METHODS AND COMPOSITIONS FOR THE TREATMENT OF FIBROTIC CONDITIONS AND IMPAIRED LUNG FUNCTION AND TO ENHANCE LYMPHOCYTE PRODUCTION

PROCEDES ET COMPOSITIONS SERVANT A TRAITER DES ETATS FIBREUX ET L'ALTERATION DE LA FONCTION PULMONAIRE, ET A AMELIORER LA PRODUCTION DE LYMPHOCYTE

English Abstract

The present invention provides methods and compositions to treat fibrotic
conditions, to increase lymphocyte production in vivo, and to improve and/or
normalize lung function, pulmonary compliance, blood oxygenation, and blood pH
to inhibit inflammatory processes to stimulate or inhibit pro-inflammatory and
immune cells, and to inhibit migration of vascular endothelial cells. The
invention contemplates the administration of human uteroglobin, native or
recombinant, as a means of achieving these ends. Specifically, it has been
found that uteroglobin inhibits cell adhesion to fibronectin, increases
lymphocyte production in vivo, and improves and/or normalizes lung function,
pulmonary compliance, blood oxygenation, and blood pH, and inhibits
inflammatory process. In addition it has been found that uteroglobin can
stimulate or inhibit pro-inflammatory and immune cells and inhibitor migration
of vascular endothelial cells.

French Abstract

L'invention concerne des procédés et compositions servant à traiter des états fibreux, à augmenter <i>in vivo</i> la production de lymphocytes et à améliorer et/ou normaliser la fonction pulmonaire, la compliance pulmonaire, l'oxygénation sanguine et le pH sanguin, de manière à inhiber des processus inflammatoires afin de stimuler ou inhiber des cellules pro-inflammatoires et immunes, et à inhiber la migration des cellules endothéliales vasculaires. A cette fin, l'invention consiste à administrer de l'utéroglobine humaine, naturelle ou recombinée. On a notamment trouvé que l'utéroglobine inhibait l'adhésion cellulaire à la fibronectine, augmentait la production de lymphocytes in vivo et améliorait et/ou normalisait la fonction pulmonaire, la compliance pulmonaire, l'oxygénation sanguine et le pH sanguin, et inhibait le processus inflammatoire. En outre, on a trouvé que l'utéroglobine pouvait stimuler ou inhiber des cellules pro-inflammatoires et immunes et inhiber la migration de cellules endothéliales vasculaires.

Claims

84

CLAIMS:

1. A method of treatment comprising improving and/or normalizing lung
function in a patient in need of said treatment, said method comprising
administering
an amount of uteroglobin to said patient sufficient to improve lung function
relative to
lung function in the absence of said treatment.

2. The method of claim 1 wherein said uteroglobin is recombinant human
uteroglobin.

3. The method of claim 1 wherein said amount of uteroglobin is 10 ng/kg
-25 mg/kg.

4. A method of treatment comprising improving and/or normalizing
pulmonary compliance in a patient in need of said treatment, said method
comprising
administering an amount of uteroglobin to said patient sufficient to improve
and/or
normalize pulmonary compliance relative to pulmonary compliance in the absence
of
said treatment.

5. The method of claim 4 wherein said patient suffers from reduced
pulmonary compliance as a result of a pulmonary challenge or insult resulting
from
exposure to one or more substances selected from the group consisting of non-
atmospheric gases, non-atmospheric ratios of atmospheric gases, inhaled
chemicals,
pollutant, irritants, inhaled pollens, allergens, particulate matter, and an
airborne
infectious agent.

6. The method of claim 4 wherein said uteroglobin is recombinant human
uteroglobin.

7. The method of claim 4 wherein said amount of uteroglobin is 10 ng/kg
-25 mg/kg.

8. A method of treatment comprising improving blood oxygenation
and/or normalizing blood pH in a patient in need of said treatment, said
method
comprising administering to said patient an amount of uteroglobin sufficient
to
improve blood oxygenation and/or normalize blood pH relative to blood
oxygenation
and/or blood pH in the absence of said treatment.

9 The method of claim 8 wherein said uteroglobin is recombinant human
uteroglobin.




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The method of claim 8 wherein said amount of uteroglobin is 10 ng/kg
- 25 mg/kg
11. A composition comprising uteroglobin in an amount sufficient to
improve and/or normalize lung function, and a pharmaceutically acceptable
carrier or
diluent.
12. The composition of claim 11 wherein said uteroglobin is recombinant
human uteroglobin.
13. The composition of claim 11 wherein said amount of uteroglobin is 10
ng/kg - 25 mg/kg.
14. A composition comprising uteroglobin in an amount sufficient to
improve and/or normalize pulmonary compliance, and a pharmaceutically
acceptable
carrier or diluent.
15. The composition of claim 14 wherein said uteroglobin is recombinant
human uteroglobin.
16. The composition of claim 4 wherein said amount of uteroglobin is 10
ng/kg - 25 mg/kg.
17. A composition comprising uteroglobin in an amount sufficient to
improve blood oxygenation and/or normalize blood pH, and a pharmaceutically
acceptable carrier or diluent.
18. The method of claim 17 wherein said uteroglobin is recombinant
human uteroglobin.
19. The method of claim 17 wherein said amount of uteroglobin is 10
ng/kg - 25 mg/kg
20. A method of treatment comprising increasing lymphocyte production
in vivo in a patient in need of said treatment, said method comprising
administering to
said patient an amount of uteroglobin sufficient to increase lymphocyte
production in
said patient relative to lymphocyte production in the absence of said
treatment.
21. The method of claim 20 wherein said uteroglobin is recombinant
human uteroglobin.




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22. The method of claim 20 wherein said amount of uteroglobin is 1ng/kg
- 100 mg/kg.
23. The method of claim 20 wherein said lymphocytes are selected from
the group consisting of effector lymphocytes and cytotoxic T cells.
24. The method of claim 20 wherein said effector lymphocytes are selected
from the group consisting of helper T cells, plasma B cells, and memory B
cells.
25. The method of claim 20 wherein said patient suffers from decreased
lymphocyte production as a result of an autoimmune disease or allergy.
26. The method of claim 25 wherein said autoimmune disease is acquired
immunodeficiency syndrome.
27. A method of treatment comprising increasing production of suppressor
T cells in a patient in need of said treatment, said method comprising
administering to
said patient an amount of uteroglobin sufficient to increase production of
suppressor T
cells relative to suppressor T cell production in said patient in the absence
of said
treatment.
28. The method of claim 27 wherein said amount of uteroglobin is 1 ng/mg
- 100 mg/kg.
29. The method of claim 27 wherein said uteroglobin is recombinant
human uteroglobin.
30. A method of treatment comprising enhancing a lymphocyte-mediated
response in vivo in a patient in need of said treatment, said method
comprising
administering to said patient an amount of uteroglobin sufficient to enhance
said
lymphocyte-mediated response relative to a response observed in the absence of
said
treatment.
31. The method of claim 30 wherein said amount of uteroglobin is 1 ng/mg
- 100 mg/kg.
32. The method of claim 30 wherein said uteroglobin is recombinant
human uteroglobin.
33. The method of claim 30 wherein said lymphocyte-mediated response
results from administration of a vaccine.




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34. The method of claim 33 wherein said vaccine is selected from the
group consisting of B cell and T cell vaccines.
35. The method of claim 30 wherein said lymphocyte-mediated response
results from administration of a tolerance-inducing treatment.
36. The method of claim 35 wherein said tolerance-inducing treatment is
selected from the group consisting of oral tolerance and allergy shots.
37. A method of treatment comprising decreasing the production of
polymorphonuclear leukocytes (PMN) in a patient in need of said treatment,
said
method comprising administering to said patient an amount of uteroglobin
sufficient
to decrease the production of PMN relative to PMN levels in the absence of
said
treatment.
38. The method of claim 37 wherein said amount of uteroglobin is 1 ng/mg
- 100 mg/kg.
39. The method of claim 37 wherein said uteroglobin is recombinant
human uteroglobin.
40. A composition comprising uteroglobin in an amount sufficient to
increase lymphocyte production in vivo, and a pharmaceutically acceptable
carrier or
diluent.
41. The composition of claim 40 wherein said uteroglobin is recombinant
human uteroglobin.
42. The composition of claim 40 wherein said amount of uteroglobin is
1 ng/kg - 100 mg/kg.
43. A composition comprising uteroglobin in an amount effective to
increase production of suppressor T cells, and a pharmaceutically acceptable
carrier or
diluent.
44 The composition of claim 43 wherein said amount of uteroglobin is 1
ng/mg - 100 mg/kg.
45. The composition of claim 43 wherein said uteroglobin is recombinant
human uteroglobin.




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46. A composition comprising uteroglobin in an amount effective to
enhance a lymphocyte-mediated response in vivo, and a pharmaceutically
acceptable
carrier or diluent.
47. The composition of claim 46 wherein said amount of uteroglobin is 1
ng/mg - 100 mg/kg.
48. The composition of claim 46 wherein said uteroglobin is recombinant
human uteroglobin.
49. A composition comprising uteroglobin in an amount effective to
decrease production of polymorphonuclear leukocytes (PMN), and a
pharmaceutically
acceptable carrier or diluent.
50. The method of claim 49 wherein said amount of uteroglobin is 1 ng/mg
- 100 mg/kg.
51. The method of claim 49 wherein said uteroglobin is recombinant
human uteroglobin.
52. A method of treatment comprising inhibiting fibronectin-dependent
cell adhesion to fibronectin in a patient in need of said treatment, said
method
comprising administering to said patient an amount of uteroglobin sufficient
to inhibit
fibronectin-dependent cell adhesion to fibronectin in vivo.
53. The method of claim 52 wherein said uteroglobin is recombinant
human uteroglobin.
54. The method of claim 52 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
55. A method of treatment comprising inhibiting cell adhesion to type III
domains of fibronectin in a patient in need of said treatment, said method
comprising
administering to said patient an amount of uteroglobin sufficient to inhibit
cell
adhesion to type III domains of fibronectin.
55. The method of claim 55 wherein said uteroglobin is recombinant
human uteroglobin.
56. The method of claim 55 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.




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57. A method of treatment comprising inhibiting an interaction between
fibronectin and cells dependent on fibronectin binding in a patient in need of
said
treatment, said method comprising administering to said patient an amount of
uteroglobin sufficient to inhibit said interaction.
58. The method of claim 57 wherein said uteroglobin is recombinant
human uteroglobin.
59. The method of claim 57 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
60. The method of claim 57 wherein said cells dependent on fibronectin
binding are selected from the group consisting of neural cells, muscle cells,
hematopoietic cells, fibroblasts, neutrophils, eosinophils, basophils,
macrophages,
monocytes, lymphocytes, platelets, red blood cells, endothelial cells, stromal
cells,
dendritic cells, mast cells, and epithelial cells.
61. The method of claim 57 wherein said patient is suffering from a
condition selected from the group consisting of the formation of adhesions
following
surgery, atheroschlerosis, thrombosis, heart disase, vasculitis, formation of
scar tissue,
restenosis, phlobitis, COPD, pulmonary hypertension, pulmonary fibrosis,
pulmonary
inflammation, bowel adhesions, bladder fibrosis and cystitis, fibrosis of the
nasal
passages, sinusitis, inflammation mediated by neutrophils, and fibrosis
mediated by
fibroblasts.
62. A method of treatment comprising inhibiting inflammatory cell and
fibroblast migration on fibronectin already deposited in vivo in a patient in
need of
said treatment, said method comprising administering to said patient an amount
of
uteroglobin sufficient to inhibit fibronectin-dependent cell adhesion to
fibronectin,
thereby inhibiting inflammatory cell and fibroblast migration.
63. The method of claim 62 wherein said uteroglobin is recombinant
human uteroglobin.
64. The method of claim 62 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
65. The method of claim 62 wherein said inflammatory cell is selected
from the group consisting of neutrophils, eosinophils, basophils, macrophages,





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monocytes, lymphocytes, platelets, red blood cells, dendritic cells, mast
cells, and
fibroblasts.
66. A method of treatment comprising inhibiting fibronectin
polymerization, deposition, and/or cell adhesion in vivo in a patient in need
of said
treatment, said method comprising administering to said patient an amount of
uteroglobin sufficient to bind to at least one type III domain of fibronectin,
thereby
inhibiting fibronectin polymerization, deposition, and/or cell adhesion.
67. The method of claim 66 wherein said uteroglobin is recombinant
human uteroglobin.
68. The method of claim 66 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
69. A composition comprising uteroglobin in an amount effective to
inhibit fibronectin-dependent cell adhesion to fibronectin, and a
pharmaceutically
acceptable carrier or diluent.
70. The composition of claim 69 wherein said uteroglobin is recombinant
human uteroglobin.
71. The composition of claim 69 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
72. A composition comprising uteroglobin in an amount effective to
inhibit an interaction between fibronectin and cells dependent on fibronectin
binding,
and a pharmaceutically acceptable carrier or diluent.
73. The composition of claim 72 wherein said uteroglobin is recombinant
human uteroglobin.
74. The composition of claim 72 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
75. A composition comprising uteroglobin in an amount effective to
inhibit inflammatory cell and fibroblast migration on fibronectin already
deposited in
vivo, and a pharmaceutically effective carrier or diluent.
76. The composition of of claim 75 wherein said uteroglobin is
recombinant human uteroglobin.




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77. The composition of claim 75 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
78. A composition comprising uteroglobin in an amount effective to
inhibit fibronectin-dependent cell adhesion in vivo, and a pharmaceutically
acceptable
carrier or diluent.
79. The composition of claim 78 wherein said uteroglobin is recombinant
human uteroglobin.
80. The composition of claim 78 wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
81. A composition comprising uteroglobin in an amount effective to
inhibit an interaction between a cell having a PLA2 receptor and an
extracellular
matrix protein and/or a membrane bound protein comprising at least one
fibronectin
type III domain protein, and a pharmaceutically acceptable carrier or diluent.
82. The composition of claim 81 wherein said uteroglobin is recombinant
human uteroglobin.
83. The composition of claim 81, wherein said amount of uteroglobin is 25
ng/200 µl to 10 µg/200 µl.
84. A method of inhibiting an LPS-dependent inflammatory processes in a
patient infected with a bacterium comprising administering to said patient an
amount
of recombinant human uteroglobin sufficient to inhibit said inflammatory
processes.
85. The method of claim 84 wherein said patient is diagnosed with septic
shock.
86. The method of claim 84 wherein said patient is diagnosed with
pneumonia.
87. The method of claim 84 wherein said patient is diagnosed with a
condition selected form the group consisiting of: peritonitis, colitis,
inflammatory
bowel disease, pancreatitis, nephritis, vasculitis, hepatitis, sinusitis,
cystitis, peridontal
disease, and myocarditis.
88. The method of claim 84 wherein said patient is diagnosed with asthma.





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89. A composition comprising recombinant human uteroglobin in an
amount sufficient to inhibit LPS-dependent inflammatory processes in a
patient, and a
pharmaceutically acceptable carrier or diluent.
90. The composition of claim 89 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
91. A method of decreasing TNF-alpha concentrations in vivo in a patient
in need of such treatment comprising administering to said patient an amount
of
recombinant human uteroglobin sufficient to decrease said TNF-alpha
concentrations.
92. The method of claim 91 wherein said patient is diagnosed with a
bacterial infection.
93. The method of claim 91 wherein said patient is diagnosed with
inflammatory disease.
94. The method of claim 91 wherein said patient is diagnosed with
Crohn's disease.
95. The method of claim 91 wherein said patient is diagnosed with a
condition selected form the group consisiting of peritonitis, colitis,
inflammatory
bowel disease, pancreatitis, nephritis, vasculitis, hepatitis, sinusitis,
cystitis, peridontal
disease, and myocarditis.
96. A composition comprising recombinant human uteroglobin in an
amount sufficient to decrease TNF-alpha concentrations and a pharmaceutically
acceptable carrier or diluent.
97. The composition of claim 96 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
98. A method of regulating the nitric oxide pathway for relaxing smooth
muscle cells in a patient in need of such treatment comprising administering
to said
patient an amount of recombinant human uteroglobin sufficient to regulate said
nitric
oxide pathway.
99. The method of claim 98 wherein said patient is diagnosed with
abnormal blood pressure.
100. The method of claim 98 wherein said patient is diagnosed with high
blood pressure.




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101. The method of claim 98 wherein said patient is diagnosed with
bronchoconstriction.
102. The method of claim 98 wherein said patient is diagnosed with
respiratory distress syndrome.
103. The method of claim 98 wherein said patient is diagnosed with
esophageal dysphagia.
104. The method of claim 98 wherein said patient is diagnosed with ileus.
105. The method of claim 98 wherein said patient is diagnosed with rectal
prolapse.
106. A composition comprising recombinant human uteroglobin in an
amount sufficient to regulate the nitric oxide pathway of a patient, and a
pharmaceutically acceptable carrier or diluent.
107. The composition of claim 106 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
108. A method of regulating vascular permeability in a patient in need of
such treatment comprising administering to said patient an amount of
recombinant
human uteroglobin sufficient to regulate said vascular permeability.
109. The method of claim 108 wherein said patient is diagnosed with
abnormal blood pressure.
110. The method of claim 108 wherein said patient is diagnosed with high
blood pressure.
111. The method of claim 108 wherein said patient is diagnosed with
primary pulmonary hypertension.
112. The method of claim 108 wherein said patient is diagnosed with
congestive heart failure.
113. The method of claim 108 wherein said patient suffers from edema.
114. A composition comprising recombinant human uteroglobin in an
amount sufficient to regulate vascular permeability of a patient, and a
pharmaceutically acceptable carrier or diluent.
115. The composition of claim 114 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.




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116. A method of suppressing proliferation of CD71-positive cells in a
patient in need of such treatment comprising administering to said patient an
amount
of recombinant human uteroglobin sufficient to suppress proliferation of said
cells.
117. A method of claim 116 wherein said patient is diagnosed with a
leukemia.
118. A method of claim 116 wherein said patient is diagnosed with a
lymphoma.
119. A method of claim 116 wherein said patient is diagnosed with an
inflammatory disease.
120. The method of claim 116 wherein said patient is diagnosed with an
infectious disease.
121. A method of claim 116 wherein said patient is diagnosed with a
fibrotic disease.
122. A method of claim 116 wherein said patient is diagnosed with an
autoimmune disease.
123. A method of claim 116 wherein said patient is diagnosed with cancer.
124. A method of claim 116 wherein said cells are selected from the group
consisting of: neutrophils, band cells, stab cells, granulocytes, eosinophils,
basophils,
monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes, T cells, B
cells, NK cells, lymphoid precursors, and myeloid precursors.
125. A composition comprising recombinant human uteroglobin in an
amount sufficient to suppress proliferation of CD71 positive cells in a
patient, and a
pharmaceutically acceptable carrier or diluent.
126. The composition of claim 125 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
127. A method of suppressing proliferation of CD71-positive cells in vitro
comprising exposing said CD71-positive cells to an amount of recombinant human
uteroglobin sufficient to suppress proliferation of said cells in vitro.
128. The method of claim 127 wherein said CD71-positive cells are
hematopoietic stem cells.




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129. The method of claim 127 wherein said hematopoietic stem cells are
transplanted from a donor to a recipient in need of such cells.
130. The method of claim 127 wherein said hematopoietic stem cells must
be stored for a period of time prior to transplant.
131. The method of claim 127 wherein said CD71-positive cells are
lymphoid precursor cells.
132. The method of claim 127 wherein said CD71-positive cells are
myeloid precursor cells.
133. The method of claim 127 wherein said cells are selected from the
group consisting of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
134. A composition comprising recombinant human uteroglobin in an
amount sufficient to suppress proliferation of CD71 positive cells in vitro.
135. The composition of claim 134 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
136. A method of suppressing proliferation of CD71-positive cells in vitro
comprising exposing said CD71-positive cells to an amount of recombinant human
uteroglobin and and an amount of fibronectin sufficient to suppress
proliferation of
said cells in vitro.
137. The method of claim 136 wherein said CD71-positive cells are
hematopoietic stem cells.
138. The method of claim 136 wherein said hematopoietic stem cells are
transplanted from a donor to a recipient in need of such cells.
139. The method of claim 136 wherein said hematopoietic stem cells must
be stored for a period of time prior to transplant.
140. The method of claim 136 wherein said CD71-positive cells are
lymphoid precursor cells.
141. The method of claim 136 wherein said CD71-positive cells are
myeloid precursor cells.




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142. The method of claim 136 wherein said cells are selected from the
group consisting of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
143. A composition comprising recombinant human uteroglobin and
fibronectin, each present in an amount sufficient to suppress proliferation of
CD71
positive cells in a patient, and a pharmaceutically acceptable carrier or
diluent.
144. The composition of claim 143 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
145. A method of suppressing activation of CD71-positive cells in a patient
in need of such treatment comprising administering to said patient an amount
of
recombinant human uteroglobin sufficient to suppress activation of said cells.
146. The method of claim 145 wherein said patient is diagnosed with an
inflammatory disease.
147. The method of claim 145 wherein said patient is diagnosed with an
infectious disease.
148. The method of claim 145 wherein said patient is diagnosed with an
autoimmune disease.
149. The method of claim 145 wherein said patient is diagnosed with
cancer.
150. The method of claim 145 wherein said patient is diagnosed with a
fibrotic disease.
151. A method of claim 145 wherein said cells are selected from the group
consisting of: neutrophils, band cells, stab cells, granulocytes, eosinophils,
basophils,
monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes, T cells, B
cells, NK cells, lymphoid precursors, and myeloid precursors.
152. A composition comprising recombinant human uteroglobin in an
amount sufficient to suppress activation of CD71 positive cells in a patient,
and a
pharmaceutically acceptable carrier or diluent.
153. The composition of claim 152 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.




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154. A method of suppressing activation of CD71-positive cells in vitro
comprising exposing said cells to an amount of recombinant human uteroglobin
sufficient to suppress activation of said cells in vitro.
155. The method of claim 154 wherein said CD71-positive cells axe
hematopoietic stem cells.
156. The method of claim 155 wherein said hematopoietic stem cells axe to
be transplanted from a donor to a recipient in need of such cells.
157. The method of claim 156 wherein said hematopoietic stem cells are
stored for a period of time prior to transplant.
158. The method of claim 154 wherein said CD71-positive cells are
lymphoid precursor cells.
159. The method of claim 154 wherein said CD71-positive cells are
myeloid precursor cells.
160. The method of claim 154 wherein said cells are selected from the
group consisting of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
161. A composition comprising recombinant human uteroglobin in an
amount sufficient to suppress activation of CD71 positive cells in vitro.
162. The composition of claim 161 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
163. A method of enhancing proliferation of CDllb-positive cells in a
patient in need of such treatment comprising administering to said patient an
amount
of recombinant human uteroglobin sufficient to enhance proliferation of said
cells.
164. The method of claim 163 wherein said patient is diagnosed with a
leukemia.
165. The method of claim 163 wherein said patient is diagnosed with a
lymphoma.
166. The method of claim 163 wherein said patient is diagnosed with an
inflammatory disease.


