Patent US794998A successfully refreshed from Google Patents.
Patent EP0272489A2 successfully refreshed from Google Patents.
EP0272489A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Antimicrobial proteins, compositions containing same and uses thereof
Assignee: Rockefeller University
•
Filing: 1987-11-25
•
Granted: —
•
Expires: 2007-11-25
Abstract
Abstract The present invention provides a composition of matter useful as an antimicrobial agent. This composition of matter comprises at least two polypeptides selected from the group consisting of human polymorphonuclear leukocyte polypeptides having apparent molecular weights of about 3,500 daltons, about 13,000 daltons, about 18,000 daltons, about 29,000 daltons, and about 54,000 daltons. The composition of matter has respiratory burst-independent, antimicrobial activity for bacteria and fungi at a pH from about 5.0 to about 8.0 and at calcium ion concentrations up to about 10 mM, bactericidal activity at sodium chloride concentrations up to about 0.3M, and fungicidal activity at sodium chloride concentrations up to about 0.15M. Methods for preparing this composition of matter are also provided. Further provided are purified polypeptides useful as antimicrobial agents, and methods for producing these polypeptides.
Inventors
- 👤 Joelle E. Gabay
- 👤 Carl F. Nathan
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 21:55
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/4701; C07K14/4723; C07K14/4742; A; A61; A61K; A61K38/00; Y; Y02; Y02A; Y02A50/00; Y02A50/30
Claims Analysis
Total Claims: 100
Claim 1
Independent Claim
99 words
A composition of matter useful as an antimicrobial agent which comprises at least two polypeptides selected from the group consisting of human polymorphonuclear leukocyte polypeptides having apparent molecular weights of about 3,500 daltons, about 13,000 daltons, about 18,000 daltons, about 29,000 daltons, and about 54,000 daltons, the composition of matter having respiratory burst-independent, antimicrobial activity against bacteria and fungi at a pH from about 5.0 to about 8.0 and at calcium ion concentrations up to about 10 mM, bactericidal activity at sodium chloride concentrations up to about 0.3M, and fungicidal activity at sodium chloride concentrations up to about 0.15M.
Claim 2
Dependent Claim
18 words
A composition of matter of claim 1, wherein the human polymorphonuclear leukocyte polypeptides are derived from HL-60 cells.
Claim 3
Dependent Claim
18 words
A composition of matter of claim 1, wherein the human polymorphonuclear leukocyte polypeptides are derived from KG-1 cells.
Claim 4
Dependent Claim
18 words
A composition of matter of claim 1 wherein the human polymorphonuclear leukocyte polypeptides are derived from azurophil granules.
Claim 5
Dependent Claim
23 words
A composition of matter of claim wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 3,500 daltons is defensin.
Claim 6
Dependent Claim
39 words
A composition of matter of claim 1 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 13,000 daltons comprises the 12,867 dalton polypeptide shown in Figure 20 from amino acid 105 to amino acid 220.
Claim 7
Dependent Claim
31 words
A composition of matter of claim 1 which comprises a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 10,000 daltons which is present in an azurophil granule extract.
Claim 8
Dependent Claim
30 words
A composition of matter of claim 1 which comprises a human polymorphonuclear leukocyte polypeptide having a molecular weight of about 29,000 daltons which is present in an azurophil granule extract.
Claim 9
Dependent Claim
24 words
A composition of matter of claim 8 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons is elastase.
Claim 10
Dependent Claim
25 words
A composition of matter of claim 8 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons is cathepsin G.
Claim 11
Dependent Claim
31 words
A composition of matter of claim 1 which comprises a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54,000 daltons which is present in an azurophil granule extract.
Claim 12
Dependent Claim
29 words
A composition of matter of claim 11 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54,000 daltons comprises the N-terminal amino acid sequence Val-Asn-Pro-Gly-Val-Val-Val-Arg-Ile-Ser-Gln-Lys-Gly-Leu-Asp-Tyr-Ala-Ser-Gln-Gln-Gly-Thr-Ala-Ala-Leu-Gln-X-X-Leu-Lys-His-Ile-Lys-Ile-Pro-Asp-Tyr-Leu.
