U.S. patent application number 10/470568 was filed with the patent office on 2004-08-12 for rapid production of autologous tumor vaccines.
Invention is credited to Federoff, Howard, Fong, Yuman, Rosenblatt, Joseph D..
Application Number | 20040157299 10/470568 |
Document ID | / |
Family ID | 21930021 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157299 |
Kind Code |
A1 |
Fong, Yuman ; et
al. |
August 12, 2004 |
Rapid production of autologous tumor vaccines
Abstract
An analogous vaccine to tumor cells is produced by transducing
the tumor cells with a herpes simplex virus amplicon containing the
gene for an immunomodulatory protein to provide transient
expression of the immunomodulatory protein by the cells. The tumor
cells may transduced with the herpes simplex amplicons ex vivo or
in vivo. Suitable immunomodulatory proteins include cytokines, for
example, interleukins, interferons, and chemokines such as RANTES;
intercellular adhesion molecules, for example ICAM-1 and
costimulatory factors such as B7.1. The tumor cells may also be
transduced with one or more species of amplicon containing genes
for two or more different immunomodulatory proteins.
Inventors: |
Fong, Yuman; (New York,
NY) ; Federoff, Howard; (Rochester, NY) ;
Rosenblatt, Joseph D.; (Rochester, NY) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
21930021 |
Appl. No.: |
10/470568 |
Filed: |
March 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10470568 |
Mar 24, 2003 |
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09550892 |
Apr 17, 2000 |
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09550892 |
Apr 17, 2000 |
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09045476 |
Jan 20, 1998 |
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6051428 |
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60044005 |
Mar 21, 1997 |
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Current U.S.
Class: |
435/69.5 ;
435/320.1; 435/325; 435/456; 530/351; 536/23.5 |
Current CPC
Class: |
C12N 2710/16643
20130101; A61K 2039/55516 20130101; C12N 15/86 20130101; C12N
2830/60 20130101; A61K 2039/5156 20130101; A61K 48/00 20130101;
A61P 37/04 20180101; C12N 2830/00 20130101; C12N 2840/203 20130101;
A61P 35/00 20180101; A61K 39/0011 20130101 |
Class at
Publication: |
435/069.5 ;
435/456; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C12P 021/02; C07H
021/04; C07K 014/52; C12N 015/86 |
Goverment Interests
[0002] The work described in this application was supported in part
by NIH Grants Nos. CA76416, CA72632, HD 31300, DK53160, and POI
CA59326. The United States government may have certain rights in
this invention.
Claims
1. A method for production of an autologous vaccine to tumor cells
comprising transducing the tumor cells with a herpes simplex virus
amplicon containing the gene for an immunomodulatory protein to
provide transient expression of the immunomodulatory protein by the
cells.
2. The method according to claim 1, wherein the tumor cells are
transduced with the herpes simplex amplicons ex vivo.
3. The method according to claim 1, wherein the tumor cells are
transduced with the herpes simplex cell in vivo.
4. The method according to claim 1, wherein the immunomodulatory
protein is a cytokine.
5. The method according to claim 4, wherein the cytokine is
interleukin-2.
6. The method according to claim 4, wherein the cytokine is
granulocyte macrophage colony stimulating factor.
7. The method according to claim 4, wherein the immunomodulatory
protein is a chemokine.
8. The method according to claim 7, wherein the chemokine is
RANTES.
9. The method according to claim 1, wherein the immunomodulatory
protein is a intercellular adhesion molecule.
10. The method according to claim 9, wherein the intracellular
adhesion molecule is ICAM-1.
11. The method according to claim 1, wherein the immunomodulatory
protein is a costimulatory factor.
12. The method according to claim 11, wherein the costimulatory
factor is B7.1.
13. The method according to claim 1, wherein a population of tumor
cells is transduced with one or more species of amplicon containing
the genes for more than one kind of immunomodulatory protein and
expressing more than one kind of immunomodulatory protein.
14. The method according to claim 13, wherein the tumor cells are
transduced with amplicons encoding and expressing at least two
species of cytokines.
15. The method according to claim 14, wherein tumor cells are
transduced with amplicons containing the genes for interleukin-2
and interleukin-12.
16. The method according to claim 13, wherein the tumor cells are
transduced with amplicons encoding and expressing a cytokine and a
costimulatory factor.
17. The method according to claim 16, wherein tumor cells are
transduced with amplicons containing the genes for RANTES and
B7.1.
18. The method according to claim 1, wherein the tumor cells are
hepatoma cells or lymphoma cells.
19. A method for inducing a protective immune response to tumor
cells in a patient comprising the step of transducing the tumor
cells with a herpes simplex virus amplicon containing the gene for
a immunomodulatory protein to provide transient expression of the
immunomodulatory protein by the cells.
20. The method according to claim 19, wherein the tumor cells are
transduced with the amplicon ex vivo, further comprising the step
of introducing the transduced tumor cells into the patient.
21. The method according to claim 19, wherein the amplicons are
injected into the site of the tumor cells in vivo.
22. The method according to claim 19, wherein the immunomodulatory
protein is a cytokine.
23. The method according to claim 22, wherein the cytokine is
interleukin-2.
24. The method according to claim 22, wherein the cytokine is
granulocyte macrophage colony stimulating factor.
25. The method according to claim 22, wherein the immunomodulatory
protein is a chemokine.
26. The method according to claim 25, wherein the chemokine is
RANTES.
27. The method according to claim 19, wherein the immunomodulatory
protein is a intercellular adhesion molecule.
28. The method according to claim 27, wherein the intracellular
adhesion molecule is ICAM-1.
29. The method according to claim 19, wherein the immunomodulatory
protein is a costimulatory factor.
30. The method according to claim 29, wherein the costimulatory
factor is B7.1.
31. The method according to claim 19, wherein a population of tumor
cells is transduced with one or more species of amplicon containing
the genes for more than one kind of immunomodulatory protein and
expressing more than one kind of immunomodulatory protein.
32. The method according to claim 31, wherein the tumor cells are
transduced with amplicons encoding and expressing at least two
species of cytokines.
33. The method according to claim 32, wherein tumor cells are
transduced with amplicons containing the genes for interleukin-2
and interleukin-12.
34. The method according to claim 31, wherein the tumor cells are
transduced with amplicons encoding and expressing a cytokine and a
costimulatory factor.
35. The method according to claim 34, wherein tumor cells are
transduced with amplicons containing the genes for RANTES and
B7.1.
36. The method according to claim 19, wherein the tumor cells are
hepatoma cells or lymphoma cells.
37. A herpes simplex virus amplicon containing the gene for at
least one immunomodulatory protein.
38. The amplicon according to claim 37, wherein the
immunomodulatory protein is a cytokine.
39. The amplicon according to claim 38, wherein the cytokine is
interleukin-2.
40. The amplicon according to claim 38, wherein the cytokine is
granulocyte macrophage colony stimulating factor.
41. The amplicon according to claim 38, wherein the
immunomodulatory protein is a chemokine.
42. The amplicon according to claim 41, wherein the chemokine is
RANTES.
43. The amplicon according to claim 37, wherein the
immunomodulatory protein is a intercellular adhesion molecule.
44. The amplicon according to claim 43, wherein the intracellular
adhesion molecule is ICAM-1.
45. The amplicon according to claim 37, wherein the
immunomodulatory protein is a costimulatory factor.
46. The amplicon according to claim 45, wherein the costimulatory
factor is B7.1.
47. The amplicon according to claim 37, wherein the amplicon
contains genes encoding at least two immunomodulatory proteins.
48. The amplicon according to claim 47, wherein the amplicon
contains genes encoding for at least two species of cytokines.
49. The amplicon according to claim 48, wherein the amplicon
containing genes encoding for interleukin-2 and interleukin-12.
50. Tumor cells transduced with a herpes simplex virus amplicon
containing the gene for at least one immunomodulatory protein and
expressing said immunomodulatory protein.
51. The cells according to claim 50, wherein the immunomodulatory
protein is a cytokine.
52. The cells according to claim 51, wherein the cytokine is
interleukin-2.
53. The cells according to claim 51, wherein the cytokine is
granulocyte macrophage colony stimulating factor.
54. The cells according to claim 51, wherein the immunomodulatory
protein is a chemokine.
55. The cells according to claim 54, wherein the chemokine is
RANTES.
56. The cells according to claim 50, wherein the immunomodulatory
protein is a intercellular adhesion molecule.
57. The cells according to claim 56, wherein the intracellular
adhesion molecule is ICAM-1.
58. The cells according to claim 50, wherein the immunomodulatory
protein is a costimulatory factor.
59. The cells according to claim 58, wherein the costimulatory
factor is B7.1.
60. The cells according to claim 50, wherein the tumor cells are
transduced with one or more species of amplicon containing the
genes for more than one kind of immunomodulatory protein and
expressing more than one kind of immunomodulatory protein.
61. The cells according to claim 60, wherein the tumor cells are
transduced with amplicons encoding and expressing at least two
species of cytokines.
62. The cells according to claim 61, wherein tumor cells are
transduced with amplicons containing the genes for interleukin-2
and interleukin-12.
63. The cells according to claim 60, wherein the tumor cells are
transduced with amplicons encoding and expressing a cytokine and a
costimulatory factor.
64. The cells according to claim 63, wherein tumor cells are
transduced with amplicons containing the genes for RANTES and
B7.1.
65. The cells according to claim 50, wherein the tumor cells are
hepatoma cells or lymphoma cells.
66. A method for production of an autologous vaccine to tumor cells
comprising transducing the tumor cells with one or more species
herpes simplex virus amplicon containing the gene for an
immunomodulatory protein and at least one additional therapeutic
gene to provide transient expression of the immunomodulatory
protein and the therapeutic gene product by the cells.
67. The method according to claim 66, wherein the tumor cells are
transduced with the herpes simplex amplicons ex vivo.
68. The method according to claim 66, wherein the tumor cells are
transduced with the herpes simplex cell in vivo.
69. A method for inducing a protective immune response to tumor
cells in a patient comprising the step of transducing the tumor
cells with one or more species herpes simplex virus amplicon
containing the gene for an immunomodulatory protein and at least
one additional therapeutic gene to provide transient expression of
the immunomodulatory protein and the therapeutic gene product by
the cells.
70. A mixture containing a plurality of species of herpes simplex
virus amplicons, including at least a first species of amplicon
containing the gene for at least one immunomodulatory protein and a
second species of amplicon containing the gene for an additional
therapeutic gene product.
Description
[0001] This application is a regular application filed under 35 USC
.sctn. 111(a), claiming priority from U.S. Provisional Application
60/044,005 filed Mar. 21, 1997.
BACKGROUND OF THE INVENTION
[0003] Cytokine gene transfer to tumor cells has been used to
increase local production of these immune modulating proteins, with
the aim of enhancing tumor immunogenicity and consequent host
recognition and elimination of tumor (Dranoff et al. 1993;
Gansbacher et al. 1992). Production of irradiated, non-dividing
tumor cells secreting cytokines such as interleukin-2 (IL-2),
gamma-interferon (.gamma.-IFN), or granulocyte macrophage-colony
stimulating factor (GM-CSF) represents a potential therapeutic
strategy for treatment of malignant disease (Saito et al. 1994;
Dranoff et al. 1993; Gansbacher et al. 1992), and one that is
currently being evaluated in clinical trials (Lotze et al. 1994;
Seigler et al. 1994; Rosenberg et al. 1992). Many methods have been
examined for gene transfer (Davidson et al. 1993; Drazan et al.
1994; Yang et al. 1995; Paquereau & Le Cam, 1992; Jarnagin et
al. 1992); the most successful have been those using retroviral
vectors (Dranoff et al. 1993; Gansbacher et al. 1992).
