Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer.

Tumor vaccines composed of autologous tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) (GVAX) have demonstrated clinical activity in advanced-stage non-small-cell lung cancer (NSCLC). In an effort to remove the requirement for genetic transduction of individual tumors, we developed a 'bystander' GVAX platform composed of autologous tumor cells mixed with an allogeneic GM-CSF-secreting cell line. We conducted a phase I/II trial of this vaccine (3-12 biweekly vaccinations) in advanced-stage NSCLC. Tumors were harvested from 86 patients, tumor cell processing was successful in 76, and 49 proceeded to vaccination. The most common toxicity was local vaccine injection site reactions. Serum GM-CSF pharmacokinetics were consistent with secretion of GM-CSF from vaccine cells for up to 4 days with associated transient leukocytosis confirming the bioactivity of vaccine-secreted GM-CSF. Evidence of vaccine-induced immune activation was demonstrated; however, objective tumor responses were not seen. Compared with autologous GVAX vaccines prepared by transduction of individual tumors with an adenoviral GM-CSF vector, vaccine GM-CSF secretion was approximately 25-fold higher with the bystander GVAX vaccine used in this trial. However, the frequency of vaccine site reactions, tumor response, time to disease progression, and survival were all less favorable in the current study.

Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents.

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

A mutation in adenylosuccinate lyase associated with mental retardation and autistic features.

We have examined the molecular basis of three cases of severe mental retardation with autistic features in one family. A point mutation in a purine nucleotide biosynthetic enzyme, adenylosuccinate lyase (ASL), segregates with the disorder. The affected children are homozygous for the point mutation while the parents and all four unaffected children are heterozygous. The point mutation is absent in control subjects. The point mutation results in a Ser413Pro substitution which leads to structural instability of the recombinant mutant enzyme, and this instability lowers ASL levels in lymphocytes. These observations suggest that the instability of ASL underlies the severe developmental disorder in the affected children, and that mutations in the ASL gene may result in other cases of mental retardation and autistic features.

Cloning and characterization of the rat complementary deoxyribonucleic acid and gene encoding the neuroendocrine peptide 7B2.

A 7B2 cDNA clone was isolated from a rat insulinoma cDNA library. The 1.1-kilobase (kb) cDNA insert contained 1 open reading frame of 630 nucleotides which encoded a 210-amino acid sequence. The deduced rat 7B2 precursor peptide of approximately 24 kDa would yield an approximately 21-kDa peptide upon cleavage of the signal sequence. The rat 7B2 nucleotide and amino acid sequences were highly homologous to those of mouse, human, pig, toad, and salmon. Northern analysis revealed 7B2 expression in rat pituitary and brain and in the PC12 and RIN5F cell lines. A DNA clone containing the 5' end of the rat 7B2 gene was isolated from a rat genomic DNA library. A 3.5-kb fragment isolated from this clone contained 1.5 kb of 5' nontranscribed DNA and 2 kb of the transcriptional unit. Nucleotide sequence and primer extension analyses revealed a TATA-like sequence approximately 25 basepairs up-stream of the transcription start site. Sequence analysis also revealed three putative cis-acting elements in the 5' flanking region. The second exon encoded the first 73 amino acids of the 7B2 prepeptide. Plasmids containing the 7B2 promoter fused with a reporter gene were transiently transfected into LAN-5 cells. Expression was evident only when the first intron was included with the 5' flanking sequences in the construct and only when cells were treated with forskolin or a phorbol ester. The rat 7B2 gene and cDNA will be valuable tools for the identification of factors that regulate 7B2 gene expression.

De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. coli.

The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E. coli mutant using an avian liver cDNA expression library. In E. coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins. The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities. Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced. The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins. Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS. The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis. Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli.

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.

Cloning, characterization, and sequence of a porcine cDNA encoding a secreted neuronal and endocrine protein.

This report describes the cloning, sequence, and characterization of a cDNA which encodes a protein synthesized in the brain and endocrine tissue, including pituitary, adrenal medulla, and ovary. The deduced 207-amino-acid sequence of the 23-kD protein contains a hydrophobic signal peptide suggesting that it is secreted. Northern blot analysis utilizing the cDNA clone identifies a single RNA of approximately 1400 nucleotides in porcine brain, adrenal medulla, pituitary, and ovary, as well as in human endocrine tumors. Very high levels of RNA were observed in one human pancreatic tumor. Southern blot analysis suggests that sequences homologous to the porcine cDNA are present in human, cow, rat, and salmon DNA, indicating that the gene(s) have been highly conserved during evolution.

Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage.

The localization of mRNA encoding preproatrial natriuretic peptide was investigated in tissue sections and cultures of rat heart and in sections of human right atrial appendage using the technique of in situ hybridization with 32P- and 35S-labeled RNA probes. Rat atrial natriuretic peptide (ANP) transcripts were demonstrated in numerous atrial myocytes and, to a lesser extent, in ventricular myocytes in both tissue sections and newborn rat heart cultures. These findings are consistent with those obtained by RNA blot analysis of rat heart total RNA, indicating that a single prepro-ANP transcript of approximately 900 nucleotides was present in the ventricles as well as the atria. Using a 35S-labeled RNA probe for human ANP mRNA, ANP transcripts were also localized to the majority of myocytes in the human right atrial appendage. Only background levels of autoradiographic labeling were obtained when RNA probes identical to the coding sequence of rat or human ANP mRNA were used. A close correlation was found between the distribution of ANP immunoreactivity and prepro-ANP mRNA in these preparations. These results provide unequivocal evidence for the expression of the ANP gene in the rat ventricles, as well as the atria, because myocytes in these tissues have been established as the sites of both ANP localization and precursor biosynthesis. The combined use of cardiac cultures and in situ hybridization may be of value in future studies investigating the regulation of ANP synthesis in cardiac myocytes.