EP0164965A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Lymphotoxin, nucleic acid encoding it, vectors incorporating the nucleic acid and cells transformed therewith, methods of obtaining lymphotoxin, and lymphotoxin neutralizing antibody
Assignee: Genentech Inc
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Filing: 1985-05-30
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Granted: —
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Expires: 2005-05-30
Abstract
Abstract Biologically active lymphotoxin polypeptides are synthesized in recombinant cell culture. Novel nucleic acid and vectors incorporating same are provided. The compositions and processes herein enable the economical preparation of compositions containing uniform lymphotoxin polypeptides and variant lymphotoxins having amino acid sequences that differ from those found in nature. The lymphotoxins are purified to a specific activity of 2-10x10 7 units/mg of protein by purification using a novel immobilized, lymphotoxin-neutralizing monoclonal antibody.
Inventors
- 👤 Bharat Bhushan Aggarwal
- 👤 Patrick William Gray
- 👤 Timothy Scott Bringman
- 👤 Glenn Evan Nedwin
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 09:15
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/18; C07K16/24; C07K16/241; C07K16/242; A; A61; A61P; A61P35/00; C07K14/00; C07K14/435; C07K14/52; C07K14/525; C07K14/5255; A61K; A61K38/00
Claims Analysis
Total Claims: 77
Claim 1
Independent Claim
13 words
Lymphotoxin absolutely free of homologous proteins which-are unidentified or present in unknown amounts.
Claim 2
Dependent Claim
13 words
Lymphotoxin absolutely free of homologous proteins which-are unidentified or present in unknown amounts.
Claim 3
Dependent Claim
18 words
The lymphotoxin of claim 1 having specific activity of about from 2 to 10x10 7 units/mg of protein.
Claim 4
Dependent Claim
8 words
The lymphotoxin of claim 1 which is prelymphotoxin.
Claim 5
Dependent Claim
2 words
Unglycosylated lymphotoxin.
Claim 6
Dependent Claim
2 words
Unglycosylated lymphotoxin.
Claim 7
Dependent Claim
60 words
Variant lymphotoxin wherein an amino acid residue in the lymphotoxin sequence of Fig. 2a has been (a) deleted, (b) substituted by another residue, or (c) another residue has been inserted into the sequence of Fig. 2a; provided, however, that such variant lymphotoxin excludes lymphotoxin having the Fig. 2a sequence with amino terminii at Leu + 1 or His + 23.
Claim 8
Dependent Claim
60 words
Variant lymphotoxin wherein an amino acid residue in the lymphotoxin sequence of Fig. 2a has been (a) deleted, (b) substituted by another residue, or (c) another residue has been inserted into the sequence of Fig. 2a; provided, however, that such variant lymphotoxin excludes lymphotoxin having the Fig. 2a sequence with amino terminii at Leu + 1 or His + 23.
Claim 9
Dependent Claim
16 words
The variant lymphotoxin of claim 5 which is a human allele of the Fig. 2a sequence.
Claim 10
Dependent Claim
11 words
The variant lymphotoxin of claim 5 which is an animal lymphotoxin.
Claim 11
Dependent Claim
12 words
The variant lymphotoxin of claim 7 wherein the animal lymphotoxin is bovine.
Claim 12
Dependent Claim
26 words
The variant lymphotoxin of claim 5 wherein residues -34 to -1 have been deleted and additionally containing an insertion of at least one amino acid residue.
Claim 13
Dependent Claim
21 words
The variant lymphotoxin of claim 9 wherein the insertion is a fusion of a polypeptide with the carboxyl terminus of lymphotoxin.
Claim 14
Dependent Claim
19 words
The variant lymphotoxin of claim 10 wherein the polypeptide is fused to lymphotoxin through a proteolytic enzyme hydrolysis site.
Claim 15
Dependent Claim
13 words
The variant lymphotoxin of claim 11 wherein the site comprises lys-lys or lys-arg.
Claim 16
Dependent Claim
17 words
The variant lymphotoxin of claim 11 wherein the variant lymphotoxin is cytolytically inactive prior to proteolytic hydrolysis.
Claim 17
Dependent Claim
18 words
The variant lymphotoxin of claim 9 wherein a hydrophobic di- or tripeptide is fused to leu + 171.
Claim 18
Dependent Claim
18 words
The variant lymphotoxin of claim 9 wherein ala-lys is inserted between glu + 127 and pro + 128.
Claim 19
Dependent Claim
20 words
The variant lymphotoxin of claim 9 wherein a hydrophobic amino acid residue is inserted between thr + 163 and val+164.
Claim 20
Dependent Claim
25 words
The variant lymphotoxin of claim 5 wherein at least one amino acid residue of the Fig. 2a lymphotoxin sequence has been substituted by another residue.
Claim 21
Dependent Claim
23 words
The variant lymphotoxin of claim 17 wherein residues -34 to -1 inclusive of Fig. 2a have been substituted by methionyl or formyl methionyl.