[0004] An impediment to the production of autologous tumor vaccines
has been logistic problems surrounding gene transfer to freshly
harvested tumors. The most widely utilized approach for gene
transfer to tumors relies on retroviral vectors, which are
relatively inefficient and require replicating cells for gene
expression (Wilson et al. 1988). The production of an autologous
vaccine using retroviral vectors requires placing harvested tumor
in tissue culture before in vitro transduction, selection, and
isolation of the minority of cells in which gene transfer was
successful. Such a process is therefore lengthy, expensive, and
fraught with technical problems of establishing and maintaining
primary cell culture. These difficulties have forced investigators
to examine as alternative vaccine strategies the administration of
established allogeneic cytokine secreting tumor cell lines (Patel
et al. 1994), use of other vectors for gene transfer such as
adenoviral vectors (Drazan et al. 1994; Yang et al. 1995), or the
administration of cytokine-producing fibroblast cell lines along
with the autologous tumor cells (Lotze et al. 1994).
[0005] It is an object of the present invention to provide a method
for rapid production of autologous tumor vaccines which can be
completed within hours, for example in less than four hours,
permitting rapid treatment of tumor patients.
[0006] It is a further object of the invention to provide a method
for rapid production with autologous tumor vaccines which can be
applied to tumor cells in vivo without requiring surgical removal
of tumor material.
[0007] It is still a further object of the present invention to
provide compositions useful in the methods of the invention.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention an autologous
vaccine to tumor cells is produced by transducing the tumor cells
with a herpes simplex virus amplicon containing the gene for an
immunomodulating protein to provide transient expression of the
immunomodulating protein by the cells. The tumor cells may be
transduced with the herpes simplex amplicons ex vivo or in vivo.
Preferred immunomodulating protein used in the method of the
invention include cytokines such as RANTES (a chemokine),
interleukin-2 and GM-CSF, intracellular adhesion molecules such as
ICAM-1, and costimulatory factors such as B7.1.
[0009] A particularly important aspect of the present invention is
the fact that tumor cells may be readily transduced with a
combination of amplicons containing genes for two or more different
immunomodulating proteins, for example interleukin-2 and
interleukin 12 or RANTES and B7.1. This greatly facilitates the
production of multiply-transduced cells for multi-targeted
therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A-E summarize the results of studies on the
efficiency of gene transfer using HSV amplicons according to the
invention;
[0011] FIGS. 2A-C summarize the effects of irradiation on gene
transfer efficiency;
[0012] FIGS. 3A-C illustrate the tumoricidal activity splenocytes
from mice treated by intrasplenic injection with HSV amplicon
transduced tumor cells;
[0013] FIG. 4 summarizes the results of studies on the efficiency
of gene transfer using HSV amplicons according to the
invention;
[0014] FIG. 5 illustrates the effect of transduced cells on tumor
growth;
[0015] FIG. 6 illustrates the effects of transduced cells on
hepatectomy induced tumor formation;
[0016] FIG. 7 shows the amount of human ICAM-1 found in cell
culture supernatants for transduced cells;
[0017] FIG. 8 shows the adhesion index for adhesion of lymphocytes
to hepatoma cells transduced with HSVhlCAM1 versus controls;
[0018] FIG. 9 shows tumor growth in rats injected with hepatoma
cells transduced with HSV-hlCAM1 versus controls;
[0019] FIG. 10 shows tumor nodules formed in rat liver after
vaccination with radiated (nonviable) HSVhlCAM1-transduced hepatoma
cells followed by challenge with viable hepatoma cells;
[0020] FIG. 11 shows the structure of several HSV-immunomodulatory
protein amplicons in accordance with the invention;
[0021] FIGS. 12A-C show B7.1 expression in EL4 cells transduced
with HSVB7.1 versus controls;
[0022] FIGS. 13A and B show tumor size in intratumorally-treated
tumors and contralateral tumors, respectively; and
[0023] FIGS. 14A-D show CTL activity observed in splenocytes from
mice receiving HSVB7.1 or HSVrantes alone or in combination, versus
an HSVlac control.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Herpes simplex virus (HSV) is a DNA virus capable of rapidly
and efficiently infecting a wide variety of cell types (Leib &
Olivo, 1993; Geller & Federoff, 1991). Plasmid-based viral
vectors derived form HSV, termed amplicons, are easily constructed
and packaged into viral particles. The present invention uses
herpes simplex virus amplicons containing genes encoding for
immunomodulating proteins to transduce tumor cells with high
efficiency either ex vivo or in vivo.
[0025] As used herein, the term "immunomodulating proteins" refers
to a class of protein or peptide molecules which, when expressed by
a target cell, enhance the development of an immune response to
that cell. The term includes cytokines, including chemokines;
intercellular adhesion molecules, and costimulatory factors
necessary for activation of B or T cells.
[0026] Cytokines which may be used as immunomodulating proteins in
the invention include but are not limited to interleukins, such as
interleukin-2 (IL-2), interleukin-12 (IL-12); interferons, for
example gamma interferon (.gamma.-IFN), granulocyte macrophage
colony stimulating factor (GM-CSF) and tumor necrosis factor alpha
(TNF-.alpha.). The immunomodulating protein may also be a chemokine
such as RANTES, which is a .beta. or C-C chemokine, that functions
as a chemoattractant and activator for monocytes and macrophages.
Other C-C chemokines, such as MCP-1, -2, and -3, DC-CK1 and
MIP-1.alpha., -3.alpha., -.beta. and -3.beta., and .alpha. or C-X-C
chemokines such as IL-8, SDF-1.beta., SDF-1.alpha., GRO, PF-4 and
MIP-2 could also be used. Other chemokines useful in the method are
C family, for example lympotactin and CX3C family, for example
fractal kine, chemokines.
[0027] Intercellular adhesion molecules are transmembrane proteins
within the Ig superfamily that act as mediators of adhesion of
leukocytes to vascular endothelium and to one another. A preferred
intercellular adhesion molecule for use in the invention is ICAM-1
(also known as CD54), although other cell adhesion molecules that
binds to T or B cells, including ICAM-2 and -3 could also be
used.
[0028] Costimulatory factors which may be used as the
immunomodulatory protein in the present invention are cell surface
molecules other than an antigen receptor and its ligand that are
required for an efficient response of lymphocytes to an antigen.
Examples of such costimulatory factors include B7 (also known as
CD80).
[0029] HSV vector systems are efficient vehicles for gene transfer
to tumor cells. In experiments using HSVlac, over 50% of the target
cells are transduced using an MOI of 1. The efficiency of
transduction is further reflected by the high levels of IL-2
produced by HSVil2-transduced cells. Production of levels greater
than 1 .mu.g/10.sup.6 cells/24 hour was found, which is more than
30 times that achieved by retrovirally-produced vaccines (Patel et
al. 1994; Gransbacher et al. 1992). Additionally, the data from the
experiments with HSVil2-transduced human tumors demonstrate that
successful HSV-mediated gene transfer to freshly-isolated tumor
cells can also be used to produce genetically-engineered cells that
secrete significant amounts of bioactive IL-2.
[0030] A major advantage of using HSV vectors for gene transfer is
the ability to transduce non-replicating or slowly replicating
cells (Geller & Federoff, 1991). This physical property of HSV
translates into important clinical advantages. Freshly isolated
tumor cells may be transduced without the need to provide a tissue
culture environment conducive to cell replication. This advantage
is clearly demonstrated by the rapidity with which freshly
harvested human tumors were transduced in the current experiments.
Within 20 min, efficient gene transfer was produced, suggesting
that vaccines prepared by this method could be ready for
administration to patients within a single operative procedure.
That HSV-mediated gene transfer is independent of cell division and
is supported by a transduction efficiency that was not reduced by
prior irradiation of tumor cells. Thus, gene transfer to tumor
cells may be performed either before or after radiation according
to irradiation source availability, providing greater flexibility
in the clinical care of patients.
[0031] HSV-immunomodulatory protein amplicons and cells transduced
with such amplicons are able to confer specific antitumor immunity
that protects against tumor growth in vivo. The amplicons may be
introduced indirectly by administration of transduced cells into a
living organism or patient (mammalian, including human).
Alternatively, the HSV-immunomodulatory protein amplicon may be
introduced directly into tumor tissue (e.g. by peritumoral
injection) within a living organism or patient to generate an
antitumor immunity which leads to reduction in tumor size. This
latter approach is useful, for example, in the case of inoperable
tumors.
[0032] In accordance with the present invention,
HSV-immunomodulatory protein amplicons may be administered,
directly or indirectly, as individual species in order to provide a
therapeutic and/or prophylactic benefit. For example, as described
in the examples set forth herein, it has been determined that
administration of HSV-immunomodulatory protein amplicons encoding
cytokines such as IL-2, GM-CSF and RANTES, intercellular adhesion
molecules such as ICAM-1 and costimulatory factors such as B7.1 all
provide therapeutic benefit in the form of reduction or preexisting
tumor size, a vaccine-effect protecting against tumor growth after
a subsequent challenge, or both.
[0033] HSV-immunomodulatory protein amplicons may also be
administered, directly or indirectly, with other species of
HSV-immunomodulatory transduced cells or in combination with
cytokine therapy. Such administrations may be concurrent or they
may be done sequentially. Thus, in one embodiment of the invention,
HSV amplicons or cells transformed with an HSV amplicon encoding an
immunomodulatory protein are injected into a living organism or
patient, after a pre-treatment with a therapeutically effective
amount of a cytokine. Both HSVil2 and HSVgm-csf have been shown to
have increased efficacy when administered following a pretreatment
of .gamma.-IFN.
[0034] In another embodiment of the invention, populations of HSV
amplicons or cells transduced with HSV amplicons encoding a
plurality of different immunomodulatory proteins may be
coadministered to the subject. For example, populations of tumor
cells transduced with HSVil2 and HSVil12 may be coadministered. As
shown in the examples, such coadministration is somewhat more
effective than administration of individual populations.
Coadministration of cells expressing these two cytokines appears to
be most effective, however, when a single population of cells that
has been transduced with two different cytokine-encoding amplicons
is used. Such populations can be made either with separate
amplicons species, one encoding each immunomodulatory protein, or
which a single amplicon species encoding a plurality of
immunomodulatory proteins.
[0035] The ability to use separate amplicon species to transduce
cells to produce multiple immunomodulatory proteins is a major
advantage over prior methods, such as use of retroviral vectors,
for introduction of genetic material into target cells. In these
prior methods, the frequency of transduction is so low that no
reasonable percentage of cells would be transduced with multiple
genes if two or more separate viral vectors were used. Therefore,
therapies of this type require the preparation of a unique and
complicated construct containing multiple genes for each separate
form of multi-targeted gene therapy. Using the method of the
present invention, however, each target gene can be constructed in
its own amplicon, and multi-transduced cells produced by simply
mixing combinations of desired amplicon species.
[0036] Another example of the benefits of coadministration of a
plurality of HSV-immunomodulatory protein amplicons is seen with
the chemokine RANTES and the costimulatory factor B7.1. Although
peritumoral administration of either HSVB7.1 or HSVrantes resulted
in tumor rejection is a significant number of test subjects, when
HSV amplicons encoding these two immunomodulatory proteins are
combined, an increased number of animals reject the tumors.
[0037] Thus, the present invention provides a method for production
of an autologous vaccine to tumor cells comprising transducing the
tumor cells with a herpes simplex virus amplicon containing the
gene for an immunomodulatory protein to provide transient
expression of the immunomodulatory protein by the cells. The tumor
cells may be transduced with the herpes simplex amplicons ex vivo
or the may be transduced with the herpes simplex amplicons in vivo.
The tumor cells may be transduced with one or more species of
amplicon containing the genes for more than one kind of
immunomodulatory protein and expressing more than one kind of
immunomodulatory protein.
[0038] The invention also provides a method for inducing a
protective immune response to tumor cells in a patient (animal or
human) comprising the step of transducing the tumor cells with a
herpes simplex virus amplicon containing the gene for at least one
immunomodulatory protein to provide transient expression of the
immunomodulatory protein by the cells. The tumor cells may be
transduced with the amplicon ex vivo, in which case the method
further comprises the step of introducing the transduced tumor
cells into the patient. The tumor cells may also be transduced in
vivo by injecting the HSV amplicons into the site of the tumor
cells.
