U.S. patent application number 10/222722 was filed with the patent office on 2002-12-12 for methods and compositions for reducing immune response.
Invention is credited to LaFace, Drake M., Rahman, Amena, Shabram, Paul W., Tsai, Van T..
Application Number | 20020187143 10/222722 |
Document ID | / |
Family ID | 22543805 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187143 |
Kind Code |
A1 |
LaFace, Drake M. ; et
al. |
December 12, 2002 |
Methods and compositions for reducing immune response
Abstract
The present invention provides an apparatus and method to
diminish the pre-existing immune response to the administration of
a therapeutic virus by the selective elimination of antiviral
antibodies from the serum. The present invention provides a
chromatographic material for the elimination of such antibodies.
The invention further provides plasmapheresis apparatus comprising
this material. The invention further provides methods for the
employment of such apparatus as part of therapeutic treatment
regiments.
Inventors: |
LaFace, Drake M.; (San
Diego, CA) ; Rahman, Amena; (San Diego, CA) ;
Shabram, Paul W.; (Olivenhain, CA) ; Tsai, Van
T.; (San Diego, CA) |
Correspondence
Address: |
Richard B. Murphy
Canji, Inc.
3525 John Hopkins Court
San Diego
CA
92121
US
|
Family ID: |
22543805 |
Appl. No.: |
10/222722 |
Filed: |
August 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10222722 |
Aug 16, 2002 |
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09653474 |
Aug 31, 2000 |
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6464976 |
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60152650 |
Sep 7, 1999 |
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Current U.S.
Class: |
424/140.1 ;
435/320.1; 536/23.72 |
Current CPC
Class: |
A61P 31/12 20180101;
A61K 35/16 20130101; C12N 15/86 20130101; A61M 1/3496 20130101;
A61K 48/00 20130101; C12N 2710/10343 20130101; Y10S 514/885
20130101; A61P 31/20 20180101; A61K 35/30 20130101; A61M 1/3486
20140204 |
Class at
Publication: |
424/140.1 ;
435/320.1; 536/23.72 |
International
Class: |
A61K 039/395; A61K
039/00; C07H 021/04; C12N 015/00; C12N 015/09; C12N 015/63; C12N
015/70; C12N 015/74 |
Claims
We claim:
1. A method for reducing the concentration of antiviral antibodies
in a sample of plasma by contacting said isolated plasma with an
immunoaffinity chromatographic material comprising an
immunoaffinity material linked to a chromatographic support such
that antibodies are retained on the immunoaffinity material.
2. The method of claim 1 wherein the immunoaffinity material is
selected from the group consisting of Protein A, Protein G and a
viral coat protein.
3. The method of claim 2 wherein the immunoaffinity material is a
viral coat protein of an adenovirus selected from the group
consisting of hexon, penton, protein IX, protein IIIA fiber, knob
and penton base.
4. The method of claim 2 wherein the immunoaffinity material is
protein A.
5. The method of claim 2 wherein the immunoaffinity material is
protein G.
6. A method of reducing the concentration of antiviral antibodies
in a mammalian organism, said method comprising the steps of: a)
obtaining a sample of blood from said mammalian organism; b)
isolating the plasma from the cellular components from said blood
sample; c) contacting said isolated plasma with an immunoaffinity
chromatographic material comprising an immunoaffinity material
linked to a chromatographic support such that antibodies are
retained on the immunoaffinity material; d) reintroducing the
cellular components isolated from step (b) and the purified plasma
from step (c) to the mammal.
7. The method of claim 6 wherein the immunoaffinity material is
selected from the group consisting of Protein A, Protein G and a
viral coat protein.
8. The method of claim 7 wherein the immunoaffinity material is a
viral coat protein of an adenovirus selected from the group
consisting of hexon, penton, protein IX, protein IIIA fiber, knob
and penton base.
9. The method of claim 8 wherein the immunoaffinity material is
protein A.
10. The method of claim 8 wherein the immunoaffinity material is
protein G.
11. An improved method of treating a mammal with a therapeutic
virus, the improvement comprising reducing the concentration of
antiviral antibodies in said mammal prior to or in conjunction with
the administration of said therapeutic virus by: a) obtaining a
sample of blood from said mammalian organism; b) isolating the
plasma from the cellular components from said blood sample; c)
contacting said isolated plasma with an immunoaffinity
chromatographic material comprising an immunoaffinity material
linked to a chromatographic support such that antibodies are
retained on the immunoaffinity material; d) reintroducing the
cellular components isolated from step (b) and the purified plasma
from step (c) to the mammal.
12. The method of claim 11 wherein the therapeutic virus is an
adenovirus.
13. The method of claim 12 wherein the immunoadsorbent material is
selected from the group consisting of Protein A, Protein G and a
SAVID material.
14. The method of claim 13 wherein the adenovirus is a replication
deficient adenovirus.
15. The method of claim 14 wherein the virus is expresses the p53
tumor suppressor gene.
16. The method of claim 5 wherein the virus is A/C/N/53.
17. The method of claim 12 wherein the adenovirus is a replication
competent adenovirus.
18. The method of claim 17 wherein said replication competent virus
is a conditionally replicating adenovirus.
19. The method of claim 18 wherein said conditionally replicating
virus comprises a tumor specific promoter driving expression of a
early adenovirus gene.
20. The method of claim 19 wherein said tumor specific promoter is
the prostate specific antigen promoter.
21. The method of claim 17 wherein said replication competent
adenovirus contains a deletion of in the E1b-55K gene so as to
disrupt the ability of the E1B-55K gene product to bind to p53.
22. The method of claim 21 wherein said virus is dl1520.
23. A composition of matter comprising SAVID immunoaffinity
chromatographic material.
Description
RELATION OF OTHER APPLICATIONS
[0001] The present application claims the benefit of U.S. Patent
Provisional Application Serial No. 60/152,650 filed Sep. 7, 1999
pursuant to 35 U.S.C. .sctn.119(e). The present application is a
division of co-pending U.S. patent application 09/653,474 filed
Aug, 31, 2000.
BACKGROUND OF THE INVENTION
[0002] The therapeutic utility of wild type or recombinantly
modified viruses are well known in the art. Early reports on the
therapeutic use of viruses date from the 1950's. For example, in
1952, Southam and Moore reported on the use of Vaccinia, Newcastle
Disease, West Nile, Ilheus and Bunyamwera, and Egypt 101 viruses
for the treatment of a variety of cancers. Cancer 5:1025-1034
(1952). In 1956, Newman and Moore summarized the results of the
treatment of fifty-seven cancer patients with a variety of viruses.
Cancer 7:106-118. In 1956, Smith, et al. reported on the
therapeutic use of adenovirus for the treatment of cervical cancer.
Cancer 9: 1211-1218. Southam presented a summary of the clinical
experience obtained at the Sloan Kettering Institute on the
efficacy of viruses as anti-neoplastic agents in 1960. Transactions
of the New York Academy of Sciences 22:657-673. More recent reports
demonstrate a continuing interest in the therapeutic use of
viruses. Taylor, et al. presented results suggesting the
therapeutic use of bovine enterovirus-1 for the treatment of solid
and ascites tumors based on experiments conducted in mice. PNAS
(USA) 68:836-840 (1971). Additional human clinical trials continued
to demonstrate promise in this field as illustrated by the use of
Mumps virus to treat a variety of cancers. Asada, T. (1974) Cancer
34:1907-1928.
[0003] An increased understanding the viral genome and the advent
of recombinant DNA techniques permitted the manipulation of viruses
to possess particular desirable features. For example, a
recombinant adenovirus containing a modification to the E1B-55K
region is currently in Phase II clinical trials in human beings.
Additionally, recombinant viral vectors have been employed for the
delivery of a variety of therapeutic substances. Most notably,
recombinant adenoviral vectors have been employed in anti-cancer
therapies where the viral genome has been modified to encode a
tumor suppressor gene. In particular, a replication deficient virus
expressing the p53 tumor suppressor gene has successfully completed
Phase I and is currently in Phase II/III clinical development.
[0004] However, the clinical experience with such vectors has
demonstrated that a significant fraction of the therapeutic virus
which is administered to a patient is disabled by the presence of
neutralizing antibodies in the serum and reticular endothelial
system (RES). This obstacle is particularly acute when an
adenovirus is used as the vehicle for delivery of a transgene since
a significant portion of the human population has naturally been
exposed to adenoviruses vectors and possesses pre-existing
immunity. Consequently, the administration of a significant excess
of the recombinant adenoviral vector is administered to the patient
to "dose through" the pre-existing immune response. Additionally,
even if no pre-existing immune response was present, following
administration of a therapeutic virus the mammal will generally
produce an immune response to the virus. This "induced" antiviral
immune response complicates additional courses of therapy with the
therapeutic virus in a manner similar to the pre-existing immune
response induced by exposure to the virus in the environment. This
is not desirable from a clinical standpoint in that it may present
complications to the already ill patient. Furthermore, from a
commercial standpoint a large quantity of material is wasted in an
attempt to dose through the preexisting immune response.
Consequently, there is a need in the art to reduce the pre-existing
immune response to therapeutic viral vectors.
[0005] A variety of methods have been employed in an attempt to
cope with this problem. In one method, the virus is coated with
masking agents such as polyethylene glycol (so called "PEGylation")
to coat the virus and mask the immunological determinants of the
virus. This is a cumbersome process requiring that the virus be
coated with an agent and the long-term stability of the PEGylated
virus has yet to be demonstrated as commercially feasible. Other
avenues include co-administration of immunosuppressive agents.
However, the administration of broad spectrum immunosuppressive
agents is not desirable. In particular, there is mounting evidence
to suggest that a significant complement to anti-cancer therapy is
that the immune response augments the anti-tumor activity of the
therapeutic virus. This phenomenon has been observed for some time
and many individuals have suggested that amplification of the
immune response to tumor antigens may be of therapeutic benefit.
Consequently, it is not generally desirable to broadly suppress the
immune system of a cancer patient.