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167. The method of claim 163 wherein said patient is diagnosed with an
infectious disease.
168. The method of claim 163 wherein said patient is diagnosed with a
fibrotic disease.
169. The method of claim 163 wherein said patient is diagnosed with an
autoimmune disease.
170. The method of claim 163 wherein said patient is diagnosed with
cancer.
171. The method of claim 163 wherein said cells are selected from the
group consisting of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
172. A composition comprising recombinant human uteroglobin in an
amount sufficient to enhance proliferation of CD11b-positive cells in a
patient, and a
pharmaceutically acceptable carrier or diluent.
I73. The composition of claim 172 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
174. A method of enhancing proliferation of CD11b-positive cells in vitro
comprising exposing said cells to an amount of recombinant human uteroglobin
sufficient to enhance proliferation of said cells in vitro.
175. The method of claim 174 wherein said CD11b-positive cells are
hematopoietic stem cells.
176. The method of claim 175 wherein said hematopoietic stem cells are to
be transplanted from a donor to a recipient in need of such cells.
177. The method of claim 176 wherein said hematopoietic stem cells are
stored fox a period of time prior to transplant.
178. The method of claim 174 wherein said CD11b-positive cells are
lymphoid precursor cells.
179. The method of claim 174 wherein said CD11b-positive cells are
myeloid precursor cells.


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180. The method of claim 174 wherein said cells are selected from the
group consisting of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
181. A composition comprising recombinant human uteroglobin in an
amount sufficient to enhance proliferation of CD11b-positive cells in vitro.
182. The composition of claim 181 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
183. A method of enhancing activation of CD11b-positive cells in a patient
in need of such treatment comprising administering to said patient an amount
of
recombinant human uteroglobin sufficient to enhance activation of said cells.
184. The method of claim 183 wherein said patient is diagnosed with an
inflammatory disease.
185. The method of claim 183 wherein said patient is diagnosed with an
infectious disease.
186. The method of claim 183 wherein said patient is diagnosed with an
autoimmune disease.
187. The method of claim 183 wherein said patient is diagnosed with
cancer.
188. The method of claim 183 wherein said patient is diagnosed with a
fibrotic disease.
189. A method of claim 183 wherein said cells are selected from the group
consisting of: neutrophils, band cells, stab cells, granulocytes, eosinophils,
basophils,
monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes, T cells, B
cells, NK cells, lymphoid precursors, and myeloid precursors.
190. A composition comprising recombinant human uteroglobin in an
amount sufficient to enhance activation of CD11b-positive cells in a patient,
and a
pharmaceutically acceptable carrier or diluent.
191. The composition of claim 190 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.


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192. A method of enhancing activation of CD11b-positive cells in vitro
comprising exposing said cells to an amount of recombinant human uteroglobin
sufficient to enhance activation of said cells in vitro.
193. The method of claim 192 wherein said CD11b-positive cells are
hematopoietic stem cells.
194. The method of claim 193 wherein said hematopoietic stem cells are to
be transplanted from a donor to a recipient in need of such cells.
195. The method of claim 194 wherein said hematopoietic stem cells are
stored for a period of time prior to transplant.
196. The method of claim 192 wherein said CD71-positive cells are
lymphoid precursor cells.
197. The method of claim 192 wherein said CD71-positive cells are
myeloid precursor cells.
198. The method of claim 192 wherein said cells are selected from The
group consistin of: neutrophils, band cells, stab cells, granulocytes,
eosinophils,
basophils, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes,
T
cells, B cells, NK cells, lymphoid precursors, and myeloid precursors.
199. A composition comprising recombinant human uteroglobin in an
amount sufficient to enhance activation of CD11b-positive cells in vitro.
200. The composition of claim 199 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
201. A method of inhibiting migration of vascular endothelial cells
comprising administering recombinant human uteroglobin to a patient in need of
such
treatment in an amount sufficient to inhibit migration of said cells.
202. The method of claim 201 wherein said patient has been diagnosed with
a primary cancer.
203. The method of claim 202 wherein the recombinant human uteroglobin
inhibits or prevents metastatis of the primary cancer.
204. The method of 201 wherein said patient has been diagnosed with a
diabetic condition.


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205. The method of 201 wherin the recombinant human uteroglobin inhibits
or prevents retinopathy.
206. A composition comprising recombinant human uteroglobin in an
amount sufficient to suppress migration of vascular endothelial cells in a
patient, and
a pharmaceutically acceptable carrier or diluent.
207. The composition of claim 206 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
208. A method of inhibiting angiogenesis in a patient in need of such
treatment comprising administering to said patient an amount of recombinant
human
uteroglobin sufficient to inhibit angiogenesis.
209. A composition comprising recombinant human uteroglobin in an
amount sufficient to inhibit angiogenesis in a patient, and a pharmaceutically
acceptable carrier or diluent.
210. The composition of claim 209 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
211. A method of inhibiting migration of vascular endothelial cells in a
patient in need of such treatment comprising administering to said patient
recombinant human uteroglobin and fibronectin or a fragment derived from
fibronectin in amounts sufficient to inhibit migration of said cells.
212. A composition comprising recombinant human uteroglobin and
fibronectin, or a fragment derived from fibronectin, in amounts sufficient to
suppress
migration of vascular endothelial cells in a patient, and a pharmaceutically
acceptable
carrier or diluent.
213. The composition of claim 212 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
214. A method of inhibiting angiogenesis in a patient in need of such
treatment comprising administering to said patient recombinant human
uteroglobin
and fibronectin, or a fragment derived from fibronectin, in amounts sufficient
to
inhibit angiogenesis.


102

215. A composition comprising recombinant human uteroglobin and
fibronectin or a fragment derived from fibronectin in amounts sufficient to
inhibit
angiogenesis in a patient, and a pharmaceutically acceptable carrier or
diluent.
216. The composition of claim 215 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
217. A method of inhibiting extracellular matrix invasion by vascular
endothelial cells in a patient in need of such treatment comprising
administering to
said patient an amount of recombinant human uteroglobin sufficient to inhibit
extracellular matrix invasion of said cells.
218. A composition comprising recombinant human uteroglobin in an
amount sufficient to extracellular matrix invasion by vascular endothelial
cells in a
patient, and a pharmaceutically acceptable carrier or diluent.
219. The composition of claim 218 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
220. A method of inhibiting extracellular matrix invasion by vascular
endothelial cells in a patient in need of such treatment comprising
administering to
said patient recombinant human uteroglobin and fibronectin or a fragment
derived
from fibronectin in amounts sufficient to inhibit extracellular matrix
invasion.
221. A composition comprising recombinant human uteroglobin and
fibronectin or a fragment derived from fibronectin in amounts sufficient to
inhibit
extracellular matrix invasion by vascular endothelial cells in a patient, and
a
pharmaceutically acceptable carrier or diluent.
222. The composition of claim 221 wherein said amount of recombinant
human uteroglobin is 10 ng/kg - 25 mg/kg.
223. A method of regulating signal transduction in uteroglobin-responsive
cells said method comprising exposing said cells to recombinant human
uteroglobin,
wherein said signal transduction is mediated by CD148 and CD148 immunoreactive
proteins.
224. The method of claim 223 further comprising exposing said cells to
fibronectin or a fibronectin immunoreactive protein.


103

225. The method of claim 223 wherein arachidonic acid metabolism is
regulated.
226. The method of claim 223 wherein nitric oxide metabolism is regulated.
227. The method of claim 223 wherein the cell cycle is regulated
228. The method of claim 223 wherein cell adhesion molecule and/or
integrin expression is regulated.
229. A method of regulating cellular activities mediated by CD148 and
CD148 immunoreactive proteins comprising exposing the cells to recombinant
human
uteroglobin.
230. The method of claim 229 further comprising exposing said cells to
fibronectin or a fibronectin immunoreactive protein.
231. The method of claim 229 wherein cellular adhesion is regulated.
232. The method of claim 229 wherein cellular metabolism is regulated.
233. The method of claim 229 wherein cellular migration is regulated.
234. The method of claim 229 wherein cellular proliferation is regulated.
235. The method of claim 229 wherein cellular extracellular matrix invasion
is regulated.
236. The method of claim 229 wherein angiogenesis is regulated.
237. The method of claim 229 wherein cellular differentiation is regulated.
238. A method of regulating signal transduction in uteroglobin-responsive
cells said method comprising exposing said cells to recombinant human
uteroglobin,
wherein said signal transduction is mediated by PLA2 receptors and PLA2
immunoreactive proteins.
239. The method of claim 238 further comprising exposing said cells to
fibronectin or a fibronectin immunoreactive protein.
240. The method of claim 238 wherein arachidonic acid metabolism is
regulated.
241. The method of claim 238 wherein nitric oxide metabolism is regulated.
242. The method of claim 238 wherein the cell cycle is regulated
243. The method of claim 238 wherein cell adhesion molecule and/or
integrin expression is regulated.


104

244. A method of regulating cellular activities mediated by CD148 and
CD148 immunoreactive proteins comprising exposing the cells to recombinant
human
uteroglobin.
245. The method of claim 244 further comprising exposing said cells to
fibronectin or a fibronectin immunoreactive protein.
246. The method of claim 244 wherein cellular adhesion is regulated.
247. The method of claim 244 wherein cellular metabolism is regulated.
248. The method of claim 244 wherein cellular migration is regulated.
249. The method of claim 244 wherein cellular proliferation is regulated.
250. The method of claim 244 wherein extracellular matrix invasion is
regulated.
251. The method of claim 244 wherein angiogenesis is regulated.
252. The method of claim 244 wherein cellular differentiation is
regulated.
253. A method of identifying proteins that interact with each other, in
which
at least one protein contains at least one four helical bundle motif and at
least one
protein having at least one fibronectin Type III domain comprising mapping a
pathway involving one or more protein interactions.
254. The method of claim 253 wherein the pathway is physiological.
255. The method of claim 253 wherein the pathway is pathological.
256. The method of claim 253 wherein the pathway is pharmacological.
257. The method of claim 170 wherein receptors for rhUG and UG-like
proteins are identified.
258. The method of claim 253 wherein receptors for fibronectin and
fibronectin immunoreactive proteins are identified.
259. The method of claim 253 wherein receptors for proteins containing a
four helical bundle motif are identified.
260. The method of claim 253 wherein receptors for proteins containing a
fibronectin Type III domain are identified.
261. The method of claim 253 wherein ligands for proteins containing a
four helical bundle motif are identified.


105

262. The method of claim 253 wherein ligands for proteins containing a
fibronectin Type III domain are identified.
263. The method of claim 253 whereinligands for CD148 and CD148
immunoreactive proteins are identified.
264. The method of claim 253 wherein proteins with which rhUG and
rhUG-like proteins can form a complex are identified.
265. The method of claim 253 wherein proteins with which fibronectin and
fibronectin immunoreactive proteins can form a complex are identified.
266. The method of claim 253 wherein proteins bearing fibronectin Type III
repeats are identified, wherein said proteins are selected from the group
consisting of:
fibronectin, CD 148, collagens, titins, tenascins, cytotactins, fibrin, cell
adhesion
molecules, integrins, protein tyrosine phosphatases, and others.
267. The method of claim 253 wherein proteins bearing four helical bundle
motifs are identified, wherein said proteins are selected from the group
consisting of:
UG-like proteins, the secretory PLA2 protein family (including all subtypes),
the
annexins, and others.

Description

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METHODS AND COMPOSITIONS FOR THE TREATMENT OF FIBROTIC
CONDITIONS & IMPAIRED LUNG FUNCTION & TO ENHANCE
LYMPHOCYTE PRODUCTION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of the following applications: U.S.
Application Serial No. 09/549,926, filed April 14, 2000, which is a
continuation-in-
part of U.S. Application Serial No. 09/120,264, filed July 21, 1998, which is
a
continuation-in-part of U.S. Application Serial No. 09/087,210, filed May 28,
1998,
which is a continuation-in-part of U.S. Application Serial No. 08/864,357,
filed May
28, 1997. The disclosures of each of the aforementioned applications are
incorporated
herein by reference.
FIELD OF THE INVENTION
The present invention relates to the use of human uteroglobin or recombinant
human uteroglobin in the treatment of fibrotic conditions, to increase
lymphocyte
production in vivo, to improve and/or normalize lung function, pulmonary
compliance, blood oxygenation, and blood pH to inhibit inflammatory processes,
to
stimulate or inhibit pro-inflammatory and immune cells, and to to inhibit
migration of
vascular endothelial cells. Novel physiological roles and therapeutic targets
for
uteroglobin have been identified. Specifically, the invention provides a
method of
inhibiting cell adhesion to fibronectin by administering human uteroglobin or
recombinant human uteroglobin. The invention also provides a method of
increasing
lymphocyte production ih vivo by administering human uteroglobin or
recombinant
human uteroglobin. In addition, the invention provides a method of improving
lung
function by administering human uteroglobin or recombinant human uteroglobin.
Further the invention provides a method of inhibiting inflammatory processes
by
administering human uteroglobin or recombinant human uteroglobin. The
invention
also provides a method of stimulating or inhibiting pro-inflammatory and
immune
cells by administering human uteroglobin. In addition the invention provides a
method of inhibiting the migration of vascular endothelial cells by
administering
human uteroglobin or recombinant human uteroglobin. The invention also
provides a


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2
bioinformatics approach to the identification of human uteroglobin
receptor(s).
Documents cited in this application relate to the state-of the-art to which
this
invention pertains. The disclosures of each of these references are
incorporated
herein by reference.
BACKGROUND OF THE INVENTION
Uteroglobin (also known as UG, GC10, CC16, CC17, urine protein-1, P-1,
progesterone binding protein, PCB-binding protein, Clara cell secretory
protein
(CCSP), blastokinin, retinol-binding protein, phospholipid-binding protein,
and
alpha2-microglobulin) is a highly conserved mammalian protein that is
primarily
produced by the pulmonary epithelia. It is present in the mucosal fluid of the
respiratory tract, circulates in the blood, and is excreted in the marine.
Uteroglobin is a
small globular homodimeric protein that consists of two identical seventy
amino acid
peptides that complex in an anti-parallel orientation. It has a molecular
weight of 15.8
kDa, but it migrates in electrophoretic gels at a size corresponding to 10
kDa. Two
disulfide bonds spontaneously form to covalently link the monomers as a dimer.
Human uteroglobin is abundant in the adult human lung, and comprises up to
about
7% of the total soluble protein. However, its expression is not fully
activated in the
developing human fetus until late in gestation. Consequently, the
extracellular lung
fluids of pre-term infants contain far less human uteroglobin than those of
adults.
Uteroglobin is also expressed by the pancreas.
Amino acid analysis of purified human uteroglobin reveals that it is
structurally similar but not identical to other uteroglobin-like proteins,
e.g. rabbit
uteroglobin; 39 of 70 amino acids are identical between human and rabbit
uteroglobin. The uteroglobin-like proteins, including human uteroglobin, rat
uteroglobin, mouse uteroglobin, and rabbit uteroglobin, exhibit species-
specific and
tissue-specific antigenic differences, as well as differences in their tissue
distribution
and biochemical activities in vitro. Uteroglobin-like proteins have been
described in
many different contexts with regard to tissue and species of origin, including
rat lung,
human urine, sputum, blood components, rabbit uterus, rat and human prostate,
and
human lung.


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The absence of structural identity among uteroglobin-like proteins makes it
impossible to predict whether a protein will possess ih vivo therapeutic
function in
humans based on in vitro or other activity exhibited by a structurally related
protein.
For example, human uteroglobin binds less than 5% of the amount of
progesterone as
rabbit uteroglobin binds in the same assay. In addition, human uteroglobin has
a
lower isoelectric point (4.7) than rabbit uteroglobin (5.4).
Uteroglobin is known to inhibit the enzymatic activity of secretory (soluble)
phospholipases A2 (sPLA2s) which hydrolyze phospholipids, sometimes releasing
axachidonic acid in the process. Arachidonic acid is a precursor for several
pro-
inflammatory and anti-inflammatory eicosanoids. The role of uteroglobin as an
anti-
inflammatory agent ih vivo was confirmed by the discovery of an inflammatory
phenotype in the organs of a transgenic uteroglobin knockout mouse (USSN
08/864,357). The renal fibrotic phenotype of the uteroglobin lcnockout mouse
also led
to the discovery that uteroglobin forms a complex with fibronectin, preventing
fibronectin aggregation and deposition in vivo (USSN 08/864,357). In addition,
it
was found that uteroglobin prevents the formation of a complex between
fibronectin
and IgA. However, this animal exhibits no pulmonary phenotype.
The renal fibrotic phenotype of the uteroglobin knockout mouse first disclosed
in USSN 08/864,357 led to the discovery that uteroglobin may play a
significant role
in controlling fibronectin aggregation and deposition. Fibronectin is a 200
lcDa
glycoprotein which exists in several different forms and is secreted by
different
tissues. Fibronectin is an essential protein and targeted disruption of the
fibronectin
gene in mice showed that it has a central role in embryogenesis. Fibronectin
also
plays a lcey role in inflammation, cell adhesion, tissue repair and fibrosis,
and is
deposited at the site of injury. Plasma fibronectin is secreted by the liver
and
circulates in the plasma. In the lung, cellular fibronectin is secreted upon
inflammation and injury. Both types of fibronectin are chemotactic factors for
inflammatory cells and fibroblasts. They also interact with cell surface
proteins,
called integrins, as well as cell adhesion molecules to anchor cells during
adhesion
and extravasation. Large numbers of inflammatory cells and fibroblasts
infiltrate the
lung during inflammatory episodes, which can lead to pulmonaxy fibrosis and


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4
ultimately death. Elevated levels of fibronectin have been detected in human
clinical
conditions such as neonatal respiratory distress syndrome and bronchopulnonary
disease of the lung, and glomerular nephropathy of the kidney.
However, the physiological role of uteroglobin remains a source of
controversy in the art. Stripe et al. (1996) also generated a uteroglobin
knockout
mouse in which the expression of uteroglobin was eliminated. The mouse has
Clara
cells which exhibit odd intracellular structures in place of uteroglobin
secretion
granules, but there is no other life-threatening phenotype. This knockout
mouse also
showed no evidence of renal, pancreatic, or reproductive abnormality. These
results
are completely at odds with the observations made from the uteroglobin
knockout
mouse described in USSN 08/864,357. This mouse does, however, exhibit
exacerbated pulmonary inflammation when challenged with pulmonary insult.
Leyton et al. (1994) reported the anti-metastatic properties of uteroglobin
which were attributed to its inhibition of the release of arachidonic acid by
tumor
cells. (See also U.S. Patent No. 5,696,092 to Patierno et al.) Kundu et al.
(1996)
continued this work with the observation of inhibition of extracellular matrix
invasiveness by a variety of tumor cell types. Extracellular matrix invasion
correlated
with the presence of a 190 lcDa uteroglobin binding protein in responsive cell
types.
The extracellular matrix invasion activity of cells lacking this protein could
not be
inhibited by uteroglobin.
New investigations into the therapeutic properties of uteroglobin in non-
murine animal models has led to the discovery of novel mechanisms of action ih
vivo
that are distinct from the effects of uteroglobin on inflammation and fibrosis
previously observed by slcilled artisans in the field.
OBJECT OF THE INVENTION
It is an object of the present invention to provide a method of improving
and/or normalizing lung function, pulmonary compliance, blood oxygenation,
and/or
blood pH by administering an effective amount of human uteroglobin or
recombinant
human uteroglobin.
It is also an object of the invention to provide a composition consisting of
an
amount of human uteroglobin or recombinant human uteroglobin sufficient to


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improve and/or normalize lung function, pulmonary compliance, blood
oxygenation,
and/or blood pH. Such a composition should include a pharmaceutically
acceptable
carrier or diluent, and the composition should preferably consist of dimeric
recombinant human uteroglobin containing two disulfide bridges.
5 Further, it is an object of the invention to provide a method of increasing
lymphocyte production ih vivo by administering an amount of human uteroglobin
or
recombinant human uteroglobin sufficient to increase lymphocyte production
and/or
decrease polymorphonuclear leukocyte proliferation. Preferably, the
concentration of
effector lymphocytes and/or cytotoxic T cells is increased by the
administration of
uteroglobin. Moreover, it is an object of the invention to administer
uteroglobin to
increase lymphocyte production and/or decrease polymorphonuclear leukocyte
proliferation in patients suffering from an autoimmune disease or allergy.
It is an additional object of the invention to provide a composition
consisting
of an amount of human uteroglobin or recombinant human uteroglobin sufficient
to
increase lymphocyte production and/or decrease polymorphonuclear leukocyte
proliferation, together with a pharmaceutically acceptable carrier or diluent.
Still further, it is an object of the present invention to provide a method of
inhibiting cellular adhesion to fibronectin by administering an amount of
human
uteroglobin or recombinant human uteroglobin sufficient to inhibit cellular
adhesion
to fibronectin ih vivo. It is a further object of the invention to inhibit
inflammatory
cell and fibroblast migration on fibronectin already deposited in vivo, and to
inhibit
the interaction between a cell and an extracellular matrix protein and/or
membrane
bound protein.
Another object of the present invention to provide a composition consisting of
an amount of human uteroglobin or recombinant human uteroglobin sufficient to
inhibit cellular adhesion to fibronectin ih vivo. Such compositions should
consist of a
pharmaceutically acceptable carrier or diluent.
It is an additional object of the invention to provide a method of inhibiting
inflammatory processes by administering to a patient an amount of human
uteroglobin
or recombinant uteroglobin to inhibit inflammatory processes. Further, it is
an object
of the invention to provide a composition consisting of an amount of human


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6
uteroglobin or recombinant human uteroglobin inflammatory processes.
Still further, it is an object of the invention to provide a method of method
of
stimulating or inhibiting pro-inflarmnatory and immune cells by ih vivo or in
vitro
using an amount of human uteroglobin or recombinant human uteroglobin to
stimulate or inhibit such cells.
It is another object of the invention to provide a composition consisting of
an
amount of human uteroglobin or recombinant human uteroglobin sufficient to
stimulate or inhibit pro-inflammatory and immune cells ih vivo or in vitro.
Still further, it is an object of the invention to provide a method of
inhibiting
the migration of vascular endothelial cells by administering an amount of
human
uteroglobin or recombinant human uteroglobin sufficient to inhibit vascular
endothelial cell migration. Another object of the invention is to provide a
composition consisting of an amount of human uteroglobin or recombinant human
uteroglobin sufficient to inhibit endothelial cell migration.
An additional object of the invention is to provide a bioinformatics approach
to identify human uteroglobin receptor(s).
SUMMARY OF THE INVENTION
It has now been found that uteroglobin plays a central physiological role in
fibronectin deposition, lymphocyte production, smooth muscle function, and
lung
function in vivo.
In a first experiment it was found that the administration of uteroglobin to
neonatal lambs delivered by caesarian section, an accepted model for
surfactant-
dependent neonatal respiratory distress syndrome in humans, led to improved
and/or
normalized blood oxygenation and pH. These effects are indicative of improved
lung
function. This observation, discussed in more detail below, is the first
observation of
the direct effect of uteroglobin on lung tissue and the first indication that
uteroglobin
may be used to improve and/or normalize lung function.
Further, in a second experiment using newborn piglets, it was found that the
administration of recombinant human uteroglobin increased pulmonary
compliance.
The newborn piglet is an excellent model for neonatal lung injury mediated by


