EP0267463A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Tissue-derived tumor growth inhibitors, methods of preparation and uses thereof
Assignee: OSI Pharmaceuticals LLC
•
Filing: 1987-10-20
•
Granted: —
•
Expires: 2007-10-20
Abstract
Abstract The invention provides a composition of matter designated tissue-derived growth inhibitor-1 (TGI-1) which comprises a protein which is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds. TGI-1 has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts and has an apparent molecular weight of about 26,000 daltons. The invention additionally provides a composition of matter designated tissue-derived growth inhibitor-2 (TGI-2) which is a protein which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts. TGI-2 has an apparent molecular weight of about 26,000 daltons and is a dimer composed of two polypeptides each of which has an apparent molecular weight of about 13,000 daltons and each is joined to the other by disulfide bonds under nonreducing conditions. This invention also provides a protein having tumor growth activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at positon 1 and ending with serine at position 112. A nucleic acid molecule encoding the protein is also provided. A polypeptide designated CM-1 and a polypeptide recoverable from conditioned media of A431 cells are also disclosed.
Inventors
- 👤 Kenneth K. Iwata
- 👤 Leslie I. Gold
- 👤 John R. Stephenson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 16:09
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; A; A61; A61K; A61K35/00; A61K35/12; A61K35/44; A61K35/48; A61K35/50; A61P; A61P17/00; A61P35/00; A61P43/00; C07K14/475; C07K14/495; C07K16/00; C07K16/18; C07K16/22; C07K16/24; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/74; A61K38/00; C07K2319/00; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 136
Claim 1
Independent Claim
154 words
An acidified, ethanol extract derived from human tissue which comprises a plurality of proteins, each of which has an apparent molecular weight of about 26,000 daltons and is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds, each of the extracts having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the inhibitory activity against human tumor cell growth not being destroyed upon increasing the temperature of the acidified, ethanol extract to about 100°C for about 3 minutes or upon adding acetic acid until the acidified, ethanol extract is up to about 1.0 molar in acetic acid and the inhibitory activity being enhanced when the acidified, ethanol extract is prepared at about 4°C rather than at about 23°C.
Claim 2
Dependent Claim
154 words
An acidified, ethanol extract derived from human tissue which comprises a plurality of proteins, each of which has an apparent molecular weight of about 26,000 daltons and is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds, each of the extracts having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the inhibitory activity against human tumor cell growth not being destroyed upon increasing the temperature of the acidified, ethanol extract to about 100°C for about 3 minutes or upon adding acetic acid until the acidified, ethanol extract is up to about 1.0 molar in acetic acid and the inhibitory activity being enhanced when the acidified, ethanol extract is prepared at about 4°C rather than at about 23°C.
Claim 3
Dependent Claim
14 words
An acidified, ethanol extract of claim 1, wherein the human tissue is umbilical cord.
Claim 4
Dependent Claim
13 words
An acidified, ethanol extract of claim 1, wherein the human tissue is placenta.
Claim 5
Dependent Claim
145 words
An acidified, ethanol extract derived from human umbilical cord which has been treated to remove substantially all blood, and all extracellular soluble components which comprises at least two acidic proteins having apparent molecular weights of about 26,000 daltons and being a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds, each of the proteins having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the inhibitory activity against human tumor cell growth not being destroyed upon increasing the temperature of the acidified, ethanol extract to about 100°C for about 3 minutes or upon adding acetic acid until the acidified, ethanol extract is up to about 1.0 molar in acetic acid.
Claim 6
Dependent Claim
145 words
An acidified, ethanol extract derived from human umbilical cord which has been treated to remove substantially all blood, and all extracellular soluble components which comprises at least two acidic proteins having apparent molecular weights of about 26,000 daltons and being a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds, each of the proteins having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the inhibitory activity against human tumor cell growth not being destroyed upon increasing the temperature of the acidified, ethanol extract to about 100°C for about 3 minutes or upon adding acetic acid until the acidified, ethanol extract is up to about 1.0 molar in acetic acid.
Claim 7
Dependent Claim
77 words
A protein designated tissue-derived growth inhibitor-1 (TGI-1) which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts, has an apparent molecular weight of about 26,000 daltons and is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds under nonreducing conditions.
Claim 8
Dependent Claim
77 words
A protein designated tissue-derived growth inhibitor-1 (TGI-1) which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts, has an apparent molecular weight of about 26,000 daltons and is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds under nonreducing conditions.