[0006] Consequently there remains a need in the art to reduce the
pre-existing humoral immune response to a therapeutic viral vector.
The present invention addresses this need.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions, devices and
methods to remove antiviral antibodies from the blood of mammals.
The compositions and methods of the present invention may be
practiced in conjunction with administration of a therapeutic
virus. The invention further provides an immunoaffinity material
comprising a chromatographic support material derivatized with
antigenic determinants of viral coat. The invention further
provides an improved apheresis apparatus to remove antiviral
antibodies from the blood and, in particular from plasma. The
present invention further provides therapeutic methods involving
the use of such apparatus.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 is a histogram representing ID50 values of
anti-adenoviral neutralizing antibodies following subcutaneous
administration of adenovirus. The first column (solid black)
represents antibody titer observed at 7-10 days following a single
adenovirus injection. The second column (white) represents antibody
titer observed at 14 days following a single adenovirus injection.
The third column (gray) represents antibody titer at 70 days
following two subcutaneous injections of recombinant adenovirus.
Panel A represents the antibody titer of unpurified serum. Panel B
represents antibody titer of serum purified over a column
containing adenoviral capsid epitopes. Panel C represents a vPBS
control. As can be seen from the data presented, antibody titer is
significantly higher following re-dosing (injections, 70 days).
[0009] FIG. 2 is a graphical representation of the data
demonstrating that removal of anti-adenoviral antibodies results in
an increased transduction efficiency of adenovirus in vitro. The
vertical axis is a measure of transduction efficiency with rAd/GFP
relative to untransduced control HeLa cells. Solid circles
represent serum from rAd primed animals. Triangles show
transduction efficiently of virus mixed with serum from vPBS
injected animals. Open circles indicate serum from rAd primed
animals that was depleted of anti-Ad antibodies and squares show
transduction efficiency of virus mixed with anti-Ad antibodies,
which were eluted from the column used to deplete the anti-Ad
antibodies from the rAd primed animals.
[0010] FIG. 3 is a line graph showing that the passive immunization
procedure does result in serum neutralizing antibodies in the
recipient host animal. The vertical axis represents antibody titer.
The horizontal axis represents time (hours) following passive
immunization. As can be seen from the data presented, the presence
of serum neutralizing antibodies is observed within three hours of
passive immunization and remains stable over a period of at least
72 hours. Note that the injected material (0.5 ml) is essentially
diluted to the normal blood volume of the mouse and then remains
stable for at least 72 hours.
[0011] FIG. 4, is a graphical representation of serum neutralizing
antibody capacity (ID-50) following passive immunization and
subsequent dosing with an adenovirus expressing
.beta.-galactosidase (BGCG). The vertical axis represents antibody
titer and the horizontal axis represents time in hours following
administration of virus. Each panel represents the material which
was used for passive immunization (i.e. vPBS control, Ad-Antibody
undepleted serum, Ad-Ab depleted serum: Ad-Ab). The black bars show
titer of neutralizing one hour following passive immunization. The
gray bars show neutralizing antibody titer two hours following
virus dosing after passive immunization. The hatched columns show
neutralizing antibody titer three days following virus dosing after
passive immunization. The white bars reflect neutralizing antibody
titer of the materials used for passive immunization. The data show
that an antibody response by the host is mounted within 3 days.
Moreover, the data show that the injection of rAd by intravenous
route further depletes neutralizing antibodies from the sera within
2 hours. The data also suggest that almost all the high affinity
antibody in the major coat proteins are depleted following
injection of rAd by intravenous route, eluted from the column. (see
the data relating to Ad-antibody at 40 .mu.l).
[0012] FIG. 5 are photographs of x-gal stained liver cross sections
of BALB/c mice following administration with 1.times.10.sup.10 PN
of BGCG. As can be seen with the vPBS control (A), there is
substantial staining of the liver due to the absence of a
preexisting immune response in mice. Panel B represents the x-gal
liver staining of those animal receiving passive immunization of
serum depleted of antibodies by passing over an Ad-capsid protein
column. Panel C represents .beta.-galactosidase staining of mice
receiving passive immunization of unpurified Ad-primed serum. As
can be seen from the data presented, a substantial increase in
.beta.-galactosidase expression (indicating increased transduction
efficiency) was observed in those animals receiving passive
immunization of purified serum (B) relative those receiving
unpurified serum (C).
[0013] FIG. 6 is a graphical representation of the results of
experiments conducted to compare the efficiency of different
columns in removing adenoviral neutralizing antibodies obtained
from the exposure of pooled human sera exhibiting a high
neutralizing anti-adenoviral titer (ID50>1280). The vertical
axis is percent transduction which is inversely correlated with
percentage of neutralizing capacity. The horizontal axis represents
neutralizing antisera titer. The filled squares represent primed
serum; the shaded circles represent serum depleted over a
Prosorba.RTM. protein A column; the diamonds represent serum
depleted over an adenoviral capsid SAVID column; the filled
triangles represent bound antibodies eluted from a Prosorba.RTM.
column; the open circles represent hela cells transduced with a
recombinant adenovirus encoding the green fluorescent protein
(rAd-GFP) in the absence of antisera; the open squares represent
antibodies eluted from a capsid SAVID column; and the open
triangles represent HeLa cells as a background control (no virus,
no antibodies).
[0014] FIG. 7 is a graphical representation of the results of
experiments conducted to compare the efficiency of different
columns in removing adenoviral neutralizing antibodies obtained
from the exposure of pooled human sera exhibiting a low
neutralizing anti-adenoviral titer (ID50<320). The vertical axis
is percent transduction which is inversely correlated with
percentage of neutralizing capacity. The horizontal axis represents
neutralizing antisera titer. The shaded squares represent primed
serum; the shaded circles represent serum depleted over a
Prosorba.RTM. protein A column; the shaded diamonds represent serum
depleted over an adenoviral capsid SAVID column; the shaded
triangles represent bound antibodies eluted from a Prosorba.RTM.
column; the open circles represent HeLa cells transduced with
rAd-GFP in the absence of antisera; the open squares represent
bound antibodies eluted from the SAVID adenoviral capsid column;
and the open triangles represent HeLa cells as a background control
(no rAd-GFP virus, no antibodies).
[0015] FIG. 8 is a graphical representation of the results of
experiments conducted to compare the efficiency of a protein G
column relative to a capsid SAVID column in removing adenoviral
neutralizing antibodies obtained from the exposure of pooled human
sera exhibiting a high neutralizing anti-adenoviral titer
(ID50>1280) or low neutralizing antibody titre (ID50<320).
The vertical axis is percent transduction. The horizontal axis
represents neutralizing titer. The shaded squares represent
antibodies eluted from an adenoviral capsid SAVID column using high
titer antiserum; the shaded circles represent antibodies eluted
from an adenoviral capsid SAVID column using low titer antiserum;
the shaded triangles represent antibodies eluted from a protein G
column using high titer serum; the shaded diamonds represent
antibodies eluted from a protein G column using low titer serum;
the open circles represent HeLa cells transduced with a rAd-GFP in
the absence of antisera; the open squares represent HeLa cells
transduced with rAd-GFP in presence of antisera and the open
triangles represent HeLa background control with normal serum; and
the open diamonds represent HeLa background control without
serum.
[0016] FIG. 9 is a schematic representation of one orientation of
the apparatus used in the practice of the present invention. The
arrows represent the direction of the flow of blood materials
through the apparatus. Item 1 represents a plasmaphersis apparatus.
Item 2 represents an immunoaffinity chromatographic material in
column format. Item 3 represents a 3-way valve. Item 4 represents a
storage vessel for purified plasma. Item 5 represents a filter
apparatus to remove microaggregates from the purified plasma prior
to reintroduction. Item 6 represents a drip chamber.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a method for reducing the
concentration of antiviral antibodies in a sample of plasma by
contacting said isolated plasma with an immunoaffinity
chromatographic material comprising an immunoaffinity material
linked to a chromatographic support such that antibodies are
retained on the immunoaffinity material.
[0018] The invention further provides a method of reducing the
concentration of antiviral antibodies in a mammalian organism, said
method comprising the steps of:
[0019] a) obtaining a sample of blood from said mammalian
organism;
[0020] b) isolating the plasma from the cellular components from
said blood sample;
[0021] c) contacting said isolated plasma with an immunoaffinity
chromatographic material comprising an immunoaffinity material
linked to a chromatographic support such that antibodies are
retained on the immunoaffinity material;
[0022] d) reintroducing the cellular components isolated from step
(b) and the purified plasma from step (c) to the mammal.
[0023] It will be readily understood by those skilled in the art
that the procedures of step (d), i.e. the reintroduction of the
cellular components and purified plasma, may occur simultaneously
or be separated by some period of time. However, it is preferred
that these procedures be performed contemperaneously.
[0024] The invention further provides an improved method of
treating a mammal with a therapeutic virus wherein the improvement
comprises reducing the concentration of antiviral antibodies in
said mammal prior to the administration of said therapeutic virus
by:
[0025] a) obtaining a sample of blood from said mammalian
organism;
[0026] b) isolating the plasma from the cellular components from
said blood sample;
[0027] c) contacting said isolated plasma with an immunoaffinity
chromatographic material comprising an immunoaffinity material
linked to a chromatographic support such that antibodies are
retained on the immunoaffinity material;
[0028] d) reintroducing the cellular components isolated from step
(b) and the purified plasma from step (c) to the mammal.
[0029] The invention further provides immunoaffinity
chromatographic materials comprising components of the viral
coat.
[0030] The invention further provides apparatus useful in the
practice of the present invention.
[0031] The invention further provides an improvement to a method of
treatment of a mammal with a therapeutic virus wherein the
foregoing procedure is performed in advance of the administration
of a therapeutic virus.