[0039] The invention also provides a method for production of an
autologous vaccine to tumor cells comprising transducing the tumor
cells with one or more species herpes simplex virus amplicon
containing the gene for an immunomodulatory protein and at least
one additional therapeutic gene to provide transient expression of
the immunomodulatory protein and the therapeutic gene product by
the cells. As noted from the specific examples in this application,
the additional gene may be a gene encoding a second
immunomodulatory protein. However, the therapeutic gene product is
not limited to immunomodulatory proteins, and may include any
protein or peptide which it is desirable to have expressed by
autologous tumor vaccine cells. Thus, for example, the gene might
code for an enzyme which is used for pro-drug conversion (for
example, thymidine kinase), or for a protein which promotes
apoptosis (BAX or BCLX.sub.s).
[0040] The invention also provides HSV amplicons which contain the
gene for one or more immunomodulatory proteins, and cells
transduced with such amplicons.
[0041] The invention will now be further described with reference
to the specific examples which follow. It should be understood,
however, that these are merely offered as examples and are not
intended to limit the scope of the invention. Thus, other
immunomodulatory proteins not specifically mentioned, and other
combinations of immunomodulatory proteins, including combination of
three or more immunomodulatory proteins may be used and are
considered to be with in the scope of the present invention as
defined in the claims of this application.
EXAMPLE 1
[0042] Herpes viral vectors: construction and packaging: The
replication defective HSV amplicon vector expressing human IL-2 was
constructed by directionally cloning the gene, excised from r-IL-2
(Saito et al. 1994) with Sac I and EcoRI, into HSV PrPUC (Bergold
et al. 1993) digested with the same enzymes. The HSV vector
expressing .beta.-galactosidase (HSVlac) has been previously
described (Geller & Breakefield, 1988). Both amplicon vectors
were packaged as previously described (Federoff, 1996; Geller &
Breakefield, 1988). HSVPrPUC contains the HSV immediate early 4/5
promoter, a multiple cloning site and SV40 A sequence and has been
described previously (Paterson & Everett, 1990; Johnson et al.
1992; Xu et al. 1994; Linnik et al. 1995; Bergold et al. 1993). The
RR1 cells used for packaging HSV amplicons were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing high glucose
(HG, 4.5 g/l), 10% FCS, 1% penicillin/streptomycin and 400 .mu.g/ml
of bioactive geneticin (G418, Gibco) at 37.degree. C., 5% CO.sub.2.
RR1 cells are BHK cells stably transfected with the HSV IE3 gene
and were obtained from Dr. Paul Johnson (Johnson et al. 1992). D30
EBA helper virus was prepared by growth on RR1 cells. D30EBA is a
strain 17 derived IE3 mutant deleted from codons 83 to 1236 and was
obtained from Dr. Roger Everett (Paterson & Everett, 1990). To
package amplicon vectors, 3.times.10.sup.6 RR1 cells were plated in
media containing 10% FCS and 4 h later were transfected by adding
40 .mu.l of Lipofectin (Gibco-BRL), waiting 5 min and then adding
the amplicon DNA solution dropwise (30 .mu.g at 1 .mu.g/.mu.l in
DMEM). Six hours later, plates were fed with media containing 5%
FCS. Approximately 20 h after transfection, D30 EBA virus in 50-100
.mu.l was added to achieve a multiplicity of infection MOI) of 0.2.
Five ml of compete media with 5% FCS were added to each plate after
1 h. Amplicon virus stocks were harvested 2 days later. After
overnight storage at -70.degree. C., fresh RR1 cells
(.times.10.sup.6 cells/60 mm plate) were infected with sonicated
and warmed (34.degree. C.) virus stock. Two days later, the stocks
were harvested and stored for subsequent use. HSVlac virus stocks
were titered by an expression assay. In brief, NIH 3T3 cells were
plated (2.times.10.sup.5 cells per well of 24 well plate) and
infected with increasing volumes of an HSV amplicon virus stock in
duplicate. Twenty-four h after infection, cells were fixed and
stained with the chromogenic substrate 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (X-gal) using standard methods (Geller &
Breakefield, 1988). The number of X-gal+ (blue) cells were counted.
Titers were expressed as the number of blue forming units/ml. The
D30 EBA helper virus in each stock was titered by plaque assay
.sub.GH RR1 cells, and HSVil2 was titered by a slot blot assay as
described previously (Geschwind et al. 1994). For slot blot
analysis, viral DNA was extracted from packaged virus by
phenol/chloroform twice, ethanol precipitated with single strand
calf thymus DNA as carrier, denatured at room temperature with 0.2
N NaOH, 0.5 M NaCl for ten minutes and loaded on nylon membrane
with a slot blot apparatus. The membrane was then baked for 2 hours
at 65.degree. C., and probed with a [.sup.32P]-labeled 435 bp Sspl
and Pvul fragment containing part of the .beta.-lactamase gene from
pBR322 (nucleotides 3733-4168). After stringent washing
(0.1.times.SSC twice for 15 minutes), blots were exposed to X-Ray
film and various timed exposures taken and densitometrically
scanned (LKB Ultrascan). Band densities between HSVlac and
HSVil2were compared and the titer of HSVil2 calculated from the
density relative to HSVlac given that this latter amplicon was
titered by an expression assay (blue forming units on NIH 3T3
cells). The titers of HSVil2 are expressed as particles/ml.
[0043] Titers of amplicon stocks: HSVlac titers were between
2.times.10.sup.6 blue forming units/ml as titered by expression and
X-gal histochemistry on NIH 3T3 cells. The HSVil2 titers,
determined by slot blot (described above), were between 0.8 and
2.times.10.sup.6 particles/ml. D30EBA titers in stocks ranged
between 5.times.10.sup.6 to 6.times.10.sup.7 plaque forming
units/ml. Recombination for wildtype revertants was monitored by
plaque assay on Vero cells and occurred at a frequency of
1.times.10.sup.6.
EXAMPLE 2
[0044] Murine hepatoma cells were transduced ex vivo using
amplicons prepared as in example 1. Murine HEPA 1-6 hepatoma cells
(ATCC, Rockville, Md.) were maintained in DMEM+HG+10% FCS. This is
a non-immunogenic hepatoma cell line (Engvall et al. 1977). Cells
were plated at either 2 or 10.times.10.sup.5 cells/well for all
virus expression studies. In some experiments, cells were
irradiated 2 h after plating and then infected with HSV amplicon
stocks. In other experiments, cells were irradiated 1 h after
infection with HSV amplicon stocks. Hepatoma cells were irradiated
at room temperature with a 6 mV Varian CL6-100 linear accelerator
at a dose-rate of 100 rads/min. To assess the rigidity of HSV
amplicon gene transfer, hepatoma cells were exposed to vector
stocks for either 20 or 60 min, washed extensively and cultured.
After an additional 48 h, cells were histochemically stained with
X-gal (HSVlac) or media assayed for IL-2 (HSVil2). In some
experiments tumor cell lysates were prepared by suspension in a
solution containing 0.15 M NaCl, 50 mM Tris, 1% NP-40, 4 mM NaF,
pH=8, and assayed for IL-2. Additionally, representative samples
were harvested 48 hours after treatment and viable tumor cells
counted.
[0045] The results of these experiments on the efficiency of gene
transfer according to the invention are summarized in FIGS. 1A and
B. As shown, both the HSVlac and HSV-IL-2 amplicon stocks gave
maximum transfer efficiencies at an MOI of 1 or greater. In HSVlac
infected cultures (FIG. 1 A), greater than 50% of the hepatoma
cells expressed the reporter gene, .beta.-galactosidase. Fewer
cells (30%) expressed .beta.-galactosidase when infected at an MOI
of 0.5. HSVil2 infected cultures (FIG. 1 B, MOI 1.0) secreted
1,200.+-.160 ng/10.sup.6 cells/24 hours. The immunoreactive IL-2
detected by ELISA was confirmed to be bioactive by the CML assay.
Each 50 pg of immunoreactive IL-2 was equivalent to approximately 1
unit of bioactivity. The extent of gene transfer was equivalent at
whether virus exposure was 20 or 60 minutes (FIGS. 1C and D),
indicating that virtually all infections HSV virions adsorb to
cells within 20 min. In addition, rapid gene transfer was not a
function of MOI, since expression was comparable in 20 and 60 min
exposures periods at both MOIs tested (0.5 and 1.0, FIGS. 1C and
D).
[0046] Although IL-2 secretory rates from HSVil2-infected hepatoma
cells were appreciable and in the range previously demonstrated to
be immunomodulatory, it was possible that additional IL-2 might
remain in an intracellular compartment. To address this issue, IL-2
measurements were made on infected cell lysates and compared with
the levels found in media conditioned by these cells (FIG. 1E). The
amount of IL-2 secreted in a 24 hour period was approximately
10-fold greater than the cellular content (media: 1400.+-.100
ng/10.sup.6 cells/24 h, lysate: 100.+-.9 ng/10.sup.6 cells/24 h),
suggesting the that the murine hepatoma cell efficiently secreted
the cytokine.
[0047] Because radiation treatment of tumor cells has been viewed
as an important part of producing non-dividing tumor vaccines, the
affects of the timing of cell irradiation relative to HSV infection
on gene transfer efficiency was investigated. As shown in FIGS. 2A
and B, irradiation prior to (broken lines) or just after (solid
lines) HSV infection produced similar gene transfer efficiencies.
Although there was a trend to higher gene transfer and expression
levels in cells infected prior to irradiation, this difference was
not significant. This trend towards higher gene transfer in cells
infected prior to irradiation was not due to a difference in cell
viability (Table 2). Of particular interest was the observation
that cells irradiated at different doses secreted levels of IL-2
that were comparable to non-irradiated cells (FIG. 2C). Moreover,
although irradiation affects cellular replication functions, it
appears to have no affect on the biogenesis of secreted IL-2.
EXAMPLE 3
[0048] Human tumor cells were transduced in vitro using an amplicon
containing the interleukin-2 gene produced in accordance with
Example 1. This study was performed with approval and under the
guidelines of the Institutional Review Board of the Memorial
Sloan-Kettering Cancer Center. Tumor biopsies of approximately 5
grams were obtained from four patients undergoing liver resection
for hepatobiliary malignancies. The patient characteristics are
listed in Table 1. All specimens were removed prior to any muscular
interruption or Pringle maneuvers. Histologic verification of tumor
was obtained:h all cases. Tumor specimens were immediately placed
in cold (4.degree. C.) RPMI-1640 for transport to the laboratory.
Each specimen was then minced into fine pieces and treated with
0.125% trypsin/0.125% EDTA in PBS without Ca.sup.41 or Mg.sup.11
for 5 min. The treated tumor was then teased apart and filtered
through a sterile 85 .mu.m nylon mesh into RPMI-1640 medium
(4.degree. C.) containing 0% human serum. Freshly-isolated cells in
suspension were irradiated at 10,000 rads at room temperature with
a 6-mV Varian CL6-100 linear accelerator at a dose-rate of 100
rads/min. Aliquots of 10.sup.6 tumor cells were then infected with
HSV amplicon stocks for 20 min. Aliquots non-irradiated cells were
treated similarly and served as controls. After exposure to virus,
tumor cells were washed twice and cultured at 3.degree. C., 5%
CO.sub.2. Forty-eight h after transduction, media from each well
was harvested and assayed for IL-2.
[0049] While no IL-2 was produced by any of these tumor cells prior
to HSVil2 infection (Table 1), infection with HSVil2 resulted in
IL-2 production by cells from all four of the tumors. In addition,
as with the murine hepatoma cell lines, efficiency of gene
expression was unaffected by irradiation with 10,000 rads. Finally,
it is noteworthy that the culture procedure, including the
radiation time, required less than 4 h, a time period that would be
commensurate with intraoperative autologous vaccine generation,
allowing potential reimplantation into exposed tumor sites during
the same operative procedure.