Claim 13
Dependent Claim
16 words
A composition of matter of claim 1, wherein the bacteria are Gram positive or Gram negative.
Claim 14
Dependent Claim
37 words
A composition of matter of claim 13, wherein the Gram positive bacteria are Staphylococcus aur eas , β-hemolytic Streptococci, or Listeria and the Gram negative bacteria are Pseudomonas aeruginosa , Escherichia coli , or Salmonella typhimurium .
Claim 15
Dependent Claim
13 words
A composition of matter of claim 1, wherein the fungus is a yeast.
Claim 16
Dependent Claim
14 words
A composition of matter of claim 15, wherein the yeast is Candida albicans .
Claim 17
Dependent Claim
30 words
A method for killing bacteria or fungi which comprises contacting the bacteria or fungi with an effective bacterial or fungal killing amount of the composition of matter of claim 1.
Claim 18
Dependent Claim
24 words
A pharmaceutical composition useful for treating bacterial or fungal infections which comprises the composition of matter of claim 1 and a pharmaceutically acceptable carrier.
Claim 19
Dependent Claim
32 words
A method for treating a subject having a bacterial or fungal infection which comprises administering to the subject an effective bacterial or fungal killing amount of the pharmaceutical composition of claim 18.
Claim 20
Independent Claim
197 words
A method for preparing a composition of matter useful as an antimicrobial agent which comprises: (a) substantially separating polymorphonuclear leukocytes from blood so as to obtain a polymorphonuclear leukocyte enriched preparation; (b) suspending the polymorphonuclear leukocyte enriched preparation in a suitable buffer; (c) treating the resulting polymorphonuclear leukocyte suspension with a suitable protease inhibitor; (d) treating the polymorphonuclear leukocyte suspension so as to lysed the suspended leukocytes; (e) treating the resulting suspension of lysed leukocytes with a suitable chelating agent; (f) centrifuging the treated suspension of lysed leukocytes so as to obtain a nuclei/unbroken cell phase and a postnuclear supernatant; (g) recovering the postnuclear supernatant; (h) fractionating the postnuclear supernatant on a density gradient; (i) collecting the fraction of the density gradient which includes azurophil granules; (j) suspending the collected azurophil granules in a buffer having a pH of about 7; (k) treating the resulting azurophil granule suspension with an extracting reagent having a pH less than about 8.0 and capable of solubilizing azurophil membrane proteins so as to obtain an extracting reagent/azurophil membrane suspension; (l) separating the extracting reagent/azurophil membrane suspension so as to form a solid phase and a supernatant; and (m) recovering the supernatant.
Claim 21
Dependent Claim
12 words
A method according to claim 20, wherein the protease inhibitor is diisopropylfluorophosphate.
Claim 22
Dependent Claim
15 words
A method according to claim 20, wherein the blood sample is obtained from a human.
Claim 23
Dependent Claim
25 words
A method according to claim 20, wherein the polymorphonuclear leukocyte enriched preparation comprises greater than about 95% polymorphonuclear leukocytes and less than about 3% eosinophils.
Claim 24
Dependent Claim
22 words
A method according to claim 20, wherein the polymorphonuclear leukocyte suspension is treated by nitrogen cavitation to obtain a lysed leukocyte suspension.
Claim 25
Dependent Claim
16 words
A method according to claim 20, wherein the density gradient comprises a discontinuous Percoll density gradient.
Claim 26
Dependent Claim
24 words
A method according to claim 20, wherein prior to step (j) the fractionating material of the density gradient is separated from the azurophil granules.
Claim 27
Dependent Claim
45 words
A method according to claim 20, wherein prior to step (k) the azurophil granule suspension is treated so as to lyse the azurophil granules and the membrane fragments are recovered and treated with an extracting reagent so as to obtain an extracting reagent/azurophil membrane suspension.