[0032] Antiviral Antibodies
[0033] The term antiviral antibodies is used to describe antibodies
which bind to viruses. These antibodies are generally produced by
the mammalian humoral immune system by exposure of the mammal to a
virus, either environmentally or therapeutically (e.g. vaccination
or administration of a therapeutic virus). Antiviral antibodies
comprise both neutralizing and non-neutralizing antibodies. The
term neutralizing antibodies is used in its conventional sense to
refer to antibodies which prevent the proliferation of infectious
virus. When the method is used in conjunction with the
administration of a therapeutic virus, removal of neutralizing
antiviral antibodies is of greater concern than the removal of
non-neutralizing antibodies as neutralizing antibodies are
primarily responsible for inactivation of the therapeutic virus to
be administered.
[0034] Blood
[0035] The term "blood" is used in its conventional sense to refer
to the blood of a mammal. A blood sample may be obtained from a
living mammal by conventional blood collection procedures such as
venipuncture or arterial puncture techniques. The lower risks and
ease of access associated with venous collection procedures make
venous blood the preferred source of blood for use in the present
procedure. Venous blood may be collected by syringe or evacuated
collection vessels such as the Vacutainer.RTM. brand blood
collection system (commercially available from Becton-Dickinson
Corporation, Franklin Lakes, N.J.) for batchwise treatment
according to the method of the present invention. However, the
quantities of blood generally treated to provide dimunition of
antiviral antibody response in a living mammal, in the preferred
practice of the invention the blood is collected in a continuous
process for introduction to a plasmapheresis apparatus by venous
catheter. The proximal end of a catheter is inserted into the vein
of the mammal using conventional venipuncture techniques and the
distal end of the catheter is connected to the inlet port of a
plasmapheresis apparatus. A plasmapheresis apparatus generally
comprises a pump mechanism to drive the isolated blood through a
means for isolating plasma from cellular blood components, wherein
the cell rich blood is returned to the mammal while the plasma is
directed for storage or further processing.
[0036] The range of mammals subject to therapeutic virus therapy
are varied and certain sources are preferred for the isolation of
venous blood. In cattle or sheep, jugular catheterization is
preferred. In rabbits, the median ear artery, ear vein, or carotid
artery are preferred. In pigs, the ear vein is preferred. In
non-human primates, the saphenous, cephalic, and femoral veins are
preferred sources of venous blood. In cats, the cephalic, femoral,
jugular and saphenous veins are preferred. In dogs, the cephalic,
jugular, and saphenous veins are preferred. In human beings, the
cephalic and saphenous veins are preferred sources of venous blood,
cephalic being most preferred.
[0037] Isolation of Plasma: Plasmapheresis
[0038] Blood is a complex mixture of cellular and solubilized fluid
components. The fluid portion of blood (plasma) comprises a variety
of dissolved nutrients, waste products and solubilized proteins
such as antibodies. The term "plasmapheresis" refers to an
apheresis procedure in whereby blood removed from a mammal is
separated into plasma and cellular blood components, the plasma
being isolated for further processing. The principles and practice
of apheresis are well known in the art. Standard procedures for
apheresis are described in Apheresis: Principles and Practice
commercially available from the American Association of Blood
Banks, 8101 Glenbrook Road, Bethesda, Md. 20814-2749 as Stock
#PC98-972003.
[0039] Plasmapheresis is currently performed in clinical arena
using continuous flow centrifugal separators, which separate cells
by density; flat-sheet and intra-lumenal hollow fiber membrane
devices, which operate by tangential flow microfiltration; and
rotating membrane devices, which enhance microfiltration flux by
inducing Taylor vortices. Such devices are commercially available
and are well known in the literature, see, e.g. Plasmapheresis:
Therapeutic Applications and New Techniques, Nose Y, et al., Raven
Press, New York (1983); Kessler S. B., Blood Purif., 11: 150-157
(1993);. See, also Fischel, U.S. Pat. No. 5,783,085 issued Jul. 21,
1998; Kessler, et al., U.S. Pat. No. 5,846,427 issued Dec. 8, 1998;
Ash, U.S. Pat. No. 5,919,369 issued Jul. 6, 1999, Ball, et al. U.S.
Pat. No. 5,914,042 issued Jun. 22, 1999; Fischel, U.S. Pat. No.
5,464,534 issued Nov. 7, 1995; Bensinger, U.S. Pat. No. 4,614,513
issued Sep. 30, 1986; Terman, et al. U.S. Pat. No. 4,215,688 issued
Aug. 5, 1980; and Pollard, Jr., U.S. Pat. No. 4,464,165 issued Aug.
7, 1984, the teachings of which are herein incorporated by
reference.
[0040] Immunoadsorbent/Immunoaffinity Material
[0041] The terms "immunoaffinity material" or "immunoadsorbent
material" are used interchangeably herein to refer to material
which binds to antibodies. A variety of immunoadsorbent materials
are well known in the art such as Staphyloccus aureus protein A,
recombinant protein A and G, KappaLock.TM. (Zymed). The use of
generalized immunoadsorbent material (such as protein A) or protein
G results in general elimination of circulating antibodies and is
not necessarily specific with relation to the potentially antigenic
therapeutic virus to be administered. Protein A binds preferably to
IgG class antibodies (and to some extent to IgA and IgM antibodies)
and is not selective for any particular antibody type. Although
these general immunoadsorbent materials are not necessarily
specific to a particular type of antibody, they are nonetheless
useful immunoadsorbent materials which may be employed in the
practice of the present invention as shown in the data presented in
FIGS. 6, 7, and 8 of the attached drawings.
[0042] In the preferred practice of the invention the
immunoadsorbent material is prepared with respect to the particular
therapeutic virus to be employed in order to achieve selective
elimination of antiviral antibodies specific to that virus by the
linkage of a viral epitope or epitopes to the chromatographic
support. As previously discussed, the broad elimination of serum
antibodies has the potential to eliminate any circulating
anti-tumor antibodies which are a potentially important adjunct to
successful chemotherapy and potentially leaves one more susceptible
to opportunistic infections. Consequently, selective removal of
antibodies to the particular therapeutic virus being employed is
preferred. In particular, the present invention provides a
selective antiviral immuno-depletion ("SAVID") chromatographic
material comprising a viral epitope conjugated to a chromatographic
support. The acronym SAVID is employed as shorthand for selective
antiviral immuno-depletion. Selective, emphasizing the nature of
the process whereby the antibodies against the therapeutic virus to
be used are selectively eliminated from the plasma. Antiviral,
emphasizing that the methods and devices are designed to eliminate
the pre-existing or induced immune response to therapeutic viruses.
Immuno-Depletion, emphasizes the feature of the invention in that
the immune response, particularly the level of plasma antiviral
antibodies, is transiently reduced. Through the use of a viral
epitope as the immunoadsorbent material, selective reduction of
antiviral antibodies from the plasma is achieved, the antiviral
antibodies present in the bloodstream being retained on the SAVID
chromatographic material. As a result of this process, the
pre-existing or induced humoral immune response to a specific
type(s) of therapeutic viral vector(s) is selectively diminished
resulting in increased transduction efficiency from a given dosage
of the viral vector. Concomitantly, this permits a lower viral
dosage to achieve an equivalent therapeutic response in the absence
of this procedure.
[0043] The term "epitope" is used herein in its conventional sense
to refer to the structure on an antigen that interacts with the
combining site of an antibody or T-cell receptor as a result of
molecular complementarity. The term "viral epitope" is used to
refer to epitopes of viral surface proteins. Epitopes may be
naturally occurring or synthetic mimetics. The epitope employed may
represent the entire viral coat protein or antigenic determinant
fraction thereof. For example, in order to present the viral
epitope, a viral coat protein may be conjugated to the column.
Alternatively, a fragment of the viral coat protein may be employed
which retains the primary antibody binding site. The epitope may
also be a synthetic peptide or protein and is used to collectively
refer to naturally occurring or synthetic peptides which comprise
the antigenic determinants of viral surface proteins. The
determination of those regions of a protein most responsible for
antibody binding can be determined by procedures known in the art.
For example, the DIRECT.TM. methodology, instrumentation and
procedures commercialized by Argonex, Inc., 2044 India Road,
Charlottesville Va. 22901 provides a means to identify the
individual peptides that stimulate a CTL response. These peptides
may be modeled using conventional molecular modeling software to
generate non-peptidyl small molecule epitope mimetics. These
individual peptides or small molecule mimetics representing a
particular epitope of a protein may be conjugated to the
chromatographic support in lieu of using the entire viral surface
protein.
[0044] In the preferred embodiment as exemplified herein, the viral
surface antigenic moieties comprise the viral coat proteins of the
adenovirus. These may be readily obtained as by-products of the
production of recombinant virus or be produced by recombinant DNA
techniques for the production of proteins well known to those of
skill in the art. Methods for the high titer production of viruses
are provided in Giroux, et al., U.S. Pat. No. 5,994,134 issued Nov.
30, 1999 the entire teaching of which is herein incorporated by
reference. For example, a recombinant adenovirus preparation was
subjected to column chromatography as described in Shabram, et al.
(U.S. Pat. No. 5,837,520 issued Nov. 17, 1998 the entire teaching
of which is hereby incorporated by reference). The chromatography
eluent provides the purified adenovirus with a variety of
contaminants comprising viral coat proteins such as penton, hexon,
3A, fiber proteins of the adenovirus. These additional
"contaminants" representing useful epitopes may be purified to
homogeneity from the column eluent by conventional chromatographic
procedures. The identities of these proteins may be easily
confirmed by the use of commercially available antibodies specific
against the adenoviral capsid. Such antibodies are commercially
available from a variety of sources Chemicon and Lee BioMolecular.
Alternatively, monoclonal antibodies to such proteins may be
generated by techniques well known to those of skill in the
art.
[0045] Linked
[0046] The term "linked" is used herein to describe a kinetically
stable association with and is used interchangeably with the terms
conjugated or cross-linked. A stable interaction between the
immunoadsorbent material and the chromatographic support may be
achieved be by ionic, affinity, covalent cross-linking, kinetically
labile coordinate covalent cross-linkages (Smith, et al. U.S. Pat.
No. 4,569,794), or kinetically inert coordinate covalent
cross-linkages as described in Anderson, et al. (U.S. Pat. No.
