U.S. patent application number 11/062354 was filed with the patent office on 2005-08-11 for formulation of adenovirus for gene therapy.
Invention is credited to Wu, Zheng, Zhang, Shuyuan.
Application Number | 20050175592 11/062354 |
Document ID | / |
Family ID | 27380509 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050175592 |
Kind Code |
A1 |
Wu, Zheng ; et al. |
August 11, 2005 |
Formulation of adenovirus for gene therapy
Abstract
The present invention addresses the need to improve the
long-term storage stability (i.e. infectivity) of vector
formulations. In particular, it has been demonstrated that for
adenovirus, the use of bulking agents, cryoprotectants and
lyoprotectants imparts desired properties that allow both
lyophilized and liquid adenovirus formulations to be stored at
4.degree. C. for up to 6 months and retain an infectivity between
60-100% of the starting infectivity.
Inventors: |
Wu, Zheng; (Sugar Land,
TX) ; Zhang, Shuyuan; (Media, PA) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
27380509 |
Appl. No.: |
11/062354 |
Filed: |
February 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11062354 |
Feb 18, 2005 |
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09941296 |
Aug 28, 2001 |
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09941296 |
Aug 28, 2001 |
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09441410 |
Nov 16, 1999 |
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6689600 |
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60108606 |
Nov 16, 1998 |
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60133116 |
May 7, 1999 |
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Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
A61K 35/761 20130101;
A61K 47/32 20130101; C12N 2710/10351 20130101; C12N 2710/10332
20130101; A61K 47/38 20130101; A61K 47/42 20130101; A61K 9/0019
20130101; A61K 9/19 20130101; C12N 15/86 20130101; A61K 47/46
20130101; C12N 2710/10343 20130101; A61K 47/02 20130101; C12N
2710/10051 20130101; A61K 47/26 20130101; C12N 7/00 20130101; A61K
47/183 20130101; A61K 47/20 20130101; A61K 48/00 20130101; A01N
1/02 20130101 |
Class at
Publication: |
424/093.2 ;
435/456 |
International
Class: |
C12N 015/861; A61K
048/00 |
Claims
1-60. (canceled)
61. A pharmaceutical composition in an oral dosage form, wherein
the oral dosage form is a tablet, a troche, a capsule, an elixir, a
suspension, a syrup, a solution, a wafer, a pill, a microsphere, a
nanoparticle, a mouthwash, a dentrifice, or a spray, wherein the
composition comprises a viral vector comprising an expression
cassette, wherein the expression cassette comprises a promoter
active in eukaryotic cells, wherein the promoter is operatively
coupled to a nucleic acid encoding a therapeutic gene.
62. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a tablet.
63. The pharmaceutical composition of claim 62, wherein the tablet
is an ingestible tablet or a buccal tablet.
64. The pharmaceutical composition of claim 62, wherein the tablet
is formulated to dissolve in the mouth or under the tongue.
65. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a troche.
66. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a capsule.
67. The pharmaceutical composition of claim 66, wherein the capsule
is a hard-shell capsule or a soft-shell capsule.
68. The pharmaceutical composition of claim 61, wherein the oral
dosage form is an elixir.
69. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a suspension.
70. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a syrup.
71. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a solution.
72. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a wafer.
73. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a pill.
74. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a microsphere.
75. The pharmaceutical composition of claim 74, wherein the
microsphere comprises a polymer that can adhere to a mucosal
surface.
76. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a nanoparticle.
77. The pharmaceutical composition of claim 76, wherein the
nanoparticle is formulated with a stabilizer and/or a linear
poly(ethylene oxide) polymer.
78. The pharmaceutical composition of claim 76, wherein the
nanoparticle is formulated for controlled release.
79. The pharmaceutical composition of claim 76, wherein the
nanoparticles comprise a biodegradable polycyanoacrylate
polymer.
80. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a mouthwash.
81. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a dentrifice.
82. The pharmaceutical composition of claim 81, wherein the
dentrifice is a gel, a paste, a powder, or a slurry.
83. The pharmaceutical composition of claim 82, further comprising
water, a binder, an abrasive, a flavoring agent, a foaming agent,
and/or a humectant.
84. The pharmaceutical composition of claim 61, wherein the oral
dosage form is a spray.
85. The pharmaceutical composition of claim 61, further comprising
an inert diluent, an assimilable edible carrier, a binder, an
excipient, a disintegrating agent, a lubricant, a sweetening agent,
a flavoring agent, a liquid carrier, a coating, a preservative, a
dye, a solvent, water, an abrasive, a foaming agent, a humectant,
and/or an adhesive.
86. The pharmaceutical composition of claim 85, wherein the binder
is gum tragacanth, acacia, constarch, or gelatin.
87. The pharmaceutical composition of claim 85, wherein the
excipient is dicalcium phosphate, glycerin, or potassium
bicarbonate.
88. The pharmaceutical composition of claim 85, wherein the
disintegrating agent is corn starch, potato starch, or alginic
acid.
89. The pharmaceutical composition of claim 85, wherein the
lubricant is magnesium stearate.
90. The pharmaceutical composition of claim 85, wherein the
sweetening agent is sucrose, lactose, or saccharin.
91. The pharmaceutical composition of claim 85, wherein the
flavoring agent is peppermint, oil of wintergreen, or cherry
flavoring.
92. The pharmaceutical composition of claim 85, wherein the oral
dosage form is a tablet, pill, or capsule, and wherein the coating
is shellac or sugar.
93. The pharmaceutical composition of claim 61, wherein the
adenovirus vector is formulated to be incorporated directly into
food.
94. The pharmaceutical composition of claim 61, further defined as
a sustained release preparation or controlled release
preparation.
95. The pharmaceutical composition of claim 85, wherein the solvent
comprises sodium borate solution.
96. The pharmaceutical composition of claim 61, wherein the viral
vector is an adenoviral vector, a retroviral vector, an
adeno-associated virus vector, a vaccinia virus vector, a
herpesvirus vector, or an oncolytic virus vector.
97. The pharmaceutical composition of claim 96, wherein the viral
vector is an adenoviral vector.
98. The pharmaceutical composition of claim 97, wherein the
adenoviral vector is a replication-deficient adenoviral vector.
99. The pharmaceutical composition of claim 98, wherein the
replication-deficient adenoviral vector is lacking at least a
portion of the E1 region.
100. The pharmaceutical composition of claim 96, wherein the
oncolytic virus is a reovirus, OYNX-015, or CN706.
101. The pharmaceutical composition of claim 61, wherein the
promoter is a CMV IE, SV40 early, or RSV LTR.
102. The pharmaceutical composition of claim 61, wherein the viral
vector is comprised within a liposome.
103. The pharmaceutical composition of claim 61, wherein the
therapeutic gene is a tumor suppressor gene, a gene encoding an
inducer of apoptosis, a gene encoding an enzyme, a gene encoding a
hormone, a gene encoding an interleukin, or a gene encoding a
cytokine.
104. The pharmaceutical composition of claim 103, wherein the tumor
suppressor gene is p53, CDK4 or other cyclin-dependent kinase;
p16.sup.INK4, p16.sup.B, .sup.p21WAF1, CIP1, SDI1, p27.sup.KIP1 or
other CDK-inhibitory protein; C-CAM, RB, APC, DCC, NF-1, NF-2,
WT-1, MEN-I, MEN-II, zac1, p73, BRCA1, VHL, FCC, MMAC1, MCC, p16,
p21, p57, p27, and BRCA2.
105. The pharmaceutical composition of claim 103, wherein the
inducer of apoptosis is Bax, Bak, Bcl-X.sub.s, Bik, Bid, Harakiri,
Ad E1B, Bad, or an ICE-CED2 protease.
106. The pharmaceutical composition of claim 103, wherein the
enzyme is cytosine deaminase, hypoxanthine-guanine
phosphoribosyltransferase, galactose-1-phosphate uridyltransferase,
phenylalanine hydroxylase, glucocerebrosidase, sphingomyelinase,
.alpha.-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV
thymidine kinase, or human thymidine kinase.
107. The pharmaceutical composition of claim 103, wherein the
hormone is growth hormone, prolactin, placental lactogen,
luteinizing hormone, follicle-stimulating hormone, chorionic
gonadotropin, thyroid-stimulating hormone, leptin,
adrenocorticotropin (ACTH), angiotensin I, angiotensin II,
.beta.-endorphin, .beta.-melanocyte stimulating hormone,
cholecystokinin, endothelin I, galanin, gastric inhibitory peptide,
glucagon, insulin, a lipotropin, a neurophysin, somatostatin,
calcitonin, calcitonin gene related peptide, .beta.-calcitonin gene
related peptide, hypercalcemia of malignancy factor, parathyroid
hormone-related protein, glucagon-like peptide, pancreastatin,
pancreatic peptide, peptide YY, PHM, secretin, vasotocin,
enkephalinamide, metorphinamide, alpha melanocyte stimulating
hormone, atrial natriuretic factor, amylin, amyoid P component
(SAP-1), corticotropin releaseing hormone (CRH), growth hormone
releasing factor (GHRH), luteinizing hormone releasing hormone
(LHRH), neuropeptide Y, substance K (neurokinin A), substance P, or
thryrotropin releasing hormone (TRH).
108. The pharmaceutical composition of claim 103, wherein the
interleukin or cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, or G-CSF.
109. A pharmaceutical composition in an nasal dosage form
comprising a viral vector comprising an expression cassette,
wherein the expression cassette comprises a promoter active in
eukaryotic cells, wherein the promoter is operatively coupled to a
nucleic acid encoding a therapeutic gene.
110. The pharmaceutical composition of claim 109, wherein the nasal
dosage form is an intranasal aerosol spray.
111. The pharmaceutical composition of claim 110, wherein the
composition is further defined as being comprised in a
liposome.
112. The pharmaceutical composition of claim 110, wherein the
intranasal aerosol spray comprises microspheres.
113. The pharmaceutical composition of claim 112, wherein the
microspheres are bioadhesive microspheres.
114. The pharmaceutical composition of claim 113, wherein the
microspheres comprise starch, gelatin, dextran, collagen, an
adsorption enhancer, and/or albumin.
115. The pharmaceutical composition of claim 114, wherein the
absorption enhancer is a surfactant.
116. The pharmaceutical composition of claim 109, wherein the nasal
dosage form is an aerosol.
117. The pharmaceutical composition of claim 109, wherein the nasal
dosage form is a microparticle resin.
118. The pharmaceutical composition of claim 117, wherein the
microparticle resin is fractionated sodium polystyrene sulfonate
powder or styrene-divinylbenzene copolymer.
119. The pharmaceutical composition of claim 109, wherein the
composition is formulated to be delivered via transmucosal
delivery.
120. The pharmaceutical composition of claim 119, further
comprising an absorption enhancer.
121. The pharmaceutical composition of claim 120, wherein the
absorption enhancer is a lysophosphatidyl-glycerol compound.
122. The pharmaceutical composition of claim 119, wherein the
composition further comprises a polytetrafluoroethylene support
matrix.
123. The pharmaceutical composition of claim 109, wherein the viral
vector is an adenoviral vector, a retroviral vector, an
adeno-associated virus vector, a vaccinia virus vector, a
herpesvirus vector, or an oncolytic virus vector.
124. The pharmaceutical composition of claim 123, wherein the viral
vector is an adenoviral vector.
125. The pharmaceutical composition of claim 124, wherein the
adenoviral vector is a replication-deficient adenoviral vector.
126. The pharmaceutical composition of claim 125, wherein the
replication-deficient adenoviral vector is lacking at least a
portion of the E1 region.
127. The pharmaceutical composition of claim 123, wherein the
oncolytic virus is a reovirus, OYNX-015, or CN706.
128. The pharmaceutical composition of claim 109, wherein the
promoter is a CMV IE, SV40 early, or RSV LTR.
129. The pharmaceutical composition of claim 109, wherein the viral
vector is comprised within a liposome.
130. The pharmaceutical composition of claim 109, wherein the
therapeutic gene is a tumor suppressor gene, a gene that encodes an
inducer of apoptosis, a gene encoding an enzyme, a gene encoding a
hormone, a gene encoding an interleukin, or a gene encoding a
cytokine.
131. The pharmaceutical composition of claim 130, wherein the tumor
suppressor gene is p53, CDK4 or other cyclin-dependent kinase;
p16.sup.INK4, p16.sup.B, .sup.p21WAF1, CIP1, SDI1, p27.sup.KIP1 or
other CDK-inhibitory protein; C-CAM, RB, APC, DCC, NF-1, NF-2,
WT-1, MEN-I, MEN-II, zacl, p73, BRCA1, VHL, FCC, MMAC1, MCC, p16,
p21, p57, p27, and BRCA2.
132. The pharmaceutical composition of claim 130, wherein the
inducer of apoptosis is Bax, Bak, Bcl-X.sub.s, Bik, Bid, Harakiri,
Ad E1B, Bad, or an ICE-CED2 protease.
133. The pharmaceutical composition of claim 130, wherein the
enzyme is cytosine deaminase, hypoxanthine-guanine
phosphoribosyltransferase, galactose-1-phosphate uridyltransferase,
phenylalanine hydroxylase, glucocerebrosidase, sphingomyelinase,
.alpha.-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV
thymidine kinase, or human thymidine kinase.
134. The pharmaceutical composition of claim 130, wherein the
hormone is growth hormone, prolactin, placental lactogen,
luteinizing hormone, follicle-stimulating hormone, chorionic
gonadotropin, thyroid-stimulating hormone, leptin,
adrenocorticotropin (ACTH), angiotensin I, angiotensin II,
.beta.-endorphin, .beta.-melanocyte stimulating hormone,
cholecystokinin, endothelin I, galanin, gastric inhibitory peptide,
glucagon, insulin, a lipotropin, a neurophysin, somatostatin,
calcitonin, calcitonin gene related peptide, .beta.-calcitonin gene
related peptide, hypercalcemia of malignancy factor, parathyroid
hormone-related protein, glucagon-like peptide, pancreastatin,
pancreatic peptide, peptide YY, PHM, secretin, vasotocin,
enkephalinamide, metorphinamide, alpha melanocyte stimulating
hormone, atrial natriuretic factor, amylin, amyoid P component
(SAP-1), corticotropin releaseing hormone (CRH), growth hormone
releasing factor (GHRH), luteinizing hormone releasing hormone
(LHRH), neuropeptide Y, substance K (neurokinin A), substance P, or
thryrotropin releasing hormone (TRH).
135. The pharmaceutical composition of claim 130, wherein the
interleukin or cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, or G-CSF.
Description
[0001] The present application claims priority to the contents of
U.S. Provisional Patent Application Ser. No. 60/108,606, filed Nov.
16, 1998 and U.S. Provisional Patent Application Ser. No.
60/133,116, filed May 7, 1999. The entire text of the
above-referenced disclosure is specifically incorporated by
reference herein without disclaimer.
BACKGROUND OF THE INVENTION
[0002] A. Field of the Invention
[0003] The present invention relates generally to the fields of
molecular biology, virus production and gene therapy. More
particularly, it concerns methods for the formulation of highly
purified lyophilized and liquid adenovirus particles stable for
long-term storage. An important embodiment of the present invention
is the use of such long-term storage virus preparations for gene
therapy treatments of viral disease, genetic disease and
malignancies.
[0004] B. Description of Related Art
[0005] Viruses are highly efficient at nucleic acid delivery to
specific cell types, while often avoiding detection by the infected
hosts immune system. These features make certain viruses attractive
candidates as gene-delivery vehicles for use in gene therapies
(Robbins and Ghivizzani; 1998; Cristiano et al., 1998). Retrovirus,
adenovirus, adeno-associated virus (AAV), and herpes simplex virus
are examples of commonly used viruses in gene therapies. Each of
the aforementioned viruses has its advantages and limitations, and
must therefore be selected according to suitability of a given gene
therapy (Robbins and Ghivizzani; 1998).
[0006] A variety of cancer and genetic diseases currently are being
addressed by gene therapy. Cardiovascular disease (Morishita et
al., 1998), colorectal cancer (Fujiwara and Tanaka, 1998), lung
cancer (Roth et al., 1998), brain tumors (Badie et al., 1998), and
thyroid carcinoma (Braiden et al., 1998) are examples of gene
therapy treatments currently under investigation. Further, the use
of viral vectors in combination with other cancer treatments also
is an avenue of current research (Jounaidi et al., 1998).
[0007] Viral particles must maintain their structural integrity to
be infectious and biologically active. The structural integrity of
a viral vector often is compromised during the formulation process,
thus precluding its use as a gene therapy vector. Adenoviruses for
gene therapy traditionally have been formulated in buffers
containing 10% glycerol. Formulated adenovirus is stored at
<-60.degree. C. to ensure good virus stability during storage.
This ultra-low temperature storage not only is very expensive, but
creates significant inconvenience for storage, transportation and
clinic use. There is an urgent need to develop new formulation for
adenovirus that can be stored at refrigerated condition.
[0008] Lyophilization has been used widely to improve the stability
of various viral vaccine and recombinant protein products. It is
expected that the long-term storage stability of adenovirus can be
improved by reducing the residual water content (moisture) in the
formulated product through lyophilization. However, there have not
been reported studies on the lyophilization of live adenovirus for
gene therapy.
[0009] Generally it is assumed that adenovirus will not maintain
its infectivity when stored at refrigerated condition in a liquid
form for extended period of time. As a result, there are no
reported studies on formulating and storing adenovirus at
refrigerated condition in a liquid form. Thus, there remains a need
for long-term storage stable formulations of viral
preparations.
SUMMARY OF THE INVENTION
[0010] The present invention addresses the need for improved,
storage stable viral formulations, and methods for the production
thereof, for use in gene therapy. In particular embodiments, a
pharmaceutical adenovirus composition comprising adenovirus
particles and pharmaceutical excipients, the excipients including a
bulking agent and one or more protectants, wherein the excipients
are included in amounts effective to provide an adenovirus
composition that is storage stable. In preferred embodiments, the
adenovirus composition has an infectivity of between 60 and 100% of
the starting infectivity, and a residual moisture of less than
about 5%, when stored for six months at 4.degree. centigrade.
