U.S. patent application number 09/994965 was filed with the patent office on 2003-11-27 for method of reducing an immune response to a recombinant virus.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Trinchieri, Giorgio, Wilson, James M., Yang, Yiping.
Application Number | 20030219435 09/994965 |
Document ID | / |
Family ID | 23612314 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219435 |
Kind Code |
A9 |
Wilson, James M. ; et
al. |
November 27, 2003 |
Method of reducing an immune response to a recombinant virus
Abstract
A method of reducing immune response during gene therapy is
provided which involves co-administration of the viral vector
bearing a therapeutic transgene and a selected immune modulator
capable of inhibiting the formation of neutralizing antibodies
and/or CTL elimination of the vectors upon repeated
administration.
Inventors: |
Wilson, James M.; (Gladwyne,
PA) ; Yang, Yiping; (Philadelphia, PA) ;
Trinchieri, Giorgio; (Wynnewood, PA) |
Correspondence
Address: |
HOWSON AND HOWSON
ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
The Wistar Institute of Anatomy and Biology
Philadelphia
PA
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 0081297 A1 |
June 27, 2002 |
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Family ID: |
23612314 |
Appl. No.: |
09/994965 |
Filed: |
November 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09994965 |
Nov 27, 2001 |
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09407490 |
Sep 28, 1999 |
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6372208 |
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09407490 |
Sep 28, 1999 |
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08894488 |
Aug 22, 1997 |
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6251957 |
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08894488 |
Aug 22, 1997 |
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PCT/US96/03035 |
Feb 23, 1996 |
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PCT/US96/03035 |
Feb 23, 1996 |
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08585397 |
Jan 11, 1996 |
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08585397 |
Jan 11, 1996 |
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08394032 |
Feb 24, 1995 |
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5872154 |
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Current U.S.
Class: |
424/144.1 ;
424/85.2; 424/93.2 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2875 20130101; C12N 15/86 20130101; C07K 16/2812 20130101;
A61K 2300/00 20130101; C12N 2710/10343 20130101; A61K 39/39541
20130101; A61K 48/00 20130101; A61K 39/39541 20130101 |
Class at
Publication: |
424/144.1 ;
424/93.2; 424/85.2 |
International
Class: |
A61K 048/00; A61K
039/395; A61K 038/20 |
Goverment Interests
[0002] This invention was supported by the National Institutes of
Health Grant Nos. DK 47757-02 and AI 34412-02. The United States
government has certain rights in this invention.
Claims
What is claimed is:
1. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said virus and an immune
modulator which inhibits the formation of neutralizing antibodies
to the recombinant virus.
2. The method according to claim 1, further comprising the step of
re-administering said virus.
3. The method according to claim 1, wherein said immune modulator
is administered simultaneously with said virus.
4. The method according to claim 1, wherein said immune modulator
is administered prior to administration of said virus.
5. The method according to claim 1, wherein said immune modulator
is administered subsequently to administration of said virus.
6. The method according to claim 1, wherein said immune modulator
is interleukin-12.
7. The method according to claim 1, wherein said immune modulator
comprises an anti-CD4 antibody.
8. The method according to claim 1, wherein said agent is selected
from the group consisting of a soluble CD40 molecule and an
anti-CD40 ligand antibody.
9. The method according to claim 1, wherein said agent is selected
from the group consisting of a soluble CD28 and an anti-CD28
antibody.
10. The method according to claim 1, wherein said immune modulator
is gamma-interferon.
11. The method according to claim 1, wherein said immune modulator
is selected from the group consisting of a soluble CTLA4 and an
anti-CTLA4 antibody.
12. In a method for repeated delivery of a recombinant virus to a
mammal, said method comprising the step of administering to said
mammal a recombinant virus comprising a transgene, wherein the
improvement comprises the steps of co-administering with said
recombinant virus an immune modulator which inhibits the formation
of neutralizing antibodies against said virus; and re-administering
the recombinant virus to said mammal, whereby the inhibition of
formation of neutralizing antibodies to the recombinant virus by
the immune modulator permits re-administration of the recombinant
virus.
13. The method according to claim 12, wherein said immune modulator
is selected from the group consisting of: an agent which inhibits
the formation of neutralizing antibodies by depleting or inhibiting
CD4+ cells; an anti-T cell antibody which inhibits the formation of
neutralizing antibodies; an agent which inhibits the formation of
neutralizing antibodies by blocking the interaction between CD40
ligand on a T cell and CD40 on a B cell; and an agent which
inhibits the formation of neutralizing antibodies by blocking the
interaction between the CD28 molecule or CTLA4 ligand on a T cell
and B7 on a B cell.
14. The method according to claim 12, wherein said immune modulator
is a cytokine which inhibits a T.sub.H1-mediated neutralizing
antibody or T.sub.H2-mediated neutralizing antibody.
15. The method according to claim 12 wherein said immune modulator
is interleukin-12.
16. The method according to claim 12 wherein said immune modulator
comprises an anti-CD4 antibody.
17. The method according to claim 12, wherein said immune modulator
binds to the CD40 ligand on a T cell.
18. The method according to claim 12, wherein said immune modulator
is selected from the group consisting of a soluble CD40 molecule
and an anti-CD40 ligand antibody.
19. The method according to claim 12, wherein said immune modulator
binds to the CD28 molecule or CTLA4 ligand on a T cell.
20. The method according to claim 19, wherein said immune modulator
is selected from the group consisting of a soluble CD28 molecule
and an anti-CD28 antibody.
21. The method according to claim 13 wherein said immune modulator
comprises a DNA molecule.
22. The method according to claim 13 wherein said immune modulator
comprises a protein.
23. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said recombinant virus and an
agent which deplete or inhibits CD4+ cells.
24. The method according to claim 23, further comprising the step
of re-administering said recombinant virus.
25. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said recombinant virus and an
anti-T cell antibody.
26. The method according to claim 25, further comprising the step
of re-administering said recombinant virus.
27. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said recombinant virus and an
agent which blocks the interaction between CD40 ligand on a T cell
and CD40 on a B cell.
28. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said recombinant virus and an
immune modulator which binds to the CD40 ligand on the T cell,
thereby inhibiting formation of neutralizing antibodies.
29. The method according to claim 28, further comprising the step
of re-administering said recombinant virus.
30. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant virus comprising the step
of co-administering to said mammal said recombinant virus and an
immune modulator which binds to the CD28 or CTLA4 ligand on a T
cell, thereby inhibiting formation of neutralizing antibodies.
31. The method according to claim 30, wherein said agent binds to
the CD28 or CTLA4 ligand on the T cell.
32. The method according to claim 31, wherein said immune modulator
is selected from the group consisting of soluble CD28, a soluble
CTLA4, an anti-CD28 antibody and an anti-CTLA4 antibody.
33. The method according to claim 30, further comprising the step
of re-administering said recombinant virus.
34. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant adeno-associated virus
comprising the step of co-administering to said mammal said
recombinant adeno-associated virus and a cytokine which inhibits
the formation of neutralizing antibodies against said virus.
35. The method according to claim 34, further comprising the step
of re-administering said recombinant virus.
36. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant adeno-associated virus
comprising the step of co-administering to said mammal said
recombinant adeno-associated virus and an agent which depletes or
inhibits CD4+ cells.
37. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant adeno-associated virus
comprising the step of co-administering to said mammal said
recombinant adeno-associated virus and an anti-T cell antibody.
38. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant adeno-associated virus
comprising the step of co-administering to said mammal said
recombinant adeno-associated virus and an immune modulator which
binds to the CD40, thereby inhibiting formation of neutralizing
antibodies.
39. A method of inhibiting in a mammal formation of neutralizing
antibodies directed against a recombinant adeno-associated virus
comprising the step of co-administering to said mammal said
recombinant adeno-associated virus and an immune modulator which
binds the CD28 or CTLA4 ligand on a T cell, thereby inhibiting
formation of neutralizing antibodies.
40. The method according to claim 39, wherein said immune modulator
is selected from the group consisting of a soluble CD28, a soluble
CTLA4, an anti-CD28 antibody and an anti-CTLA4 antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/407,490, filed Sep. 28, 1999, which is a
continuation of U.S. patent application Ser. No. 08/894,488, filed
Aug. 22, 1997, which is a 35 USC .sctn.371 of PCT/US96/03035, filed
Feb. 23, 1996, which claims the benefit of the priority of U.S.
patent application Ser. No. 08/585,397, filed Jan. 11, 1996, now
abandoned, and U.S. patent application Ser. No. 08/394,032, filed
Feb. 24, 1995, now U.S. Pat. No. 5,872,154.
BACKGROUND OF THE INVENTION
[0003] The present invention relates generally to gene therapy, and
more specifically, to methods of administering viral vectors used
in gene therapy.
[0004] Recombinant adenoviruses have emerged as attractive vehicles
for in vivo gene transfer to a wide variety of cell types. The
first generation vectors, which are rendered replication defective
by deletion of the immediate early genes E1a and E1b, are capable
of highly efficient in vivo gene transfer into nondividing target
cells [M. Kay et al, Proc. Natl. Acad. Sci. USA 91:2353-2357
(1994); S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993); B.
Quantin et al, Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); M.
Rosenfeld et al, Cell, 68:143 (1992); R. Simon et al, Hum. Gene
Thera., 4:771 (1993); Rosenfeld et al, Science, 252:431-434 (1991);
Stratford-Perricaudet et al, Hum. Gene Ther., 1:241-256
(1990)].
[0005] Immune responses of the recipient to the viral vector, the
transgene carried by the vector, and the virus-infected cells have
emerged as recurring problems in the initial application of this
technology to animals and humans [Yang et al, J. Virol.,
69:2004-2015 (1995) (Yang I)]. In virtually all models, including
lung-directed and liver-directed gene therapy, expression of the
transgene is transient and associated with the development of
pathology at the site of gene transfer.
[0006] The transient nature of transgene expression from
recombinant adenoviruses is due, in part, to the development of
antigen specific cellular immune responses to the virus-infected
cells and their subsequent elimination by the host. Specifically,
first generation vectors, although deleted in the E1a region of the
vector, express viral proteins in addition to the transgene. These
viral proteins activate cytotoxic T lymphocytes (CTL) [Y. Dai et
al, Proc. Natl. Acad. Sci. USA 92: 1401-1405 (1995); Y. Yang et al,
Proc. Natl. Acad. Sci. USA, 91:4407-4411 (1994) (Yang II); and Y.
Yang et al, Immunity, 1:433-442 (1994) (Yang III)]. The
collaboration of CTLs directed against newly synthesized viral
proteins and viral specific T helper cells [Zabner et al, Cell
75:207-216 (1993); Crystal et al, Nat. Genet. 8:42-51 (1994)] leads
to the destruction of the virus-infected cells.
[0007] Another antigenic target for immune mediated clearance of
virally-infected cells can be the product of the transgene when
that transgene expresses a protein that is foreign to the treated
host. CTLs are thus an important effector in the destruction of
target cells, with activation occurring in some cases in the
context of the transgene product, or of the viral-synthesized
proteins, both of which are presented by MHC class I molecules
[Yang I; and Zsengeller et al, Hum. Gene Thera., 6:457-467 (1995)].
These immune responses have also been noted to cause the occurrence
of associated hepatitis that develops in recipients of in vivo
liver directed gene therapy within 2-3 weeks of initial
treatment.