Claim 9
Dependent Claim
46 words
A composition of claim 5, wherein the protein is recoverable as a defined activity on hydrophobic interaction chromatography on phenyl-Sepharose of an acidified, ethanol extract of human tissue, after ether-ethanol preparation, with a linear gradient at about 1.5 M ammonium acetate and about 31% ethylene glycol.
Claim 10
Dependent Claim
67 words
A composition of claim 5, wherein the protein is recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract of human tissue with a separating gradient of acetonitrile containing 0.05% trifluoracetic acid and is recoverable as a defined activity on high performance liquid chromatography of such acidified, ethanol extract of human tissue with a separating gradient of 2-propanol containing 0.05% trifluoracetic acid.
Claim 11
Dependent Claim
37 words
A composition of claim 7, wherein the high performance liquid chromatography of the acidified, ethanol extract is carried out on a linear gradient of acetonitrile on a C18 column containing 0.05% trifluoracetic acid at about 27% acetonitrile.
Claim 12
Dependent Claim
37 words
A composition of claim 7, wherein the high performance liquid chromatography of the acidified ethanol extract is carried out on a linear gradient of 2-propanol on a CN column containing 0.05% trifluoracetic acid at about 40-41% 2-propanol.
Claim 13
Dependent Claim
148 words
A composition of matter designated tissue-derived growth inhibitor (TGI) which comprises at least two polypeptides having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts and having an apparent molecular weight of about 26,000 daltons, the composition a) being resolved as a single peak of defined activity from a cation exchange resin when eluted by a linear NaCl gradient at about 0.6-0.7 M NaCl b) being recoverable as a defined activity on high peformance liquid chromatography of the acidified, ethanol extract of claim 4 with a linear gradient of acetonitrile containing 0.05% trifluroacetic acid at about 28-34% acetonitrile and c) being recoverable as a defined activity from phenyl-Sepharose of the acidified, ethanol extract with a linear separating gradient at about 1.5 M ammonium acetate containing 31% ethylene glycol.
Claim 14
Dependent Claim
75 words
A protein designated tissue-derived growth inhibitor-2 (TGI-2) which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts, has an apparent molecular weight of 26,000 daltons and is a dimer of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds under nonreducing conditions.
Claim 15
Dependent Claim
75 words
A protein designated tissue-derived growth inhibitor-2 (TGI-2) which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts, has an apparent molecular weight of 26,000 daltons and is a dimer of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds under nonreducing conditions.
Claim 16
Dependent Claim
45 words
A composition of claim 11, wherein the protein is recoverable as a defined activity on hydrophobic interaction chromatography on phenyl-Sepharose of an acidified, ethanol extract of human tissue, after ether-ethanol preparation, with a linear gradient at about 1.5 M ammonium acetate and 31% ethylene glycol.
Claim 17
Dependent Claim
67 words
A composition of claim 12, wherein the protein is recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract of human tissue with a separating gradient of acetonitrile containing 0.05 trifluoracetic acid and is recoverable as a defined activity on high performance liquid chromatography of such acidified, ethanol extract of human tissue with a separating gradient of 2-propanol containing 0.05% trifluoracetic acid.
Claim 18
Dependent Claim
37 words
A composition of claim 13, wherein the high performance liquid chromatography of the acidified, ethanol extract is carried out on a linear gradient of acetonitrile on a C18 column containing 0.05% trifluoracetic acid at about 28-30% acetonitrile.
Claim 19
Dependent Claim
37 words
A composition of claim 13, wherein the high performance liquid chromatography of the acidified, ethanol extract is carried out on a step gradient of 2-propanol on a CN column containing 0.05% trifluoracetic acid at about 44% 2-propanol.
Claim 20
Dependent Claim
44 words
A protein designated CM-I, which has the property of inhibiting the growth of human tumor cells but not the growth of an established mink lung cell line (CCL 64) and being recoverable by cation exchange chromatography of an acidified ethanol extract of human tissue.
Claim 21
Dependent Claim
44 words
A protein designated CM-I, which has the property of inhibiting the growth of human tumor cells but not the growth of an established mink lung cell line (CCL 64) and being recoverable by cation exchange chromatography of an acidified ethanol extract of human tissue.