EXAMPLE 4
[0050] Media and cell lysate from HSVil2-transduced tumor cells
were harvested at 48 h and immediately frozen at -7.degree. C.
until assay. Immunoreactive IL-2 levels were determined by standard
sandwich ELISA (Biosource International, , Calif.). The total IL-2
produced in the forty-eight hours of cell culture were divided by
two to arrive at average production per twenty-four hours.
Interleukin-2 bioactivity in the supernatant or cell lysate was
also determined by assessing their ability to induce proliferation
of CTLL-2 cells in a standard cell mediated lympholysis (CML) assay
(Zier, 1982). Briefly, 5.times.10.sup.5 CTLL-2 cells were mixed
with serial dilutions of test samples and cultured at 37.degree.
C., 5% CO.sub.2. After 24 h, cell viability was measured by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylium bromide: 5 mg/ml)
incorporation. Recombinant human IL-2 (Chiron Corporation,
Emeryville, Calif.) is used as an internal standard. Units are
given as Cetus units.
EXAMPLE 5
[0051] To evaluation transduction efficiency, histochemical
analysis was performed on tumor cells transduced with HSVlac. The
cells were fixed at 48 h and histochemically stained with X-gal
(Dannenberg & Suga, 1981). Briefly, plates containing
transduced cells were fixed for 5 min with 1% glutaraldehyde,
washed 3 times with PBS, then incubated with X-gal solution (X-gal
(pH=7.4)[1 mg/ml] in PBS containing 2 mM MgCl.sub.2, 5 mM
KFc(CN).sub.6, and 5 mM K.sub.4Fc(CN).sub.6-3H.sub.2O). Total cells
and blue cells were counted and transduction efficiency expressed
as percent of total cells that were blue.
EXAMPLE 6
[0052] To determine the in vivo effects of tumor vaccines produced
using HSV-mediated gene transfer, syngeneic C57Bl/6j mice were
immunized using murine HEPA 1-6 hepatoma cells radiated with 10,000
rads and then exposed to HSVil2 at an multiplicity of infection
(MOI) of 1 for twenty minutes. The hepatoma cells (10.sup.6 cells)
were washed thrice with media after the twenty-minute viral
exposure and immediately injected either 1) subcutaneously, 2)
intraperitoneally, or 3) intrasplenicly. Animals were given either
a single injection or a daily injections on three consecutive days
(three injections total). As controls, animals were injected with
1) media (media-control), or 2) a similar number of radiated tumor
cells exposed to HSVlac (MOI=1), namely HSV carrying no cytokine
genes (HSV-control). Animals were sacrificed three weeks later and
splenocytes harvested for assessment of specific and non-specific
tumor cell kill by coincubation with hepatoma for assessment of
specific tumoricidal activity, K562 erythroblastic cell line for
assessment of NK activity, or a syngeneic colorectal tumor cell
line CO51 (ATCC; Rockville, Md.) for further assessment of
non-specific tumoricidal activity.
[0053] In order to determine if vaccinations with HSV-modified
tumor vaccine may alter in vivo response to tumor, C57Bl/6j mice
were immunized by intrasplenic injection with 1) 10.sup.6 radiated
tumor cells exposed to HSV carrying no cytokine genes
(HSV-control), or 2) 10.sup.6 radiated, IL-2 secreting hepatoma
cells. Three weeks later, the animals were injected intraportally
with 10.sup.6 replicating hepatoma cells to determine host response
to tumor. Three weeks after this tumor challenge, all animals were
sacrificed, and tumor growth in the liver assessed.
[0054] Splenocyte isolation was carried out as follows. Spleens
were harvested from pentobarbital anesthetized animals under
sterile conditions. Each spleen was placed in a petri dish
containing 10 ml of PBS, brought into the hood and transferred to a
new petri dish with 10 ml of RPMI+10% FCS+50 .mu.g/ml gentamicin.
Splenocytes were washed from the spleen by repeated injection with
media. Cells will be spun (300 g, 5 min) and resuspended in 5 ml of
red blood cell lysis solution (pH=7.4) (0.15 M NH.sub.4Cl, 1.0 mM
KHCO.sub.3, 0.1 mM Na.sub.2EDTA). After 1 min, solution were
diluted with 5 ml of RPMI, 10% FCS. Cells will be spun (300 g, 10
min) and washed 2.times. with media. Cells were then resuspended in
30 ml of RPMI+10% FCS+50 .mu.g/ml gentamicin+30 U/ml IL-2 (Chiron
Corp, Emeryville, Calif.) and kept in culture for 2 d prior to use.
Prior to assay, cells were spun, resuspended, counted and volume
adjusted to form the appropriate concentration.
[0055] The experiments summarized above examining the effects of
the route and number of injections on immunization, by the
subcutaneous route or intraperitoneal route, showed that three
injections were necessary for specific tumor immunity. However, for
the intrasplenic route, the hepatoma cell line tested elicited
specific immunity with a single injection (FIGS. 3A-C, FIG. 3A
presents data for HEPA 1-6 targets; FIG. 3B for K562 targets and
FIG. 3C for CO51 targets). This is the reason that the intrasplenic
route was used for the subsequent experiment examining the effects
of immunization in vivo tumor growth.
[0056] Mice pretreated by intrasplenic injection of either 1)
irradiated, HSV-treated tumor (HSV-control) or 2) irradiated,
HSVil2 treated tumor were subsequently challenged with intraportal
injection of 10.sup.6 replicating tumor cells to determine the
effects of immunization on tumor growth. Immunization using
irradiated IL-2 secreting tumor cells produced by HSV-mediated gene
transfer conferred in vivo antitumor effects. In animals treated
with HSV-control, seven of the ten animals challenged with 10.sup.6
hepatoma cells developed liver tumors, with mean tumor size being
1.5:0.4 gm (2% body weight). For animals pretreated with HSVil2
however, only one of ten animals developed tumor (p-0.02 vs.
HSV-control) with the size of that tumor being 0.2 gm (0.9% body
weight).
EXAMPLE 7
[0057] K562 or tumor cells served as targets in in vitro europium
release cytotoxicity assays. 5.times.10.sup.6 cells from culture
were washed 2.times. with Buffer 1 (pH=7.4) (50 mM Hepes, 93 mM
NaCl, 5 mM KCl, 2 mM MgCl.sub.3) then incubated in labeling
solution (K562: 30 ml EuCl.sub.3, 10 ml DTPA, 250 ml Dextran
Sulfate in Buffer 1; Hep: 35 ml EuCl.sub.3, 10 ml DTPA, 100 ml
Dextran Sulfate in Buffer 1) for 15 min in an ice bath, mixing
gently every 5 min. After 15 min. 20 ml of 100 mM CaCl.sub.2 was
added and the mixture incubated for 5 min. Nine ml of Repair Buffer
(Buffer 1, 2 mM CaCl.sub.2, 10 mM glucose) was added. Cells were
spun (200 g, 10 min) and washed 4.times. with Repair Buffer and
3.times. with media. Cells then were resuspended and plated at a
concentration of 5.times.10.sup.4 cells/100 ml per well in a 96
well U-Bottom plate (Costar Corp., Cambridge, Mass.) containing
effector cells in wells at effector to target ratios of 100:1,
50:1, 25:1, and 12.5:1. The plate was spun (10 g, 5 min), incubated
(4-6 hr, 37.degree. C.), and spun (100 g, 5 min). 20 .mu.l of
supernatant were transferred to a 96 well Flat Bottom plate (Costar
Corp) already containing 180 .mu.l Delfia Enhancement Solution
(Wallac Oy, Turku, Finland). The plate was read in a 1232 Delfia
Fluorometer (Wallac Oy). Maximum release was measure by lysing
cells with 1% Triton X. Percentage specific lysis was equal to
(experimental-spontaneous release)/(maximum release) spontaneous
release).times.100. Spontaneous release varied between 5 and 15% of
max.
EXAMPLE 8
[0058] HSV vectors containing the gene for either IL-2 (HSVil2) or
LacZ (HSVlac) were constructed in accordance with Example 1.
Twenty-five Fischer rats with bilateral flank squamous cell lung
tumors were randomized to receive left flank injections of either
HSVil2, HSVlac, saline or no injection on weeks 5, 7 and 9
post-implantation. Tumor volume was measured 3 times weekly for 6
weeks. There were no significant differences in tumor growth and
volume among the HSVlac, saline and non-injected groups. At 6
weeks, the HSVil2 group had and 81% reduction in mean tumor volume
in the injected left flank compared to controls. There was also an
88% reduction in mean tumor volume in the opposite, non-injected
flank, thus indicating that in vivo transfection of tumor by HSV
vectors containing cytokine genes is effective to stimulate a
systemic antitumor response. Four of the 5 HSVil2-treated animals
were clinical responders. Staining studies for LacZ revealed
transfection of tumor and surrounding stromal cells only on the
treated side.
EXAMPLE 9
[0059] Murine GM-CSF, human IL-2 and LacZ genes were cloned
directionally into HSVprPUC which contains the HSV immediate early
4/5 promoter, a multiple cloning site, and an SV40 A sequence, and
packaged as previously described by Geller et al. (1990). RR1 cells
(BHK cells stably transfected with the HSV IE3 gene) (20), along
with D30 EBA helper virus (a strain 17-derived IE3 mutant deleted
from codons 83 to 1236 and maintained in Dulbecco's modified Eagle
medium (DME) containing high glucose [HG, 4.5 g/liter], 10% FCS, 1%
penicillin/streptomycin, and 400 .mu.g/ml of bioactive geneticin
[G418; Gibco BRL, Gaithersburg, Md.] at 37.degree. C. and 5% CO2)
were used for packaging HSV amplicons. To package amplicon vectors,
3.times.106 RR1 cells were plated in media containing 0% FCS and
transfected 4 h later by adding 40 .mu.l of Lipofectin (Gibco),
waiting 5 min, and adding amplicon DNA solution dropwise (30 .mu.g
at 1 .mu.g/.mu.l in DME). 6 h later, plates were fed with media
containing 5% FCS. 20 h after transfection, D30 EBA virus in 50-100
.mu.l was added to achieve an moi of 0.2. 5 ml of complete media
with 5% FCS were added to each plate after 1 h, and amplicon virus
stocks were harvested 2 d later. After overnight storage at
70.degree. C., fresh RR1 cells (4.times.10.sup.6 cells/60 mm plate)
were infected with warmed (34.degree. C.), sonicated virus stock. 2
d later, stocks were harvested and stored for subsequent use.
HSVlac stocks were titered by an expression assay using NIH3T3
cells plated (2.times.105 cells/well of a 24-well plate) and
infected with increasing volumes of virus stock in duplicate. 24 h
after infection, cells were fixed and stained with
5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal) using standard
methods. The number of X-gal+ (blue) cells were counted, and titers
were expressed as the number of blue forming units/ml. The D30 EBA
helper virus in each stock was titered by plaque assay on RR1
cells, and the cytokine-containing vectors were titered by slot
blot analysis. For slot blot analysis, viral DNA was extracted
twice from packaged virus by phenol/chloroform,
ethanol-precipitated with single-strand calf thymus DNA as carrier,
denatured at room temperature with 0.2 N NaOH, 0.5 M NaCl for 10
min, and loaded on a nylon membrane with a slot blot apparatus. The
membrane was baked for 2 h at 65.degree. C. and probed with a
[32P]-labeled 435 bp Sspl and Pvul fragment containing part of the
.beta.-lactamase gene from pBR322 (nucleotides 3733-4168). After
stringent washing (0.1.times.SSC 2.times. for 15 min), blots were
exposed to x-ray film, and various timed exposures taken and
densitometrically scanned (LKB Ultrascan; Pharmacia LKB
Biotechnology Inc., Piscataway, N.J.). Band densities and the
titers of HSVil2 and HSV GM-CSF (expressed as particles/ml)
calculated from the density relative to HSVlac given that this
latter amplicon was titered by an expression assay, were compared.