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7
oxygen toxicity arising from the use of positive pressure ventilation and
elevated
oxygen delivery in respiratory distress syndrome rescue. This significant
observation
was the first indication that uteroglobin may be used to increase pulmonary
compliance. This effect was independent of any effects of uteroglobin on
surfactant
function. These data show that uteroglobin may be used to treat patients
suffering
from reduced pulmonary compliance as a result of a pulmonary challenge or
insult
resulting from exposure to non-atmospheric gases, inhaled chemicals,
pollutants,
irritants, pollens, allergens, particulate matter, and airborne infectious
agents. It was
also found that a single dose of recombinant human uteroglobin to newborn
piglets
significantly increased lymphocyte proliferation and decreased
polymorphonuclear
leukocyte proliferation. The increase in lymphocyte proliferation was
significant, up
to 2.5 fold, and the decrease in polymorphonuclear leukocyte proliferation of
up to 2.3
fold persisted for a period exceeding one month.
In addition, using two new assay formats designed to specifically detect
uteroglobin-fibronectin binding, it was found that recombinant human
uteroglobin
binds to portions of fibronectin that are important in cell adhesion and not
known to
be relevant to fibrillogenesis. Fibronectin consists of eight type I domains
in the N-
terminal third of the protomer, three type I domains at the C-terminus, two
type II
domains clustered in the middle of the protomer, and 15-17 type III domains,
depending on the tissue of origin. One or more of the type III domains have
been
implicated in cell adhesion, fibronectin-fibronectin interactions, and
deposition in
vitro. Using two commercially available chymotryptic fragments of fibronectin,
each
containing type III domains involved in fibronectin-dependent cell adhesion
and/or
polymerization, and a recombinant fragment of fibronectin, termed
"superfibronectin"
(so named because of its ability to promote fibronectin-fibronectin
interactions,
polymerization, deposition, and cell adhesion ih vitro), the interaction
between
uteroglobin and the various regions of fibronectin was examined.
A clear dose-response relationship in binding between recombinant human
uteroglobin and "superfibronectin" was observed. This indicates that
recombinant
human uteroglobin binds to the type III domain of fibronectin which is
represented by
"superfibronectin". Further, it was also found that recombinant human
fibronectin


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binds to more than one type III domain of fibronectin because a dose-response
relationship was observed for binding,between uteroglobin and a chymotryptic
fragment that does not contain the "superfibronectin" domain. Still further,
because
fibronectin type III domains are present in nearly all components of the
extracellular
matrix, e.g.; laminin, collagens, vitronectin, and fibrin, as well as in
numerous
membrane bound proteins, e.g., adhesion molecules, integrins, and receptors,
these
domains may play a central role in cell-cell and cell-extracellular matrix
interactions.
The observation that uteroglobin interacts with these domains shows that it
may be
used to mediate these interactions and physiological conditions affected by
such
interactions.
Therefore, according to one aspect of the present invention, the invention
provides a method of treatment including improving and/or normalizing lung
function
in a patient in need of such treatment, wherein the method consists of
administering
an amount of uteroglobin effective to improve and/or normalize lung function
relative
to that observed in the absence of such treatment. In a preferred embodiment,
the
uteroglobin is recombinant human uteroglobin, and the preferred dosage range
is 10
ng/kg - 25 mg/kg.
According to a further aspect, the invention provides a method of improving
and/or normalizing pulmonary compliance in a patient in need of such
treatment,
wherein the method consists of administering an amount of uteroglobin to the
patient
sufficient to improve and/or normalize pulmonary compliance relative to that
observed in the absence of such treatment. In a preferred embodiment, the
uteroglobin
is recombinant human uteroglobin, and the preferred dosage range is 10 ng/kg -
25
mg/kg. The reduced pulmonary compliance may have resulted from pulmonary
challenge or insult resulting from exposure to non-atmospheric gases, inhaled
chemicals, pollutants, irritants, inhaled pollens, allergens, particulate
matter, and
airborne infectious agents.
An additional aspect of the invention provides a method of treating a patient
suffering from reduced blood oxygenation and/or blood pH, wherein the method
includes administering an amount of uteroglobin effective to improve and/or
normalize blood oxygenation and/or blood pH relative to that observed in the
absence


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of such treatment. In a preferred embodiment, the uteroglobin is recombinant
human
uteroglobin, and the preferred dosage range is 10 ng/kg - 25 mg/kg.
Another aspect of the invention provides compositions consisting of
uteroglobin effective for the improvement and/or normalization of lung
function,
pulmonary compliance, blood oxygenation, and/or blood pH. Such compositions
preferably contain a dosage of 10 ng - 500 mg, in combination with a
pharmaceutically acceptable carrier or diluent, wherein the amount of
uteroglobin
contained in the composition is commensurate with the administration of 10
ng/kg -
25 mg/kg by the method of the present invention. In a preferred embodiment,
the
uteroglobin is recombinant human uteroglobin.
According to a further aspect, the invention provides a method of increasing
lymphocyte production in a patient in need of such treatment, wherein the
method
includes administering an amount of uteroglobin sufficient to increase
lymphocyte
production in the patient relative to that observed in the absence of such
treatment. In
a preferred embodiment, recombinant human uteroglobin is used at a dosage
range of
1 ng/kg - 100 mg/kg. Preferably, the method increases the production of
effector
lymphocytes and/or cytotoxic T cells. The patient may be suffering from
decreased
lymphocyte production as a result of an autoimmune disease, such as acquired
immunodeficiency syndrome, or an allergy. In addition, uteroglobin may be used
to
enhance a lymphocyte response to a vaccine.
In an additional aspect, the invention provides a method of increasing the
production of suppressor T cells in a patient in need of such treatment,
wherein the
method includes administering an amount of uteroglobin sufficient to increase
production of suppressor T cells in the patient relative to that observed in
the absence
of such treatment. In a preferred embodiment, recombinant human uteroglobin is
used at a dosage range of 1 ng/kg - 100 mg/kg.
In accordance with an additional aspect, the invention provides a method of
enhancing a lymphocyte-mediated response in a patient in need of such
treatment,
wherein the method includes administering an amount of uteroglobin sufficient
to
enhance such a response in the patient relative to that observed in the
absence of such
treatment. In a preferred embodiment, recombinant human uteroglobin is used at
a


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dosage range of 1 ng/kg - 100 mg/kg.
A further aspect of the invention is to provide a method of decreasing the
production of polymorphonuclear leukocytes in a patient in need of such
treatment,
wherein the method includes administering an amount of uteroglobin sufficient
to
5 decrease polymorphonuclear leukocyte production in the patient relative to
that
observed in the absence of such treatment. In a preferred embodiment,
recombinant
human uteroglobin is used at a dosage range of 1 ng/kg - 100 mg/kg.
An aspect of the invention is to provide compositions including uteroglobin in
an amount sufficient to increase production of lymphocytes and/or suppressor T
cells,
10 enhance a lymphocyte-mediated response, and/or to decrease the production
of
polymorphonuclear leukocytes in a patient in need of such treatment. In a
preferred
embodiment, recombinant human uteroglobin is used in the composition at a
dosage
range of 1 ng/kg - 100 mg/kg, together with a pharmaceutically acceptable
carrier or
diluent.
According to an additional aspect, the invention provides a method of
treatment including inhibiting fibronectin-dependent cell adhesion to
fibronectin in a
patient in need of such treatment, wherein the method consists of
administering an
amount of uteroglobin to the patient sufficient to inhibit fibronectin-
dependent cell
adhesion to fibronectin relative to that observed in the absence of such
treatment. In a
preferred embodiment, the uteroglobin is recombinant human uteroglobin, and
the
preferred dosage range is 8 ~g - 3.5 g dose per 70 kg patient. Most
preferably, the
dosage range is 25 ng/200 ~,1- 10 ~.g/200 ~,1. In an additional preferred
embodiment,
uteroglobin blocks cell adhesion to type III domains of fibronectin.
An aspect of the invention provides a method of treatment including inhibiting
an interaction between fibronectin and cells dependent on fibronectin binding,
wherein the method includes administering an amount of uteroglobin effective
to
inhibit such interactions relative to that observed in the absence of such
treatment. In
a preferred embodiment, the uteroglobin is recombinant human uteroglobin, and
the
preferred dosage range is 8 ~,g - 3.5 g dose per 70 kg patient. Most
preferably, the
dosage range is 25 ng/200 ~,l - 10 ~,g/200 ~,1.


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According to a fuxther aspect, the invention provides a method of inhibiting
inflammatory cell and fibroblast migration on fibronectin already deposited ih
vivo in
a patient in need of such treatment, wherein the method includes administering
an
amount of uteroglobin sufficient to inhibit fibronectin-dependent cell
adhesion to
fibronectin in the patient relative to that observed in the absence of such
treatment. In
a preferred embodiment, the uteroglobin is recombinant human uteroglobin, and
the
preferred dosage range is 8 ~,g - 3.5 g dose per 70 kg patient. Most
preferably, the
dosage range is 25 ng/200 ~1- 10 ~,g/200 ~1.
In another aspect, the invention provides a method of inhibiting fibronectin-
dependent cell adhesion in a patient in need of such treatment, wherein the
method
includes administering an amount of uteroglobin sufficient to inhibit
fibronectin-
dependent cell adhesion in the patient relative to that observed in the
absence of such
treatment.
In accordance with an aspect, the invention provides a method of inhibiting an
interaction betweeen a cell and an extracellular matrix protein and/or
membrane
bound protein in a patient in need of such treatment, wherein the method
includes
administering an amount of uteroglobin sufficient to inhibit such interactions
in the
patient relative to that observed in the absence of such treatment. In a
preferred
embodiment, the uteroglobin is recombinant human uteroglobin, and the
preferred
dosage range is 8 ~,g - 3.5 g dose per 70 lcg patient. Most preferably, the
dosage range
is 25 ng/200 p,1 - 10 ~.g/200 ~.1.
Another aspect of the invention is to provide compositions including
uteroglobin in an amount sufficient to inhibit fibronectin-dependent cell
adhesion to
fibronectin, an interaction between fibronectin and cells dependent on
fibronectin
binding, inflammatory cell and fibroblast migration on fibronectin deposited
in vivo,
fibronectin-dependent cell adhesion, and an interaction between a cell and an
extracellular matrix protein and/or membrane bound protein in a patient in
need of
such treatment. In a preferred embodiment, recombinant human uteroglobin is
used in
the composition, together with a pharmaceutically acceptable carrier or
diluent, and
the preferred dosage range is 8 ~,g - 3.5 g dose per 70 kg patient. Most
preferably, the


CA 02405946 2002-10-15
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12
dosage range is 25 ng/200 ~,1- 10 ~g/200 ~,1.
Another aspect of the invention is to provide methods and compositions to
inhibit an LPS-dependent inflammatory processes in a patient infected with a
bacterium by administering to a patient an amount of recombinant human
uteroglobin
sufficient to inhibit the inflammatory processes.
An additional aspect of the invention is to provide methods and compositions
to decrease TNF-alpha concentrations in vivo in a patient in need of such
treatment by
administering to a patient an amount of recombinant human uteroglobin
sufficient to
decrease TNF-alpha concentrations.
A further aspect of the invention is to provide methods and compositions to
regulate the nitric oxide pathway for relaxing smooth muscle cells in a
patient in need
of such treatment by administering to the patient an amount of recombinant
human
uteroglobin sufficient to regulate the nitric oxide pathway.
Another aspect of the invention is to provide methods and compositions to
regulate vascular permeability in a patient in need of such treatment by
administering
to the patient an amount of recombinant human uteroglobin sufficient to
regulate
vascular permeability.
A further aspect of the invention is to provide methods and compositions to of
suppress proliferation of CD71-positive cells in a patient in need of such
treatment by
administering to the patient an amount of recombinant human uteroglobin
sufficient
to suppress proliferation of such cells.
An additional aspect of the invention is to provide methods and compositions
to suppress proliferation of CD71-positive cells in vitro by exposing CD71-
positive
cells to an amount of recombinant human uteroglobin sufficient to suppress
proliferation of the cells in vitro.
Another aspect of the invention is to provide methods and compositions to of
suppress proliferation of CD71-positive cells in vitro by exposing CD71-
positive cells
to an amount of recombinant human uteroglobin and and an amount of fibronectin
sufficient to suppress proliferation of the cells in vitro.
A further aspect of the invention is to provide methods and compositions to
suppress activation of CD71-positive cells in a patient in need of such
treatment by


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13
administering to the patient an amount of recombinant human uteroglobin
sufFcient
to suppress activation of such cells.
An additional aspect of the invention is to provide methods and compositions
to suppress activation of CD71-positive cells in vitro by exposing the cells
to an
amount of recombinant human uteroglobin sufficient to suppress activation of
the
cells in vitro.
An additional aspect of the invention is to provide methods and compositions
to enhance proliferation of CD 1 lb-positive cells in a patient in need of
such treatment
by administering to the patient an amount of recombinant human uteroglobin
sufficient to enhance proliferation of such cells.
Another aspect of the invention is to provide methods and compositions to
enhance proliferation of CD1 lb-positive cells in vitro by exposing the cells
to an
amount of recombinant human uteroglobin sufficient to enhance proliferation of
the
cells in vitro.
A further aspect of the invention is to provide methods and compositions to
enhance activation of CDl lb-positive cells in a patient in need of such
treatment by
administering to the patient an amount of recombinant human uteroglobin
sufficient
to enhance activation of the cells.
An additional aspect of the invention is to provide methods and compositions
to enhance activation of CD1 lb-positive cells in vitro by exposing said cells
to an
amount of recombinant human uteroglobin sufficient to enhance activation of
the cells
in vitro.
Another aspect of the invention is to provide methods and compositions to
inhibit migration of vascular endothelial cells by administering recombinant
human
uteroglobin to a patient in need of such treatment in an amount sufficient to
inhibit
migration of such cells.
A further aspect of the invention is to provide methods and compositions to
inhibit angiogenesis in a patient in need of such treatment by administering
to the
patient an amount of recombinant human uteroglobin sufficient to inhibit
angiogenesis.


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14
An additional aspect of the invention is to provide methods and compositions
to inhibit migration of vascular endothelial cells in a patient in need of
such treatment
by administering to the patient recombinant human uteroglobin and fibronectin
or a
fragment derived from fibronectin in amounts sufficient to inhibit migration
of said
cells.
Another aspect of the invention is to provide methods and compositions to
inhibit angiogenesis in a patient in need of such treatment by administering
to the
patient recombinant human uteroglobin and fibronectin, or a fragment derived
from
fibronectin, in amounts sufficient to inhibit angiogenesis.
A further aspect of the invention is to provide methods and compositions to
inhibit extracellular matrix invasion by vascular endothelial cells in a
patient in need
of such treatment by administering to the patient an amount of recombinant
human
uteroglobin sufficient to inhibit extracellular matrix invasion of such cells.
An additional aspect of the invention is to provide methods and compositions
to inhibit extracellular matrix invasion by vascular endothelial cells in a
patient in
need of such treatment by administering to the patient recombinant human
uteroglobin and fibronectin or a fragment derived from fibronectin in amounts
sufficient to inhibit extracellular matrix invasion.
Another aspect of the invention is to provide methods and compositions to
regulate signal transduction mediated by CD 148 and CD 148 immunoreactive
proteins
in uteroglobin-responsive cells by exposing the cells to recombinant human
uteroglobin.
A further aspect of the invention is to provide methods and compositions to
regulate cellular activities mediated by CD 148 and CD 148 immunoreactive
proteins
by exposing the cells to recombinant human uteroglobin.
An additional aspect of the invention is to provide methods and compositions
to regulate signal transduction mediated by PLA2 receptors and PLA2
immunoreactive proteins in uteroglobin-responsive cells by exposing the cells
to
recombinant human uteroglobin.


CA 02405946 2002-10-15
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Another aspect of the invention is to provide methods and compositions to
regulate cellular activities mediated by CD 148 and CD 148 immunoreactive
proteins
by exposing the cells to recombinant human uteroglobin.
An additional aspect of the invention is to provide methods to identify
proteins
5 that interact with each other, in which at least one protein contains at
least one four
helical bundle motif and at least one protein having at least one fibronectin
Type III
domain by mapping a pathway involving one or more protein interactions.
BRIEF DESCRIPTION OF THE DRAWINGS
10 The invention will now be described in more detail, with reference to the
accompanying drawings, in which:
Figure 1 shows the standard curve obtained using the uteroglobin (UG)
immunoassay described below.
Figure 2 shows the bicarbonate excess (BE) exhibited in pre-term lambs upon
15 intratracheal administration of recombinant human uteroglobin.
Figure 3 shows the decrease in C02 exhibited in pre-term lambs upon
intratracheal administration of recombinant human uteroglobin.
Figure 4 shows the increase in blood pH exhibited in pre-term lambs upon
intratracheal administration of recombinant human uteroglobin.
Figure 5 shows the increase in pa02/Fi02 exhibited in pre-term lambs upon
intratracheal administration of recombinant human uteroglobin.
Figure 6A shows the concentration of recombinant human uteroglobin (CC10)
in serum as a function of time after intratracheal administration of
recombinant human
uteroglobin to newborn piglets.
Figure 6B shows the total protein concentration in BAL fluids obtained from
piglets in each of the eight treatment groups.
Figure 7 shows the pressure-volume relationship observed upon administration
of recombinant human uteroglobin to newborn piglets.
Figure 8 shows the mean pressure-volume relationship observed upon
administration of recombinant human uteroglobin to newborn piglets ventilated
with
100% oxygen.


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16
Figure 9 shows the mean pressure-volume relationships observed for all
animals upon administration of recombinant human uteroglobin to newborn
piglets
ventilated with room air and 100% oxygen.
Figure 10A shows the mean pressure-volume curve among four treatment
groups of newborn piglets administered recombinant human uteroglobin.
Figure lOB shows the mean pressure-volume curve among five treatment
groups of newborn piglets administered recombinant human uteroglobin.
Figure lOC shows changes in PMN and lymphocyte cell counts over a 28-day
period.
Figure 11 shows radioactive counts as a function of time for each group of
Wistar rats administered recombinant human' uteroglobin via intravenous
administration.
Figure 12 shows radioactive counts as a function of time for each group of
Wistar rats administered recombinant human uteroglobin via intranasal
administration.
Figure 13 shows radioactive counts as a function of time for each group of
Wistar rats administered recombinant human uteroglobin via stomach gavage.
Figure 14 shows the concentration of recombinant human uteroglobin as a
function of time for each group of Wistar rats administered recombinant human
uteroglobin via intravenous administration.
Figure 15 shows the concentration of recombinant human uteroglobin as a
function of time for each group of Wistar rats administered recombinant human
uteroglobin via intranasal administration.
Figure 16 shows the concentration of recombinant human uteroglobin as a
function of time for each group of Wistar rats administered recombinant human
uteroglobin via stomach gavage.
Figures 17A-17B are schematic representations of two ELISA-based assay
formats for the uteroglobin-fibronectin binding interaction. Format A, shown
in
Figure 17A represents an assay based on immunodetection, wherein CC10 is
uteroglobin and HRP is horse radish peroxidase. Format B, shown in Figure 17B
represents a competitive binding assay format in which CC10 is uteroglobin,
HRP is


CA 02405946 2002-10-15
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17
horse radish peroxidase, and rhFn is recombinant human fibronectin, and the
free
uteroglobin in the same competes with the HRP-labeled uteroglobin for binding
sites
on recombinant human fibronectin.
Figure 18 shows a map of the human fibronectin protomer.
Figure 19 shows the results obtained from binding assays between uteroglobin
(CC10) and intact and fragmented fibronectin using format A, wherein hFn is
human
fibronectin and SuperFn is superfibronectin.
Figures 20A-20B show the dose response curves for uteroglobin binding to
fibronectin and its fragments using format A, wherein rhUG is recombinant
human
uteroglobin, hFn is human fibronectin, and Fn is fibronectin.
Figure 21 shows the mean change in airway resistance in perfused rat lung.
Figure 22 shows the mean concentration of TNF-alpha in BAL fluid from
perfused rat lung.
Figure 24 shows a flow diagram of the maturation of hemopoietic stem cells.
Figure 25 shows ATPase activity in CD71-positive cells from rats treated with
recombinant human uteroglobin.
Figure 26 shows ATPase activity in CDllb-positive cells from rats treated
with recombinant human uteroglobin.
Figure 27 shows an in vitro wound healing assay with recombinant human
uteroglobin.
Figure 29 shows the inhibition of endothelial cell migration by recombinant
human uteroglobin.
Figure 30 shows an extracellular matrix invasion assay with endothelial cells
and recombinant human uteroglobin.
Figure 31A shows the endothelial cell growth curve with and without
recombinant human uteroglobin.
Figure 31B shows the dose dependent response of endothelial cell VEGF
stimulated proliferation assay.
Figure 32 shows a Western blot of proteins from three cell lines probed with
anti-fibronectin antibody.
Figure 33 shows colony counts of A549 soft agar assay.