5,439,829 issued Aug. 8, 1995). In one embodiment of the invention
as exemplified herein, the viral epitopes are adsorbed onto the
surface of an affinity gel (Affi-Gel.RTM.). The Affi-Gel.RTM.
product is available in two different versions: Affi-Gel.RTM. 10
(BioRad Catalog No. 153-6046) and Affi-Gel.RTM. 15 (BioRad Catalog
No. #153-6052). The proper use of either product (or a mix of the
two) is determined by the pI of the epitope to be coupled. The
Affi-Gel column couples free alkyl or amino groups. A commonly
employed buffer, Tris, will couple to the column effectively.
Consequently, it is important when using such columns to avoid the
use of Tris buffers. Alternative to the use of Tris, cell lysis,
wash, column loading/washing and elution is all performed using
buffers made with MOPS or HEPES rather than Tris. Detailed
instructions on performing the coupling of the protein in 0.1 M
MOPS to the matrix are available from the manufacturer.
Approximately 0.5 ml (packed volume) of matrix binds approximately
20 mg of protein. Coupling is achieved by mixing and allowing the
reaction to proceed for approximately 4 hours at 4.degree. C. The
efficiency of coupling is determined by comparing the free protein
concentration before and after the coupling reaction. Following
coupling reaction, it is preferred to block any unreacted esters.
This can be readily achieved by removing the excess protein and
adding 1M ethanolamine pH 8.0, with mixing at 4.degree. C. for an
hour. Excess ethanolamine is rinsed from the column with TBS. The
column may be stored for extended periods at 4.degree. C. in TBS
plus 0.2% sodium azide.
[0047] Alternative to linking through the free amino groups, one
may also link the epitope to the column matrix via free cysteine
residues. For example, a cysteine residue can provide a stable
thio-ether bond to Epoxy-Sepharose.RTM.-6B (commercially available
from Pharmacia). This may be employed to link the C-terminus of the
epitope by the addition of a C-terminal cysteine residue. The
coupling reaction may be achieved by addition of the epitope to 1.0
ml of pre-swollen Epoxy-Sepharose-6B.RTM. in 0.1M NaHCO.sub.3 pH9.0
in a small reaction volume with constant mixing at 37.degree. C.
for 24 hours. Washing with 50 ml of 0.1 M NaHCO.sub.3 pH9.0, then
block unreacted groups on the matrix by incubating the 1.0 ml of
resin in 5.0 ml of 0.1M 2-mercaptoethanol with gentle
agitation.
[0048] Chromatographic Support
[0049] The term "chromatographic support" is used in its commonly
accepted definition as the basic element of an affinity
chromatography matrix. The principles of immunoaffinity
chromatography are well known in the art. See, e.g. Mohr, et al.
(1992) Immunosorption Techniques: Fundamentals and Applications,
ISBN# 3055013506. Common chromatographic supports include natural
or synthetic polymers in the form of a membrane, bead
(microparticle), resins, or tube. Common materials include agarose
(a polymer of D-galactose and 3,6-anhydroL-galactose), polystyrene,
polyethylene, etc. and are well known to those of skill in the art.
In the preferred practice of the invention, the chromatographic
support is a cross-linked activated agarose gel such as the
Affi-Gel.RTM. 10 or Affi-Gel.RTM. 15 supports commercially
available from Bio-Rad.
[0050] Contacting
[0051] The isolated serum is may be brought into contact with the
with the immunoaffinity chromatographic material in a variety of
contexts. The immunoaffinity chromatographic material may be used
in batch preparations whereby the blood is exposed to a quantity of
the material, but generally will be formed into a column for use
with a conventional plasmapheresis apparatus. The column may either
be a packed bed column comprising a stationary phase in a granular
form and packed so as to form a homogenous bed wherein the
stationary phase completely fills the column. Alternatively, open
tubular columns may be employed wherein the stationary phase
comprising the viral epitope is deposited as a thin film or layer
on the column wall and possessing a central passage to permit
passage of the mobile phase. In such instances, a plurality of
small diameter tubular materials are employed to maximize the
exposure of the viral epitopes to the mobile phase.
[0052] Alternatively the immunoaffinity chromatographic material
may be in liquid form but possessing a greater or lesser density
than blood products such that the blood products can be exposed to
the chromatographic material and easily separated. For example,
capsid proteins could be bound to a dense material conjugated to a
liquid polymer whose density would be, for example, 1.35. The
materials could be mixed in batch mode with the isolated blood
product. The blood components binding to the material could then be
separated by centrifugation. Alternatively, the immunoaffinity
chromatographic material capable of phase separation could be mixed
with the blood and separated in a centrifugal partition
chromatography device. Centrifugal countercurrent chromatographic
devices and procedures are known in the art. Either the
immunoaffinity chromatographic material or the blood or plasma
could be employed as the mobile phase in such procedures.
[0053] The antibodies retained on immunoaffinity chromatographic
support may be removed to regenerate the column for repeated use.
However, the immunoaffinity chromatographic material is preferably
made from disposable materials and is disposed of following use to
ensure that the materials isolated from one patient and retained on
the column by ineffective washing procedures are not leeched from
the column and introduced into the bloodstream of a second
patient.
[0054] It will be readily apparent to those of skill in the art
that the plasma produced following passage over the immunoadsorbent
material may be stored for later use. However, in the preferred
practice of the invention, the purified plasma is reintroduced into
the circulation system of the subject contemporaneously with the
purification process. The term "contemporaneously" generally means
less than about three hours, preferably less than one hour. In the
most preferred practice of the invention to avoid the complications
associated with losses in blood volume in live mammals, the
foregoing process is run in a continuous mode using an apparatus
such as the apparatus schematically represented in FIG. 9 of the
attached drawings.
[0055] In order to demonstrate that the present method is useful in
the reduction of serum neutralizing antiviral antibodies, serum of
mice exposed to human adenovirus was subjected to purification on a
Protein A column and a KappaLock.TM. sepharose column (commercially
available from Zymed Laboratories, Inc., South San Francisco,
Calif. as catalog No. 10-1841) which retains primarily IgG, IgM,
and IGA class immunoglobulins and assayed for neutralizing anti-Ad
antibodies. In particular, the blood of mice possessing
pre-existing anti-adenoviral antibodies was isolated, the serum
separated and exposed to a Protein A chromatographic resin. The
levels of serum neutralizing anti-adenoviral antibodies before and
after the procedure was determined in substantial accordance with
the teaching of Example 3 herein. Moreover, neutralizing antibody
could be effectively depleted in-vitro with a column consisting of
rAd proteins linked to a bead rAd affinity column). In this
experiment, serum purified on a Protein A column or
KappaLock.TM.-Sepharose column showed a reduction in serum
neutralizing antibody titer.
[0056] The utility of a SAVID immunoadsorbent material was further
demonstrated in an in vivo mouse passive immunization model. Since
conventional plasmapheresis procedures are not feasible in mice and
mice do not innately possess anti-human adenoviral antibodies,
"passive immunization" procedure was used to create a model system
to mimic plasmapheretic removal of anti-adenoviral antibodies.
Passive immunization refers to a procedure whereby the serum of one
animal is introduced into a second animal. In this instance, a
group of BALB/c mice were injected with recombinant human
adenovirus (hAd) in order to develop anti-hAd antibodies. These
mice were sacrificed and the sera were collected. This serum was
then injected intraperitoneally into a second group of synergenic
BALB/c mice. In order to ensure that this passive immunization
procedure was effective at transmitting the ant-hAd antibodies to
the serum of the intraperitoneally injected mice in the second
group, a pilot study was conducted to show that 10 .mu.g of
anti-adenoviral antibody eluted from the rAd -column, or equivalent
amount of serum (in a total volume of 500 .mu.l) injected
intraperitoneally can be detected in the serum of naive BALB/C
mice. The results of these experiments are presented in FIG. 3 of
the attached drawings. As can be seen from the data presented,
anti-hAd antibodies were present in the serum as soon as 3 hours
post IP administration and were still detectable 72 hours after IP
injection. The serum (500 .mu.l) was diluted by the normal serum
volume in the mouse and then remained stable for at least 72 hours.
These results indicate that mice injected intraperitoneally with
serum containing anti-adenoviral antibodies effectively transfer
humoral immunity (i.e. neutralizing antibodies to adenovirus) and
provides a valid model system for demonstrating the effectiveness
of the present invention.
[0057] A recombinant adenovirus (ZZCB) was used to prime the mice
with a recombinant human adenovirus to generate a "pre-existing"
immune response for later evaluation. The method of preparation of
the ZZCB and BGCG vector is described in Gregory, et al., supra,
and are referred to in that reference as the A/C and A/C/.beta.-gal
viruses respectively. BALB/c mice were injected with
5.times.10.sup.10 particles of ZZCB. The mice were then given a
booster injection of 5.times.10.sup.10 particles 28 days following
the first injection. The mice were sacrificed 14 days following the
second injection and the serum isolated and pooled. A cross-linked
agarose column was prepared in substantial accordance with the
teaching of Example 1 herein using multiple human adenoviral capsid
proteins cross-linked to the column as a source of epitopes. One
fraction of the pooled serum was allowed to equilibrate with the
column material and eluted (referred to hereinafter as "purified
serum") and the remaining fraction of the pooled serum was retained
as a control (hereinafter referred to as "unpurified serum").
[0058] Five mice were each injected intraperitoneally with 40 .mu.g
of anti-hAd antibodies eluted from the rAD-protein column, 80 .mu.g
of anti-hAd antibodies eluted from rAd-protein column,
column-depleted serum, untreated serum from primed animals and
serum from vPBS (phosphate buffered saline further containing 2 mM
MgCl.sub.2, 3% sucrose) injected animals and a sample of blood
obtained one hour following passive immunization (to use as a
standard) and allowed to rest overnight. The following day
(approximately 12 hours post passive immunization), the mice were
injected via the tail vein with 5.times.10.sup.10 particles of the
BGCG recombinant human adenovirus. Serum was collected at 2 hours
post injection and the serum was analyzed for the presence of serum
neutralizing anti-adenoviral antibodies. The mice were sacrificed
at 3 days following injection of BGCG and the serum analyzed for
the presence of serum neutralizing anti-adenoviral antibodies and
liver tissue was analyzed for B-gal expression. The results of
these experiments can be seen in FIG. 4 of the attached drawings.