[0011] In one embodiment, the adenovirus composition is a freeze
dried composition. In particular embodiments, the bulking agent in
the freeze dried adenovirus composition forms crystals during
freezing, wherein the bulking agent is mannitol, inositol,
lactitol, xylitol, isomaltol, sorbitol, gelatin, agar, pectin,
casein, dried skim milk, dried whole milk, silcate,
carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methhylcellulose or methylcellulose.
[0012] In certain embodiments, the bulking agent in the freeze
dried adenovirus composition is mannitol. In other embodiments the
composition is further defined as an aqueous composition comprising
mannitol in a concentration of from about 1% to about 10% (w/v). In
another embodiment, the aqueous composition comprises the mannitol
in a concentration of from about 3% to 8%. In a preferred
embodiment, the aqueous composition comprises mannitol in a
concentration of from about 5% to 7%.
[0013] In certain embodiments, the freeze dried composition is
prepared from an aqueous composition comprising a bulking agent in
a concentration of from about 1% to 10% (w/v). In other embodiments
the freeze dried composition is prepared from an aqueous
composition comprising a bulking agent in a concentration of from
about 3% to 8%. In yet other embodiments, the freeze dried
composition is prepared from an aqueous composition comprising a
bulking agent in a concentration of from about 5% to 7%.
[0014] In particular embodiments, pharmaceutical excipients serve
as a protectants. In one embodiment, the protectant is further
defined as a cryoprotectant. In certain embodiments, the
cryoprotectant is a non-reducing sugar. In particularly defined
embodiments the non-reducing sugar is sucrose or trehalose. In
preferred embodiments the non-reducing sugar is sucrose.
[0015] In certain embodiments, the composition is further defined
as an aqueous composition comprising a non-reducing sugar in a
concentration of from about 2% to about 10% (w/v). In other
embodiments, the aqueous composition comprises the sugar in a
concentration of from about 4% to 8%. In still other embodiments,
the aqueous composition comprises the sugar in a concentration of
from about 5% to 6%.
[0016] In one embodiment, the freeze dried composition is prepared
from an aqueous composition comprising a non-reducing sugar in a
concentration of from about 2% to 10% (w/v). In other embodiments,
the freeze dried composition is prepared from an aqueous
composition comprising a non-reducing sugar in a concentration of
from about 4% to 8%. In yet other embodiments, the freeze dried
composition is prepared from an aqueous composition comprising a
non-reducing sugar in a concentration of from about 5% to 6%.
[0017] In another embodiment, the cryoprotectant is niacinamide,
creatinine, monosodium glutamate, dimethyl sulfoxide or sweet whey
solids.
[0018] In certain embodiments, the protectant includes a
lyoprotectant, wherein the lyoprotectant is human serum albumin,
bovine serum albumin, PEG, glycine, arginine, proline, lysine,
alanine, polyvinyl pyrrolidine, polyvinyl alcohol, polydextran,
maltodextrins, hydroxypropyl-beta-cyclode- xtrin, partially
hydrolysed starches, Tween-20 or Tween-80. In a preferred
embodiment, the lyoprotectant is human serum albumin.
[0019] In certain embodiments, the composition is further defined
as an aqueous composition comprising the lyoprotectant in a
concentration of from about 0.5% to about 5% (w/v). In another
embodiment, the aqueous composition comprises the lyoprotectant in
a concentration of from about 1% to about 4%. In still another
embodiment, the aqueous composition comprises the lyoprotectant in
a concentration of from about 1% to about 3%.
[0020] In particular embodiments, the freeze dried composition is
prepared from an aqueous composition comprising a lyoprotectant in
a concentration of from about 0.5% to 5% (w/v). In other
embodiments, the freeze dried composition is prepared from an
aqueous composition comprising a lyoprotectant in a concentration
of from about 1% to 4%. In another embodiment, the freeze dried
composition is prepared from an aqueous composition comprising a
lyoprotectant in a concentration of from about 1% to 3%.
[0021] In one embodiment, pharmaceutical excipients defined as
protectants, comprise both a lyoprotectant and a
cryoprotectant.
[0022] Also contemplated in the present invention is an aqueous
pharmaceutical adenovirus composition comprising a polyol in an
amount effective to promote the maintenance of adenoviral
infectivity. In one embodiment, adenoviral infectivity of the
adenovirus polyol composition is further defined as maintaining an
infectivity of about 70% PFU/mL to about 99.9% PFU/mL of the
starting infectivity when stored for six months at 4.degree.
centigrade. In preferred embodiments, adenoviral infectivity is
about 80% to 95% PFU/mL of the starting infectivity when stored for
six months at 4.degree. centigrade.
[0023] In the context of the present invention, a polyol is defined
as a polyhydric alcohol containing two or more hydroxyl groups. In
certain embodiments, the polyol is glycerol, propylene glycol,
polyethylene glycol, sorbitol or mannitol, wherein the polyol
concentration is from about 5% to about 30% (w/v). In other
embodiments, the polyol concentration is from about 10% to about
30%. In yet other embodiments, the polyol concentration is about
25%.
[0024] In a preferred embodiment, the aqueous pharmaceutical
adenovirus composition comprises a polyol in an amount effective to
promote the maintenance of adenoviral infectivity, wherein the
polyol is glycerol, included in a concentration of from about 5% to
about 30% (w/v).
[0025] In other embodiments, the aqueous pharmaceutical adenovirus
composition comprising a polyol in an amount effective to promote
the maintenance of adenoviral infectivity further comprises an
excipient in addition to the polyol, wherein the excipient is
inositol, lactitol, xylitol, isomaltol, gelatin, agar, pectin,
casein, dried skim milk, dried whole milk, silicate,
carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methhylcellulose, methylcellulose,
sucrose, dextrose, lactose, trehalose, glucose, maltose,
niacinamide, creatinine, monosodium glutamate dimethyl sulfoxide,
sweet whey solids, human serum albumin, bovine serum albumin, PEG,
glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,
polyvinyl alcohol, polydextran, maltodextrins,
hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches,
Tween-20 or Tween-80.
[0026] In further defined embodiments, the aqueous pharmaceutical
adenovirus composition comprising a polyol further comprises in
addition to the polyol at least a first and a second excipient,
wherein the second excipient is different the first excipient, and
the excipient is inositol, lactitol, xylitol, isomaltol, gelatin,
agar, pectin, casein, dried skim milk, dried whole milk, silicate,
carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methhylcellulose, methylcellulose,
sucrose, dextrose, lactose, trehalose, glucose, maltose,
niacinamide, creatinine, monosodium glutamate dimethyl sulfoxide,
sweet whey solids, human serum albumin, bovine serum albumin, PEG,
glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,
polyvinyl alcohol, polydextran, maltodextrins,
hydroxypropyl-beta-cyclode- xtrin, partially hydrolysed starches,
Tween-20 or Tween-80.
[0027] In another embodiment of the present invention, a method for
the preparation of a long-term, storage stable adenovirus
formulation, comprising the steps of providing adenovirus and
combining the adenovirus with a solution comprising a buffer, a
bulking agent, a cryoprotectant and a lyoprotectant; and
lyophilizing the solution, whereby lyophilization of the solution
produces a freeze-dried cake of the adenovirus formulation that
retains high infectivity and low residual moisture.
[0028] In particular embodiments, the bulking agent used for
preparing the freeze dried adenovirus formulation is mannitol,
inositol, lactitol, xylitol, isomaltol, sorbitol, gelatin, agar,
pectin, casein, dried skim milk, dried whole milk, silcate,
carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methhylcellulose or methylcellulose. In
preferred embodiments, the bulking agent is mannitol, wherein
mannitol comprises about 0.5% to about 8% (w/v) of the
formulation.
[0029] In other embodiments, the cryoprotectant used for preparing
the freeze dried adenovirus formulation is sucrose, dextrose,
lactose, trehalose, glucose, maltose, niacinamide, creatinine,
monosodium glutamate dimethyl sulfoxide or sweet whey solids. In
preferred embodiments, the cryoprotectant is sucrose, wherein
sucrose comprises about 2.5% to about 10% (w/v) of said
formulation.
[0030] In further embodiments, the lyoprotectant used for preparing
the freeze dried adenovirus formulation is human serum albumin,
bovine serum albumin, PEG, glycine, arginine, proline, lysine,
alanine, polyvinyl pyrrolidine, polyvinyl alcohol, polydextran,
maltodextrins, hydroxypropyl-beta-cyclodextrin, partially
hydrolysed starches, Tween-20 or Tween-80. In preferred
embodiments, the lyoprotectant is human serum albumin.
[0031] In other embodiments, the buffer used for preparing the
freeze dried adenovirus formulation is Tris-HCl, TES, HEPES,
mono-Tris, brucine tetrahydrate, EPPS, tricine, or histidine,
wherein the buffer is present in the formulation at a concentration
at about 1 mM to 50 mM. In one preferred embodiment, the buffer
used for preparing the freeze dried adenovirus formulation is
Tris-HCl, wherein the Tris-HCl is included in a concentration of
from about 1 mM to about 50 mM. In another embodiment, the Tris-HCl
is included in a concentration of from about 5 mM to about 20 mM.
In still other embodiments, the freeze dried adenovirus formulation
further comprises a salt selected from the group consisting of
MgCl.sub.2, MnCl.sub.2, Ca Cl.sub.2, ZnCl.sub.2, NaCl and KCl.
[0032] In one embodiment, lyophilizing the adenovirus formulation
is carried out in the presence of an inert gas.
[0033] In certain embodiments, the method for preparing the freeze
dried adenovirus formulation, wherein lyophilizing the solution
comprises the steps of, freezing the solution, subjecting the
solution to a vacuum and subjecting the solution to at least a
first and a second drying cycle, whereby the second drying cycle
reduces the residual moisture content of the freeze-dried cake to
less than about 2%.
[0034] In another embodiment, a method for the preparation of a
long-term storage, stable adenovirus liquid formulation, comprising
the steps of providing adenovirus and combining the adenovirus with
a solution comprising a buffer and a polyol, whereby the adenovirus
liquid formulation retains high infectivity.
[0035] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various modifications within the spirit and scope of
the invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0037] FIG. 1. Lyophilization Cycle of Adenovirus.
[0038] FIG. 2. Residual Moisture of Lyophilized Adenovirus After
Secondary Drying at 10.degree. C.
[0039] FIG. 3. Stability of Lyophilized Adenovirus after Secondary
Drying at 110.degree. C.
[0040] FIG. 4. Residual Moisture of Lyophilized Adenovirus After
Secondary Drying at 30.degree. C.
[0041] FIG. 5. Stability of Lyophilized Adenovirus after Secondary
Drying at 30.degree. C.
[0042] FIG. 6. HPLC Analysis of Lyophilized Adenovirus Stored at
Room Temperature.
[0043] FIG. 7. HPLC Analysis of Lyophilized Adenovirus Stored at
4.degree. C.
[0044] FIG. 8. HPLC Analysis of Lyophilized Adenovirus Stored at
-20.degree. C.
[0045] FIG. 9A and FIG. 9B. Addition of DMSO to the formulation for
an adenoviral vector increases the transduction efficiency. Human
NSCLC xenografts were established on the flanks of nude mice.
Animals received intratumoral injection of 2.times.10.sup.10 viral
particles (vp) of Ad-.beta.gal formulated in either PBS+glycerol
(FIG. 9A and FIG. 9B, top panels) or in PBS+glycerol+5% DMSO (FIG.
9A and FIG. 9B, lower panels). Tumors were excised at either 24
(FIG. 9A) or 48 hours (FIG. 9B) post-injection and sectioned for
histochemical analysis of reporter gene expression. Histochemical
analysis was done on multiple sections from the tumor block to
analyze vector transduction and distribution. Two sections for each
formulation are illustrated: one from the tumor periphery (FIG. 9A
and FIG. 9B, left panels) and one from the center of the tumor
(FIG. 9A and FIG. 9B, right panels). In each section both
transduction (as indicated by intensity of blue staining) and
distribution (as indicated by extent of blue staining) were
improved by addition of DMSO to the formulation.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0046] The need for long-term stable virus formulations that can be
stored at or above refrigerated temperatures without losing
infectivity is highly desirable. Traditional methods of ultra-low
temperature storage (<60.degree. C.) of virus preparations often
limit the storage, transportation and clinical applications of
viruses. The inventors have developed optimal lyophilization
formulations for freeze-drying adenovirus in which the freeze-dried
products maintain their stability (i.e., infectivity of 60-100% of
the starting infectivity) and have a residual moisture of less than
about 5% when stored for 6 months at 4.degree. C.
[0047] In another embodiment, the inventors have developed
long-term stable adenovirus formulations for storing adenovirus at
4.degree. C. in a liquid form that maintains stability (i.e.,
infectivity of 60-100% of the starting infectivity) for at least 6
months.
[0048] A. Purification Techniques
[0049] A large scale process for the production and purification of
adenovirus is described in U.S. Ser. No. 08/975,519 filed Nov. 20,
1997 (specifically incorporated herein by reference without
disclaimer). This production process offers not only scalability
and validatability but also virus purity comparable to that
achieved using CsCl gradient ultracentrifugation. This process
involves the preparation of recombinant adenovirus particles, the
process comprising preparing a culture of producer cells by seeding
producer cells into a culture medium, infecting cells in the
culture after mid-log phase growth with a recombinant adenovirus
comprising a selected recombinant gene operably linked to a
promoter, harvesting recombinant adenovirus particles from the cell
culture and removing contaminating nucleic acids. An important
aspect of this process is the removal of contaminating nucleic
acids using nucleases. Exemplary nucleases include Benzonase.RTM.,
Pulmozyme.RTM.; or any other DNase or RNase commonly used within
the art.
[0050] Enzymes such as Benzonaze.RTM. degrade nucleic acid and have
no proteolytic activity. The ability of Benzonase.RTM. to rapidly
hydrolyze nucleic acids makes the enzyme ideal for reducing cell
lysate viscosity. It is well known that nucleic acids may adhere to
cell derived particles such as viruses. The adhesion may interfere
with separation due to agglomeration, change in size of the
particle or change in particle charge, resulting in little if any
product being recovered with a given purification scheme.
Benzonase.RTM. is well suited for reducing the nucleic acid load
during purification, thus eliminating the interference and
improving yield.
[0051] As with all endonuclease, Benzonase.RTM. hydrolyzes internal
phosphodiester bonds between specific nucleotides. Upon complete
digestion, all free nucleic acids present in solution are reduced
to oligonucleotides 2 to 4 bases in length.
[0052] The present invention further employs a number of different
purification techniques to purify viral vectors of the present
invention. Such techniques include those based on sedimentation and
chromatography and are described in more detail herein below.
[0053] 1. Density Gradient Centrifugation
[0054] There are two methods of density gradient centrifugation,
the rate zonal technique and the isopycnic (equal density)
technique, and both can be used when the quantitative separation of
all the components of a mixture of particles is required. They are
also used for the determination of buoyant densities and for the
estimation of sedimentation coefficients.
[0055] Particle separation by the rate zonal technique is based
upon differences in size or sedimentation rates. The technique
involves carefully layering a sample solution on top of a performed
liquid density gradient, the highest density of which exceeds that
of the densest particles to be separated. The sample is then
centrifuged until the desired degree of separation is effected,
i.e., for sufficient time for the particles to travel through the
gradient to form discrete zones or bands which are spaced according
to the relative velocities of the particles. Since the technique is
time dependent, centrifugation must be terminated before any of the
separated zones pellet at the bottom of the tube. The method has
been used for the separation of enzymes, hormones, RNA-DNA hybrids,
ribosomal subunits, subcellular organelles, for the analysis of
size distribution of samples of polysomes and for lipoprotein
fractionations.
[0056] The sample is layered on top of a continuous density
gradient which spans the whole range of the particle densities
which are to be separated. The maximum density of the gradient,
therefore, must always exceed the density of the most dense
particle. During centrifugation, sedimentation of the particles
occurs until the buoyant density of the particle and the density of
the gradient are equal (i.e., where p.sub.p=p.sub.m in equation
2.12). At this point no further sedimentation occurs, irrespective
of how long centrifugation continues, because the particles are
floating on a cushion of material that has a density greater than
their own.
[0057] Isopycnic centrifugation, in contrast to the rate zonal
technique, is an equilibrium method, the particles banding to form
zones each at their own characteristic buoyant density. In cases
where, perhaps, not all the components in a mixture of particles
are required, a gradient range can be selected in which unwanted
components of the mixture will sediment to the bottom of the
centrifuge tube whilst the particles of interest sediment to their
respective isopycnic positions. Such a technique involves a
combination of both the rate zonal and isopycnic approaches.
[0058] Isopycnic centrifugation depends solely upon the buoyant
density of the particle and not its shape or size and is
independent of time. Hence soluble proteins, which have a very
similar density (e.g., p=1.3 g cm.sup.-3 in sucrose solution),
cannot usually be separated by this method, whereas subcellular
organelles (e.g., Golgi apparatus, p=1.11 g cm.sup.-3,
mitochondria, p=1.19 g cm.sup.-3 and peroxisomes, p=1.23 g
cm.sup.-3 in sucrose solution) can be effectively separated.
[0059] As an alternative to layering the particle mixture to be
separated onto a preformed gradient, the sample is initially mixed
with the gradient medium to give a solution of uniform density, the
gradient `self-forming`, by sedimentation equilibrium, during
centrifugation. In this method (referred to as the equilibrium
isodensity method), use is generally made of the salts of heavy
metals (e.g., cesium or rubidium), sucrose, colloidal silica or
Metrizamide.
[0060] The sample (e.g., DNA) is mixed homogeneously with, for
example, a concentrated solution of cesium chloride. Centrifugation
of the concentrated cesium chloride solution results in the
sedimentation of the CsCl molecules to form a concentration
gradient and hence a density gradient. The sample molecules (DNA),
which were initially uniformly distributed throughout the tube now
either rise or sediment until they reach a region where the
solution density is equal to their own buoyant density, i.e. their
isopycnic position, where they will band to form zones. This
technique suffers from the disadvantage that often very long
centrifugation times (e.g., 36 to 48 hours) are required to
establish equilibrium. However, it is commonly used in analytical
centrifugation to determine the buoyant density of a particle, the
base composition of double stranded DNA and to separate linear from
circular forms of DNA.
