Note: Descriptions are shown in the official language in which they were submitted.
CA 02597865 2007-09-13
FIELD OF THE INVENTION
The present invention relates to compositions for using preferably as
therapeutic
vaccines in the treatment of human papillomavirus infection related conditions
or
diseases, and also to the methods for preparing such compositions.
BACKGROUND OF THE INVENTION
Human Papillomaviruses (I-IPV) are small nonenveloped DNA viruses involved in
many conditions and diseases, which are generally specific either for the skin
or
mucosal surfaces. The skin HPV types cause warts on hands and feet, and
usually
warts can persist for several months or years. Such benign tumours may be
distressing
for the individuals concerned but tend not to be life threatening, with a few
exceptions.
The mucosal HPV types infect the anogenital region and the oral cavity.
Approximately 100 different types of HPV have been characterized to date, and
approximately 40 HPV types specifically infect the genital and oral mucosa.
Infection
with these types of HPV may not cause any symptoms and does not always produce
visible genital warts. When symptoms do develop, they usually occur 2 to 3
months
after infection with the virus. Symptoms have been known to develop, however,
from
3 weeks to many years after infection occurs. As such, HPV may be spread
unknowingly. Infection of Mucosal HPV types are considered one of the most
common sexually transmitted diseases (STDs) throughout the world with an
incidence
roughly twice that of herpes simplex virus infection, many have placed the
lifetime
likelihood of getting genital HPV to be in the range of 75-90%. While a
majority of
infections are asymptomatic, infection can lead to development of genital
warts and
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CA 02597865 2007-09-13
cancer of anogenital tract. Cervical carcinoma, the second most widespread
gynecological cancer worldwide, is associated (>99%) with the detection of
mostly
HPV16 and HPV18 DNA. About one percent of women worldwide are afflicted with
cervical cancer, with about 500,000 new cases occurring in the world each
year, which
is the most common cause of death in women under the age of 50. In addition to
cervical cancer, HPV are associated with a number of anal and perianal cancers
(1-8,
16).
Prophylactic vaccines have been developed against HPV. The major capsid
protein of
HPV can be recombinantly expressed in eukaryotic cells and the expressed major
capsid protein can form authentic virus-like particles (VLPs) in the
expression hosts.
The purified VLPs are effective prophylactic vaccines against HPV and they are
available for preventing HPV infection (9-15). However, there are no effective
therapeutic compositions available to cure infected individuals. Since viruses
use so
much of the host-cell machinery for their replication, it has been much more
difficult
to develop drugs that specifically inhibit viral replication without harming
the host. It
is known that the immune system can play a critical role in controlling HPV
infection.
It is well known that the incidence of HPV-induced skin warts and HPV-
associated
diseases increases in those who are receiving immunosuppressive treatment,
suggesting that in many cases virus infection is kept under control by
immunological
mechanisms. Further evidence for the capacity of the immune system to control
infection has come from study of spontaneous wart regression. A common
observation
is that in some individuals with genital warts, the warts suddenly disappear.
Such
regression warts have been studied histologically, revealing a substantial
influx of T
lymphocytes in the lesions. Regression is believed to be mediated by the
immune
system. Effective immune responses to HPV infections are thought to be mainly
cell-mediated since disease can persist in individuals with serum antibodies
against
HPV. Moreover, it is known that spontaneous regression of warts is often
accompanied by Iymphocytic infiltration, itching, reddening of the affected
area and
other symptoms characteristic of cell-mediated immune reaction. HPV infections
are
also common in patients with impaired cell-mediated immunity, where
persistence of
viral disease suggests poor immune surveillance. Therefore the induction of
effective
cell-mediated immune response against protein antigens of HPV is of major
importance in therapeutic vaccine strategies against HPV infection related
conditions
or diseases (16-25).
Although the main target for prophylactic vaccines have been focussed on the
L1
protein, which has been shown to be effective in generating both humoral and
cell-mediated immune responses, due to its restricted expression, it will not
be able to
induce significant therapeutic effects for HPV infection related conditions or
diseases
(16-24). There is however evidence that the two HPV oncoproteins, E6 and E7,
are
ideal targets for designing therapeutic medicines. E6 and E7, are critical to
the
induction and maintenance of cellular transformation and are co-expressed in
the
majority of HPV-containing carcinomas (16-24). Thus, therapeutic compositions
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CA 02597865 2007-09-13
targeting E6 and E7 may provide the best option for controlling HPV infection
related
conditions or diseases. By using appropriate antigen delivery systems (or
antigen
carriers), E6 and E7 antigens or their epitopes can be delivered to a host to
elicit a
strong, long-lasting and specific cell-mediated immune response, which can
potentially have the HPV related conditions or diseases cured by eliminating
infected
HPV, cells infected with HPV and HPV-containing carcinomas, such as cervical
cancel cells.