CA 02405946 2002-10-15
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18
Figure 34 shows a silver-stained SDS-Page gel indicating UG affinity purified
bands that cross react with specific antibodies.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
Native and recombinant human uteroglobin may be used in the present
invention. In a preferred embodiment, however, recombinant human uteroglobin
is
employed in the methods and compositions of the invention. The recombinant
form
of uteroglobin preferably has substantially the same amino acid sequence as
that of
the native human uteroglobin protein. An amino acid sequence having
"substantially
the same" amino acid sequence as that of the native human protein includes
recombinant human uteroglobin having at least 75% identity to the native human
protein. In a preferred embodiment, recombinant human uteroglobin has at least
85%
identity, and in a most preferred embodiment, recombinant human uteroglobin
has at
least 98% identity to the native uteroglobin. In a further preferred
embodiment,
dimeric recombinant human uteroglobin is used in the methods and compositions
of
the present invention (with respect to the various forms of uteroglobin,
reference is
made to USSN 09/120,264).
Also included in the method of the present invention is the use of fragments
or
derivatives of uteroglobin, native or recombinant. A "fragment" of uteroglobin
refers
to a portion of the native uteroglobin amino acid sequence having six or more
contiguous amino acids of the native protein sequence. The term "derivative"
refers
to peptide analogs of uteroglobin, including one or more amino acid
substitutions
and/or the addition of one or more chemical moieties, e.g., acylating agents,
sulfonating agents, carboxymethylation of the disulphide bonds, or complexed
or
chelated metal or salt ions, e.g. Mg+Z, Ca+2, or Na 1, with the proviso that
the
derivative retains the biological activity of the parent molecule. In
addition, the
present invention also contemplates the use of small molecule mimetics and
chemical
structural derivatives of uteroglobin.
A "uteroglobin-like" protein includes those isolated from mouse, rat, rabbit,
etc., having substantially the same amino acid sequences and/or substantial
sequence


CA 02405946 2002-10-15
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19
similarity, termed conservative substitutions, with native human uteroglobin.
With
regard to sequence similarity, like-amino acids may be substituted in a
uteroglobin-
like protein, e.g. tyrosine for phenylalanine or glycine for alanine.
Uteroglobin-like
proteins which are considered substantially similar have approximately 30%
sequence
similarity, preferably 50% sequence similarity, more preferably at least 75%
sequence
similarity, and most preferably at least 90-95% sequence similarity.
Uteroglobin-
receptor ligands are peptide, protein or chemical moieties (e.g. organic
ligands) that
bind to the uteroglobin receptor and mediate all or part of its activities.
Uteroglobin
structural analogs are compounds, peptides or proteins, or fragments or
derivatives
thereof having substantially similar secondary and tertiary structural
characteristics
when compared to native uteroglobin, such that a structural analog retains at
least
50% and preferably at least 75% of the activity of native protein. In a most
preferred
embodiment, a structural analog retains at least 90% of the activity of the
native
protein and retains the ability to interact with the uteroglobin receptor and
to mediate
all or part of its activities. As used herein, ther term "recombinant human
uteroglobin" includes recombinant CC10.
Further, the uteroglobin used in the method of the present invention is
substantially pure. The term "substantially pure" refers to uteroglobin having
a purity
of about 75% to about 100%. In a preferred embodiment, uteroglobin has a
purity of
about 90% to about 100%, and in the most preferred embodiment, uteroglobin has
a
purity of at least 95%.
In addition, as used herein "fibronectin immunoreactive protein" includes
proteins, protein fragments, glycosylated or otherwise modified, peptides or
derivatives that react with anti-human fibronectin monoclonal antibody.
Further, "CD 148 immunoreactive protein" includes protein, protein fragments,
peptides, or derivatives that react with anti-CD 148 polyclonal antibody.
In addition, as used herein "PLA2 receptor immunoreactive protein" includes
protein, protein fragments, peptides, or derivatives that react with an anti M
type
PLA2 receptor antibody.
In so much as the present invention provides a method of treating or
preventing a disease condition associated with fibronectin deposition, lung
damage,


CA 02405946 2002-10-15
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and/or decreased lymphocyte production, the term "prevention" refers to
preventing
the development of disease in a susceptible or potentially susceptible
population, or
limiting its severity or progression, whereas the term "treatment" refers to
the
amelioation of a disease or pathological condition. As used herein "regulate"
includes
5 increasing or decreasing in a pharmacologically, therapeutically or
physiologically
beneficial manner or purpose, and to enhance or re-assert therapeutically
beneficial
cellular and tissue control pathways.
To map a pathway means to identify the molecular participants in the pathway
and to eventually determine the sequence of molecular interactions that result
in a
10 quantifiable net effect at the molecular, cellular or tissue level.
UG-like proteins are members of the UG protein family, and includes
mammoglobin, lymphoglobin, lipophilins, and others, all bearing the
characteristic
dimeric structure with at least one monomer containing a four helical bundle
motif.
Proteins that contain four helical bundle motifs include the UG and UG
15 family, the sPLA2 protein family, the annexin family, and others.
Proteins that contain fibronectin Type III repeats include collagens, titins,
tenascins, cytotactins, fibrin, cell adhesion molecules, integrins, protein
tyrosine
phosphatases, and others.
The Effect of Uteroglobin in
20 Lung Damage, Lymphocyte Production, and Fibronectin
Using neonatal lambs delivered by caesarean section as a model of surfactant-
dependent neonatal respiratory distress syndrome (RDS), it was found that
recombinant human uteroglobin did not interfere with surfactant replacement
therapy.
In fact, an animal with severe meconium aspiration which potently inactivates
surfactant responded to the administration of recombinant human uteroglobin by
a
marked increase in blood oxygenation and pH, both of which are indicators of
improved lung function. This was the first observation of a direct effect of
uteroglobin on lung tissue.
It was also unexpectedly found that the bioavailability of recombinant human
uteroglobin in this experiment was excellent. These results show that
recombinant


CA 02405946 2002-10-15
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21
human uteroglobin may be administered systemically via the lungs, for the
purpose of
raising circulating levels of the protein, to deliver the protein to tissues
and organs,
and to raise the concentration of uteroglobin in the urine. Therefore, it is
possible to
treat various internal organs and tissues, including the vasculature, muscle,
connective
tissue, bone, blood cells, stomach, kidneys, pancreas, liver, intestines,
colon, heart,
spleen, thymus, uterus, and bladder, by administering uteroglobin topically to
the
lungs through intratracheal deposition or through an inhaler or nebulizer.
Further,
results obtained from the intravenous, intranasal, and stomach gavage
administration
of uteroglobin to adult Wistar rats indicated that these routes may be
practical for the
systemic administration of the protein in humans. The presence of radioactive
recombinant human uteroglobin in protein extracts of the trachea, bronchi,
esophagus,
and thyroid in an animal administered uteroglobin via each of these routes
show that
these tissues take up uteroglobin from the circulatory system. This
demonstrates that
one route of administration may be effective in the specific delivery of
protein to
target delivery for another system, e.g., the digestive system.
Next, using a ventilated newborn piglet, a well-characterized model of
neonatal lung injury, one can readily observe significant decreases in
pulmonary
compliance and lung function, as well as increases in inflammatory markers
which are
indicative of pulmonary inflammation. These animals were administered
uteroglobin,
then broncho-alveolar lavage fluid (BAL), serum, and urine were collected in
order to
monitor the half life and elimination of uteroglobin following intratracheal
administration. Further, the total protein concentration in BAL samples was
measured and total soluble protein was calculated. These parameters are
significant
indicators of lung injury. The data show that administration of recombinant
human
uteroglobin increased pulmonary compliance in the newborn piglets sampled, and
that
the effect was independent of surfactant function. Complete blood counts (CBC)
and
differential cell counts were taken on the blood samples collected prior to
and
following uteroglobin administration to the ventilated newborn piglets. It was
found
that a single dose of recombinant human uteroglobin in the newborn piglets
significantly enhanced lymphocyte proliferation and decreased
polymorphonuclear
leukocyte production.


CA 02405946 2002-10-15
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22
Therefore, the present invention provides methods and compositions for
improving and/or normalizing lung function, pulmonary compliance, blood
oxygenation and/or blood pH. Suitable compositions include uteroglobin, and
preferably recombinant human uteroglobin, in a dosage of 10 ng/kg - 2S mg/kg.
The
S methods may be used to treat patients suffering from reduced lung function
and/or
pulmonary compliance as a result of exposure to non-atmospheric gases, non-
atmospheric ratios of atmospheric gases, inhaled chemicals, pollutants,
irritants,
inhaled pollens, allergens, particulate matter, and airborne infectious
agents.
The present invention also provides methods and compositions for increasing
lymphocyte production in vivo, increasing the production of suppressor T
cells,
enhancing a lymphocyte-mediated response in vivo, and decreasing the
production of
polymorphonuclear leukocytes. The method of the present invention contemplates
a
dosage of uteroglobin, preferably recombinant human uteroglobin, of 1 ng/kg -
100
mg/kg. The Lymphocytes that are typically affected by this method are effector
1 S lymphocytes and cytotoxic T cells, and more particularly, helper T cells,
suppressor T
cells, NK cells, plasma B cells, memory B cells, and their precursers.
Further, in so
much as the present invention provides a method of enhancing a lymphocyte
mediated response in vivo, such a method may be used to enhance the effects of
the
administration of a vaccine, such as a B cell or T cell vaccine, or a
tolerance-inducing
treatment, such as oral tolerance or allergy shots.
Finally, the interaction between uteroglobin and fibronectin was examined. It
was found that recombinant human uteroglobin was a potent inhibitor of
cellular
adhesion to fibronectin and that it specifically bound to type III domains of
fibronectin. Such domains are present in nearly all protein components of the
2S extracellular matrix, e.g., laminin, collagens, vitronectin, and fibrin, as
well as in
numerous membrane bound proteins, including adhesion molecules, integrins, and
receptors. Thus, the inhibition of cellular adhesion by uteroglobin indicates
that
uteroglobin can play a critical role in cell-cell and cell-extracellular
matrix
interactions.
Therefore, the present invention provides methods and compositions for
inhibiting the following processes: (1) fibronectin-dependent cell adhesion to


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23
fibronectin, (2) interactions between fibronectin and cells dependent on
fibronectin
binding, (3) inflammatory cell and fibroblast migration on fibronectin already
deposited ih vivo, (4) fibronectin-dependent cell adhesion in vivo, (5) an
interaction
between a protein cell containing a four helical bundle motif and a
protein/ECm
protein containing at least one FnIII domain and (6) an interaction between a
cell
having a PLA2 receptor and an extracellular matrix protein and/or membrane
bound
protein comprising at least one fibronectin type III domain. The compositions
contain
uteroglobin, and preferably recombinant human uteroglobin, in a dosage of 8 ~g-
3.5 g
total dose per 70 kg patient, and more preferably, 25 ng/200 ~1- 10 ~,g/200
~.1.
1 p Cells dependent on fibronectin binding include, but are not limited to,
neural
cells, muscle cells, hematopoietic cells, fibroblasts, neutrophils,
eosinophils,
basophils, macrophages, monocytes, lymphocytes, platelets, red blood cells,
endothelial cells, stromal cells, dendritic cells, mast cells, and epithelial
cells. A
patient suffering from one of the following conditions may benefit from
therapies
which inhibit the formation of vascular adhesions following surgery,
atheroschlerosis,
thrombosis, heart disease, vasculitis, formation of scar tissue, restenosis,
phlobitis,
COPD (chronic obstructive pulmonary disease), pulmonary hypertension,
pulmonary
fibrosis, pulmonary inflammation, bowel adhesions, bladder fibrosis and
cystitis,
fibrosis of the nasal passages, sinusitis, inflammation mediated by
neutrophils, and
fibrosis mediated by fibroblasts.
In so much as the invention provides a method of inhibiting inflammatory cell
and fibroblast migration on fibronectin already deposited ih vivo,
inflammatory cells
targeted by this method include, but are not limited to, neutrophils,
eosinophils,
basophils, macrophages, monocytes, lymphocytes, platelets, red blood cells,
dendritic
cells, mast cells, stem cells and fibroblasts.
Finally, extracellular matrix proteins affected by the method of the present
invention include, but are not limited to, laminin, collagen, vitronectin, and
fibrin, and
membrane bound proteins affected by the instant method include, but are not
limited
to, adhesion molecules, integrins, and receptors.
Without wislung to be bound by any particular theory, the interactions
between uteroglobin and lung damage, lymphocyte production, and fibronectin do
not


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24
appear to be discrete and insular occurrences. Rather, they are indicative of
a
complex network of interactions between a variety of physiological events.
First, the
primary site of action of surfactant phospholipids in the lungs is thought to
be the
alveoli, which are membranous sacs at the end of small passages called
bronchioles.
The surfactant allows the alveoli to expand and fill with air in response to
the
expansion of the chest wall which is mediated by smooth muscle. Surfactant
also
mediates O2-CO2 gas exchange across the mucosal fluid layer and alveolar
membranes. The smooth muscle components of the lungs, i.e., the endothelial
cell
layers of bronchi, bronchioles, blood vessels, and capillaries are not known
to be
affected by surfactant.
The effect of uteroglobin on pulmonary compliance and serum protein leakage
into BAL fluids in piglets indicate that a second mechanism of action for
uteroglobin
is at worlc, and one that is distinct from the inhibition of PLAZ-mediated
digestion of
surfactant phospholipids. First, the observation that piglets ventilated with
100%
oxygen that received uteroglobin had normalized lung compliance in comparison
to
those similarly ventilated but not administered uteroglobin indicates that
uteroglobin
mediates an entirely unanticipated effect on the lungs that is distinct from
its
protective effects on exogenous surfactant. Further, there was no significant
difference in surfactant function in BAL of animals that did and did not
receive
uteroglobin. This shows that the uteroglobin-mediated protection of surfactant
phospholipids from digestion by soluble, secreted PLA2s did not persist 48
hours after
administration in this model. In contrast, the uteroglobin-mediated effect on
pulmonary compliance did persist 4~ hours after administration. Thus, at the
4~-hour
endpoint, surfactant function did not correlate with the uteroglobin-mediated
differences in pulmonary compliance. Therefore, uteroglobin mediates a
surfactant-
independent effect on pulmonary compliance. In accordance with these
observations,
uteroglobin could be described as a bronchodilator.
Because surfactant improves pulmonary compliance primarily by making the
alveolar sacks moxe elastic, it follows that uteroglobin affects the
flexibility of the
other primary structures involved in pulmonary compliance, i.e., the bronchi
and
bronchioles. Bronchi and bronchioles are composed of three main cellular
layers: the


CA 02405946 2002-10-15
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surface epithelia, the stroma, and the endothelia. The endothelial layers
contain the
smooth muscle responsible for the changes in volumetric capacity of the
bronchi and
broncluoles. Therefore, uteroglobin most likely affects pulmonary compliance
by
increasing the ability of smooth muscle to expand and contract. This
explanation is
5 supported by the observation that the protein content of the BAL of
uteroglobin-
treated piglets is significantly lower than that of untreated piglets. The
source of
excess protein in BAL fluids is generally the serum. The amount of protein
that leaks
from the serum into the BAL depends upon local vascular permeability. Pro-
inflammatory treatments to the lungs, such as 100% oxygen exposure, generally
10 increase vascular permeability, resulting in excess protein in BAL fluids.
The
administration of uteroglobin countered this effect. Vasculax permeability is,
in part,
dependent upon the degree of smooth muscle contraction in blood vessels and
smooth
muscle contraction is controlled by the autonomic nerve system.
Fibronectin type III repeats are found in structural proteins of the
musculature,
15 such as collagen, titins, and tenascins, as well as in several cellular
adhesion
molecules, such as ICAM-1 (intercellular cell adhesion molecule), LFA
(leukocyte
function associated antigens), VCAM-1 (vascular cell adhesion molecule), and
NCAMs (neural cell adhesion molecules).
There are 17 type III repeats in fibronectin that can form intramolecular and
20 intermolecular bridges and their formation may be calcium dependent. Thus,
the type
III repeats can act like building blocks that fit together. When fibronectin
mediates
fibrillogenesis, these repeats may interact via hydrophobic and ionic
interactions,
acting like beads on a strand that can interlock and align. The strings may be
locked
into threads by the action of transglutaminase which can covalently crosslink
the
25 fibronectin strands together and build the fibril. Therefore, one can
assume that
fibronectin type III repeats in molecules other than fibronectin may also
interact non-
covalently to affect communication. For example, the interaction between
fibronectin
type III repeats in neural cell adhesion molecules and fibronectin type III
repeats in
muscle structural proteins like collagen, titins, and tenascins, could help to
trigger the
signal for muscle contraction. Alignment of the fibronectin type III repeats,
therefore,
provides a physical connection between nerve and muscle, and interactions
between


CA 02405946 2002-10-15
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26
fibronectin type III repeats may be important to the normal function of the
lungs. The
enhanced pulmonary compliance in uteroglobin-treated piglets suggests that
uteroglobin binds fibronectin type III repeats and may block the alignment of
type III
repeats in both nerve and muscle in vivo, perhaps regulating the degree of
muscle
contraction by sterically blocking the alignment of excess fibronectin type
III repeats,
in muscle tissue and at the interface of muscle tissue and neural tissue.
Preferred Routes of Administration and Formulations for Utero~lobin
Uteroglobin may be administered either alone or in combination with other
active agents or compositions typically used in the treatment or prevention of
the
above-identified disease conditions. Such active agents or compositions
include, but
are not limited to steroids, non-steroidal anti-inflammatories drugs.
(NSAIDs),
chemotherapeutics, analgesics, immunotherapeutics, antiviral agents,
antifungal
agents, vaccines, immunosuppressants, hematopoietic growth factors, hormones,
cytokines, antibodies, antithrombotics, cardiovascular drugs, or fertility
drugs. Also
included are vaccines, oral tolerance drugs, vitamins and minerals.
Uteroglobin may be administered to target a uteroglobin-receptor. Targeting
of a uteroglobin receptor refers to inducing specific binding of a ligand to a
receptor
to mediate effects on cell growth and/or activity.
As discussed above, the data show that recombinant human uteroglobin may
be administered systemically via the lungs, for the purpose of raising
circulating
levels of the protein, to deliver the protein to tissues and organs, and to
raise the
concentration of uteroglobin in the urine. Therefore, one can treat various
internal
organs and tissues, including the vasculature, muscle, connective tissue,
bone, blood
cells, stomach, lcidneys, pancreas, liver, intestines, colon, heart, spleen,
thymus,
uterus, and bladder, by administering uteroglobin topically to the lungs
through
intratracheal deposition or through an inhaler or nebulizer. Further, the data
also
show that intravenous, intranasal, and stomach gavage administration of
uteroglobin
are practical for the systemic administration of the protein in humans.
Uteroglobin may be administered intravenously or, in the case of treatment of
neonatal RDSBPD and adult RDS, in the form of a liquid or semi-aerosol via the


CA 02405946 2002-10-15
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27
intratracheal tube. Other viable routes of admiustration include topical,
ocular,
dermal, transdermal, anal, systemic, intramuscular, subcutaneous, slow
release, oral,
vaginal, intraduodenal, intraperitoneal, and intracolonic. Such compositions
can be
administered to a subject or patient in need of such administration in dosages
and by
techniques well known to those skilled in the medical, nutritional or
veterinary arts
taking into consideration such factors as the age, sex, weight, and condition
of the
particular subject or patient, and the route of administration. The
compositions of the
present invention may also be administered in a controlled-release
formulation. The
compositions can be co-administered or sequentially administered with other
active
agents, again, taping into consideration such factors as the age, sex, weight,
and
condition of the particular subject or patient, and, the route of
administration.
Further, the data show that recombinant human uteroglobin may be
administered systemically via the digestive tract (orally), for the purpose of
raising
circulating levels of the protein, to deliver the protein to tissues and
organs, and to
raise the concentration of uteroglobin in the urine. Uteroglobin could be
formulated
with a gel or matrix for sustained delivery of a high localized dose, such as
at the site
of a surgical procedure, particulary vascular surgery to prevent scarring,
fibrosis, and
reclosing of the arteries. Therefore, one can treat various internal organs
and tissues,
including the lungs, vasculature, muscle, connective tissue, bone, blood
cells,
stomach, kidneys, pancreas, liver, intestines, colon, heart, spleen, thymus,
uterus,
prostate, and bladder by administering uteroglobin topically to the lungs
through a
drink, pill, nutraceutical, or suppository.
Examples of compositions of the invention include edible compositions for
oral administration such as solid or liquid formulations, for instance,
capsules, tablets,
pills, and the like liquid preparations for orifice, e.g., oral, nasal, anal,
vaginal etc.,
formulation such as suspensions, syrups or elixirs; and, preparations for
parenteral,
subcutaneous, intradermal, intramuscular or intravenous administration (e.g.,
injectable administration), such as sterile suspensions or emulsions. However,
the
active ingredient in the compositions may complex with proteins such that when
administered into the bloodstream, clotting may occur due to precipitation of
blood
proteins; and, the skilled artisan should take this into account.


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28
In such compositions uteroglobin may be in admixture with a suitable carrier,
diluent, or excipient such as sterile water, physiological saline, glucose,
DMSO,
ethanol, or the like. Uteroglobin could be provided in lyophilized form for
reconstituting, for instance, in isotonic aqueous, saline, glucose, or DMSO
buffer. In
certain saline solutions, some precipitation of recombinant human uteroglobin
has
been observed; and this observation may be employed as a means to isolate
inventive
compounds, e.g., by a "salting out" procedure.
Further, the invention also comprehends a kit wherein uteroglobin is provided.
The kit can include a separate container containing a suitable carrier,
diluent or
excipient. The kit can include an additional agent which reduces or alleviates
the ill
effects of the above-identified conditions foi- co- or sequential-
administration. The
additional agents) can be provided in separate containers) or in admixture
with
uteroglobin. Additionally, the kit can include instructions for mixing or
combining
ingredients and/or administration.
EXAMPLES
The invention will now be further described with reference to the following
non-limiting examples. Parts and percentages are by weight unless otherwise
stated.


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29
Example 1
Recombinant human uteroglobin was administered to several mammalian
species via several routes of administration to determine the safety and
biological
activity of the protein. The protein was given to rats in order to assess
pharmacokinetics, bioavailability, and tissue distribution when administered
intravenously, intranasally, and by stomach gavage. It was also given
intratracheally
to very young animals of three large animal species, including premature
baboons,
premature lambs and newborn piglets. The biological activity of recombinant
human
uteroglobin and its effect on various aspects of lung function was evaluated
in these
animal studies. The concentrations of recombinant human uteroglobin in all
species
were determined using an ELISA assay that is specific for human uteroglobin.
a. Purification of Recombinant Human Uteroglobin
Recombinant human uteroglobin was cloned and expressed by a method
similar to that described in copending U.S. application serial no. 08/864,357.
Protein
was purified by proprietary method under FDA guidelines for use as a
pharmaceutical
agent.
In the alternative, the protein was extracted from the E coli cell paste by
high
pressure shear and clarified by centrifugation. The first crude fraction was
separated
by tangential flow filtration at 100,000 daltons, followed by chromatography
on anion
exchange and hydroxyapatite supports. The purified protein was further
purified by
tangential flow filtration at 30,000 daltons.
b. ELISA for uteroglobin
A quantitative competitive ELISA was developed that provides the necessary
sensitivity and precision required for measurement of recombinant human
uteroglobin
in human samples for use in clinical trials. The assay utilizes a rabbit
polyclonal anti-
human urine protein-1 (uteroglobin) antibody obtained from Dako, USA. Coating


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and blocking conditions, antibody dilution, and the type of microtiter plate
have been
optimized for signal reproducibility and for stability in storage.
Antibody (supplied as a 2mg/ml stock solution) was used to coat the wells of
microtiter plates at a 1:2,500 dilution in a O.1M carbonate/bicarbonate
buffer, pH 9.5.
5 Pipetting of all reagents into microtiter wells was done with an 8-or 12-
multichannel
pipetter. The diluted antibody solution was pitpetted into the wells of Nunc
Maxisorp
strip plates (100 microliters/well), then the plate strips was sealed in
Ziploc bags, and
incubated overnight at room temperature (18-25°C). The next day, the
coating
solution was removed from each well by aspiration and 200 microliters of
blocking
10 buffer (5% sucrose, 5% Bovine serum albumin in phosphate buffered saline)
was
added to each well. Plate strips were placed back into Ziploc bag and onto a
benchtop
rotator and rotated gently for 2 hours at room temperature. After the two hour
blocl~ing step, the contents of all wells were aspirated and the plate strips
were placed
upside down in a biosafety cabinet with the fan on, to dry for two hours. Dry
plates
15 were stored at 4°C until ready to use for the assay. Plates prepared
in this manner are
stable for at least ten weeks.
In this competitive immunoassay format, the anti-uteroglobin antibody is used
as the capture reagent for any uteroglobin in the sample. A conjugate of horse
radish
peroxidase (HRP) to recombinant human uteroglobin was generated using a Pierce
20 HRP-labelling kit. The uteroglobin-HRP conjugate bound to the anti-
uteroglobin
antibody coating the wells, and generated a signal (A49o) proportional to the
amount of
HRP-uteroglobin conjugate bound. Signal was generated using a standard HRP
enzymatic colorimetric reaction (Pierce OPD substrate). An optimized amount of
the
uteroglobin-HRP conjugate was mixed with a sample to be assayed (which may be
25 pre-diluted with PBS, if necessary). Typically, two different dilutions
(1:2-3 and
1:10) of each sample were run in duplicate, requiring ~ 100 microliters of
sample.
The assay thus revealed a decrease in signal as the uteroglobin in the sample
competes
with the uteroglobin-HRP conjugate for antibody binding sites, as shown in
Figure 1.
A standard curve, using carefully quantitated recombinant human uteroglobin
30 calibrators, was always run in duplicate with each set of samples to assess
reproducibility and quantitate uteroglobin in samples.