HSVlac titers were between 1-2.times.106 blue forming units/ml as
titered by expression and X-gal biochemistry on NIH 3T3 cells. The
HSVil2 and HSVGM-CSF titers were between 1-2.times.106
particles/ml. The ratio of D30 EBA helper virus to amplicon varied
from 2:1 to 5:1 moi refers to the amplicon. Recombination for
wild-type revertants was monitored by plaque assay on Vero cells
and occurred at a frequency of 1.times.10.sup.6.
[0060] To assess in vitro production of cytokines, 10.sup.6
hepatoma cells per 2 ml were plated in six-well plates (Costar),
irradiated with 10,000 rad, and rested for 1 h. Cells were then
exposed to HSV-IL12, HSVGM-CSF, HSVlac, or Media for 20 min at
moi's of one and two and washed 2.times. with media. Cell culture
supernatants were harvested on days 1, 2, 4, and 7 post-exposure,
and cytokine levels were measured by ELISA (IL-2, R & D
Systems, Minneapolis, Minn.; GM-CSF, Genzyme Corp., Cambridge,
Mass.).
[0061] As shown in FIG. 4, control cells not exposed to cytokine
gene-containing vectors do not produce cytokines, and no cytokines
are seen immediately after transduction with HSVil2 and HSV gm-csf
and washing, indicating that proteins are not injected along with
the tumor cells. Cells exposed to HSVil2 or HSV gm-csf produce
nanogram quantities of these cytokines per 10.sup.6 cells after
vaccination, peaking on day 1 and decreasing thereafter.
EXAMPLE 10
[0062] Hepatoma cells in culture were irradiated with 10,000 rad,
allowed to rest for 1 h, then exposed to HSVil2, HSVGM-CSF, HSVlac
or media for 20 min at an moi of one. Cells were then washed
2.times. with media, and 10.sup.6 cells/200 .mu.l were injected
intrasplenically. An additional control group underwent injection
of media alone. On day 18, half the animals in each group received
either 5.times.104 U of .gamma.-IFN i.p. or normal saline for 3
day. On day 21, all animals received a challenge of 5.times.105
hepatoma cells/200 .mu.l intrasplenically followed by splenectomy
10 min later, allowing sufficient time for the hepatoma cells to
migrate to the liver. Animals were killed 20 d later, and tumor
nodules were counted. Additional animals were vaccinated, killed on
d 2 and 18 post-vaccination, and heart, lung, liver, kidney and
serum harvested for assessment of in vivo production of cytokines
by ELISA.
[0063] There was no significant effect on tumor growth as a result
of vaccination with irradiated cells or vaccination with irradiated
cells transduced with HSVlac compared to vaccination with medium
alone. As shown in FIG. 5, animals immunized with IL-2 or
GM-CSF-secreting cells or pretreated with .gamma.-IFN had
significantly fewer tumor nodules that all three control groups.
Combination treatment with IL-2 or GM-CSF secreting cells and
pretreatment with .gamma.-IFN was more effective than any single
treatment. Complete responses were seen in 8 of 11 IL-2 animals and
4 of 12 GM-CSF animals. No animal treated with .gamma.-IFN alone
was without tumor.
EXAMPLE 11
[0064] To assess the effects of vaccination on tumor growth
following a partial hepatectomy (shown to be immunosuppressive and
to accelerate the growth of hepatic tumors), animals were immunized
intrasplenically with hepatoma vaccines (HSVil2, HSVGM-CSF, HSVlac)
produced as above. On day 18, half the animals in each group
received either 5.times.104 U of IFN intraperitoneally, or normal
saline for 3 d. On day 21, all animals received a challenge of
5.times.105 hepatoma cells/200 .mu.l intrasplenically followed by
splenectomy 10 min later. Half the animals in each group underwent
70% partial hepatectomy 1 h after tumor injection. One control
group did not undergo vaccination or partial hepatectomy. Animals
were killed 18 d after tumor challenge, and nodules were counted.
In previous experiments, the number of surface nodules was shown to
correlate directly with tumor volume as measured by water
displacement.
[0065] As shown in FIG. 6, treatment with IL-2 or GM-CSF secreting
cell lines or pretreatment with .gamma.-IFN reduced the growth of
hepatectomy-induced tumors. The best results, comparable to the
results for animals with no hepatectomy, were obtained using a
combination of either IL-2 or GM-CSF secreting cell lines and
pretreatment with .gamma.-IFN.
EXAMPLE 12
[0066] To assess the effect of vaccination and IFN on splenocyte
and Kupfer cell (KC) function, animals underwent vaccination and
IFN treatment as described in Example 11, and splenocytes and KC
were harvested on day 21 post-vaccination. Tumoricidal activity was
assessed by mixing effectors with Europium-labeled tumor cells in
an in vitro assay. Labeled cells were plated at a concentration of
5.times.10.sup.4 cells/100 .mu.l per well in a 96-well U-Bottom
plate (Costar) containing effector cells in wells at varying
effector to target ratios. The plate was spun (200 rpm, 5 min),
incubated (4 h, 37.degree. C.), and respun (500 rpm, 5 min). 20
.mu.l of supernatant were transferred to a 96-well Flat Bottom
plate (Costar) already containing 180 .mu.l /well of Delfia
Enhancement Solution (Wallac Oy, Turku, Finland). The plate was
read in a 1232 Delfia Fluorometer (Wallac Oy). Maximum lysis was
measured by lysing cells with 1% Triton X. Percent specific lysis
is equal to experimental-spontaneous release/max.
release+spontaneous release.times.100. Spontaneous release varied
between 5 and 15% of max. Assays were performed in triplicate.
[0067] Vaccination with HSVlac or irradiated cells had no
significant effect on either KC function or splenocyte activity.
Splenocytes from animals vaccinated with HSVil2 or HSVgm-csf
exhibited significantly greater killing of targets than splenocytes
from control or .gamma.-IFN-treated animals. .gamma.-IFN did not
appear to affect splenocyte activity. KC from rats pretreated with
.gamma.-IFN had significantly greater killing of targets than KC
from controls. KC from rats vaccinated with HSVil2 also had
significantly greater killing of targets than KC from controls, but
not as great as KC from .gamma.-IFN-treated rats. Vaccines
secreting GM-CSF did not appear to affect KC activity.
EXAMPLE 13
[0068] Murine IL12m35, murine IL12m40, human IL2 and LacZ genes
were cloned directionally into HSV/PRPuc and packaged as previously
described. (Geller et al. (1990), Geller and Breakefield (1988),
Federoff (1996). To produce HSVm75, the m35 and m40, genes were
cloned directionally using appropriate restriction enzymes into
HSV/PRPuc separated by an IRES fragment. HSVPrPUC contains the HSV
immediate early 4/5 promoter, a multiple cloning site and SV40 A
sequence. The RR1 cells used for packaging HSV amplicons were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
high glucose (HG, 4.5 g/l), 10% FCS, 1% penicillin/streptomycin and
400 .mu.g/ml of bioactive geneticin (G418, Gibco) at 37 C. 5%
CO.sub.2. RR1 cells are BHK cells stably transfected with the HSV
IE3 gene and were obtained form Dr. Paul Johnson. Johnson et al.
(1992). D30 EBA helper virus was prepared by growth on RR1 cells
D30EBA is a strain 17 derived IE3 mutant deleted from codons 83 to
1236 and was obtained from Dr. Roger Everett. Paterson and Everett
(1990). To package amplicon vectors, 3.times.10.sup.4 RR1 cells
were plated in media containing 10% FCS and 4 h later were
transfected by adding 40 .mu.l of Lipofectin (Gibco-BRL), waiting 5
min and then adding the amplicon DNA solution dropwise (30 .mu.g at
1 .mu.g/.mu.l in DMEM). Six hours later, plates were fed with media
containing 5% FCS. Approximately 20 h after transfection, D30 EBA
virus in 50-100 .mu.l was added to achieve a multiplicity of
infection (MOI) of 0.2. Five ml of complete media with 5% FCS were
added to each plate after 1 h. Amplicon virus stocks were harvested
2 days later. After overnight storage at -70 C, fresh RR1 cells
(4.times.10.sup.6 cells/60 mm plate) were infected with sonicated
and warmed (34 C) virus stock. Two days later, the stocks were
harvested and stored for subsequent use. HSVlac virus stocks were
titered by an expression assay. In brief, NIH 3T3 cells were plated
(2.times.10.sup.5 cells per well of 24 well plate) and infected
with increasing volumes of an HSV amplicon virus stock in
duplicate. Twenty-four h after infection, cells were fixed and
stained with the chromogenic substrate 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (X-gal) using standard methods. (Geller and
Breakefield (1990)) The number of X-gal+ (blue) cells were counted.
Titers are expressed as the number of blue forming units/ml. The
D30 EBA helper virus in each stock was titered by plaque assay on
RR1 cells, and HSVil2 was titered by a slot blot assay. For slot
blot analysis, viral DNA was extracted from packaged virus by
phenol/chloroform twice, ethanol precipitated with single strand
calf thymus DNA as carrier, denatured at room temperature with 0.2
N NaOH, 0.5 M NaCl for ten minutes and loaded on nylon membrane
with a slot blot apparatus. The membrane was then baked for 2 hours
at 65 C, and probed with a [.sup.32P]-labeled 435 bp Sspl and Pvul
fragment containing part of the .beta.-lactamase gene from pBR322
(nucleotides 3733-4168). After stringent washing (0.1.times.SSC
twice for 15 minutes), blots were exposed to X-Ray film and various
timed exposures taken and densitometrically scanned (LKB
Ultroscan). Band densities between HSVlac and HSVil2 were compared
and the titer of HSVil2 calculated from the density relative to
HSVlac given that this latter amplicon was titered by an expression
assay (blue forming units on NIH 3T3 cells). The titers of HSVil2
are expressed as particles/ml.
[0069] HSVlac titers were between 2.times.10.sup.6 blue forming
units/ml as titered by expression and X-gal histochemistry on NIH
3T3 cells. The HSVil2 titers, determined by slot blot (described
above), were between 0.8 and 2.times.10.sup.4 particles/ml. D30EBA
titers in stocks ranged between 5.times.10.sup.6 to
6.times.10.sup.7 plaque forming units/ml. Recombination for
wildtype revertants was monitored by plaque assay on Vero cells and
occurred at a frequency of 1.times.10.
EXAMPLE 14
[0070] Efficiency of transduction with HSVm35+HSVm40 vs. HSVm75 was
assessed by measuring in vitro production of cytokines. To assess
in vitro production of cytokines, 10.sup.6 hepatoma cells per 2 ml
were plated in 6-well plates (Costar), radiated with 10,000 rads
and rested for 1 h. Cells were then exposed to HSVm35, HSVm40,
HSVm35+HSVm40, HSVm75, HSVlac or Media for 20 min at a multiplicity
of infection (MOI) of between 1 and 4 and then washed 2.times. with
media. Cell culture supernatants were harvested on days 1, 2, 4, 5
and 7 post-exposure, and cytokine levels were measured by ELISA
specific for the heterodimeric protein.
[0071] Control cells not exposed to cytokine gene-containing
vectors do not produce cytokines. IL12 production was not detected
in cells transduced with either HSVm35 or HSVm40 alone.
Transduction using 2 vectors produced levels of IL12 similar to
transduction using a single vector carrying both genes, which peak
on day 1 and decrease thereafter
EXAMPLE 15
[0072] To determine the effect of vaccination on hepatic tumor
growth, hepatoma cells in culture were radiated with 10000 rads,
rested for 1 h, then exposed to HSVil2, HSVm75, HSVil2+HSVm75,
HSVm35+HSVm40, or media for 20 min at an MOI of 1-4. Cells were
washed 2.times. with media, and 10.sup.6 cells/200 .mu.l were
injected intrasplenically. An additional group received 2
populations of cells: 10HSVil2-transduced cells and
10HSVm75-transduced cells. On day 21, all animals received a
challenge of 5.times.10.sup.5 hepatoma cells/200 .mu.l
intrasplenically followed by splenectomy 10 min later. This model
produces uniform numbers of tumors within the liver that can be
counter on day 20 after tumor challenge. Operative procedures were
performed under pentobarbital anesthesia (25 mg/kg i.p.) via
midline abdominal incision. Animals were sacrificed 20 days later
and tumor nodules counted.