Claim 28
Dependent Claim
17 words
A method according to claim 20, wherein the extracting reagent has a pH of less than 4.0.
Claim 29
Dependent Claim
16 words
A method according to claim 28, wherein the extracting reagent has a pH of about 2.0.
Claim 30
Dependent Claim
20 words
A method according to claim 20, wherein the extracting reagent is a non-ionic detergent having a pH of about 7.0.
Claim 31
Dependent Claim
37 words
A purified polypeptide useful as an antimicrobial agent which comprises a human polymorphonuclear leukocyte polypeptide having a predicted molecular weight of 24,823 daltons and the amino acid sequence shown in Figure 20, or a fragment derived therefrom.
Claim 32
Dependent Claim
23 words
A DNA molecule which encodes the polypeptide of claim 31 comprising at least a portion of the nucleotide sequence shown in Figure 20.
Claim 33
Dependent Claim
15 words
A purified polypeptide of claim 31 having an apparent molecular weight of about 13,000 daltons.
Claim 34
Dependent Claim
14 words
A purified polypeptide of claim 33 having a predicted molecular weight of 12,867 daltons.
Claim 35
Dependent Claim
24 words
A purified polypeptide of claim 34 which comprises the amino acid sequence shown in Figure 20 from amino acid 105 to amino acid 220.
Claim 36
Dependent Claim
24 words
A DNA molecule which encodes the polypeptide of claim 35 comprising the DNA sequence shown in Figure 20 from nucleotide 369 to nucleotide 716.
Claim 37
Dependent Claim
15 words
A purified polypeptide of claim 31 having an apparent molecular weight of about 18,000 daltons.
Claim 38
Dependent Claim
10 words
A DNA molecule which encodes the polypeptide of claim 37.
Claim 39
Dependent Claim
23 words
A method for killing a microorganism which comprises contacting the microorganism with an effective microorganism killing amount of the polypeptide of claim 31.
Claim 40
Dependent Claim
10 words
A vector which comprises the DNA molecule of claim 38.
Claim 41
Dependent Claim
9 words
A vector of claim 40 which comprises a plasmid.
Claim 42
Dependent Claim
36 words
A host vector system for the production of a human polymorphonuclear leukocyte polypeptide having a predicted molecular weight of 24,823 daltons, or a fragment thereof, which comprises the plasmid of claim 41 in a suitable host.
Claim 43
Dependent Claim
49 words
A method for producing a human polymorphonuclear leukocyte polypeptide having a molecular weight of 24,823 daltons, or a fragment thereof, which comprises growing the host vector system of claim 42 under suitable conditions permitting production of the human polymorphonuclear leukocyte polypeptide and recovering the resulting human polymorphonuclear leukocyte polypeptide.
Claim 44
Dependent Claim
170 words
A method for preparing the purified polypeptide of claim 31 which comprises: (a) culturing neutrophil precursor cells; (b) harvesting the cultured neutrophil precursor cells; (c) suspending the harvested, cultured neutrophil precursor cells in a suitable buffer; (d) treating the resulting neutrophil precursor cell suspension so as to obtain a suspension of lysed neutrophil precursor cells; (e) separating the treated suspension of lysed neutrophil precursor cells so as to obtain a nuclei and unbroken cell phase and a postnuclear supernatant; (f) recovering the postnuclear supernatant; (g) treating the postnuclear supernatant with an extracting reagent having a pH less than about 8.0 and capable of solubilizing membrane proteins so as to obtain an extracting reagent phase and an insoluble membrane phase; (h) separating the extracting reagent phase from the insoluble membrane phase so as to obtain a soluble protein phase and an insoluble membrane phase; (i) recovering the soluble protein phase; and (j) purifying the recovered soluble protein phase so as to obtain a purified 24,823 dalton polypeptide or a fragment thereof.
Claim 45
Dependent Claim
14 words
A method according to claim 44, wherein the neutrophil precursor cells are HL60 cells.