The L1 and L2 capsid proteins of HPV have been used as delivery systems to
carry
HPV E6 or E7 protein antigens, for inducing cell-mediated immune response (29-
30).
As infected individuals may have developed certain immunity to HPV, or as HPV
can
establish latent or persistent infection in an infected individual, and by
employing
numerous decoy strategies, HPV can induce immune tolerance in the host (31-
32).
Indeed, despite the potent immunogenicity of the HPV capsid, only half of
cervical
cancer patients generate capsid-specific immunoglobulin G (IgG). The pre-
existing
immunity or existing immune tolerance may limit the usefulness of therapeutic
vaccines developed based on HPV Ll or L2 capsid proteins.
Capsid proteins from other viruses can also self-assemble into VLPs in a
suitable host
when recombinantly expressed in an appropriate expression system. These VLPs
can
also be used as antigen delivery systems (or antigen carriers) for carrying E6
or E7
antigens or their epitopes for inducing cell-mediated immune response.
However,
there are still problems related to the method of purification of VLPs (26-
27), and if
the VLPs are from viruses with human as hosts, the therapeutic vaccines may
also
possibly face the problems of preexisting immunity or existing inunune
tolerance.
Thus there exists a need in the art to provide therapeutic compositions which
can be
used to treat HPV related conditions or diseases.
SUMMARY OF THE INVENTION
Generally speaking, the present invention resides in the discovery that the
macro-molecules derived from fusion capsid-chaperone protein (FCCP) can be
used
as antigen delivery system (or antigen carrier) for inducing a cell-mediated
immune
response against carried antigens (US patent application #60944780). In this
invention,
the macro-molecules are used as a delivery system or vehicle to carry HPV
antigen
for inducing immune responses, particularly a cell-mediated immune response
against
HPV antigen. Thus, the present invention relates to compositions comprising
macro-molecules carrying HPV antigen. In one embodiment, the compositions can
be
prepared from single fusion protein, the HPV antigen and FCCP can be joined at
the
nucleotide level permitting expression and purification of a single fusion
protein
containing both HPV antigen and FCCP sequences. The separation and
purification
process is conducted in one or more steps in denatured condition using high
concentration of chaotropic agents, such as urea or guanidine hydrochloride
(Gu.HCI)
CA 02597865 2007-09-13
solution or buffered solution. The denatured FCCP-HPV antigen fusion protein
is
subjected to refolding and self-assembling process to form macro-molecules
containing multi-units of FCCP-HPV antigen fusion protein by a process
involving
gradually removing out chaotropic agents presented in the denatured FCCP-HPV
antigen fusion protein sample. As the chaperone protein in the FCCP is to
facilitate
the capsid protein refolding and reassembling into macro-molecules after the
purification process involving using protein denaturants, such as urea and
Gu.HCI, it
might be desirable in some situations to have the chaperone protein clipped
off by an
enzymatic method and removed from the macro-molecules after refolding and
reassembling process. In another embodiment, the HPV antigen can also be
chemically linked or conjugated to the macro-molecules by well known methods,
or
HPV antigen can also join macro-molecules noncovalently. Any of several known
high-affinity interactions can be adapted to nonconvalently connect HPV
anrigen with
macro-molecules. In particular embodiments, the immunostimulatory substances,
preferable unmethylated CpG-DNA or double strand RNA can be packaged into
macro-molecules. The compositions in this invention may be used for
therapeutic
treatment of HPV related conditions or diseases by inducing immune responses,
particularly a cell-mediated immune response against HPV antigen.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions that can be used to induce a
cell-mediated immune response against HPV antigen, therefore the compositions
may
be used therapeutically to treat HPV related conditions or diseases. The
method of
preparation the compositions in this invention and the use of such
compositions reside
in the discovery that the macro-molecules derived from fusion capsid-chaperone
protein (FCCP) can be used as antigen delivery system (or antigen carrier) for
inducing a cell-mediated immune response against carried antigen. (US patent
application #60944780). In this invention, the method of preparation of the
compositions comprise macro-molecules carrying HPV antigen and the use of the
compositions for therapeutic treatment of HPV infection related conditions and