[0073] Animals immunized with cells transduced by HSVm35+HSVm40,
HSVm75 or HSVil2 had significantly fewer tumor nodules than
control. Vaccination with 2 tumor cell populations, one secreting
IL2 and one secreting IL12, was more effective than vaccination
with a single population of cytokine-secreting cells. Vaccination
with a single population of cells transduced by both HSVil2 and
HSVm75 was the most effective treatment, significantly better than
any single treatment or two population treatment.
EXAMPLE 16
[0074] To access the effect of vaccination on splenocyte and KC
Function, animals underwent vaccination as described in Example 15,
and splenocytes and KC were harvested on day 21 post-vaccination
and assessed for tumoricidal activity by standard Europium-release
assay. Briefly, tumoricidal activity was assessed by mixing
effectors with Europium-labeled tumor cells in vitro. Labeled cells
were plated at a concentration of 5.times.10.sup.4 cells/100 .mu.l
per well in a 96 well U-Bottom plate (Costar) containing effector
cells in wells at varying effector to target ratios. The plate was
spun (200 rpm, 5 min), incubated (4 hr, 37.degree. C.), and spun
(500 rpm, 5 min). 20 .mu.l of supernatant were transferred to a 96
well Flat Bottom plate (Costar Corp) already containing 180
.mu.l/well of Delfia Enhancement Solution (Wallac Oy, Turku,
Finland). The plate was read in a 1232 Delfia Fluorometer (Wallac
Oy). Maximum lysis was measured by lysing cells with 1% Triton
X-100. Percent specific lysis is equal to (experimental-spontaneous
release)/(max. release+spontaneous release).times.100. Spontaneous
release varied between 5 and 15% of max. Assays were performed in
triplicated.
[0075] Splenocytes from animals vaccinated by either HSVil2 or
HSVm75 had significantly greater killing of targets than
splenocytes from animals vaccinated by radiated cells. Splenocytes
from animals vaccinated by cells transduced by HSVm75 and HSVil2
had significantly greater killing of targets than splenocytes from
animals vaccinated by a single cytokine at an effector to target
ratio of 100:1.
[0076] KC from rats vaccinated with HSVil2 or HSVm75 had
significantly greater tumoricidal activity than KC from controls
(p<0.05) at effector to target ratio of 50:1. KC from animals
vaccinated by cells transduced by HSVm75 and HSVil2 had
significantly greater killing of targets than KC from animals
vaccinated by a single cytokine at an effector to target ratio of
100:1.
EXAMPLE 17
[0077] Human ICAM-1 and E. coli .beta.-galactosidase cDNA was
directionally cloned into HSVPrPuc (HSVhicam1 and HSVlac
respectively) which contains the HSV immediate early 4/5 promoter,
a multiple cloning site, and an SV40 A sequence, and packaged as
previously described in Example 1. RR1 cells (BHK cells stably
transfected with the HSV IE3 gene), along with D30 EBA helper virus
(a strain 17-derived IE3 mutant deleted from codons 83 to 1236 and
maintained in Dulbecco's modified Eagle medium (DME) containing
high glucose [HG, 4.5 g/liter], 10% FCS, 1%
penicillin/streptomycin, and 400 .mu.g/ml of bioactive geneticin
[G418: Gibco BRL, Gaithersburg, Md.] at 37.degree. C. and 5% CO2)
were used for packaging HSV amplicons.
[0078] To package amplicon vectors, 3.times.10.sup.6 RR1 cells were
plated in media containing 10% FCS and transfected 4 hours later by
adding 40 .mu.l of Lipofectin (Gibco), waiting 5 min, and adding
amplicon DNA solution dropwise (30 .mu.g at 1 .mu.g/.mu.l in DME).
Six hours later, plates were fed with media containing 5% FCS.
Twenty hours after transfection, D30 EBA virus in 50-100 .mu.l was
added to achieve a multiplicity of infection (MOI) of 0.2. Five ml
of complete media with 5% FCS were added to each plate after 1
hours, and amplicon virus stocks were harvested 2 days later. After
overnight storage at 70.degree. C., fresh RR1 cells
(4.times.10.sup.6 cells/60 mm plate) were infected with warmed
(34.degree. C.), sonicated virus stock. Two days later, stocks were
harvested and stored for subsequent use. HSVlac stocks were titered
by an expression assay using NIH3T3 cells plated (2.times.10.sup.5
cells/well of a 24-well plate) and infected with increasing volumes
of virus stock in duplicate. Twenty four hours after infection,
cells were fixed and stained with 5-bromo-4-chlor-3-indolyl
Beta-D-galactosidase (X-gal) using standard methods. The number of
X-gal+ (blue) cells were counted, and titers were expressed as the
number of blue forming units/ml.
[0079] The D30 EBA helper virus in each stock was titered by plaque
assay on RR1 cells, and the cytokine-containing vectors were
titered by slot blot analysis. For slot blot analysis, viral DNA
was extracted twice from packaged virus by phenol/chloroform,
ethanol-precipitated with single-strand calf thymus DNA as carrier,
denatured at room temperature with 0.2 N NaOH, 0.5 M NaCl for 10
minutes and loaded on a nylon membrane with a slot blot apparatus.
The membrane was backed for 2 hours at 64.degree. C. and probed
with a [32p]-labeled 435 by Sspl and Pvul fragment containing part
of the Beta-lactamase gene from pBR322 (nucleotides 3733-4168).
After stringent washing (0.1.times.SSC 2.times. for 15 min), blots
were exposed to x-ray film, and various timed exposures taken and
densitometrically scanned (LKB Ultroscan: Pharmacia LKB
Biotechnology Inc., Piscataway, N.J.). Band densities and the
titers of HSVhicam1 (expressed as particles/ml calculated from the
density relative to HSVlac given that this later amplicon was
titered by an expression assay, were compared. HSVlac titers were
between 1-2.times.10.sup.6 blue forming units/ml as titered by
expression and X-gal biochemistry on NIH3T3 cells. The HSVhicam1
titers were between 1-2.times.10.sup.6 particles/ml. The ratio of
D30 EBA helper virus to amplicon varied from 2:1 to 5:1. MOI refers
to the amplicon. Recombination for wild-type revertants was
monitored by plaque assay on Vero cells and occurred at a frequency
of 1.times.10.
EXAMPLE 18
[0080] The tumor cell line Morris Hepatoma McA-RH7777 (ATCC CRL
1601) was maintained in culture (DME, 6.25% FCS, 20% Horse serum, 2
mM L-Glutamine) and periodically implanted into buffalo rat flanks
to ensure tumorigenicity. This cell line was tested to be free of
mycoplasma and viral infection.
[0081] Hepatoma cells from culture were radiated with 10,000 rats
and rested for 1 hour. Cells were then exposed to HSVhicam1, HSVlac
or nothing at an MOI of 1 for 20 minutes at 37.degree. C. Cells
were then washed with media twice and maintained in culture until
analysis. To assess the cell surface expression of hlCAM1, cells
were harvested at 1, 2, 5 and 7 days after transduction and washed
twice with HBSS containing 10 mM HEPES. Separate aliquots of cells
were then incubated on ice for 20 minutes with anti-human ICAM-1
(Clone MEM111, Caltag, Burlingame, Calif.) and anti-rat ICAM-1
(Clone IA29, Caltag, Burlingame, Calif.) antibodies conjugated to
PE or FITC. Additional aliquots of cells were incubated with
isotype controls (Caltag, Burlingame, Calif.) to account for
nonspecific binding of antibodies. Cells were then analyzed with a
FACscanner (Becton Dickinson) for the presence of human and rat
ICAM.
[0082] With PE labeling, greater than 90% of normal untreated rat
hepatoma cells expressed rat ICAM on the cell surface with mean
fluorescent intensities ranging from 200 to 288. There was no
difference in rat ICAM expression between transduced and non
transduced cells. Cells transduced with HSVlac or nothing had no
detectable surface human ICAM-1. Flow cytometric analysis of rat
hepatoma cells transduced with HSVhicam1 showed that a 20 minute
exposure, at an MOI=1 resulted in high level expression of human
ICAM on the surface of tumor cells. Peak cell surface positivity
for human ICAM-1 was found 24 hours after transduction and tapered
off by 1 week (Percent of cells positive for hlCAM1 was 25%, 16%,
and 9% on days 1, 2 and 5 post transduction). Mean fluorescent
intensity of human ICAM-1 on HSVhicam1-transduced cells was 450,
271, and 124 on days 1, 2 and 5 respectively. On day 7 post
transduction with HSVhicam1, cell viability was limited, but
approximately 4% of viable cells were positive for surface
hlCAM1.
[0083] FIG. 7 illustrates the quantitation of soluble human ICAM
found in cell culture supernatants of transduced cells. No soluble
human ICAM was detectable in supernatants of cells transduced with
HSVlac or nothing. Levels in supernatants of transduced cells
peaked at 48 hours after transduction and approached the level of
detection by day 7.
EXAMPLE 19
[0084] To determine if ICAM-1 transduced hepatoma cells bound
lymphocytes more avidly, a modification of previously reported
adhesion assays (Miki, et al., 1993) was performed. Briefly,
hepatoma cells were radiated with 10,000 rads, exposed to
HSVhicam1, HSVlac or nothing for 20 minutes at 37.degree. C. and
washed with media twice. Cells were then plated in nearly confluent
monolayers in 96 well plates. Splenocytes were harvested from
normal Buffalo rats one day prior to each assay and cultured
overnight in Complete RPMI (0.01 mM NEAA, 1 mM NaPyruvate, 2 mM
L-Glutamine, 50 .mu.M 2-ME, Pcn/Step) containing 10% FCS, 50 U/ml
IL2 (Chiron Corporation, Emeryville, Calif.), 5 .mu.g/ml Con A
(Sigma, St. Louis, Mo.), and 50 .eta.g/ml PMA (Phorbol 12-Myristate
13-Acetate) (Sigma, St. Louis, Mo.). On the day of the assay,
nonadherent splenocytes were harvested at a concentration of
10.sup.6/cc, and labeled with MTT (5 mg/ml PBS) in a v:v ratio of
3:1 (splenocytes:MTT). Splenocytes were incubated with MTT for 6
hours at 37.degree. C. with gentle agitation every 30 minutes.
Labeled lymphocytes were then plated at a concentration of
10.sup.6/100 .mu.l in the wells containing the hepatoma targets.
The cells were then co-incubated at 37.degree. C. for 30 minutes.
Nonadherent splenocytes were then gently washed off with PBS.
Adherent lymphocytes were lysed with DMSO and read by
spectrophotometry at 570 .eta.m. Representative wells were used to
count the number of hepatoma targets present for each experimental
group. Additional labeled splenocytes were plated at varying
concentrations, lysed and read by spectrophotometry in order to
create a standard curve for the number of splenocytes per well. An
adhesion index calculated as the number of adherent lymphocytes per
hepatoma target cell and the mean of 8 wells was recorded.
[0085] In order to determine if hlCAM1 gene transfer would alter
lymphocyte binding by tumor, an in vitro lymphocyte binding assay
was used. There was a significant increase in the number of
adherent lymphocytes per hepatoma target cell in wells containing
HSVhicam1-transduced cells compared to lac-transduced and untreated
cell (FIG. 8). This doubling of lymphocyte binding was
statistically significant (p<0.05).