Claim 46
Dependent Claim
23 words
A method according to claim 44, wherein prior to step (d), the neutrophil precursor cell suspension is treated with a suitable protease inhibitor.
Claim 47
Dependent Claim
23 words
A method according to claim 44, wherein prior to step (e), the suspension of lysed neutrophils is treated with a suitable chelating agent.
Claim 48
Dependent Claim
17 words
A method according to claim 44, wherein the soluble protein phase is purified by ion exchange chromatography.
Claim 49
Dependent Claim
17 words
A method according to claim 44, wherein the soluble protein phase is purified by size exclusion chromatography.
Claim 50
Dependent Claim
16 words
A method according to claim 44, wherein the soluble protein phase is purified by affinity chromatography.
Claim 51
Dependent Claim
16 words
A method according to claim 44, wherein the soluble protein phase is purified by immuno-affinity chromatography.
Claim 52
Dependent Claim
27 words
A pharmaceutical composition useful for treating microorganism infections which comprises an effective microorganism killing amount of the purified polypeptide of claim 31 and a pharmaceutically acceptable carrier.
Claim 53
Dependent Claim
28 words
A method for treating a subject having a microorganism infection which comprises administering to the subject an effective microorganism killing amount of the pharmaceutical composition of claim 52.
Claim 54
Dependent Claim
25 words
A purified polypeptide useful as an antimicrobial agent which comprises a human polymorphonuclear leukocyte polypeptide having an apparent molecu lar weight of about 18,000 daltons.
Claim 55
Dependent Claim
57 words
A purified polypeptide of claim 54 which has respiratory burst-independent, antimicrobial activity against bacteria and fungi at a pH from about 5.0 to about 8.0 and at calcium ion concentrations up to about 10 mM, bactericidal activity at sodium chloride concentrations up to about 0.3M, and fungicidal activity at sodium chloride concentrations up to about 0.15 M.
Claim 56
Dependent Claim
10 words
A DNA molecule which encodes the polypeptide of claim 54.
Claim 57
Dependent Claim
23 words
A method for killing a microorganism which comprises contacting the microorganism with an effective microorganism killing amount of the polypeptide of claim 54.
Claim 58
Dependent Claim
10 words
A vector which comprises the DNA molecule of claim 56.
Claim 59
Dependent Claim
9 words
A vector of claim 58 which comprises a plasmid.
Claim 60
Dependent Claim
33 words
A host vector system for the production of a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 18,000 daltons which comprises the plasmid of claim 59 in a suitable host.
Claim 61
Dependent Claim
47 words
A method for producing a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 18,000 daltons which comprises growing the host vector system of claim 60 under suitable conditions permitting production of the human polymorphonuclear leukocyte polypeptide and recovering the resulting human polymorphonuclear leukocyte polypeptide.
Claim 62
Dependent Claim
166 words
A method for preparing the purified polypeptide of claim 54 which comprises: (a) culturing neutrophil precursor cells; (b) harvesting the cultured neutrophil precursor cells; (c) suspending the harvested, cultured neutrophil precursor cells in a suitable buffer; (d) treating the resulting neutrophil precursor cell suspension so as to obtain a suspension of lysed neutrophil precursor cells; (e) separating the treated suspension of lysed neutrophil precursor cells so as to obtain a nuclei and unbroken cell phase and a postnuclear supernatant; (f) recovering the postnuclear supernatant; (g) treating the postnuclear supernatant with an extracting reagent having a pH less than about 8.0 and capable of solubilizing membrane proteins so as to obtain an extracting reagent phase and an insoluble membrane phase; h) separating the extracting reagent phase form the insoluble membrane phase so as to obtain a soluble protein phase and an insoluble membrane phase; (i) recovering the soluble protein phase; and (j) purifying the recovered soluble protein phase so as to obtain a purified 18,000 dalton polypeptide.
Claim 63
Dependent Claim
14 words
A method according to claim 62, wherein the neutrophil precursor cells are HL60 cells.
Claim 64
Dependent Claim
23 words
A method according to claim 62, wherein prior to step (d), the neutrophil precursor cell suspension is treated with a suitable protease inhibitor.
