Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1994-01-24
2001-07-03
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
C435S069100, C514S008100, C514S012200, C530S300000, C530S350000, C530S324000, C530S351000, C530S395000
Reexamination Certificate
active
06254861
ABSTRACT:
The present invention relates in general to colony stimulating factors and specifically relates to colony stimulating factors derived from T cells.
BACKGROUND
Mediators of proliferation and differentiation for hematopoietic progenitor cells of all lineages are frequently present in the same conditioned medium. Although these secreted growth factors have similar molecular mass [usually between 25 to 35 kilo Daltons (“Kd”)] and may have apparently overlapping functional properties, they are products of distinct genes.
Hematopoietic growth factors include factors which stimulate development of certain hematopoietic cell lineages: IL-3, which has multi-lineage activity; GM-CSF, which predominantly stimulates granulocyte and macrophage colony formation, although, under certain conditions, it may stimulate megakaryocytic lineage cells as well; CSF-1, which stimulates only macrophage colonies; and G-CSF, which stimulates only granulocyte colony formation. None of these growth factors have a molecular mass (“IMW”) exceeding about 30 Kd.
Hematopoietic growth factors may be used in treating immune-compromised patients, including Acquired Immunodeficiency Syndrome “AIDS” patients and certain types of cancer patients. Accordingly, it is desirable to obtain additional hematopoietic growth factors for existing and new applications.
SUMMARY OF THE INVENTION
The present invention provides a purified and isolated nucleic acid encoding a T cell colony stimulating factor (“TC-CSF”), which term is defined by a nucleotide sequence selected from the group consisting of: a nucleotide sequence encoding a TC-CSF (such as SEQ ID NO: 1) or the nucleotide sequence in SEQ ID NO: 5; a nucleotide sequence which encodes the sequence of amino acids of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; a nucleotide sequence which hybridizes under stringent conditions with any 20 sequential nucleotides encoding a sequence of amino acids in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or with the complement thereof; a nucleotide sequence which hybridizes under stringent conditions with any 20 sequential nucleotides in a nucleic acid encoding a TC-CSF (such as SEQ ID NO: 1 or the nucleotide sequence in SEQ ID NO: 6) or with the complement thereof; and a nucleotide sequence which encodes an epitope encoded by 6 sequential amino acids in a TC-CSF or in the specific amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6, and preferably SEQ ID NO:7, or SEQ ID NO: 8.
The present invention further provides a vector including a nucleic acid according to the present invention, and a cell, preferably a eukaryotic cell, including such a vector or including a nucleic acid according to the present invention at a location or in a multiplicity in which it does not occur in nature. Such a vector may be the eukaryotic cell vector deposited as ATCC Accession No. 68824 on Oct. 28, 1991, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852. Other vectors according to the present invention may be prokaryotic or eukaryotic expression vectors, including baculovirus vectors.
The present invention also provides an isolated polypeptide having a biological or immunological property of a T cell-derived colony stimulating factor and having an amino acid sequence in which at least 6 sequential amino acids are identical to 6 sequential amino acids in a TC-CSF, particularly wherein the polypeptide includes an epitope of TC-CSF in native or denatured conformation.
An isolated TC-CSF polypeptide according to the present invention may be selected from the group consisting of human TC-CSF, mouse TC-CSF, rat TC-CSF, bovine TC-CSF, canine TC-CSF, feline TC-CSF, ovine TC-CSF, ape TC-CSF, avian TC-CSF, and porcine TC-CSF, especially a TC-CSF having a molecular mass of about 2.5-3.5 Kd, about 11-13 Kd, about 20-23 Kd, about 25-30 Kd or about 55 Kd, and may include an amino acid sequence comprising a sequence of 6 or more amino acids in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and preferably, SEQ ID NO:7 or SEQ ID NO: 8. The TC-CSF may be an expression product of a cell including a vector having DNA encoding TC-CSF or of a cell including a nucleic acid according to the present invention at a location or in a multiplicity in which it does not occur in nature. The isolated TC-CSF polypeptide may further include a diluent, adjuvant or carrier, preferably wherein the diluent comprises an isotonic buffer or pharmaceutical grade water for injection to form a pharmaceutical composition.
Any TC-CSF or nucleic acid according to the present invention not directly provided herein may be obtained according to procedures well known to those skilled in the art, including obtaining the screening of DNA libraries with polynucleotide probes based on the nucleotide sequences and deposited vector provided herein, and including identifying cells expressing TC-CSF by using a labeled antibody or oligonucleotide according to the present invention, isolating mRNA therefrom and preparing cDNA from the TC-CSF mRNA.
The isolated polypeptide may also include a label or reporter group or may be bound to a support. The isolated TC-CSF polypeptide according to the present invention may be associated with a support and an anti-TC-CSF antibody in a diagnostic kit for an immunoassay.
In addition, the present invention provides an isolated ligand specifically binding a TC-CSF, which ligand may be a TC-CSF receptor. Such a TC-CSF receptor may be identified using labeled TC-CSF according to the present invention and may be isolated using purified TC-CSF according to the present invention by techniques well known to those skilled in the art. Alternatively, the ligand may be serum or an isolated monoclonal or polyclonal antibody exhibiting a specific immunoreactivity with a TC-CSF, in particular exhibiting a specific immunoreactivity with an expression product of a cell containing a vector having DNA encoding a TC-CSF or a cell including a nucleic acid according to the present invention at a location or in a multiplicity in which it does not occur in nature.
A purified and isolated antigen is provided according to the present invention exhibiting immunological characteristics such that it specifically reacts with a monoclonal or polyclonal antibody against a TC-CSF, and the present invention also includes an immortalized cell line, such as a hybridoma, producing a monoclonal antibody to a TC-CSF.
A process for purifying TC-CSF according to the present invention includes the steps of applying cell culture supernatant containing TC-CSF to an anion exchange column, and collecting a fraction containing a TC-CSF from the anion exchange column, especially wherein the applying step includes the step of introducing the TC-CSF onto a column having a quaternary ammonium group bound to a support. The purification process may further include the steps of introducing the TC-CSF onto a gel filtration column and retaining fractions comprising TC-CSF. The purification process may also include the steps of: concentrating the eluate from the anion exchange column; introducing a TC-CSF-containing solution onto a rpHPLC column, and pooling fractions eluted therefrom; and dialyzing a TC-CSF containing solution.
A method for production of a T cell-derived colony stimulating factor (TC-CSF) according to the present invention includes the step of culturing a medium enriched in cells containing a gene encoding TC-CSF under conditions which permit expression of the gene and isolating TC-CSF from the medium or cells.
A method of treatment according to the present invention includes administering to a patient in need of therapy an effective amount of a pharmaceutical composition according to the present invention, and preferably includes administering to a patient in need of therapy an effective amount of a pharmaceutical composition including a human TC-CSF.
Also part of the present invention
Kunz Gary L.
Landsman Robert S.
Marshall O'Toole Gerstein Murray & Borun
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