and tumor S values were obtained by assuming uniform distribution of the 131I-labeled monoclonal antibody in spherical tumor regions considering all emitted electrons, beta-particles, and photons. The following mean absorbed doses in rads/mCi injected were obtained: lungs, 2.3 +/- 1.6 (SD); liver, 1.4 +/- 0.7; spleen, 2.6 +/- 1.4; kidneys, 3.1 +/- 1.5; total body, 0.7 +/- 0.5; red marrow from blood sampling, 2.9 +/- 1.9; red marrow from sacral scintigraphy, 1.7 +/- 1.2; and liver tumors, 69.3 +/- 92.5. Tumor volumes ranged from 1 to 216 g and the percentage of uptake/g of monoclonal antibody into these tumors ranged from 0.0006 to 1.040. There was a statistically significant difference between the two techniques for estimation of red marrow dose (P less than 0.01). This methodology, permits calculation of tumor, red marrow, and organ dosimetry using planar gamma camera imaging.

Sacral scintigraphy for bone marrow dosimetry in radioimmunotherapy.

Myelosuppression has been identified as the dose-limiting toxicity in radioimmunotherapy studies. Accurate bone marrow dosimetry is, therefore, necessary to evaluate bone marrow toxicity which may result from systemic cancer treatment with radiolabeled monoclonal antibodies. Dose to the red marrow was determined in 20 patient studies with 131I labeled anti-carcinoembryonic antigen, anti-alpha-feto-protein, or anti-human chorionic gonadotropin monoclonal antibody for diagnosis or treatment of diverse metastatic carcinomas, using a new technique involving sacral scintigraphy and a previously reported blood-based methodology. For the sacral technique, anterior and posterior gamma camera images of the pelvis were obtained at multiple times. Regions of interest were drawn around the sacrum in order to quantitate activity uptake as a function of time using the conjugate view counting method. Cumulated activity in red marrow was determined by curve integration and division by 0.099, since it has been estimated that 9.9% of the total red marrow is contained in the sacrum of the adult. Red marrow doses were then obtained by multiplying the cumulated activities by the appropriate S factor. These doses were compared to red marrow doses obtained from serial whole blood samples taken from these patients. Cumulated activity in the red marrow was determined from the blood with the assumption that the activity concentration in the blood and red marrow were equal. The mean red marrow dose per injected activity was 2.0 +/- 0.9 rad/mCi using the sacral data and 2.7 +/- 1.3 rad/mCi using the blood data (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)