Claim 22
Dependent Claim
72 words
A protein recoverable from condition media of A431 cells, having an apparent molecular weight of less than about 30,000 daltons, having the property of substantially inhibiting the growth of a human tumor cell line (A549) but not of an established mink lung cell line (CCL 64) and retaining said property of insensitivity to reduction of disulfide bonds by 65 mM dithiothreitol in 0.1 M ammonium bicarbonate for 2 hours at room temperature.
Claim 23
Dependent Claim
72 words
A protein recoverable from condition media of A431 cells, having an apparent molecular weight of less than about 30,000 daltons, having the property of substantially inhibiting the growth of a human tumor cell line (A549) but not of an established mink lung cell line (CCL 64) and retaining said property of insensitivity to reduction of disulfide bonds by 65 mM dithiothreitol in 0.1 M ammonium bicarbonate for 2 hours at room temperature.
Claim 24
Dependent Claim
93 words
A protein having the following characteristics: a) an apparent molecular weight of about 26,000 daltons; b) being a dimer composed of two polypeptides having an apparent molecular weight of about 13,000 daltons and being joined by disulfide bonds; c) demonstrating tumor growth inhibitory activity of human tumor cell line (A549) and of an established mink lung cell line (CCL 64); d) being acid soluble in 1.0 M acetic acid and 0.1% trifluoracetic acid; e) being stable in heat to about 100°C; and f) being stable in up to 39% acetonitrile and 45% 2-propanol.
Claim 25
Dependent Claim
93 words
A protein having the following characteristics: a) an apparent molecular weight of about 26,000 daltons; b) being a dimer composed of two polypeptides having an apparent molecular weight of about 13,000 daltons and being joined by disulfide bonds; c) demonstrating tumor growth inhibitory activity of human tumor cell line (A549) and of an established mink lung cell line (CCL 64); d) being acid soluble in 1.0 M acetic acid and 0.1% trifluoracetic acid; e) being stable in heat to about 100°C; and f) being stable in up to 39% acetonitrile and 45% 2-propanol.
Claim 26
Dependent Claim
7 words
A protein of claim 18 designated TGI-1.
Claim 27
Dependent Claim
7 words
A protein of claim 18 designated TGI-2.
Claim 28
Dependent Claim
29 words
A protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
Claim 29
Dependent Claim
29 words
A protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
Claim 30
Dependent Claim
6 words
A purified protein of claim 21.
Claim 31
Dependent Claim
9 words
A protein of claim 22 having 112 amino acids.
Claim 32
Dependent Claim
26 words
A protein claim 21 comprising 205 amino acids shown in Figure 29 beginning with serine at position - 93 and ending with serine at position 112.
Claim 33
Dependent Claim
10 words
A nucleic acid molecule encoding the protein of claim 21.
Claim 34
Dependent Claim
10 words
A nucleic acid molecule encoding the protein of claim 23.
Claim 35
Dependent Claim
10 words
A nucleic acid molecule encoding the protein of claim 24.
Claim 36
Dependent Claim
9 words
A cDNA of claim 25 shown in Figure 29.
Claim 37
Dependent Claim
28 words
A cDNA of claim 26 shown in Figure 29 beginning with guanine of the codon at position 1 and ending with cytosine of the codon at position 112.
Claim 38
Dependent Claim
29 words
A cDNA of claim 27 shown in Figure 29 beginning with thymine of the codon at position - 93 and ending with cytosine of the codon at position 112.
Claim 39
Dependent Claim
11 words
A plasmid which comprises the nucleic acid molecule of claim 25.
Claim 40
Dependent Claim
15 words
A host vector system, comprising a plasmid of claim 31 in a suitable host cell.
Claim 41
Dependent Claim
16 words
A host vector system of claim 32, wherein the suitable host cell is a bacteria cell.
Claim 42
Dependent Claim
16 words
A host vector system of claim 32, wherein the suitable host cell is a eucaryotic cell.
Claim 43
Dependent Claim
30 words
A method for producing a protein comprising growing the host vector system of claim 32 so as to produce the protein in the host and recovering the protein so produced.
Claim 44
Dependent Claim
31 words
A polypeptide derived from the protein of claim 21, comprising the 20 amino acids shown in Figure 29 beginning with arginine at position 9 and ending with leucine at position 28.
Claim 45
Dependent Claim
15 words
An antibody which specifically binds to an epitope contained with the protein of claim 21.