Animals receiving passive immunization with unpurified serum or 40
.mu.g or 80 .mu.g of anti-hAd antibodies eluted from the column
demonstrated a high titer of serum neutralizing anti-hAd antibodies
one hour following IP injection. However, those animals which were
passively immunized with column-depleted serum, had a no detectable
serum neutralizing antibodies at one hour post injection. The
presence of neutralizing antibodies in all five groups of mice on
the third day following BGCG injection is due to a primary humoral
response to BGCG by the. Consequently, the level of serum anti-hAd
neutralizing antibodies in those animals passively immunized with
purified serum was substantially diminished relative to those
animals receiving unpurified serum indicating that removal of
antiviral antibodies is useful in vivo to minimize the pre-existing
immune response to such viruses. The serum purified over a SAVID
column showed a improved reduction in serum neutralizing anti-hAd
antibodies relative to the Protein A and KappaLock.TM.-Sepharose
columns in vivo. Also evident from the data in FIG. 4 was that the
intravenous injection of BGCG resulted in a further decrease in
neutralizing antibodies in the sera (see undepleted serum two hour
post virus). Moreover, in the group that received rAd antibody
eluted from columns, virtually all the neutralizing antibody was
depleted from the serum following injection of the BGCG by
intravenous route. Thus systemic administration of rAd depletes
serum neutralizing antibody and suggests that high affinity
antibodies are depleted very efficiently. Thus readministration one
hour or one day after injection of rAd may further promote
redosing.
[0059] Perhaps more important to the clinician and from a
commercial standpoint is the fact that the diminution of the
pre-existing or induced humoral immune response permits the use of
a lower dose of virus to achieve the same level of therapeutic
transgene expression. This was demonstrated by examining
.beta.-galactosidase expression in the livers using the animals
from the previous experiment. It is known from previous experiments
that the majority of expression of systemically administered
replication deficient adenovirus results in transduction of the
cells of the liver. Consequently, upon sacrifice of the animals
from the previous experiment, the livers were removed and assayed
for .beta.-galactosidase activity as an indicator of BGCG
transduction efficiency. The results are presented in the photo
micrographs in FIG. 5 of the attached drawings. These data provide
evidence of the relative levels of .beta.-galactosidase expression
in livers from animals receiving passive immunization of
column-depleted serum, undepleted serum, anti-hAd antibodies eluted
from the rAd protein column serum from animals tests with vPBS. The
data demonstrate that the level of expression of
.beta.-galactosidase expression in mice receiving column depleted
the serum is substantially greater than those mice receiving
undepleted sera. Challenging these passively immunized mice with a
dose of virus showed that transduction of virus was not detectable
in livers of mice passively immunized with Ad-unabsorbed serum,
however virus transduction was readily detected in livers of mice
passively immunized with Ad-preadsorbed serum. Consequently, these
experimental results demonstrate that the present invention is
useful in vivo to increase transduction efficiency from recombinant
adenoviral vectors in mammalian systems possessing pre-existing
serum neutralizing antibodies. In particular, these data
demonstrate that effective immunodepletion of anti-adenoviral
antibodies can reduce the dosage of virus administration for
transduction to achieve equivalent levels of transgene expression
in the target tissues.
[0060] In order to determine the effects of this procedure in the
human population, a series of experiments were conducted using
human blood containing anti-adenoviral antibodies. Human serum from
normal donors was obtained from the San Diego Blood Bank. The
neutralizing capacity of each sample was determined by the GFP
assay described in Example 3 herein. The serum was characterized as
having high or low neutralizing capacity. Low neutralizing capacity
was defined as a titer of 1:320 or less. High neutralizing capacity
was defined as a titer of 1:2500 or more. The serum samples
exhibiting either "high" or "low" neutralizing capacity were pooled
and subjected to column chromatography over a Protein G column
(commercially available from Zymed) and SAVID adenoviral capsid
column (prepared in substantial accordance with the teaching of
Example 2 herein). The results are presented in FIGS. 6, 7, and 8
of the attached drawings. The results demonstrate a significant
reduction in serum neutralizing capacity following passage over the
SAVID column. Furthermore, the results demonstrate that the
antibodies eluted from the protein G column exhibited the same
titer as the antibody eliminated form the capsid column suggesting
that the efficiency of the protein G column and capsid column was
similar in removing antiadenoviral antibodies.
[0061] It will be readily apparent to the skilled artisan that a
combination of the above immunoadsorbent materials may also be
employed. Such materials may be used together in a heterogenous
mixture or sequentially to achieve improved reduction in antibodies
from the serum or to remove antibodies of various types in a single
plasmapheresis procedure.
[0062] Apparatus
[0063] The present invention further provides a plasmapheresis
apparatus wherein the plasma filtration element of such apparatus
comprises an immunoaffinity chromatographic material comprising a
viral epitope conjugated a chromatographic support. The plasma
filtration elements of such devices is modified to incorporate an
immunoaffinity chromatographic material in column format which is
used to eliminate antiviral antibodies from blood plasma. Such
apparatus is provided in schematic form in FIG. 9 of the attached
drawings.
[0064] The immunaodsorbent material linked to the chromatographic
support is generally to be supplied in kit of parts comprising the
immunoadsorbent material linked to the chromatographic support
packaged with instructions for the proper use of the material. In
the preferred embodiment, the immunoadsorbent material linked to
the chromatographic support is contained in an aseptic vessel
constructed capable of sterilization such as glass or plastic (e.g.
polycarbonate plastic). Preferably, the vessel is substantially
cylindrical in shape defining a top and bottom surface, each of
which top and bottom are provided with a centrally located nipple
for attachment to the flexible tubing conventionally used in
apheresis procedures to permit passage of the plasma through the
vessel. The vessel optionally comprises a membrane element of pore
diameter sufficient to permit the free flow of plasma but
insufficient to allow passage of the immunoadsorbent material. The
vessel containing the immunoadsorbent chromatographic material is
generally to be provided in sterile packaging materials
facilitating its sterility immediately prior to use. The kit of
parts may optionally comprise a means for attaching the vessel to a
support means and to maintain the vessel in a substantially
vertical position during its use. Examples of such attachment means
are well known in the art such as the Catalog No. 05-769 adjustable
3-prong clamp commercially available from Fisher Scientific. The
kit of parts may optionally include flexible sterile tubing
facilitating connection to conventional apheresis apparatus. The
kit of parts may optionally include sterile heparinized luer lock
3-way valves to facilitate the draining of unwanted material from
the essentially closed loop apparatus. The kit of parts may
optionally include a leukocyte filter (e.g. Pall PL100.RTM. filter
commercially available from Pall Corporation, 2200 Northern
Boulevard, East Hills, N.Y. 11548) to facilitate the removal of
microaggregates. The kit of parts may optionally include a drip
chamber to avoid the introduction of air to the line returning to
the patient's venous supply.
[0065] Applications
[0066] As previously described, the method and apparatus of the
present invention may be used in combination with the
administration of therapeutic viruses to a mammal wherein the
process for removing antiviral antibodies from the subject is
performed in advance of the administration of a therapeutic virus.
It should be noted that the present invention may be used to remove
pre-existing antiviral antibodies (i.e. antibodies in a naive
patient who has not been exposed to the therapeutic virus) or
antiviral antibodies induced through previous exposure to the
therapeutic virus. The method of the present invention and the
materials provided are suitable for use in the clinical
environment, particularly in conjunction with the administration of
therapeutic products comprising engineered therapeutic viruses.
[0067] Whether or not an individual possesses pre-existing
neutralizing antibodies to a given virus may be readily determined
by conventional assay procedures. Example 2 herein provides an
example of such an assay which may readily be employed in the
clinical environment. Similar assays relating to the detection of
other antiviral antibodies are known in the scientific literature
may readily be employed.
[0068] Although a subject may not possess antibodies to a given
therapeutic virus, following the administration of the first dose
of such virus, the immune system of the mammal will mount an immune
response against the vector. Consequently, the method of the
present invention is particularly useful in the context where
multiple doses of the virus are administered to the subject over a
prolonged period of time. The fact that the present invention
facilitates the redosing of an individual with a given therapeutic
virus is of particular value.
[0069] Apheresis procedures are commonly practiced in the clinical
environment and the adjustments to the following general procedure
for the individual patient will be readily apparent to the
ordinarily skilled clinician. Conventional aseptic technique should
be employed throughout the procedure. Certain precautions commonly
evaluated in the practice of apheresis procedures should be
observed. For example, patients who are receiving angiotensin
converting enzyme (ACE) inhibitor medications should not use such
medications for a period of approximately 72 hours prior to
initiation of the apheresis procedure. Additionally, patients
exhibiting significant vascular or intracranial diseases where
minor fluid or pressure shifts could result in harmful effects,
patients with impaired renal function, severe anemia or systemic
infections should be carefully evaluated by the clinician for their
suitability for apheresis procedures.
[0070] The blood volume of a typical human being is 69 ml/kg of
body weight for human males and 65 ml/kg for human females. Of this
volume 39 ml/kg (males) and 40 ml/kg (females) is attributable to
plasma volume. Consequently a typical male human patient weighing
75 kg possesses a plasma volume of approximately 3 liters. It is
not essential that the entire plasma volume of the patient be
isolated and subjected to the column procedure in order to achieve
a therapeutically significant reduction in the preexisting
immunity. A reduction of serum neutralizing titer of approximately
one log results in significant increase in transduction efficiency
of recombinant adenoviral constructs. A reduction in titer of
approximately two logs is more preferred. Additional reductions in
serum neutralizing titer are preferable, but are not believed to be
clinically necessary. However, it is preferred that a substantial
fraction of the plasma volume (1 liter or more) be isolated for
treatment. This volume of serum is routinely isolated by
conventional apheresis techniques.