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31
The uteroglobin ELISA involves a single antibody binding step to capture
uteroglobin antigens in each sample. Therefore, the sample must be pre-mixed
with
the uteroglobin-HRP in an untreated microtiter dish, prior to addition to
antibody-
coated wells. A 110 microliter volume of recombinant human uteroglobin
calibrator,
pre-diluted sample or control (PBS only) was mixed with 110 microliter of the
uteroglobin-HRP conjugate (1:200 dilution in PBS of 1 mg/ml stock solution) in
appropriate wells of a pre-labeled microtiter dish). Once the samples were
prepared,
the entire 220 microliters was transferred to labelled antibody-coated wells.
The
microtiter plate strips were incubated at room temperature for 60-75 minutes
on a
benchtop rotator to allow itteroglobin antigen to bind to the wells. Plates
were then
washed three times using a microplate washer (Biotek Instruments model) with
0.05%
Tween-20 in PBS. The HRP substrate (Pierce OPD) was prepared according to the
manufacturer's instructions. Substrate (100 microliters) was then added to all
test
wells using a multi-channel pipetter and the plate was incubated for 30
minutes at
room temperature. Color development was stopped at 30 minutes by adding 50
microliters of 1.2N sulfuric acid to each well. Absorbance was read in a
microtiter
plate reader (Biotelc Instruments EL800) at a wavelength of 490 nm.
A uteroglobin standard curve, uteroglobin sample concentration and test
statistics were generated with KC4 software (Biotek Instruments) through a
direct
interface were between the microplate reader and PC computer. Coefficients of
variation (CV) in this assay between less than 8% at concentrations down to 25
ng/ml
and were generally lower than 15% between concentrations of 1-25 ng/ml with
recombinant human uteroglobin calibrators and in human sera.
c. Intratracheal Administration of Recombinant
Human Uteroglobin to Pre-Term Lambs
This study used neonatal lambs delivered by cesarean section as a model of
surfactant-dependent neonatal respiratory distress syndrome (RDS). The primary
objective of the study was to determine whether the administration of
recombinant
human uteroglobin would in any way decrease the benefit of surfactant
administration
in these animals. Since uteroglobin is known to bind to phospholipids, it
could


CA 02405946 2002-10-15
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32
thereby interfere with the action of surfactant, which is mainly composed of
phospholipids. The severity of RDS in the animals required very aggressive
ventilation by human clinical standards, and thus provided a sensitive
indication of
any effects of recombinant human uteroglobin on the action of surfactant in
RDS
rescue.
Four lambs were delivered between 135-138 days of gestation by cesarean
section and were in respiratory distress at birth. One dose of surfactant was
administered after each animal was stabilized on ventilation, and 15 minutes
later
approximately Smg/kg recombinant human uteroglobin (in two animals) or control
vehicle (in the remaining two animals) was administered.
Although all animals were of similar gestational age, body weight ranged from
2.6 to 3.6 kg, and condition at birth ranged from mild to severe respiratory
distress.
The three lowest weight animals (#1, #2 and #4) had severe or very severe RDS,
while animal #3 had milder RDS as judged from the initial chest x-ray. This
was also
reflected in the pre-treatment blood oxygenation (pa02), which was
considerably
better in animal #3 than in the other three animals. Animal #4 was exceptional
in that
it was born with very severe meconium aspiration. The amniotic fluid was a
thick
brown consistency and tracheal aspirates from this animal were also brown and
very
viscous. There were no apparent physical defects but the animal was not active
prior
to sedation. Chest X-ray showed that the animal had the most severe
respiratory
distress, with mostly opaque lungs. The two animals treated with recombinant
human
uteroglobin had the most severe RDS, despite the fact that one of them had
severe
meconium aspiration with mostly opaque lungs.
All animals received the same surfactant treatment, except that animal #1
received only one dose of surfactant (shortly after birth), whereas the others
received
two (the first shortly after birth and the second six hours later). Animal #1
required
very high ventilator pressure (PIP > 50) to maintain blood oxygenation and
developed
pneumothorax (confirmed by chest X-ray) at 10.5 hours after delivery. When
final
fluid samples were taken the animal was euthanized. After treatment of this
animal,
the protocol was altered to provide for a second dose of surfactant and
recombinant
human uteroglobin six hours after the first doses in accordance with
prescribing


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33
information for Survanta. The treated animals were #2 (5 mg/kg x 2) and #4
(6.25
mg/kg x 2). Animals #1 and #3 did not receive the study drug; animal #1
received no
treatment and animal #3 received a volume of normal saline equal to the volume
of
recombinant human uteroglobin solution given to the treated animals (See Table
1
below).
All four animals responded as anticipated to surfactant with increases in
pa02/Fi02, decreases in paC02 and increases in blood pH and bicarbonate
excess,
BE(B). These data are shown in the Figures 2-5 representing blood values as a
function of time in each animal. The animal (#2) treated with recombinant
human
uteroglobin (but without meconium aspiration) showed the greatest response to
surfactant administration based on blood gases, even though it had more severe
respiratory distress than the animals not treated with recombinant human
uteroglobin.
The other animal treated with recombinant human uteroglobin, which had
meconium
aspiration (#4), had significantly delayed responses in pa02 relative to the
other
animals (about 180 minutes versus 15-30 minutes), but eventually responded
better
than the untreated animals. The two treated lambs also developed a higher
blood pH
relative to the untreated animals, and were the only animals in the study to
reach a
positive bicarbonate excess. None of the animals received electrolytes or any
fluids
that might have resulted in induction of a metabolic alkalosis, so the
increases in
blood pH and bicarbonate excess were due solely to improvements in pulmonary
function mediated by recombinant human uteroglobin.


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34
Table 1. Summar
of Animals
Treated


Single/twin Animal #1 Animal #2 Animal #3 Animal #4
Sin 1e Twin (with Twin (with Twin (other
#3 #2) dead)


Sex Male female Female Female


Gest. A a (da I34 136 ~ 136 135
s


Wei t k ) 3.1 2.7 _ 2.6
~ 3.6


Condition at Vigorous SevereActive SevereActive Mild Not active
birth RDS RDS RDS Severe
RDS


Amniotic fluidClear Clear Clear Meconium


Pre-treatment 50 80 160 90
Paoz
(Tory)


Pre-treatment Significant Significant Mostly clear Mostly opaque
chest x- opacity opacity Good InflationPoor Inflation
ra Poor inflationPoor inflation


Survanta @ 200 mg 200 mg 200 mg 200 mg
birth (65 m /kg) (74 mg/k ) (55 mg/k ') (77m c )


@6 hrs Not treated 200 mg 200 mg 200 mg
(74 m /k ) (55 m ) (77 m )


Recombinant Not treated 6.25 m /k 0 m ' k (saline)5.0 m lc
human


uteroglobin Not treated 6.25 mg/kg 0 mg/kg (saline)5.0 mg/kg
*@ birth
@ 6 hrs


Post-treatment200 @ 120 280 @ 100 380 @ 100 min.450 @ 300
Pao2 mini min. min.
(Torr)


Survival 11 hrs 12 hrs 12 hrs 12 hrs


Cause of deathPneumothorax Euthanized Euthanized Ethuanized


It is clear from this data that recombinant human uteroglobin did not
interfere
with surfactant replacement therapy and is thus safe to use during surfactant
rescue
therapy. The animal with severe meconium aspiration responded remarkably well
with respect to blood oxygenation and pH, indicators of improved lung
function, in
the presence of recombinant human uteroglobin. Meconium is known to inactivate
surfactant and this animal did not respond to the exogenous surfactant in the
normal
timeframe of 15-30 minutes, as did the other animals. Therefore, the delayed
blood
gas responses that each occurred about 180 minutes after each administration
of
surfactant and recombinant human uteroglobin can be attributed to an
independent
action of recombinant human uteroglobin on the lung tissue. This is the first
observation of the direct effect of uteroglobin on lung tissue.


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Lambs were treated and monitored for twelve hours post-delivery. Bodily
fluid samples were taken from each animal for analysis of uteroglobin
concentration
by ELISA as listed in Table 2 below.
5 Table 2; Samples for Utero~lobin Pharmacokinetics
Serum Plasma Tracheal As Urine
irate


Pre-TreatmentX X X


2 Hours X


6 Hours X X X


12 Hours X X


At death, necropsy tissue specimens focusing on lungs were examined for
gross pathology and preserved in formalin for histopathological analysis.
Animals
treated with recombinant human uteroglobin showed no evidence of drug-related
10 toxicity during treatment. Tissues and organs isolated from the animals
showed no
gross or microscopic abnormalities resulting from recombinant uteroglobin
administration. Therefore, intratracheal administration of recombinant human
uteroglobin in at least two separate doses of up to 6.25 mg/kg was safe and
non-toxic.
The concentration of recombinant human uteroglobin in the lamb fluids was
15 quantitated by uteroglobin ELISA and results are shown in Table 3 below.
The
. recombinant protein followed the same pattern of distribution in bodily
fluids as the
native protein in humans. That is, it was taken up from the site of
administration to
the extracellular lung fluids into the blood, and was excreted in the urine.
Therefore,
the recombinant human protein behaves the same way ivy vivo as does the native
20 protein.


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36
Table 3. Recombinant
Human Uteroglobin
Concentration in
Bodily Fluids
n lmL


Sam 1e Animal #1 Animal #2 Animal #3 Animal
#4


Tracheal As irates


Pretreatment <5 <5 <5 47


1-2 Hrs Post-treatmentnd >68,200 10 19,400


2-4 Hrs Post-treatment<5 >68,200 I1 nd .


4-6 Hrs Post-treatmentnd nd nd 11,900


6-8 Hrs Post-treatmentnd >68,200 nd nd


8-10 Hrs Post-treatmentnd 13,000 nd 58,200


Plasma


Pretreatment nd <5 <5 <5


6 Hrs Post-treatment <5 4,090 <5 584


8 Hrs Post-treatment nd nd nd 2,932


12 Hrs Post-treatment<5 3,795 <5 1,964


Serum


Pretreatment <5 <5 <5 nd


2 Hrs Post-treatment nd nd nd 7_23


6 H'rs Post-treatment<5 3,455 <5 447


8 Hrs Post-treatment nd nd nd 1,730


12 Hrs Post-treatment<5 3,758 <5 1,563


Mother of animal nd nd 5.3 <5


Urine


Pretreatment nd 5.3 <5 8


6 Hrs Post-treatment nd 31 <5 nd


Post-mortem (12 Hrs) <5 27 <5 121


nd=not done



The bioavailability of intratracheal recombinant human uteroglobin was
excellent. The recombinant human uteroglobin-treated animals showed 12-60
~,g/mL
recombinant human uteroglobin in tracheal aspirates at two hours after the
first
recombinant human uteroglobin administration, 0.5-4.0 ~.g/mL in serum and
plasma
that peaked at four hours after the first recombinant human uteroglobin
administration, and 5-100 ng/mL in urine at ten hours after the first dose of
recombinant human uteroglobin. This demonstrates that recombinant human
uteroglobin can be administered systemically via the lungs, for the purpose of
raising
circulating levels of the protein, for the purpose of delivering recombinant
human
uteroglobin to tissues and organs, and for the purpose of raising the
concentration of


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37
uteroglobin in the urine. Thus, it is possible to treat various internal
organs and
tissues, including the vasculature, muscle, connective tissue, bone, blood
cells,
stomach, kidneys, pancreas, liver, intestines, colon, heart, spleen, thymus,
ureters and
bladder, etc. by administering uteroglobin topically to the lungs by
intratracheal
deposition or through an inhaler device or nebulizer.
d. Intratracheal administration of Recombinant Human
Uteroglobin to newborn piglets ventilated for 48 hours
The ventilated newborn piglet was selected for this study because it is a well
characterized model of neonatal lung injury. While the newborn piglet is not a
surfactant dependent model, it is an excellent model for neonatal lung injury
mediated
by oxygen toxicity arising from the use of positive pressure ventilation and
elevated
oxygen delivery in RDS rescue. Significant decreases in pulmonary compliance,
as
well as increases in inflammatory markers, indicative of pulmonary
inflammation, are
observed within 48 hours in this model. Although the model is not surfactant
dependent, it is quite responsive to the administration of exogenous
surfactant. Thus,
the linkage between increased pulmonary inflammation and decreased pulmonary
mechanical function that occurs in human neonates who develop chronic lung
disease
is preserved in this model. Further, direct injury to pulmonary surfactant is
measured
by analyzing the surface tension properties of surfactant collected by BAL
from the
lungs of treated and untreated animals. Therefore, this model is well suited
to the
evaluation of the effect of uteroglobin on lung function and safety of
intratracheal
recombinant human uteroglobin for the treatment of RDS and the prevention of
chronic lung disease in ventilated human neonates.
In this study, piglets were treated with combinations of artificial surfactant
and
recombinant human uteroglobin or control vehicle and then ventilated with
either
room air or 100% oxygen for 48 hours. Exogenous surfactant consisted of
Survanta
(Ross Labs) given intratracheally, in a single dose, via the endotracheal tube
and at
the recommended human dosage of 100 mg/kg. Recombinant human uteroglobin,
formulated in sterile saline (0.9%), was given in a single intratracheal dose,
within 30


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38
minutes following the surfactant and at concentrations of 1, 5, and 25 mg/kg.
Control
groups received comparable volumes of sterile saline only.
A total of 55 newborn piglets were sedated and ventilated for 48 hours with
either room air or 100% oxygen, according to Davis, et al. (1993) as shown in
Table
4. Twenty-one piglets were ventilated with room air and thirty-four were
ventilated
with 100% oxygen. Human neonates in respiratory distress always receive
supplemental oxygen but it is often less than 100%, depending upon the degree
of
severity of the RDS and the medical practitioners' individual approach to
ventilation
management. Therefore, the use of room air and 100% oxygen allows the
comparison
of treatment extremes in this safety evaluation.
Tabl e 4. Grou s in the
48 Hour Stud


Recombinant human Ventilated with Room Ventilated with 100%
uteroglobin dose group Air ~ Oxygen


Control N = 4 N = 4


Control vehicle + SurvantaN = 5 ~ N = 5


1 mg/kg recombinant humanN = 4 N = 6
utero lobin + Survanta


5 mg/kg recombinant humanN = 4 N = 12
utero lobin + Survanta


25 mg/kg recombinant N = 4 N = 7
human
utero lobin + Survanta


i. Pharmacokinetics of Recombinant Human
Uteroglobin in newborn piglets
In humans, a considerable body of evidence indicates that endogenous
uteroglobin is produced in the pulmonary and tracheal epithelia, enters the
blood by
an unknown mechanism, and is eliminated from the blood via the kidney. Three
types
of fluid samples were collected in order to monitor the half life and
elimination of
recombinant human uteroglobin following intratracheal administration. The
first type
was broncho-alveolar lavage fluid (BAL) collected at 48 hours, after
sacrifice, lung
excision and pulmonary function testing. This is as close to the site of
administration
as was practical to sample. The second type was serum collected before
treatment


CA 02405946 2002-10-15
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' 39
and at 2, 4, 8, 12, 24, 36, and 48 hours after administration, from which an
estimate of
circulating half life can be made. The third type of sample was urine,
collected on the
same schedule as serum. Overall, the distribution of recombinant human
uteroglobin
in piglet fluid was consistent with the distribution of endogenous uteroglobin
in
humans.
The concentration of recombinant human uteroglobin was measured in BAL,
sera, and urine using the competitive ELISA described above. Only three sets
of sera
from seven untreated piglets were tested in order to establish the background
level for
the uteroglobin ELISA. The four remaining sets were not tested in the
interests of
conserving valuable ELISA reagents. Likewise, not all urine samples from
untreated
piglets were tested. Background immunoreactivity for the uteroglobin ELISA in
serum ranged from undetectable to about 100 nanograms/ml and the highest
background level in urine was 26 nanograms/ml.
The serum data were fairly consistent among the eight piglets dosed with
recombinant human uteroglobin. Recombinant human uteroglobin was detected in
all
animals at the two hour timepoint. The recombinant human uteroglobin level
peaked
between two and eight hours post-administration. This shows that either the
distribution of the drug in the lungs was variable or that the ability of the
lungs to
convey recombinant human uteroglobin to' the blood was variable, or both. Peak
serum levels of 4-17 ~.g/mL (mean 8.6~6.1 ~.g/mL) were measured within 2-8
hours
of drug administration. Elimination of recombinant human uteroglobin from the
serum corresponded well to first-order kinetics between 8 and 48 hours after
drug
administration (R2=0.97) with a half life of 7.9 hours. The half life of
uteroglobin in
humans has not yet been accurately assessed. At 48 hours, serum uteroglobin
levels
were still elevated (0.24~0.16 ~.g/mL) relative to control animals, in which
uteroglobin was usually less than the assay detection limit of approximately
0.01 ~,g/mL Data showing the concentration of recombinant human uteroglobin in
serum as a function of time after intratracheal administration are shown in
Figure 6A.
A comparison of the urine recombinant human uteroglobin concentrations in
the treated animals in the 100% oxygen group versus the room air groups shows
that


CA 02405946 2002-10-15
WO 01/79285 PCT/USO1/12126
there may be a difference in the renal handling of circulating recombinant
human
uteroglobin. Animals #7, 31 and 42 seemed to excrete a much greater amount of
recombinant hmnan uteroglobin in their urine during the study period than did
animals #12, 39, and 44. Piglet #22 appears to have been dehydrated since it
did not
5 produce much urine during the study period, was noted to have bloating and
diarrhea
within the first twelve hours of the study period, and the investigators noted
difficulties extracting blood samples from the animal. It is not known whether
this
apparent difference in the renal handling of recombinant human uteroglobin
reflects a
difference in the molecular form of recombinant human uteroglobin or a
difference in
10 the kidneys' ability to process recombinant human uteroglobin. The kidney
is known
to respond to the level of oxygen in the blood and is part of the homeostatic
regulatory
system, with potential feedback mechanisms to the lungs. An example of an
altered
molecular form of recombinant human uteroglobin that affects renal handling
may
involve complexing to a high molecular weight protein like fibronectin, which
does
15 not pass through the renal glomeruli. When homodimeric uteroglobin passes
through
the glomeruli, it is thought to be reabsorbed by the tubules, such that there
is
approximately a twenty to one hundred-fold steady state difference between the
circulating concentration and the urine concentration in normal humans.
A considerable quantity of recombinant human uteroglobin also remained in
20 the BAL fluid after 48 hours. The background level in the BAL of untreated
piglets
was about 200 nanograrns/ml. This relatively high level of background was
probably
due to cross-reactivity with endogenous porcine uteroglobin or some other
protein(s),
as well as the matrix effects of this particular fluid in the uteroglobin
ELISA. The
BAL concentrations in treated piglets ranged from background levels of about
200
25 nanograms/ml (piglet #42) to over 16 micrograms/ml (piglet #31). The
concentration
of uteroglobin immunoreactivity in BAL from normal adult humans was 3-8
micrograms/ml with occasional concentrations up to 25 micrograms/ml in
asymptomatic normal adults.
It is clear that a significant amount of recombinant human uteroglobin,
30 remains in the extracellular lung fluids for an extended period of time
(ie. two days).
In particular, piglets #31 and #44 had significantly higher recombinant human


CA 02405946 2002-10-15
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41
uteroglobin in BAL than the rest of the treated piglets. The reason for this
is not
understood but there is no indication from either the lung pathology or the
pulmonary
function tests that these animals suffered any toxic effects as a result of
high
recombinant human uteroglobin immunoreactivity remaining in their lung fluids.
These results also indicate that recombinant form of human uterglobin is most
likely
utilized and processed by the same pathways as the endogenous protein in the
lungs,
kidneys and circulatory system, based on parallels with published adult human
data.
Further, there is no indication that there is a significant difference between
the
phannacokinetics of recombinant human uteroglobin in the newborn piglet versus
that
of endogenous uteroglobin in the adult human.
ii. Total Protein in BAL
A significant indicator of lung injury is the concentration of total protein
in
lung lavage fluids. Serum proteins are known to leak into extracellular lung
fluids
when the alveolar-capillary barrier is impaired and vascular permeability is
increased.
Total protein concentration in BAL samples was measured in each of the
animals.
The data are shown in Figure 6B. A major characteristic of this piglet model
of
oxygen-induced lung injury is nicely illustrated in that the room air (RA)
groups all
have lower protein content, and less injury, than the groups that received
100%
oxygen. There was also a significant difference in total protein in BAL
between the
treatment groups receiving surfactant with and without rhUG. The groups that
received rhUG all contained lower mean protein concentrations, indicating
lower
levels of lung injury and lower vascular permeability. Thus, rhUG had a
significant
effect on regulation of vascular permeability, perhaps as a result of
interactions with
the smooth muscle component of the vasculature, or perhaps as result of an
effect on
the nitric oxide signaling pathway. This observation was consistent with the
apparent
benefit of rhUG in pulmonary function tests, in that rhUG-treated animals
exhibited
better lung compliance that their untreated counterparts.
iii. Pulmonary function testing


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42
Pulmonary function testing was successfully performed on two thirds of the
animals studied, including at least three animals in each of the ten groups.
It was
apparent after completing the 0 and 25 mg/kg groups that CC10 conferred a
benefit.
The data are shown in the accompanying figures. Figure 7 shows the pressure-
s volume relationships. While most of the animals (5) fall into a single main
group, the
animals with lower pulmonary compliance (piglet #11 and #43) are both in the 0
-
100 group. Of the two animals (piglet #23 and #31) that are above the main
group
with better lung compliance, both were ventilated with room air, one received
recombinant human uteroglobin and one did not.
Figure 8 shows the mean pressure-volume relationships measured for animals
ventilated with room air (error bars indicate standard deviation). There was
no
significant difference in lung compliance between room air-ventilated animals
that did
and did not receive the study drug (p=0.90).
Figure 9 shows the mean pressure-volume relationships measured for all
animals ventilated with room air and 100% 02 (error bars indicate standard
deviation). There was clearly an improvement in mean pulmonary compliance in
animals that were treated with recombinant human uteroglobin compared to
saline
controls.
In this model, most of the pulmonary damage manifested as decreased
compliance is caused by hyperoxia rather than barotrauma. This effect can be
seen in
Figure 10A, in which the mean pulmonary compliance among all four groups is
compared. For animals not receiving the study drug, pulmonary compliance was
considerably lower in animals ventilated with 100% oxygen (0100) relative to
those
ventilated with room air (0 RA) (p=0.09). For animals receiving the study
drug,
however, pulmonary compliance was similar whether or not the animals were
ventilated with room air (25 R.A) or 100% oxygen (25 100). Thus, recombinant
human uteroglobin has countered the negative effects of 100% oxygen on
pulmonary
compliance.
Further analysis of the 1 mg/kg and 5 mg/kg rhUG dose groups showed that
rhUG produced a marked improvement in pulmonary compliance, elasticity and
distensibility in groups injured by ventilation with 100% oxygen. These
results are


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43
illustrated in Figure 10B. The most effective dose was 5 mg/lcg, but 1 mg/kg
and 25
mg/kg were also beneficial. This is an extremely important finding in terms of
the
clinical development of rhUG. We have uncovered a previously unsuspected
property
of rhUG in relaxing smooth muscle in the lung tissue and vasculature, in the
presence
of ventilation with oxygen. Inspired elevated oxygen is used clinically in
patients
with respiratory insufficiency, in acute respiratory distress syndrome, as
well as with
many chronic lung diseases that result in respiratory insufficiency, including
idiopathic pulmonary fibrosis, chronic obstructive lung disease, cystic
fibrosis, and
other lung diseases. Our results indicate that rhUG can be used to alleviate
breathing
difficulties in patients requiring elevated oxygen inspiration, and may offset
or reduce
the pulmonary toxicity due to the oxygen itself.
e. Long term effects of intratracheal administration
of Recombinant Human Uteroglobin to newborn piglets
Twenty newborn piglets were sedated, entubated and dosed according to the
groupings shown in the Table 5 below. They were then allowed to recover and
were
maintained for one month. (Animals were bottle-fed for the first two weeks.)
After
28 days the animals were sacrificed and necropsied for a full toxicological
evaluation.
Several analyses were performed on piglets samples as shown in the Table 6.
No evidence of toxicity was observed, even at the highest dose of recombinant
human
uteroglobin. There was no evidence of an anti-recombinant human uteroglobin
antibody production at 28 days. Recombinant human uteroglobin did not persist
in the
circulation of the animals for 28 days, consistent with the 7.9 hour half life
measured
in the 48 hour study. There was no long-term toxicity observed for
intratracheal
recombinant human uteroglobin administration in the newborn piglet.
Table 5
28 Dav Piglet Studv Groups
Number of Piglets Recombinant human Survanta dose
uteroglobin dose


