Practical blood dendritic cell vaccination for immunotherapy of multiple myeloma.

Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio-process to purify CMRF-56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF-56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.

Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells.

The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and flow cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells.

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO.

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitute pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells.

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specificß subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with bothα2,3 andα2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

Super-CHO-A cell line capable of autocrine growth under fully defined protein-free conditions.

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6) cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6) cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.

Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Expression of FSH in CHO cells. II. Stimulation of hFSH expression levels by defined medium supplements.

A Chinese Hamster Ovary (CHO-K1) derived cell line, which expresses human follicle stimulating hormone (hFSH) under the control of a beta actin promoter, was used as a model system to study heterologous glycoprotein expression. It has been shown previously that specific productivities in this cell line were three times higher in the presence of serum than in its absence. In this paper, a systematic study was made of the affects of various serum components of product levels in order to determine if the affect of serum on FSH expression could be duplicated by defined medium supplements. Culture media were supplemented with growth factors, direct activators of secondary messengers, steroids, lipids and various sugars. It was shown that the components with the most stimulatory affect of hFSH expression were sodium butyrate, mevalonate and hydrocortisone. Although butyrate has been shown to elevate transcription of some genes, it was concluded that this could not have been the only mechanism of action, since mevalonate and hydrocortisone are both implicated in the lipid pathway of protein glycosylation, but not with transcriptional activation of the beta actin promoter. Conversely, actual supply of dolichol-linked oligosaccharide for glycosylation was probably not rate limiting, since butyrate has never been reported to affect the supply of this comerabolite, but glycosylation is likely to be implicated in some way.

Expression of FSH in CHO cells, I. Comparison of promoter types and effects of their respective inducers.

The expression levels of recombinant human follicle stimulating hormone (r-hFSH) were studied in several CHO-Kl derived cell lines. The cell lines varied in the promoter used to control r-hFSH expression and in the subunit gene copy ratio. FSH is a heterodimeric molecule, with 2 N-glycosylation sites per peptide chain, and shares a common alpha subunit with the other gonadotropins. Serum stimulated FSH production in the beta actin promoter cell lines 2-3 times over the 7-10 ng/10(6) cells/h levels obtained in protein-free medium. Serum seemed to have roles other than purely at the transcriptional level judging by the increased free alpha to dimer ratio secreted from cells cultured in serum-free medium. Zinc induced FSH expression in metallothionein cell lines, with a 3-fold induction at 50 microM concentrations compared to 0 microM zinc, giving specific productivities of about 7-10 ng/10(6) cells/h, but the induction kinetics were complicated, and suggested other roles for zinc in addition to activation of the metallothionein promoter. Evidence suggested significant post-transcriptional regulation of FSH expression.

Role of environmental conditions on the expression levels, glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells.

A recombinant CHO cell line in which the expression of human follicle stimulating hormone (hFSH) was under the control of the beta actin promoter was maintained in steady state perfusion cultures on a protein free medium. The level of expression of the hFSH was controlled by varying the steady state level of dissolved oxygen (10-90% of air saturation) and of sodium butyrate (0-1.5mM). Under these conditions, the specific productivity of hFSH (qFSH) varied from 0.7 to 4.8 ng hFSH/10(6) cells/h. As the specific productivity of hFSH increased, there was a shift in the FSH isoforms to the lower pI fractions, corresponding to increased sialic acid content. As the specific productivity of hFSH increased, shifting the isoform distribution towards the lower pI isoforms, that the sialyltransferase enzymic activity also increased.

Growth and protein production kinetics of a murine myeloma cell line transfected with the human growth hormone gene.

A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.

Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements.

We describe the development of metallothionein-based vectors with low basal levels of expression that are hyperinducible upon treatment with heavy metals. Vectors were constructed by substituting a region in the hMTIIA promoter (bp -70 to -129) containing an element (BLE) involved in basal level expression with multiple metal responsive elements (MREs). In expression studies utilizing cat as a reporter gene, heavy metal inducibility was examined in both transiently transfected and permanently transformed Chinese hamster ovary (CHO) cells. Our results demonstrate that, within the same promoter structure, inducibility can be increased by altering the ratio of MREs to BLEs. Optimal induction of expression in permanently transformed CHO cells was achieved by exposure to heavy metals for 48 h prior to cell harvest, with an additional boost 12 h before harvest. These vectors have the potential to be used for production of proteins in cultured mammalian cells and in gene expression in transgenic animals.

Optimization of steam explosion as a method for increasing susceptibility of sugarcane bagasse to enzymatic saccharification.