[0071] Human serum contains a variety of antibodies only a fraction
of which are related to neutralizing activity. For example human
plasma contains approximately 10 mg/ml of IgG, only a fraction of
which is associated with neutralizing anti-adenoviral activity.
Consequently, the immunoaffinity chromatographic column preferably
comprises approximately 100-500 mg of adenoviral capsid protein or
protein G (more preferably 200-400 mg) bound to the chromatographic
support per each liter of plasma to be treated. Adenoviral capsid
proteins may be obtained by culture of adenoviruses in accordance
with known procedures. Immunoadsorption column supports comprising
cross-linked protein G are available from commercial sources such
as Zymed Laboratories, Inc., 458 Carlton Court, South San
Francisco, Calif. 94080.
[0072] Prior to clinical use, the immunoaffinity chromatographic
material should be thoroughly washed by passing approximately three
bed volumes of clinical sterile phosphate buffered saline through
the column. Following the washing procedure, one bed volume of
heparin sulfate (approximately 5000 units in a volume 500 ml)
should be introduced into the column. The column is then placed
upright and should not be agitated or air allowed to enter the
column bed or feed lines. A conventional arrangement of the
apparatus used in the practice of the method of the present
invention is represented schematically in FIG. 9 of the attached
drawings.
[0073] Venous access to the patient is established and the venous
whole blood enters the apheresis apparatus where plasma components
are isolated from other non-plasma components. Non-plasma
components are returned to the patient's bloodstream. Plasma
components are directed to the immunoaffinity chromatographic
column. The isolated serum should be passed through the
immunoaffinity chromatographic column at a flow rate of from
approximately 5 ml/min to approximately 25 ml/min. More preferably,
a flow rate of from 10-20 ml/min is maintained throughout the
course of the procedure. The first volume equivalent to at least
the heparin volume should be discarded to avoid the introduction of
heparin to the patient. Remaining column purified serum is directed
to the vessel for receiving treated plasma. Prior to reintroduction
to the patient, it is preferred that the treated plasma components
may be passed through a leukocyte filter (e.g. Pall PL100.RTM.
filter commercially available from Pall Corporation, 2200 Northern
Boulevard, East Hills, N.Y. 11548) to remove microaggregates.
Additionally, in order to avoid any air volume entering the line
returning to the patient's venous supply, a drip chamber should be
employed.
[0074] The reduction of serum neutralizing capacity resulting from
the present procedure results in a reduction of serum neutralizing
capacity persisting for at least 5 days. Consequently, in order to
maximize the therapeutic benefit of the foregoing procedure, it is
preferred that the administration of the virus should occur within
this period of time following the procedure. Since the depletion of
antiviral antibodies is observed relatively quickly following the
plasmapheresis procedure and the immune system immediately attempts
to regenerate this response, it is preferred that the
plasmapheresis procedure be performed relatively soon (preferably
hours) prior to administration(s) of the therapeutic virus.
Additionally, since injection of the therapeutic virus results in a
further depleted serum neutralizing antibody, further
administrations of virus (serial injection) may be performed from
about one hour to about one day after the initial dose to take
maximal effect of this secondary antibody depletion. Although the
plasmapheresis procedure is well tolerated by human patients there
is no technical limitation to daily performance of this procedure
to achieve the maximal effect. However, as previously stated, the
effect produced is of sufficient duration that daily repetition of
the process is not generally required.
[0075] It should be noted that it is not essential to completely
deplete the antiviral antibodies from the circulation in order to
provide a beneficial effect. Consequently, it is not necessary that
the entire plasma volume of the individual be subjected to the
present chromatographic procedure. Although the effect of the
procedure on minimizing immune response to a therapeutic virus is
enhanced with increased dimunition of the levels of antiviral
antibodies, even a relatively modest reduction in the concentration
of antiviral antibodies can provide a significant therapeutic
effect. In the preferred practice of the invention, in accordance
with conventional apheresis procedure, approximately one liter of
serum is isolated for purification over the immunoaffinity column
in a single procedure. If greater reduction of antiviral antibodies
is required, the procedure may be expanded to cope with larger
volumes or be performed repeatedly.
[0076] The terms "therapeutic virus" and "therapeutic viral vector"
are used interchangeably herein to refer to viruses used as
therapeutic agents (e.g. wild-type viruses, attenuated viruses),
vaccine vectors or recombinant viruses containing modifications to
the genome to enhance therapeutic effects. The use of viruses or
"viral vectors" as therapeutic agents are well known in the art as
previously discussed. Additionally, a number of viruses are
commonly used as vectors for the delivery of exogenous genes.
Commonly employed vectors include recombinantly modified enveloped
or non-enveloped DNA and RNA viruses, preferably selected from
baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae,
poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors
may also be employed which exploit advantageous elements of each of
the parent vector properties (See e.g., Feng, et al.(1997) Nature
Biotechnology 15:866-870). Such viral vectors may be wild-type or
may be modified by recombinant DNA techniques to be replication
deficient, conditionally replicating or replication competent.
[0077] Therapeutic viruses are currently administered to mammalian
subjects by a variety of routes of administration including local
administration (e.g. intratumoral injection), regional
administration (e.g. intraperitoneal, intravesicular, or
intrahepatic arterially) and systemic administration (e.g.
intramuscular and intravenous).
[0078] Preferred vectors are derived from the adenoviral,
adeno-associated viral and retroviral genomes. In the most
preferred practice of the invention, the vectors are derived from
the human adenovirus genome. Particularly preferred vectors are
derived from the human adenovirus serotypes 2 or 5. The replicative
capacity of such vectors may be attenuated (to the point of being
considered "replication deficient") by modifications or deletions
in the E1a and/or E1b coding regions. Other modifications to the
viral genome to achieve particular expression characteristics or
permit repeat administration or lower immune response are
preferred. Most preferred are human adenoviral type 5 vectors
containing a DNA sequence encoding the p53 tumor suppressor gene.
In the most preferred practice of the invention as exemplified
herein, the vector is replication deficient vector adenoviral
vector encoding the p53 tumor suppressor gene A/C/N/53 as described
in Gregory, et al., U.S. Pat. No. 5,932,210 issued Aug. 3, 1999
(the entire teaching of which is herein incorporated by
reference).
[0079] Alternatively, the viral vectors may be conditionally
replicating or replication competent. Conditionally replicating
viral vectors are used to achieve selective expression in
particular cell types while avoiding untoward broad spectrum
infection. Examples of conditionally replicating vectors are
described in Pennisi, E. (1996) Science 274:342-343; Russell, and
S. J. (1994) Eur. J. of Cancer 30A(8):1165-1171. Additional
examples of selectively replicating vectors include those vectors
wherein an gene essential for replication of the virus is under
control of a promoter which is active only in a particular cell
type or cell state such that in the absence of expression of such
gene, the virus will not replicate. Examples of such vectors are
described in Henderson, et al., U.S. Pat. No. 5,698,443 issued Dec.
16, 1997 and Henderson, et al., U.S. Pat. No. 5,871,726 issued Feb.
16, 1999 the entire teachings of which are herein incorporated by
reference.
[0080] Additionally, the viral genome may be modified to include
inducible promoters which achieve replication or expression only
under certain conditions. Examples of inducible promoters are known
in the scientific literature (See, e.g. Yoshida and Hamada (1997)
Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J.
Virol. 70(9):6054-6059; Hwang, et al.(1997) J. Virol
71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol. 17(9):
5097-5105; and Dreher, et al.(1997) J. Biol. Chem
272(46);29364-29371.
[0081] The viruses may also be designed to be selectively
replicating viruses. Particularly preferred selectively replicating
viruses are described in Ramachandra, et al. PCT International
Publication No. WO 00/22137, International Application No.
PCT/US99/21452 published Apr. 20, 2000 and Howe, J., PCT
International Publication No. WO WO0022136, International
Application No. PCT/US99/21451 published Apr. 20, 2000. A
particularly preferred selectively replicating recombinant
adenovirus is the virus dl01/07/309 as more fully described in
Howe, J.
[0082] It has been demonstrated that viruses which are attenuated
for replication are also useful in the therapeutic arena. For
example the adenovirus dl1520 containing a specific deletion in the
E1b55K gene (Barker and Berk (1987) Virology 156: 107) has been
used with therapeutic effect in human beings. Such vectors are also
described in McCormick (U.S. Pat. No. 5,677,178 issued Oct. 14,
1997) and McCormick, U.S. Pat. No 5,846,945 issued Dec. 8, 1998.
The method of the present invention may also be used in combination
with the administration of such vectors to minimize the
pre-existing or induced humoral immune response to such
vectors.
[0083] Additionally, the therapeutic virus may incorporate a
therapeutic transgene for expression in an infected cell. The term
"therapeutic transgene" refers to a nucleotide sequence the
expression of which in the target cell produces a therapeutic
effect. The term therapeutic transgene includes but is not limited
to tumor suppressor genes, antigenic genes, cytotoxic genes,
cytostatic genes, pro-drug activating genes, apoptotic genes,
pharmaceutical genes or antiangiogenic genes. The vectors of the
present invention may be used to produce one or more therapeutic
transgenes, either in tandem through the use of IRES elements or
through independently regulated promoters.
[0084] The term "tumor suppressor gene" refers to a nucleotide
sequence, the expression of which in the target cell is capable of
suppressing the neoplastic phenotype and/or inducing apoptosis.
Examples of tumor suppressor genes useful in the practice of the
present invention include the p53 gene, the APC gene, the DPC-4
gene, the BRCA-1 gene, the BRCA-2 gene, the WT-1 gene, the
retinoblastoma gene (Lee, et al. (1987) Nature 329:642), the MMAC-1
gene, the adenomatous polyposis coli protein (Albertsen, et al.,
U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), the deleted in colon
carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,
nasopharyngeal carcinoma tumor suppressor gene that maps at
chromosome 3p21.3. (Cheng, et al. 1998. Proc. Nat. Acad. Sci.