EXAMPLE 20
[0086] In order to determine if transduction of hepatoma cells with
the ICAM-1 gene altered in vitro growth properties, cell
proliferation assays were performed. Replicating rat hepatoma cells
were exposed to HSVhicam1, HSVlac or nothing at an MOI of 1 for 20
minutes at 37.degree. C. Cells were then plated in 24 well plates
at a concentration of 10.sup.4 viable cells/ml/well. Cells were
harvested by trypsin disaggregation at 1, 2 and 4 days after
plating and counted by trypan blue exclusion. The mean count of 8
wells per time point was compared. Cells transduced with HSVhicam1
grew similarly in culture compared to HSVlac-transduced cells and
untreated cells, indicating that changes in in vivo tumor growth
(see Example 21) cannot be accounted for by changes in intrinsic
growth rate of the modified tumor.
EXAMPLE 21
[0087] Male Buffalo rats (Harlan Sprague Dawley) were housed 2 per
cage in a temperature (22.degree. C.) and humidity controlled
environment and were given water and standard rat chow (PMI Mills,
St. Louis, Mo.) ad libitum. They were maintained in 12 hour
light/dark cycles. All surgical procedures were carried out through
a midline laparotomy under i.p. pentobarbital (50 mg/kg)
anesthesia. For major abdominal operations, 3 ml of 0.9% saline was
administered i.p. for resuscitation post operatively. All animals
received care under approved protocols in compliance with Memorial
Sloan-Kettering Cancer Centers Institutional Animal Care and Use
Committee guidelines.
[0088] Tumorigenicity Experiments
[0089] In order to analyze the effects of ICAM-1 overexpression on
the in vivo growth characteristics of hepatoma cells, flank
tumorigenicity experiments were performed. Animals (n=5 per group)
were randomized to receive subcutaneous left flank injections of
10.sup.6 viable rat hepatoma cells transduced with HSVhicam1,
HSVlac or nothing (MOI of 1). On the opposite right flank, all
animals received subcutaneous flank injections of 10.sup.6 viable
non-transduced cells. Animals were weighed and tumors measured with
external calipers twice weekly. Tumor measurements were made in two
perpendicular dimensions and averaged. Tumor volume was calculated
using the equation 4/3.pi.m.sup.3.
[0090] There was significantly decreased tumor growth in the left
flanks of animals injected with HSVhicam1-transduced cells compared
to controls (FIG. 9). Tumor volumes at the termination of the
experiment were compared. Tumors transduced with HSVhicam1 had a
significantly (p<0.05) smaller volume (1,397+/-1296 mm.sup.3)
compared to tumors transduced with HSVlac (7,109+/-2118 mm.sup.3)
and untreated tumors (13,556+/-3354 mm.sup.3). On the contralateral
untreated side, all groups had progressive tumor growth that was
not significantly different.
[0091] Immunohistochemistry
[0092] In order to assess potential immunologic mechanisms of tumor
regression, Immunohistochemical analysis of cell infiltrates in
tumors was carried out. Animals from additional tumorigenicity
experiments had tumors excised at 1 week and 3 weeks after
injection of cells (n=5 per time point) and placed immediately in
10% buffered formalin. Twenty four hours later, tumors were
embedded in paraffin using standard techniques. Five .mu.m sections
were made. Hematoxylin and Eosin staining was performed using
standard techniques. The following antibodies were used for
immunohistochemical analysis; mouse monoclonal anti-rat CD4
(IgG.sub.1, clone W3/25, Serotec, Oxford, England), mouse
monoclonal anti-rat CD8 (IgG.sub.1, clone OX-8, Caltag, Burlingame,
Calif.), and mouse monoclonal anti-rat 1-A (IgG.sub.1, clone OX-6,
Serotec, Oxford, England) which recognizes rat MHC Class II. The
secondary antibody used was Biotinylated anti-mouse IgG, rat
adsorbed (Vector, Burlingame, Calif.). Slides used for CD4 and CD8
staining were pretreated with 1 mM EDTA (ph 8) in a microwave for
10 minutes. For MHC II staining slides were pretreated for 10
minutes with a 0.05% Protease XXIV (Sigma, St. Louis, Mo.) in
Tris-HCl buffer, ph 7.6. Endogenous peroxide was then quenched with
a five minute incubation in 3% H.sub.2O.sub.2. After washed with
PBS, slides are then placed in 0.05% bovine serum albumin for 1
minute. Slides were then dried and whole horse serum applied at a
1:20 dilution in 2% bovine serum albumin and incubated for 10
minutes. Serum was then suctioned off and 150 .mu.l of primary
antibody applied. The primary antibody was incubated for 16-18
hours at 4.degree. C. in a humidity chamber. After PBS washed,
secondary antibody was applied to the slides at a 1:500 dilution in
1% bovine serum albumin and incubated for 60 minutes at room
temperature in a humidity chamber. Slides were then washed in PBS
and peroxidase-conjugated streptavidin was applied at a dilution of
1:500 in 1% bovine serum albumin. Slides were then washed with PBS
and transferred to a bath of 0.06% diaminobenzidine (Sigma, St.
Louis, Mo.) for 5 to 15 minutes Slides were then washed in water
and decolorized with 1% acid alcohol and blue in ammonia water.
Dehydration with ethanol and xylene were carried out with standard
techniques and slides were mounted with Permount (Fisher,
Pittsburgh, Pa.) mounting media.
[0093] A single pathologist blinded to the experiment reviewed
slides and graded them in the following way. Tumor cells were
assessed for the presence or absence of MHC II staining. The degree
of tumor infiltration with MHC II staining non-tumor cells was
graded from 1 to 4. The degree of infiltration of tumors with the
total amount of CD4 and CD8 positive lymphocytes was graded from 1
to 4. The relative percentage of CD4 and CD8 positive cells was
then assessed and expressed as a ratio. Rat splenic tissue was used
as a positive control for each experiment.
[0094] The amount of infiltration of tumors with both CD4 and CD8
positive T lymphocytes did not differ between treatment groups at 1
and 3 weeks. The ratio of CD4 to total CD4 and CD8 positive T cells
did not differ between groups at 1 week, but at 3 weeks, there was
a significant increase in this ratio in the HSVhicam1-treated
animals compared to HSVlac and untreated animals (0.42 vs. 0.25 and
0.24, p<0.05). There was no significant difference in the degree
of infiltration of tumors with MHC II staining immune cells between
treatment groups at 1 and 3 weeks. Tumor cells did not stain
positively for MHC II expression in any case.
EXAMPLE 22
[0095] In order to determine whether previous exposure to ICAM-1
transduced hepatoma cells would protect against future challenges
with the parental tumor, vaccination experiments were performed.
Whole tumor cell vaccines were prepared as follows. Rat hepatoma
cells were radiated with 10,000 rads, exposed to HSVhlCAM1, HSVlac
or nothing at an MOI of 1 for 20 minutes at 37.degree. C. and
washed twice with media. Animals (n=19 per group) were then
randomized to receive either cell type by intrasplenic injections
of 10.sup.6 cells in 200 .mu.l of media on day 1. Control animals
received 200 .mu.l of media intrasplenically. Three weeks after
vaccination, animals were challenged with 5.times.10.sup.5
replicating hepatoma cells by intrasplenic injection. After 10
minutes, a splenectomy was performed in all animals. Three weeks
after challenge, animals were sacrificed and liver surface tumor
nodules counted. Body weights were recorded and grooming habits
monitored twice a week throughout the experiment.
[0096] Throughout the experiment, there was no difference in weight
gain in all treatment groups and all animals maintained normal
grooming habits. As illustrated in FIG. 10, there was significantly
decreased uptake and growth of hepatic metastases in animals
vaccinated with HSVhicam1 cells compared to all controls
(p.ltoreq.0.05). There was no difference between animals vaccinated
with HSVlac-transduced cells, radiated cells alone or media.
EXAMPLE 22
[0097] The coding sequences for human B7.1 or human RANTES were
cloned into the polylinker region of the pHSVPrPUC plasmid. To form
the HSV-B7.1 amplicon, pBJ.huB7.1 plasmid (kindly provided by Dr.
Lewis Lanier, DNAX, Palo Alto, Calif.) was digested with HindIII
and was filled in to generate a blunt end and. Subsequently, this
plasmid was digested with Xbal. A The HindIII blunt/Xbal fragment
encoding the for the human B7.1 cDNA was gel purified and used as
insert in the ligation with the vector. The HSV amplicon vector
pHSVPrPUC plasmid was digested with EcoRI and filled in with Klenow
to make a blunt end, followed by Xbal digestion. The
EcoRIblunt/Xbal vector fragment was gel purified and ligated with
the insert. The constructed amplicon plasmid was analyzed for the
orientation of the coding sequences of huB7.1 with respect to the
HSV-1 IE4/5 promoter, and the amplicon used in the generation of
the HSVB7.1 amplicon virus.
[0098] To form the HSV-RANTES amplicon, SK+pBS-RANTES plasmid
(kindly provided by Dr. Tom Schall, ChemoCentryx, Mountain View,
Calif.) was partially digested with Kpnl followed by digestion with
Xbal. The Kpnl/Xbal fragment encoding human RANTES cDNA was gel
purified and used as insert in the ligated to the HSV amplicon
vector pHSVPrPUC plasmid digested with Kpnl and Xbal. Orientation
of the coding sequences for huRANTES with respect to the HSV-1
IE4/5 promoter was verified, and the amplicon used in the
generation of the HSVrantes amplicon virus. The HSV amplicons are
shown schematically in FIG. 11.
EXAMPLE 23
[0099] Amplicon DNA was packaged into HSV-1 particles by
transfecting 5 .mu.g of plasmid DNA into RR1 cells with
lipofectamine as recommended by the manufacturer (GIBCO-BRL).
Following incubation for 24 hours the transfected monolayer was
superinfected with the HSV strain 17, IE3 deletion mutant virus
D30EBA (Paterson et al., 1990) at a multiplicity of infection (MOI)
of 0.2. Once cylopathic changes were observed in the infected
monolayer, the cells were harvested, freeze-thawed, and sonicated
using a cup sonicator (Misonix, Inc.). Viral supernatants were
clarified by centrifugation at 5000 g for 10 min prior to repeat
passage on RR1 cells. This second viral passage was harvested as
above and concentrated overnight by ultracentrifugation in a 25%
sucrose gradient as previously described (Tung et al., 1996). Viral
pellets were resuspended in PBS (Ca2+ and MG2+ free) and stored at
-80.degree. C. for future use. Stocks were titered for helper virus
by standard plaque assay methods. Amplicon titers were determined
as follows: NIH 3T3 cells were plated in a 24-well plate at a
density of 1.times.10.sup.3 cells/well and infected with the virus.
Twenty-four hours after viral infection the monolayers were washed
twice in PBS and either fixed with 4% paraformaldehyde and stained
by X-gal histochemistry (5 mM Potassium Ferricyanide; 5 mM
Potassium Ferrocyanide; 0.02% NP-40; 0.01% sodium deoxycholic acid;
2 mM MgCl.sub.2 and 1 mg/ml Xgal dissolved in PBS) or harvested for
total DNA using lysis buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 25
mM EDTA, 0.5% SDS) followed by subsequent phenol-chloroform
extraction and ethanol precipitation. PCR was performed on
duplicate samples using primers corresponding to the
.beta.-lactamase gene present in the amplicon plasmid under the
following conditions: 94.degree. C., 2 min; then 20, 23 or 26
cycles of 94.degree. C. (30 sec), 58.degree. C. (30 sec), followed
by 72.degree. C. (7 min). PCR products from early and late cycles
were run on a 1% ethidium bromide gel, and the 450 bp band
intensities were assessed using the FOTDODYNE FOTO/ECLIPSE.TM.
system (Fotodyne, Inc, Hartland, Wis.) and COLLAGE.TM. Image
Analysis Software. HSVB7.1 and HSVrantes titers were estimated by
comparison with HSVlac virus as standards. Plaque forming unit
(pfu/ml) and amplicon (bfu/ml) titers obtained form these
measurements, were used to calculate amplicon titer and thus
standardize experimental viral delivery. Amplicon titer in the
different virus preparations ranged from 1-10.times.10.sup.7 bfu/ml
and the helper titers were in the range of 5-15.times.10.sup.7
pfu/ml.