4 0 mg/k 0 mg/k


4 0 m /kg 100 mg/kg


4 1 m /kg 100 mg/kg


4 5 mg/kg 100 mg/kg




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44
4 2S mg/kg 100 mg/k~
Table 6
Summary of Samples and Analyses for 28 Dav Piglet Study
Data or Sam 1e T pe of Analysis Pre-Rx 28 Days
Type


Whole blood CBC & Differentials X X


Sexum uteroglobin ELISA X X


Serum Anti-utero lobin antibodyX X
titer


Whole animal Overall rowth X X
weights


Necxo sy Gross atholo y X


Organs in formalin:Weights and histopathology X


Lungs (both)


Heart


Liver


Thyroid


Adrenals (both)


Spleen


Kidneys (both)


Brain


Lym h nodes


S
f. Piglet lymphocyte data
CBC (complete blood counts) and differential white counts were done in the
hospital lab on whole blood collected prior to uteroglobin administration and
at 28
days post-administration, immediately prior to sacrifice. The mean cell counts
before
rhUG treatment and at 28 days were calculated for each group, then the
differences
between the mean initial and final cell counts were calculated. There was a
significant difference in the changes in PMN (mature neutrophils) and
lymphocyte (T
cells, B cells, NK cells) cell counts between the animals receiving rhUG and
the
control groups. These data are shown in Figure l OC. (The control group that
did not
1 S receive either surfactant or rhUG was comparable to the surfactant only
group and is
not shown.)
The data show a powerful effect of a single dose of rhUG in the
newborn piglets. In the control group, the lymphocyte counts went up and the
PMN
counts went down over the one month period. This is probably normal in the
development of the immune system aftex birth. The rhUG significantly enhanced


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both of these changes. At a dose of 5 mg/kg, rhUG nearly doubled the increase
in
lymphocytes, and more than doubled the decrease in PMN. It is clear that the
rhUG
significantly enhances lymphocyte proliferation and decreases PMN production
over
the long term. It has been postulated that a hemopoietic stem cell exists that
can be
5 committed to either the myeloid or lymphoid lineage. Our data further
indicates that
such a stem cell exists and that recombinant human uteroglobin is the
differentiation
factor that channels the development of stem cell precursors from the PMN
lineage
(myeloid) to the lymphocyte lineage (lymphoid). This is a powerful
pharmacologic
effect and there is no known agent with equivalent activity. This discovery
enables
10 the application of rhUG to several clinical indications involving long term
imbalances
between myeloid and lymphoid cell counts, treating patients with an excess of
myeloid cells, or a deficiency of functional lymphoid cells. This mechanism,
for
example, may be important in maintaining immune surveillance, mediated by
lymphoid cells, in preventing or treating cancer. It will also be important in
15 controlling conditions characterized by an excess of myeloid cells, such as
asthma (an
excess of eosinophils), or leukemia.
g. Administration of recombinant human uteroglobin to Wistar rats
The primary purposes of the animal pharmacology and toxicology models
described in the Examples thusfar is to support the clinical trials of
recombinant
20 human uteroglobin in neonatal lung disease. However, they are also
applicable to
studies of adult lung disease and provide certain basic information about the
distribution, metabolism and excretion of the protein.
Uteroglobin is a naturally occurring mammalian protein for which there are no
known post-secretory modifications, with the possible exception of alterations
in
25 oxidation state, depending upon whether zero, one or two of the possible
disulfide
bonds between the monomers of the dimeric protein are present. Therefore, it
is very
unlikely that biotransformation is a consideration in the pharmacodynamics of
the
recombinant human uteroglobin drug. The main source of endogenous uteroglobin
protein in the body is the lungs and it is eliminated from the circulation by
the
30 kidneys. The tissue distribution or kinetics of elimination of recombinant
human


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46
uteroglobin from the body has not been determined when the recombinant protein
drug is administered in doses representing a much higher level than the
endogenous
uteroglobin circulating in the body. But once the normal, circulating
physiological
concentration is reached (about 150 nanograms/ml in serum), then the
elimination
kinetics for recombinant protein drugs can be expected to follow the pattern
of the
endogenous protein, provided the drug is biologically equivalent to the native
protein.
The steady-state level of endogenous uteroglobin in the blood is 80-150
nanograms/ml and 2-50 nanograms/ml in the urine of healthy individuals.
Bernard, et
al., showed that the kidney mediates the excretion of recombinant human
uteroglobin
from the blood into the urine by comparing serum and urine concentrations of
native
uteroglobin in normal healthy humans to those of patients with different types
of renal
impairment. The glomeruli of the kidney filter out small molecular weight
waste
products and proteins, with a size cutoff of approximately 40 kilodaltons.
Uteroglobin is a very compact globular protein with a Stokes radius of only
18.4
Angstroms, despite a molecular weight of 16 kilodaltons. Crystallization
studies
verify the very compact structure of this protein. Tn addition, uteroglobin
will pass
through a dialysis membrane with a molecular weight cutoff of 8.0 kilodaltons.
Patients with either glomerular or tubular disease had high urine
concentrations of
uteroglobin, which was comparable to that of the serum in patients with the
most
severe renal disease, and human albumin injected intravenously in rats was
reported
to competitively inhibit tubular reabsorption of uteroglobin. Together, these
results
indicate that the uteroglobin homodimer may pass through the renal glomeruli,
but is
selectively reabsorbed by the renal tubules prior to urinary excretion.
Therefore, a
significant proportion, as much as 30%, of the circulating endogenous
uteroglobin
may be removed by healthy kidneys and is excreted in the urine.
A pharmacokinetic study of recombinant human uteroglobin administration to
rats was undertaken to provide basic information on half life, tissue
distribution,
metabolism and excretion of the protein. A single dose of radiolabeled
recombinant
human uteroglobin was administered to a total of 12 Wistar rats by three
routes of
administration: intravenous, intranasal, and stomach gavage. Fluid samples
were


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47
taken over a period of 24 hours, after which the aiumals were euthanized and
dissected. Radioactivity was measured in all fluids and tissues.
Adult Wistar rats, ages 8-10 weeks, each received approximately 25 million
dpm of highly-purified 1~SI-labeled recombinant human uteroglobin (Lofstrand
Labs,
Inc.), corresponding to 1.9 ~,g of recombinant human uteroglobin or 13.6 ~.Ci
of l2sl
(specific activity: 7.18 ~.Ci/~,g protein). Four rats, two male and two
female, were
included in each of the three groups that received the lasI-labeled
recombinant human
uteroglobin by the three routes of administration. Each animal received 7.17 -
8.64
~,g/kg of recombinant human uteroglobin, corresponding to 51.3 - 61.6 ~.Ci/kg
of lasl.
Doses of recombinant human uteroglobin varied slightly between animals due to
differences in body weight. Animals were housed in metabolism cages so that
urine
and feces could be collected for analysis at the end of the 24-hour study
period.
Blood (200-300 ~,L) was collected at 1, 2, 4, 8, 12 and 24 hours after
administration
of the radiolabeled protein, and sera and plasma were prepared. The stomach
gavage
group were also sampled at 30 minutes after administration. Animals had free
access
to food and water throughout the study period. After final blood samples were
collected, animals were exsanguinated and necropsied. In addition to the blood
samples, 25 different tissue samples representing all major organs and tissues
were
collected. Samples were frozen at -80°C to preserve them for radiation
measurements
and protein extraction and analysis. Two animals died during the study, one
apparently due to blood loss and the other apparently due to handling trauma.
Neither
death appeared related to the study drug.
Fluid and tissue samples were counted in a gamma counter to determine the
amount of radioactivity present. Ten microliters of each sample of serum,
plasma and
urine were added to 3 mls of scintillation cocktail and counted for one minute
each.
The samples are analyzed using the competitive ELISA described above to
determine
concentrations of uteroglobin antigen in all fluid samples and protein
extracts of
tissues. Likewise, frozen organs were bisected and the intact half was placed
in a
scintillation vial containing 3 mls of scintillation cocktail and counted for
one minute
each.


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48
The remaining half of each organ sample was ground up in a 1.5 ml Eppendorf
tube, using a motorized pestle (Fisher brand) and the powdered sample was
resuspended in a protein extraction buffer (50 mM Tris-HCI, pH 7.6, 1% Triton
X-
100, 150 mM NaCI, 2 mM PMSF, 10 mM EDTA, 20 mglml each aprotinin, leupeptin,
and pepstatin A) and vortexed briefly to mix. The mixture was then centrifuged
in a
microfuge at 4°C for 15 minutes. The supernatant was transferred to a
clean tube and
the pellet was discarded. SDS-PAGE analysis was performed on the supernatants,
followed by autoradiogram to analyze the pattern of radioactive protein in the
tissue
samples. .
Figures 11-13 show radioactive counts as a function of time for each of the
three administration groups: intravenous, intranasal, and stomach gavage,
respectively. The amount of radioactivity in the blood decreases over time, as
expected, and appears to follow first-order elimination kinetics in the
intravenous
administration group. The elimination patterns in the intranasal and stomach
gavage
groups are not as distinct as that of the intravenous administration group,
presumably
due to absorption effects in the sinuses and gastrointestinal tract.
Radiation counts corresponding to recombinant human uteroglobin protein in
these samples was confirmed by analyzing them with the uteroglobin ELISA that
does
not recognize free iodine or degradation products. These data are shown in
Figures
14-16. In contrast to the radiation counts, there is a delay of 1-4 hours in
reaching the
peals uteroglobin concentration in the serum for the intranasal and oral
routes. These
data demonstrate that there is an uptake phase for uteroglobin across the
mucosal
surfaces of the stomach and upper respiratory tract. It is clear, however,
that both the
intranasal and oral routes of administration were effective in delivering a
significant
dose of the protein to the circulation. It was remarkable and unexpected that
these
routes would be so effective for uteroglobin administration. The nasal mucosa
is a
barrier between the external flora containing bacteria and viruses, as well as
inhaled
antigens, and the internal system. Likewise, the gut (via the stomach) is also
a
selective barrier that distiguishes between food molecules and other ingested
materials
that are not food. These barriers are generally not permeable to molecules as
large as
uteroglobin and the specific and efficient uptake shows that there is an
active or


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49
passive transport system that allows entry of uteroglobin in both the
respiratory tract
and the digestive tract.
I Results in the intranasal group are also consistent with pharmacokinetics
observed in both the neonatal lamb and piglet studies, in which uteroglobin
persists
for an extended period of time in the extracellular lung fluids. This
indicates that the
extracellular mucosal fluid of the mammalian respiratory tract may serve as a
reservoir from which the protein enters the blood. Inhalation may thus be a
practical
route for systemic administration of the protein in humans. The presence of
radioactive recombinant human uteroglobin homodimer in protein extracts of
trachea,
bronchi, esophagus, and thyroid in an animal from each administration route
show
that these tissues take up uteroglobin from the circulatory system. This
demonstrates
that one route may be effective in the specific delivery of protein to target
delivery for
another system. For example, the intravenous route may be used to deliver a
large
dose of uteroglobin specifically to the gut via the esophagus or to the lungs
via the
trachea. Likewise, inhaled uteroglobin may be used to deliver uteroglobin to
the gut
and kidneys and oral uteroglobin may be used to deliver the protein to the
lungs and
kidneys. In summary, the data indicate that recombinant human uteroglobin
enters
the circulation by all three routes of administration tested proving the
feasibility of
intranasal and oral uteroglobin administration in humans.
Example 2
Binding of UG to Fibronectin
The demonstration of the binding interaction between human fibronectin and
recombinant human uteroglobin prompted the development of a non-radioactive
assay
for this interaction that could be used as a measure of recombinant human
uteroglobin
biological activity. Therefore, two ELISA-based assay formats for the
uteroglobin-
fibronectin binding interaction were tested, as shown in Figures 17A-17B.
Briefly, in
the first of these assay methods recombinant human uteroglobin was used to
coat the
wells of a microtiter dish, which was followed by fibronectin binding and
detection of
the bound fibronectin with an anti-fibronectin monoclonal antibody (Life


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S0
Technologies, Inc.; product #12062-014). In the second assay, purified human
fibronectin was used to coat the wells of a rnicrotiter dish, followed by
recombinant
human uteroglobin binding and detection of the bound recombinant human
uteroglobin with an anti-uteroglobin antibody (Dako, USA). Both formats gave
comparable results with a 2-3 fold signal increase over controls when similar
concentrations of recombinant human uteroglobin and hFn were used. There being
no
apparent advantage of one format over the other, the second format was chosen
fox
further development and analyses due to the relative ease of reagent
availability. A
detailed description of the second ELISA assay follows.
Purified human fibronectin (hFn) was obtained as a frozen lyophil from Life
Technologies, Inc. (Gaithersburg, MD), product #33016-015. According to the
supplier's product specifications, this hFn preparation was purified from
human
plasma. The hFn was resuspended in sterile water to a concentration of 1
mg/ml.
This stock solution was then aliquoted and unused aliquots were frozen at -
80°C. An
aliquot of the 1 mg/ml stock solution was diluted to 10 micrograms/ml with
phosphate
buffered saline (PBS) (pH 7.4). Fifty microliters of the diluted hFn were used
to coat
wells of a microtiter plate (Falcon) with 5 micrograms/well. Further dilutions
were
made to generate a set of wells containing 1 microgram and 500 nanograms of
hFn as
well. The hFn coating was performed overnight at room temperature. After
coating,
the hFn was removed by aspiration and the wells were blocked with a 1:1
dilution of
Pierce blockerTM in PBS (pH 7.4), with a final concentration of 5% BSA in 1X
PBS
(pH 7.4). The blocking reagent was diluted immediately prior to the addition
of 320
microliters per well. The plate was incubated for two hours at room
temperature with
gentle shaking. The blocking reagent was removed by aspiration and the wells
were
washed three times with 1X PBS (pH 7.4).
Purified recombinant human uteroglobin, diluted from a 10 mg/ml stock
solution in PBS, was added to the wells in a constant volume of 50 microliters
but in
different concentrations, ranging from 1 ng/well to 1 microgram/well. After
the
addition of recombinant human uteroglobin or PBS controls, the plates were
gently
shaken for one hour at room temperature. The recombinant human uteroglobin was
then aspirated off and the wells were washed three times with PBS and blocked
again


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51
for an hour with undiluted Pierce blockerTM at room temperature. After re-
blocking
for an hour, the block buffer was removed by aspiration and the wells washed
three
times with 1X PBS, pH 7.4. Two hundred microliters of a 1:2,000 dilution of a
rabbit
polyclonal anti-human urine protein-1 (uteroglobin) antibody (Dako, USA) in
PBS,
were then added to each well. The plate was gently shaken for 1 hour at room
temperature, then the antibody solution was removed by aspiration the plate
was
washed three times with PBS, and blocked again for one hour as described
above.
A goat anti-rabbit IgG antibody, conjugated to horse radish peroxidase (HRP),
was used to quantitate the amount of anti-uteroglobin antibody in the wells.
Two
hundred microliters of a 1:20,000 dilution of the HRP-conjugate in PBS was
added to
each well and the plate was shaken gently for 1 hour at room temperature. The
conjugate solution was removed by aspiration and the plate was washed three
times
with PBS. A HRP substrate (Pierce ODP, made to the manufacturer's
instructions)
was added (320 microliters) to each well and the colorimetric reaction
proceeded fox
30 minutes. The HRP reaction was stopped at 30 minutes by pipetting 50
rnicroliters
of 1.2N sulfuric acid into each well using a mufti-channel pipetter. The
absorbance at
490 nm was read in a microtiter plate reader. In order to compare results in
separate
experiments, data was normalized by expressing the signal generated in
uteroglobin-
fibronectin test wells as a percent of the signal from fibronectin alone.
Fibronectin is a >200 kDa glycoprotein with three types of repeating elements,
called domains, which share a highly conserved secondary structure and a
moderately
conserved primary amino acid sequence. There are eight type I domains located
in
the N-terminal third of the human fibronectin protomer and three type I
domains at
the C-terminus of hFn. There are two type II domains, clustered in the middle
of the
protomer. There are between 15-17 type III domains in hFn, depending upon the
tissue of origin of the fibronectin. In plasma fibronectin, the circulating
type
produced by Life Technologies Inc. the liver, Type III domains EDA and EDB are
not
present.
An N-terminal proteolytic fragment of hFn was not available and could not be
tested. However, the 120 kDa and 40 kDa chymotryptic fragments of hFn
(obtained
from Life Technologies Inc.) encompass about 70% of the length of the intact


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52
fibronectin molecule, as shown in the map of fibronectin in Figure 18. Type
III
domains #1-11 are present in the 120 kDa chymotryptic fragment as shown in the
diagram. Type III domains #12-17 are present in the 40 kDa chymotryptic
fragment.
The clear dose-response curve for the 40 kDa chymotryptic fragment shows that
recombinant human uteroglobin binds to at least one other hFn type III domain
present in this fragment (#12-17). The recombinant type III domain #1
(referred to as
III.1) is found within the 120 kDa chymotryptic fragment.
Reports that recombinant human uteroglobin and human fibronectin form a
complex in solution with a measured binding constant of 13 nM and demonstrated
biological activity in vivo in mice indicate that an uteroglobin fibronectin
complex
could exist in vivo in humans. Indeed, high background readings for the
purified
human fibronectin using the anti-uteroglobin antibody (DAKO) in this
uteroglobin-
fibronectin binding assay led to speculation that there may be endogenous
native
uteroglobin that copurified with the fibronectin from human plasma. However,
this
solution phase interaction occurs at much lower concentrations of uteroglobin
and Fn
than the interaction seen between recombinant human uteroglobin and insoluble
fibronectin. In addition, the solution phase interaction was shown to be
relevant to
fibronectin polymerization, conversion to the insoluble form, the initial
fibronectin
deposition in the extracellular matrix or on cells, and in the process of
fibrillogenesis.
At low concentrations of plated fibronectin and recombinant human uteroglobin
no
interaction can be detected in this assay. On the basis of this apparent
difference, it
can be inferred that the downstream process of cell adhesion to the deposited
fibronectin during inflammatory cell and fibroblast migration may also be
effected by
the presence of uteroglobin. Therefore, the following hypotheses were tested
in the
experiment below: (1) is there a uteroglobin-like antigen in the intact hFn
preparation,
and (2) does recombinant human uteroglobin binds to portions of fibronectin
that are
important in cell adhesion and not relevant to fibrillogenesis.
Two commercially available chymotryptic fragments of hFn were selected for
experimentation. These fragments were more highly purified than the hFn,
having
gone through several stages of chromatographic purification after proteolytic
cleavage. These chymotryptic fragments axe referred to as the 120 kDa fragment
and


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53
the 40 kDa fragment, both type III domains that are involved in cell adhesion.
However, only the 120 kDa fragment contains the region of fibronectin required
for
polymerization. Fibronectin self polymerization activity has been localized to
an
N-terminal 70 kDa proteolytic fragment of human fibronectin, specifically a
region
including the type L9-type IIL1 domains. The C-terminal 70 amino acid part of
the
type III.1 domain was produced recombinantly (by Morla et al., 1994) and
dubbed
"Superfibronectin" because of its tremendous ability to promote
fibronectin-fibronectin interactions, polymerization, deposition, and cell
adhesion in
vitro. Superfibronectin also has the advantage that no endogenous human
uteroglobin
could possibly be present in the protein preparation because it was purified
from
bacteria.
The three pieces of fibronectin were tested in parallel with the intact
purified
human fibronectin. Five micrograms per well of each Fn species were tested as
described above, for binding to 250 nanograms of recombinant human
uteroglobin/well. Two such experiments were performed, in duplicate, and the
results
are shown in Figure I9.
In the absence of recombinant human uteroglobin the two chymotryptic
fragments and the recombinant type III domain, did not show an appreciable
signal.
Therefore, endogenous uteroglobin is present in the purified intact
fibronectin
preparation at an approximate concentration of 250 nanograms of uteroglobin
per 5
micrograms of hFn. This represents a molar ratio of 17:1, using molecular
weights of
16 kDa for the uteroglobin homodimer and 200 kDa for the fibronectin protomer.
Second, the binding of the recombinant human uteroglobin to both the 120 kDa
and
the 40 kDa fragments shows that there is more than one recombinant human
uteroglobin binding site in plated fibronectin. The binding of recombinant
human
uteroglobin to superfibronectin localizes this multiple binding phenomena to
the Fn
type III repeats, which are present in both the 120 kDa and the 40 kDa
fragments.
A titration of recombinant human uteroglobin was also done with each
fibronectin preparation, in parallel. All four of these preparations were used
to coat
plates as described, using one microgram of protein per well. Titration curves
were
done in which various amounts of recombinant human uteroglobin were bound to
the


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54
intact purified hFn, the two fragments and the recombinant superfibronectin as
shown
in Figures 20A and 20B. This experiment was done in duplicate and the
uteroglobin-fibronectin binding step was done in the presence of 2.7 mM
calcium
chloride, in 1X PBS (pH 7.4) (which is consistent with the normal
concentration of
calcium in the serum).
There are three conclusions to be drawn from these results: first, the clear
dose-response relationship in binding between recombinant human uteroglobin
and
superfibronectin shows that recombinant human uteroglobin binds to the domain
of
fibronectin which is also contained in superfibronectin; second, recombinant
human
uteroglobin binds to more than one Fn Type III domain, because there is a
clear
dose-response relationship for recombinant human uteroglobin binding to the 40
kDa
chymotryptic fragment, which does not contain the superfibronectin peptide
sequence,
i.e., the type IIL1 domain.
The Fn IIL 10 domain is known as the cell adhesion domain and it contains the
only "RGDS" cell adhesion motif present in the entire hFn molecule.
Mutagenesis of
this sequence to "RGES" severely diminishes adhesion of fibroblasts and
pro-inflammatory immune cells, such as neutrophils, to fibronectin. A
monoclonal
antibody against the cell adhesion domain, clone 3E3, is capable of blocking
cell
adhesion to fibronectin. Since recombinant human uteroglobin binds to the
insoluble
cell adhesion domain and to insoluble intact hFn and its chymotryptic
fragments, it
may be capable of blocking cell adhesion to fibronectin, via an anti-
inflammatory,
anti-fibrotic and anti-metastatic mechanism.
This hypothesis was tested in cell culture by assaying adhesion of NIH-3T3
cells (ATCC deposit # CRL-6589), an immortalized mouse fibroblast cell line,
to
hFn-coated wells (BioCoat plates, supplier). The average results from two of
experiments are shown in Table 7 below and clearly demonstrate that
recombinant
human uteroglobin is a potent inhibitor of cellular adhesion to fibronectin.
Table 7
Recombinant human ~ Anti-Fn mAb ~ Myoglobin
utero ~lobin I 0 a a 10 u,g


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5S
SOn


Inhibition 54% 60% 21


Clearly, the recombinant human uteroglobin was a potent inhibitor of cellular
adhesion to fibronectin. It was nearly as effective as the anti-Fn mAb, but at
a
200-fold lower concentration. Myoglobin is a protein that is thought to be
irrelevant to
the physiology of both uteroglobin and fibronectin and was selected as a non-
specific
protein control because it is also a circulating protein that is roughly the
same size as
uteroglobin.
Both the uteroglobin four helical bundle motif and the Fn Type III domain
represent fundamental protein structural motifs present in many different
proteins and
it is reasonable to infer that these motifs share a certain affinity for each
other,
independent of the context of each individual protein. In fact, all 17
isolated hFn
Type III domains mediate cell adhesion to some degree. It, therefore, follows
that it is
the availability of these domains for binding to other moieties in the
surrounding
environment that determines whether hFn interacts with other proteins, the
extracellular matrix, or with cells. Indeed, the conformation of hFn is known
to
change from an elongated disk-like globular shape in solution to a stretched-
out Y
shape upon deposition onto a surface (Erickson & Carrell, 1983). The Fn Type
III
domains are present in nearly all protein components of the extracellulax
matrix (e.g.,
laminin, collagens, vitronectin, fibrin) as well as numerous membrane bound
proteins,
including adhesion molecules, integrins, and receptors. This Fn type III
repeat
domain, as well as the four helical bundle, takes on a characteristic
secondary
structure that is thought to be highly conserved, despite differences in
primary amino
acid sequence.
Fibronectin Type III repeats are found in a large number of extracellular
matrix proteins, as well as in a number of cell surface receptors. Based on
their
distribution in certain proteins, these domains would seem to play an integral
role in
cell-cell and cell-extracellular matrix interactions. Our discovery that the
four helical
bundle of UG and the Fn Type III repeat domains broadly interact enables us to
more
rapidly identify proteins with these motifs that are likely to interact in
physiological


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56
and disease processes. As such, this discovery represents a powerful tool for
drug
discovery and the elucidation of pharmacologic pathways. For example,
secretory
phospholipases are proteins that contain four helical bundle motifs. Very
little is
known about how these enzymes act as signalling molecules. While some
receptors
have been identified, they do not appear to be the receptors through which
these
enzymes mediate some of their biological effects such as increasing cell
proliferation.
It is possible to identify novel candidate receptors for these proteins using
our
discovery, by searching existing publically-accessible databases such as
Genebank.
This is done through protein-based similarity searches to uncover new
sequences with
similarity or partial identity with the query sequence (an FN Type III repeat
from
fibronectin for example). Alternatively, one can perform a keyword search of
the
entries for the annotation of Fn Type III repeat or four helical bundle motif.
Such
candidates would be membrane bound proteins with one or more Fn Type III
repeat
domains in their extracellular components. Likewise, the converse is also
possible.
Indeed, we have used this bioinformatics/proteomics approach to identify the
membrane bound signalling protein, CD148, containing 8-10 Fn Type III repeats,
as a
possible receptor for UG. This enables us to directly test whether rhUG
interacts with
CD148 to mediate some or all of its biological activities.
Examule 3
Inhibition of Cell Adhesion to Fibronectin by rhUG
The discovery that recombinant human uteroglobin binds to human fibronectin
in solution has profound implications (See USSN 08/864,357). In addition, the
ability
of recombinant human uteroglobin to prevent fibronectin aggregation ih vitro,
fibronectin-mediated fibrillogenesis in cell culture, and renal fibronectin
deposition in
vivo, demonstrates the important physiological role of endogenous uteroglobin
in all
mammals. Fibronectin is one of the most well characterized mediators of cell
adhesion, and is involved in several physiologic processes, including platelet
aggregation (thrombosis), wound healing, fibrosis, inflammatory cell and
fibroblast
adhesion, tumor metastases, and extracellular basement membrane formation.