Claim 22
Dependent Claim
21 words
The variant lymphotcxin of claim 17 wherein residues -34 to + 22 inclusive have been substituted by methionyl or formyl methionyl.
Claim 23
Dependent Claim
186 words
The variant lymphotoxin of claim 17 wherein residues -34 to -1 have been deleted and (a) histidyl substituted for lysyl + 89; (b) valyl, isoleucyl or leucyl for alanyl + 163 ; (c) tyrosyl substituted for threonyl + 163; (d) lysyl substituted for seryl +82; (e) isoleucyl, leucyl, phenyalanyl, valyl or histidyl substituted for seryl +4 2 ; (f) glutamyl, tryptophanyl, seryl or histidyl substituted for lysyl + 84; (g) aspartyl or lysyl substituted for threonyl + 163; (h) lysyl or glycyl substituted for seryl + 70; (i) tyrosyl substituted for threonyl + 69; (j) arginyl or histidyl substituted for lysyl + 28; (k) arginyl or lys y l substituted for histidyl + 32; (1) prolyl, seryl, threonyl, tyrosyl or glutanyl substituted for aspartyl + 36; (m) tyrosyl, methionyl, or glutamyl substituted for seryl + 38; (n) threonyl, tyrosyl, histidyl or lysyl substituted for seryl + 61; (o) aspartyl, seryl or tyrosyl substituted for glycyl +124; (p) arginyl, lysyl, tyrosyl, tryptophanyl or prolyl substituted for histidyl +1 35 ; (q) aspartyl substituted for threonyl + 142; or (r) lysyl or threonyl substituted for glutamyl + 146.
Claim 24
Dependent Claim
12 words
The variant lymphotoxin of claim 5 which is a hybrid animal-human lymphotoxin.
Claim 25
Dependent Claim
28 words
The variant l y mphotoxin of claim 21 wherein methionyl residues + 20 , + 120 and + 133 are substituted, respectively by threonyl, seryl and valyl residues.
Claim 26
Dependent Claim
12 words
The variant lymphotoxin of claim 5 comprising a tumor necrosis factor fragment.
Claim 27
Dependent Claim
32 words
The variant lymphotoxin of claim 23 wherein the first 27, 26 or 25 amino-terminal residues of leucyl amino terminal lymphotoxin are substituted for, respectively, by a polypeptide selected from the group of
Claim 28
Dependent Claim
16 words
The variant lymphotoxin of claim 5 which contains a substitution of a single amino acid residue.
Claim 29
Dependent Claim
19 words
The variant lymphotoxin of claim 5 containing a single deletion of from 1 to about 30 amino acid residues.
Claim 30
Dependent Claim
14 words
The variant lymphotoxin of claim 5 containing an insertion of a single amino residue.
Claim 31
Dependent Claim
21 words
The variant lymphotoxin of claim 5 wherein the substitution, deletion or insertion is within about 30 residues of leucyl + 1.
Claim 32
Dependent Claim
22 words
The variant lymphotoxin of claim 5 containing a predetermined amino acid residue substitution within 25 residues of the carboxy terminal amino acid.
Claim 33
Dependent Claim
43 words
The variant lymphotoxin of claim 5 wherein'the substitution is a substitution of a residue from the classes of neutral, acid, or basic amino acid residues for a residue which is not a member of the class to which the substituted amino acid belongs.
Claim 34
Dependent Claim
9 words
The variant lymphotoxin of claim 5 which is cytotoxic.
Claim 35
Dependent Claim
71 words
Nucleic acid encoding lymphotoxin selected from the group of (a) DNA encoding lymphotoxin which is free of untranslated intervening sequences; (b) DNA encoding lymphotoxin which is free of DNA encoding other proteins of the organism from which the DNA originated; and (c) nucleic acid capable of hybridizing to nucleic acid encoding lymphotoxin provided, however, that such hybridizing nucleic acid does not have the nucleotide sequence of-natural DNA or RNA encoding lymphotoxin.
Claim 36
Dependent Claim
71 words
Nucleic acid encoding lymphotoxin selected from the group of (a) DNA encoding lymphotoxin which is free of untranslated intervening sequences; (b) DNA encoding lymphotoxin which is free of DNA encoding other proteins of the organism from which the DNA originated; and (c) nucleic acid capable of hybridizing to nucleic acid encoding lymphotoxin provided, however, that such hybridizing nucleic acid does not have the nucleotide sequence of-natural DNA or RNA encoding lymphotoxin.
Claim 37
Dependent Claim
9 words
The nucleic acid of claim 32 encoding human lymphotoxin.
Claim 38
Dependent Claim
20 words
The nucleic acid of claim 33 wherein the DNA is free of an intron present between nucleotides 284 and 285.
Claim 39
Dependent Claim
9 words
The nucleic acid of claim 34 which is cDNA.
Claim 40
Dependent Claim
14 words
The nucleic acid of claim 32 which is covalently labelled with a detectable substance.