diseases are disclosed. The HPV antigen in the compositions of this invention
can be
any HPV gene-encoded polypeptide. Two HPV oncogenic proteins, E6 and E7, are
critical to the induction and maintenance of cellular transformation and are
co-expressed in the majority of HPV-containing carcinomas such as cervical
cancel
cells and cells within warts (17-24). Thus, it is highly desirable to use E6,
E7 or both
proteins in the compositions of this invention. In addition, Those skilled in
the art will
recognize and appreciate that in the compositions of this invention, the said
HPV
antigen can be a portion of an E6 or E7 protein, provided that portion, when
joined to
macro-molecules (being carried by macro-molecules), retains the ability to
induce a
cell-mediated immune response against E6 or E7 antigen. Compositions comprise
various portions of E6 or E7 rather than a complete HPV protein antigen can be
produced by routine methods by those skilled in the art, such as gene-encoding
E6 or
E7 antigen can be deleted part of its sequence or chemically synthesized only
part of
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CA 02597865 2007-09-13
its sequence, then in an expression system, recombinantly express altered DNA
sequence to produce only portion of the E6 or E7 polypeptide. In the
compositions of
this invention, E6 and E7 polypeptides also can be combined, such as it can be
the
combination of E6 and E7 proteins. Those skilled in the art will recognize and
appreciate that the combination of the E6 and E7 polypeptides can be achieved
by
recombinantly expressing a fusion protein, or by chemically conjugating
polypeptides
together, furthermore, E6 or E7 polypeptide can be chemically conjugated to
macro-molecules separately. In addition, in the compositions of this
invention, it can
be the combination of a portion of E6 and a portion of E7 proteins, provided
the
portion, when joined with macro-molecules, retains the ability to induce a
cell-mediated immune response against E6 or E7 antigens. Each composition have
a
particular portion of HPV antigen or epitopes can be tested and assayed for
the degree
and quality of the immune responses, particularly cell-mediated immune
response
against HPV antigen by well known methods, such as by in vitro assay of CTL
activity, and by animal experiments described in the examples hereinafter.
The compositions of this invention can be prepared using following steps.
1. A nucleic acid which encodes FCCP and desired HPV antigen described above
is
incorporated into a suitable vector system as a single open reading frame with
any
accessory sequences proper for its expression in a chosen system. Preferably,
HPV antigen is linked to the capsid protein of the FCCP as a single fusion
protein.
The host cell is transformed with the vector. Transformed host cells are then
cultured and the desired fusion protein is recombinantly expressed;
2. the separation and purification process is conducted in high concentration
of
chaotropic agents such as urea or Gu.HC1 in one or more steps; the
concentration
of urea or Gu.HCI solution used in the purification process can be up to 10M
urea
or Gu.HCI, preferably from 4M-8M for urea and 3-6M for Gu.HCI;
3. the purified homogenous FCCP-HPV antigen fusion protein will be refolded
with
a process involving gradually removing out chaotropic agents presented in the
purified sample by dialysis;
4. in the refolding process, the fusion protein of FCCP-HPV antigen will
self-assemble into macro-molecules containing multi-units of FCCP-HPV
antigen.
The capsid protein in the FCCP-HPV antigen fusion protein can be utilized to
package
nucleic acids. Some nucleic acids such as double strand RNA or unmethylated
CpG-DNA are well known for their ability to greatly enhance the inunune
responses.
Thus it is highly desirable to have double strand RNA or unmethylated CpG-DNA
be
packaged into the macro-molecules containing multi-units of FCCP-HPV antigen
by
adding desired double strand RNA or unmethylated CpG-DNA into purified and
denatured fusion protein of FCCP-HPV antigen, then reassembling FCCP-HPV
antigen into macro-molecules by a process involving gradually removing out
chaotropic agents presented in the denatured fusion protein sample.
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CA 02597865 2007-09-13
In the FCCP fusion protein, the chaperone protein is to facilitate the
refolding of the
denatured capsid protein, in some situations, it may be desirable to have the
chaperone
protein being removed after refolding and reassembling process. The
composition can
be prepared by another method to have the chaperone protein being removed from
the
final preparation using following steps.