[0008] Another limitation of recombinant adenoviruses for gene
therapy has been the difficulty in obtaining detectable gene
transfer upon a second administration of virus. This limitation is
particularly problematic in the treatment of single gene inherited
disorders or chronic diseases, such as cystic fibrosis (CF), that
will require repeated therapies to obtain life-long genetic
reconstitution. Diminished gene transfer following a second therapy
has been demonstrated in a wide variety of animal models following
intravenous or intratracheal delivery of virus [T. Smith et al,
Gene Thera., 5:397 (1993); S. Yei et al, Gene Thera., 1:192-200
(1994); K. Kozarsky et al, J. Biol. Chem., 269:13695 (1994)]. In
each case, resistance to repeated gene therapy was associated with
the development of neutralizing anti-adenovirus antibody, which
thwarted successful gene transfer following a second administration
of virus.
[0009] Potential solutions for these problems have been directed
towards the development of second generation recombinant viruses
[Y. Yang et al, Nat. Genet., 7:362-369 (1994) (Yang IV); and J.
Engelhardt et al, Hum. Gene Thera. 5:1217 (1994)] designed to
diminish the expression of newly synthesized viral proteins, and
the use of non-immunogenic transgenes, to prevent CTL
activation.
[0010] Thus, there remains a need in the art for a method and
composition for improving the efficiency of gene transfer during
repeated administrations of viral gene therapy.
SUMMARY OF THE INVENTION
[0011] The present invention provides a method of gene therapy and
compositions for use therein which result in a reduced immune
response to the recombinant viral vector used to accomplish the
therapy. The method involves co-administering with the gene therapy
viral vector a selected immune modulator, which substantially
reduces the occurrence of neutralizing antibody responses directed
against the vector encoded antigens and/or cytolytic T cell
elimination of the viral protein containing cells. This method is
particularly useful where readministration of the recombinant virus
is desired. According to this method the immune modulator may be
administered prior to or concurrently with the recombinant viral
vector bearing the transgene to be delivered.
[0012] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is a graph summarizing neutralizing antibody titer
present in brochoalveolar lavage fluid (BAL) samples of C57BL-6
mice adenovirus-infected on day 0 and necropsied on day 28 as
described in Example 2. Control represents normal mice ("control");
CD4 mAB represents CD4.sup.+ cell-depleted mice; IL-12 represents
mice treated with IL-12 on days 0 and +1 and IFN-7 represent mice
treated with IFN-.gamma. on days 0 and +1. Data are presented as
the mean .+-.1 standard deviation for three independent
experiments.
[0014] FIG. 1B is a graph summarizing the relative amounts
(OD.sub.405) of IgG present in BAL samples. The symbols are as
described in FIG. 1A.
[0015] FIG. 1C is a graph summarizing the relative amounts
(OD.sub.405) of IgA present in BAL samples. The symbols are as
described in FIG. 1A.
[0016] FIG. 2 is a graph summarizing neutralizing antibody titer,
expressed as reciprocal dilution of serum samples for the animals
of Example 4. The symbols representing the mice are described as
follows: C57BL-6 mice infected with H5.010CMVlacZ on day 0 and with
H5.010CBALP at day 28 ("B6 mice"); C57BL-6 mice infected with
UV-inactivated H5.010CMVlacZ on day 0 and with H5.010CBALP at day
28 ("B6-UV mice"); MHC class II-deficient mice infected with
H5.010CMVlacZ at day 0 and with H5.010CBALP on day 28 ("class
II.sup.- mice"); .beta.2 microglobulin deficient mice infected with
H5.010CMVlacZ at day 0 and with H5.010CBALP at day 28
(".beta.2m.sup.- mice"); and C57BL/6 mice treated with GKI1.5
(anti-CD4) mAb and infected with H5.010CMVlacZ at day 0 and with
H5.010CBALP at day 28 ("CD4Ab mice").
[0017] FIG. 3A is a graph summarizing neutralizing antibody titer,
expressed as reciprocal dilution of serum samples for C57BL/6 mice
infused into the tail vein on day 0 with H5.010CMVLDLR and on day
21 with H5.010CMVLacZ, and on day 42 with H5.010CBALP, and
administered with saline on days -3, 0 and 3. The titer is reported
as a function of days post-infection.
[0018] FIG. 3B is a graph similar to that of FIG. 3A for C57BL/6
mice infused into the tail vein on day 0 with H5.010CMVLDLR and on
day 21 with H5.010CMVLacZ, and on day 42 with H5.010CBALP, and
administered with anti-CD4 mAb GK1.5 on days -3, 0 and 3.
[0019] FIG. 3C is a graph similar to that of FIG. 3A for C57BL/6
mice infused into the tail vein on day 0 with H5.010CMVLDLR and on
day 21 with H5.010CMVLacZ, and on day 42 with H5.010CBALP, and
administered with anti-CD4 mAb GK1.5 on days -3, 0, 3, 18, 21 and
24.
[0020] FIG. 3D is a graph similar to that of FIG. 3A for C57BL/6
mice infused into the tail vein on day 21 with H5.010CMVLacZ, and
on day 42 with H5.010CBALP, and administered with anti-CD4 mAb
GK1.5 on days -3, 0, and 3.
[0021] FIG. 4A is a graph summarizing the relative amounts
(OD.sub.405) of IgG1 present in serum samples as a function of
sample dilutions, as described in Example 6.
[0022] FIG. 4B is a graph summarizing the relative amounts
(OD.sub.405) of IgG2a present in the serum samples as a function of
sample dilutions as described in Example 6.
[0023] FIG. 5 is a graph illustrating percentage of specific lysis
on mock-infected ("mock") and H5.010CBALP ("ALP")-infected C57SV
cells as a function of effector to target ratios for a .sup.51Cr
release assay of Example 6B. Splenocytes from C57BL/6 mice ("B6")
and IL-12 treated C57BL/6 ("B6+IL12") mice 10 days after
administration of H5.010CBALP, were restimulated in vitro with
H5.010CMVlacZ for 5 days.
[0024] FIG. 6A is a bar chart providing the neutralizing antibodies
to Ad5 obtained in the BAL (lung experiment) of Example 7. The
results in column 1 are from C57BL/6 mice (control), column 2
results are from CD40L-deficient knockout mice (CD40L-KO), and
column 3 results are from C57BL/6 mice treated with CD40L antibody
(CD40L Ab). Data are presented as the mean neutralizing antibody
titer of three samples +/-1 S.D.
[0025] FIG. 6B is a bar chart as described in FIG. 6A, with data
obtained from serum for the liver experiment of Example 7.
[0026] FIG. 7A is a line graph comparing the percentage of specific
lysis in lymphocytes harvested from control C57BL/6 mice (open
circles) and C57BL/6 mice treated with antibody to CD40L (filled
circles). Seven days after virus administration, the lymphocytes
were restimulated in vitro for 5 days and tested for specific lysis
on mock-infected C57SV cells in a 6 hour .sup.51Cr release assay.
Percentage of specific lysis is expressed as a function of
different effector to target ratios (6:1, 12:1, 25:1, and 50:1).
Splenocytes were used for this lung experiment.
[0027] FIG. 7B is a line graph as described in FIG. 7A, using
virus-infected C57SV cells.
[0028] FIG. 7C is a line graph as described in FIG. 7A, using
mock-infected cells. Mediastinal lymph node (MLN) cells were used
for these liver experiments.
[0029] FIG. 7D is a line graph as described in FIG. 7A, using
virus-infected cells. MLN cells were used for these liver
experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides methods and compositions for
improving an animal or human's ability to tolerate administration
of gene therapy viral vectors. The invention provides methods to
transiently prevent activation of CD4.sup.+ T cells which are
involved in both cellular and humoral immune barriers to gene
therapy. The methods involve administering to an individual
receiving a gene therapy vector a suitable amount of a preferably
short-acting immune modulator. The immune modulator is preferably
administered concurrently with administration of the gene therapy
vector, i.e., a recombinant virus, used to deliver a therapeutic
transgene desired for gene therapy. The immune modulator may also
be administered before or after administration of the vector.
[0031] The method of this invention, which prevents the development
of adverse cellular and humoral immune responses to gene therapy
viral vectors, is based on immune suppression to block activation
of T helper cells, specifically CD4 function, and B cells. In
contrast to the prior art, which involved chronic immune
suppression by continuous administration of non-specific immune
suppressing drugs, the present invention uses a transient approach
to immunosuppression. Without wishing to be bound by theory, the
inventors theorize that the primary stimulus for immune activation
is viral capsid proteins from the recombinant vector. Chronic
immune suppression is not necessary in such a scenario.
Specifically, transient ablation of CD4 function at or near the
time of recombinant virus administration according to this
invention prevents the formation of neutralizing antibody, thereby
allowing efficient gene transfer following at least two subsequent
administrations of gene therapy viral vectors.
[0032] As illustrated below, administration of immune modulators
according to the method of this invention is preferably conducted
only at the time of virus administration. However, more prolonged
immune modulation than is necessary in the following examples of
gene transfer to mouse liver and/or lung may be needed, depending
upon the manner in which the antigens are presented in different
gene therapy protocols. Thus, the method of transient immune
modulation of the invention may, in certain circumstances, be
combined with long term immuno-suppression or other
immunomodulatory therapies.
[0033] I. Immune Modulators
[0034] The selected immune modulator is defined herein as an agent
capable of inhibiting the formation by activated B cells of
neutralizing antibodies directed against the recombinant viral
vector and/or capable of inhibiting cytolytic T lymphocyte (CTL)
elimination of the vector. The immune modulator may be selected to
interfere with the interactions between the T helper subsets
(T.sub.H1 or T.sub.H2) and B cells to inhibit neutralizing antibody
formation. Alternatively, the immune modulator may be selected to
inhibit the interaction between T.sub.H1 cells and CTLs to reduce
the occurrence of CTL elimination of the vector. More specifically,
the immune modulator desirably interferes with or blocks the
function of the CD4 T cells.
[0035] Immune modulators for use in inhibiting neutralizing
antibody formation according to this invention may be selected
based on the determination of the immunoglobulin subtype of any
neutralizing antibody produced in response to the viral vector. The
neutralizing antibody that develops in response to administration
of a gene therapy viral vector is frequently based on the identity
of the virus, the identity of the transgene, what vehicle is being
used to deliver the vector and/or the target location or tissue
type for viral vector delivery.
[0036] For example, T.sub.H2 cells are generally responsible for
interfering with the efficient transfer of genes administered
during gene therapy. This is particularly true when the viral
vector is adenovirus-based. More particularly, the inventors have
determined that neutralizing antibodies of the subtypes, IgG.sub.1
and IgA, which are dependent upon the interaction between T.sub.H2
cells and B cells, appear to be the primary cause of major
neutralizing antibodies against adenoviral vectors.
[0037] The identity of the neutralizing antibody induced by
administering a specific gene therapy recombinant viral vector is
readily determined in animal trials. See, e.g., Example 6. For
example, administration of adenoviral vectors via the lungs
generally induces production of IgA neutralizing antibody, while
administration of adenoviral vectors via the blood generally
induces IgG, neutralizing antibody. In these cases, a
T.sub.H2-dependent immune response interferes with transfer of the
adenovirus-based viral vector carrying a therapeutic transgene.