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However, these processes involve the insoluble form of fibronectin, not the
soluble
form. It would be desirable to prevent the conversion of fibronectin from its
soluble
form to its insoluble form, which could be exploited to prevent the initiation
of the
processes listed above, or in limiting the extent of such processes. It has
now been
found that recombinant human uteroglobin also binds to insoluble fibronectin.
When fibronectin converts to its insoluble form, it changes conformation and
deposits on the surfaces of cells and on the extracellular matrix, where it
may act as
an anchor for polymorphonuclear leukocytes, macrophages, monocytes, and
fibroblasts during an inflammatory episode. The receptors for fibronectin on
cell
surfaces are numerous and fall into different classes of molecules, including
cell
adhesion molecules (ie. ICAM-1) and integrin complexes (ie. alb4. integrin).
These
types of molecules mediate not only cell adhesion but also provide a means
through
which the cell senses, and reacts to, its environment. Recombinant human
uteroglobin
specifically binds to fragments of fibronectin that contain type III domains,
and the
IS RGD-containing type III domain (#10), in particular. The RGD peptide is
well known
as a mediator of cell adhesion in leukocyte extravasation during inflammation.
RGD-
containing peptides are potent inhibitors of cell adhesion ih vitro, for many
types of
mammalian cells. Commercial interests have attempted to use RGD peptides and
peptidomimetics as anti-inflammatory agents ih vivo, but have met with limited
success due to the instability of these types of compounds. Therefore, the
potential
use of recombinant human uteroglobin as an inhibitor of cell adhesion to
fibronectin
in vitro was investigated.
Cellular adhesion assays were performed essentially as described by Retta, et
al. ("Adhesion to Matrix Proteins" in Methods in Molecular Biology, Dejana, E.
and
M. Corada, Eds., 96: 125-130, Humana Press, Totowa, NJ; 1999). Briefly, a 96-
well
fibronectin-coated plate was blocked (to prevent non-specific binding of cells
or
proteins to the plastic) with 1X Pierce BlockerTM, containing 5% BSA, at
37°C in
incubator for at least 1 hour. The blocking reagent was then removed and the
wells
were washed three times with PBS (phosphate buffered saline). The prepared
plate
were stored in the incubator while preparing the cells. Two cell lines, NIH
3T3 and
Hela cells (ATCC # CCL-2) were selected for the assay. NIH 3T3 cells have been


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58
shown to have a high density of the uteroglobin receptor and Hela cells do not
have
the uteroglobin receptor. Both lines were grown with standard tissue culture
techniques in Dulbecco's Modified Eagle's Medium containing 10% fetal calf
serum.
The cells were removed from culture flasks using 5 mM EDTA and spun down
gently. The cells were then washed three times by alternately resuspending in
serum-
free medium and repeat centrifugation. The final cell pellet was resuspended
to an
estimated density of 2.5 - 7.5 x 104 cells per 200 ml. Cells were partitioned
into
aliquots to receive either uteroglobin, a monoclonal antibody control, Type 1b
secretory PLA2 (porcine pancreatic) (ppPLA2), myoglobin (dog heart) control,
or PBS
control. Because uteroglobin and sPLA2s share structural similarity, PLA2 was
tested
as well.
Cells were then added to each well of the fibronectin-coated plate as quickly
as possible. The plates were incubated at 37°C in a C02 incubator for 1
hour. While
the plated cells were incubating, live cells in the pipetted suspension were
counted by
trypan blue dye exclusion using a hemacytometer. At one hour the wells of the
plate
were aspirated and washed one time with PBS to remove non-adhered cells. The
adhered cells were then fixed to the plate with 3.7% paraformaldehyde in PBS
for 10
minutes at room temperature. The plates were then washed once with PBS and the
adherent cells were stained by adding 200 ml of 0.25% 8250 Coomassie blue to
each
well and allowing the cells to stand at room temperature for 1 hour. The stain
solution
was then aspirated and wells washed thxee times with PBS. Adherent cells were
quantitated by reading the optical density at 540 nm in microtiter plate
reader.
The tabulated results shown in Tables 8A & 8B below are reported as the
percent inhibition of cellular adhesion. Percent inhibition was calculated as
100%
minus the ratio of the mean OD of the test protein over the mean OD of the PBS
alone
(no protein) control. All protein groups were run in triplicate in each
experiment. All
numbers represent the mean percent inhibition for three separate experiments.
Table 8A - Effects of Uteroglobin on Cellular Adhesion to Fibronectin;
NIH-3T3 Cell line (high density of utero~lobin receutor(s); mouse lung
fibroblastsl


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Uteroglobin Anti-fibronectinppPLA2 (50 Control (10
ng) ~,g)


(50 ng) monoclonal


antibody (10
~,g)


Percent Inhibition54% 60% 68% ~ 31


Table 8B - Effects of Uteroglobin on Cellular Adhesion to Fibronectin;
Hela Cell line (low density of utero~lobin receptor(s); human cervical
carcinoma)
Uteroglobin Anti-fibronectinppPLA2 (50 Control (50
ng) ng)


(50 ng) monoclonal


antibody (10
g


Percent Inhibition-5% ND 28.5% 23%


These results show that rhUG and ppPLA2 mediate a similar anti-adhesive
S effect, suggesting a common signalling pathway. Both proteins showed a
significant
effect at inhibiting adherence of NIH-3T3 cells to plated fibronectin and no
significant
effect on Hela cells. This indicates that rhUG may bind to the receptor that
recognizes
ppPLA2 in NIH-3T3 cells. Both the N-type (neural type) and the M-type (muscle
type) PLA2 receptors are known to be expressed on NIH-3T3 cells, and since
both
rhUG and ppPLA2 share common structural patterns, it is possible that the UG
receptor is a PLA2 receptor. This hypothesis is supported by the fact that the
M-type
PLA2 receptor is known to play a role in muscle contraction. Together with our
discovery of the effects of rhUG on smooth muscle in the lungs of ventilated
newborn
piglets, these data strongly implicate the M-type PLA2 receptor in the rhUG
signalling pathway. Thus, we believe that the M-type PLA2 receptor acts as a
UG
receptor in certain cellular aszd tissue-specific contexts. In addition,
inhibition of
cellular adhesion to fibronectin is a powerful pharmacologic effect that has
been used
as a screen for anti-inflammatory compounds that can inhibit the adherence of
inflammatory cells such as neutrophils to the vasculature at sites of
inflammation, as
well as inhibit the extravasation of these cells into the inflamed tissues.
Thus this is a
powerful anti-inflammatory property of rhUG and would be expected to lead to
the
regulation and/or inhibition of neutrophil infiltration of damaged tissue.
Example 4
Effects of rhUG in a Perfused Rat Lung Model of Endotoxin-induced Inflammation


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Another study of the effects of rhUG on pulmonary inflammation was done in
a perfused rat lung model of lipopolysaccharide (LPS)-induced inflammatory
response developed by Uhlig, et al (1996) and Ljungman, et al. (1996). This
model
S simulates one of the major pathways through which bacterial infection causes
pulmonary dysfunction and is often used to explore the mechanisms of LPS-
induced
pulmonary inflammation, as well as to determine the ability of potential
therapeutic
agents to counter these mechanisms. In this model, lungs were excised from
adult
Norway rats and perfused with saline buffer for two hours. A total of 16 rats
were
10 treated in four groups of four animals each, as shown in Table 9.


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Table 9
Group 1 Vehicle controlN = 4


Group 2 LPS only N = 4


Group 3 rhUG only N = 4


Group 4 LPS + rhUG N = 4


The lungs were initially ventilated and perfused for about S minutes with SO
milliliters of sterile physiologic saline to wash out remaining blood cells.
Then the
S perfusate buffer was changed and bacterial endotoxin (LPS) and/or rhUG were
administered via the perfusate. The concentration of LPS used was S mg/kg and
the
dose of rhUG was 20 mg/kg. After two hours of perfusion, the perfusates were
collected and the perfusion was halted. Perfusates were stored frozen at -
80°C. The
airway resistance of the ventilated lungs was measured as described. The
tracheal
clamps were removed and broncho-alveolar lavage performed in order to recover
extracellular lung fluids and proteins. The broncho-alveolar lavage (BAL)
fluid was
then stored at -80°C. BAL fluids were analyzed for rhUG content as well
as the pro-
inflammatory cytokine (TNF-alpha). Human UG concentration in BAL fluid was
measured with the competitive UG ELISA as previously described. Rat TNF-alpha
1 S was measured by ELISA using a commercially available kit (R&D Systems,
Inc.)
Total protein in BAL fluids was measured using the micro-BCA protocol
according to
the manufacturer's instructions (Pierce Chemical Co.).
In this perfused lung system, the LPS and study compound can be given either
by intratracheal instillation or via the circulatory system by addition to the
perfusate.
The LPS and the rhUG were admiustered via the perfusates in order to insure an
even
distribution of the agents in the lungs. When the LPS is administered via the
circulatory route, the model is more representative of sepsis than it is of a
local
pulmonary infection. However, LPS is a potent pro-inflammatory compound and
induces similar changes to the lungs and airways, regardless of the route of
2S administration.


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One of the changes that is common to the LPS response, irrespective of route
of administration is an increase in airway resistance. The effect of LPS on
airway
resistance is shown in Figure 21. The group receiving only LPS exhibits a
large
increase in airway resistance compared to the control. The group that received
rhUG
shows a modest increase in airway resistance that is probably due to the fact
that the
rhUG preparation itself, derived from a recombinant bacterial extract,
contained a
small quantity of endotoxin and other impurities. However, the group that
received
both the LPS and the rhUG shows only the same modest increase, demonstrating
that
the rhUG effectively reversed the effect of the LPS on airway resistance.
The effect of rhUG to mediate an inhibition of the release of the pro-
inflammatory cytokine, TNF-alpha, by lung tissue into the extracellular lung
fluid was
also investigated. Elevations in TNF-alpha are a well-known sequelae to
infection,
and TNF-alpha is, in general, a marker of ongoing inflammation. The results
are
shown in Figure 22. As expected, LPS mediated a large increase, over ten-fold,
in the
mean TNF-alpha concentration. The rhUG and control groups had comparable mean
TNF-alpha concentrations, showing that rhUG mediated a significant decrease in
the
mean TNF-alpha concentrations in BAL fluid in LPS treated lungs.
These results on airway resistance are remarkable since very few, if any,
agents have been shown to be so effective in countering this effect of LPS.
The effect
was achieved in the absence of any type of blood cell in the circulation and,
therefore,
is an effect of both agents directly on the lungs, airways, and vasculature.
This is
particularly unexpected for rhUG because the anti-inflammatory pulmonary
effects of
UG have previously been attributed to its inhibitory effects on white blood
cells.
This result in perfused rat lungs is in agreement with the study in a newborn
piglet model of oxygen toxicity where rhUG showed a similar effect on
normalizing
pulmonary compliance and distensibility (see Example 1). Without being bound
by
theory, it is believed that the beneficial effects of rhUG in this perfused
rat lung model
are not likely due to enhancement or protection of surfactant, as was also the
conclusion in the ventilated newborn piglet model. These two experiments taken
together also indicate that rhUG acts on the smooth muscle component of the


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63
vasculature and the airways to mediate relaxation, regardless of the
particular type of
inflammatory insult that triggers inappropriate or deleterious muscle
contraction.
The concentration of rhUG in BAL fluid samples was determined. The mean
rhUG concentration in each group is given in Table 10 below. Both of the
groups
receiving rhUG had substantial quantities of rhUG in the BAL fluids. The
groups that
did not receive rhUG had nothing but background. The limit of sensitivity for
the
assay is 5 nanograms/ml and anything less than that value is noise. Comparison
of the
values for CC10 concentration versus total protein concentration shows that
these
differences are not due to differences in the dilution factor inherent in the
BAL fluids
technique. The presence of rhUG in BAL demonstrates that UG is taken up from
the
blood by the airways and lungs.
Without being bound by theory, this suggests that a second source of UG in
the body may replenish UG in the lungs when acute depletion of UG or Clara
cells is
caused by some external insult. It is possible to interpret the high
circulating levels of
UG in ALI and CLD as a physiologic response to compensate for decreased local
pulmonary production of this essential protein.


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Table 10
Group [CC 10] [Total protein]
nanograms/mlmilligrams/ml


LPS 1.3 +/- 0.3 6.2 +/- 8.5


LPS + CC10 3,064 +/- 9 +/- 13
874


CC10 2,590 +/- 1.6 +/- 0.4
362


Control 1.5 +/- 0.3 12 +/- 16


An additional treatment benefit shown in this study is that rhUG can be
administered via the circulation and still mediate a therapeutic effect in the
lungs,
providing a practical route of administration to treat non-entubated patients
with an
acute or chronic lung condition. Thus, an intravenous injection could treat or
prevent
ARDS, even in sepsis patients with circulating LPS. An intravenous injection
of
rhUG could likewise treat bronchoconstriction in an asthma patient who is
physically
unable to inhale medication. Similarly, many patients with pulmonary fibrosis
(IPF or
CF) or chronic obstructive pulmonary disease (COPD) may have difficulty taking
a
breath, or inhaling other medication, relieved by an intravenous dose of rhUG.
Finally, this study illustrates the potential for rhUG as an agent for the
lowering of
blood pressure caused by rigid, non-compliant, or non-elastic blood
vessels,,including
primary pulmonary hypertension (PPH).
Example 5:
Elevation of neutrophils in UGKO mice
In the course of determining baseline levels of complete blood counts and
differential white blood cell counts in uteroglobin knock-out (UGKO) mice, a
new
phenotype of these mice was discovered. Whole blood was collected by retro-
orbital
bleed from six UGKO mice and six normal C57B16 mice (the parental strain). The
numbers of blood cells of different types were determined by automated cell
counting,
as well as by manual counting of cell types on differentially stained
microscope


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slides. The data are shown in Table 11 below. Each number represents the mean
of
five determinations in five animals. In instances where determinations were
not made
in five animals, the numbers in parentheses indicate the number of animals for
which
determinations were made for that type of blood cell. There was a significant
increase
5 in the number of circulating neutrophils in the UGKO mice compared to the
non-
lcnocl{out parental strain. The UGKO mice had three times the normal level of
neutrophils. This observation is significant in terms of determining the basis
for the
inflammatory necrotic foci in pancreas, spleen, and thymus, as well as the
high level
of circulating lysophosphatidic acid (LPA) and sPLA2 activity (Zhang, 1997; US
10 08/864,357) previously observed in these mice. A greater number of
neutrophils
could result in a greater tendency towards inflammation.
Table 11
Cell Type UG -/- (n=5)UG +/+ (n=5) Normal mouse
Range


WBC 13.46 K/~.1 5.8 K/~,l 4-12 K/~.1


RBC 10.6 M/~.1 9.4 M/p,l 7-11 Mlp,l
(3) (4)


hematocrit 51.3% (4) 46.2% (4) 35-45%


neutrophils 19.2 % 9.5% (4) 5-40%


lymphocytes 74% 75.6% 30-90%


monocytes 4% 2.4% 0-10%


eosinophils 0.32% 7.2% 0-5%


basophils 0% 0% 0-1


15 Taken together with the piglet data that demonstrated trends towards higher
numbers of lymphocytes and lower numbers of PMN (quantitated one month after a
single intratracheal dose of rhUG) this observation of high neutrophils in
UGKO mice
further shows that rhUG affects white blood cell distribution. In addition,
there is
another mouse model of IgA nephropathy, the DDY mouse, in which the renal
20 fibrotic phenotype is due to abnormal B cell development. The necrotic foci
in the


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spleen and thymus of the UGKO mice, organs where white blood cells normally
differentiate into mature T and B cells, indicate that the absence of UG
results in a
defect in T or B cell maturation, not just granulocyte maturation, as shown in
Figure
24. Likewise, necrotic foci in the pancreas of the UG knockout mouse suggest a
role
for UG in maturation of resident lymphoid cells. Therefore, long term
immunomodulatory effects of rhUG are due to alterations in distribution of
white
blood cells, both lymphoid and hemopoietic lineages, in the body. Long term
anti-
inflammatory effects of rhUG may be due to its regulation of production of
hemopoietic precursors, including neutrophils (PMN). Long term effects of UG
in
preventing tumor formation are due at least in part to the increase in the
number of
lymphocytes that mediate immune surveillance.
These long term effects of UG are distinct from short term anti-inflammatory
or immunomodulatory effects in that the long term effects involve immature
precursors, rather than mature cells. Short term anti-inflammatory effects of
rhUG are
due to its immediate direct effects on mature pro-inflammatory leukocytes,
such as
inhibition of release of TNF-alpha and inhibition of chemotaxis. Furthermore,
the
nature of the effect is different than has been previously described because
UG affects
the maturation process itself, rather than suppressing a response to an
external
stimulus in the mature cells. These long term effects of UG indicate that the
application of rhUG, and other UG supplementation strategies such as gene
therapy
and glucocorticoid administration, would be successful in the treatment of
hemopoietic stem cell disorders.
Example 6
Short term effects of rhUG on lymphoid and hemopoietic cell metabolism in
vivo.
Based on the piglet study and the phenotypes of the UG knockout (UGKO)
mice, it was postulated that rhUG could induce a growth arrest in certain
types of
circulating lymphoid and hemopoietic cells in the body. The term hemopoietic
refers
to several cell types and their precursors as indicated in Figure 24,
specifically
including granulocytes (neutrophils, basophils, eosinophils), erythrocytes,
monocytes


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and macrophages, and megakaryocytes. The term lymphoid refers to T cells and B
cells, specifically including T helper cells (CD4), T suppressor cells (CD8),
NIA
(natural killer) cells, B cells, dendritic cells, and their precursors, as
indicated in
Figure 24. The term lineage includes not only the mature, fully differentiated
forms
of these cells, but also their precursor cells that are not yet committed to a
differentiation pathway.
In order to determine whether rhUG could mediate a growth arrest in
circulating granulocytes and lymphocytes, the short-term effects of rhUG on
white
blood cell metabolism in vivo were investigated. rhUG was adx-ninstered
intravenously to 24 young adult male Wistar rats in six dose groups of four
animals
each, as shown in Table 12. The rhUG was given by tail vein injection in a
constant
volume of 200 microliters. Animals were housed in metabolism cages and given
free
access to food and water. The animals were anesthetized and sacrificed twelve
hours
after administration of a single dose of rhUG. During sacrifice, five
milliliters of
whole blood was collected into EDTA collection tubes. The blood from all
animals in
each group was pooled and kept at room temperature for 1-3 hours during
processing.
Table 12
Dose Group Number


0 ~,g/kg 4


10 ~,g/kg 4


50 ~,g/kg 4


200 ~,g/kg 4


500 ~,g/kg 4


2,000 ~,g/kg


Specific populations of white blood cells were purified from each pool of
blood.
Cells bearing surface epitopes for CDllb and CD71 were selected out and
analyzed.


