Oncogen

Bristol-myers Squibb Compnay

Oncogen Limited Partnership

Molecular Genetics Research and Development Limited Partnership

Molecular Genetics, Inc.

Matrix Pharmaceuticals, Inc.

Bristol-myers Squibb Pharmaceutical Research Institute-seattle

Anthony F Purchio

Timothy M Rose

Linda Madisen

Gregory D Plowman

Karl E Hellstrom

Ingegerd E Hellstrom

Joseph P Brown

Shiu-Lok Hu

Mario N Lioubin

Marc S Collett

Charles D Estin

Sridhar Pennathur

Peter S Linsley

Dennis M Brown

Daniel R Twardzik

Hans Marquardt

Mohammed Shoyab

Thomas J Brown

Michael G Neubauer

Joyce M Zarling

Edward E Luck

Marcia B Hanson

John E Skonier

Vera Brankovan

Nancy Webb

June R Merwin

Najma Malik

Larry Gentry

Amy M Brunner

Tomas Kempe

William J DeLorbe

Recombinant transforming growth factor-.beta.1 (TGF-.beta.1) is expressed to high levels in Chinese hamster ovary (CHO) cells using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-.beta.1 and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the SV40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines expressing amplified TGF-.beta.1 nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate selected transfectants. The major proteins secreted by these cells consisted of latent TGF-.beta.1 and TGF-.beta.1 precursor polypeptides as judged by immunoblots using site-specific anti-peptide antibodies derived from various regions of the TGF-.beta.1 precursor. Levels of recombinant TGF-.beta.1 protein secreted by these cells approached 30 ug/24 hour/10.sup.7 cells. Expression vectors encoding TGF-.beta.1 precursor variants which direct the synthesis and secretion of TGF-.beta.1 in transfected mammalian cells are also described.

Cloning and expression of simian transforming growth factor .beta.1

Novel compositions comprising Oncostatin M and congeners thereof, as well as methods for their preparation and methods for their use are provided. The compositions may be prepared by isolation from natural sources, or by recombinant means in either prokaryotic or eukaryotic host cells. In addition, the DNA and polypeptide sequences for Oncostatin M are disclosed. The compositions find use in modulating growth of cells, in particular inhibition of tumor cell proliferation, and stimulation of normal cell growth, especially cells involved in hematopoiesis. Cell growth inhibition compositions may additionally include an adjunctive agent comprising at least one of a transforming growth factor, tumor necrosis factor, or an interferon. Receptors having high affinity for Oncostatin M may additionally be used to screen polypeptides for Oncostatin M-like activity. Methods for use of antibodies to the compositions and probes specific for Oncostatin M mRNA as a means for detecting tumor cells are also provided.

Oncostatin M and novel compositions having anti-neoplastic activity

Antiproliferative compositions are provided which are capable of sustained release of an antiproliferative agent, particularly a TGF-.beta., at a site proximal to a target cell. The compositions are effective in inhibiting proliferation of the target cell, particularly when used in combination with a vasoconstrictive agent.

TGF-.beta. protein compositions for inhibition of cell proliferation

A new TGF-.beta. induced gene and protein is described. Treatment of TGF-.beta. growth arrested cells induces the production of a novel gene which encodes a 683 amino acid protein, designated BIG-H3, that contains four homologous repeat regions and which may represent a cell surface recognition molecule. This gene and protein is induced in mammalian cells, and specifically human cells, upon treatment with TGF-.beta., and is shown to inhibit the growth of tumor cells.

TGF-.beta. induced gene

Type III TGF-.beta. receptor is identified in and purified from normal human embryonic palatal mesenchyme (HEPM) cells and the purified product characterized structurally and functionally. HEPM cells were found to express high levels of the type III TGF-.beta. receptor and were found to significantly down-regulate two classes of TGF-.beta. receptor binding site. Purification of the type III TGF-.beta. receptor from solubilized HEPM cell membranes by affinity chromatography yielded a biologically active protein of about 205 kd which specifically binds both the recombinant and natural forms of TGF-.beta.1 and TGF-.beta.2, with affinity dissociation constants in the picomolar range.