Where the neutralizing antibody induced by viral vector
administration is a T.sub.H2 mediated antibody, such as IgA or
IgG.sub.1, the immune modulator selected for use in this method
desirably suppresses or prevents the interaction of T.sub.H2 cells
with B cells. Alternatively, if the induced neutralizing antibody
is found to be a T.sub.H1 mediated antibody, such as IgG.sub.2A,
the immune modulator desirably suppresses or prevents the
interaction of T.sub.H1 cells with B cells. Where the reduction of
CTL elimination of the viral vectors is desired as well as the
blocking of neutralizing antibody formation, the immune modulator
is selected for its ability to suppress or block CD4.sup.+ T.sub.H1
cells to permit prolonged residence of the viral vector in
vitro.
[0038] The immune modulators may comprise soluble or naturally
occurring proteins, including cytokines and monoclonal antibodies.
The immune modulators may comprise other pharmaceuticals. In
addition, the immune modulators according to the invention may be
used alone or in combination with one another. For example,
cyclophosphamide and the more specific immune modulator anti-CD4
monoclonal antibody may be co-administered. In such a case,
cyclophosphamide serves as an agent to block T.sub.H1 activation
and to stabilize transgene expression beyond the period of
transient immune blockade induced by anti-CD4 MAb treatment.
[0039] A suitable amount or dosage of the selected immune modulator
will depend primarily on the identity of the modulator, the amount
of the recombinant vector bearing the transgene that is initially
administered to the patient, and the method and/or site of delivery
of the vector. These factors can be evaluated empirically by one of
skill in the art using the procedures described herein. Other
secondary factors such as the condition being treated, the age,
weight, general health, and immune status of the patient, may also
be considered by a physician in determining the dosage of immune
modulator to be delivered to the patient in conjunction with a gene
therapy vector according to this invention.
[0040] Generally, for example, a therapeutically effective human
dosage of a protein immune modulator, e.g., IL-12 or IFN-.gamma.,
is administered in the range of from about 0.5 .mu.g to about 5 mg
per about 1.times.10.sup.7 pfu/ml virus vector. Various dosages may
be determined by one of skill in the art to balance the therapeutic
benefit against any adverse side effects.
[0041] A. Monoclonal Antibodies and Soluble Proteins
[0042] Preferably, the method of inhibiting an adverse immune
response to the gene therapy vector involves non-specific
inactivation of CD4.sup.+ cells. One such method comprises
administering an appropriate monoclonal antibody. Preferably, such
blocking antibodies are "humanized" to prevent the recipient from
mounting an immune response to the blocking antibody. A "humanized
antibody" refers to an antibody having its complementarily
determining regions (CDRs) and/or other portions of its light
and/or heavy variable domain framework regions derived from a
non-human donor immunoglobulin, the remaining
immunoglobulin-derived parts of the molecule being derived from one
or more human immunoglobulins. Such antibodies can also include
antibodies characterized by a humanized heavy chain associated with
a donor or acceptor unmodified light chain or a chimeric light
chain, or vice versa. Such "humanization" may be accomplished by
methods known to the art. See, for example, G. E. Mark and E. A.
Padlan, "Chap. 4. Humanization of Monoclonal Antibodies", The
Handbook of Experimental Pharmacology, vol. 113, Springer-Verlag,
New York (1994), pp. 105-133, which is incorporated by reference
herein.
[0043] Other suitable antibodies include those that specifically
inhibit or deplete CD4.sup.+ cells, such as an antibody directed
against cell surface CD4. Depletion of CD4+cells has been shown by
the inventors to inhibit the CTL elimination of the viral vector.
Such modulatory agents include but are not limited to anti-T cell
antibodies, such as anti-OKT3+[see, e.g., U.S. Pat. No. 4,658,019;
European Patent Application No. 501,233, published Sep. 2, 1992].
See Example 2 below, which employs the commercially available
antibody GK1.5 (ATCC Accession No. TIB207) to deplete CD4.sup.+
cells.
[0044] Alternatively, any agent that interferes with or blocks the
interactions necessary for the activation of B cells by T.sub.H
cells, and thus the production of neutralizing antibodies, is
useful as an immune modulator according to the methods of this
invention. For example, B cell activation by T cells requires
certain interactions to occur [F. H. Durie et al, Immunol. Today,
15(9):406-410 (1994)], such as the binding of CD40 ligand on the T
helper cell to the CD40 antigen on the B cell, and the binding of
the CD28 and/or CTLA4 ligands on the T cell to the B7 antigen on
the B cell. Without both interactions, the B cell cannot be
activated to induce production of the neutralizing antibody.
[0045] The CD40 ligand (CD40L)-CD40 interaction is a desirable
point to block the immune response to gene therapy vectors because
of its broad activity in both T helper cell activation and function
as well as the absence of redundancy in its signaling pathway. A
currently preferred method of the present invention thus involves
transiently blocking the interaction of CD40L with CD40 at the time
of adenoviral vector administration. This can be accomplished by
treating with an agent which blocks the CD40 ligand on the T.sub.H
cell and interferes with the normal binding of CD40 ligand on the T
helper cell with the CD40 antigen on the B cell. Blocking
CD40L-CD40 interaction prevents the activation of the T helper
cells that contributes to problems with transgene stability and
readministration.
[0046] Thus, an antibody to CD40 ligand (anti-CD40L) [available
from Bristol-Myers Squibb Co; see, e.g., European patent
application 555,880, published Aug. 18, 1993] or a soluble CD40
molecule can be a selected immune modulator in the method of this
invention.
[0047] Alternatively, an agent which blocks the CD28 and/or CTLA4
ligands present on T helper cells interferes with the normal
binding of those ligands with the antigen B7 on the B cell. Thus, a
soluble form of B7 or an antibody to CD28 or CTLA4, e.g., CTLA4-Ig
[available from Bristol-Myers Squibb Co; see, e.g., European patent
application 606,217, published Jul. 20, 1994] can be the selected
immune modulator in the method of this invention. This method has
greater advantages than the below-described cytokine administration
to prevent TH2 activation, because it addresses both cellular and
humoral immune responses to foreign antigens.
[0048] B. Cytokines
[0049] Still other immune modulators which inhibit the T.sub.H cell
function may be employed in the methods of this invention.
[0050] Thus, in one embodiment, an immune modulator for use in this
method which selectively inhibits the function of the T.sub.H1
subset of CD4.sup.+ T helper cells may be administered at the time
of primary administration of the viral vector. One such immune
modulator is interleukin-4 (IL-4). IL-4 enhances antigen specific
activity of T.sub.H2 cells at the expense of the T.sub.H1 cell
function [see, e.g., Yokota et al, Proc. Natl. Acad. Sci., USA,
83:5894-5898 (1986); U.S. Pat. No. 5,017,691]. It is envisioned
that other immune modulators that can inhibit T.sub.H1 cell
function will also be useful in the methods of this invention.
[0051] In another embodiment, the immune modulator can be a
cytokine that prevents the activation of the T.sub.H2 subset of T
helper cells. The success of this method depends on the relative
contribution that T.sub.H2 dependent Ig isotypes play in virus
neutralization, the profile of which may be affected by strain, the
species of animal as well as the mode of virus delivery and target
organ.
[0052] A desirable immune modulator for use in this method which
selectively inhibits the CD4.sup.+ T cell subset T.sub.H2 function
at the time of primary administration of the viral vector includes
interleukin-12 (IL-12). IL-12 enhances antigen specific activity of
T.sub.H1 cells at the expense of T cell function [see, e.g.,
European Patent Application No. 441,900; P. Scott, Science
260:496-497 (1993); R. Manetti et al, J. Exp. Med., 177:1199
(1993); A. D'Andrea et al, J. Exp. Med., 176:1387 (1992)]. IL-12
for use in this method is preferably in protein form. Human IL-12
may be recombinantly produced using known techniques or may be
obtained commercially. Alternatively, it may be engineered into a
viral vector (which optionally may be the same as that used to
express the transgene) and expressed in a target cell in vivo or ex
vivo.
[0053] T.sub.H2 specific ablation with IL-12 is particularly
effective in lung-directed gene therapies where IgA is the primary
source of neutralizing antibody. In liver-directed gene therapy,
both T.sub.H1 and T.sub.H2 cells contribute to the production of
virus specific antibodies. However, the total amount of
neutralizing antibody can be diminished with IL-12.
[0054] Another selected immune modulator which performs a similar
function is gamma interferon (IFN-.gamma.) [S. C. Morris et al, J.
Immunol. 152:1047-1056 (1994); F. P. Heinzel et al, J. Exp. Med.,
177:1505 (1993)]. IFN-.gamma. is believed to mediate many of the
biological effects of IL-12 via secretion of activated macrophages
and T helper cells. IFN-.gamma. also partially inhibits IL-4
stimulated activation of T.sub.H2. IFN-.gamma. may also be obtained
from a variety of commercial sources.
[0055] Alternatively, it may be engineered into a viral vector and
expressed in a target cell in vivo or ex vivo using known genetic
engineering techniques.
[0056] Preferably, such cytokine immune modulators are in the form
of human recombinant proteins. These proteins may be produced by
methods extant in the art. Active peptides, fragments, subunits or
analogs of the known immune modulators described herein, such as
IL-12 or gamma interferon, which share the T.sub.H2 inhibitory
function of these proteins, will also be useful in this method when
the neutralizing antibodies are T.sub.H2 mediated.
[0057] As illustrated in the examples below, the cytokines IL-12
(which activates T.sub.H1 cells to secrete IFN-.gamma.) and
IFN-.gamma. are shown to ablate humoral immunity only (i.e., they
inhibit T.sub.H2 differentiation). Co-administration of either
cytokine at the time of virus instillation prevented formation of
IgA and allowed efficient re-administration of virus.
[0058] To permit an effective second administration of virus in
liver-directed gene therapy, the method may preferably comprise
administration of more than one cytokine, specific dosing regimens
and/or co-administration of an additional immune modulator, such as
one or more of the antibodies discussed above. The use of cytokines
is advantageous because cytokines are natural products, and thus
not likely to generate any adverse immune responses in the patient
to which they are administered.
[0059] C. Other Pharmaceuticals
[0060] Other immune modulators or agents that non-specifically
inhibit immune function, i.e., cyclosporin A or cyclophosphamide,
may also be used in the methods of the invention. For example, a
short course of cyclophosphamide has been demonstrated to
successfully interrupt both CD4 and CD8 T helper cell activation to
adenovirus capsid protein at the time of virus delivery to the
liver. As a result, transgene expression was prolonged and, at
higher doses, formation of neutralizing antibody was prevented,
allowing successful vector readministration. In the lung,
cyclophosphamide prevented formation of neutralizing antibodies at
all doses and stabilized transgene expression at high dose.
[0061] II. Viral Vectors
[0062] Suitable viral vectors useful in gene therapy are well
known, including retroviruses, vaccinia viruses, poxviruses,
adenoviruses and adeno-associated viruses, among others. The method
of this invention is anticipated to be useful with any virus which
forms the basis of a gene therapy vector. However, exemplary viral
vectors for use in the methods of the invention are adenovirus
vectors [see, e.g., M. S. Horwitz et al, "Adenoviridae and Their
Replication", Virology, second edition, pp. 1712, ed. B. N. Fields
et al, Raven Press Ltd., New York (1990); M. Rosenfeld et al, Cell,
68:143-155 (1992); J. F. Engelhardt et al, Human Genet. Ther.,
4:759-769 (1993); Yang IV; J. Wilson, Nature 365:691-692 (October
1993); B. J. Carter, in "Handbook of Parvoviruses", ed. P. Tijsser,
CRC Press, pp. 155-168 (1990).
