Differential effect of isotype on efficacy of anti-tumor necrosis factor alpha chimeric antibodies in experimental septic shock.

Immune complexes containing human gamma (g)1 or murine g2a antibodies generate secondary effector mechanisms via Fc receptor binding or complement activation, whereas those containing human g4 or murine g1 antibodies generally do not. Therefore, isotype selection of therapeutic antibodies may have important clinical consequences. In a rabbit model of human tumor necrosis factor (rhuTNF)-induced pyrexia, a murine/human chimeric g4 anti-human TNF-alpha monoclonal antibody (mAb) (cCB0011) showed a dose-dependent inhibition of pyrexia, whereas a g1 isotype variant of the same mAb gave a marked pyrexia that was seen at all doses indicative of an immune complex-mediated response. To investigate whether isotype difference could influence mAb efficacy in pathological disease states, hamster/murine chimeric g1 and g2a anti-murine TNF-alpha mAbs (TN3g1, TN3g2a) were studied in experimental shock in mice and rats. In lipopolysaccharide-induced shock in mice, treatment with TN3g1 mAb at 30 and 3 mg/kg resulted in 90% survival by 72 h (p < or = 0.004), and prolonged survival to 45 h (p < or = 0.05), respectively, compared with 100% mortality by 27 h in controls. In contrast, a g2a isotype variant of the same mAb (30 mg/kg) resulted in only 10% survival by 72 h (p < or = 0.05). In a neutropenic sepsis model in rats there was greater survival in animals receiving the g1 isotype of TN3 compared with g2a isotype variant (70 vs. 27%; p < or = 0.005) with 100% mortality in the controls. These differences were not due to the pharmacokinetic profiles of the mAbs. In models of experimental shock antibody isotype can affect outcome with inactive isotypes (human g4 and murine g1) being more efficacious than active isotypes (human g1 and murine g2a).

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator.

Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

A 6-fold difference in the half-life of immunoglobulin mu heavy chain mRNA in cell lines representing two stages of B cell differentiation.

When B cells differentiate into plasma cells, there is a large increase in the cellular content of mRNA coding for immunoglobulin. This increase cannot be fully accounted for by the increase in rate of transcription of the genes. We have investigated the possibility that the half-life of mu heavy chain mRNA increases during B cell differentiation, by measuring the rates of decay of the same endogenous mu gene in two cell lines representing the B cell and the plasma cell. Using a pulse-chase protocol, it was found that there was a significant increase in the half-life of mu mRNA between the B cell and the plasma cell, and no detectable difference in the average half-life of total poly(A)+ RNA in the two cell lines. The reduced rate of decay of mu mRNA in the more differentiated cell type is almost sufficient to account for the difference in steady state mu mRNA levels between the two cell lines.

Expression of heterologous genes in E. coli.

The strategies for the expression of cloned foreign genes in E. coli are described, and some of the tactics that can be employed to ensure high levels of expression are identified. More fundamental problems associated with scale-up, such as plasmid stability and protein processing, are also addressed.

Vitamin D in the avian egg. Its molecular identity and mechanism of incorporation into yolk.

The chemical identity of vitamin D in the egg of the domestic fowl was studied by analysing radioactivity in eggs from hens injected with [3H]cholecalciferol. Labelled molecules were found throughout the egg, but the concentration of total radioactivity in albumin was only 5-7% of that in yolk. In lipid extracts of yolk, more than 90% of the radioactivity was as unchanged cholecalciferol and 5% as 25-hydroxycholecalciferol. Only about 3% of the radioactivity in albumin was chloroform-soluble, and of this 40% was 25-hydroxycholecalciferol and 15% was cholecalciferol. Evidence is presented to support the idea that the specific transport of cholecalciferol into yolk is mediated by a cholecalciferol-binding protein in blood. This protein forms a complex with yolk proteins in transit from liver to ovary via the blood. A cholecalciferol-binding protein, chromatographically similar to that from blood, was found in egg yolk. It is postulated that cholecalciferol forms part of a complex with its specific binding protein, Ca2+ and the yolk phosphoprotein, phosvitin. This complex is then incorporated into yolk by the thecal cells of the ovarian follicle.

Polyadenylation and reverse transcription of influenza viral RNA.

The polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E. coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported. We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase. The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA. Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels. These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end. Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8.

Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.

Structural features of encephalomyocarditis virus RNA from analysis of reverse transcription products.