The technique of autohydrolysis steam explosion was examined as a means for pretreatment of sugarcane bagasse. Treatment conditions were optimized so that following enzymatic hydrolysis, pretreated bagasse would give 65.1 g sugars/100 g starting bagasse. Released sugars comprised 38.9 g glucose, 0.6 g cellobiose, 22.1 g xylose, and 3.5 g arabinose, and were equivalent to 83% of the anhydroglucan and 84% of the anhydroxylan content of untreated bagasse. Optimum conditions were treatment for 30 S with saturated steam at 220 degrees C with a water-to-solids ratio of 2 and the addition of 1 g H(2)SO(4)/100 g dry bagasse. Bagasse treated in this manner was not inhibitory to fermentation by Saccharomyces uvarum except at low inoculum levels when fermentation time was extended by up to 24 h. Pretreated saccharified bagasse was inhibitory to Pachysolen tannophilus and this was attributed to the formation of acetate from the hydrolysis of acetyl groups present in hemicellulose. The major advantage of the pretreatment is the achievement of high total sugar yield with moderate enzyme requirement and only minor losses due to sugar decomposition.

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture.

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production.

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h and a maximum prodigiosin concentration of 80 ng of prodigiosin . mg of cell protein were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h), and maximum prodigiosin concentrations were higher (1,500 ng . mg of protein). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH(2)PO(4) above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion . liter, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng . mg protein) was observed in chemostat cultures at a dilution rate of 0.057 h. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.

Continuous-culture studies on the regulation of tylosin biosynthesis.

The metabolic regulation of tylosin synthesis by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture a medium which diminished the trophophase-idiophase kinetic pattern was used to assess the activities of the enzymes involved in tylosin synthesis. The enzymes methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) showed early enzymatic derepression, both enzymes reaching their highest specific activities after 72-96 fermentation. The activity of macrocin 3' -O-methyltransferase, the enzyme catalyzing the conversion of macrocin (tylosin C) to tylosin (tylosin A). also peaked at 72 h. The specific activities of the three enzymes showed close correlation with the q(tylosin) value. In chemostat cultures the activities of the enzymes and the intracellular level of the adenylate pool and energy charge were studied as a function of dilution rate. Under steady-state conditions, increases in the specific growth rate repressed the enzymes activities with a concomitant increase in the intracellular level of the adenylate pool, while the adenylate energy charge remained almost constant and in the range 0.5-0.52. The highest specific activities of the enzymes were observed when D = 0.008 h (-1). The specific rate of tylosin synthesis was inversely proportional to the specific growth rate and the intracellular level of adenylate pool. The pool of adenylate could be a nutritional parameter which had a considerable influence on the biosynthesis of tylosin.

Metabolic regulation in tylosin-producing Streptomyces fradiae: phosphate control of tylosin biosynthesis.

The effects of increased concentration of inorganic phosphate on the biosynthesis of tylosin, the level of the intracellular adenylates, the energy charge, and the activities of enzymes involved in the synthesis of tylonolide precursors were studied in Streptomyces fradiae NRRL 2702. No metabolic response was observed when elevated levels of inorganic phosphate were added in idiophase. Increased initial levels of inorganic phosphate suppressed tylosin production and markedly increased the levels of the adenylates, although the adenylate energy charge was unchanged. Higher growth and glucose uptake rates were also observed. The activities of methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) were suppressed by the increased concentration of inorganic phosphate. The results indicated that the rate of tylosin synthesis was inversely related to the absolute level of the adenylates rather than to the energy charge.

Metabolic regulation in tylosin-producing Streptomyces fradiae: regulatory role of adenylate nucleotide pool and enzymes involved in biosynthesis of tylonolide precursors.

The relationship was studied between the level of the intracellular adenylates and the biosynthesis of tylosin by Streptomyces fradiae NRRL 2702. The adenylate level was observed to be inversely related to the rate of tylosin biosynthesis and hence the final concentration of the antibiotic. The concentrations of the adenylates were maximal during the trophophase, dropped quickly before the onset of tylosin biosynthesis, and remained at low levels throughout the idiophase. The adenylate energy charge was almost constant throughout the fermentation and was in the range of 0.4 to 0.55. Glucose addition in the idiophase suppressed tylosin biosynthesis, accompanied by a rapid increase in the adenylate levels. The biosynthesis of tylosin resumed after a rapid drop in the adenosine triphosphate concentration. Two enzymes catalyzing the interconversion of propionyl-coenzyme A and methylmalonyl-coenzyme A were found in this organism: methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3). The activity of the former was two orders of magnitude higher than that of the latter. The activities of both enzymes were affected by the increased glucose addition in the idiophase.