[0063] Particularly desirable are human type C adenoviruses (Ad),
including serotypes Ad2 and Ad5, which have been rendered
replication defective for gene therapy by deleting the early gene
locus that encodes E1a and E1b. There has been much published on
the use of E1 deleted adenoviruses in gene therapy. See, K. F.
Kozarsky and J. M. Wilson, Curr. Opin. Genet. Dev. 3:499-503
(1993). The DNA sequences of a number of adenovirus types are
available from Genbank, including type Ad5 [Genbank Accession No.
M73260]. The adenovirus sequences may be obtained from any known
adenovirus type, including the presently identified 41 human types
[Horwitz et al, Virology, 2d ed., B. N. Fields, Raven Press, Ltd.,
New York (1990)]. A variety of adenovirus strains are available
from the American Type Culture Collection, Rockville, Md., or
available by request from a variety of commercial and institutional
sources. In the following embodiment, an adenovirus type 5 (Ad5) is
used for convenience.
[0064] The selection of the virus useful for engineering the
recombinant vectors, including the viral type, e.g., adenovirus,
and strain is not anticipated to limit the following invention.
[0065] Similarly, selection of the transgene contained within the
viral vector is not a limitation of this invention. This method is
anticipated to be useful with any transgene. Suitable transgenes
for delivery to a patient in a viral vector for gene therapy may be
selected by those of skill in the art. These therapeutic nucleic
acid sequences typically encode products for administration and
expression in a patient in vivo or ex vivo to replace or correct an
inherited or non-inherited genetic defect or treat an epigenetic
disorder or disease. Such therapeutic genes which are desirable for
the performance of gene therapy include, without limitation, a very
low density lipoprotein receptor gene (VLDL-R) for the treatment of
familial hypercholesterolemia or familial combined hyperlipidemia,
the cystic fibrosis transmembrane regulator gene (CFTR) for
treatment of cystic fibrosis, DMD Becker allele for treatment of
Duchenne muscular dystrophy, and a number of other genes which may
be readily selected by one of skill in the art to treat a
particular disorder or disease. Thus, the selection of the
transgene is not considered to be a limitation of this invention,
as such selection is within the knowledge of those skilled in the
art.
[0066] The viral vector bearing a therapeutic gene may be
administered to a patient, preferably suspended in a biologically
compatible solution or pharmaceutically acceptable delivery
vehicle. A suitable vehicle includes sterile saline. Other aqueous
and non-aqueous isotonic sterile injection solutions and aqueous
and non-aqueous sterile suspensions known to be pharmaceutically
acceptable carriers and well known to those of skill in the art may
be employed for this purpose.
[0067] The viral vector is administered in sufficient amounts to
transfect the desired cells and provide sufficient levels of
transduction and expression of the selected transgene to provide a
therapeutic benefit without undue adverse or with medically
acceptable physiological effects which can be determined by those
skilled in the medical arts. Conventional and pharmaceutically
acceptable routes of administration include direct delivery to the
target organ, tissue or site, intranasal, intravenous,
intramuscular, subcutaneous, intradermal, oral and other parental
routes of administration. Routes of administration may be combined,
if desired.
[0068] Dosages of the viral vector will depend primarily on factors
such as the condition being treated, the selected gene, the age,
weight and health of the patient, and may thus vary among patients.
For example, a therapeutically effective human dosage of the viral
vectors is generally in the range of from about 20 to about 50 ml
of saline solution containing concentrations of from about
1.times.10.sup.7 to 1.times.10.sup.10 pfU/ml viruses. A preferred
adult human dosage is about 20 ml saline solution at the above
concentrations. The dosage will be adjusted to balance the
therapeutic benefit against any side effects. The levels of
expression of the selected gene can be monitored to determine the
selection, adjustment or frequency of dosage administration.
[0069] III. The Method of the Invention
[0070] The method of this invention involves the co-administration
of the selected immune modulator with the selected recombinant
viral vector. The co-administration occurs so that the immune
modulator and vector are administered within a close time proximity
to each other. It is presently preferred to administer the
modulator concurrently with or no longer than one to three days
prior to the administration of the vector. The immune modulator may
be administered separately from the recombinant vector, or, if
desired, it may be administered in admixture with the recombinant
vector.
[0071] As illustrated by the examples below, the immune modulator,
whether it is an anti-CD40L antibody, anti-CD4 antibody or
cytokine, is desirably administered in close time proximity to the
administration of the viral vector used for gene therapy.
Particularly, administration of IL-12 or IFN-.gamma. causes
reduction in T.sub.H2 cell levels for about 2-3 days. Therefore,
IL-12 and/or IFN-.gamma. are desirably administered within a day of
the administration of the viral vector bearing the gene to be
delivered. Preferably, however, the IL-12 and/or IFN-.gamma. are
administered essentially simultaneously with the viral vector.
[0072] The immune modulator may be administered in a
pharmaceutically acceptable carrier or diluent, such as saline. For
example, when formulated separately from the viral vector, the
immune modulator is desirably suspended in saline solution. Such a
solution may contain conventional components, e.g. pH adjusters,
preservatives and the like. Such components are known and may be
readily selected by one of skill in the art.
[0073] Alternatively, the immune modulator may be itself
administered as DNA, either separately from the vector or admixed
with the recombinant vector bearing the transgene. Methods exist in
the art for the pharmaceutical preparation of the modulator as
protein or as DNA [See, e.g., J. Cohen, Science 259:1691-1692
(1993) regarding DNA vaccines]. Desirably, the immune modulator is
administered by the same route as the recombinant vector.
[0074] The immune modulator may be formulated directly into the
composition containing the viral vector administered to the
patient. Alternatively, the immune modulator may be administered
separately, preferably shortly before or after administration of
the viral vector. In another alternative, a composition containing
one immune modulator, such as IL-12, may be administered separately
from a composition containing a second immune modulator, such as
anti-CD40L antibody, and so on depending on the number of immune
modulators administered. These administrations may independently be
before, simultaneously with, or after administration of the viral
vector.
[0075] The administration of the selected immune modulator may be
repeated during the treatment with the recombinant viral vector
carrying the transgene during the period of time that the transgene
is expressed (as monitored by assays that detect transgene
expression or its intended effect), or with every booster of the
recombinant vector. Alternatively, each re-injection of the same
viral vector may employ a different immune modulator.
[0076] One advantage of the method of this invention is that it
represents a transient manipulation, necessary only at the time of
administration of the gene therapy vector. This strategy is
anticipated to be safer than strategies based on induction of
tolerance, which may permanently impair the ability of the
recipient to respond to viral infections.
[0077] Furthermore, the preferred use of immune modulators such as
the above-mentioned cytokines or antibodies is anticipated to be
safer than the use of agents such as cyclosporin or
cyclophosphamide (which cause generalized immune suppression)
because the transient immune modulation is selective (i.e.,
CTL-mediated responses are retained, as are humoral responses
dependent on T.sub.H1 function).
[0078] In one example of efficient gene transfer according to the
methods of this invention, the selected immune modulators are
IL-12, which causes the selective induction of T.sub.H1 cells,
and/or IFN-.gamma., which suppresses induction of T.sub.H2 cells.
Another preferred immune modulator is the anti-CD4.sup.+ antibody,
GK1.5, which depletes the T.sub.H1 cells and reduces CTL
elimination of the vector. Yet another preferred immune modulator
is the anti-CD40 ligand monoclonal antibody, MR1, available from
the American Type Culture Collection, Rockville, Md.
[0079] As exemplified below, the use of the above-identified immune
modulators permitted efficient gene transfer, as well as repeated
use of the same viral vector. In conjunction with gene therapy
which utilized an adenovirus vector containing either an alkaline
phosphatase ("ALP") transgene, a beta-galactosidase ("lacZ")
transgene, or a low density lipoprotein receptor ("LDLR")
transgene.
[0080] The following examples illustrate the preferred methods for
preparing suitable viral vectors useful in the gene therapy methods
of the invention. These examples are illustrative only and do not
limit the scope of the invention.
EXAMPLE 1
Construction and Purification of Exemplary Recombinant Adenovirus
Vectors
[0081] The recombinant adenovirus H5.010CMVlacZ, was constructed as
follows. The plasmid pAd.CMVlac [described in Kozarsky et al, J.
Biol. Chem., 269(18):13695-13702 (1994)], which contains adenovirus
map units 0-1, followed by a cytomegalovirus enhancer/promoter
[Boshart et al, Cell, 41:521-530 (1985)], an E. coli
beta-galactosidase gene (lacZ), a polyadenylation signal (pA),
adenovirus 5 map units 9.2-16 (Ad 9.2-16) and generic plasmid
sequences including an origin of replication and ampicillin
resistance gene was used. pAd.CMVlacZ was linearized with NheI and
co-transfected into 293 cells [ATCC CRL1573] with sub360 DNA
(derived from adenovirus type 5) which had been digested with XbaI
and Clal as previously described [K. F. Kozarsky, Somatic Cell Mol.
Genet., 19:449-458 (1993) and Kozarsky (1994), cited above]. The
resulting recombinant virus, H5.010CMVlacZ, contains adenovirus map
units 0-1, followed by a CMV enhancer/promoter, a lacZ gene, a
polyadenylation signal (pA), adenovirus map units 9.2-100, with a
small deletion in the E3 gene at 78.5 to 84.3 mu from the Ad 5
sub360 backbone. The recombinant adenovirus H5.010CBALP contains
the adenovirus map units 0-1, followed by a CMV enhanced, chicken
cytoplasmic .beta.-actin promoter [T. A. Kost et al, Nucl. Acids
Res., 11(23):8287 (1983)], a human placental ALP gene, a
polyadenylation signal (pA), and adenovirus type 5 map units 9-100,
with a small deletion in the E3 gene at 78.5 to 84.3 mu from the Ad
5 sub360 backbone. This recombinant adenovirus was constructed
substantially similarly to the H5.010CMVlacZ adenovirus described
above. See, also, Kozarsky (1994), cited above.
[0082] These recombinant adenoviruses, H5.010CMVlacZ and
H5.010CBALP, were isolated following transfection [Graham, Virol.,
52:456-467 (1974)], and were subjected to two rounds of plaque
purification. Lysates were purified on two sequential cesium
chloride density gradients as previously described [Englehardt et
al, Proc. Natl. Acad. Sci. USA, 88:11192-11196 (1991)]. Cesium
chloride was removed by passing the virus over BioRad DG10 gel
filtration columns using phosphate-buffered saline (PBS).
[0083] For mouse experiments, virus was either used fresh, or after
column purification, glycerol was added to a final concentration of
10% (v/v), and virus was stored at -70.degree. C. until use.
EXAMPLE 2
Enhancement of Adenovirus Mediated Gene Transfer upon Second
Administration by IL-12 and IFN-.gamma. in Mouse Lung.
[0084] The recombinant adenoviruses H5.010CMVlacZ and H5.010CBALP
were used in this example. Each virus expresses a different
reporter transgene whose expression can be discriminated from that
of the first reporter transgene.
