Monoclonal antibodies for affinity purification of IL-6/IFN- beta 2 and for neutralization of HGF activity.

Two types of recombinant human IL-6 (rIL-6) were used for the development of specific monoclonal antibodies. The first was produced in E. coli and used for immunization, the second was produced in Chinese Hamster Ovary Cells (CHO) and used for screening. The complete translated sequence of the cDNA coding for human IL-6 was fused, in phase, to protein-A and the hybrid gene was fused to the strong lambda PR promoter. This protein was purified from bacterial extracts by chromatography on rabbit IgG-Sepharose columns. After six injections of the purified protein into mice, sera were tested for their binding titer in a solid phase radioimmunassay (sRIA) and for the specificity of binding by Western blots. In the sRIA, crude supernatants of CHO cells (harboring a plasmid containing the human IL-6 gene and expressing high levels of IL-6 but no protein-A or any bacterial antigen) were bound to a solid support, reacted with supernatants of the hybridomas and finally detected with [125I]-goat anti-mouse antibodies. Spleen cells derived from a mouse showing the highest binding titer were fused to mouse myeloma cells. The hybridomas were screened by the sRIA and several positive clones were isolated and characterized. One of the clones was found to neutralize the hybridoma growth factor activity of the rIL-6 from both sources. The same clone was also used for Western blots and for affinity purification of both natural and recombinant IL-6 (E. coli and CHO).

Production of two human 2',5'-oligoadenylate synthetase enzymes in Escherichia coli.

We have isolated and characterized two types of cDNA clones corresponding to interferon (IFN)-induced 1.6- and 1.8-kb mRNAs, as encoding two different forms of the 2',5'-oligoadenylate (2'-5')A synthetase enzyme. Direct expression of the two cDNAs was obtained in Escherichia coli under the control of a trp-lac hybrid promoter strongly inducible in E. coli by IPTG. Bacterial extracts were tested for 2'-5'A synthetase activity after adsorption to immobilized poly(I).poly(C) or in solution. With either one of the cDNA constructions, IPTG induced 2'-5'A synthetase activity in the bacteria to levels 10 times higher per microgram of protein than those in SV80 cells treated by 500 U/ml of IFN-beta1 for 24 h. Both bacterially produced enzymes bind to double-stranded (ds)RNA and are maximally active at 100 micrograms/ml of poly(I).poly(C). Both enzymes synthesized similar 2'-5'(Ap)nA oligomers of 2 to 8 residues in length. Antibodies against a synthetic peptide common to the two enzymes were used to characterize the bacterial products on immunoblots and confirmed that the 1.6-kb RNA produces a 39-kD protein, whereas the 1.8-kb RNA encodes a 45- to 46-kD protein. The E. coli enzyme coded by the 1.6-kb mRNA was purified to nearly homogeneity. When immobilized on poly(I).poly(C) agarose, the enzyme produces, per milliliter of poly(I).poly(C), 10(3) times more 2'-5'(Ap)nA oligomer than the most active cellular extracts. Moreover, the immobilized enzyme remains stable for several months at 4 degrees C.

Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-beta 2.

Recombinant human interferon-beta 2 was produced in Escherichia coli by direct expression of cDNA encoding the mature protein sequence. At concentrations that stimulate DNA synthesis and growth in B-cell hybridomas and plasmacytomas, the cytokine was found to exert a strong inhibition on the growth of a number of carcinoma and leukemia/lymphoma cell lines. This antigrowth effect was observed in clonogenic assays and by measurements of cell number and thymidine incorporation in growing cultures. The effect was blocked by antibodies to a synthetic peptide from the N terminus of the molecule. Normal diploid fibroblasts were inhibited at concentrations higher than those needed for breast carcinoma cells.

Cloning of genomic DNA for tumor necrosis factor and efficient expression in CHO cells.

