EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Inducible vectors for expression in mammalian cells.

The most recent developments in mammalian cell inducible expression systems have involved the use of bacterial gene control elements and viral transactivator proteins. The combination of hybrid viral transactivator and bacterial repressor proteins, and simple chemical inducers can provide induction ratios of over 1000-fold. These developments will have applications in both cell-based research and the generation of transgenic animals.

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes.

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the lambda pR promoter, the cI857 temperature-sensitive repressor gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of greater than 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.

Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL.

The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular lambda pL. Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30 degrees C and readily amplified by thermal induction of strains carrying a thermolabile lambda repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.

High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification.

We have used a glutamine synthetase (GS) gene as an amplifiable marker in Chinese hamster ovary (CHO) cells. GS was combined with an efficient transcription unit to produce tissue inhibitor of metalloproteinases (TIMP). Initial transfectant cell-lines selected using a GS gene secreted up to 9 micrograms TIMP/10(6) cells/24h. After one round of GS gene amplification expression levels of 110 micrograms TIMP/10(6) cells/24h were achieved. These GS gene amplified CHO cells, when adapted to grow in suspension, accumulated 180mg/l in shake flask culture. This system therefore provides a rapid method of achieving high level gene expression in mammalian cells.

High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.

A DNA polymerase from Ustilago maydis. Evidence of proof-reading by the associated 3' leads to 5' deoxyribonuclease activity.

The 3' leads to 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.

Inducible DNA repair in Ustilago maydis.

Maximum survival of UV-irradiated U. maydis required a 2-3 h period of post-irradiation RNA and protein synthesis. Split dose experiments showed that this requirement correlated with the development of a radio-resistant cell state induced by UV doses above 200 Jm-2. Once induced, the radio-resistant state precluded the need for further RNA and protein synthesis for proficient repair of DNA damage caused by a second UV dose. Such radio-resistance was retained for up to 15 hours and it is concluded that this phenomenon represents the expression of an inducible DNA repair process, which is under transcriptional control.

Cloned truncated recA genes in E. coli. I. Effect on radiosensitivity and recA+ dependent processes.

Plasmids pMH1 and pDR1461, possessing the control region and 22% or 73% of the E. coli recA gene, conferred UV sensitivity to wild-type uvrA, and umuC bacteria. Sensitization was less in recA441 (tif-1) mutants and absent in lexA cells. Radiosensitization correlated with inhibition of recombinational repair, even through induced recA protein synthesis and recombination in Hfr matings were normal. Plasmids pMH1 and pDR1461 also prevented induction of some, but not all, SOS functions. Mutagenic reversion to tryptophan prototrophy and induced reactivation of UV-irradiated phage lambda were eliminated, and the efficiency of lambda lysogenic induction reduced. However, naladixic acid induced filamentous growth, mitomycin-C induced uvrA gene expression and post UV-irradiation DNA degradation control were little changed. Explanations of these effects are discussed which involve the presence of either truncated recA protein or multiple copies of the recA gene control sequence.

Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity.

The ability of plasmids carrying truncated recA genes to sensitize recA+ cells to UV-irradiation was dependent upon the size of the cloned recA gene fragment. Radiosensitization correlated with the inhibition of recombinational repair, and the in vivo reduction of recA protein recombinase activity, as measured by lambda bio 11 plating efficiency. W-reactivation was also abolished by the radiosensitizing plasmids, whilst DNA degradation control, naladixic acid induced filamentation and lambda induction were unaffected. UV-induced mutagenesis in excision proficient E. coli was unaffected, whilst excision deficient strains were hypermutable. It is suggested that these effects of plasmids bearing 22% or more of the recA gene are the result of the interaction of full-sized and truncated protein subunits to generate multimers unable to catalyze recombination.

A DNA polymerase from Ustilago maydis. 2. Properties of the associated deoxyribonuclease activity.

The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3' leads to 5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.

Enzyme-catalyzed DNA unwinding. A DNA-dependent ATPase from E. coli.

We have isolated a new DNA-dependent ATPase from E. coli. The enzyme has been purified to greater than 90% purity. It appears to be composed of two identical polypeptide chains of molecular weight 20,000. The enzyme catalyzed the hydrolysis of ATP in the presence, but not in the absence, of single-stranded DNA. Double-stranded DNA is not a cofactor. The products of hydrolysis are ADP and Pi. The enzyme also catalyzed strand separation of duplex DNA in the presence of ATP and E. coli DNA binding protein. Two E. coli proteins capable of promoting strand separation have been reported previously and have been termed helicase I and II (Abdel-Monem, M., and Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38). Accordingly, this protein is named helicase III.

Enzyme-catalyzed DNA unwinding. The role of ATP in helicase III activity.

The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G. T., Das, R. H., and Gefter, M. L. (1979) J. Biol. Chem. 254, 11997-12001). The free enzyme is able to bind to double- and single-stranded DNA. In the presence of ATP the enzyme can bind single- but not double-stranded DNA. The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA. It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA. These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis.

The influence of DNA binding protein on the substrate affinities of DNA polymerase from Ustilago maydis: one polymerase implicated in both DNA replication and repair.

The DNA polymerase of Ustilago maydis is stimulated by a DNA binding protein from the same organism. Analysis of this stimulation shows that there is an increase in affinity for both substrates of the reaction. The apparent Km for deoxynucleoside triphosphates is decreased 3 fold, and that for denatured DNA by 4 fold. In both cases the maximum velocity (Vmax) is increased 1.2 to 1.4 fold. It is suggested that the variability in the affinity of the enzyme for deoxynucleoside triphosphates mediated by the binding protein may provide the basis for the UV sensitivity of pyrimidine auxotrophs in this organism.

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli.

UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage lambda, lambda i21, lambda i434 and phi 80. Maximal induction in lexA3 lysogens needed less UV than in lexA+ bacteria and gave 25-100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective lambda prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA+ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.

The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity.

The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.