[0085] Female C57BL/6 mice (6.about.8 week old) were infected with
suspensions of H5.010CBALP (1.times.10.sup.9 pfu in 50 .mu.l of
PBS) via the trachea at day 0 and similarly with H5.010CMVlacZ at
day 28. One group of such mice was used as a control. Another group
of mice were acutely depleted of CD4.sup.+ cells by i.p. injection
of antibody to CD4.sup.+ cells (GK1.5; ATCC No. TIB207, 1:10
dilution of ascites) at the time of the initial gene therapy (days
-3, 0, and +3). A third group of mice was injected with IL-12 (1
.mu.g intratracheal or 2 .mu.g, i.p. injections) at the time of the
first administration of virus (days 0 and +1). A fourth group of
mice was injected with gamma interferon (1 .mu.g intratracheal or 2
.mu.g, i.p. injections) at the time of the first administration of
virus (days 0 and +1).
[0086] When mice were subsequently euthanized and necropsied at
days 3, 28, or 31, lung tissues were prepared for cryosections,
while bronchial alveolar lavage (BAL) and mediastinal lymph nodes
(MLN) were harvested for immunological assays.
[0087] A. Cryosections
[0088] The lung tissues were evaluated for ALP expression at day 3
and day 28 by histochemical staining following the procedures of
Yang I, cited above. .beta.-galactosidase expression was assayed at
day 31 by X-gal histochemical staining. The results described below
were obtained from the alkaline phosphatase histochemical stains
(magnification .times.100) or .beta.-galactosidase X-gal stains
(magnification .times.100).
[0089] Instillation of ALP virus (10.sup.9 pfu) into the airway of
all groups of the C57BL/6 mice resulted in high level transgene
expression in the majority of conducting airways that diminished to
undetectable levels by day 28. Loss of transgene expression was
shown to be due to CTL-mediated elimination of the genetically
modified hepatocytes [Yang I, cited above].
[0090] In the control mice, no recombinant gene expression was
detected three days after the second administration of virus, i.e.,
at day 31.
[0091] Administration of virus to the CD4+-depleted animals was
associated with high level recombinant transgene expression that
was stable for a month. Expression of the second virus was
detectable on day 31. Thus, depletion of the CD4.sup.+ cells
effectively permits readministration of the vector without
immediate CTL elimination.
[0092] Initial high level gene transfer diminished after about one
month in the IL-12 treated mice. However, in contrast to the
control, high level gene transfer to airway epithelial cells was
achieved when virus was readministered to IL-12 treated animals at
day 28, as seen in the day 31 results.
[0093] The gamma-interferon treated animals were virtually
indistinguishable from the animals treated with IL-12 in that
efficient gene transfer was accomplished upon a second
administration of virus.
[0094] Thus, the use of these cytokines as immune modulators
enabled the repeated administration of the vector without its
immediate elimination by neutralizing antibody. In other
experiments, T.sub.H2 cells were not inhibited at the expense of
increased T.sub.H1 activation. In mice treated with the ALP virus
parenterally and IL-12 i.p., the IL-12 did not increase
adenovirus-specific CTL activity as shown by chromium release
assays. More importantly, treatment of the animals with IL-12 at
the time of intratracheal instillation of virus did not enhance
inflammation or diminish transgene persistence following a second
administration of virus.
[0095] B. Immunological Assays--MLN
[0096] Lymphocytes from MLN of the control group and IL-12 treated
group of C57BL/6 mice were harvested 28 days after administration
of H5.010CBALP and restimulated in vitro with UV-inactivated
H5.010CMVlacZ at 10 particles/cell for 24 hours. Cell-free
supernatants were assayed for the presence of IL-2 or IL-4 on HT-2
cells (an IL-2 or IL-4-dependent cell line) [Yang I, cited above].
Presence of IFN-.gamma. in the same lymphocyte culture supernatant
was measured on L929 cells as described [Yang I, cited above].
Stimulation index (S.I.) was calculated by dividing
.sup.3H-thymidine cpm incorporated into HT-2 cells cultured in
supernatants of lymphocytes restimulated with virus by
.sup.3H-thymidine cpm incorporated into HT-2 cells cultured in
supernatants of lymphocytes incubated in antigen-free medium.
[0097] The results are shown in Table I below.
1 TABLE I .sup.3H-Thymidine IFN-.gamma. Incorporation (cpm .+-. SD)
titre Medium H5.010CMVlacZ S.I. (IU/ml).sup.d C57BL/6 175 .+-. 40
2084 .+-. 66 11.91 80 anti-IL2 523 .+-. 81 2.98 (1:5000) anti-IL4
1545 .+-. 33 8.83 (1:5000) C57BL/6 247 .+-. 34 5203 .+-. 28 21.07
160 + IL12 anti-IL2 776 .+-. 50 3.14 (1:5000) anti-IL4 4608 .+-. 52
18.66 (1:5000)
[0098] Stimulation of lymphocytes from regional lymph nodes with
both recombinant adenoviruses led to secretion of cytokines
specific for the activation of both T.sub.H1 (i.e., IL-2 and
IFN-.gamma.) and T.sub.H2 (i.e., IL-4) subsets of T helper cells
(Table I).
[0099] Analysis of lymphocytes from the IL-12 treated animals
stimulated in vitro with virus revealed an increased secretion of
IL-2 and IFN-.gamma. relative to the production of IL-4, when
compared with animals that did not receive IL-12 (i.e., ratio of
IL-2/IL-4 was increased from 3 to 6 when IL-12 was used; Table
I).
[0100] C. Immunological Assays--BAL
[0101] BAL samples obtained from animals 28 days after primary
exposure to recombinant virus were evaluated for neutralizing
antibodies to adenovirus and anti-adenovirus antibody isotypes as
follows. The same four groups of C57BL/6 mice, i.e., control,
CD4.sup.+ depleted, IL-12 treated and IFN-.gamma. treated, were
infected with H5.010CBALP. Neutralizing antibody was measured in
serially-diluted BAL samples (100 .mu.l) which were mixed with
H5.010CBlacZ (1.times.10.sup.6 pfu in 20 .mu.l), incubated for 1
hour at 37.degree. C., and applied to 80% confluent Hela cells in
96 well plates (2.times.10.sup.4 cells per well). After 60 minutes
of incubation at 37.degree. C., 100 .mu.l of DMEM containing 20%
FBS was added to each well. Cells were fixed and stained for
P-galactosidase expression the following day.
[0102] All cells were lacZ positive in the absence of
anti-adenoviral antibodies.
[0103] Adenovirus-specific antibody isotype was determined in BAL
by using an enzyme-linked immunosorbent assay (ELISA). Briefly,
96-well plates were coated with 100 .mu.l of PBS containing
5.times.10.sup.9 particles of H5.010CBlacZ for 18 hours at
4.degree. C. The wells were washed 5 times with PBS. After blocking
with 200 .mu.l of 2% BSA in PBS, the plates were rinsed once with
PBS and incubated with 1:10 diluted BAL samples for 90 minutes at
4.degree. C. Thereafter, the wells were extensively washed and
refilled with 100 .mu.l of 1:1000 diluted ALP-conjugated anti-mouse
IgG or IgA (Sigma). The plates were incubated, subsequently washed
5 times, and 100 .mu.l of the substrate solution (p-nitrophenyl
phosphate, PNPP) was added to each well. Substrate conversion was
stopped by the addition of 50 .mu.l of 0.1 M EDTA, and reactions
were read at 405 nm.
[0104] The results are shown graphically in FIGS. 1A through 1C,
which summarize neutralizing antibody titer, and the relative
amounts (OD.sub.405) of IgG and IgA present in BAL samples. The
titer of neutralizing antibody for each sample was reported as the
highest dilution with which less than 50% of cells stained
blue.
[0105] As demonstrated in the first bar of FIGS. 1A through 1C, the
cytokines identified in Table 1 above were associated in the
control mice with the appearance of antibodies to adenovirus
proteins in BAL of both the IgG and IgA isotypes that were capable
of neutralizing the human Ad5 recombinant vector in an in vitro
assay out to a 1:800 dilution.
[0106] As shown in the second bar of the graphs of FIGS. 1A through
1C, transient CD4.sup.+ cell depletion inhibited the formation of
neutralizing antibody (FIG. 1A) and virus specific IgA antibody
(FIG. 1C) by 80-fold, thereby allowing efficient gene transfer to
occur following a second administration of virus. FIG. 1B shows a
slight inhibition of IgG as well.
[0107] As shown in the third bar of the three graphs, IL-12
selectively blocked secretion of antigen specific IgA (FIG. 1C),
without significantly impacting on formation of IgG (FIG. 1B). This
was concurrent with a 20-fold reduction in viral-specific
neutralizing antibody (FIG. 1A).
[0108] The gamma-interferon treated animals (fourth bar of FIGS. 1A
and 1B) were virtually indistinguishable from the animals treated
with IL-12 in that virus specific IgA (FIG. 1C) and neutralizing
antibody (FIG. 1A) were decreased as compared to the control
animals not treated with cytokine, but not to the extent obtained
with those treated with IL-12.
[0109] These studies demonstrate that the administration of
selected immune modulators to recipients of gene therapy
recombinant viral vectors at or about the time of primary exposure
to the vector can prevent the formation of blocking antibodies
and/or CTL elimination of the vector both initially and at the time
of repeated exposure to the viral vector. The concordant reduction
of neutralizing antibody with antiviral IgA suggests that
immunoglobulin of the IgA subtype is primarily responsible for the
blockade to gene transfer.
EXAMPLE 3
Enhancement of Adenovirus Mediated Gene Transfer upon Second
Administration by IL-12 and IFN-.gamma. in Mouse Liver
[0110] Experiments substantially identical to those described in
Example 2 above were conducted in which viral vectors were
administered into the blood for introduction of the transgene into
the liver (rather than intratracheal delivery into the lung).
[0111] The recombinant adenoviruses H5.010CMVlacZ and H5.010CBALP
were used in this example.
[0112] Female C57BL/6 mice (6-8 weeks old) were injected with
suspensions of H5.010CBALP (1.times.10.sup.9 pfu in 50 .mu.l of
PBS) i.p. at day 0 and similarly with H5.010CMVlacZ at day 28. One
group of such mice was used as a control. Another group of mice was
acutely depleted of CD4.sup.+ cells by i.p. injection of antibody
to CD4.sup.+ cells (GK1.5; ATCC No. TIB207, 1:10 dilution of
ascites) at the time of the initial gene therapy (days -3, 0, and
+3). A third group of mice was injected with IL-12 (2 .mu.g, i.p.
injections) at the time of the first administration of virus (days
0 and +1). A fourth group of mice was injected with gamma
interferon (2 .mu.g, i.p. injections) at the time of the first
administration of virus (days 0 and +1).
[0113] When mice were subsequently euthanized and necropsied at
days 3, 28, or 31, liver tissues were prepared for cryosections
according to the procedures used above for lung tissue in Example
2.
[0114] The cryosection results were substantially similar for
liver-directed gene therapy according to this method as for the
lung-directed therapy of Example 2 above. The results described
below were obtained from the alkaline phosphatase histochemical
stains (magnification .times.100) or .beta.-galactosidase X-gal
stains (magnification .times.100).