Claim 41
Dependent Claim
11 words
- The nucleic acid of claim 32 encoding a variant lymphotoxin.
Claim 42
Dependent Claim
9 words
A replicable vector comprising the DNA of claim 32.
Claim 43
Dependent Claim
10 words
The vector of claim 38 which is replicable in prokaryotes.
Claim 44
Dependent Claim
10 words
The vector of claim 38 which is replicable in eukaryotes.
Claim 45
Dependent Claim
18 words
The vector of claim 39 additionally comprising a bacterial promoter operably linked to the nucleic acid encoding lymphotoxin.
Claim 46
Dependent Claim
18 words
The vector of claim 39 wherein the DNA encodes a fusion of a bacterial secretory leader and lymphotoxin.
Claim 47
Dependent Claim
1 words
pLTtrpl.
Claim 48
Dependent Claim
1 words
pLTtrpl.
Claim 49
Dependent Claim
1 words
p20KLT.
Claim 50
Dependent Claim
1 words
p20KLT.
Claim 51
Dependent Claim
11 words
A heterologous cell transformed with the nucleic acid of claim 32.
Claim 52
Dependent Claim
11 words
A cell transformed with the vector of claim 3 ' 8.
Claim 53
Dependent Claim
9 words
The cell of claim 46 which is a prokaryote.
Claim 54
Dependent Claim
23 words
A method comprising culturing the cell of claim 46, allowing lymphotoxin to accumulate in the culture, and recovering the lymphotoxin from the culture.
Claim 55
Dependent Claim
32 words
The method of claim 48 wherein the cell is a yeast or mammalian, nonhuman cell, the nucleic acid encodes human pre lymphotoxin and lymphotoxin having variant glycosylation is recovered from the culture.
Claim 56
Dependent Claim
11 words
The method of claim 48 wherein the cell is a prokaryote.
Claim 57
Dependent Claim
12 words
The method of claim 50 wherein the lymphotoxin is a lymphotoxin variant.
Claim 58
Dependent Claim
8 words
The product of the process of claim 49.
Claim 59
Dependent Claim
8 words
A recombinant cell culture comprising leucyl amino-terminal lymphotoxin.
Claim 60
Dependent Claim
8 words
A recombinant cell culture comprising leucyl amino-terminal lymphotoxin.
Claim 61
Dependent Claim
26 words
A method comprising administering to an animal a composition comprising lymphotoxin selected from the group of variant lymphotoxin, unglycosylated lymphotoxin, or the product of claim 52.
Claim 62
Dependent Claim
16 words
An antibody which neutralizes the cytolytic activity of lymphotoxin and which is free of non-neutralizing antibodies.
Claim 63
Dependent Claim
16 words
An antibody which neutralizes the cytolytic activity of lymphotoxin and which is free of non-neutralizing antibodies.
Claim 64
Dependent Claim
9 words
The antibody of claim 55 labelled with detectable substance.
Claim 65
Dependent Claim
8 words
The antibody of claim 55 which is immobilized.
Claim 66
Dependent Claim
39 words
A method for separating lymphotoxin from a composition comprising contacting the antibody of claim 55 with the composition under conditions whereby lymphotoxin is adsorbed thereon, separating the antibody-adsorbed lymphotoxin from the composition and separating the lymphotoxin from the antibody.
Claim 67
Dependent Claim
10 words
The antibody of claim 55 which is a monoclonal antibody.
Claim 68
Dependent Claim
51 words
The antibody of claim 55 wherein the lymphotoxin is a polypeptide (1) capable either of cytotoxic activity towards tumor cells in vitro or tumor necrosis in vivo and (2) having a region demonstrating functional amino acid homology with the lymphotoxin amino acid sequence shown in Fig. 2a or a fragment thereof.
Claim 69
Dependent Claim
13 words
The antibody of claim 55 which neutralizes the cytolytic activity of human lymphotoxin.
Claim 70
Dependent Claim
16 words
The antibody of claim 56 wherein the detectable substance is a fluorescent, chemiluminescent or radioisotopic label.
Claim 71
Dependent Claim
20 words
The antibody of claim 57 which is immobilized by adsorption or by a covalent bond to a surface or matrix.
Claim 72
Independent Claim
20 words
A method comprising immunizing an animal against glutaraldehyde-polymerized lymphotoxin and thereafter immunizing the same animal against a lymphotoxin-alum adsorption complex.
Claim 73
Independent Claim
20 words
A method comprising immunizing an animal against glutaraldehyde-polymerized lymphotoxin and thereafter immunizing the same animal against a lymphotoxin-alum adsorption complex.
Claim 74
Dependent Claim
2 words
Glutaraldehyde-polymerized lymphotoxin.
Claim 75
Dependent Claim
2 words
Glutaraldehyde-polymerized lymphotoxin.
Claim 76
Dependent Claim
7 words
An adsorption complex of lymphotoxin and alum.
Claim 77
Dependent Claim
7 words
An adsorption complex of lymphotoxin and alum.
References Cited (Backward Citations)
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