The presence in encephalomyocarditis (EMC) virus RNA of homonucleotide tracts 10 nucleotides or more in length has been investigated by testing the ability of homo-oligodeoxynucleotides to prime DNA synthesis in the reverse transcriptase from avian myeloblastosis virus. Neither (dC)10 nor (dA)10 promoted incorporation of [3H]deoxynucleotides into acid-insoluble material but (dG)10 and (dT)12-18 were effective primers and produced DNA products approximately 2000 nucleotides in length. We conclude that there are single-stranded oligo(rC) and oligo(rA) tracts in native EMC virus RNA at 37 degrees C. Kinetic analysis indicated that oligo(dT) priming is similar to priming on ovalbumin mRNA and that it gives rise to only one DNA product per template molecule. Oligo(dG) priming appears to be complicated by self-aggregation of the primer. Oligo(dT)-primed and oligo(dG)-primed DNA have both been separated on alkaline-sucrose gradients into two peaks of which only the 'heavier' will hybridise to EMC virus RNA. Competitive hybridisation experiments indicate that the 'heavy' oligo(dT)-primed and oligo(dG)-primed DNA fractions hybridise to overlapping sequences of EMC virus RNA and place the priming regions of EMC virus RNA approximately 500 nucleotides apart during reverse transcription.

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes.

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.

The response of the small intestine to vitamin D. Correlation between calcium-binding-protein production and increased calcium absorption.

1. The synthesis of calcium-binding protein, a protein produced in the small intestine in response to vitamin D, was investigated with a view to determining whether calcium-binding-protein production could be correlated with the stimulation of calcium absorption by vitamin D. 2. A radioimmunological assay, which can quantitatively estimate calcium-binding-protein concentrations as low as 1mug/g wet wt., was used to detect the synthesis of soluble calcium-binding protein. 3. When used on intestinal supernatants from chicks dosed with vitamin D, calcium-binding protein was not detectable at 8h but was present after 12h at a concentration of 8.6mug/g wet wt.; in agreement with this an increase in calcium absorption due to vitamin D was detected at 12h but not at 8h. 4. The synthesis of calcium-binding protein was also monitored directly by making use of the ability of the iodinated antiserum to bind specifically to nascent calcium-binding protein chains on intestinal polyribosomes; in this way calcium-binding-protein synthesis could be detected 8h after dosage with vitamin D. Further, the binding reaction indicated a near linear increase in the calcium-binding-protein-synthesizing capacity over a 16h period. 5. From the amount of calcium-binding protein present 12 and 24h after vitamin D administration it is calculated that calcium-binding-protein mRNA is produced at approx. 1mol/min per intestinal cell. 6. It is concluded that the high correlation between the initiation of calcium-binding-protein synthesis and the stimulation of calcium absorption by vitamin D strengthens the proposal that calcium-binding protein plays an important role in calcium transport.

Production and properties of vitamin-D-induced mRNA for chick calcium-binding protein.

1. Calcium-binding protein synthesis on chick intestinal polysomes is induced within 2 h of injecting vitamin-D-deficient birds with 1,25-dihydroxycholecalciferol. The induction is short-lived: the maximum output of the binding protein is reached by 13 h after hormone injection, and declines rapidly thereafter. 2. This induction of calcium-binding protein synthesis occurs by the production of active mRNA for the protein. The sedimentation coefficient of this mRNA in denaturing conditions is 18 S, equivalent to a molecular weight of approximately 700000, and the molecule contains a tract of polyadenylate. 3. Both polysomal and poly(A)-containing RNA extracted from intestinal polysomes stimulate the synthesis of a range of proteins (up to 70000 molecular weight) by the wheat germ cell-free system. Immunoprecipitable calcium-binding protein is translated from RNA obtained from 1,25-dihydroxycholecalciferol-dosed birds but not from control birds. This calcium-binding protein is the same size (27000 molecular weight) as authentic chick calcium-binding protein; No other proteins are specifically precipitated by the antiserum. Thus in chickens 1,25-dihydroxycholecalciferol-induced calcium-binding protein is not synthesised via any precursor molecule. The implications of this result are discussed.

Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli.

Genes for a murine mu heavy chain and a lambda light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosome binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.

The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli.

A gene for murine mu heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Escherichia coli trp promoter and ribosome binding site. A low level expression of mu protein was detected. Secondary structure analysis showed the presence of a hairpin loop burying the mu initiation codon. Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between mu expression levels and accessibility of the ribosome binding site. Abolition of secondary structure increased mu protein expression over ninety-fold, to a level approximately equal to that of a trpE -mu fusion protein using the native trpE ribosome binding site.

Influenza antigenic determinants are expressed from haemagglutinin genes cloned in Escherichia coli.

A gene sequence for the fowl plague virus (FPV) haemagglutinin molecule has been inserted into a bacterial plasmid such that its transcription is under the control of a promoter derived from the tryptophan operon. Such plasmids direct the synthesis of a protein that reacts specifically with antisera to FPV haemagglutinin. Evidence is also presented that in some cases DNA inserted at the HindIII site of pBR322 is expressed.

Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA.

A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.

Synthesis of calf prochymosin (prorennin) in Escherichia coli.

A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.

The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.

The synthesis and in vivo assembly of functional antibodies in yeast.

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.