[0115] Administration of ALP virus (10.sup.9 pfu) into the veins of
all groups of the C57BL/6 mice resulted in high level transgene
expression in liver tissue that diminished to undetectable levels
by day 28. Loss of transgene expression was shown to be due to CTL
mediated elimination of the genetically modified hepatocytes [see
also, Yang I, cited above].
[0116] In the control mice, no recombinant gene expression was
detected three days after the second administration of virus, i.e.,
day 31.
[0117] Administration of virus to the CD4.sup.+ depleted animals
was associated with substantially lower neutralizing antibodies and
high level recombinant transgene expression that was stable for a
month. Expression of the second virus was detectable on day 31.
[0118] Initial high level gene transfer diminished after about one
month in the IL-12 treated mice; however, in contrast to the
control, some gene transfer to the liver via the blood was achieved
when virus was readministered to IL-12 treated animals at day 28
and the level of neutralizing antibody was reduced.
[0119] The gamma-interferon treated animals were virtually
indistinguishable from the animals treated with IL-12 in that
efficient gene transfer was accomplished upon a second
administration of virus.
[0120] Thus, the use of these cytokines and the anti-CD4+antibodies
as immune modulators enabled the repeated liver-directed
administration of the vector without its immediate elimination by
neutralizing antibodies.
EXAMPLE 4
Adenovirus Mediated Gene Transfer in Mouse Liver
[0121] Immune responses to primary administration of recombinant
virus were characterized further using different strains of
mice.
[0122] Recombinant virus (H5.010CMVLacZ or H5.010CBALP) was
inactivated with ultraviolet light in the presence of
8-methoxypsoralen. Briefly, purified virus was resuspended in 0.33
mg/ml of 8-methoxypsoralen solution and exposed to a 365 nm UV
light source on ice at 4 cm from the lamp filter for 30 min. The
virus was then passed over a Sephadex G-50 column equilibrated with
PBS. Limiting dilution transduction assays of inactivated stocks of
virus demonstrated less than one functional virus per 10.sup.5
particles of inactivated virus. Suspensions of the viruses
(2.times.10.sup.9 pfu in 100 .mu.l of PBS) were infused into the
tail vein of 6 to 8 week old female mice as detailed in the
following experiments. Each experiment was performed with a minimum
of three mice in which transgene expression was quantified in a
section of each of 5 lobes. The minimum analysis was 15 sections
per experimental condition.
[0123] a. C57BL/6 mice [H-2.sup.b; Jackson Laboratories, Bar
Harbor, Me.] were injected with suspensions of H5.010CMVlacZ at day
0 and similarly with H5.010CBALP at day 28 ("B6 mice");
[0124] b. C57BL/6 mice were injected with UV-inactivated
H5.010CMVlacZ at day 0 and H5.010CBALP at day 28 ("B6-UV
mice");
[0125] c. MHC class II-deficient (II.sup.-) mice [GenPharm
International, Mountain View, Calif.], bred into the C57BL/6
background (between 5-10 generations) and which carry the H-2.sup.b
haplotype, are unable to express I-A.sup.b determinants and cannot
develop CD4.sup.+ T cell mediated responses [Grusby et al, Science,
253:1417-1420 (1991)]. These mice were infected with H5.010CBALP at
day 0 and H5.010CMVlacZ at day 28 ("class II.sup.- mice");
[0126] d. .beta.2 microglobulin deficient (.beta.2m.sup.-) mice
[GenPharm International, Mountain View, Calif.], bred onto the
C57BL/6 background (between 5-10 generations) and which carry the
H-2.sup.b haplotype are unable to develop MHC class I associated
responses. These mice were infected with H5.010CMVlacZ at day 0 and
H5.010CBALP at day 28 (".beta.2m.sup.- mice");
[0127] e. C57BL/6 mice were inoculated i.p. with 0.5 ml aliquots of
1:10 dilution of mouse ascites fluid containing the GK1.5 (anti-CD4
MAb, ATCC TIB207) at days -3, 0 and +3 as described in Example 2.
This was equivalent to 100 .mu.g of purified monoclonal antibody
per injection. These CD4.sup.+ cell-depleted mice were infected
with H5.010CMVlacZ at day 0 and H5.010CBALP at day 28 ("CD4Ab
mice"); and
[0128] f C57BL/6 mice, treated with IL-12 (2 .mu.g in 200 .mu.l
PBS) i.p. on day 0 and day +1 as described in Example 2, were
infected with H5.010CMVlacZ at day 0 and H5.010CBALP at day 28
("IL-12 mice").
[0129] Mice from each group were subsequently euthanized and liver
tissues evaluated for lacZ expression by X-gal histochemistry
(magnification .times.100) at day 3 and day 28; and for ALP
expression by histochemical staining at day 31 (magnification
.times.100). Development of neutralizing antibody to adenovirus in
each group of mice was examined in serum samples obtained at day
28.
[0130] In fusion of lacZ virus into C57BL/6 mice was associated
with high level, but transient, expression of the reporter gene and
the eventual development of neutralizing antibody directed against
adenoviral antigen (FIG. 2). No gene transfer was detected when the
ALP virus was subsequently infused into these animals. In contrast,
the class II.sup.- mice did not produce neutralizing antibody (FIG.
2) and were receptive to high level gene transfer from a second
administration of virus. Similarly, animals transiently depleted of
CD4 partially stabilized lacZ expression and did not develop
neutralizing antibody, allowing efficient readministration of
virus.
[0131] In the B6-UV mice which received UV-inactivated recombinant
virus, the inactivated virus generated a full neutralizing antibody
response (FIG. 2) that completely prevented subsequent gene
transfer. This result demonstrates that capsid proteins of the
input virus are sufficient to activate a blocking T helper cell and
B cell-mediated humoral immune response.
[0132] The experiment with .beta.2m.sup.- mice was performed to
evaluate the role of CD8 cells and class I MHC expression in the
primary response to virus [Zijlstra et al, Nature, 344:742-746
(1990)]. Transgene expression was stable in these animals
consistent with prior reported data [Yang III, cited above].
However, gene transfer occurred at a significant level in the
setting of a readministration of virus. This was unexpected because
this strain of mice should have all components of the immune
response necessary to produce neutralizing antibody, (i.e., CD4
cells, MHC class II expression and B cells). These animals failed
to mount a significant neutralizing antibody response to adenoviral
antigens. These results suggest dysregulation of T helper and/or B
cell activation (FIG. 2).
[0133] Analysis of lymphocytes from .beta.2m.sup.- animals
demonstrated antigen-activated secretion of IFN-.gamma. in excess
of that measured in C57BL/6 mice, possibly due to the persistence
of virus-infected cells and the chronic activation of T.sub.H1
cells. The amplified T.sub.H1 response in .beta.2m.sup.- mice could
lead to an inhibition of T.sub.H2 cells resulting in diminished
production of anti-viral antibodies.
[0134] Transgene expression was found to be stabilized in animals
deficient in CD8 cells and MHC class I by virtue of a germ line
.beta.2m.sup.- interruption. Specific ablation of perforin, the
molecule on CTLs and natural killer (NK) cells that mediates
cytolysis, similarly prolongs of transgene expression (data not
shown).
EXAMPLE 5
Effect of CD4 Antibody on Ad-mediated Gene Transfer Upon Repeated
Administrations
[0135] Suspensions of the recombinant adenoviruses expressing
different transgenes were infused into the tail vein of C57BL/6
(H-2.sup.b) mice at 21 day intervals.
[0136] H5.010CMWLDLR is an adenovirus deleted of the E1a and E1b
genes and has a deletion in E3 gene at 78.5 to 84.3 mu from the Ad
5 sub360 backbone, with a LDL receptor gene in place of the E1
deletion [described in Kozarsky et al, J. Biol. Chem. 269:1-8
(1994)].
[0137] H5.010CMVLDLR was administered at day 0; H5.010CMVlacZ at
day 21; and H5.010CBALP at day 42. A control group of mice was
administered i.p. injections of saline at days -3, 0 and +3, with
respect to each infusion of virus. A second group of mice was given
i.p. injections of CD4 depleting antibody to block T helper cell
activation (GK1.5 mAb) in the same protocol. A third group of mice
was given i.p. injections of GK1.5 mAb at days -3, 0, 3, 18, 21 and
24. A fourth group of mice, which did not receive the initial
administration of H5.010CMVLDLR, was treated with GK1.5 mAb at days
-3, 0 and 3.
[0138] The mice were subsequently euthanized, and the liver tissues
were evaluated for LDLR expression by immunohistochemistry at day
3, for lacZ expression by X-gal histochemistry at day 24, and for
ALP expression by histochemical staining at day 45. The assays were
performed as follows:
[0139] A. Immunofluorescent staining for LDLR expression was
performed as follows: Frozen sections (6 .mu.m) were fixed in
methanol as described in Morris et al, cited above. After blocking
with 10% goat serum in PBS (GS/PBS), sections were incubated with a
polyclonal antibody to LDLR (1:200) for 60 minutes, and then with
goat anti-rabbit IgG-FITC for 30 minutes. Sections were washed and
mounted with Citiflour (Citifluor, UK).
[0140] B. X-gal histochemistry was performed as follows: Sections
of fresh frozen tissue (6 .mu.m) were fixed in 0.5% glutaraldehyde
for 10 minutes, rinsed twice for 10 minutes in PBS containing 1 mM
MgCl.sub.2 and incubated in 1 mg/ml of 5-bromo-4-chloro-3-indolyl
.beta.-D-galactopyranoside (X-gal), 5 mM K.sub.3Fe(CN).sub.6, 5 mM
K.sub.4Fe(CN).sub.6, and 1 mM MgCl.sub.2 in PBS for 3 hours.
[0141] C. ALP histochemistry was performed as follows: Frozen
sections (6 .mu.m) were fixed in 0.5% glutaraldehyde for 10
minutes, rinsed in PBS, incubated at 65.degree. C. for 30 minutes
to inactivate endogenous ALP activity, washed in 100 mM Tris (pH
9.5), 100 mM NaCl and 50 MM MgCl.sub.2, and stained in the same
buffer containing 0.165 mg/ml of 5-bromo-4-chloro-3-indolyl
phosphate (BCIP) and 0.33 mg/ml Nitroblue Tetrazolium (NBT) at
37.degree. C. for 30 minutes.
[0142] The results discussed below were obtained from analyses of
the cytochemical stains of liver tissue three days after each virus
infusion, including LDLR virus on day 0, lacZ virus on day 21 and
ALP virus on day 42 (magnification .times.150).
[0143] Serum samples were also collected at days 0, 3, 7, 14, 21,
28, 35 and 42 from each group of animals and assayed for
neutralizing antibody titer. Serum samples were incubated at
56.degree. C. for 30 minutes and then diluted in DMEM in twofold
steps starting from 1:20. Each serum dilution (100 .mu.l) was mixed
with H5.010CMVlacZ (2.times.10.sup.6 pfu in 20 .mu.l), incubated
for 1 hour at 37.degree. C., and applied to 80% confluent Hela
cells in 96-well plates (2.times.10.sup.4 cells per well). After 60
minutes incubation at 37.degree. C., 100 .mu.l of DMEM containing
20% FBS were added to each well. Cells were fixed and stained for
P-galactosidase expression on the following day. All of the cells
stained blue in the absence of serum samples. The titer of
neutralizing antibody for each sample was reported as the highest
dilution with which less than 50% of cells stained blue. FIGS.