1. Designing a fusion protein with the capsid protein linked to HPV antigen
and a
chaperone protein via peptide bonds, designing an unique enzyme cleavage site
at
the joint of the capsid protein and the chaperone protein, the unique enzyme
cleavage site is a thrombin cleavage site or an enterokinase cleavage site, or
any
other unique enzyme cleavage site;
2. recombinantly producing the said fusion protein containing a specific
enzyme
cleavage site at the joint of the capsid protein and the chaperone protein by
an
expression system;
3. separation and purification of the recombinantly expressed fusion protein
in one
or more steps in denatured form involving using high concentration of
chaotropic
agents, such as using up to 10M urea or Gu.HCI solutions or buffered solutions
in
the separation and purification process, preferably 2-8M for urea and 1-6M for
Gu.HCI;
4. refolding and reassembling of the said fusion protein into macro-molecules
by a
process involving gradually removing out chaotropic agents presented in the
denatured fusion protein sample;
5. clipping off the chaperone protein from the macro-molecules by using the
desired
enzyme, such as using thrombin or enterokinase;
6. separating macro-molecules from the clipped off chaperone protein;
7. the final macro-molecules comprising many subunits of mostly capsid protein
fused to HPV antigen.
The compositions of this invention can also be prepared using another
different
approach. The desired HPV antigen, for example, from E6 or E7, or both E6 and
E7,
or portion of E6 and E7 can be recombinantly expressed and then purified or
chemically synthezed; the macro-molecules derived from FCCP can prepared
according to the US patent application # 60944780. The prepared HPV antigen
and
macro-molecules can be conjugated or joined chemically using known techniques,
such as, standard techniques involving covalent attachment for example to
exposed
tyrosine residues or to the epsilon-amino groups of lysine residues or the
carboxyl
groups of aspartate and glutamate residues. The purified HPV antigen and
macro-molecules can also be joined noncovalently by affinity interactions. Any
of
several known high-affinity interactions can be adapted to nonconvalently
connect
HPV antigen with macro-molecules. For examples, a biotin group can be added to
macro-molecules, the HPV antigen can be expressed as an avidin-HPV antigen
fusion
protein. The avidin-HPV antigen fusion protein will strongly bind the
biotinlated
macro-molecules.
s
CA 02597865 2007-09-13
The capsid protein in the FCCP can be utilized to package nucleic acids. Some
nucleic
acids such as double strand RNA or unmethylated CpG-DNA are well known for
their
ability to greatly enhance the immune responses. Thus it is highly desirable
to have
double strand RNA or unmethylated CpG-DNA be packaged into the
macro-molecules carrying FCCP-HPV antigen.
The compositions comprising macro-molecules carrying HPV antigen described
herein can be used to induce enhanced immune responses, particularly cell-
mediated
immune response, against HPV antigen, which is capable of mediating the
regression
of chronic HPV infection resulted conditions or diseases, these conditions and
diseases include, but are not limited to: genital warts (especially where the
compositions and the infections are based on HPV of types 6 and/or 11) or
cervical
intra-epithelial neoplasia (especially where the compositions and the
infections are
based on HPV of types 16 and/or 18) in infected patients. Thus the invention
provides
compositions comprising macro-molecules carrying HPV antigen, which can be
used
for therapeutically treating HPV infection associated conditions or diseases.
The
therapeutic treatment comprises administering to a patient an effective amount
of
compositions as described herein.
Cross-reactivity between HPV of different types has been observed, and
according to
such observable cross-reactivity the compositions produced hereby can be used
in
eliciting useful immune responses against papillomavirus types other than the
types
from which they were derived.
The compositions of this invention described above, are preferably used for
therapeutic purpose for HPV related conditions or diseases, however, the
compositions may be used for prophylactic purpose. The compositions of this
invention are suitable for injection and routes and procedures of
administration
include, but are not limited to standard intramuscular, subcutaneous,
intradermal,
intravenous, oral or rectal routes and procedures. In addition, the
compositions of this
invention can contain and be administered together with other
pharmacologically
acceptable components. The compositions of this invention can also be
formulated by
combining with an adjuvant or other accessory substance such as an
immunostimulatory molecule in order to enhance its effect as a therapeutic
vaccine,
and also to stimulate a preferred type of immune response in the recipient
host. Useful
adjuvant include, but are not limited to: double strand RNA, unmethylated CpG-
DNA,
aluminum hydroxide. Such adjuvant and/or other accessory substances can be
used
separately or in combinations as desired.