[0061] Many of the separations can be improved by increasing the
density differences between the different forms of DNA by the
incorporation of heavy isotopes (e.g., .sup.15N) during
biosynthesis, a technique used by Leselson and Stahl to elucidate
the mechanism of DNA replication in Esherichia coli, or by the
binding of heavy metal ions or dyes such as ethidium bromide.
Isopycnic gradients have also been used to separate and purify
viruses and analyze human plasma lipoproteins.
[0062] 2. Chromatography
[0063] Purification techniques are well known to those of skill in
the art. These techniques tend to involve the fractionation of the
cellular milieu (e.g., density gradient centrifugation) to separate
the adenovirus particles from other components of the mixture.
Having separated adenoviral particles from the other components,
the adenovirus may be purified using chromatographic and
electrophoretic techniques to achieve complete purification.
Analytical methods particularly suited to the preparation of a pure
adenoviral particle of the present invention are ion-exchange
chromatography, size exclusion chromatography and polyacrylamide
gel electrophoresis. A particularly efficient purification method
to be employed in conjunction with the present invention is
HPLC.
[0064] Certain aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an adenoviral particle. The term "purified" as
used herein, is intended to refer to a composition, isolatable from
other components, wherein the adenoviral particle is purified to
any degree relative to its naturally-obtainable form. A purified
adenoviral particle therefore also refers to an adenoviral
component, free from the environment in which it may naturally
occur.
[0065] Generally, "purified" will refer to an adenoviral particle
that has been subjected to fractionation to remove various other
components, and which composition substantially retains its
expressed biological activity. Where the term "substantially
purified" is used, this designation will refer to a composition in
which the particle, protein or peptide forms the major component of
the composition, such as constituting about 50% or more of the
constituents in the composition.
[0066] Various methods for quantifying the degree of purification
of a protein or peptide will be known to those of skill in the art
in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number". The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0067] There is no general requirement that the adenovirus, always
be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater -fold purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0068] Of course, it is understood that the chromatographic
techniques and other purification techniques known to those of
skill in the art may also be employed to purify proteins expressed
by the adenoviral vectors of the present invention. Ion exchange
chromatography and high performance liquid chromatography are
exemplary purification techniques employed in the purification of
adenoviral particles and are described in further detail herein
below.
[0069] a. Ion-Exchange Chromatography
[0070] The basic principle of ion-exchange chromatography is that
the affinity of a substance for the exchanger depends on both the
electrical properties of the material and the relative affinity of
other charged substances in the solvent. Hence, bound material can
be eluted by changing the pH, thus altering the charge of the
material, or by adding competing materials, of which salts are but
one example. Because different substances have different electrical
properties, the conditions for release vary with each bound
molecular species. In general, to get good separation, the methods
of choice are either continuous ionic strength gradient elution or
stepwise elution. (A gradient of pH alone is not often used because
it is difficult to set up a pH gradient without simultaneously
increasing ionic strength.) For an anion exchanger, either pH and
ionic strength are gradually increased or ionic strength alone is
increased. For a cation exchanger, both pH and ionic strength are
increased. The actual choice of the elution procedure is usually a
result of trial and error and of considerations of stability. For
example, for unstable materials, it is best to maintain fairly
constant pH.
[0071] An ion exchanger is a solid that has chemically bound
charged groups to which ions are electrostatically bound; it can
exchange these ions for ions in aqueous solution. Ion exchangers
can be used in column chromatography to separate molecules
according to charge; actually other features of the molecule are
usually important so that the chromatographic behavior is sensitive
to the charge density, charge distribution, and the size of the
molecule.
[0072] The principle of ion-exchange chromatography is that charged
molecules adsorb to ion exchangers reversibly so that molecules can
be bound or eluted by changing the ionic environment. Separation on
ion exchangers is usually accomplished in two stages: first, the
substances to be separated are bound to the exchanger, using
conditions that give stable and tight binding; then the column is
eluted with buffers of different pH, ionic strength, or composition
and the components of the buffer compete with the bound material
for the binding sites.
[0073] An ion exchanger is usually a three-dimensional network or
matrix that contains covalently linked charged groups. If a group
is negatively charged, it will exchange positive ions and is a
cation exchanger. A typical group used in cation exchangers is the
sulfonic group, SO.sub.3.sup.-. If an H.sup.+ is bound to the
group, the exchanger is said to be in the acid form; it can, for
example, exchange on H.sup.+ for one Na.sup.+ or two H.sup.+ for
one Ca.sup.2+. The sulfonic acid group is called a strongly acidic
cation exchanger. Other commonly used groups are phenolic hydroxyl
and carboxyl, both weakly acidic cation exchangers. If the charged
group is positive--for example, a quaternary amino group--it is a
strongly basic anion exchanger. The most common weakly basic anion
exchangers are aromatic or aliphatic amino groups.
[0074] The matrix can be made of various material. Commonly used
materials are dextran, cellulose, agarose and copolymers of styrene
and vinylbenzene in which the divinylbenzene both cross-links the
polystyrene strands and contains the charged groups. Table 1 gives
the composition of many ion exchangers.
[0075] The total capacity of an ion exchanger measures its ability
to take up exchangeable groups per milligram of dry weight. This
number is supplied by the manufacturer and is important because, if
the capacity is exceeded, ions will pass through the column without
binding.
1TABLE 1 Matrix Exchanger Functional Group Tradename Dextran Strong
Cationic Sulfopropyl SP-Sephadex Weak Cationic Carboxymethyl
CM-Sephadex Strong Anionic Diethyl-(2- QAE-Sephadex hydroxypropyl)-
aminoethyl Weak Anionic Diethylaminoethyl DEAE-Sephadex Cellulose
Cationic Carboxymethyl CM-Cellulose Cationic Phospho P-cel Anionic
Diethylaminoethyl DEAE-cellulose Anionic Polyethylenimine
PEI-Cellulose Anionic Benzoylated- DEAE(BND)-cellulose
naphthoylated, deiethylaminoethyl Anionic p-Aminobenzyl
PAB-cellulose Styrene- Strong Cationic Sulfonic acid AG 50 divinyl-
benzene Strong Anionic AG 1 Strong Cationic + Strong Sulfonic acid
+ AG 501 Anionic Teramethylammonium Acrylic Weak Cationic
Carboxylic Bio-Rex 70 Phenolic Strong Cationic Sulfonic acid
Bio-Rex 40 Expoxyamine Weak Anionic Tertiary amino AG-3
[0076] The available capacity is the capacity under particular
experimental conditions (i.e., pH, ionic strength). For example,
the extent to which an ion exchanger is charged depends on the pH
(the effect of pH is smaller with strong ion exchangers). Another
factor is ionic strength because small ions near the charged groups
compete with the sample molecule for these groups. This competition
is quite effective if the sample is a macromolecule because the
higher diffusion coefficient of the small ion means a greater
number of encounters. Clearly, as buffer concentration increases,
competition becomes keener.
[0077] The porosity of the matrix is an important feature because
the charged groups are both inside and outside the matrix and
because the matrix also acts as a molecular sieve. Large molecules
may be unable to penetrate the pores; so the capacity will decease
with increasing molecular dimensions. The porosity of the
polystyrene-based resins is determined by the amount of
cross-linking by the divinylbenzene (porosity decreases with
increasing amounts of divinylbenzene). With the Dowex and AG
series, the percentage of divinylbenzene is indicated by a number
after an X--hence, Dowex 50-X8 is 8% divinylbenzene.
[0078] Ion exchangers come in a variety of particle sizes, called
mesh size. Finer mesh means an increased surface-to-volume ration
and therefore increased capacity and decreased time for exchange to
occur for a given volume of the exchanger. On the other hand, fine
mesh means a slow flow rate, which can increase diffusional
spreading. The use of very fine particles, approximately 10 .mu.m
in diameter and high pressure to maintain an adequate flow is
called high-performance or high-pressure liquid chromatography or
simply HPLC.
[0079] Such a collection of exchangers having such different
properties--charge, capacity, porosity, mesh--makes the selection
of the appropriate one for accomplishing a particular separation
difficult. How to decide on the type of column material and the
conditions for binding and elution is described in the following
Examples.
[0080] There are a number of choice to be made when employing ion
exchange chromatography as a technique. The first choice to be made
is whether the exchanger is to be anionic or cationic. If the
materials to be bound to the column have a single charge (i.e.,
either plus or minus), the choice is clear. However, many
substances (e.g., proteins, viruses), carry both negative and
positive charges and the net charge depends on the pH. In such
cases, the primary factor is the stability of the substance at
various pH values. Most proteins have a pH range of stability
(i.e., in which they do not denature) in which they are either
positively or negatively charged. Hence, if a protein is stable at
pH values above the isoelectric point, an anion exchanger should be
used; if stable at values below the isoelectric point, a cation
exchanger is required.
[0081] The choice between strong and weak exchangers is also based
on the effect of pH on charge and stability. For example, if a
weakly ionized substance that requires very low or high pH for
ionization is chromatographed, a strong ion exchanger is called for
because it functions over the entire pH range. However, if the
substance is labile, weak ion exchangers are preferable because
strong exchangers are often capable of distorting a molecule so
much that the molecule denatures. The pH at which the substance is
stable must, of course, be matched to the narrow range of pH in
which a particular weak exchanger is charged. Weak ion exchangers
are also excellent for the separation of molecules with a high
charge from those with a small charge, because the weakly charged
ions usually fail to bind. Weak exchangers also show greater
resolution of substances if charge differences are very small. If a
macromolecule has a very strong charge, it may be impossible to
elute from a strong exchanger and a weak exchanger again may be
preferable. In general, weak exchangers are more useful than strong
exchangers.
[0082] The Sephadex and Bio-gel exchangers offer a particular
advantage for macromolecules that are unstable in low ionic
strength. Because the cross-links in these materials maintain the
insolubility of the matrix even if the matrix is highly polar, the
density of ionizable groups can be made several times greater than
is possible with cellulose ion exchangers. The increased charge
density means increased affinity so that adsorption can be carried
out at higher ionic strengths. On the other hand, these exchangers
retain some of their molecular sieving properties so that sometimes
molecular weight differences annul the distribution caused by the
charge differences; the molecular sieving effect may also enhance
the separation.
[0083] Small molecules are best separated on matrices with small
pore size (high degree of cross-linking) because the available
capacity is large, whereas macromolecules need large pore size.
However, except for the Sephadex type, most ion exchangers do not
afford the opportunity for matching the porosity with the molecular
weight.
[0084] The cellulose ion exchangers have proved to be the best for
purifying large molecules such as proteins and polynucleotides.
This is because the matrix is fibrous, and hence all functional
groups are on the surface and available to even the largest
molecules. In may cases however, beaded forms such as DEAE-Sephacel
and DEAE-Biogel P are more useful because there is a better flow
rate and the molecular sieving effect aids in separation.
[0085] Selecting a mesh size is always difficult. Small mesh size
improves resolution but decreases flow rate, which increases zone
spreading and decreases resolution. Hence, the appropriate mesh
size is usually determined empirically.
[0086] Because buffers themselves consist of ions, they can also
exchange, and the pH equilibrium can be affected. To avoid these
problems, the rule of buffers is adopted: use cationic buffers with
anion exchangers and anionic buffers with cation exchangers.
Because ionic strength is a factor in binding, a buffer should be
chosen that has a high buffering capacity so that its ionic
strength need not be too high. Furthermore, for best resolution, it
has been generally found that the ionic conditions used to apply
the sample to the column (the so-called starting conditions) should
be near those used for eluting the column.
[0087] b. High Performance Liquid Chromatography
[0088] High performance liquid chromatography (HPLC) is
characterized by a very rapid separation with extraordinary
resolution of peaks. This is achieved by the use of very fine
particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most
an hour. Moreover, only a very small volume of the sample is needed
because the particles are so small and close-packed that the void
volume is a very small fraction of the bed volume. Also, the
concentration of the sample need not be very great because the
bands are so narrow that there is very little dilution of the
sample.
[0089] B. Viral Formulation
[0090] Retrovirus, adenovirus, adeno-associated virus, and herpes
simplex virus are the most commonly used viruses in gene therapy
(Robbins and Ghivizzani; 1998). It is contemplated in the present
invention that the preparation of long-term stable adenovirus
vectors that can be stored at or above refrigerated temperatures
would be useful as gene therapy vectors. Viral particles must
maintain their structural integrity to remain infective and
biologically active for use as gene therapy vectors. Current virus
formulations do not readily make it feasible to store or transport
viral vector at or above refrigerated temperatures without
significant loss of viral infectivity.
[0091] The present invention describes long-term stable adenovirus
formulations that can be stored at 4.degree. C. for periods up to 6
months. In one embodiment of the present invention, adenovirus
preparations are formulated for lyophilization and long-term
storage at 4.degree. C. as freeze-dried adenovirus. In another
embodiment, the adenovirus is prepared as a liquid formulation that
is long-term stable at 4.degree. C. An important aspect of both the
lyophilized and liquid adenovirus formulations is the addition of
at least one or more compounds that improve the long-term, storage
stability of the adenovirus.
[0092] The term "compound" in the context of the present invention
includes pharmaceutically acceptable carriers such as bulking
agents, cryoprotectants, lyoprotectants, preservatives, solvents,
solutes and any additional pharmaceutical agents well known in the
art. Buffering agents and other types of pH control can also be
added simultaneously in order to provide for maximum buffering
capacity for the adenovirus formulation. For example, pH changes
that deviate from physiological conditions often result in
irreversible aggregation of proteins (Wetzel, 1992) and viral
capsids (Misselwitz et al., 1995) due to complete or partial
denaturation of the protein. Thus, buffering agents are
particularly important for virus preparations that aggregate or
denature at sub-optimal pH ranges.
[0093] 1. Lyophilized Formulations
[0094] The formulation of lyophilized, long-term storage stable
adenovirus in the present invention requires the presence of one or
more excipients. More particularly, for optimal long-term stability
of lyophilized adenovirus formulations, a bulking agent and one or
more protectants are desirable. It is well known in the art that
loss in virus infectivity often is directly related to
denaturation, self association and aggregation of the viral
particles (Misselwitz et al., 1995; Vanlandschoot et al., 1998;
Sagrera et al., 1998; Lu et al., 1998). In fact, the E. coli heat
shock proteins GroEL/GroES have been shown to both stabilize viral
particles from denaturation and aggregation during high stress
cellular conditions and to facilitate capsid assembly during
non-stressed, normal cellular conditions (Polissi et al., 1995;
Nakonechny and Teschke, 1998).
[0095] The use of bulking agents, cryoprotectants, lyoprotectants
and salts in the present invention are included in the formulation
of lyophilized adenovirus to improve long-term stability (i.e.
infectivity) of the adenovirus freeze-dried products. The
stabilizing effect of the cryoprotectant sucrose against
irreversible denaturation and aggregation has been described
previously as an excluded volume effect (Hall et al., 1995).
Similarly, bulking agents, cryo- and lyoprotectants such as
polyacrylamide gels, agaorse gels, dextran and polyethylene glycol
(PEG) have demonstrated enhanced stabilities of proteins and
nucleic acids in part by excluded volume effects (Fried and
Bromberg, 1997; Vossen and Fried, 1997). The exact mechanistic
details of excluded volume effects are still not clear. A currently
accepted theory is that many of these compounds result in the
preferential hydration of protein molecules (i.e. volume of
exclusion), which tends to stabilize the native versus the
denatured conformation of proteins, and therefore prevents
aggregation. In addition, the presence of low concentrations of
cosolvents (e.g., salts) result in charge screening of proteins and
viral protein coats increasing their solubility in water.
[0096] The use of bulking agents, cryoprotectants, lyoprotectants
and salts in the present invention are contemplated and
demonstrated experimentally to improve the storage stability of
lyophilized adenovirus products. In one embodiment, a bulking agent
and protectants are combined with a buffer comprising
adenovirus.
[0097] Bulking agents, cryoprotectants and lyoprotectants are well
known in the art (Lueckel et al., 1998; Herman et al., 1994; Croyle
et al., 1998; Corveleyn and Remon, 1996). Bulking agents considered
in the present invention are mannitol, inositol, lactitol, xylitol,
isomaltol, sorbitol, gelatin, agar, pectin, casein, dried skim
milk, dried whole milk, silcate, carboxypolymethylene, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methhylcellulose,
methylcellulose and other bulking agents well known in the art.
Cryoprotectants considered are sucrose, dextrose, lactose,
trehalose, glucose, maltose, niacinamide, creatinine, monosodium
glutamate, dimethyl sulfoxide, sweet whey solids, as well as other
known cryoprotectants. Lyoprotectants contemplated for use in the
present invention are human serum albumin, bovine serum albumin,
PEG, glycine, arginine, proline, lysine, alanine, polyvinyl
pyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,
hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches,
Tween-20 and Tween-80. Certain lyoprotectants are also classified
as cryoprotectants and vice versa. For the purpose of the present
invention, cryoprotectants and lyoprotectants are represented as
independent classes of compounds. However, this classification is
only for clarity of the invention and should not limit the person
skilled in the art from using any excipient that stabilizes the
adenovirus formulation. In other embodiments of the present
invention, the term excipient encompasses bulking agents, cryo- and
lyoprotectants. In certain embodiments, salts are included in the
formulation in addition to the aforementioned excipients. The
following salts are considered for use in the present invention
MgCl.sub.2, MnCl.sub.2, CaCl.sub.2, ZnCl.sub.2, NaCl, and KCl, but
should not preclude the use of other salts that improve stability
of the adenovirus formulation.