95:3042-3047), the MTS1 gene, the CDK4 gene, the NF-1 gene, the NF2
gene, and the VHL gene. A particularly preferred adenovirus for
therapeutic use is the ACN53 vector encoding the p53 tumor
suppressor gene as more fully described in Gregory, et al., U.S.
Pat. No. 5,932,210 issued Aug. 3, 1999, the entire teaching of
which is herein incorporated by reference.
[0085] The term "antigenic genes" refers to a nucleotide sequence,
the expression of which in the target cells results in the
production of a cell surface antigenic protein capable of
recognition by the immune system. Examples of antigenic genes
include carcinoembryonic antigen (CEA), p53 (as described in
Levine, A. PCT International Publication No. WO94/02167 published
Feb. 3, 1994). In order to facilitate immune recognition, the
antigenic gene may be fused to the MHC class I antigen.
[0086] The term "cytotoxic gene" refers to nucleotide sequence, the
expression of which in a cell produces a toxic effect. Examples of
such cytotoxic genes include nucleotide sequences encoding
pseudomonas exotoxin, ricin toxin, diptheria toxin, and the
like.
[0087] The term "cytostatic gene" refers to nucleotide sequence,
the expression of which in a cell produces an arrest in the cell
cycle. Examples of such cytostatic genes include p21, the
retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin
dependent kinase inhibitors such as P16, p15, p18 and p19, the
growth arrest specific homeobox (GAX) gene as described in
Branellec, et al. (PCT Publication WO97/16459 published May 9, 1997
and PCT Publication WO96/30385 published Oct. 3, 1996).
[0088] The term "cytokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. Examples of
such cytokines include GM-CSF, the interleukins, especially IL-1,
IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, interferons of the .alpha.,
.beta. and .gamma. subtypes, consensus interferons and especially
interferon .alpha.-2b and fusions such as interferon
.alpha.-2.alpha.-1.
[0089] The term "chemokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. The term
chemokine refers to a group of structurally related low-molecular
cytokines weight factors secreted by cells are structurally related
having mitogenic, chemotactic or inflammatory activities. They are
primarily cationic proteins of 70 to 100 amino acid residues that
share four conserved cysteine. These proteins can be sorted into
two groups based on the spacing of the two amino-terminal
cysteines. In the first group, the two cysteines are separated by a
single residue (C-x-C), while in the second group, they are
adjacent (C-C). Examples of member of the `C-x-C` chemokines
include but are not limited to platelet factor 4 (PF4), platelet
basic protein (PBP), interleukin-8 (IL-8), melanoma growth
stimulatory activity protein (MGSA), macrophage inflammatory
protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig
alveolar macrophage chemotactic factors I and II (AMCF-I and -II),
pre-B cell growth stimulating factor (PBSF),and IP10. Examples of
members of the `C-C` group include but are not limited to monocyte
chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2
(MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte
chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1
.alpha. (MIP-1-.alpha.), macrophage inflammatory protein 1 .beta.
(MIP-1-.beta.), macrophage inflammatory protein 1-.gamma.
(MIP-1-.gamma.), macrophage inflammatory protein 3.alpha.
(MIP-3-.alpha., macrophage inflammatory protein 3 .beta.
(MIP-3-.beta.), chemokine (ELC), macrophage inflammatory protein-4
(MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 .beta.,
RANTES, SIS-epsilon (p500), thymus and activation-regulated
chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-2, human
protein HCC-3, mouse protein C10.
[0090] The term "pharmaceutical protein gene" refers to nucleotide
sequence, the expression of which results in the production of
protein have pharmaceutically effect in the target cell. Examples
of such pharmaceutical genes include the proinsulin gene and
analogs (as described in PCT International Patent Application No.
WO98/31397, growth hormone gene, dopamine, serotonin, epidermal
growth factor, GABA, ACTH, NGF, VEGF (to increase blood perfusion
to target tissue, induce angiogenesis, PCT publication WO98/32859
published Jul. 30, 1998), thrombospondin etc.
[0091] The term "pro-apoptotic gene" refers to a nucleotide
sequence, the expression thereof results in the induction of the
programmed cell death pathway of the cell. Examples of
pro-apoptotic genes include p53, adenovirus E3-11.6K(10.5K), the
adenovirus E4orf4 gene, p53 pathway genes, and genes encoding the
caspases.
[0092] The term "pro-drug activating genes" refers to nucleotide
sequences, the expression of which, results in the production of
protein capable of converting a non-therapeutic compound into a
therapeutic compound, which renders the cell susceptible to killing
by external factors or causes a toxic condition in the cell. An
example of a prodrug activating gene is the cytosine deaminase
gene. Cytosine deaminase converts 5-fluorocytosine to 5
fluorouracil, a potent antitumor agent). The lysis of the tumor
cell provides a localized burst of cytosine deaminase capable of
converting 5FC to 5FU at the localized point of the tumor resulting
in the killing of many surrounding tumor cells. This results in the
killing of a large number of tumor cells without the necessity of
infecting these cells with an adenovirus (the so-called bystander
effect"). Additionally, the thymidine kinase (TK) gene (see e.g.
Woo, et al. U.S. Pat. No. 5,631,236 issued May 20, 1997 and
Freeman, et al. U.S. Pat. No. 5,601,818 issued Feb. 11, 1997) in
which the cells expressing the TK gene product are susceptible to
selective killing by the administration of gancyclovir may be
employed.
[0093] The term "anti-angiogenic" genes refers to a nucleotide
sequence, the expression of which results in the extracellular
secretion of anti-angiogenic factors. Anti-angiogenesis factors
include angiostatin, inhibitors of vascular endothelial growth
factor (VEGF) such as Tie 2 (as described in PNAS(USA)(1998)
95:8795-8800), endostatin.
[0094] It will be readily apparent to those of skill in the art
that modifications and or deletions to the above referenced genes
so as to encode functional subfragments of the wild type protein
may be readily adapted for use in the practice of the present
invention. For example, the reference to the p53 gene includes not
only the wild type protein but also modified p53 proteins. Examples
of such modified p53 proteins include modifications to p53 to
increase nuclear retention, deletions such as the .DELTA.13-19
amino acids to eliminate the calpain consensus cleavage site
(Kubbutat and Vousden (1997) Mol. Cell. Biol. 17:460-468,
modifications to the oligomerization domains (as described in
Bracco, et al. PCT published application WO97/0492 or U.S. Pat. No.
5,573,925, etc.).
[0095] It will be readily apparent to those of skill in the art
that the above therapeutic genes may be secreted into the media or
localized to particular intracellular locations by inclusion of a
targeting moiety such as a signal peptide or nuclear localization
signal(NLS). Also included in the definition of therapeutic
transgene are fusion proteins of the therapeutic transgene with the
herpes simplex virus type 1 (HSV-1) structural protein, VP22.
Fusion proteins containing the VP22 signal, when synthesized in an
infected cell, are exported out of the infected cell and
efficiently enter surrounding non-infected cells to a diameter of
approximately 16 cells wide. This system is particularly useful in
conjunction with transcriptionally active proteins (e.g. p53) as
the fusion proteins are efficiently transported to the nuclei of
the surrounding cells. See, e.g.Elliott, G. & O'Hare, P. Cell.
88:223-233:1997; Marshall, A. & Castellino, A. Research News
Briefs. Nature Biotechnology. 15:205:1997; O'Hare, et al. PCT
publication WO97/05265 published Feb. 13, 1997. A similar targeting
moiety derived from the HIV Tat protein is also described in Vives,
et al. (1997) J. Biol. Chem. 272:16010-16017.
[0096] It may be valuable in some instances to utilize or design
vectors to achieve introduction of the exogenous transgene in a
particular cell type. Certain vectors exhibit a natural tropism for
certain tissue types. For example, vectors derived from the genus
herpesviridiae have been shown to have preferential infection of
neuronal cells. Examples of recombinantly modified herpesviridiae
vectors are disclosed in U.S. Pat. No. 5,328,688 issued Jul. 12,
1994. Cell type specificity or cell type targeting may also be
achieved in vectors derived from viruses having characteristically
broad infectivities by the modification of the viral envelope
proteins. For example, cell targeting has been achieved with
adenovirus vectors by selective modification of the viral genome
knob and fiber coding sequences to achieve expression of modified
knob and fiber domains having specific interaction with unique cell
surface receptors. Examples of such modifications are described in
Wickham, et al.(1997) J. Virol 71(11):8221-8229 (incorporation of
RGD peptides into adenoviral fiber proteins); Arnberg, et al.(1997)
Virology 227:239-244 (modification of adenoviral fiber genes to
achieve tropism to the eye and genital tract); Harris and Lemoine
(1996) TIG 12(10):400-405; Stevenson, et al.(1997) J. Virol. 71(6):
4782-4790; Michael, et al.(1995) Gene Therapy 2:660-668
(incorporation of gastrin releasing peptide fragment into
adenovirus fiber protein); and Ohno, et al.(1997) Nature
Biotechnology 15:763-767 (incorporation of Protein A-IgG binding
domain into Sindbis virus). Other methods of cell specific
targeting have been achieved by the conjugation of antibodies or
antibody fragments to the envelope proteins (see, e.g. Michael, et
al. (1993) J. Biol. Chem 268:6866-6869, Watkins, et al. (1997) Gene
Therapy 4:1004-1012; Douglas, et al.(1996) Nature Biotechnology 14:
1574-1578. Alternatively, particularly moieties may be conjugated
to the viral surface to achieve targeting (See, e.g. Nilson, et al.
(1996) Gene Therapy 3:280-286 (conjugation of EGF to retroviral
proteins)). Additionally, the virally encoded therapeutic transgene
also be under control of a tissue specific promoter region allowing
expression of the transgene preferentially in particular cell
types.