Claim 65
Dependent Claim
23 words
A method according to claim 62, wherein prior to step (e), the suspension of lysed neutrophils is treated with a suitable chelating agent.
Claim 66
Dependent Claim
17 words
A method accoding to claim 62, wherein the soluble protein phase is purified by ion exchange chromatography.
Claim 67
Dependent Claim
17 words
A method according to claim 62, wherein the soluble protein phase is purified by size exclusion chromatography.
Claim 68
Dependent Claim
16 words
A method according to claim 62, wherein the soluble protein phase is purified by affinity chromatography.
Claim 69
Dependent Claim
16 words
A method according to claim 62, wherein the soluble protein phase is purified by immuno-affinity chromatography.
Claim 70
Dependent Claim
31 words
A pharmaceutical composition useful for treating bacterial or fungal infections which comprises an effective bacterial or fungal killing amount of the purified polypeptide fo claim 54 and a pharmaceutically acceptable carrier.
Claim 71
Dependent Claim
32 words
A method for treating a subject having a bacterial or fungal infection which comprises administering to the subject an effective bacterial or fungal killing amount of the pharmaceutical composition of claim 70.
Claim 72
Dependent Claim
55 words
A purified polypeptide useful as an antimicrobial agent which comprises a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54,000 daltons, respiratory burst-independent, antibacterial activity at a pH from about 5.0 to about 8.0, at calcium ion concentrations up to about 10 mM, and at sodium chloride concentrations up to about 0.3M.
Claim 73
Dependent Claim
35 words
A purified polypeptide of claim 72 having antifungal activity at a pH from about 5.0 to about 8.0, at calcium ion concentrations up to about 10mM, and at sodium chloride concentrations up to about 0.15M.
Claim 74
Dependent Claim
10 words
A DNA molecule which encodes the polypeptide of claim 72.
Claim 75
Dependent Claim
14 words
A purified polypeptide of claim 72 which comprises the N-terminal amino acid sequence Val-Asn-Pro-Gly-Val-Val-Val-Arg-Ile-Ser-Gln-Lys-Gly-Leu-Asp-Tyr-Ala-Ser-Gln-Gln-Gly-Thr-Ala-Ala-Leu-Gln-X-X-Leu-Lys-His-Ile-Lys-Ile-Pro-Asp-Tyr-Leu.
Claim 76
Dependent Claim
28 words
A method for killing bacteria or fungi which comprises contacting the bacteria or fungi with an effective bacterial or fungal killing amount of the polypeptide of claim 73.
Claim 77
Dependent Claim
10 words
A vector which comprises the DNA molecule of claim 74.
Claim 78
Dependent Claim
9 words
A vector of claim 77 which comprises a plasmid.
Claim 79
Dependent Claim
64 words
A host vector system for the production of a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54,000 daltons, respiratory burst-independent, antibacterial activity at a pH from about 5.0 to about 8.0, at calcium ion concentrations up to about 10 mM, and at sodium chloride concentrations up to about 0.3M, which comprises the plasmid of claim 78 in a suitable host.
Claim 80
Dependent Claim
79 words
A method for producing a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 54,000 daltons, respiratory burst-independent, antibacterial activity at a pH from about 5.0 to about 8.0, at calcium ion concentrations up to about 10 mM, and at sodium chloride concentrations up to about 0.3M, which comprises growing the host vector system of claim 79 under suitable conditions permitting produc tion of the human polymorphonuclear leukocyte polypeptide and recovering the resulting human polymorphonuclear leukocyte polypeptide.