Normal human growth regulatory receptor for TGF-.beta.

The present invention relates to the production of large quantities of a novel chimeric TGF-.beta., termed TGF-.beta.1/.beta.2, by eucaryotic host cells transfected with recombinant DNA vectors containing the TGF-.beta.1/.beta.2 precursor coding sequence controlled by expression regulatory elements. Simian TGF-.beta.1 cDNA (Sharples et al., 1987, DNA 6:239-244) was modified so that the nucleotides encoding amino acid residue numbers 9-13, 17, 19, 25 and 26 of the mature TGF-.beta.1 sequence were hanged to the nucleotides encoding the corresponding amino acids of the mature TGF-.beta.2 structure. Simian codon usage was maintained. The chimeric TGF-.beta.1/.beta.2 of the invention induces effects on the proliferation of vascular endothelial cells equivalent to those induced by TGF-.beta.1.

Methods using novel chimeric transforming growth factor-.beta.1/.beta.2

Peptides or proteins related to a melanoma associated antigen are described. These are produced in large quantities via recombinant DNA techniques and/or by chemical synthetic methods. The peptides or proteins can be used as immunogens in vaccine formulations which can induce an immune response that selectively destroys melanoma cells in a vaccinated individual. Where the peptides or proteins are expressed by a recombinant virus, inactivated or live virus vaccine formulations may be prepared.

Recombinant viruses encoding the human melanoma-associated antigen

A chimeric transforming growth factor-beta termed TGF-.beta.1/.beta.2, a DNA molecule encoding TGF-.beta.1/.beta.2, a mammaliam cell transformed with said DNA molecule, and a method for producing chimeric TGF-.beta.1/.beta.2 are disclosed.

TGF-.beta.1/.beta.2: a novel chimeric transforming growth factor-beta

The use of TGF-.beta. to inhibit HIV infection and/or replication is described. Both mature and precursor forms of TGF-.beta. are efficacious in inhibiting production of HIV. The TGF-.beta. used to inhibit HIV may be obtained from natural sources or may be produced by recombinant DNA or chemical synthetic techniques. TGF-.beta.1 and/or TGF-.beta.2 may be used. Additionally, hybrid TGF-.beta.1/.beta.2 molecules may also be utilized.

In vitro method of inhibition of HIV using transforming growth factor-.beta

cDNA clones coding for TGF-.beta.2 which are used to construct expression vectors capable of directing the high-level expression of mature, biologically active TGF-.beta.2, as well as precursor TGF-.beta.2 forms, in transfected Chinese Hamster Ovary cells (CHO cells) and transfected COS cells are described. CHO and COS transfectants secreting TGF-.beta.2 at high levels are also described. CHO cells transfected with a plasmid vector carrying the complete 414 amino acid simian TGF-.beta.2 precursor secrete approximately 5 .mu.g per ml culture media.

Cloning and expression of transforming growth factor .beta.2

Vaccines against melanoma

Methods and compositions are provided for the cloning and expression of Rift Valley Fever Virus genes in single cell host organisms. Also described are methods for culturing these novel single-cell organisms to produce Rift Valley Fever Virus gene gene products which may be formulated for use as products which may be formulated for use as immunogens in vaccines to protect against Rift Valley Fever Virus infections.

Vaccines against rift valley fever virus

Methods and compositions for the insertion and molecular cloning of RNA in DNA cloning vectors are described. RNA molecules to be cloned are modified by the ligation of oligonucleotide linkers onto the termini of the RNA molecule using T4 RNA ligase. Such linkers may be composed of DNA, RNA, or mixtures of each and facilitate the insertion and ligation of the RNA species into a DNA cloning vector. The resulting recombinant vectors are used to transform host cells which provide for the generation of multiple DNA copies of the RNA molecule.

Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides

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Seattle, WA, United States of America

Anthony F Purchio