[0097] In the preferred practice of the invention, this procedure
is employed in conjunction with recombinant adenoviral therapy for
the treatment of human cancers. In accordance with conventional
oncology practice, patients are dosed at the maximum tolerated dose
of the therapeutic agent. In the course of clinical investigation,
a dose of 1.5.times.10.sup.13 recombinant adenoviral particles is
well tolerated in human subjects. Clinical experience with
replication deficient recombinant adenoviruses expressing p53 has
indicated that a course of therapy of injection of approximately
1.times.10.sup.13 recombinant viral particles for a period of 5
days repeated weekly for a period of three weeks is effective in
the treatment of ovarian cancer in human beings. The therapeutic
treatment regimen preferred in the present invention would involve
removal of antiviral antibodies using the plasmapheresis technique
described above followed by this course of therapy. Moreover, the
injection of recombinant adenoviruses in such quantity may further
deplete antibodies thus enhancing subsequent transduction.
[0098] The following is a description of procedures and parameters
for the conventional application of this procedure in conjunction
with the administration of a replication deficient recombinant
adenovirus encoding p53 ACN53 for the treatment of ovarian cancer.
A typical course of therapy with this agent involves administration
of 1.5.times.10.sup.13 viral particles each day for a period of 5
days. An FDA Phase III approved clinical protocol for the treatment
of ovarian cancer using the ACN53 virus calls involves a typical 5
day course of therapy described above in conjunction with the
chemotherapeutic agents cisplatin and paclitaxel. Patients receive
three courses of therapy with intervening rest periods.
Modifications to this procedure for therapeutic viruses other than
adenovirus will be readily apparent to the skilled artisan
[0099] Prior to the initiation of treatment with the therapeutic
virus, the patient may optionally be assayed for the presence of
pre-existing antiviral antibodies in accordance with standard assay
procedures well known in the art. An assay for the determination of
antiadenoviral antibodies is provided in Example 3 herein. This
pre-screening may be more indicated in the naive patient as the
patient who has been previously exposed to the therapeutic virus in
previous courses of therapy may generally be assumed to possess
such antibodies. In the event that the patient possesses
pre-existing or induced neutralizing antibodies, an immunoaffinity
chromatographic material comprising 250 mg of adenoviral capsid
proteins in a column format is a vessel of 300 ml volume is
prepared in substantial accordance with the teaching of Example 2
herein and sterilized for use. The vessel containing the
chromatographic material is affixed to a support in an upright
fashion. An apparatus as described above is prepared and arranged
in substantial accordance with the schematic representation in FIG.
9 of the attached drawings. The column is washed with three bed
volumes of sterile phosphate buffered saline. 500 ml of heparin are
introduced into the vessel and the excess discarded. Venous blood
supply to the apheresis apparatus is established by catheterization
of the cephalic vein. The plasma output port of the apheresis
apparatus is connected via flexible tubing to the bottom inlet port
of the vessel containing the chromatographic material. Plasma is
allowed to enter the vessel containing the chromatographic
material. Plasma flow should be established initially at
approximately 10 ml/min. The first 400 ml of serum emanating from
the top of the column should be discarded as it is substantially
polluted with heparin. The remaining volume is directed to the
holding vessel for reintroduction to the patient. If possible, the
plasma flow should be increased to approximately 15-20 ml/min.
Following the treatment of approximately 1-1.5 liters of serum, the
procedure should be discontinued. If additional volumes are to be
treated, a new chromatographic resin should be employed.
[0100] Use of Viral Epitopes
[0101] The present invention further provides a improved method for
treating a mammalian subject with a therapeutic virus, the method
comprising administering to said mammal a viral epitope or viral
epitope mimetic followed by the administration of a therapeutically
effective dose of a therapeutic virus. Alternatively to the
plasmapheresis procedure described above to remove the antibodies,
one may also administer a quantity of the antiviral epitope into
the bloodstream of the mammal to be treated. In some instances,
these viral coat proteins are toxic (such as adenoviral hexon and
fiber protein) in which case it is preferred to administer a
quantity of an epitope mimetic (either peptidyl or small molecule
organic compound). These agents are then able to bind up the
pre-existing antiviral antibodies in circulation. As described
above, the injection of rAd vector will also deplete neutralizing
antibody from the serum, and thus is useful for in vivo antibody
depletion and redosing. The data presented in FIG. 4 further
demonstrates that high affinity antibodies are essentially
completely depleted by administration of virus. This indicates that
pre-treatment of an individual with the virus will deplete the
pre-existing or induced immune response to a virus of that type.
Thus administration of a virus or immunological subfragments
thereof can reduce pre-existing or induced immune response to a
therapeutic virus. This procedure may then be followed by the
administration of the therapeutic virus. As shown from the previous
data, the duration of immunodepletion is limited. Consequently, it
will be preferred to administer this epitope or epitope mimetic
relatively soon (within hours) in advance of the therapeutic virus
to maximize the efficiency of viral delivery
EXAMPLES
[0102] The following examples provide the methodology and results
of experiments demonstrating the construction of exemplary hybrid
vectors of the invention. It will be apparent to those of skill in
the art to which the invention pertains, the present invention may
be embodied in forms other than those specifically disclosed in
these examples, without departing from the spirit or essential
characteristics of the invention. The particular embodiments of the
invention described below, are therefore to be considered as
illustrative and not restrictive of the scope of the invention. In
the following examples, "g" means grams, "ml" means milliliters,
".degree. C." means degrees Centigrade, "min." means minutes, "FBS"
means fetal bovine serum.
Example 1
Priming Animals for the Induction of Neutralizing Antibodies in
Vivo
[0103] 15 BALB/C mice were injected subcutaneously with
5.times.10.sup.10 particles of ZZCB. After 28 days, the mice
received a booster injection of 5.times.10.sup.10 particles
subcutaneously. 2 weeks following the second injection, the mice
were sacrificed and the serum was isolated. The serum (referred to
as Ad-undepleted serum) from all 15 mice were pooled for subsequent
experiments. The data is presented in FIG. 1 of the attached
drawings. This serum was shown to have a much higher titer than
serum from animals that were dosed once with adenovirus. The mice
were injected twice with adenovirus to illicit a strong antibody
response and mimic a scenario in people pre-exposed to virus and
undergoing further therapy with adenovirus.
Example 2
Preparation of the Adenoviral Capsid Protein Column
[0104] In order to purify the antibodies from the pooled serum, a
chromatography column was prepared in which adenoviral capsid
proteins were coupled to Affi-Gel.RTM. 15 (commercially available
from BioRad as Catalog # 1536051). Adenoviral capsid proteins
(hexon, penton, fiber and 3A) were purified chromatographically in
accordance with the teaching of Shabram, et al. supra. and purified
to homogeneity using. Known amounts of hexon (210 .mu.g), penton
(522 .mu.g), fiber (146 .mu.g) and 3A (105 .mu.g) were coupled to
Affi-Gel.RTM. 15 in substantial accordance with the instructions
provided by the manufacturer. Coupling efficiency was determined to
be approximately 71% by Bradford assay, (as determined by the
user). Bradford assay is based on a blue dye (Coomassie Brilliant
Blue) that binds to free amino groups in the side chains of amino
acids, especially lysine and was performed using a Bio-Rad Protein
Assay Kit (commercially available from Bio-Rad) in substantial
accordance with the manufacturer's instructions.
Example 3
Purification of Anti-Adenoviral Antibodies from Serum of Primed
Animals
[0105] 200-1500 .mu.l of serum obtained from ZZCB primed animals
prepared in substantial accordance with the teaching of Example 1
above was introduced to the column prepared from Example 2. The
serum was allowed to bind the adenoviral capsid protein column at
four degrees for 2-4 hours with gentle rotation. If less than 1000
.mu.l of serum was used on the column, the serum was diluted to
1000 .mu.l with PBS.
[0106] Ad-column adsorbed serum (referred to as column-depleted
serum) was collected from the column and anti-adenoviral capsid
antibodies (referred to as Ad-Ab) were eluted from the column with
0.2M glycine pH2. The antibodies obtained from the column were
neutralized in 1/3 volume of 1M Tris pH8 and then subsequently
dialyzed in PBS and stored. The concentration of antibodies
depleted from the serum was quantitated by using the Bradford
assay.
[0107] In vitro neutralizing assay was performed to show that
removal of anti-adenoviral antibodies decreased the neutralizing
activity of the serum (FIG. 2). Neutralizing assays were performed
by serial dilution of serum of interest (starting at 1:20 dilution
and increasing in increments of 2, i.e., 1:40, 1:80, etc.) in a 96
well format. The appropriately diluted serum is allowed to incubate
with a recombinant adenovirus (GFCB) expressing the Green
Fluorescent Protein (GFP) at a final concentration of
4.times.10.sup.8 particles/ml for 1 hr at 37.degree. C. The serum,
virus mixture is transferred onto HeLa cells plated at
9.times.10.sup.3/well in a 96 well format and the virus is allowed
to infect the cells overnight. Fluorescence reading for GFP was
evaluated approximately 12 hours later using a fluorometer to assay
neutralizing capacity.
Example 4
Gene Transduction in the Absence of Neutralizing Antibodies
[0108] For the experimental procedure to test whether removal of
anti-adenoviral antibodies increased the transduction efficiency of
virus, 6 mice were passively immunized with either 40 .mu.g of
anti-adenoviral antibodies eluted from rAd protein column, or
equivalent amounts of column depleted or undepleted serum. An
additional 5 mice were passively immunized with serum injected with
vPBS and two extra mice were passively immunized with 80 .mu.g of
anti-adenoviral antibodies eluted from rAd protein column. Small
quantities of sera were collected individually from each mouse 1-3
hours after IP administration to determine if the passive
immunization was effective for each particular mouse. The mice were
allowed to rest overnight.
[0109] The following day, the mice were injected via tail vein
injection with 5.times.10.sup.10 particles of virus (BGCG). BGCG
was used so transduction efficiency of the virus could be monitored
by staining for .beta.-gal activity in the livers. Two hours
following virus injection, the mice were bled a second time to
collect serum to observe neutralizing antibody levels. The mice
were sacrificed 3 days post virus injection and both serum and
livers were isolated from each mouse. The serum was analyzed for
neutralizing activity (FIG. 4) and livers were assayed for
.beta.-gal activity as an indicator of successful virus
transduction (FIG. 5).
* * * * *