[0098] In other embodiments of the invention, the lyophilized
adenovirus formulation is dried in the presence of an inert gas or
a combination of inert gasses. The purging of the lyophilization
vessel with an inert gas or gasses, the presence of the inert gas
or gasses during lyophilization of the adenovirus solution and
during the capping of the lyophilization vial after the drying
step, are contemplated to minimize the deleterious effects of
O.sub.2. It is known that residual O.sub.2 leads to oxidation and
degradation of proteins. It is contemplated that purging and
capping of the freeze-dried adenovirus product improves the
long-term storage stability of the adenovirus product. The use of
antioxidants such as .beta.-mercapto ethanol, DTT, citric acid and
the like may also be considered for use in formulations.
[0099] An important aspect of the lyophilization process is a
second drying cycle. The second drying cycle is at a temperature of
30.degree. C. for at least 3.5 hours, which is demonstrated to
reduce the residual moisture of the adenovirus freeze-dried product
to less than 2% water immediately after drying. It is contemplated
that the reduced residual moisture improves the long-term storage
stability of the adenovirus freeze-dried product. Longer drying
times up to 20 hours are thus contemplated to further reduce
residual moisture.
[0100] 2. Liquid Formulations
[0101] The formulation of liquid, long-term storage stable
adenovirus in the present invention requires the presence of a
polyol. A polyol is a polyhydric alcohol containing two or more
hydroxyl groups. For optimal long-term stability of liquid
adenovirus formulations in the present invention, glycerol is used.
In particular embodiments of the invention, the presence 20%
glycerol results in adenovirus stability (80% PFU/mL) for periods
of time at least up to 6 months days when stored at 4.degree.
C.
[0102] Glycerol (glycerin) is one of the oldest and most widely
used excipients in pharmaceutical products. It is a clear,
colorless liquid which is miscible with water and alcohol. Glycerol
is hygroscopic, stable to mild acidic and basic environments and
can be sterilized at temperatures up to 150.degree. C. It is well
known as both a taste masking and cryoprotective agent, as well as
an antimicrobial agent. It has good solubilizing power and is a
commonly used solvent in parenteral formulations. It is considered
to be one of the safest excipients used since it is metabolized to
glucose, or to substances which are involved with triglyceride
synthesis or glycolysis (Frank et al., 1981). It is a GRAS listed
excipient and typically used at levels up to 50% in parenteral
formulations.
[0103] The stabilizing effects of glycerol on protein structure is
well known in the art (Hase et al., 1998; Juranville et al., 1998).
Several studies indicate that glycerol has a similar effect of
viral particles. For example, when competent Haemophilus influenza
bacteria were exposed to purified phage and plated for
transfectants, a 100-fold increase in transfectants was observed
when 32% glycerol was present in the solution (Stuy, 1986) In yet
another study, glycerol was demonstrated to preserve the integrity
of vaccinia virus (Slonin and Roslerova, 1969).
[0104] Other polyols contemplated for use in the present invention
are polyethylene glycol, propylene glycol, sorbitol, mannitol, and
the like. Polyethylene glycols are polymers of ethylene oxide with
the general formula:
HO--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.n--CH.sub.2OH
[0105] where n represents the number of oxyethylene groups. The
PEG's are designated by a numerical value, which is indicative of
the average molecular weight for a given grade. Molecular weights
below 600 are liquids, and molecular weights above 1000 are solids
at room temperature. These polymers are readily soluble in water,
which make them quite useful for parenteral dosage forms. Only PEG
400 and PEG 300 are utilized in parenteral products, typically at
concentrations up to 30% v/v. These polymers are generally regarded
as non-toxic and non-irritating. There are numerous reviews
regarding the pharmaceutical and toxicological characteristics of
these polyols (Smyth et al., 1950; Rowe and Wolf, 1982, Swarbrick
and Boylan, 1990).
[0106] Propylene glycol, a dihydroxy alcohol, is one of the more
common solvents encountered in pharmaceutical cosolvent
formulations, for both parenteral and non-parenteral dosage forms.
PG is generally regarded as non-toxic. It is more hygroscopic than
glycerin, and has excellent solubilizing power for a wide variety
of compounds. In addition, it has excellent bacteriocidal and
preservative properties (Heine et al., 1950). PG is metabolized to
carbon dioxide and water via lactic and pyruvic acid intermediates
and, therefore, not prone to the severe toxicities.
[0107] Sorbitol and mannitol are hexahydric alcohols, consisting of
white, crystalline powders, that are soluble in water. Both are
commonly used excipients in pharmaceutical products with little or
no toxicity associated, as approved by the FDA for food use. The
mechanistics of sorbitol and mannitol protein and viral
stabilization is still not completely understood. Current theories
suggest at least part of the effect is osmotic diuretic (Vanholder,
et al., 1984; de Rizzo, et al., 1988). The use of the polyols
described above are considered exemplary, but should not limit the
skilled artist from selecting other polyols that confer viral
stability for liquid formulations.
[0108] It is also contemplated, in addition to a polyol in the
liquid formulation, that one or possibly two excipients may also be
included. Excipients considered for use in the present invention
are inositol, lactitol, xylitol, isomaltol, gelatin, agar, pectin,
casein, dried skim milk, dried whole milk, silcate,
carboxypolymethylene, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methhylcellulose, methylcellulose,
sucrose, dextrose, lactose, trehalose, glucose, maltose,
niacinamide, creatinine, monosodium glutamate, dimethyl sulfoxide,
sweet whey solids, human serum albumin, bovine serum albumin,
glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,
polyvinyl alcohol, polydextran, maltodextrins,
hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches,
Tween-20 and Tween-80. The choice of a particular excipient is
dependent in some instances on the desired properties of the viral
formulation.
[0109] In particular embodiments, dimethyl sulfoxide (DMSO) is
contemplated for use in the present invention. DMSO has been
demonstrated to enhance the infectivity of adenovirus preparations
by increasing the efficiency of gene transfer (Chikada and Jones,
1999). For example, the infectivity of adenovirus type 2 DNA in 293
cells was increased up to five-fold by the brief treatment of cell
monolayers with 25% DMSO (Chinnadurai et al., 1978) The
stabilization of virus particles via DMSO also has been reported
(Wallis and Melnick, 1968). The present inventors demonstrate that
the intratumoral administration of Ad-p53 is improved when DMSO is
added to 5 or 10% (see FIG. 9). Adenovirus studies via intravesical
administration indicate that an adenoviral vector may be stable in
up to 50% DMSO (WO 98/35554). In other embodiments, a polyol
contemplated for use in the present invention as an enhancer of
adenovirus gene transduction is a polyoxyalkene (U.S. Pat. No.
5,552,309, specifically incorporate herein by reference in its
entirety).
[0110] Thus in particular embodiments, an adenoviral formulation
according to the present invention may also contain DMSO. The
concentration for intratumoral administration may contain from
about 2% to 67% DMSO, preferably from about 5% to 20%. The
concentration for intravesical administration may contain from
about 2% to 67% DMSO, preferably from about 20% to 50%. The
concentration for topical administration may contain from about 2%
to 67% DMSO, preferably from about 10% to 40%. The concentration
for intra-articular administration may contain from about 2% to 67%
DMSO, preferably, from about 5% to 40%. The concentration for
systemic administration may contain from about 2% to 75% DMSO,
preferably from about 50% to 67%.
[0111] Adenovirus polyol formulations of the invention may future
comprise a polyoxamer, such as Polyoxamer 407, at concentrations of
from about 0.5% to 20%, preferably from about 10% to 20%. The
formulation storage stable adenovirus may also contain from about
5% to 40% dimethylacetamide, preferably from about 10% to 25%, Or
it may contain from about 10% to 50% of a polyethylene glycol, such
as polyethylene glycol 400, preferably from about 15% to 50%. Of
course, the formulation of said adenovirus also may contain
combinations of the above components.
[0112] C. Viral Transformation
[0113] The present invention employs, in one example, adenoviral
infection of cells in order to generate therapeutically significant
vectors. Typically, the virus will simply be exposed to the
appropriate host cell under physiologic conditions, permitting
uptake of the virus. Though adenovirus is exemplified, the present
methods may be advantageously employed with other viral vectors, as
discussed below.
[0114] 1. Viral Infection
[0115] a. Adenovirus
[0116] One method for delivery of the recombinant DNA involves the
use of an adenovirus expression vector. Although adenovirus vectors
are known to have a low capacity for integration into genomic DNA,
this feature is counterbalanced by the high efficiency of gene
transfer afforded by these vectors. "Adenovirus expression vector"
is meant to include those constructs containing adenovirus
sequences sufficient to (a) support packaging of the construct and
(b) to ultimately express a recombinant gene construct that has
been cloned therein.
[0117] The vector comprises a genetically engineered form of
adenovirus. Knowledge of the genetic organization or adenovirus, a
36 kb, linear, double-stranded DNA virus, allows substitution of
large pieces of adenoviral DNA with foreign sequences up to 7 kb
(Grunhaus and Horwitz, 1992). In contrast to retrovirus, the
adenoviral infection of host cells does not result in chromosomal
integration because adenoviral DNA can replicate in an episomal
manner without potential genotoxicity. Also, adenoviruses are
structurally stable, and no genome rearrangement has been detected
after extensive amplification.
[0118] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target-cell range and high
infectivity. Both ends of the viral genome contain 100-200 base
pair inverted repeats (ITRs), which are cis elements necessary for
viral DNA replication and packaging. The early (E) and late (L)
regions of the genome contain different transcription units that
are divided by the onset of viral DNA replication. The E1 region
(E1A and E1B) encodes proteins responsible for the regulation of
transcription of the viral genome and a few cellular genes. The
expression of the E2 region (E2A and E2B) results in the synthesis
of the proteins for viral DNA replication. These proteins are
involved in DNA replication, late gene expression and host cell
shut-off (Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by the
major late promoter (MLP). The MLP, (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the mRNA's issued from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNA's for
translation.
[0119] In a current system, recombinant adenovirus is generated
from homologous recombination between shuttle vector and provirus
vector. Due to the possible recombination between two proviral
vectors, wild-type adenovirus may be generated from this process.
Therefore, it is critical to isolate a single clone of virus from
an individual plaque and examine its genomic structure.
[0120] Generation and propagation of the current adenovirus
vectors, which are replication deficient, depend on a unique helper
cell line, designated 293, which was transformed from human
embryonic kidney cells by Ad5 DNA fragments and constitutively
expresses E1 proteins (Graham et al., 1977). Since the E3 region is
dispensable from the adenovirus genome (Jones and Shenk, 1978), the
current adenovirus vectors, with the help of 293 cells, carry
foreign DNA in either the E1, the D3 or both regions (Graham and
Prevec, 1991). In nature, adenovirus can package approximately 105%
of the wild-type genome (Ghosh-Choudhury et al., 1987), providing
capacity for about 2 extra kb of DNA. Combined with the
approximately 5.5 kb of DNA that is replaceable in the E1 and E3
regions, the maximum capacity of the current adenovirus vector is
under 7.5 kb, or about 15% of the total length of the vector. More
than 80% of the adenovirus viral genome remains in the vector
backbone.
[0121] Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells or
other human embryonic mesenchymal or epithelial cells.
Alternatively, the helper cells may be derived from the cells of
other mammalian species that are permissive for human adenovirus.
Such cells include, e.g., Vero cells or other monkey embryonic
mesenchymal or epithelial cells. As stated above, the preferred
helper cell line is 293.
[0122] Racher et al. (1995) have disclosed improved methods for
culturing 293 cells and propagating adenovirus. In one format,
natural cell aggregates are grown by inoculating individual cells
into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the
cell viability is estimated with trypan blue. In another format,
Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is
employed as follows. A cell inoculum, resuspended in 5 ml of
medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer
flask and left stationary, with occasional agitation, for 1 to 4 h.
The medium is then replaced with 50 ml of fresh medium and shaking
initiated. For virus production, cells are allowed to grow to about
80% confluence, after which time the medium is replaced (to 25% of
the final volume) and adenovirus added at an MOI of 0.05. Cultures
are left stationary overnight, following which the volume is
increased to 100% and shaking commenced for another 72 h.
[0123] Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be of
any of the 42 different known serotypes or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the present invention. This is because Adenovirus
type 5 is a human adenovirus about which a great deal of
biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector.
[0124] As stated above, the typical vector according to the present
invention is replication defective and will not have an adenovirus
E1 region. Thus, it will be most convenient to introduce the
transforming construct at the position from which the E1-coding
sequences have been removed. However, the position of insertion of
the construct within the adenovirus sequences is not critical to
the invention. The polynucleotide encoding the gene of interest may
also be inserted in lieu of the deleted E3 region in E3 replacement
vectors as described by Karlsson et al. (1986) or in the E4 region
where a helper cell line or helper virus complements the E4
defect.
[0125] Adenovirus growth and manipulation is known to those of
skill in the art, and exhibits broad host range in vitro and in
vivo. This group of viruses can be obtained in high titers, e.g.,
10.sup.9-10.sup.11 plaque-forming units per ml, and they are highly
infective. The life cycle of adenovirus does not require
integration into the host cell genome. The foreign genes delivered
by adenovirus vectors are episomal and, therefore, have low
genotoxicity to host cells. No side effects have been reported in
studies of vaccination with wild-type adenovirus (Couch et al.,
1963; Top et al., 1971), demonstrating their safety and therapeutic
potential as in vivo gene transfer vectors.
[0126] Adenovirus vectors have been used in eukaryotic gene
expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and
vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec,
1992). Adenoviral vectors also have been described for treatment of
certain types of cancers (U.S. Pat. No. 5,789,244, specifically
incorporated herein by reference in its entirety). Animal studies
have suggested that recombinant adenovirus could be used for gene
therapy (Stratford-Perricaudet and Perricaudet, 1991;
Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in
administering recombinant adenovirus to different tissues include
trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al.,
1992), muscle injection (Ragot et al., 1993), peripheral
intravenous injections (Herz and Gerard, 1993) and stereotactic
inoculation into the brain (Le Gal La Salle et al, 1993).
[0127] b. Retrovirus
[0128] The retroviruses are a group of single-stranded RNA viruses
characterized by an ability to convert their RNA to double-stranded
DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into
cellular chromosomes as a provirus and directs synthesis of viral
proteins. The integration results in the retention of the viral
gene sequences in the recipient cell and its descendants. The
retroviral genome contains three genes, gag, pol, and env that code
for capsid proteins, polymerase enzyme, and envelope components,
respectively. A sequence found upstream from the gag gene contains
a signal for packaging of the genome into virions. Two long
terminal repeat (LTR) sequences are present at the 5' and 3' ends
of the viral genome. These contain strong promoter and enhancer
sequences and are also required for integration in the host cell
genome (Coffin, 1990).
[0129] In order to construct a retroviral vector, a nucleic acid
encoding a gene of interest is inserted into the viral genome in
the place of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol, and env genes but without the
LTR and packaging components is constructed (Mann et al., 1983).
When a recombinant plasmid containing a cDNA, together with the
retroviral LTR and packaging sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the
packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then
secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and stable expression require the division of host cells (Paskind
et al., 1975).
[0130] Concern with the use of defective retrovirus vectors is the
potential appearance of wild-type replication-competent virus in
the packaging cells. This can result from recombination events in
which the intact sequence from the recombinant virus inserts
upstream from the gag, pol, env sequence integrated in the host
cell genome. However, new packaging cell lines are now available
that should greatly decrease the likelihood of recombination
(Markowitz et al., 1988; Hersdorffer et al., 1990).
[0131] c. Adeno-Associated Virus
[0132] Adeno-associated virus (AAV) is an attractive vector system
for use in the present invention as it has a high frequency of
integration and it can infect nondividing cells, thus making it
useful for delivery of genes into mammalian cells in tissue culture
(Muzyczka, 1992). AAV has a broad host range for infectivity
(Tratschin, et al., 1984; Laughlin, et al., 1986; Lebkowski, et
al., 1988; McLaughlin, et al., 1988), which means it is applicable
for use with the present invention. Details concerning the
generation and use of rAAV vectors are described in U.S. Pat. No.
5,139,941 and U.S. Pat. No. 4,797,368, each incorporated herein by
reference.
[0133] Studies demonstrating the use of AAV in gene delivery
include LaFace et al. (1988); Zhou et al. (1993); Flotte et al.
(1993); and Walsh et al. (1994). Recombinant AAV vectors have been
used successfully for in vitro and in vivo transduction of marker
genes (Kaplitt et al., 1994; Lebkowski et al., 1988; Samulski et
al., 1989; Shelling and Smith, 1994; Yoder et al., 1994; Zhou et
al., 1994; Hermonat and Muzyczka, 1984; Tratschin et al., 1985;
McLaughlin et al., 1988) and genes involved in human diseases
(Flotte et al., 1992; Luo et al., 1994; Ohi et al., 1990; Walsh et
al., 1994; Wei et al., 1994). Recently, an AAV vector has been
approved for phase I human trials for the treatment of cystic
fibrosis.
[0134] AAV is a dependent parvovirus in that it requires
coinfection with another virus (either adenovirus or a member of
the herpes virus family) to undergo a productive infection in
cultured cells (Muzyczka, 1992). In the absence of coinfection with
helper virus, the wild-type AAV genome integrates through its ends
into human chromosome 19 where it resides in a latent state as a
provirus (Kotin et al., 1990; Samulski et al., 1991). rAAV,
however, is not restricted to chromosome 19 for integration unless
the AAV Rep protein is also expressed (Shelling and Smith, 1994).
When a cell carrying an AAV provirus is superinfected with a helper
virus, the AAV genome is "rescued" from the chromosome or from a
recombinant plasmid, and a normal productive infection is
established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin
et al., 1990; Muzyczka, 1992).
[0135] Typically, recombinant AAV (rAAV) virus is made by
cotransfecting a plasmid containing the gene of interest flanked by
the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et
al., 1989; each incorporated herein by reference) and an expression
plasmid containing the wild-type AAV coding sequences without the
terminal repeats, for example pIM45 (McCarty et al., 1991;
incorporated herein by reference). The cells are also infected or
transfected with adenovirus or plasmids carrying the adenovirus
genes required for AAV helper function. rAAV virus stocks made in
such fashion are contaminated with adenovirus which must be
physically separated from the rAAV particles (for example, by
cesium chloride density centrifugation). Alternatively, adenovirus
vectors containing the AAV coding regions or cell lines containing
the AAV coding regions and some or all of the adenovirus helper
genes could be used (Yang et al., 1994a; Clark et al., 1995). Cell
lines carrying the rAAV DNA as an integrated provirus can also be
used (Flotte et al., 1995).