The amount of the compositions in this invention used for therapeutic or
prophylactic
purposes is an amount which can induce effective immune responses in a subject
when administered. In addition, the amount of the compositions administered to
the
subject will vary depending on a variety of factors, including but not
limiting to: the
formulation of the compositions, adjuvant and its amount, the size, age, body
weight,
sex, general health and immunological responses of the subject. Effective
amounts
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CA 02597865 2007-09-13
can be determined in subjects and adjustment and manipulation of established
dose
range are well within the ability of those skilled in the art. For example,
the effective
amount of compositions can be from 10 micrograms to about 1 gram, preferably
from
50 micrograms to 50 milligrams. One or more doses of the vaccine may be
administered at intervals. This regime can readily be optimized in subjects by
those
skilled in the art.
The use of the macro-molecules derived from FCCP instead of the VLPs derived
from
capsid proteins as HPV antigen carrier to produce the compositions described
above
has the following advantages:
1. FCCP or FCCP-HPV antigen fusion protein can be purified in denatured forms
using high concentration of chaotropic agents such as urea or Gu.HCI. The
concentration of urea or Gu.HCI solution used in the purification process can
be
up to lOM, preferably up to 8M for urea and 6M for Gu.HCI. The purified and
denatured homogenous FCCP or FCCP-HPV antigen fusion protein subsequently
can be refolded and reassembled into macro-molecules by a process involving
gradually removing chaotropic agents out from the denatured FCCP or
FCCP-HPV antigen fusion protein sample. Denatured capsid proteins normally
cannot be correctly refolded and normally form aggregates instead of VLP
(26-27).
2. The macro-molecules formed by the self-assembling of FCCP or FCCP-HPV
antigen fusion protein may be morphologically different from authentic VLP.
The
peptides displayed or exposed by authentic VLPs would be the same as the
viruses, but because of the differences of the morphology, the macro-molecules
may display or expose a different set of epitope peptides compare to authentic
VLPs, and may have the following advantages over authentic VLPs: (1)
pre-existing immunity to authentic VLPs might be circumvented by the use of
macro-molecules with different morphology; (2) existing immune tolerance to
authentic capsid proteins might be circumvented by the use of the
macro-molecules with different morphology; (3) the use of macro-molecules with
different morphology might circumvent the problem of interference with
commercial anti-capsid protein assays.
3. After the purification, refolding and reassembling process, the chaperone
protein
might be cliped off from FCCP with a chemical method, or by an enzyemtic
method, such as, a specific enzyme cleavage site can be designed at the joint
of
the capsid protein and chaperone protein. For example, asp asp asp asp lys can
be
recognized by enterokinase, and this sequence can be introduced into the joint
of
the capsid protein and chaperone protein, after refolding and reassembling,
the
enterokinase can be used to clip off the chaperone protein.
The following examples are provided in order to demonstrate and further
illustrate the
present invention, and are not to be construed as limiting the scope thereof.
EXAMPLES
CA 02597865 2007-09-13
Example 1
A chaperone protein-Hsp65 derived from Mycobacterium bovis BCG hsp65 gene
(35-36) is fused to the C-terminal of the capsid protein (nucleocapsid protein
or core
antigen) of Hepatitis B virus (HBV) subtype ADW2 (37-38) to form a FCCP
molecule.
An E7 protein from human papillomavirus type 16 (39) is fused to the N-
terminal of
the FCCP molecule. The single fusion protein starting from N-terminal is E7
protein,
the C-terminal of E7 protein is fused to the N-terminal of capsid protein, the
C-terminal of capsid protein is fused to the N-terminal of Hsp65 protein, and
can be
represented as E7-Core-Hsp65. The 2479 bp of DNA sequence encoding
E7-Core-Hsp65 fusion protein was chemically synthesized according to DNA
sequences from GenBank (36, 38-39).
The synthesized DNA sequence was named Ankegens 2479bp and cloned into Smal
digested pBluescript II SK (+/-) from Stratagene (40) to produce
pB S K-Ankeg ens -2479bp .