Claim 46
Dependent Claim
6 words
A monoclonal antibody of claim 37.
Claim 47
Dependent Claim
16 words
An antibody of which specifically binds to an epitope contained with the polypeptide of claim 36.
Claim 48
Dependent Claim
6 words
A monoclonal antibody of claim 39.
Claim 49
Dependent Claim
50 words
A method for diagnosing a tumor which comprises contacting a sample from a human subject with an antibody of claim 37 under suitable conditions so as to form a complex between the antibody and an epitope contained with the protein and detecting the complex so formed, thereby diagnosing a tumor.
Claim 50
Dependent Claim
14 words
A pharmaceutical composition comprising the antibody of claim 37 and a pharmaceutically acceptable carrier.
Claim 51
Dependent Claim
23 words
A method of treating a tumor which comprises administering to the subject an effective tumor treating amount of the composition of claim 42.
Claim 52
Dependent Claim
27 words
A method of treating a proliferative type disorder which comprises administering to the subject an effective proliferative type disorder treating amount of the composition of claim 42.
Claim 53
Dependent Claim
26 words
A pharmaceutical composition comprising an effective amount of the acidified, ethanol extract of claim 1 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 54
Dependent Claim
26 words
A pharmaceutical composition comprising an effective amount of the acidified, ethanol extract of claim 4 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 55
Dependent Claim
25 words
A pharmaceutical composition comprising an effective amount of the protein of claim 5 or a biolog ically active fragment thereof and a suitable pharmaceutical carrier.
Claim 56
Dependent Claim
26 words
A pharmaceutical composition comprising an effective amount of the composition of matter of claim 10 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 57
Dependent Claim
24 words
A pharmaceutical composition comprising an effective amount of the protein of claim 11 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 58
Dependent Claim
24 words
A pharmaceutical composition comprising an effective amount of the protein of claim 16 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 59
Dependent Claim
24 words
A pharmaceutical composition comprising an effective amount of the protein of claim 17 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 60
Dependent Claim
24 words
A pharmaceutical composition comprising an effective amount of the protein of claim 18 or a biologically active fragment thereof and a suitable pharmaceutical carrier.
Claim 61
Dependent Claim
24 words
A pharmaceutical composition comprising an effective amount of the protein of claim 21 or a biologically active fragment thereof in a suitable pharmaceutical carrier.
Claim 62
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth inhibiting amount of composition of claim 45.
Claim 63
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 46.
Claim 64
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 47.
Claim 65
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 48.
Claim 66
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 49.
Claim 67
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 50.
Claim 68
Dependent Claim
27 words
A method for inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 51.
Claim 69
Dependent Claim
27 words
A method of inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 52.
Claim 70
Dependent Claim
27 words
A method of inhibiting the growth of human tumor cells which comprises contacting the cells with an effective tumor growth-inhibiting amount of the composition of claim 53.
Claim 71
Dependent Claim
32 words
A method of treating a proliferative type disorder in a subject which comprises administering to the subject an amount of the composition of claim 53 effective to treat the proliferative type disorder.
Claim 72
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 46.
Claim 73
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 47.
Claim 74
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 48.
Claim 75
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 49.
Claim 76
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 50.
Claim 77
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 51.
Claim 78
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 52.
Claim 79
Dependent Claim
24 words
A method for treating a burn or healing a wound which comprises contacting the burn or wound with a pharmaceutical composition of claim 53.
Claim 80
Independent Claim
168 words
A method for preparing an acidified, ethanol extract from human tissue, the acidified, ethanol extract comprising a plurality of proteins, each of which has a molecular weight of less than 30,000 daltons and each of which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. treating the tissue to solubilize the proteins and removing the solubilized proteins derived from the tissue; b. recovering the solubilized proteins; c. separately recovering from the solubilized tissue, proteins having an apparent molecular weight of less than about 30,000 daltons; d. assaying the separately recovered proteins to identify those which either inhibit the growth of human tumor cells or inhibit the growth of an established mink lung cell line (CCL 64) or enhance the growth of normal human foreskin fibroblasts; and e. recovering an acidified, ethanol extract containing the proteins so identified.