[0136] d. Other Viral Vectors
[0137] Other viral vectors may be employed as constructs in the
present invention. Vectors derived from viruses such as vaccinia
virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al.,
1988) and herpesviruses may be employed. They offer several
attractive features for various mammalian cells (Friedmann, 1989;
Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988;
Horwich et al.,-1990).
[0138] With the recent recognition of defective hepatitis B
viruses, new insight was gained into the structure-function
relationship of different viral sequences. In vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggested that large
portions of the genome could be replaced with foreign genetic
material. Chang et al. recently introduced the chloramphenicol
acetyltransferase (CAT) gene into duck hepatitis B virus genome in
the place of the polymerase, surface, and pre-surface coding
sequences. It was cotransfected with wild-type virus into an avian
hepatoma cell line. Culture media containing high titers of the
recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al., 1991).
[0139] Also contemplated for use in the present invention is a
fairly new class of viruses termed oncolytic virus (Pennisi, 1998).
Some of the viruses included in this group are reovirus, the
genetically modified adenovirus OYNX-015 and CN.sub.7O.sub.6. These
oncolytic viruses, which have not been genetically altered to
prevent their replication, destroy certain types of cancer cells by
multiplying and spreading, killing only the cancer cells. Each of
the above oncolytic viruses are proposed to operate via different
pathways involved in cancers.
[0140] For example, human reovirus requires an activated Ras
signaling pathway for infection of cultured cells. Thus, in certain
tumors with an overactive ras gene, reovirus readily replicates. In
a study on reovirus, severe combined immune deficient mice bearing
tumors established from v-erbB-transformed murine NIH 3T3 cells or
human U87 glioblastoma cells were treated with the virus. A single
intratumoral injection of virus resulted in regression of tumors in
65% to 80% of the mice. Treatment of immune-competent C3H mice
bearing tumors established from a ras-transformed C3H-10T1/2 cells
also resulted in tumor regression, although a series of injections
were required (Coffey et al., 1998).
[0141] 2. Vectors and Regulatory Signals
[0142] Vectors of the present invention are designed, primarily, to
transform cells with a gene under the control of regulated
eukaryotic promoters (i.e., inducible, repressable, tissue
specific). Also, the vectors usually will contain a selectable
marker if, for no other reason, to facilitate their production in
vitro. However, selectable markers may play an important role in
producing recombinant cells and thus a discussion of promoters is
useful here. Table 2 and Table 3 below, list inducible promoter
elements and enhancer elements, respectively.
2TABLE 2 Inducible Elements Element Inducer References MT II
Phorbol Ester (TFA) Palmiter et al., 1982; Haslinger and Heavy
metals Karin, 1985; Searle et al., 1985; Stuart et al., 1985;
Imagawa et al., 1987; Karin .RTM., 1987; Angel et al., 1987b;
McNeall et al., 1989 MMTV (mouse Glucocorticoids Huang et al.,
1981; Lee et al., 1981; mammary tumor virus) Majors and Varmus,
1983; Chandler et al., 1983; Lee et al., 1984; Fonta et al., 1985;
Sakai et al., 1986 .beta.-Interferon poly(rI)X Tavernier et al.,
1983 poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins, 1984
Collagenase Phorbol Ester (TPA) Angle et al., 1987a Stromelysin
Phorbol Ester (TFA) Angle et al., 1987b SV40 Phorbol Ester (TFA)
Angel et al., 1987b Murine MX Gene Interferon, Newcastle Disease
Virus GRP78 Gene A23187 Resendez et al., 1988
.alpha.-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum
Rittling et al., 1989 MHC Class I Gene H-2.kappa.b Interferon
Blanar et al., 1989 HSP70 Ela, SV40 Large T Taylor et al, 1989;
Taylor and Antigen Kingston, 1990a, b Proliferin Phorbol Ester-TPA
Mordacq and Linzer, 1989 Tumor Necrosis Factor FMA Hensel et al.,
1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989
Hormone .alpha. Gene
[0143]
3TABLE 3 Other Promoter/Enhancer Elements Promoter/Enhancer
References Immunoglobulin Heavy Chain Hanerji et al., 1983; Gilles
et al., 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry,
1986, 1987; Imler et al., 1987; Weinberger et al., 1988; Kiledjian
et al., 1988; Porton et al., 1990 Immunoglobulin Light Chain Queen
and Baltimore, 1983; Picard and Schaffner, 1984 T-Cell Receptor
Luria et al., 1987, Winoto and Baltimore, 1989; Redondo et al.,
1990 HLA DQ .alpha. and DQ .beta. Sullivan and Peterlin, 1987
.beta.-Interferon Goodbourn et al., 1986; Fujita et al., 1987;
Goodbourn and Maniatis, 1985 Interleukin-2 Greene et al., 1989
Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC
Class II 5 Koch et al., 1989 MHC Class II HLA-DR.alpha. Sherman et
al., 1989 .beta.-Actin Kawamoto et al., 1988; Ng et al., 1989
Muscle Creatine Kinase Jaynes et al., 1988; Horlick and Benfield,
1989; Johnson et al., 1989a Prealbumin (Transthyretin) Costa et
al., 1988 Elastase I Omitz et al., 1987 Metallothionein Karin et
al., 1987; Culotta and Hamer, 1989 Collagenase Pinkert et al.,
1987; Angel et al., 1987 Albumin Gene Pinkert et al., 1987, Tronche
et al., 1989, 1990 .alpha.-Fetoprotein Godbout et al., 1988;
Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987;
Perez-Stable and Constantini, 1990 .beta.-Globin Trudel and
Constantini, 1987 e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986;
Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell
Adhesion Molecule Hirsch et al., 1990 (NCAM) a.sub.1-Antitrypain
Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse or
Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang
et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al.,
1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I
(TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et
al., 1989 Duchenne Muscular Dystrophy Klamut et al., 1990 SV40
Banerji et al., 1981; Moreau et al., 1981; Sleigh and Lockett,
1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and
Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek
et al., 1987; Kuhl et al., 1987 Schaffner et al., 1988 Polyoma
Swartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et
al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;
deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988;
Campbell and Villarreal, 1988 Retroviruses Kriegler and Botchan,
1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b,
1988; Bosze et al., 1986; Miksicek et al., 1986; Celander and
Haseltine, 1987; Thiesen et al., 1988; Celander et al., 1988; Chol
et al., 1988; Reisman and Rotter, 1989 Papilloma Virus Campo et
al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz
et al., 1985; Lusky and Botchan, 1986; Cripe et al., 1987; Gloss et
al., 1987; Hirochika et al., 1987, Stephens and Hentschel, 1987;
Glue et al., 1988 Hepatitis B Virus Bulla and Siddiqui, 1986;
Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and
Lee, 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber
and Cullan, 1988; Jakobovits et al., 1988; Feng and Holland, 1988;
Takebe et al., 1988; Rowen et al, 1988; Berkhout et al., 1989;
Laspia et al., 1989; Sharp and Marciniak, 1989; Braddock et al.,
1989 Cytomegalovirus Weber et al., 1984; Boshart et al., 1985;
Foecking and Hofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook et
al., 1987; Quinn et al., 1989
[0144] Another signal that may prove useful is a polyadenylation
signal (hGH, BGH, SV40).
[0145] The use of internal ribosome binding sites (IRES) elements
are used to create multigene, or polycistronic, messages. IRES
elements are able to bypass the ribosome scanning model of
5'-methylated cap-dependent translation and begin translation at
internal sites (Pelletier and Sonenberg, 1988). IRES elements from
two members of the picornavirus family (polio and
encephalomyocarditis) have been described (Pelletier and Sonenberg,
1988), as well an IRES from a mammalian message (Macejak and Samow,
1991). IRES elements can be linked to heterologous open reading
frames. Multiple open reading frames can be transcribed together,
each separated by an IRES, creating polycistronic messages. By
virtue of the IRES element, each open reading frame is accessible
to ribosomes for efficient translation. Multiple genes can be
efficiently expressed using a single promoter/enhancer to
transcribe a single message.
[0146] As discussed above, in certain embodiments of the invention,
a cell may be identified and selected in vitro or in vivo by
including a marker in the expression construct. Such markers confer
an identifiable change to the cell permitting easy identification
of cells containing the expression construct. Usually, the
inclusion of a drug selection marker aids in cloning and in the
selection of transformants, for example, genes that confer
resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin,
tetracycline and histidinol are useful selectable markers.
Alternatively, enzymes such as herpes simplex virus thymidine
kinase (tk) or chloramphenicol acetyltransferase (CAT) may be
employed.
[0147] The promoters and enhancers that control the transcription
of protein encoding genes in eukaryotic cells are composed of
multiple genetic elements. The cellular machinery is able to gather
and integrate the regulatory information conveyed by each element,
allowing different genes to evolve distinct, often complex patterns
of transcriptional regulation.
[0148] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator proteins.
[0149] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV 40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0150] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between elements
is flexible, so that promoter function is preserved when elements
are inverted or moved relative to one another. In the tk promoter,
the spacing between elements can be increased to 50 bp apart before
activity begins to decline. Depending on the promoter, it appears
that individual elements can function either co-operatively or
independently to activate transcription.
[0151] Enhancers were originally detected as genetic elements that
increased transcription from a promoter located at a distant
position on the same molecule of DNA. This ability to act over a
large distance had little precedent in classic studies of
prokaryotic transcriptional regulation. Subsequent work showed that
regions of DNA with enhancer activity are organized much like
promoters. That is, they are composed of many individual elements,
each of which binds to one or more transcriptional proteins.
[0152] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Aside from this
operational distinction, enhancers and promoters are very similar
entities.
[0153] Promoters and enhancers have the same general function of
activating transcription in the cell. They are often overlapping
and contiguous, often seeming to have a very similar modular
organization. Taken together, these considerations suggest that
enhancers and promoters are homologous entities and that the
transcriptional activator proteins bound to these sequences may
interact with the cellular transcriptional machinery in
fundamentally the same way.
[0154] In any event, it will be understood that promoters are DNA
elements which when positioned functionally upstream of a gene
leads to the expression of that gene. Most transgene constructs of
the present invention are functionally positioned downstream of a
promoter element.
[0155] D. Engineering of Viral Vectors
[0156] In certain embodiments, the present invention further
involves the manipulation of viral vectors. Such methods involve
the use of a vector construct containing, for example, a
heterologous DNA encoding a gene of interest and a means for its
expression, replicating the vector in an appropriate helper cell,
obtaining viral particles produced therefrom, and infecting cells
with the recombinant virus particles. The gene could simply encode
a protein for which large quantities of the protein are desired,
i.e., large scale in vitro production methods. Alternatively, the
gene could be a therapeutic gene, for example to treat cancer
cells, to express immunomodulatory genes to fight viral infections,
or to replace a gene's function as a result of a genetic defect. In
the context of the gene therapy vector, the gene will be a
heterologous DNA, meant to include DNA derived from a source other
than the viral genome which provides the backbone of the vector.
Finally, the virus may act as a live viral vaccine and express an
antigen of interest for the production of antibodies they are
against. The gene may be derived from a prokaryotic or eukaryotic
source such as a bacterium, a virus, a yeast, a parasite, a plant,
or even an animal. The heterologous DNA also may be derived from
more than one source, i.e., a multigene construct or a fusion
protein. The heterologous DNA may also include a regulatory
sequence which may be derived from one source and the gene from a
different source.
[0157] 1. Therapeutic Genes
[0158] p53 currently is recognized as a tumor suppressor gene
(Montenarh, 1992). High levels of mutant p53 have been found in
many cells transformed by chemical carcinogenesis, ultraviolet
radiation, and several viruses, including SV40. The p53 gene is a
frequent target of mutational inactivation in a wide variety of
human tumors and is already documented to be the most
frequently-mutated gene in common human cancers (Mercer, 1992). It
is mutated in over 50% of human NSCLC (Hollestein et al., 1991) and
in a wide spectrum of other tumors.
[0159] The p53 gene encodes a 393-amino-acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The protein is found in normal tissues and cells, but at
concentrations which are generally minute by comparison with
transformed cells or tumor tissue. Interestingly, wild-type p53
appears to be important in regulating cell growth and division.
Overexpression of wild-type p53 has been shown in some cases to be
anti-proliferative in human tumor cell lines. Thus, p53 can act as
a negative regulator of cell growth (Weinberg, 1991) and may
directly suppress uncontrolled cell growth or directly or
indirectly activate genes that suppress this growth. Thus, absence
or inactivation of wild-type p53 may contribute to transformation.
However, some studies indicate that the presence of mutant p53 may
be necessary for full expression of the transforming potential of
the gene.
[0160] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0161] Casey and colleagues have reported that transfection of DNA
encoding wild-type p53 into two human breast cancer cell lines
restores growth suppression control in such cells (Casey et al.,
1991). A similar effect has also been demonstrated on transfection
of wild-type, but not mutant, p53 into human lung cancer cell lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene
and will select against proliferation when transfected into cells
with the mutant gene. Normal expression of the transfected p53 is
not detrimental to normal cells with endogenous wild-type p53.
Thus, such constructs might be taken up by normal cells without
adverse effects. It is thus proposed that the treatment of
p53-associated cancers with wild-type p53 expression constructs
will reduce the number of malignant cells or their growth rate.
Furthermore, recent studies suggest that some p53 wild-type tumors
are also sensitive to the effects of exogenous p53 expression.
[0162] The major transitions of the eukaryotic cell cycle are
triggered by cyclin-dependent kinases, or CDK's. One CDK,
cyclin-dependent kinase 4 (CDK4), regulates progression through the
G.sub.1 phase. The activity of this enzyme may be to phosphorylate
Rb at late G.sub.1. The activity of CDK4 is controlled by an
activating subunit, D-type cyclin, and by an inhibitory subunit,
e.g. p16.sup.INK4, which has been biochemically characterized as a
protein that specifically binds to and inhibits CDK4, and thus may
regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,
1995). Since the p16.sup.INK4 protein is a CDK4 inhibitor (Serrano,
1993), deletion of this gene may increase the activity of CDK4,
resulting in hyperphosphorylation of the Rb protein. p16 also is
known to regulate the function of CDK6.
[0163] p16.sup.INK4 belongs to a newly described class of
CDK-inhibitory proteins that also includes p16.sup.B, p21.sup.WAF1,
CIP1, SDI1, and p27.sup.KIP1. The p16.sup.INK4 gene maps to 9p21, a
chromosome region frequently deleted in many tumor types.
Homozygous deletions and mutations of the p16.sup.INK4 gene are
frequent in human tumor cell lines. This evidence suggests that the
p16.sup.INK4 gene is a tumor suppressor gene. This interpretation
has been challenged, however, by the observation that the frequency
of the p16.sup.INK4 gene alterations is much lower in primary
uncultured tumors than in cultured cell lines (Caldas et al., 1994;
Cheng et al, 1994; Hussussian et al., 1994; Kamb et al., 1994a;
Kamb et al, 1994b; Mori et al, 1994; Okamoto et al., 1994; Nobori
et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration
of wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994; Arap, 1995).
[0164] C-CAM is expressed in virtually all epithelial cells (Odin
and Obrink, 1987). C-CAM, with an apparent molecular weight of 105
kD, was originally isolated from the plasma membrane of the rat
hepatocyte by its reaction with specific antibodies that neutralize
cell aggregation (Obrink, 1991). Recent studies indicate that,
structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily
and its sequence is highly homologous to carcinoembryonic antigen
(CEA) (Lin and Guidotti, 1989). Using a baculovirus expression
system, Cheung et al. (1993a; 1993b and 1993c) demonstrated that
the first Ig domain of C-CAM is critical for cell adhesion
activity.
[0165] Cell adhesion molecules, or CAMs are known to be involved in
a complex network of molecular interactions that regulate organ
development and cell differentiation (Edelman, 1985). Recent data
indicate that aberrant expression of CAMs may be involved in the
tumorigenesis of several neoplasms; for example, decreased
expression of E-cadherin, which is predominantly expressed in
epithelial cells, is associated with the progression of several
kinds of neoplasms (Edelman and Crossin, 1991; Frixen et al., 1991;
Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al.,
1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing expression of .alpha..sub.5.beta..sub.1 integrin by gene
transfer can reduce tumorigenicity of Chinese hamster ovary cells
in vivo. C-CAM now has been shown to suppress tumor growth in vitro
and in vivo.
[0166] Other tumor suppressors that may be employed according to
the present invention include RB, APC, DCC, NF-1, NF-2, WT-1,
MEN-I, MEN-II, zac1, p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21,
p57, C-CAM, p27 and BRCA2. Inducers of apoptosis, such as Bax, Bak,
Bcl-X.sub.s, Bik, Bid, Harakiri, Ad E1B, Bad and ICE-CED3
proteases, similarly could find use according to the present
invention.
[0167] Various enzyme genes are of interest according to the
present invention. Such enzymes include cytosine deaminase,
hypoxanthine-guanine phosphoribosyltransferase,
galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,
glucocerbrosidase, sphingomyelinase, .alpha.-L-iduronidase,
glucose-6-phosphate dehydrogenase, HSV thymidine kinase and human
thymidine kinase.