Claim 81
Dependent Claim
167 words
A method for preparing the purified polypeptide of claim 72 which comprises: (a) culturing neutrophil precursor cells; (b) harvesting the cultured neutrophil precursor cells; (c) suspending the harvested, cultured neutrophil precursor cells in a suitable buffer; (d) treating the resulting neutrophil precursor cell suspension so as to obtain a suspension of lysed neutrophil precursor cells; (e) separating the treated suspension of lysed neutrophil precursor cells so as to obtain a nuclei and unbroken cell phase and a postnuclear supernatant; (f) recovering the postnuclear supernatant; (g) treating the postnuclear supernatant with an extracting reagent having a pH less than about 8.0 and capable of solubilizing membrane proteins so as to obtain an extracting reagent phase and an insoluble membrane phase; (h) separating the extracting reagent phase from the insoluble membrane phase so as to obtain an a soluble protein phase and an insoluble membrane phase; (i) recovering the soluble protein phase; and (j) purifying the recovered soluble protein phase so as to obtain a purified 54,000 dalton polypeptide.
Claim 82
Dependent Claim
14 words
A method according to claim 81, wherein the neutrophil precursor cells are HL60 cells.
Claim 83
Dependent Claim
23 words
A method according to claim 81, wherein prior to step (d), the neutrophil precursor cell suspension is treated with a suitable protease inhibitor.
Claim 84
Dependent Claim
23 words
A method according to claim 81, wherein prior to step (e), the suspension of lysed neutrophils is treated with a suitable chelating agent.
Claim 85
Dependent Claim
17 words
A method according to claim 81, wherein the soluble protein phase is purified by ion exchange chromatography.
Claim 86
Dependent Claim
17 words
A method according to claim 81, wherein the soluble protein phase is purified by size exclusion chromatography.
Claim 87
Dependent Claim
16 words
A method according to claim 81, wherein the soluble protein phase is purified by affinity chromatography.
Claim 88
Dependent Claim
16 words
A method according to claim 81, wherein the soluble protein phase is purified by immuno-affinity chromatography.
Claim 89
Dependent Claim
27 words
A pharmaceutical composition useful for treating bacterial infections which comprises an effective bacterial killing amount of the purified polypeptide of claim 72 and a pharmaceutically acceptable carrier.
Claim 90
Dependent Claim
28 words
A method for treating a subject having a bacterial infection which comprises administering to the subject an effective bacterial killing amount of the pharmaceutical composition of claim 89.
Claim 91
Dependent Claim
31 words
A pharmaceutical composition useful for treating bacterial or fungal infections which comprises an effective bacterial or fungal killing amount of the purified polypeptide of claim 73 and a pharmaceutically acceptable carrier.
Claim 92
Dependent Claim
30 words
A method for treating a subject having a bacterial or fungal infection which comprises administering to the subject an effective bacterial killing amount of the pharmaceutical composition of claim 91.
Claim 93
Independent Claim
31 words
A method for killing fungi which comprises contacting the fungi with an effective fungal killing amount of a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons.
Claim 94
Dependent Claim
27 words
A method of claim 93 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons is present in an azurophil granule extract.
Claim 95
Dependent Claim
22 words
A method of claim 93 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons is elastase.
Claim 96
Dependent Claim
23 words
A method of claim 93 wherein the human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons is cathepsin G.
Claim 97
Dependent Claim
24 words
A pharmaceutical composition which comprises a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 29,000 daltons and a pharmaceutically acceptable carrier.
Claim 98
Dependent Claim
28 words
A method for treating a subject having a bacterial infection which comprises administering to the subject an effective bacterial killing amount of the pharmaceutical composition of claim 97.
Claim 99
Dependent Claim
28 words
A method for treating a subject having a fungal infection which comprises administering to the subject an effective fungal killing amount of the pharmaceutical composition of claim 97.
Claim 100
Dependent Claim
72 words
A purified polypeptide useful as an antimicrobial agent which coprises a human polymorphonuclear leukocyte polypeptide having an apparent molecular weight of about 25,000 daltons, respiratory burst-independent, antimicrobial activity against bacteria and fungi at a pH from about 5.0 to about 8.0 and at calcium ion concentrations up to about 10 mM, bacteriocidal activity at sodium chloride concentrations up to about 0.3M, and fungicidal activity at sodium chloride concentrations up to about 0.15M
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.