A genomic clone for human tumor necrosis factor (TNF-alpha) was isolated using synthetic oligonucleotide probes. The genomic DNA was cleaved to remove 5' regulatory sequences and cloned in a PSVE3 expression vector containing the SV40 early promoter. The plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary cells. Efficient expression of TNF mRNA was established by Northern analysis. Expression of TNF protein was assayed for by cytotoxic activity for cycloheximide-treated SV80 fibroblasts. Selected transfected cultures secreted as much as 50,000 units of TNF activity/ml of culture medium. Synthesis of TNF protein was confirmed by immunofluorescence of transfected cells with a monoclonal antibody to TNF and immunoprecipitation of 17 kD protein from transfected CHO culture supernates. The efficient expression of TNF from genomic DNA in transfected mammalian cells may be advantageous for biologic uses.

Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells.

DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells.

Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells.

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day.

Rapid improvement in a terminal case of hairy cell leukemia treated with a new human recombinant interferon, IFN-alpha-C.

A new type of human interferon (IFN), IFN-alpha-C, produced in Escherichia coli and purified on monoclonal antibodies, was given at a dose of 3 X 10(6) u (3 micrograms)/day to a terminally ill unsplenectomized patient with hairy cell leukemia, who had had severe recurrent infections and pancytopenia. There was marked reduction in the size of the spleen after 2 weeks, and platelet counts returned to normal after 1 month of treatment. The IFN treatment also raised the granulocyte counts and hemoglobin levels, improved the normal repopulation of the bone marrow, and restored resistance to infections. IFN-alpha-C was well tolerated, without serious side effects, and treatment has been continued for 10 months.

Structure of two forms of the interferon-induced (2'-5') oligo A synthetase of human cells based on cDNAs and gene sequences.

The (2'-5') oligo A synthetase E, one of the translational inhibitory enzymes whose synthesis is strongly induced by all interferons (IFNs), is shown to be encoded in human cells by a 13.5-kb gene. By a cell-specific differential splicing, between the seventh and an additional eighth exon of this gene, two active E mRNAs of 1.6 and 1.8 kb are produced, along with several longer transcripts. cDNA clones for the two mRNAs were obtained and their sequences indicate that the human (2'-5') oligo A synthetase gene codes for two forms of the enzyme of mol. wt. 41 000 and 46 000, which differ only by their C-terminal ends. The product of the 1.6-kb RNA (E16) has a very hydrophobic C terminus, which is replaced by a longer acidic C-terminal sequence in the 1.8-kb RNA product (E18). The transcriptional start site of the gene was identified and 200 bp of the 5' flanking region were sequenced. A strong homology was found between this region of the IFN-activated (2'-5') oligo A synthetase gene and the corresponding region of the human fibroblast IFN-beta 1 gene, whose transcription is also stimulated by IFN priming. The gene has two polyadenylation sites which share a common undecanucleotide, but are used in a cell-specific manner to give rise to the 1.6- and 1.8-kb mRNAs.

Family of human alpha-interferon-like sequences.

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.

Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter.

The coding sequence of the human interferon (IFN)-beta 1 gene, fused 60 bp downstream from the RNA start site of the SV40 early gene, was transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells together with a selectable DHFR gene. Most transformants continuously secreted IFN-beta 1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-beta 1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-beta 1 gene, or by the IFN-beta 1 coding region fused to another poly(rI):(rC)-inducible gene. In these cases, the transformants showed strictly inducible (not constitutive) IFN secretion. By selection for methotrexate resistance, CHO clones with a 10-20-fold amplification of the SV40-IFN-beta 1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 10(6) cells/24 hr, i.e., over 10 times more than fully induced human fibroblasts. In continuous culture with daily changes of medium, accumulation of IFN-beta 1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-beta 1 produced by the transformed CHO cells were purified to homogeneity by affinity chromatography on monoclonal antibodies. This IFN appears identical in size, activity, and immunospecificity to the native human IFN-beta 1 glycoprotein.

Expression of human interferon genes using the recA promoter of Escherichia coli.