[0168] Hormones are another group of gene that may be used in the
vectors described herein. Included are growth hormone, prolactin,
placental lactogen, luteinizing hormone, follicle-stimulating
hormone, chorionic gonadotropin, thyroid-stimulating hormone,
leptin, adrenocorticotropin (ACTH), angiotensin I and II,
.beta.-endorphin, .beta.-melanocyte stimulating hormone
(.beta.-MSH), cholecystokinin, endothelin I, galanin, gastric
inhibitory peptide (GIP), glucagon, insulin, lipotropins,
neurophysins, somatostatin, calcitonin, calcitonin gene related
peptide (CGRP), .beta.-calcitonin gene related peptide,
hypercalcemia of malignancy factor (1-40), parathyroid
hormone-related protein (107-139) (PTH-rP), parathyroid
hormone-related protein (107-111) (PTH-rP), glucagon-like peptide
(GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,
secretin, vasoactive intestinal peptide (VIP), oxytocin,
vasopressin (AVP), vasotocin, enkephalinamide, metorphinamide,
alpha melanocyte stimulating hormone (alpha-MSH), atrial
natriuretic factor (5-28) (ANF), amylin, amyloid P component
(SAP-1), corticotropin releasing hormone (CRH), growth hormone
releasing factor (GHRH), luteinizing hormone-releasing hormone
(LHRH), neuropeptide Y, substance K (neurokinin A), substance P and
thyrotropin releasing hormone (TRH).
[0169] Other classes of genes that are contemplated to be inserted
into the vectors of the present invention include interleukins and
cytokines. Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.
[0170] Examples of diseases for which the present viral vector
would be useful include, but are not limited to, adenosine
deaminase deficiency, human blood clotting factor IX deficiency in
hemophilia B, and cystic fibrosis, which would involve the
replacement of the cystic fibrosis transmembrane receptor gene. The
vectors embodied in the present invention could also be used for
treatment of hyperproliferative disorders such as rheumatoid
arthritis or restenosis by transfer of genes encoding angiogenesis
inhibitors or cell cycle inhibitors. Transfer of prodrug activators
such as the HSV-TK gene can be also be used in the treatment of
hyperploiferative disorders, including cancer.
[0171] 2. Antisense Constructs
[0172] Oncogenes such as ras, myc, neu, raf, erb, src, fms, jun,
trk, ret, gsp, hst, bcl and abl also are suitable targets. However,
for therapeutic benefit, these oncogenes would be expressed as an
antisense nucleic acid, so as to inhibit the expression of the
oncogene. The term "antisense nucleic acid" is intended to refer to
the oligonucleotides complementary to the base sequences of
oncogene-encoding DNA and RNA. Antisense oligonucleotides, when
introduced into a target cell, specifically bind to their target
nucleic acid and interfere with transcription, RNA processing,
transport and/or translation. Targeting double-stranded (ds) DNA
with oligonucleotide leads to triple-helix formation; targeting RNA
will lead to double-helix formation.
[0173] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. Antisense RNA constructs, or DNA encoding
such antisense RNAs, may be employed to inhibit gene transcription
or translation or both within a host cell, either in vitro or in
vivo, such as within a host animal, including a human subject.
Nucleic acid sequences comprising "complementary nucleotides" are
those which are capable of base-pairing according to the standard
Watson-Crick complementarity rules. That is, that the larger
purines will base pair with the smaller pyrimidines to form only
combinations of guanine paired with cytosine (G:C) and adenine
paired with either thymine (A:T), in the case of DNA, or adenine
paired with uracil (A:U) in the case of RNA.
[0174] As used herein, the terms "complementary" or "antisense
sequences" mean nucleic acid sequences that are substantially
complementary over their entire length and have very few base
mismatches. For example, nucleic acid sequences of fifteen bases in
length may be termed complementary when they have a complementary
nucleotide at thirteen or fourteen positions with only single or
double mismatches. Naturally, nucleic acid sequences which are
"completely complementary" will be nucleic acid sequences which are
entirely complementary throughout their entire length and have no
base mismatches.
[0175] While all or part of the gene sequence may be employed in
the context of antisense construction, statistically, any sequence
17 bases long should occur only once in the human genome and,
therefore, suffice to specify a unique target sequence. Although
shorter oligomers are easier to make and increase in vivo
accessibility, numerous other factors are involved in determining
the specificity of hybridization. Both binding affinity and
sequence specificity of an oligonucleotide to its complementary
target increases with increasing length. It is contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 or more base pairs will be used. One can readily determine
whether a given antisense nucleic acid is effective at targeting of
the corresponding host cell gene simply by testing the constructs
in vitro to determine whether the endogenous gene's function is
affected or whether the expression of related genes having
complementary sequences is affected.
[0176] In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain C-5
propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of
gene expression (Wagner et al., 1993).
[0177] As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" refers to an RNA-based
enzyme capable of targeting and cleaving particular base sequences
in oncogene DNA and RNA. Ribozymes can either be targeted directly
to cells, in the form of RNA oligo-nucleotides incorporating
ribozyme sequences, or introduced into the cell as an expression
construct encoding the desired ribozymal RNA. Ribozymes may be used
and applied in much the same way as described for antisense nucleic
acids.
[0178] 3. Antigens for Vaccines
[0179] Other therapeutics genes might include genes encoding
antigens such as viral antigens, bacterial antigens, fungal
antigens or parasitic antigens. Viruses include picomavirus,
coronavirus, togavirus, flavirviru, rhabdovirus, paramyxovirus,
orthomyxovirus, bunyavirus, arenvirus, reovirus, retrovirus,
papovavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and
spongiform virus. Preferred viral targets include influenza, herpes
simplex virus 1 and 2, measles, small pox, polio or HIV. Pathogens
include trypanosomes, tapeworms, roundworms, helminths. Also, tumor
markers, such as fetal antigen or prostate specific antigen, may be
targeted in this manner. Preferred examples include HIV env
proteins and hepatitis B surface antigen. Administration of a
vector according to the present invention for vaccination purposes
would require that the vector-associated antigens be sufficiently
non-immunogenic to enable long term expression of the transgene,
for which a strong immune response would be desired. Preferably,
vaccination of an individual would only be required infrequently,
such as yearly or biennially, and provide long term immunologic
protection against the infectious agent.
[0180] 4. Control Regions
[0181] In order for the viral vector to effect expression of a
transcript encoding a therapeutic gene, the polynucleotide encoding
the therapeutic gene will be under the transcriptional control of a
promoter and a polyadenylation signal. A "promoter" refers to a DNA
sequence recognized by the synthetic machinery of the host cell, or
introduced synthetic machinery, that is required to initiate the
specific transcription of a gene. A polyadenylation signal refers
to a DNA sequence recognized by the synthetic machinery of the host
cell, or introduced synthetic machinery, that is required to direct
the addition of a series of nucleotides on the end of the mRNA
transcript for proper processing and trafficking of the transcript
out of the nucleus into the cytoplasm for translation. The phrase
"under transcriptional control" means that the promoter is in the
correct location in relation to the polynucleotide to control RNA
polymerase initiation and expression of the polynucleotide.
[0182] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work; have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0183] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0184] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either cooperatively or independently to activate
transcription.
[0185] E. Pharmaceutical Compositions
[0186] In certain embodiments, the present invention also concerns
formulations of a viral composition for administration to a mammal.
It will also be understood that, if desired, the viral compositions
disclosed herein may be administered in combination with other
agents as well, such as, e.g., various pharmaceutically-active
agents. As long as the compositions do not cause a significant
adverse effect upon contact with the target cells or host tissues,
there is virtually no limit to other components which may also be
included.
[0187] The formulation of pharmaceutically-acceptable excipients
and carrier solutions are well-known to those of skill in the art,
as is the development of suitable dosing and treatment regimens for
using the particular compositions described herein in a variety of
treatment regimens, including e.g., oral, parenteral, intravenous,
intranasal, and intramuscular administration and formulation.
[0188] 1. Injectable Compositions and Delivery
[0189] The pharmaceutical compositions disclosed herein may be
administered parenterally, intravenously, intramuscularly, or even
intraperitoneally as described in U.S. Pat. No. 5,543,158; U.S.
Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically
incorporated herein by reference in its entirety). Solutions of the
active compounds as free base or pharmacologically acceptable salts
may be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0190] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be
prepared in such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0191] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial ad
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0192] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in 1 ml of isotonic NaCl solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site
of infusion, (see for example, "Remington's Pharmaceutical
Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0193] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0194] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug release capsules
and the like.
[0195] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0196] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0197] 2. Oral Compositions and Delivery
[0198] Alternatively, the pharmaceutical compositions disclosed
herein may be delivered via oral administration to an animal, and
as such, these compositions may be formulated with an inert diluent
or with an assimilable edible carrier, or they may be enclosed in
hard- or soft-shell gelatin capsule, or they may be compressed into
tablets, or they may be incorporated directly with the food of the
diet.
[0199] The active compounds may even be incorporated with
excipients and used in the form of ingestible tablets, buccal
tables, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S.
Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No.
5,792,451, each specifically incorporated herein by reference in
its entirety). The tablets, troches, pills, capsules and the like
may also contain the following: a binder, as gum tragacanth,
acacia, cornstarch, or gelatin; excipients, such as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic acid and the like; a lubricant, such as magnesium
stearate; and a sweetening agent, such as sucrose, lactose or
saccharin may be added or a flavoring agent, such as peppermint,
oil of wintergreen, or cherry flavoring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills, or capsules may be coated with
shellac, sugar or both. A syrup of elixir may contain the active
compounds sucrose as a sweetening agent methyl and propylparabens
as preservatives, a dye and flavoring, such as cherry or orange
flavor. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compounds may be
incorporated into sustained-release preparation and
formulations.
[0200] For oral administration the compositions of the present
invention may alternatively be incorporated with one or more
excipients in the form of a mouthwash, dentifrice, buccal tablet,
oral spray, or sublingual orally-administered formulation. For
example, a mouthwash may be prepared incorporating the active
ingredient in the required amount in an appropriate solvent, such
as a sodium borate solution (Dobell's Solution). Alternatively, the
active ingredient may be incorporated into an oral solution such as
those containing sodium borate, glycerin and potassium bicarbonate,
or dispersed in a dentifrice, including: gels, pastes, powders and
slurries, or added in a therapeutically effective amount to a paste
dentifrice that may include water, binders, abrasives, flavoring
agents, foaming agents, and humectants, or alternatively fashioned
into a tablet or solution form that may be placed under the tongue
or otherwise dissolved in the mouth.
[0201] 3. Nasal Delivery
[0202] The administration of agonist pharmaceutical compositions by
intranasal sprays, inhalation, and/or other aerosol delivery
vehicles is also considered. Methods for delivering genes, nucleic
acids, and peptide compositions directly to the lungs via nasal
aerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353
and U.S. Pat. No. 5,804,212 (each specifically incorporated herein
by reference in its entirety), and delivery of drugs using
intranasal microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
F. EXAMPLES
[0203] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Materials and Methods
[0204] Lyophilizer
[0205] A Dura-stop .mu.p lyophilizer (FTSsystems) with in process
sample retrieving device was used. The lyophilizer is equipped with
both thermocouple vacuum gauge and capacitance manometer for vacuum
measurement. Condenser temperature is programmed to reach to
-80.degree. C. Vials were stoppered at the end of each run with a
build-in mechanical stoppering device.
[0206] Residual Moisture Measurement
[0207] Residual moisture in freeze dried product was analyzed by a
Karl-Fisher type coulometer (Mettler DL37, KF coulometer).
[0208] HPLC Analysis
[0209] HPLC analysis of samples was done on a Beckman Gold HPLC
system.
[0210] Vials and Stoppers
[0211] Borosilicate 3 ml with 13 mm opening lyo vials and their
corresponding butyl rubber stoppers (both from Wheaton) were used
for both lyophilization and liquid formulation development. The
stoppered vials were capped with Flip-off aluminum caps using a
capping device (LW312 Westcapper, The West Company).
Example 2
Lyophilization: Initial Cycle and Formulation Development
[0212] There are three main process variables that can be
programmed to achieve optimal freeze-drying. Those are shelf
temperature, chamber pressure, and lyophilization step duration
time. To avoid cake collapse, shelf temperature need to be set at
temperatures 2-3.degree. C. below the glass transition or eutectic
temperature of the frozen formulation. Both the glass transition
and eutectic temperatures of a formulation can be determined by
differential scanning calorimetry (DSC) analysis. Chamber pressure
is generally set at below the ice vapor pressure of the frozen
formulation. The ice vapor pressure is dependent on the shelf
temperature and chamber pressure. Too high a chamber pressure will
reduce the drying rate by reducing the pressure differential
between the ice and the surrounding, while too low a pressure will
also slow down drying rate by reducing the heat transfer rate from
the shelf to the vials. The development of a lyophilization cycle
is closely related with the formulation and the vials chosen for
lyophilization. The goal at this stage was to develop a somewhat
conservative cycle to be able to successfully freeze dry a number
of different formulations. The developed cycles and formulations
will be further optimized when viruses are formulated in the
formulations. Formulation excipient selection was based on the
classical excipients found in most lyophilized pharmaceuticals. The
excipients in a lyophilization formulation should provide the
functions of bulking, cryoprotection, and lyoprotection. The
excipients chosen were mannitol (bulking agent), sucrose (cryo- and
lyoprotectant), and human serum albumin (HSA, lyoprotectant). These
excipients were formulated in 10 mM Tris+1 mM MgCl.sub.2, pH=7.50
at various percentages and filled into the 3 ml vials at a fill
volume of 1 ml. To start with, a preliminary cycle was programmed
to screen a variety of formulations based on the criteria of
residual moisture and physical appearance after drying. The cycle
used is plotted in FIG. 1. Extensive screening was carried out by
variation of the percentages of the individual excipients. Table 4
shows briefly some of the results.
4TABLE 4 Evaluation of Different Formulations Under the Same Cycle
Formulation Moisture M %/S %/HSA % Appearance (% weight) 10/5/0.5
good cake 0.89 5/5/0.5 good cake 1.5 3/5/0.5 loose cake (partial
collapse) 3.4 1/5/0.5 no cake (collapse) 6.4
[0213] The results suggest that a minimum amount of 3% mannitol is
required in the formulation in order to achieve pharmaceutically
elegant cake. The percentages of sucrose in the formulation were
also examined. No significant effect on freeze-drying was observed
at sucrose concentrations of <10%. HSA concentration was kept
constant to 0.5% during the initial screening stage.
[0214] After the evaluation of the formulations, freeze-drying
cycle was optimized by changing the shelf temperature, chamber
vacuum and the duration of each cycle step. Based on the extensive
cycle optimization, the following cycle (cycle #14) was used for
further virus lyophilization development.
[0215] 1. Load sample at room temperature onto shelf.
[0216] 2. Set shelf temperature to -45.degree. C. and freeze
sample. Step time 2 h.
[0217] 3. Set shelf temperature at -45.degree. C., turn vacuum pump
and set vacuum at 400 mT. Step time 5 h.
[0218] 4. Set shelf temperature at -35.degree. C., set vacuum at
200 mT. Step time 13 h.
[0219] 5. Set shelf temperature at -22.degree. C., set vacuum at
100 mT. Step time 15 h.
[0220] 6. Set shelf temperature at -10.degree. C., set vacuum at
100 mT. Step time 5 h.
[0221] 7. Set shelf temperature at 10.degree. C., set vacuum at 100
mT. Step time 4 h.
[0222] 8. Vial stoppering under vacuum.
Example 3
Cycle and Formulation Development with Virus in Formulation
[0223] Effect of Sucrose Concentration in Formulation. Cycle and
formulation were further optimized according to virus recovery
after lyophilization analyzed by both HPLC and plaque forming unit
(PFU) assays. Table 5 shows the virus recoveries immediate after
drying in different formulations using the above drying cycle.
Variation of the percentage of sucrose in the formulation had
significant effect on virus recoveries.
5TABLE 5 Recoveries of Virus After Lyophilization Formulation
Residual Recovery M %/S %/HSA % Appearance moisture (%) 6/0/0.5
good cake 0.44% 0 6/3.5/0.5 good cake 2.2% 56 6/5/0.5 good cake
2.5% 81 6/6/0.5 good cake 2.7% 120 6/7/0.5 good cake 2.8% 120
6/8/0.5 good cake 3.3% 93 6/9/0.5 good cake 3.7% 120
[0224] Residual moisture in the freeze-dried product increased as
the sucrose percentage increased. A minimum sucrose concentration
of 5% is required in the formulation to maintain a good virus
recovery after lyophilization. Similar sucrose effects in
formulation that had 5% instead of 6% mannitol were observed.
However, good virus recovery immediately after drying does not
necessary support a good long-term storage stability. As a result,
formulations having 4 different sucrose concentrations of 6, 7, 8,
and 9%, were incorporated for further evaluation.
[0225] Effect of HSA in Formulation. The contribution of HSA
concentrations in the formulation on virus recovery after drying
was examined using the same freeze drying cycle. Table 6 shows the
results.
6TABLE 6 Effects of HSA Concentration on Lyophilization Formulation
Residual Recovery M %/S %/HSA % Appearance moisture (%) 6/7/0 Good
cake 0.98 83 6/7/0.5 Good cake 1.24 120 6/7/2 Good cake 1.5 110
6/7/5 Good cake 1.7 102
[0226] The results indicate that inclusion of HSA in the
formulation had positive effect on virus recovery after drying.
Concentrations higher than 0.5% did not further improve the virus
recovery post drying. As a result, 0.5% HSA is formulated in all
the lyophilization formulations.
[0227] Cycle Optimization. As indicated in Table 5, relatively high
residual moistures were present in the dried product. Although
there has not been a known optimal residual moisture for freeze
dried viruses, it could be beneficial for long term storage
stability to further reduce the residual moisture in the dried
product. After reviewing of the drying cycle, it was decided to
increase the secondary drying temperature from 10.degree. C. to
30.degree. C. without increasing the total cycle time. As indicated
in Table 7, significant reduction in residual moisture had been
achieved in all the formulations without negative effects on virus
recoveries. With the improved drying cycle, residual moisture was
less than 2% in all the formulations immediately after drying. It
is expected that the reduced residual moisture will improve the
long-term storage stability of the dried product.
7TABLE 7 Effects of Secondary Drying Temperature on Lyophilization
Secondary Secondary drying at 10.degree. C. drying at 30.degree. C.