Example 2
Expression and purification of E7-Core-Hsp65 fusion protein
E7-Core-BCG65 DNA fragment was cut from pBSK-Ankegens-2479bp by Ndel and
EcoRI then subcloned into pET-23a (41) corresponding sites to produce
pET-23a-2479. The pET-23a-2479 was transformed into E. coli cell line
Rosetta-gami(DE3) from Novagen. E7-Core-BCG65 fusion protein was expressed in
E. coli cells by fermentation and induction of transformed Rosetta-gami(DE3)
cells
with 0.5mM isopropyl-thio-galatopyranoside according to Novagen's pET System
Manual. After fermentation, cells were harvested by centrifugation. Cells were
washed once by suspending 100g cell paste in 1000m1 of buffer A(100mM Tris-Hcl
pH 9.0; 5mM EDTA) then centrifuging at 8500rpm for 30 minutes. Discarded the
supernatant then re-suspended the pelleted cells with 1000m1 of buffer B (50mM
sodium acetate; 2mM EDTA). The suspended cells were ruptured by homogenization
process with pressure at 760bar, and then centrifuged at 8500rpm for 30
minutes. The
supernatant was collected and the volume was measured. Urea was added to the
supernatant according to 0.7g urea for lmi supernatant, and then sodium
chloride was
added to final concentration at 100 mM, L-Cysteine was added to final
concentration
at 20mM. The solution was stirred at room temperature to have all the urea
dissolved
then stirred at 4 C for overnight. After overnight stirring, the sample was
applied to
an XK-50 column (GE Health) containing 300ml of SP-Sepharose resin, which was
previously washed with 1 M sodium chloride and equilibrated with buffer C
(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine). After
sample loading, the column was washed with 10 column-volumes of buffer D(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine; 2.5%
11
CA 02597865 2007-09-13
Triton-X-100) overnight to remove endotoxin. After overnight washing with
buffer D,
the column was washed with 5 column-volumes of buffer C to remove Triton-X-
100,
and then the column was washed with 3 column-volumes of buffer E(50mM
sodium acetate; 300mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine) to remove
contaminating proteins and other contaminations. E7-Core-BCG65 fusion protein
was
eluted from the column with buffer D(50mM sodium acetate; 800mM NaCI; 2mM
EDTA; 8M urea; 10mM L-Cysteine). Pooled eluted protein was dialyzed against 4X
40 volumes of buffer F (50mM sodium acetate, 6Murea) to remove NaC1 and
L-Cysteine. After dialysis, Oxidative sulfitolysis was performed by adding
sodium
sulfite and sodium tetrathionate to final concentrations of 200mM and 50mM
respectively and incubating for overnight at room temperature. The
sulfitolyzed
sample was diluted 5 volumes with buffer F then applied to an XK-50 column
with
150m1 of Q-Sepharose resin, which was previously washed with 1M NaCt and
equilibrated with buffer F. After sample loading, the column was washed with 2
column-volumes of 95% buffer F and 5% buffer G (50mM sodium acetate; 1M NaCl;
6Murea), and then E7-Core-BCG65 fusion protein was eluted with a lineal
gradient
from 95% buffer F and 5% buffer G to 50% buffer F and 50% buffer G over 8
column-volumes. Eluted E7-Core-BCG65 fusion protein was pooled, and then
dialyzed against 1X40 volumes of Tris.HCI pH9.0, 1X40 volumes of Tris.HCl
pH7.5
with 100mM NaCI to remove urea and refold E7-Core-BCG65 fusion protein. The
endotoxin level in the final preparation (E7-Core-BCG65 in Tris.HCl pH7.5 with
100mM NaCI) was below 5EU/mg protein.
Example 3
Therapeutic and prophylactic effects of E7-Core-BCG65 treatment in mice
The E7-Core-BCG65 is an FCCP carrying an HPV16 E7 protein (antigen), and the
E7
expressing TC-1 tumor cells were used to evaluate the therapeutic and
prophylactic
applications of E7-Core-BCG65 on mice bearing TC-1 tumor or being challenged
with TC- 1 tumor.
Female C57BL/6 mice, six to eight weeks old (20.0 2.0g) were purchased from
SHANGHAI SLAC LABORATORY ANIMAL CO. LTD. Quality Control No.:
S CXK(Shanghai)2003 -0003 .
