Patent EP0433827A2 successfully refreshed from Google Patents.
EP0433827A2
Expired / Lapsed
EP
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Cytotoxic lymphocyte maturation factor and monoclonal antibodies directed thereto
Assignee: F Hoffmann La Roche AG
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Filing: 1990-12-09
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Granted: —
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Expires: 2010-12-09
Abstract
Abstract The present invention relates to a novel cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF) which is produced and synthesized by a human B lymphoblastoid cell line. CLMF synergistically induces in the presence of low concentrations of IL-2 the cytolytic activity of Lymphokine Activated Killer (LAK) cells. CLMF is also capable of stimulating T-cell growth. The present invention also relates to cloned genes coding for CLMF proteins and derivatives thereof, to recombinant vectors comprising a polynucleotide encoding a CLMF protein to microorganisms transformed with the said recombinant vectors, to antibodies directed to the said proteins as well as to processes for the preparation of the said proteins, vectors and antibodies. Furthermore the present invention relates to methods for stimulating LAK cells, T-cells or Natural Killer Cells using the said CLMF protein.
Inventors
- 👤 Richard Anthony Chizzonite
- 👤 Maurice Kent Gately
- 👤 Ulrich Andreas Gubler
- 👤 Jeffrey David Hulmes
- 👤 Yu-Ching Eugene Pan
- 👤 Frank John Podlaski
- 👤 Alvin Seth Stern
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 20:13
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/18; C07K16/24; A; A61; A61P; A61P35/00; A61P37/00; A61P37/02; A61P37/06; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/5434; C07K16/244; A61K; A61K38/00; C07K2317/00; C07K2317/30; C07K2317/34
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
43 words
A protein having Cytotoxic Lymphocyte Maturation Factor (CLMF) activity in a substantially pure form or a derivative of the said protein which derivative exhibits CLMF activity and comprises at least a part of the amino acid sequence of the natural form of CLMF.
Claim 2
Dependent Claim
25 words
The protein of claim 1 which is the natural form of CLMF having a molecular weight of 75 kDa or a biologically active fragment thereof.
Claim 3
Dependent Claim
48 words
The protein of claim 1 which is the natural form of CLMF having a molecular weight of 75 kDa or a biologically active fragment thereof and wherein the 75 kDa CLMF polypeptide is comprised of two disulfide linked subunits, a 35 kDa subunit and a 40 kDa subunit.
Claim 4
Dependent Claim
21 words
The protein of claim 1 comprising the amino acid sequence or a sub-sequence thereof, and having optionally an N-terminal Met residue.
Claim 5
Dependent Claim
21 words
The protein of claim 1 comprising the amino acid sequence or a sub-sequence thereof, and having optionally an N-terminal Met residue.
Claim 6
Dependent Claim
16 words
An isolated polynucelotide encoding a protein as claimed in any one of claims 1 to 5.
Claim 7
Dependent Claim
26 words
An isolated polynucleotide encoding a protein as claimed in any one of claims 1 to 5 which polynucleotide comprises all or part of the nucleotide sequence:
Claim 8
Dependent Claim
26 words
An isolated polynucleotide encoding a protein as claimed in any one of claims 1 to 5 which polynucleotide comprises all or part of the nucleotide sequence:
Claim 9
Dependent Claim
19 words
A recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5.
Claim 10
Dependent Claim
31 words
A recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5 having all or part of the nucleotide sequence given in claim 7.
Claim 11
Dependent Claim
31 words
A recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5 having all or part of the nucleotide sequence given in claim 8.
Claim 12
Dependent Claim
23 words
A microorganism transformed with a recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5.
Claim 13
Dependent Claim
35 words
A microorganism transformed with a recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5 having all or part of the nucleotide sequence given in claim 7.
Claim 14
Dependent Claim
35 words
A microorganism transformed with a recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5 having all or part of the nucleotide sequence given in claim 8.
Claim 15
Dependent Claim
10 words
A substantially pure polyclonal or monoclonal antibody directed to CLMF.
Claim 16
Dependent Claim
15 words
A protein according to any one of claims 1 to 5 as an antitumor agent.
Claim 17
Dependent Claim
57 words
A process for producing a protein according to any one of claims 1 to 5 which process comprises: (a) culturing a microorganism transformed with a recombinant vector comprising a polynuclotide encoding the said protein in a culture medium under conditions permitting the expression of the encoded protein; and (b) recovering the expressed protein from the culture medium.