Formulation Residual Recovery Residual M %/S %/HSA % moisture (w %)
(%) moisture Recovery 6/6/0.5 2.2 100 0.8 93 6/7/0.5 2.5 86 1.1 100
6/8/0.5 2.7 83 1.3 87 6/9/0.5 3.3 93 1.5 86 5/6/0.5 2.3 110 1.0 94
5/7/0.5 2.7 88 1.2 85 5/8/0.5 3.5 97 1.6 88 5/9/0.5 4 90 1.9 86
[0228] N.sub.2 Backfilling (Blanketing). Lyophilization was done
similarly as above except that dry N.sub.2 was used for gas
bleeding for pressure control during the drying and backfilling at
the end of the cycle. At the end of a drying run, the chamber was
filled with dry N.sub.2 to about 80% atmospheric pressure.
Subsequently, the vials were stoppered. No difference was noticed
between the air and N.sub.2 blanketing runs immediate after drying.
However, if oxygen present in the vial during air backfilling
causes damaging effect (oxidation) on the virus or excipients used
during long-term storage, backfilling with dry N.sub.2 is likely to
ameliorate the damaging effects and improve long term storage
stability of the virus.
[0229] Removal of Glycerol From Formulation. During the preparation
of virus containing formulations, stock virus solution was added to
the pre-formulated formulations at a dilution factor of 10. Because
of the presence of 10% glycerol in the stock virus solution, 1%
glycerol was introduced into the formulations. To examine any
possible effect of the presence of 1% glycerol on lyophilization, a
freeze drying run was conducted using virus diafiltered into the
formulation of 5% (M)/7% (S)/0.5% (HSA). Diafiltration was done
with 5 vol. of buffer exchange using a constant volume buffer
exchange mode to ensure adequate removal of residual glycerol (99%
removal). After diafiltration, virus solution was filled into vials
and then lyophilized similarly. Table 8 shows the lyophilization
results.
8TABLE 8 Lyophilization without Glycerol Formulation M %/S %/HSA %
Residual moisture Recovery (%) 5/7/0.5 1.0 80
[0230] No significant difference after freeze drying was observed
between formulations with and without 1% glycerol. Possible
implications of this change on long term storage will be
evaluated.
Example 4
Long Term Storage Stability Study
[0231] Adp53 virus lyophilized under different formulations and
different cycles was placed at -20.degree. C., 4.degree. C., and
room temperature (RT) under dark for long term storage stability
evaluation. Parameters measured during the stability study were
PFU, HPLC viral particles, residual moisture, and vacuum inside
vial. The plan is to be able to evaluate virus stability at various
conditions for up to one-year storage. Table 6 shows the data after
12-month storage with secondary drying at 10.degree. C. without
N.sub.2 blanketing. Lyophilized virus is stable at both -20.degree.
C. and 4.degree. C. storage for up to 12 months. However, virus was
not stable at room temperature storage. More than 50% loss in
infectivity was observed at RT after 1-month storage. The reason
for the quick loss of infectivity at RT is not clear. However, it
is likely that RT is above the glass transition temperature of the
dried formulation and resulting in the accelerated virus
degradation. A differential scanning caloremitry (DSC) analysis of
the formulation could provide very useful information. Pressure
change inside the vials during storage was not detected, which
indicates that the vials maintained their integrity. The slight
increase in residual moisture during storage can be attributed to
the release of moisture from the rubber stopper into the dried
product.
9TABLE 9 Secondary Drying at 10.degree. C. Formulation Set 10 (6-9)
PFU .times. 10.sup.9/ml HPLC Viral Particle (.times.10.sup.10/ml)
Water Content (W %) Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set
10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9
Apr. 11, 1997 5.5 6.0 5.8 6.5 24.5 24.6 24.9 26.7 2.2 2.5 2.7 3.3
May 15, 1997.sup.a 7.6 7.1 7.5 8.1 22.4 25.6 26.8 28.5 2.2 2.5 2.8
3.3 May 15, 1997.sup.b 6.6 6.3 6.5 10.0 22.0 23.0 24.0 27.5 2.4 2.6
3.0 3.4 May 15, 1996.sup.c 7.1 7.1 6.7 3.3 14.5 16.5 6.2 4.2 2.7
2.9 3.2 3.5 Jul. 18, 1997.sup.a 6.8 6.4 6.8 7.2 28.7 28.9 28.6 31.2
2.3 2.5 2.8 3.3 Jul. 18, 1997.sup.b 6.0 5.8 7.3 9.0 25.0 26.6 27.6
31.1 2.5 2.8 3.0 3.6 Jul. 18, 1997.sup.c 1.2 0.8 4.0 1.4 0.9 1.8
0.7 0.7 2.7 2.9 3.0 3.4 Oct. 22, 1997.sup.a 7.9 7.5 7.9 7.8 25.5
25.0 25.4 26.2 2.4 2.6 2.8 3.1 Oct. 22, 1997.sup.b 6.8 6.8 5.8 8.0
22.0 23.0 24.7 24.2 2.7 2.9 3.2 3.6 Oct. 22, 1997.sup.c <0.01
<0.01 <0.01 <0.01 N.D. N.D. N.D. N.D. 2.7 2.9 3.1 3.4 Apr.
16, 1998.sup.a 6.0 5.8 7.1 7.2 19.3 20.3 23.5 26.1 2.4 2.6 3.0 3.4
Apr. 16, 1998.sup.b 5.4 7.2 6.1 6.3 21.7 22.8 22.9 24.6 2.9 3.1 3.3
3.8 Apr. 16, 1998.sup.c 0.0003 0.001 0.0007 0.001 N.D. N.D. N.D.
N.D. 2.7 2.9 3.1 3.4 *Temp. .sup.a(-20.degree. C.) .sup.b(4.degree.
C.) .sup.c(r.t.) Controls PFU .times. 10.sup.9/ml HPLC Viral
Particle (.times.10.sup.10/ml) Date Set 10-6 Set 10-7 Set 10-8 Set
10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 Apr. 11, 1997 5.5 7.0 7.0
7.0 35.5 35.8 36.0 36.9 N.D.: not detectable Formulation set 10:
6%-mannitol. 0.5% HSA, 1% glycerol and different percentages of
sucrose in 10 mM-tris buffer pH = 7.5, 1 mM MgCl.sub.2 Formulation
Set 11 (6-9) PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Water Content (W %) Date* Set 11-6 Set 11-7
Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set
11-7 Set 11-8 Set 11-9 May 2, 1997 7.0 6.0 6.3 5.8 28.5 28.8 28.4
29.5 2.3 2.7 3.5 4.0 Jun. 20, 1997.sup.a 6.2 6.6 6.9 65 26.4 25.0
27.0 27.3 2.2 2.8 34 4.6 Jun. 20, 1997.sup.b 6.1 6.0 6.5 6.5 24.1
22.1 25.6 26.6 2.5 2.8 3.5 4.8 Jun. 20, 1997.sup.c 3.3 3.0 1.0
<0.1 20.5 17.4 5.2 9.1 2.7 3.1 3.5 4.7 Aug. 18, 1997.sup.a 8.0
7.2 7.5 7.6 21.6 21.8 25.3 24.9 2.3 2.8 3.7 4.9 Aug. 18, 1997.sup.b
8.0 7.3 8.0 8.0 22.7 22.7 24.9 25.0 2.6 3 3.9 4.2 Aug. 18,
1997.sup.c <0.1 <0.1 <0.1 <0.1 N.D. N.D. 0.2 13.1 2.7
3.0 3.5 4.4 Oct. 22, 1997.sup.a 79 7.5 7.9 6.7 21.0 22.0 25.1 24.0
2.4 3.0 3.9 4.4 Oct. 22, 1997.sup.b 6.0 6.9 6.8 7.3 21.4 22.0 23.1
23.1 2.6 3.0 3.3 4.6 Oct. 22, 1997.sup.c <0.01 <0.01 <0.01
<0.015 N.D. N.D. N.D. 9.0 2.7 2.9 3.9 5.0 May 8, 1998.sup.a 8.3
7.5 8.0 8.7 19.0 18.2 19.9 21.1 2.6 3.1 4.0 4.6 May 8, 1998.sup.b
7.0 7.1 7.8 6.5 17.3 17.1 18.2 17.8 2.8 3.2 4.1 5.1 May 8,
1998.sup.c 0.00033 0.000065 0.00045 0.000016 N.D. N.D. N.D. N.D.
2.7 2.9 4.0 4.9 *Temp. .sup.a(-20.degree. C.) .sup.b(4.degree. C.)
.sup.c(R.T.) Controls PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Date Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set
11-6 Set 11-7 Set 11-8 Set 11-9 May 2, 1997 6.4 6.8 6.5 6.5 37.7
35.7 37.3 36.0 N.D.: not detectable Formulation set 11:
5%-mannitol, 0.5% HSA, 1%-glycerol and different percentages of
sucrose in 10 mM-tris buffer (pH = 7.5, 1 mM MgCl.sub.2)
F11-(6-9)R1-S
[0232]
10TABLE 10 Secondary Drying at 30.degree. C. Without N.sub.2
Blanketing Formulation Set 10 (6-9) PFU .times. 10.sup.9/ml HPLC
Viral Particle (.times.10.sup.10/ml) Water Content (W %) Date* Set
10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9
Set 10-6 Set 10-7 Set 10-8 Set 10-9 May 15, 1997 6.5 5.6 6.1 6.0
18.0 18.6 21.9 23.3 0.8 1.1 1.3 1.5 Jun. 20, 1997.sup.b 5.4 5.6 5.5
5.5 14.6 14.9 17.2 16.6 0.8 1.2 1.5 1.6 Jun. 20, 1997.sup.c 4.5 5.0
5.5 6.0 10.8 11.8 15.0 15.4 1.3 1.4 1.6 1.9 Aug. 18, 1997.sup.b 7.0
6.7 6.8 7.0 15.3 17.1 17.9 17.7 1.3 1.5 1.5 1.7 Aug. 18, 1997.sup.c
2.4 2.2 4.8 5.8 4.3 7.2 11.7 14.2 1.3 1.6 1.7 2.1 Nov. 20,
1997.sup.b 5.5 5.5 5.3 5.7 21.9 21.9 27.2 26.4 1.1 1.4 1.6 1.9 Nov.
20, 1997.sup.c 0.5 0.9 2.3 3.1 1.5 6.3 8.8 13.5 1.3 1.7 1.8 2.2 May
14, 1998.sup.ab 4.9 4.7 5.4 6.5 9.7 11.9 12.6 14.2 1.2 1.6 2.2 1.4
May 14, 1998.sup.c 0.000006 0.00006 0.00004 0.000024 N.D. N.D. N.D.
N.D. 1.4 1.6 1.3 2.0 *Temp. .sup.ab(4.degree. C.) .sup.c(R.T.)
Controls PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Date Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set
10-6 Set 10-7 Set 10-8 Set 10-9 May 15, 1997 7.0 5.6 7.0 7.0 31.2
30.6 31.6 31.4 Formulation set 10: 6%-mannitol, 0.5% HSA,
1%-glycerol and different percentages of sucrose in 10 mM-tris
buffer (pH = 7.5, 1 mM MgCl.sub.2) F10(6-9)R2-S Formulation Set 11
(6-9) PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Water Content (W %) Date* Set 11-6 Set 11-7
Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set
11-7 Set 11-8 Set 11-9 May 22, 1997 7.5 6.3 7.3 6.5 17.4 16.6 20.3
24.7 1.0 1.2 1.6 1.9 Jun. 20, 1997.sup.b 5.5 6.3 6.0 7.5 14.8 16.1
17.5 21.1 1.2 1.3 1.7 1.8 Jun. 20, 1997.sup.c 5.0 6.0 6.0 7.5 12.6
14.9 17.2 20.3 1.4 1.6 1.9 2.0 Aug. 18, 1997.sup.b 6.3 6.7 68 7.5
15.7 17.2 18.5 22.6 1.2 1.5 1.8 1.9 Aug. 18, 1997.sup.c 3.3 4.5 5.5
7.0 7.4 10.5 15.8 21.2 1.6 1.7 1.9 2.2 Nov. 20, 1997.sup.b 5.3 5.6
5.3 6.6 22.6 26.4 30.0 35.0 1.2 1.4 1.9 1.9 Nov. 20, 1997.sup.c 0.8
1.9 3.0 0.1 3.2 9.6 18.3 1.3 1.6 1.7 2.0 2.1 May 14, 1998.sup.b 6.7
7.2 6.9 7.6 12.4 13.9 15.5 18.5 1.3 1.6 2.0 2.2 May 14, 1998.sup.c
0.0013 0.00005 0.00031 0.00045 N.D. N.D. N.D. N.D. 1.6 1.8 1.6 2.0
*Temp. .sup.a(-20.degree. C.) .sup.b(4.degree. C.) .sup.c(R.T.)
Controls PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Date Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set
11-6 Set 11-7 Set 11-8 Set 11-9 May 22, 1997 8.0 7.4 8.3 7.6 26.7
27.6 27.5 32.4 Formulation set 11: 5%-mannitol, 0.5% HSA,
1%-glycerol and different percentages of sucrose in 10 mM-tris
buffer (pH = 7.5, 1 mM MgCl.sub.2) F11-(6-9)R2-S
[0233]
11TABLE 11 Secondary Drying at 30.degree. C. With N.sub.2
Blanketing Formulation Set 10 (6-9) + Adp53 PFU .times. 10.sup.9/ml
HPLC Viral Particle (.times.10.sup.10/ml) Water Content (W %) Date*
Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set
10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 Jun. 13, 1997 3.4 4.3 4.1
4.2 16.0 16.5 16.1 18.1 0.8 1.1 1.3 1.4 Jul. 18, 1997.sup.b 6.3 6.3
6.0 6.0 17.9 19.5 19.9 20.6 0.9 1.2 1.4 1.6 Jul. 18, 1997.sup.c 4.1
5.5 5.0 5.5 11.4 15.5 18.2 20.6 1.2 1.4 1.7 1.8 Sep. 16, 1997.sup.b
4.2 5.5 4.5 5.1 15.3 16.1 16.4 17.8 1.0 1.3 1.5 1.7 Sep. 16,
1997.sup.c 0.7 1.2 5.0 4.0 2.9 5.0 9.5 13.0 1.3 1.5 1.8 2.0 Dec. 4,
1997.sup.b 5.5 5.3 5.4 5.9 16.1 16.2 18.1 18.5 1.1 1.4 1.6 1.7 Dec.
4, 1997.sup.c 0.3 0.5 2.5 3.4 N.D. 1.7 4.7 8.8 1.4 1.6 1.8 2.0 Jun.
29, 1998.sup.ab 3.8 5.1 5.3 5.4 10.6 10.8 12.0 12.9 1.3 1.5 1.8 1.9
Jun. 29, 1998.sup.c 0.00003 0.00006 0.0001 0.0001 N.D. N.D. N.D.
N.D. 1.4 1.6 1.7 1.8 *Temp. .sup.ab(4.degree. C.) .sup.c(R.T.)
Controls PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Date Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set
10-6 Set 10-7 Set 10-8 Set 10-9 Jun. 13, 1997 4.7 3.8 5.5 6.2 26.0
26.2 27.4 27.5 Formulation set 10: 6%-mannitol, 0.5% HSA,
1%-glycerol and different percentages of sucrose in 10 mM-tris
buffer (pH: 7.5, 1 mM MgCl.sub.2) F11-(6-9)R3-S Formulation set 11
(6-9) + Adp53 PFU .times. 10.sup.9/ml HPLC Viral Particle
(.times.10.sup.10/ml) Water Content (W %) Date* Set 11-6 Set 11-7
Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set
11-7 Set 11-8 Set 11-9 Jun. 13, 1997 3.4 4.2 3.6 4.4 16.1 16.3 18.4
19.3 0.9 1.3 1.8 1.9 Jul. 18, 1997.sup.b 5.5 6.2 6.5 6.2 18.0 19.5
23.0 23.9 1.0 1.4 1.8 2.1 Jul. 18, 1997.sup.c 3.7 6.0 6.7 7.3 13.7
18.7 21.8 22.8 1.3 1.7 2.0 2.2 Sep. 16, 1997.sup.b 3.9 4 4.6 6 15.6
17.3 19.5 20.6 1.3 1.5 1.9 2.1 Sep. 16, 1997.sup.c 0.78 2.2 4.0 5.3
3.6 6.8 13.8 14.6 1.5 1.9 2.3 2.4 Dec. 4, 1997.sup.b 4.6 5.3 8.0
6.1 15.7 18.2 21.4 21.6 1.2 1.6 2.1 2.2 Dec. 4, 1997.sup.c 0.4 0.6
0.3 0.01 N.D. N.D. 1.7 N.D. 1.6 1.8 2.1 2.1 Jun. 29, 1998.sup.ab
4.9 5.0 5.4 6.4 11.4 14.2 13.7 16.0 1.5 1.7 2.1 2.6 Jun. 29,
1998.sup.c 0.0001 0.00015 0.00085 0.0012 N.D. N.D. N.D. N.D. 1.6
1.7 2.2 2.3 *Temp. .sup.ab (4.degree. C.) .sup.c (R.T.) Controls
PFU .times. 10.sup.9/ml HPLC Viral Particle (.times.10.sup.10/ml)
Date Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8
Set 11-9 Jun. 13, 1997 4.5 5.0 4.0 5.0 26.5 26.9 26.6 27.1
Formulation set 11: 6%-mannitol, 0.5% HSA, 1%-glycerol and
different percentages of sucrose in 10 mM-tris buffer (pH = 7.5, 1
mM MgCl.sub.2) F11-(6-9)R3-S
[0234]
12TABLE 12 Aqueous Formulation Set #1 5%-T + Date 5%-S + 5%-S + 1%-
(Storage Conds.) 10%-G 5%-HSA 1%-PEG PEG PFU .times.10.sup.9/ml
Aug. 1, 1997 5.8 4.7 4.3 4.4 Aug. 28, 1997 (4.degree. C., N.sub.2)
5.8 5.8 6.4 6.3 Aug. 28, 1997 (4.degree. C.,. Air) 5.0 5.9 6.0 5.9
Aug. 28, 1997 (R.T., N.sub.2) 4.4 4.8 5.0 6.0 Aug. 28, 1997 (R.T.,
Air) 4.3 5.0 5.0 5.6 Oct. 30, 1997 (4.degree. C., N.sub.2) 3.8 4.0
4.7 3.8 Oct. 30, 1997 (4.degree. C. Air) 3.0 4.1 3.7 4.7 Oct. 30,
1997 (R.T., N2) 1.5 3.4 3.5 3.6 Oct. 30, 1997 (R.T., Air) 1.5 3.6
2.2 3.1 Jan. 12, 1998 (4.degree. C., N.sub.2) 3.2 4.1 3.3 3.4 Jan.