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The metabolic states of these cell types were then assayed by measuring total
ATPase
activity in extracts of these cells (see U.S. patent 5,773,232). Any change in
the level
of ATPase activity indicates that the cell is reacting to some signal or other
change in
its environment. In this experiment, conducted in the absence of an antigenic
or
mitogenic stimulus, the change in ATP level indicates the response of these
cell types
to exogenous intravenous rhUG only.
Pharmaceutical grade recombinant human UG, 5.5 mg/mL blood was
collected in EDTA tubes (Becton Dickinson) from treated male Wistar rats.
Tosyl-
activated magnetic particles (Dynabeads M-450) were obtained from Dynal
Biotech,
and mouse monoclonal antibodies to rat CD11B, CD4, CDB, and CD71 cells were
obtained from Research Diagnostics, Inc. Rabbit anti-mouse antibody (Pierce
Chemical Co.) was used to coat the magnetic particles. All other reagents used
are
listed and described in the product inserts for each of the methods used in
this study.
Bead coating was done according to the manufacturer's instnzctions (Dynal
Biotech
Product Nos. 140.03 and 140.04, Dynal Inc.). Measurement of ATPase
concentrations was also done according to the manufacturer's instructions
(Cylex,
Inc.) using the in vitro CMITM Assay for T Cell Activation, Universal Test
Kit, and
CD4 in vitro CMITM Assay for T Cell Activation. Protein determinations were
done
using the micro-BCA protocol from the BCA Protein Assay Kit according to the
manufacturers instructions (Pierce Chemical Co.)
CD71 is a widely distributed cell surface marker for activated or
proliferating
cells and is the transferrin receptor required for iron. uptake in all rapidly
metabolizing
cells. Proliferating lymphoid and myeloid cells in the circulation express
CD71 and
are specifically selected by the technique used. CDllb is another Cluster of
Differentiation cell surface antigen also an integrin alpha chain, that is
often present
on the surface of neutrophils, monocytes and NK cells. CD 11 b is a marker for
activated and adherent cells. It is upregulated during inflammation required
for the
firm adhesion of leukocytes to endothelial cell surfaces and subsequent
extravasation
from the blood into the tissues. Each set of cells was isolated from each pool
of blood
by aliquoting 100 ~l to each of eight wells in a microtiter plate for each CD
antigen
(e.g., eight replicates of each magnetic bead separation). The cells pulled
down by the


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69
antibody-conjugated magnetic beads were washed once with PBS and then an
ATPase
extract was prepared and analyzed. The results for each CD cell type for each
of the
six dose groups is shown in Figures 25-26.
The data in Figure 25 indicates that rhUG inhibits ATPase activity in CD71-
positive cells in a dose dependent manner. This dose dependent decrease may
reflect
either a slowdown in the metabolism or an arrest of the cell cycle, in
circulating cells
such as T and B cells, or PMN or their precursors. Alternatively, it may
indicate a
dose dependent decrease in the number of proliferating cells that enter the
circulation
over the twelve-hour study period such as neutrophils, monocytes and their
precursors. Since the high concentrations of UG in blood used in this study
are not
physiologic, except in instances of acute or chronic lung injury, this
metabolic arrest
or anti-proliferative effect may be a natural mechanism for down-regulating an
ongoing inflammatory and immune response, affecting both lymphocytes and
myeloid
cells.
It has been found that the opposite result is true of cells that express the
CD 11 b marker. In general, CD 11 b-positive cells are mature lymphocytes and
leukocytes, as opposed to the majority of the CD71-positive cells which may be
immature. Figure 26 shows that rhUG enhances ATPase activity in CDllb-positive
cells in a dose dependent manner that saturates at and above 50 ~g/kg. The
nature of
this stimulation is either an increase in metabolic activity of an unchanged
number of
circulating cells or an increase in the number of cells in the circulation.
This result
was surprising since mature neutrophils represent the majority of cells in the
circulation that rapidly become CD1 lb-positive during an inflammatory
response.
Example 7:
Effects of rhUG on Human Vascular Endothelial Cells Stimulated with VEGF
Introduction
Pathological angiogenesis .plays a critical role in the clinical progression
of
solid tumors and participates in the pathogenesis of numerous inflammatory and
ischemic disorders (Carmeliet and Jain, 2000). Considerable efforts are being
made


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by academic and industrial investigators to identify novel pharmacological
agents that
can modulate angiogenesis in vivo (Carmeliet and Jain, 2000; Eatock et al.,
2000;
Hagedorn and Bikfalvi, 2000; Klohs and Hamby, 1999; Zhu and Witte, 1999).
Vascular endothelial growth factor (VEGF) and other related growth factors
5 have been identified as critical for tumor growth and angiogenesis and as
potential
therapeutic targets (Schlaeppi and Wood, 1999; Gerwins et al., 2000; McMahon,
2000; Frelin et al., 2000; Veikkola et al., 2000; Ferrara and Alitalo, 1999b;
Ferrara,
1999a; Arii et al., 1999; Crew, 1999).
Other important factors involved in regulating pathological angiogenesis are
10 cellular integrins, especially alpha-v beta-l and alpha-v beta-3 and
extracellular
matrix (ECM) components, especially fibronectin (Beckner, 1999; Chandrasekaran
et
al., 2000; Kim et al., 2000; Ruoslahti, 1999; Maier et al., 1999; Soldi et
al., 1999;
Collo and Pepper, 1999; Grant et al., 1998; Takei et al., 1998). Fibronectin
binds
alpha-v beta-1, which in turn modulates alpha-v beta-3 (Kim et al., 2000).
Integrin
15 activation by VEGF (Byzova et al., 2000) and modulation of VEGF receptors
by
integrins (Soldi et al., 1999) have been described. This suggests that complex
autocrine loops involving ECM-integrin signaling and growth factor receptor
signaling modulate angiogenesis.
This example reports the results of an investigation to determine whether
20 rhUG had anti-angiogenic properties in standard in vitro models of
angiogenesis
(VEGF-induced migration and "lesion repair" assays). It has been found that:
1) rhUG inhibits VEGF-induced migration of primary human microvascular
endothelial cells (HMEC) at nanomolar concentrations
2) rhUG inhibits VEGF-induced migration of HMEC only in the presence of
25 fibronectin
3) rhUG inhibits invasion of soft agar by A549 cells, a transformed cell line
derived from a human non-small cell lung carcinoma (NSCLC)
Taken together, these observations indicate that rhUG inhibits human primary
endothelial cell migration, a key determinant of angiogenesis, through a novel
30 mechanism that requires the presence of fibronectin, and most likely the
formation of


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71
a fibronectin/UG/receptor complexes. These findings suggest that rhUG is a
candidate new drug for the treatment of pathological angiogenesis in cancer
and other
disorders.
Materials & Methods
Cell culture.
Human foreskin dermal microvascular endothelial cells (HMVEC) were
obtained from Cell System Corp. (Kirkland, WA), grown in EGM-2 medium
(Clonetics, San Diego, CA), and used from passages 2 to 10. A549 and HEC-lA
cell
lines were obtained purchase from ATCC and cultured in F 12 HAM medium with
10% fetal bovine serum (FBS) and McCoy's modified medium with 10% FBS,
respectively (GIBCO BRL., Grand Island, NY).
Cytotoxicity assay.
Human foreskin dermal MVEC, A549 and HEC-lA were used to study a
possible cytotoxic effect of human recombinant uteroglobin (rhUG). Each cell
type
was cultured in media containing 10% FBS. Cells were harvested by
trypsinization
and then plated in a 96-well plate at 30,000 cells per well in 2001. After
overnight
incubation, cells were treated with 1 ~M rhUG. At different time points (22 to
90
hours) cells were washed with lx PBS and fixed with 50% cold TCA (tri-
chloracetic
acid. After 1-hour incubation, wells were washed (4-5 times with ddH20), air-
dried,
and rinsed with 100~1/well sulfo-rhodamine B stain (SRB; 0.4 % in 1% acetic
acid)
for 30 minutes. After removal of SRB, wells were washed (4-5 times with 1%
acetic
acid), dried and 200,1 of Tris base was added in each well. The plates were
shaken for
5 min. Finally, the 96-well plate was read at 490 and 530 nm using an ELISA
reader.
Each point was run in quadruplicate per each cell type.
The rhUG had no effect on the viability of MVEC cells. This is shown in
Figure 31 where HMVEC, A549 and Hec-lA cells grew as well with 1 ~,M rhUG in
the medium as without any rhUG. These results prove that rhUG does not have
any
cytotoxic effects, nor does it inhibit proliferation of these cells in the
absence of a


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72
growth factor (VEGF) or other mitogen. This result implies that successful
treatment
of cancer in vivo with rhUG requires an additional apoptotic signal or
cytotoxic agent
such as a chemotherapeutic agent.
Endothelial cell VEGF stimulated proliferation assay.
To study a possible inhibitory effect of rhUG as an inhibitor of endothelial
cell
proliferation, human foreskin dermal MVEC (Cell System Corp., I~irkland, WA)
were
used. Cells were harvested by trypsinization, resuspended in EGM-2 (Clonetics,
San
Diego, CA), and plated in a 6-well plate at 80,000 cells per well in 2001.
After
overnight incubation, the media were replaced with fresh EBM-2 with 1.5% FBS.
After a 4 hour starvation period cells were treated with recombinant human
VEGF-A
(R & D Systems, Minneapolis, MN) plus a variable amount (0.05 to 1 ~,M) of
rhU.G..
The final concentration of recombinant human VEGF-A was 10 ng/ml. At 48 and 96
hours the cells were quantitated using a hematocytometer.
An alternative approach was used for the same purpose. Human foreskin
dermal MVEC were plated in a 96-well plate at a density of 10,000 cells per
well.
After the treatment described above, the cells were washed with lx PBS and
rinsed
with 50% cold TCA. After 1-hour incubation wells were washed (4-5 times with
ddH20), air-dried and rinsed with 100 ~,l/well SRB (0.4 % in 1% acetic acid)
for 30
min. After removal of SRB, wells have been washed (4-5 times with 1% acetic
acid),
air-dried, treated with 200~1/well Tris base, and were placed on on a gyratory
shaker
for 5 minutes for gentle mixing. Finally, the 96-well plate was read at 490
and 530 nm
using an ELISA reader. Each point was run in a set of eight replicates.
The results of the both approaches are consistent with each other. The results
of a representative experiment are shown in figure 31b. In contrast to the
lack of anti-
proliferative activity in the absence of VEGF-A, rhUG counteracted the
stimulation of
proliferation that was induced by VEGF. These data, together with the
cytoxicity
assay, demonstrate that rhUG does not inhibit normal growth of non-transformed
cells, but does abrogate the effects of mitogen or growth factor-stimulated
proliferation. The potent inhibitory effects of rhUG on accelerated or
abnormal


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73
proliferation without simultaneous inhibition of normal, unenhanced
proliferation.
This implies that rhUG could impart the benefits of chemotherapeutic agents
without
the toxic effects.
I~ vitro wound healing assay (proliferation-migration assay).
Human foreskin dermal MVEC were grown to confluence in 10 cm cell
culture dishes. Post-confluent HMVEC were wounded by gently scraping the floor
of
the dish with a serological pipet. Three uniform injuries were produced by a
single
pipet scrape across the cell monolayer (as examined by optical microscopy) in
each
dish. After washing with lx PBS, fresh media were added and two different
medium
conditions were tested (1.5 and 5% FBS). After 4.5 hours, post-confluent cells
were
exposed to 0.2 pM rhUG. After 30 min., cells were exposed to 100 pg/ml VEGF-A
(R & D Systems, Minneapolis, MN). At regular intervals between post-injury
hour 22
to 110, cells were examined by optical microscopy. Under each condition, each
time
point was performed in quadruplicate and the control represented by cells not
exposed
to rhUG.
In the absence of rhUG, the MVEC migrate into the injured area that was
cleared of cells by the pipet scraping technique. In the presence of 200
nanomolar
rhUG, the migration of VEGF-stimulated MVEC was significantly inhibited.
Photographs of representative injury areas showing MVEC migration were taken
at
110 hours post-injury and these results are shown in Figures 27 and 28.
Endothelial cell migration assay.
Human foreskin dermal MVEC (Cell System Corp., Kirkland, WA) were
cultured in EGM-2 medium (Clonetics, San Diego, CA) up to 80% of confluence.
The
medium was replaced and the cells starved overnight in EBM-2 containing 0.1%
FBS.
The day after, the endothelial cells were harvested, resuspended into DMEM
(Dulbecco's Modif ed Eagle's Medium) with 0.1 % FBS, plated on the bottom side
of
a modified Boyden chamber (Nucleopore Corporation, MD), and allowed to attach
in
the inverted chamber for 1.5 hours at 37°C. The chamber was then re-
inverted and
rhUG added at different concentrations in the presence of VEGF-A (R & D
system,


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74
Minneapolis, MN) to the wells of the upper chamber. The cells were allowed to
migrate for four hours at 37°C. Membranes were recovered, fixed and
stained, and
the number of cells that had migrated to the upper chamber counted in ten high
power
fields. Background migration to DMEM plus 0.1% FBS was subtracted and the data
reported as the number of cells migrated per 10 high power fields (400x) or,
when
results from multiple experiments were combined, as the percentage of maximal
migration to a positive control. Each concentration of rhUG was tested in
quadruplicate. VEGF-A was tested at 100 pg/ml and neutralizing antibodies to
VEGF-
A were used as control at 20~g/ml.
The effect of rhUG on the VEGF-A-stimulated migration of human dermal
MVEC is shown in Figure 29. This is a standard assay that is used to screen
agents
that inhibit angiogenesis, since one of the first steps in new blood vessel
development
is the migration of endothelial into a new area. Our data clearly show that
rhUG
mediates a profound effect in inhibiting the MVEC migration at nanomolar
concentrations of rhUG. This result is even more important because we've shown
in
our animal studies that these rhUG concentrations are non-toxic,
therapeutically,
pharmacokinetically approachable, deliverable by several routes of
administration,
and physiologically relevant in that they can mediate significant biological
effects.
Western blot for Fibronectin detection.
Western. blot analysis was performed on Hfl-1 cells, A549 and Hec-lA and
fibronectin cross-reactive species were detected with an anti-human
fibronectin
monoclonal antibody in order to determine the endogenous expression of
fibronectin.
Whole cells were cultured (as described in Example 8), scraped from the flask
and
transferred to an Eppendorf tube. An equal volume of SDS-PAGE buffer (Novex)
was added to each sample and mixed by vortexing. The samples were then heated
for
10 minutes at 85°C and loaded onto a 10% Tris-glycine SDS-PAGE gel
(Novex).
Rainbow markerTM (Amersham) was run as the size standard. Blocking, probing,
and
washing were done as described in Example 8, except that a monoclonal anti-
human
fibronectin antibody was used as the primary antibody at a dilution of 1:2000
and a


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7S
rabbit anti-mouse IgG-HRP conjugate was used as the secondary antibody at a
dilution of 1:5,000.
Results axe shown in Figure 32. All cells line made proteins that were
recognized by anti-human fibronectin antibody. Hfl-l, the non-transformed cell
line,
exhibited a very high molecular weight complex that may be 440 kDa which
corresponds to the size of an intact fibronectin dimer. Both of the
transformed cell
lines make immunoreactive protein, the highest band of which is about 220 kDa,
corresponding to a fibronectin monomer. There are several other immunoreactive
bands that probably represent proteolytic cleavage products of the f bronectin
monomer. Thus all three of these cell lines synthesize fibronectin, and
fragments
derived from it, or are proteins that axe recognized by anti-fibronectin mAb.
These
cell lines therefore, synthesize fibronectin and fibronectin-like proteins
that make
them capable of responding to rhUG without the addition of exogenous
fibronectin.
Soft agar assay.
Recombinant human UG-treated A549 cells were analyzed in a soft agar
assay. Two 6-well plates were poured with 3 ml of 0.6% agar lx F12 HAM medium
(GIBCO BRL, Grand Island, NY) containing 10% FBS. A concentration of 0.2 ~M
of rhUG was used in each well. The plates were allowed to equilibrate
overnight at
37°C, then 12 glass tubes were filled with 0.3% agar F12 HAM medium
with 10%
FBS. 45,000 cells per sample were added to each tube at 38°C. The cells
plus agar in
the tubes were then poured onto the agar on the plates and the plates were
incubated
in a cell incubator under standard conditions (37°C, 5% C02) for 35
days. Every 5
days, a fresh aliquot of rhUG was added to a concentration of 1 ~,M in a fixed
volume
of 1501 of fresh medium to each of the 6 test wells after expired media was
removed
by aspiration. The 6 control wells received the same amount of fresh medium
lacking
rhUG at the same times. At the end of the experiment, a 3001 aliquot of rhUG
was
added to the surface of each culture to a concentration of 1 ~.M and the cells
were
incubated at 37°C for an additional 36 hours. The excess liquid was
then aspirated off


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76
and the colony staining characteristics were examined by optical microscopy.
Colonies were stained with SRB dye as described above.
Each plate was examined, photographed, and colonies were counted. The
results are shown in Figure 33 for A549. The rhUG inhibited the invasion of
the soft
agar by A549 by over two-fold. This demonstrates the ability of rhUG to reduce
extracellular matrix invasion by A549, and cancer cells in general.
Matrigel Invasion Assay with Fibronectin
HMEC were grown in complete medium as described in the above wound
healing assay. The assay was performed in serum-free medium. Cells (5000 per
well) were incubated for 24 hours in Boyden chambers and tested for invasion
through Matrigel-coated filters (Nucleopore). The bottom chamber medium
contained
VEGF (100 pg/ml) in all wells. The top chamber medium contained CC10 at the
IC50
for migration inhibition (100 nM), plasma fibronectin (Sigma) at the same
concentration (100 nM) or a combination of Fn and CC10. After 24 hours,
attached
but non-migrated cells were wiped off the upper face of each filter with Q-
tips and
migrated cells attached to the bottom face of each filter were stained and
counted by
two blinded observers. Data represent averages of four intermediate power
fields
(magnification x 100) counts plus or minus standard deviations. Data were
analyzed
by one-way ANOVA with Tukey correction for multiple comparisons
As shown in Figure 30, CC10 alone had no significant effect on Matrigel
invasion in serum-free medium. Fn alone had a modest but statistically
significant
inhibitory effect. However, CC 10 plus Fn inhibited invasion to a
significantly higher
extent than Fn alone or CC10 alone (p <0.05). Thus, CC10 requires the presence
of
Fn to inhibit Matrigel invasion by human primary microvascular endothelial
cells.
This is consistent with the observation that no saturable cell binding by CC
10 was
observed in the absence of Fn. These data indicate that CC10 binds HMEC as a
complex with Fn and modulates Fn signaling. Qualitatively, the morphology of
these
cells incubated with Fn and CC 10 together appeared in groups with
intercellular
adhesion. Cells incubated with Fn alone showed much less pronounced
intercellular
adhesion. Cells incubated in control medium or CC10 alone appeared mostly as


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77
individual cells. This suggests that upon binding Fn/CC10, cells modulate
adhesion
molecules and increase intercellular contacts, which may participate in the
inhibitory
effects observed.
The property of intercellular adhesion to cells of the same lineage is a
property
of non-tranformed normal cells and is referred to as isotypic adhesion. Agents
that
promote isotypic adhesion are candidates for clinical drug development as anti-
cancer
agents. Isotypic adhesion of A549 cells transfected with a human uteroglobin
expression vector has been reported (Szabo, 1996), but the involvement of
fibronectin
was not previously identified. The effect in this cell line was attributed
solely to the
expression of human UG, since the effect was not observed in the mock-
transfected
controls. However, we have observed that non-transfected A549 and Hec-lA cells
grown under similar culture conditions also express proteins and protein
fragments
that are recognized by an anti-human fibronectin monoclonal antibody, as shown
in
Figure 32. Indeed, many cell lines produce fibronectin immunoreactive proteins
and
protein fragments (Ruoslahti, 1987).
Example 8:
Identification of UG receptors
In a continuation of the effort to purify and identify the human UG
receptor(s),
originally described in PCT/US98/11026 two approaches were taken. The first
was a
bioinformatics approach in which the M-type PLA2 receptor and CD 148 were
identified as candidates for the UG receptor. The second was a continuation of
the
affinity purification approach. Human fetal lung fibroblast cell line, Hfl-l,
was
selected for two reasons: 1) It is a non-transformed diploid senescent cell
line, and
therefore, closer to a normal human cell than any of the tumor cell lines
previously
characterized as expressing UG binding proteins, and 2) PDGF-induced migration
of
Hfl-1 cells was reported by Lesur et al. (1995), suggesting the presence of a
functional
UG receptor.
Hfl-1 was obtained from the American Type Culture Collection, Inc. and
cultured according to the supplier's instructions. Cells were cultured to


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78
approximately 90% confluence, then the media was decanted and the cells washed
twice with sterile saline. Cells were scraped from culture flasks and lysed
inl0 mM
Tris buffer pH 7.5 with 1% Triton X-100 and CompleteTM anti-protease (Roche).
Unlysed cells were removed by centrifugation and the supernatant was loaded
onto a
UG affinity column. The UG affinity column was prepared by coupling 50
milligrams of substantially pure rhUG to 10 mls of NHS-Sepharose, according to
the
manufacturer's instructions (Pharmacia). The column was equilibrated either
with 10
mM Tris, pH 7.5, 0.1% Triton X-100. After loading the crude cell lysate onto
the
colmml, the unbound proteins passed through the column by washing with cell
lysis
buffer. Bound proteins eluted in batch using 0.5 M NaCI in 10 mM Tris, pH 7.5
with
0.1% Triton X-100. Several proteins or protein fragments were retained by the
UG
affinity colurml. Proteins were visualized by Silver stain of a 10% Tris-
Glycine SDS-
PAGE gel (Invitrogen Corp.), as shown in Figure 34.
In order to verify the presence of the M-type PLA2 receptor and CD 148 in
various
UG-responsive cells, including Hfl-1, we generated rabbit polyclonal antisera
against
peptides derived from them. The peptides were synthesized and rabbit antisera
raised
using standard methods (Research Genetics, Inc.). The peptide derived from the
M-
type PLA2 receptor to which antisera was raised is: QNWDTGRERTVNNQSQR.
The peptide derived from the CD148 protein to which antisera was raised is:
NGTDGASQKTPSSTGPSPVFD. Both of these peptides produced high titre antisera
within three months.
The anti-M-type PLA2 receptor antisera and the anti-CD 148 antisera were then
used to analyze the crude extract and UG affinity-purified protein by Western
blot.
Equal volumes of crude lysates of Hfl-1 and UG affinity-purified proteins were
run on
10% SDS-PAGE Tris-glycine gels (Invitrogen Corp.) with a Rainbow TM marker
(Amersham Pharmacia, Corp.) using the manufacturers procedures. Gels were
blotted
to Hybond-PTM (Amersham Pharmacia, Corp.) using the Novex Xcell TM II
apparatus
according to instructions (Novex). The blots were blocked in 5% BSA (Sigma
Co.)
overnight at 4°C. Excess blocking solution was washed off with two
washes in PBS
with gentle shaking at room termperature. All following steps were performed
at
room temperature. Primary incubations with the rabbit antisera against the
human M-


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79
type PLA2 receptor and CD148 were done on separate blots containing identical
protein samples. The blots were incubated in parallel with primary antisera
dilutions
of 1/500 in 5% BSA, 1X PBS, 0.2% Tween 20 for 1.5 hours at room temperature
with
gentle shaking. The primary antisera was then washed off with three washes in
PBS,
0.1% Tween 20 with gentle shaking for 5-10 minutes each. Secondary incubations
were goat anti-rabbit IgG conjugated to horse radish peroxidase (Pierce
Chemical
Co.) at 1/5000 in 5% BSA, 1X PBS, 0.1% Tween 20 for 1.5 hours at room
temperature with gentle shaking. Three washes were then done in 1X PBS, 0.1%
Tween 20 for 5-10 minutes each. The blots were developed using the ECL TM kit
(Amersham Pharmacia Corp.) and viewed by autoradiography using Kodak Biomax
TM ML film.
Protein bands corresponding to bands detected on each Western blot are
indicated by arrows in figure 34. Similar protein staining and Western band
patterns
were obtained for three additional cell lines, also obtained from ATCC and
cultured
under the recommended conditions. These included: HL-60, HEC-lA, and A549.
These UG binding proteins include the bands previously identified as UG
receptor
binding protein (Kundu, 1998), and some new protein bands have appeared.
Proteins
that eluted from the UG affinity column include both the M-type PLA2 receptor
and
CD148, as well as fragments derived from each, or otherwise antigenically
related
proteins.
The above description of the invention is intended to be illustrative and not
limiting. Various changes or modifications in the embodiments described may
occur
to those skilled in the art. These can be made without departing from the
spirit or
scope of the invention.


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