3A-3D report the antibody titer expressed as a function of days
post-infection.
[0144] These experiments demonstrated that in the group of mice
receiving no CD4 antibodies, efficient gene transfer occurred
following the first virus. However, the development of neutralizing
antibody blocked gene transfer with the subsequent two viruses.
Neutralizing antibody rapidly appeared in serum following the
second virus if CD4 antibodies were not coadministered (FIG. 3B).
The third virus was not effective in these animals. In contrast,
administration of CD4 antibodies at the time of first infusion of
virus prevented the formation of neutralizing antibodies (FIG. 3B)
and allowed high level gene transfer with the second virus. The
appearance of neutralizing antibody following the second virus was
accelerated (FIG. 3B as compared to the time course of a primary
response in FIG. 3A), suggesting that activation of some level of
cellular immunity does occur even in the presence of CD4
antibodies. Administration of CD4 antibody with the second virus
again blocked neutralizing antibody in the group of mice receiving
CD4 antibodies and both viruses, allowing efficient gene transfer
with the third virus.
[0145] These experiments demonstrate that transient immune blockade
at the time of virus delivery, as opposed to chronic immune
suppression, is all that is necessary for efficient gene
transfer.
[0146] The fourth group of animals received CD4 antibody on day -3
without H5.010CMVLDLR administration, i.e., 21 days prior to
primary administration of virus. Neutralizing antibody that
developed subsequent to primary challenge of virus blocked gene
transfer 21 days later (FIG. 3D) in a manner indistinguishable from
that observed in naive animals not pretreated with CD4 antibody
(FIG. 3A). This result confirms the transient nature of the CD4
depletion.
EXAMPLE 6
Effect of IL-12 on Ad-specific Antibody Isotypes and CTL
Responses
[0147] A. Serum samples obtained from the C57BL/6 mice ("B6") and
IL-12 treated C57BL/6 ("B6+IL12") mice of Example 5 were tested 28
days after infection for adenovirus-specific IgG1 and IgG2a
antibody isotypes.
[0148] A solid phase enzyme linked immunosorbent assay (ELISA)
using purified H5.010CMVlacZvirus as antigen was performed.
Immunolon-2-U microtiter plates (Fisher) were coated with 200
ng/well of viral antigen in 100 ml of PBS for 6 hours at 37.degree.
C., washed three times in PBS, and blocked in PBS/1% BSA overnight
at 4.degree. C. The following day, 4-fold serial diluted serum
samples were added to antigen-coated plates and incubated for 4
hours at 37.degree. C. Plates were washed three times in PBS/1% BSA
and incubated with goat anti-mouse IgG1-biotin or goat anti-mouse
IgG2a-biotin (CALTAG Laboratories, San Francisco, Calif.) at 1:5000
dilution for 2 hours at 37.degree. C. Plates were washed as above
and Avidin-ALP (Sigma) was added to each well at 1:5000 dilution
for 1 hour at 37.degree. C. Wells were again washed as above and
PNPP substrate was added. Optical densities were read on a Biorad
model 450 microplate reader.
[0149] FIGS. 4A and 4B summarize the relative amounts (OD.sub.405)
of IgG1 and IgG2a, respectively, present in serum samples as a
function of sample dilutions. The ELISA assays of sera revealed
anti-viral antibodies of both IgG1 and IgG2a subtypes consistent
with activation of both T.sub.H1 and T.sub.H2 subsets,
respectively. Animals receiving IL-12 produced less anti-viral IgG1
at the expense of an increased production of IgG2a.
[0150] B. Splenocytes harvested from C57BL/6 mice ("B6") and
IL-12-treated C57BL/6 mice ("B6+IL12") of Example 5 were
restimulated in vitro 10 days after administration of H5.010CBALP
with H5.010CMVlacZ for 5 days in DMEM supplemented with 5% FBS and
50 mM 2-mercaptoethanol. These cells were tested for specific lysis
on mock-infected ("mock") and H5.010CBALP ("ALP")-infected C57SV
cells in a 6 hour .sup.51Cr release assay performed subsequently
using the following ratios of effector to target cells (C57SV,
H-2b) in 200 .mu.l DMEM with 10% FBS in V-bottom 96-well plates
(E:T=50:1, 25:1, 12:1, 6:1, 5:1 and 3:1).
[0151] Prior to mixing with the effector cells, target cells
(1.times.10.sup.6) were labeled with 100 .mu.Ci of .sup.51Cr after
a 24 hr-infection with H5.010CMV/acZ at an moi of 50 and used at
5.times.10.sup.3 cells/well. After incubation for 6 hr, aliquots of
100 .mu.l supernatant were removed for counting in a gamma counter.
Percentage of specific .sup.51Cr release was calculated as: [(cpm
of sample-cpm of spontaneous release)/(cpm of maximal release-cpm
of spontaneous release)].times.100. The results, reported as
percentage of specific lysis as a function of different effector to
target ratios (FIG. 5), show that CTL activity against
virus-infected target cells was unaffected by IL-12 treatment.
[0152] The net result was a 3-fold reduction in neutralizing
antibody, the magnitude of which was insufficient to allow
efficient gene transfer upon readministration of virus. Differences
in efficiency of virus readministration between the P2m- mice and
the IL-12 treated C57BL/6 mice could reflect inadequate cytokine
mediated repression following i.p. injection of IL-12 or mechanisms
other than inhibition of T.sub.H2 activation.
EXAMPLE 7
CD40L-deficient Mice Illustrate the Necessary Role of T Cell
Activation in Host Responses to Adenoviral Vectors
[0153] The role of CD40L mediated signaling of T cells in cellular
and humoral immune responses to adenoviral vectors was studied in
mice genetically deficient in CD40L. Previous studies have
demonstrated abnormalities in thymic dependent B cell responses in
these mice [J. Xu et al, Immunity, 1:423-431 (1994) and B. Renshaw
et al, J. Exp. Med., 180:1889-1900 (1994)]. CD40L-deficient mice
(CD40L KO) and their normal litter mates in a C57BL/6-129 chimeric
background [J. Xu et al, cited above] were administered lacZ
containing E1 deleted adenovirus (H5.010CMVlacZ) on day 0 into the
trachea (1.times.10.sup.9 in 50 .mu.l of PBS), to effect gene
transfer to lung, and into the peripheral circulation via the tail
vein (2.times.10.sup.9 in 100 .mu.l in PBS), to effect gene
transfer to liver. Animals were retreated with H5.010CBALP, an
adenoviral vector containing a different reporter gene (alkaline
phosphatase, ALP), on day 28. Blood was analyzed prior to the
second vector administration for neutralizing antibodies, and
tissues were harvested for analysis of reporter gene expression 3
days later (i.e., day 31). Animals were sacrificed 3 and 28 days
later to assess the efficiency and stability of transgene
expression, respectively. Table II summarizes morphometric analyses
of these tissues.
2TABLE II Quantitative Analysis of Mouse Lung and Liver for
Efficiency of Transgene Expression Day 3 Day 28 Day 31 .sup.1Lung
(% airways >25% transgene expression) Control 76 0 0 CD40L Ab 72
42 30 CD40L KO 75 30 45 .sup.2Liver (% transgene expression)
Control 90.5 .+-. 2.6 0 0 CD40L Ab 89.3 .+-. 3 1 46.7 .+-. 4.8 8.2
.+-. 4.2 CD40L KO 92.3 .+-. 4.0 60.4 .+-. 2.8 85.9 .+-. 3.4
.sup.1Data were quantified by examining a total of 100 airways from
3 mice for the presence of transgene-containing respiratory
epithelial cells using the criteria of a positive airway as the
transgene was greater than 25%. .sup.2Data are presented as the
mean .+-. 1 S.D.
[0154] Normal litter mates that were administered vectors
demonstrated high level transgene expression at day 3 in lung and
liver that diminished to undetectable levels by day 28 (similar to
what is seen in naive C57BL/6 mice; Table II). Serum and bronchial
alveolar lavage (BAL) were analyzed for neutralizing antibody to
human Ad5 as described in Y. Dai et al, Proc. Natl. Acad. Sci. USA,
92:1401-1405 (1995). Substantial neutralizing antibody developed to
adenoviral capsid proteins by day 28 in either BAL fluid of animals
that received vector intratracheally or in blood of animals that
received vector into the venous circulation (FIG. 6).
Readministration of vector on day 28 was unsuccessful as evidenced
by the lack of transgene expression in the target organ 3 days
later (similar to what is seen in C57BL/6 mice, Table II).
Substantially different results were obtained in the CD40L
deficient mice. Transgene expression was stable with little
diminution for 28 days in both lung and liver (Table II). In
addition, neutralizing antibody failed to develop (FIG. 6),
resulting in highly efficient transgene expression following a
second administration of virus (Table I).
EXAMPLE 8
Transient Blockade of CD40 Ligand with Antibody Prevents Primary T
Cell Activation and Prolongs Transgene Expression
[0155] The following example demonstrates that transient inhibition
of CD40L with antibody blocked CD4.sup.+ T cell priming in the lung
model of gene therapy and effectively eliminated CD4 T and B cell
effector responses. The persistent transgene expression and
efficiency of vector readministration into the lung was essentially
identical in animals genetically deficient in CD40L (Example 7
above) as compared to those transiently inhibited with CD40L
antibody (see below).
[0156] The encouraging results obtained in the CD40L deficient mice
as shown in Example 7 above provided a basis for developing an
adjunct gene therapy with adenoviral vectors based on pharmacologic
inhibition of CD40L signaling. This therapy is based upon findings
that the capsid proteins of the input virus are the primary source
of antigen for CD4.sup.+ T cell activation, thereby restricting the
time of costimulatory blockade to a short interval when vector is
administered.
[0157] Experiments in C57BL/6 mice (six weeks of age, females) not
treated with antibody or treated with isotype control antibody
demonstrated high level transgene expression at day 3 in lung
(Table II) and liver (Table II), that diminished to undetectable
levels by day 28 (Table II). Gene transfer experiments were also
performed in C57BL/6 animals injected with 100 .mu.g mAb to CD40L
(MR1, ATCC Hybridoma HB11048) i.p. on days -3, 0, +3 and +6
relative to the initial vector administration or equivalent
quantities of a control hamster monoclonal antibody. Studies in
murine lung demonstrated stabilization of transgene expression in
CD40L mAb treated animals: the number of airways showing transgene
in >25% of epithelial cells showed minimal decline from 72% on
day 3 to 42% on day 28 (Table II). Transgene expression was also
stabilized in liver of animals treated with CD40L mAb, in which
transgene expressing hepatocytes diminished slightly from 89% to
47% over a 28 day interval (Table II). Transgene expression is
stabilized in CD40L antibody treated animals at least 6 weeks,
which is the longest time point evaluated (data not shown).