Claim 81
Independent Claim
168 words
A method for preparing an acidified, ethanol extract from human tissue, the acidified, ethanol extract comprising a plurality of proteins, each of which has a molecular weight of less than 30,000 daltons and each of which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. treating the tissue to solubilize the proteins and removing the solubilized proteins derived from the tissue; b. recovering the solubilized proteins; c. separately recovering from the solubilized tissue, proteins having an apparent molecular weight of less than about 30,000 daltons; d. assaying the separately recovered proteins to identify those which either inhibit the growth of human tumor cells or inhibit the growth of an established mink lung cell line (CCL 64) or enhance the growth of normal human foreskin fibroblasts; and e. recovering an acidified, ethanol extract containing the proteins so identified.
Claim 82
Independent Claim
189 words
A method for preparing an acidified, ethanol extract from human umbilical cord, the acidified, ethanol extract comprising tissue-derived growth inhibitor (TGI) which is comprised of at least two proteins which have a molecular weight of about 26,000 daltons and which have the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. removing veins and arteries from the umbilical cord tissue and washing the tissue to remove all traces of blood; b. treating the tissue to produce solubilized proteins and removing soluble contaminating proteins derived from the tissue; c. recovering the acid solubilized proteins; d. separately recovering from the solubilized proteins TGI having a molecular weight of about 26,000 daltons; e. assaying the separately recovered TGI to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of an established mink lung cell line (CCL 64) and enhances the growth of normal human foreskin fibroblasts; and f. recovering the acidified, ethanol extract containing the TGI so identified.
Claim 83
Independent Claim
189 words
A method for preparing an acidified, ethanol extract from human umbilical cord, the acidified, ethanol extract comprising tissue-derived growth inhibitor (TGI) which is comprised of at least two proteins which have a molecular weight of about 26,000 daltons and which have the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. removing veins and arteries from the umbilical cord tissue and washing the tissue to remove all traces of blood; b. treating the tissue to produce solubilized proteins and removing soluble contaminating proteins derived from the tissue; c. recovering the acid solubilized proteins; d. separately recovering from the solubilized proteins TGI having a molecular weight of about 26,000 daltons; e. assaying the separately recovered TGI to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of an established mink lung cell line (CCL 64) and enhances the growth of normal human foreskin fibroblasts; and f. recovering the acidified, ethanol extract containing the TGI so identified.
Claim 84
Independent Claim
178 words
A method for preparing an acidified, ethanol extract from human umbilical cord, the acidified, ethanol extract comprising tissue-derived growth inhibitor 1 (TGI-1) and tissue-derived tumor growth inhibitor 2 (TGI-2) which have a molecular weight of about 26,000 daltons and which have the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. removing the blood constituent by washing. b. treating the tissue to produce solubilized proteins and removing contaminating soluble proteins derived from the tissue; c. recovering the acid solubilized proteins; d. separately recovering from the solubilized proteins TGI-1 and TGI-2 having a molecular weight of about 26,000 daltons; e. assaying the separately recovered TGI to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of an established mink lung cell line (CCL 64) and enhances the growth of normal human foreskin fibroblasts; and f. recovering the acidified, ethanol extract containing the TGI so identified.
Claim 85
Independent Claim
178 words
A method for preparing an acidified, ethanol extract from human umbilical cord, the acidified, ethanol extract comprising tissue-derived growth inhibitor 1 (TGI-1) and tissue-derived tumor growth inhibitor 2 (TGI-2) which have a molecular weight of about 26,000 daltons and which have the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the method comprising under suitable conditions the following steps: a. removing the blood constituent by washing. b. treating the tissue to produce solubilized proteins and removing contaminating soluble proteins derived from the tissue; c. recovering the acid solubilized proteins; d. separately recovering from the solubilized proteins TGI-1 and TGI-2 having a molecular weight of about 26,000 daltons; e. assaying the separately recovered TGI to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of an established mink lung cell line (CCL 64) and enhances the growth of normal human foreskin fibroblasts; and f. recovering the acidified, ethanol extract containing the TGI so identified.
Claim 86
Dependent Claim
57 words
A method of claim 72, wherein the step (a) treating the tissue comprises homogenizing the tissue for a suitable period of time to form homogenized tissue, suspending the homogenized tissue in a suitable acidic extraction buffer containing ethanol at about 4°C, and stirring the homogenized tissue for a suitable period at about 4°C to produce solubilized proteins.
Claim 87
Dependent Claim
25 words
A method of claim 72, wherein the step (c) the separate recovery of proteins from the solubilized proteins comprises molecular sieve chromatography of the proteins.