Claim 18
Dependent Claim
45 words
A process for producing a protein according to any one of claims 1 to 5 which process comprises: (a) preparing sub-unit peptides of the said protein by conventional peptide synthesis methods; and (b) coupling the sub-unit peptides under conditions favouring the formation of peptide bonds.
Claim 19
Dependent Claim
104 words
A process for producing a protein according-to claim 1 which protein is a Cytotoxic Lymphocyte Maturation Factor (CMLF) in a substantially pure form, which process comprises: (a) stimulating cells capable of producing CLMF such as lymphoblastoid cells to produce and secrete cytokines into a supernatant liquid; (b) collecting the supernatant liquid produced by the stimulated cells; (c) separating the supernatant liquid into protein fractions; (d) testing each protein fraction for the presence of CLMF by means of a suitable assay; (e) retaining the protein fractions which contain CLMF; and (f) isolating the CLMF from the said CLMF-containing protein fraction in a substantially pure form.
Claim 20
Dependent Claim
13 words
The process of claim 19 wherein the cells are NC-37 B lymphoblastoid cells.
Claim 21
Dependent Claim
22 words
The process of claim 19 wherein the supernatant liquid is separated into protein fractions by means of a strong cation exchange column.
Claim 22
Dependent Claim
22 words
The process of claim 19 wherein the supernatant liquid is separated into protein fractions by means of a sulfopropyl cation exchange resin.
Claim 23
Dependent Claim
80 words
The process of claim 19, wherein the supernatant liquid is separated into protein fractions by means of a strong cation exchange column and which process further comprises: (a) passing the protein fractions containing CLMF through an agarose gel, said agarose gel being comprised of a Blue B dye covalently coupled to an agarose matrix; and (b) testing the protein fractions which are eluted through said agarose gel for the presence of CLMF and retaining the protein fractions which contain CLMF.
Claim 24
Dependent Claim
139 words
The process of claim 19 wherein the supernatant liquid is separated into protein fractions by means of a strong cation exchange column and which process comprises passing the protein fractions containing CLMF through an agarose gel, said agarose gel being comprised of a Blue B dye covalently coupled to an agarose matrix; testing the protein fractions which are eluted through said agarose gel for the presence of CLMF and retaining the protein fractions which contain CLMF and which process further comprises: (a) eluting the protein fractions containing CLMF obtained in this way through a strong anion exchange in a High Performance Liquid Chromatography mode or a Fast Protein Liquid Chromatography mode to obtain protein fractions; and (b) testing the protein fractions eluted through the strong anion exchange for the presence of CLMF and retaining the protein fraction containing CLMF.
Claim 25
Dependent Claim
75 words
A process of producing a protein according to claim 1 from supernatant liquids obtained from cultured cells, which liquids contain CLMF together with other proteins which process comprises: (a) separating the supernatant liquids into protein fractions; (b) testing each protein fraction for the presence of CLMF by means of a suitable assay; (c) retaining the protein fractions which contain CLMF; and (d) isolating the CLMF from said CLMF-containing protein fraction into a substantially pure form.
Claim 26
Dependent Claim
69 words
A process for the preparation of a transformed microorganism capable of producing a protein as claimed in any one of claims 1 to 5 which process comprises: (a) transforming a microorganism with a recombinant vector comprising a polynucleotide encoding a protein as claimed in any one of claims 1 to 5; (b) growing the transformed microorganism in a culture medium; and (c) recovering the microorganism from the culture medium.
Claim 27
Dependent Claim
51 words
A process for the preparation of an antibody directed to CLMF which process comprises: (a) injecting a protein as claimed in any one of claims 1 to 5 into a warm blooded animal capable of eliciting an immune response against the said protein; and (b) recovering the antibodies from the animal.
Claim 28
Dependent Claim
23 words
Pharmaceutical composition comprising a protein as claimed in any on of claims 1 to 5 and a pharmaceutically acceptable diluent, adjuvant or carrier.
Claim 29
Dependent Claim
26 words
Pharmaceutical compositions comprising a protein as claimed in any one of claims 1 to 5, an interleukin-2 protein and a pharmaceutically acceptable diluent, adjuvant or carrier.
Claim 30
Dependent Claim
25 words
Use of a protein as claimed in claims 1 to 5 as an antitumor agent or for the manufacture of a medicament for antitumor therapy.
References Cited (Backward Citations)
No citation records indexed.
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