[0158] Recipient animals were analyzed for antigen specific
activation of CD4.sup.+ and CD8.sup.+ T cells using both in vitro
and in vivo assays. The effect of CD40L blockade on CD4.sup.+ T
cells was studied in proliferation assays of lymphocytes stimulated
with UV-inactivated adenovirus essentially as described below
(Table III). Briefly, lymphocytes of mediastinal lymph nodes (for
lung experiment) or splenocytes (for liver experiment) from mice 10
days after administration of viruses were restimulated with
UV-inactivated virus for 24 hours. Supernatants were tested on HT-2
cells (ATCC, CRL 1841) for cytokine secretion, and proliferation
was assessed 72 hours later by measuring .sup.3H-thymidine
incorporation. Activation of adenoviral specific T cells, as
quantified by the stimulation index, was documented initially at
day 7 in nonantibody treated animals that were administered via
vector into lung or liver. Activation of adenoviral specific T
cells increased progressively over the ensuing 14 days. Stimulation
index was calculated by dividing .sup.3H counts in the presence of
antigen by those in the absence of antigen. T cell activation was
substantially inhibited in both models by coadministration of CD40L
antibody. The greatest inhibition was observed in animals
administered vector into lung.
3TABLE III CD4.sup.+ T Cell Responses and Neutralizing Antibody
Neutralizing Stimulation Index.sup.1 Antibody.sup.2 Day 7 Day 21
Day 28 Lung Control 11.2 .+-. 1.1 52 .+-. 2 267 .+-. 92 CD40L Ab
1.2 .+-. 0.1 10 .+-. 1 20 .+-. 0 CD40L KO N.D. N.D. 20 .+-. 0 Liver
Control 20 .+-. 1 53 .+-. 2 533 .+-. 185 CD40L Ab 4.1 .+-. 0.5 21
.+-. 1 66 .+-. 23 CD40L KO ND. ND. 20 .+-. 0 .sup.1Data are
presented as the mean stimulation index of three determinations
.+-.1 S.D. N.D. - Not determined. .sup.2Data are presented as the
mean neutralizing antibody titer (reciprocal dilution) of three
samples .+-.1 S.D.
[0159] Activation of CD8.sup.+ T cells by virus-infected cells was
analyzed in chromium release assays using MHC H-2 compatible target
cells infected with adenoviral vectors. As shown previously,
specific lysis was demonstrated with lymphocytes harvested from
C57BL/6 recipients on day 7, which were stimulated in vitro with
adenovirus infected antigen presenting cells, and incubated with
adenoviral infected targets (FIG. 7). No lysis was demonstrated to
mock infected targets.
[0160] Lymphocytes harvested from animals treated with CD40L
antibody also demonstrated CTL activity to adenovirus infected
cells. However, the extent of lysis was consistently lower than
that obtained from immune ablated animals. The necessity to amplify
CTL by in vitro stimulation prior to the cytolytic assay may
obscure more significant differences in CTL activation that
occurred following the primary exposure in vivo.
[0161] The primary effect of CD40L inhibition on the activation of
adenoviral specific CD4.sup.+ T cells was further evaluated in vivo
using techniques of immunocytochemistry. These experiments were
restricted to the model of liver directed gene transfer because of
technical limitations of immunofluorescence in lung sections. Liver
tissues were analyzed on day 14 for infiltration of CD4.sup.+ and
CD8.sup.+ T cells by double immunofluorescence. Nonantibody treated
animals showed a typical mixed lymphocyte infiltrate that was
dominated by CD4.sup.+ T cells and associated with substantial
upregulation of MHC class I on the basolateral surface of
hepatocytes. Previous studies have suggested that secretion of
IFN-.gamma. from T.sub.H1 activated CD4.sup.+ T cells contributes
to the increase in MHC class I which may sensitize the hepatocytes
to CTL mediated elimination. Animals treated with antibody to CD40L
still mobilized a mixed lymphocyte infiltrate. However, the
proportion of CD4.sup.+ T cells is substantially lower and the
increase in MHC class I is substantially blunted. The specificity
of the immunofluorescence assays was demonstrated in mock infected
animals.
EXAMPLE 9
CD40L Antibody Prevents Formation of Blocking Antibody in Lung
Directed Gene Transfer
[0162] The impact of transient blockade of CD40L signaling at the
time of vector administration on the production of neutralizing
antibody and efficiency of repeated vector administration was
evaluated in this experiment. Animals that received vector on day 0
with or without mAb were retreated with an adenoviral vector
containing a different reporter gene on day 28. Blood was analyzed
prior to administration of the second vector for neutralizing
antibodies, and tissues were harvested for analysis of reporter
gene expression 3 days later (i.e., day 31).
[0163] The most impressive results were obtained in the model of
lung directed gene therapy. The development of neutralizing
antibody in BAL following lung-directed gene transfer of vector was
inhibited 20-fold in animals administered CD40L antibody (FIG. 7).
Gene transfer with the second vector was unsuccessful in
nonantibody treated animals or animals treated with an isotype
control mAb (data not shown), as evidenced by the complete absence
of transgene expression 3 days after vector readministration. This
contrasts with animals treated during the first vector
administration with CD40L antibody in which gene transfer was
accomplished following a second administration of vector. Transgene
expression was detected in >25% of airway epithelial cells of
30% of airways following the second vector, which is only slightly
lower than the number of airways that express transgene in a naive
animal treated with vector (i.e., 75%; Table II). Antibody to CD40L
partially blocked the production of neutralizing antibody in serum
following intravenous infusion of virus (FIG. 7). This was
sufficient to enable some gene transfer to liver with the second
vector (8% of hepatocytes), that did not occur in the absence of
antibody, but is substantially reduced from that achieved following
a primary administration of vector in a naive animals (89%).
EXAMPLE 10
Short Course of Cyclophosphamide Prevents Destructive Immune
Responses in Mouse Lung and Liver
[0164] C57BL/6 mice were given cyclophosphamide in various dosing
regimens at the time an E1-deleted lacZ virus was administered into
the blood, to study liver directed gene transfer, and into the
trachea, to study lung directed gene transfer. A second E1-deleted
virus, expressing the alkaline phosphatase reporter gene, is
readministered into the same organ that received the first vector.
Lymphocytes were isolated from regional sites and evaluated in
vitro for vector specific T cell activation. Tissues were harvested
at various times for analysis of inflammation and its consequences
as well as expression of both the lacZ and ALP reporter genes.
[0165] A. Animal Studies
[0166] C57BL/6 female mice were injected with 1.times.10.sup.9 pfu
H5.010CMVlacZ via trachea (lung studies) or tail vein (liver
studies) on day 0. Cyclophosphamide injections were given iv as
indicated (in 200 ml PBS). H5.010CBALP was injected as described
above on day 28. Animals were sacrificed on day 3, 28, 31 and 50
for analysis of transgene expression. When necropsy was performed,
lung and liver tissues were prepared for cryosections, while
spleen, bronchial alveolar lavage (BAL) and mediastinal lymph nodes
(MLN) were harvested for immunological assays.
[0167] B. Morphological Analyses
[0168] For immunocytochemical analyses, frozen liver tissue was
cryosectioned, while lungs were inflated with a 1:1 mixture of
PBS/OTC, frozen and blocks cryosectioned. For X-Gal
(5-bromo-4-chloro-3-indolyl b-D-galactopyranoside) histochemistry
frozen 6 mm tissue sections were fixed in 0.5% glutaraldehyde for
10 min, washed twice with PBS containing 1 mM MgCl.sub.2, and
incubated in 1 mg of X-Gal per ml, 5 mM K.sub.3Fe(CN).sub.6, 5 mM
K.sub.4Fe(CN).sub.6, and 1 mM MgCl.sub.2 in PBS for 4 hours. For
alkaline phosphatase staining, frozen sections were fixed in 0.5%
glutaraldehyde for 10 minutes and washed twice in PBS. The sections
were incubated at 65.degree. C. for 30 minutes to inactivate
endogenous alkaline phosphatase activity, washed once in PBS and
stained in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl.sub.2
containing 0.165 mg of BCIP (5-bromo-4-chloro-3-indolyl phosphate)
and 0.33 mg of nitroblue tetrazolium per ml at 37.degree. C. for 30
minutes.
[0169] C. Immunofluorescence
[0170] Frozen sections were fixed in -20.degree. C. methanol for 10
minutes, air dried and rehydrated in PBS twice and unspecific
binding blocked in 10% goat serum/PBS for 30 minutes. Sections were
incubated for 1 hours with either rat-anti-mouse CD4 antibody (anti
L3T4, GibcoBRL, 1:100 dilution in 2% goat serum), followed by a 30
minutes incubation with 5 mg of goat anti-rat immunoglobulin G
(IgG)-fluorescein or rat-anti mouse CD8a-fluorescein isothiocyanate
(anti-Ly-2, GibcoBRL, 1:100 dilution in 2% goat serum). For MHC
class I staining, sections were incubated with 1:50 diluted mouse
hybridoma supernatant to H-2K.sup.bD.sup.b (20-8-4S) for 60
minutes, followed by a 30 minutes incubation with 5 mg/ml goat
anti-mouse IgG-conjugated fluorescein isothiocyanate (FITC).
Sections were washed twice and mounted with the antifadent
Citifluor (Canterbury Chemical Lab., Canterbury, UK).
[0171] D. CTL Assay
[0172] For CTL assays, splenocytes from three mice or lymphocytes
from 10 mice were pooled. Cells were restimulated in vitro for 5
days with H5.010CMVlacZ (MOI 0.5) and assayed on MHC-compatible
target cells, which were previously infected with H5.010CMVIacZ and
loaded with .sup.51Cr, using different effector/target cell ratios.
The percentage of specific .sup.51Cr release was calculated as
[(cpm of sample-cpm of spontaneous release)/(cpm of maximum
release-cpm of spontaneous release)].times.100. Spontaneous release
was determined by assaying target cells without effector cells in
medium, while maximum release was estimated by adding 5% SDS to the
target cells during the 6 hours incubation time.
[0173] E. Cytokine Release Assay
[0174] 6.times.10.sup.6 splenocytes were cultured with or without
antigen (i.e., UV-inactivated H5.010CMVlacZ at a MOI of 10) for 24
h in a 24 well plate. 100 ml of cell-free supernatant were
transferred onto 2.times.10.sup.3 HT-2 cells (IL-2 and IL-4
dependent cell line) in round bottom 96 well plates. Medium and 10%
rat concanavalin A supernatant were used as negative and positive
controls. Proliferation was determined 48 h later by a 6 h
[.sup.3H]thymidine (0.35 mCi/well) pulse.
[0175] F. Neutralizing Antibody Assay
[0176] Serum and BAL were incubated for 30 min at 56.degree. C. to
inactivate complement. Serial dilutions of serum and BAL in DMEM
without FBS (50 ml, starting at 1:20) were incubated with
1.times.10.sup.6 pfu of H5.010CMVlacZ for 60 min and applied onto
2.times.10.sup.4 Hela cells (80% confluent) in 96 well plates.
After a 60 minutes incubation, 100 ml of DMEM containing 20% FBS
were added. The cells were fixed 16-18 hours later and stained for
.beta.-galactosidase activity. All of the cells stained blue when
medium was added instead of serum or BAL. The neutralizing antibody
titer was determined by the highest dilution with which less than
50% of cells stained blue.
[0177] All articles identified herein are incorporated by
reference. Numerous modifications and variations of the present
invention are included in the above-identified specification and
are expected to be obvious to one of skill in the art. Such
modifications and alterations including the specific immune
modulator selected, the manner of administration, the recombinant
vector and transgene selected, route of administration, etc. are
believed to be encompassed in the scope of the claims appended
hereto.
* * * * *