Interferon beta 1 and three alpha-interferon genes were cloned on Eco RI fragments isolated from a human genomic library into the Eco RI site of a plasmid containing the recA promoter of E. coli. Expression of interferon activity from cells carrying these plasmids was nalidixic acid inducible. The alpha-interferon genes were expressed only when in the same transcriptional orientation as the recA promoter while the beta 1 interferon gene was expressed in either orientation. Interferon activity was also inducibly expressed from the recA promoter in cells containing a plasmid carrying a fusion of the recA gene with the beta 1 interferon gene. This interferon activity was thirty-fold less sensitive to neutralization by polyclonal antibodies than authentic interferon, implying that the change near the amino terminus affects either antibody recognition or specific activity or both.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Sequences involved in the regulated expression of the human interferon-beta1 gene in recombinant SV40 DNA vectors replicating in monkey cells.

The human genomic EcoRI fragment of 1.83 kb containing the interferon (IFN) gene IFN-beta1 with 285 nucleotides of 5'-flanking sequences was transfected into monkey kidney CV-1 cells as part of an SV40-pML2 vector. Induction of the monkey cells to produce IFN led to a rapid accumulation of IFN-beta1 RNA whose 5' ends were identical to the IFN-beta1 mRNA of human fibroblasts. This induction occurred with all recombinants tested. Expression from the SV40 late promoter was also seen in non-induced cells. We conclude that the regulation of the IFN-beta1 gene is retained in the replicating episomal SV40 vectors with high copy number, even when the gene is being transcribed from an external promoter. When the 5'-flanking sequences were deleted to leave only 40 bp before the presumed cap site of the IFN-beta1 gene, inducible formation of IFN-RNA with authentic 5' ends could still be demonstrated. However, inducibility and expression depended on the position of the deleted IFN-beta1 gene in the vector. We conclude that the sequences around the TATAA box and cap site on the IFN gene are involved in the regulation of its expression. Regulated short-term expression of the human IFN-beta1 gene in SV40 vectors provides a defined system in which the structures required to maintain the regulation and the influence of known external transcription signals can be examined.

Construction of human gene libraries from small amounts of peripheral blood: analysis of beta-like globin genes.

We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood. High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E. coli host. Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia. The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.

Synthesis of human interferon beta 1 in Escherichia coli infected by a lambda phage recombinant containing a human genomic fragment.

DNA from a human adult was fragmented by partial digestion with restriction endonuclease EcoRI and cloned in lambda Charon 4A. Clone C15, with a human DNA insert of 17 X 10(3) bases, was identified as containing a gene for the fibroblast interferon, interferon beta 1. Restriction mapping shows that this gene, located on a 1840-base EcoRI fragment, is not interrupted by introns. Moreover, we show that this human genomic DNA fragment is able to direct the synthesis of active human interferon beta 1 in Escherichia coli. Interferon activity of up to 7 X 10(6) U/l was recovered from phage lysates by chromatography on Cibacron blue--Sepharose, and had the same immunological properties and species specificity as interferon produced by human fibroblasts.

Analysis of mouse embryonic gene library for the frequency of single and multiple copy genes.

A gene library was constructed from embryonic mouse DNA by ligating DNA fragments generated by partial Eco RI digestion with Charon 4A vector and in vitro packaging. A special consideration was given to randomization of target DNA. The general applicability of a gene library prepared in this manner was assessed through cloning a variety of genes of known reiteration frequency in the mouse genome. The survey included a single copy gene--C region of the immunoglobulin heavy chain, and genes that appear in more than one copy--V region of the immunoglobulin light chain genes and the endogenous retrovirus related genes. In all cases tested the frequency of clone isolation was in good agreement with the expected incidence based on the number of genome equivalents screened and the reiteration frequency of that particular gene. Moreover, we found no preference with regard to the clonability of genes contained in fragments of a wide-size range.

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.

RNA synthesis in isolated nuclei: the use of mercurated nucleotides.

We have used uridine 5' triphosphate-5-mercury (Hg-UTP) in place of UTP to study RNA synthesis in a previously described isolated nuclei system (1). Employing isopycnic density gradient centrifugation to separate RNAs based upon their relative content of Hg-U, several conclusions can be drawn. In vitro RNA synthesis consists of end addition onto pre-initiated HnRNA molecules as well as apparent initiation of new HnRNA molecules de novo. Synthesis in our system continues linearly for greater than two hours. The chain elongation rate has been measured to be about 500 nucleotides per minute. The methods used to make these measurements are generally applicable to other in vitro systems.