Claim 88
Dependent Claim
77 words
A method of claim 72, wherein the step (d) assaying the proteins comprises separately contacting the human tumor cells, an established mink lung cell line (CCL 64) or normal human foreskin fibroblasts under suitable conditions for a suitable period with the proteins so as to identify proteins which inhibit the growth of human tumor cells, inhibit the growth of the established mink lung cell line (CCL 64) or which enhance the growth of normal human foreskin fibroblasts.
Claim 89
Dependent Claim
37 words
A method of claim 73, wherein the step (b) treating the tissue comprises suspending the tissue in a suitable buffer at about 4°C and homogenizing the tissue for an suitable period of time to produce soluble proteins.
Claim 90
Dependent Claim
31 words
A method of claim 73, wherein step (c) treating the tissue comprises suspending the homogenized, washed tissue in acidified ethanol for a suitable period of time to extract acid soluble proteins.
Claim 91
Dependent Claim
80 words
A method of claim 73, wherein the step (e) assaying the TGI-1 and TGI-2 comprises separately contacting the human tumor cells, an established mink lung cell line (CCL 64) or normal human foreskin fibroblasts under suitable conditions for a suitable period with the TGI so as to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of the established mink lung cell line (CCL 64) or which enhances the growth of normal human foreskin fibroblasts.
Claim 92
Dependent Claim
33 words
A method of claim 74, wherein step (b) comprises suspending the tissue in a suitable buffer and homogenizing the tissue at about 4°C for a suitable period of time to form homogenized tissue.
Claim 93
Dependent Claim
29 words
A method of claim 74, wherein step (c) comprises suspending washed tissue in a suitable acidic extraction buffer containing ethanol at about 4°C for a suitable period of time.
Claim 94
Dependent Claim
24 words
A method of claim 74, wherein the step (d) the separate recovery of polypeptides from the solubilized proteins comprises hydrophobic interaction chromatography using phenyl-Sepharose.
Claim 95
Dependent Claim
78 words
A method of claim 74, wherein the step (e) assaying the TGI comprises separately contacting the human tumor cells, an established mink lung cell line (CCL 64) or normal human foreskin fibroblasts under suitable conditions for a suitable period with the TGI so as to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of the established mink lung cell line (CCL 64) or which enhances the growth of normal human foreskin fibroblasts.
Claim 96
Dependent Claim
49 words
A method for preparing a protein designated tissue-derived growth inhibitor-1 (TGI-1) which comprises first preparing an acidified, ethanol extract according to the method of claim 74 and then recovering TGI-1 from the acidified, ethanol extract as a defined activity by high performance liquid chromatography of the acidified, ethanol extract.
Claim 97
Dependent Claim
26 words
A method of claim 85, wherein the ethanol extract is purified by hydrophobic interaction chromatog raphy using phenyl-Sepharose prior to recovery with high performance liquid chromatography.
Claim 98
Dependent Claim
36 words
A method of claim 85, wherein the high performance liquid chromatography of the ethanol extract is carried out on a separating gradient of acetonitrile in a C18 column containing 0.05% trifluoracetic acid at about 27% acetonitrile.
Claim 99
Dependent Claim
37 words
A method of claim 85, wherein the high performance liquid chromatography of the acidified ethanol extract is carried out in a separating gradient of 2-propanol in a CN column containing 0.05% trifluoractic acid at about 40-41% 2-propanol.
Claim 100
Dependent Claim
97 words
A method for preparing a composition of matter, comprised of at least 2 proteins, designated tissue-derived growth inhibitor (TGI) which comprises first preparing an acidified, ethanol extract according to the method of claim 73 and then recovering TGI from the acidified, ethanol extract i) as a defined activity on high performance liquid chromatography of the acidified, ethanol extract with a linear gradient of acetonitrile containing 0.05% trifluroacetic acid at about 28-34% acetonitrile or ii) as a single peak of activity from a cation exchange resin when eluted with a linear NaCl gradient at about 0.6-0.7 M NaCl.
Claim 101
Dependent Claim
51 words
A method for preparing a composition of matter designated tissue-derived growth inhibitor-2 (TGI-2) which comprises first preparing an acidified, ethanol extract according to the method of claim 74 and then recovering TGI-2 from the acidified, ethanol extract as a defined activity by high performance liquid chromatography of the acidified, ethanol extract.