12, 1998 (4.degree. C., Air) 1.5 3.8 3.9 3.4 Jan. 12, 1998 (R.T.,
N.sub.2) 0.1 1.4 0.7 0.7 Jan. 12, 1998 (R.T., Air) 0.4 1.6 1.0 0.4
Apr. 30, 1998 (4.degree. C., N.sub.2) 0.08 4.3 4.0 5.3 Apr. 30,
1998 (4.degree. C., Air) 1.5 3.6 4.4 4.5 Apr. 30, 1998 (R.T.,
N.sub.2) 0.0025 0.23 0.11 0.17 Apr. 30, 1998 (R.T., Air) 0.0015
0.21 0.063 0.007 Feb. 5, 1999 (4.degree. C., N.sub.2) 0.0005 5.8
4.1 3.9 Feb. 5, 1999 (4.degree. C., Air) 0.02 4.7 4.3 4.5 Feb. 5,
1999 (R.T., N.sub.2) .sup. <10.sup.2 0.0007 .sup. <10.sup.4
0.0002 Feb. 5, 1999 (R.T., Air) 2 .times. 10.sup.2 0.0002 0.0003 2
.times. 10.sup.3 HPLC Viral Particle (.times.10.sup.10/ml) Aug. 1,
1997 16.9 14.5 16.1 16.7 Aug. 28, 1997 (4.degree. C., N.sub.2) 13.3
14.9 13.8 13.4 Aug. 28, 1997 (4.degree. C., Air) 12.9 14.2 12.9
12.9 Aug. 28, 1997 (R.T., N.sub.2) 12.6 14.5 13.5 12.9 Aug. 28,
1997 (R.T., Air) 12.3 13.7 13.0 13.0 Oct. 30, 1997 (4.degree. C.,
N.sub.2) 14.0 15.5 14.7 14.8 Oct. 30, 1997 (4.degree. C., Air) 12.6
14.9 14.3 14.4 Oct. 30, 1997 (R.T., N.sub.2) 13.8 15.1 14.6 14.4
Oct. 30, 1997 (R.T., Air) 12.7 14.7 14.8 14.4 Jan. 12, 1998
(4.degree. C., N.sub.2) 7.3 11.1 9.5 9.5 Jan. 12, 1998 (4.degree.
C., Air) 7.7 10.8 10.2 10.0 Jan. 12, 1998 (R.T., N2) 10.0 10.8 11.1
10.4 Jan. 12, 1998 (R.T., Air) 9.9 11.0 10.0 10.4 Apr. 30, 1998
(4.degree. C., N.sub.2) 5.1 12.3 12.3 12.1 Apr. 30, 1998 (4.degree.
C., Air) 5.0 11.6 11.8 11.9 Apr. 30, 1998 (R.T., N.sub.2) 11.1 12.3
12.6 12.5 Apr. 30, 1998 (R.T., Air) 11.0 12.4 12.3 11.0 Feb. 5,
1999 (4.degree. C., N.sub.2) 3.4 5.8 11.4 11.0 Feb. 5, 1999
(4.degree. C., Air) 3.9 7.1 11.0 11.2 Feb. 5, 1999 (R.T., N.sub.2)
10.1 7.9 8.5 10.9 Feb. 5, 1999 (R.T., Air) 9.7 7.1 10.3 9.3 G:
glycerol S: sucrose PEG: PEG-3500 T: trehalose Glycerol: 10%
glycerol in DPBS buffer Other formulations are in 10 mM-tris buffer
with 0.15 M-NaCl and 1 mM-MgCl.sub.2 (pH = 8.2).
[0235]
13TABLE 13 Aqueous Formulation Set #2 PFU .times. 10.sup.9/ml Date
(Temp.) AQF2-1 AQF2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6 AQF2-7* Sep. 25,
1997 2.8 2.8 2.8 3.0 2.8 2.8 2.7 Nov. 05, 1997 (4.degree. C.) 2.3
3.2 2.4 3.6 2.7 2.0 3.6 Nov. 05, 1997 (R.T.) 1.4 1.9 1.3 1.5 2.4
2.5 3.1 Dec. 12, 1997 (4.degree. C.) 2.2 0.1 2.4 2.7 2.1 2.1 3.2
Jan. 09, 1998 (R.T.) 1.2 0.1 0.2 1.2 0.2 0.1 1.3 PFU .times.
10.sup.9/ml Date (Temp.) AQF2-8* AQF2-9* AQF2-10* AQF2-11* AQF2-12
Sep. 25, 1997 2.8 2.7 3.3 3.1 2.7 Nov. 05, 1997 (4.degree. C.) 3.8
2.7 3.0 3.5 2.5 Nov. 05, 1997 (R.T.) 3.3 3.1 4.1 2.8 1.1 Dec. 12,
1997 (4.degree. C.) 2.1 3.0 3.0 3.4 2.9 Jan. 09, 1998 (R.T.) 1.1
0.2 0.1 20 1.1 HPLC viral particle (.times.10.sup.10/ml) Date
(Temp.) AQF2-1 AQF2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6 AQF2-7 Sep. 25,
1997 10.9 9.6 9.7 11.3 10.7 10.6 10.9 Nov. 05, 1997 (4.degree. C.)
7.9 7.6 8.7 8.8 8.9 7.5 8.6 Nov. 05, 1997 (R.T.) 8.2 6.6 7.6 8.6
7.7 9.3 9.0 Dec. 12, 1997 (4.degree. C.) 6.7 1.5 8.0 6.9 5.2 7.5
7.5 Dec. 17, 1997 (R.T.) 7.0 1.2 7.0 7.5 4.1 7.1 7.0 HPLC viral
particle .times.10.sup.10/ml) Date (Temp.) AQF2-8 AQF2-9 AQF2-10
AQF2-11 AQF2-12 Sep. 25, 1997 10.8 10.7 11.4 11.8 10.7 Nov. 05,
1997 (4.degree. C.) 9.1 9.2 10.3 11.2 9.6 Nov. 05, 1997 (R.T.) 8.0
9.3 10.3 11.1 9.6 Dec. 12, 1997 (4.degree. C.) 6.1 7.6 8.8 7.3 7.7
Dec. 17, 1997 (R.T.) 3.0 8.2 7.6 8.4 7.5 Aqueous Formulation Set 2
Excipients AQF2-1 AQF2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6 AQF2-7
mannitol (W %) 5 5 5 5 sucrose (W %) 5 5 5 5 glycine (M) 0.25 0.25
0.25 arginine (M) 0.25 0.25 urea (W %) 1 1 peg (w %) Excipients
AQF2-8 AQF2-9 AQF2-10 AQF2-11 AQF2-12 mannitol (W %) 5 5 5 5
sucrose (W %) 5 5 5 5 10 glycine (M) 0.25 0.25 arginine (M) 0.25
0.25 urea (W %) 1 1 peg (w %) 1 1 *Gave better recovery.
Formulations are in 10 mM-tris buffer (pH = 7.5) which consists of
1% glycerol and 1 mM MgCl.sub.2. The formulations are stored at
4.degree. C. and room temperature under nitrogen.
[0236]
14TABLE 14 Aqueous Formulation Set #3 HPLC Viral Particle PFU
.times. 10.sup.9 (.times.10.sup.9/ml) Date (temp.) F10-7 F10-8
F11-7 F11-8 F10-7 F10-8 F11-7 F11-8 Oct. 3, 1997 2.2 3.3 2.1 2.8
12.1 12.0 11.8 12.0 Nov. 6, 1997 3.4 4.0 2.8 3.4 10.6 10.5 10.1
10.3 (-20.degree. C.) Nov. 6, 1997 3.5 3.6 4.3 2.8 10.0 9.7 9.9
10.3 (4.degree. C.) Jan. 15, 1998 3.8 4.8 3.2 3.7 7.3 7.4 7.7 8.0
(-20.degree. C.) Jan. 15, 1998 3.5 3.1 2.9 3.1 7.5 7.4 7.6 7.5
(4.degree. C.) Excipients F10-7 F10-8 F11-7 F11-8 mannitol(W %) 6 6
5 5 sucrose(VV %) 7 8 7 8 HSA (W %) 0.5 0.5 0.5 0.5 glycerol(W %) I
1 1 1 MgCl.sub.2(mM) 1 1 1 1
[0237]
15TABLE 15 Liquid formulation set #4 PFU (.times.10.sup.9/ml) Date
(Temp.) AQF4-1 AQF4-2 AQF4-3 AQF4-4 AQF4-5 AQF4-6 AQF4-7 Jan. 13,
1998 3.0 2.5 3.6 3.4 2.7 3.1 3.4 Feb. 16, 1998 (4.degree. C.) 2.5
3.2 3.3 2.9 2.6 2.9 2.6 Feb. 16, 1998 (R.T.) 1.8 2.7 1.6 3.6 2.6
1.6 1.7 Apr. 10, 1998 (4.degree. C.) 2.2 2.0 2.6 3.0 2.4 1.9 2.2
Apr. 10, 1998 (R.T.) 0.4 0.4 0.3 0.5 0.4 <0.1 1.1 Jul. 24, 1998
(4.degree. C.) 2.4 2.8 2.6 3.5 1.9 2.2 2.6 Jul. 24, 1998 (R.T.)
0.002 0.005 0.006 0.005 0.005 0.005 0.001 Jan. 8, 1999 (4.degree.
C.) 2.9 2.4 2.1 2.6 2.0 2.2 2.1 Jan. 8, 1999 (R.T.) 0.0002 0.0004
0.0004 0.0002 0.0004 0.0004 0.00006 HPLC Viral Particle
(.times.10.sup.10/ml) Date (Temp.) AQF4-1 AQF4-2 AQF4-3 AQF4-4
AQF4-5 AQF4-6 AQF4-7 Jan. 13, 1998 7.2 8.8 9.2 9.0 7.8 7.9 9.1 Feb.
16, 1998 (4.degree. C.) 7.5 9.3 9.2 9.5 8.2 8.4 9.6 Feb. 16, 1998
(R.T.) 6.8 9.0 9.5 9.0 8.7 8.4 9.3 Apr. 10, 1998 (4.degree. C.) 7.1
9.2 9.6 9.6 8.9 9.1 9.9 Apr. 10, 1998 (R.T.) 7.5 9.5 10.1 9.7 8.9
8.9 9.5 Jul. 24, 1998 (4.degree. C.) 8.1 9.9 11.1 10.3 9.2 7.4 9.3
Jul. 24, 1998 (R.T.) 7.3 3.0 10.7 8.9 10.4 10.45 3.5 Jan. 8, 1999
(4.degree. C.) 7.8 10.3 10.3 10.1 8.7 1.7 9.5 Jan. 8, 1999 (R.T.)
8.4 11.0 11.3 11.0 9.7 10.4 9.4 Excipients AQF4-1 AQF4-2 AQF4-3
AQF4-4 AQF4-5 AQF4-6 AQF4-7 Mannitol (w %) 5 5 5 5 5 5 5 Sucrose (w
%) 5 5 5 5 5 5 5 Tween-80 (w %) 0.02 0.1 0.5 Chap (w %) 0.02 0.1
0.5 Buffer: 10 mM Tris + H0.15 M NaCl + 1 mMMgCl2, pH = 8.2
Formulations were blanketed with N.sub.2.
[0238] Table 10 and Table 11 show the storage stability data with
secondary drying at 30.degree. C. without and with N.sub.2
backfilling, respectively. Because of the nearly identical
stability observed at -20.degree. C. and 4.degree. C. storage
conditions, and to reduce the consumption of virus, -20.degree. C.
was not included in the long-term storage stability study. Similar
to the samples dried with secondary drying at 10.degree. C., virus
is stable at 4.degree. C. but not stable at RT. However, relative
better stability was observed at RT storage than those dried at
10.degree. C. secondary drying. This is likely to be the result of
the lower residual moisture attained at 30.degree. C. secondary
drying. This result suggests that residual moisture is an important
parameter that affects storage stability during long term
storage.
[0239] HPLC viral particle recoveries are consistently lower than
virus recoveries calculated from PFU assay immediate after drying.
The reason for the discrepancy is not clear. However, it is likely
to be related to possible virus aggregation during freeze-drying.
Electron microscopy evaluation is being carried out to examine
possible virus aggregation after lyophilization. During storage,
HPLC analysis indicates that virus is stable at both -20.degree. C.
and 4.degree. C. storage and not stable at RT, which is consistent
with the results from PFU assay.
Example 5
HSA Alternatives
[0240] The presence of HSA in the formulations could be a potential
regulatory concern. As a result, a variety of excipients have been
evaluated to substitute HSA in the formulation. The substitutes
examined included PEG, amino acids (glycine, arginine), polymers
(polyvinylpyrrolidone), and surfactants (Tween-20 and Tween-80).
These HSA substitutes are, however, suboptimal relative to HSA.
Effort on further development was minimal.
Example 6
Liquid Formulation
[0241] Concurrent with the development of lyophilization of Adp53
product, experimentation was carried out to examine the possibility
of developing a liquid formulation for Adp53 product. The goal was
to develop a formulation that can provide enough stability to the
virus when stored at above freezing temperatures. Four sets of
liquid formulations have been evaluated. In the first set of
formulation, the current 10% glycerol formulation was compared to
HSA and PEG containing formulations. In the second set of
formulation, various amino acids were examined for formulating
Adp53. In the third set of formulation, the optimal formulation
developed for lyophilization was used to formulate Adp53 in a
liquid form. In the fourth set of formulation, detergents were
evaluated for formulating Adp53. Viruses formulated with all those
different formulations are being tested for long term storage
stability at -20.degree. C., 4.degree. C., and RT.
[0242] Liquid Formulation Set #1
[0243] HSA containing formulation (5% sucrose+5% HSA in 10 mM Tris
buffer, 150 mM NaCl, and 1 mM MgCl.sub.2, pH=8.20 buffer) was
compared with 10% glycerol in DPBS buffer and sucrose/PEG and
Trehalose/PEG formulations. PEG has been recommended as a good
preferential exclusion agent in formulations (Wong and
Parasrampurita, 1997). It is included in this set of formulation to
examine whether it can provide stabilization effect on Adp53.
Formulations were filled into the 3 ml lyo vials at a fill volume
of 0.5 ml. Vials were capped under either atmospheric or N.sub.2
blanketing conditions to examine any positive effects N.sub.2
blanketing may have on long term storage stability of Adp53. To
ensure adequate degassing from the formulation and subsequent
N.sub.2 blanketing, the filled vials was partially stoppered with
lyo stoppers and loaded onto the shelf of the lyophilizer under RT.
The lyophilizer chamber was closed and vacuum was established by
turning on the vacuum pump. The chamber was evacuated to 25 in Hg.
Then the chamber was purged completely with dry N.sub.2. The
evacuation and gassing were repeated twice to ensure complete
N.sub.2 blanketing. N.sub.2 blanketed vials were placed with the
non-N.sub.2 blanketed vials at various storage conditions for
storage stability evaluation. Table 12 shows the analysis data for
up to 18 months storage at 4.degree. C. and RT.
[0244] Statistically significant drops in virus PFU and HPLC viral
particles were observed for 10% glycerol formulation after 3 months
storage at both 4.degree. C. and RT. No statistically significant
virus degradation was observed for all other formulations at
4.degree. C. storage. However, decrease in virus infectivity was
observed when stored at RT.
[0245] Liquid Formulation Set #2
[0246] Various combinations of amino acids, sugars, PEG and urea
were evaluated for Adp53 stabilization during long storage. Table
13 shows the 12-month stability data. The results indicate that
combination of 5% mannitol and 5% sucrose with other excipients
gave better storage stability at RT for one month. Adp53 is most
stable in formulation has all the excipients. In this set of
formulation, no human or animal derived excipients were included.
It is our expectation to develop a liquid formulation without
including any proteins derived from either human or animal
origins.
[0247] Liquid Formulation Set #3
[0248] The optimal formulations developed for lyophilization was
evaluated for formulating Adp53 in a liquid form. This approach
would be a good bridging between liquid formulation and
lyophilization if satisfactory Adp53 stability can be achieved
using lyophilization formulation for liquid fill. Filled samples
were stored at -20.degree. C. and 4.degree. C. for stability study.
Table 14 shows the 3-month stability data. Virus is stable at both
-20.degree. C. and 4.degree. C. for the four different
formulations. This is in agreement with the results from
formulation set #2, which suggests that better virus stability is
expected with the presence of both mannitol and sucrose in the
formulation. Longer time storage stability data is being
accrued.
[0249] Liquid Formulation Set #4
[0250] Detergents have been used in the formulations for a variety
of recombinant proteins. In this set of formulation, various
concentrations of detergents were examined for formulating Adp53.
The detergents used were non-ionic (Tween-80) and zwitterionic
(Chap). Table 15 shows the 12-month stability data. Virus is stable
at 4.degree. C. storage. No significant difference in virus
stability at 4.degree. C. was observed among the formulations
tested. Similar to formulation set #2, no exogenous protein is
included in this set of formulation.
[0251] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
REFERENCES
[0252] The following references, to the extent that they provide
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forth herein, are specifically incorporated herein by
reference.
[0253] U.S. Pat. No. 4,797,368
[0254] U.S. Pat. No. 5,139,941
[0255] U.S. Pat. No. 5,552,309
[0256] U.S. Pat. No. 5,789,244
[0257] U.S. Ser. No. 08/975,519
[0258] EPO 0273085
[0259] WO 94/17178
[0260] WO 98/35554
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* * * * *