TC-1 cell line expressing HPV16 E7 protein was derived from primary lung cells
of
C57BL/6 mice by immortalization and transformation with HPV16 E7 gene and an
activated human C-Ha-ras gene as described in Lin et al. (42).TC-1 cells were
grown
in RPMI1640 medium supplemented with 10% fetal calf serum, 2mM nonessential
amino acids, 2mM L-glutamine, 1mM pyruvate, Penicillin/Streptomycin, and the
cells
were harvested by trypsinization, the cells were washed three times with PBS
then
re-suspended in PBS. 1X105 TC-1 cells were inoculated subcutaneously into the
mice and the mice were treated with E7-Core-BCG65 or saline subcutaneously
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CA 02597865 2007-09-13
according to their experiment groups.
Animal experiment groups:
Groups Mice Dose Time of E7-Core-BCG65 Treatment
Treatment
Therapeutic 8 500ug 48h and 16 days after inoculation of TC-1
Application E7-Core-BCG6
8 100ug 48h and 16 days after inoculation of TC-1
E7-Core-BCG6
5
8 20ug 48h and 16 days after inoculation of TC-1
E7-Core-BCG6
5
Prophylactic 8 100ug Two treatments with 14 days in between
Application E7-Core-BCG6 Inoculation of TC-1 14 days after second
5 treatment
8 20ug 'ltvo treatments with 14 days in between
E7-Core-BCG6 Inoculation of TC-1 14 days after second
5 treatment
Control 6 Saline 48h and 16 days after inoculation of TC-1
The mice were monitored for the presence or absence of tumor by palpation and
the
volume of the tumor was measured with Vernier Caliber by 2 orthogonal
dimensions
twice a week; these measurements were extrapolated to mm3 and are presented as
average tumor volume standard error of the mean. The life span of the mice
was
recorded.
In control group, the presence of the tumor was observed 4 days after TC-1
inoculation; the average volume of the tumor was grown to 40mm3 10 day after
inoculation and 7499.84mm3 36 days after inoculation. All mice in the control
group
died within 60 days after inoculation.
In therapeutic group, mice were treated with E7-Core-BCG65 48h and 16 days
after
TC-1 inoculation; the average volume of the tumor was grown to 181.89mm3
(500ug),
671.34mm3 (100ug) and 2148.57mm3 (20ug) 36 days after inoculation. All mice
were alive 60 days after inoculation.
In the prophylactic group, mice were treated with E7-Core-BCG65 twice in 14
days,
and after second treatment, mice were inoculated with TC-1; the average volume
of
the tumor was grown to22.43mm3 (100ug) and 89.08mm3 (20ug) 36 days after
inoculation. All mice were alive 60 days after inoculation.
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CA 02597865 2007-09-13
Table 1 The average tumor volume in different experiment groups (mm3) (x s)
Date Therapeutic Group Prophylactic Group Control
(day) 500ug 100ug 20ug 100ug 20ug Group
(n=8) (n=8) (n=8) (n=8) (n=8) (n=6)
8.65 5.40 16.33 8.83 42.54 24.55 * 2.32 1.06 5.56 2.91 39.00 19.28
13 41.70 20.90 51.01 20.37 84.72 36.72 * 1,97 2.44 10.58f25.56 133.57 69.64
16 31.91 12.26 49.96 20.62 189.07i91.07* 1.97 1.42 5.24 2.08 320.20 149.14
19 35.69 10.52 156.28 46.49 208.49 85.46 2.85 1,49 25.65 10.43 782.65 257.69
22 43.89 21.13 224.71 107.46 357.47 159.47 2.44 1.98 35.24 80.41 1033.81
594.12
25 109.04 47.41 257.01 107.19 756.40f258.40 4.52 2.78 17.38 6.76 2414.19
1201.87
28 127.68 56.24 395.56 128.72 892.54 364.47 18.81 5.42 69.63 24.46 4432.67
1824.46
31 156.12 49.46 525.81 152.94 1527.21 510.46 20.57 10.46 85.57t25.85 6024.54
2465.46
34 181.89 75.53 671.34 301.28 2048.57 1050.57 22.42 18.60 89.08 43.09 7499.84
3722.56
37 250.52 120.59 785.69 268.85 3051.65 1253.32 56.83 25.56 173.59 89.41
9483.58 4565.74
40 396.88 208.12 921.87 368.03 3887.08 1889.08 59.81 26.79 173.92 86.39
13141.43 5077.39
Those skilled in the art will recognize, or be able to ascertain that the
basic
construction in this invention can be altered to provide other embodiments
which
utilize the process of this invention. Therefore, it will be appreciated that
the scope of
this invention is to be defmed by the claims appended hereto rather than the
specific
embodiments which have been presented hereinbefore by way of example.
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