Claim 102
Dependent Claim
25 words
A method of claim 90, wherein the ethanol extract is purified by hydrophobic interaction chromatography using phenyl-Sepharose prior to recovery with high performance liquid chromatography.
Claim 103
Dependent Claim
36 words
A method of claim 90, wherein the high performance liquid chromatography of the ethanol extract is carried out on a separating gradient of acetonitrile and a C18 column containing 0.05% trifluoracetic acid at about 28% acetonitrile.
Claim 104
Dependent Claim
37 words
A method of claim 90, wherein the high performance liquid chromatography of the acidified ethanol extract is carried out on a separating gradient of 2-propanol on a CN column containing 0.05% trifluoracetic acid at about 44% 2-propanol.
Claim 105
Dependent Claim
36 words
A method for preparing a polypeptide designated CM-I which comprises first preparing an acidified, ethanol extract according to the method claim 72 and then recovering the polypeptide from the acidified, ethanol extract by cation exchange chromatography.
Claim 106
Dependent Claim
11 words
The acidified, ethanol extract prepared by the method of claim 72.
Claim 107
Dependent Claim
11 words
The acidified, ethanol extract prepared by the method of claim 73.
Claim 108
Dependent Claim
11 words
The acidified, ethanol extract prepared by the method of claim 74.
Claim 109
Dependent Claim
8 words
TGI-1 prepared by the method of claim 85.
Claim 110
Dependent Claim
8 words
TGI prepared by the method of claim 89.
Claim 111
Dependent Claim
8 words
TGI-2 prepared by the method of claim 90.
Claim 112
Dependent Claim
8 words
CM-1 prepared by the method of claim 94.
Claim 113
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI-1 present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 114
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI-1 present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 115
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 116
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 117
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI-2 present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 118
Independent Claim
54 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of TGI-2 present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 119
Dependent Claim
58 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of the protein of claim 21 present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 120
Independent Claim
57 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of a protein designated CM-I present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 121
Independent Claim
57 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of a protein designated CM-I present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 122
Independent Claim
62 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of a protein recoverable from conditioned media of A431 cells present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 123
Independent Claim
62 words
A method for detecting the presence of a tumor which comprises quantitatively determining the amount of a protein recoverable from conditioned media of A431 cells present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor.
Claim 124
Independent Claim
86 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI-1 and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI-1 present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 125
Independent Claim
86 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI-1 and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI-1 present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 126
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI present in the sample to the amount of TGF-alpha,determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 127
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI present in the sample to the amount of TGF-alpha,determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 128
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI-2 and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI-2 present in the sample to the amount of TGF-alpha,determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 129
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of TGI-2 and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI-2 present in the sample to the amount of TGF-alpha,determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 130
Dependent Claim
93 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of the protein of claim 21 and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of the protein of claim 21 present in the sample to the amount of TGF-alpha,determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 131
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of CM-I and of transforming growth factor (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of CM-I present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 132
Independent Claim
85 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of CM-I and of transforming growth factor (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of CM-I present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 133
Independent Claim
101 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of a polypeptide recoverable from conditioned media of A431 cells and of transforming growth factor (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of the polypeptide recoverable from conditioned media of A431 cells present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 134
Independent Claim
101 words
A method for detecting the presence of a tumor which comprises separately quantitatively determining the amount of a polypeptide recoverable from conditioned media of A431 cells and of transforming growth factor (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of the polypeptide recoverable from conditioned media of A431 cells present in the sample to the amount of TGF-alpha, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject to the ratio for the normal subject, a significant variation in the ratio indicating the presence of a tumor.
Claim 135
Dependent Claim
61 words
A method for typing tumors which comprises determining for a sample from a subject with a tumor presence of one or more of TGI-1, TGI, TGI-2, the protein of claim 21, CM-I or a polypeptide recoverable from conditioned media of A431 cells in the sample, the presence or absences of a specific combination thereof being indicative of a specific tumor type.
Claim 136
Dependent Claim
62 words
A method for typing tumors which comprises quantitatively determining for a sample from a subject with a tumor the amount of each of TGI-1, TGI, TGI-2, the protein of claim 21, CM-I, or a polypeptide recoverable from conditioned media of A431 cells present in the sample, the presence of specific amounts or relative amounts thereof being indicative of a specific tumor type.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.