EXAMPLE 24
[0100] EL4 cells were infected in vitro either with HSVB7.1, or
HSVlac amplicon virus at an MOI of 0.5-1-5 pfu per cell.
Specifically, 10.sup.6 ED4 cells were adsorbed with the amplicon
virus in a volume of 0.5 ml at 37.degree. C., 5% CO.sub.2 for 4
hours. At the end of 4 hours, 0.5 ml of fresh ID-10 medium was
added and incubation continued for another 12 hours. The infected
cells were harvested at the end of 16 hours and 10.sup.6 cells in
0.1 ml of chilled PBS were stained with 1:10 diluted phycocrythrin
(PE) conjugated anti-B7.1 antibody (anti-CD80-PE, Becton-Dickinson)
for 30 minutes at 4.degree. C. Uninfected EL4 cells (as negative
control), or EL4 stably expressing B7.1 (EL4-B7.1 as cells as
positive control) were also stained simultaneously with the
anti-CD80 PE antibody. The stained cells were analyzed by flow
cytometry using an EPICS flow cytometry instrument.
[0101] Control uninfected EL4 cells or EL4 cells infected with
HSVlac were negative for the B7.1 expression (FIGS. 12A&B). In
contrast, approximately 95% of EL4 cells infected at an estimated
MOI of 1 stained positively for B7.1 expression (FIG. 12C). On a
per cell basis, HSV-B7.1 amplicon virus infected cells showed
significantly higher levels of B7.1 expression than those observed
for EL4-B7.1 cell line established by retroviral transduction.
Expression of B7.1 in HSVB7.1 infected cells was maintained for up
to 60 hours post-infection.
EXAMPLE 25
[0102] The bioactivity of HSV vector-expressed B7.1 was studied in
an in vitro proliferation assay. Murine T-cells were enriched using
a murine T-cell enrichment column (R&D Systems). 10.sup.5
T-cells were incubated in the presence of 5.times.10.sup.4
gamma-irradiated stimulator cells. EL4 or CHO cells infected with
HSV-B7.1, were used as stimulator cells. Retrovirally transduced
EL4-B7.1, or CHO-B7.1 (kindly provided by Dr. Peter Linsley) were
used as a positive control for B7.1 expression and parental EL4 or
CHO cells served as a negative controls. Stimulator cells were
irradiated to a total of 7500 rads using a Cesium-gamma source.
Either anti-CD3 antibody (2C11) used as a (2C11)1:50 dilution of
the hybridoma cell culture supernatant, or phorbol myristate (10
ng/ml) with ionophore (0.1 ng/ml) were added and the cells were
cultured for 3 days at 37 OC in 5% CO2 incubator. To assay for
proliferative responses in these stimulated cells, triplicate
cultures were labeled for 16 hours with 1 .mu.Ci .sup.3H-thymidine
(NEN, 2 Ci/mmol, 1 .mu.Ci/0.2 ml, final concentration). Cells were
harvested on glass fiber filters using a cell harvester (Packard
Instruments) and the incorporated 33H-radioactivity was measured
using a beta-counter (Packard Instruments). Results are expressed
as the mean (of triplicate cultures)+/-with the standard deviation.
T-cell proliferation index (normalized cpm) was determined as the
ratio of .sup.3H-thymidine incorporated in the stimulated versus
unstimulated control cultures.
[0103] When stimulated with anti-CD3 antibody (2C11) or a mixture
of phorbol myristate acetate (PMA) and ionophore to provide `signal
one,` a significant proliferative response was observed for T-cells
cocultured with HSVB7.1, but not HSVlac infected stimulator cells.
The B7.1-dependent T-cell proliferative response observed with the
HSVB7.1 infected EL4 cells was comparable to that seen with the
retrovirally transduced control stimulator cells EL4-B7.1 or
CHO-B7.1.
EXAMPLE 26
[0104] EL4 cells were infected with HSVrantes or HSVlac amplicon at
an MOI of 1. EL4 cells at 1.times.10.sup.6 were adsorbed with the
amplicon virus in a volume of 0.5 ml at 37.degree. C., 5% CO.sup.2
for 4 hours, then 0.5 ml of fresh medium was added and incubation
continued for another 20 hours. Cell culture supernatants were
harvested at the end of 24 hours and supernatants tested for RANTES
in a sandwich ELISA using anti-RANTES antibody (R&D Systems)
for RANTES capture of RANTES in the culture supernatants and
biotinylated anti-RANTES (R&D Systems) for detection followed
by alkaline phosphatase-conjugated avidin. Para-Nitrophenyl
phosphate was used as a substrate and absorbance developed color
read at 405 nm was read in a BIORAD ELISA reader. Serial two fold
dilutions of standard recombinant human-RANTES (R&D Systems) in
duplicates were run in parallel to quantitate the amount of RANTES
in the culture supernatant of infected cells.
[0105] In uninfected EL4 cells or cells transduced with HSVlac, no
detectable RANTES secretion was observed in culture supernatants.
Cells infected with HSVrantes at an MOI of 0.5 produced 3.1 ng of
RANTES/ml/24 hours/10.sup.6 cells. The observed levels of RANTES
were higher than those measured in pooled G418 selected
retrovirally transduced EL4-RANTES cells which secreted RANTES at a
concentration of 1.45 ng/ml/24 hours/106 cells.
EXAMPLE 27
[0106] Adult C57BL/6 (H-2.sup.b) female mice (8 weeks old) were
obtained from Charles River Laboratories (MA) and maintained at the
Animal Facility, University of Rochester Medical Center. The mice
were handled under an approved laboratory animal handling and care
protocol. Mice (6-12 per group) were shaved on the dorsal side of
the hind limb and were inoculated subcutaneously (sc) with 10.sup.6
viable EL4 cells infected ex vivo with HSVB7.1, HSVrantes, or
HSVlac amplicon virus, or with uninfected EL4 cells. In some
experiments 10.sup.6 uninfected EL4 cells were inoculated sc.
contralaterally on the other hind limb at the same time. Tumor
growth was measured every 2-3 days using a caliper and size
reported in millimeters diameter (mm). Animals were sacrificed when
the tumor size reached 22-23 mm.
[0107] The results of these experiments on growth of HSV-infected
EL4 cells an on contralateral EL4 tumors are summarized in Table 3
and FIGS. 13A and 13B. On Day 20, complete regression of tumor was
noted in 3/6 mice inoculated with EL4-HSVB7.1 transduced EL4 cells.
Two of six mice inoculated with HSVrantes-transduced EL4 cells also
showed initial tumor growth followed by complete regression in mice
inoculated with EL4 infected with HSV-RANTES. When EL4 cells were
infected with both HSVB7.1 and HSVrantes, 5/6 mice showed complete
regression following initial tumor growth. Control EL4 cells or EL4
cells infected with the HSVlac vector grew tumor in 100% of the
mice (6/6). Stably transduced EL4-B7.1 cells showed no evidence of
tumor growth in all mice by day 20. These results support the
conclusion that HSVB7.1 or HSVrantes amplicon infected cells were
rejected due to a tumor specific immune response.
[0108] Similar results were observed in the experiment to evaluate
whether inoculation of HSV vector transduced cells would inhibit
growth of concurrent contralaterally inoculated parental
non-transduced EL4 cells. In 3/5 mice, regression of ex vivo
HSVB7.1 infected EL4 tumor was concordant with regression of the
contralateral EL4 tumor (FIG. 13A). Both HSVlac infected EL4 cells
and contralateral parental EL4 cells developed into tumor in 5/5
animals studied (FIG. 13B). These data support the conclusion that
systemic tumor specific immunity to parental EL4 cells had
developed in a subset of mice inoculated with EL4-HSVB7.1
transduced EL4 cells.
[0109] To test the efficacy of HSVB7.1 and HSVrantes on
pre-established tumors using intratumoral inoculation of the HSV
amplicons, 10.sup.6 viable EL4 cells were inoculated sc. on the
dorsal side of the shaved hind limb and the tumor allowed to grow
to a size of 5-6 mm (6-7 days). At this point the mice were grouped
and either HSVB7.1, HSVrantes, HSVB7.1+HSVrantes, or HSVlac
amplicon virus diluted in PBS to a concentration of
2.times.10.sup.6 amplicon containing virus particles in 50 .mu.l
was inoculated intratumorally (10-12 mice/group). Control animals
with pre-established EL4 tumor received only the diluent PBS. A
second inoculation of the HSV amplicons was given on day 14, and
the tumor growth was measured every 2-3 days. Tumors were allowed
to grow to a maximal size of 22-23 mm size at which point the
animals were sacrificed.
[0110] Complete tumor regression was observed in 17/26 mice
injected with HSVB7.1 vector alone, in 11/22 mice injected with
HSVrantes, and in 23/26 mice injected with the combination of
HSVB7.1 and HSVrantes. Results of three independent experiments
yielded similar results as summarized in Table 4.
[0111] To determine whether regression of tumor correlated with the
development of systemic and memory T-cell immunity, mice
manifesting complete tumor regression were rechallenged with
parental EL4 cells in the on the other hind limb contralateral to
the primary inoculation. All mice the rechallenged with parental
EL4 cells shows no tumor growth (Table 4), thus indicating that
tumor specific immunity was established by the antecedent direct
intratumoral delivery of HSVB7.1 and/or HSVrantes into
pre-established tumors.
EXAMPLE 28
[0112] To examine the induction of CTL responses in mice transduced
intratumorally with the HSV amplicon vectors, splenocytes from the
mice of Example 27 were evaluated. Spleens were harvested from
C57BL/6 mice which had been inoculated with EL4 cells and injected
intratumorally with either HSVB7.1 or HSVrantes alone or in
combination. Control splenocytes were obtained from mice which were
inoculated intratumorally with HSVlac virus or mice with PBS
diluent alone. Splenocytes were prepared according to standard
procedures and red blood cells lysed using AKC lysis buffer. To
obtain cylolytic T-cells, splenocyte cell suspensions
(2.times.1066/ml in RP-10) were cultured together with
gamma-irradiated (7500 rads) EL4 cells (0.5.times.10.sup.6
cells/ml) in a 25 cm.sup.2 flask at 5% CO.sub.2, 37.degree. C. for
6 days. These in vitro cocultured splenocytes were then used as
effector cells in the CTL assays. On the day of assay, EL4 target
cells were washed with PBS and resuspended in RP10 medium 0.1 ml)
at a concentration of 1.5-2.times.10.sup.6 cells/ml and
Na.sup.51CrO.sub.4 (NEN, 100 .mu.Ci; stock concentration 1 mCi/ml)
added for 90 minutes at 37.degree. C. These cells were washed three
times with PBS, resuspended in 1 ml RP-10 and viable cell count
taken using a haemocytometer. .sup.51Cr-labeled target cells
(10.sup.4 cells/0.1 ml) were added to the wells of a V-shaped 96
well plate, and three-fold serial dilutions of effector cells were
made in triplicate, resulting in final effector-target cell ratios
(E:T ratios) of 100:1, 33:1, 11:1, 3:1, and 1:1. Spontaneous
release of radioactivity from labeled target cells was measured by
culturing the target cells with medium alone in six wells. Total
release of radioactivity was determined by lysing the target cells
with 2% Triton-X 100 detergent. Plates were spun at 1K for 2
minutes and incubated for 4 hrs at 37.degree. C., 5% CO.sub.2. The
plates were then centrifuged at 2K for 4 minutes and half of the
culture supernatant (100 .mu.l) was counted for .sup.51Cr release
in a gamma counter (Packard Instrument). Mean values are calculated
for the replicate wells and the results are expressed as % specific
lysis according to the formula: 1 % specific lysis = 100 .times.
experimental counts - spontaneous counts total counts - spontaneous
counts
[0113] The mean spontaneous release for virus-infected and
uninfected controls averaged between 10 to 20% of the total
counts.
[0114] Significant specific CTL activity was seen in splenocytes
from mice receiving HSVB7.1 or HSVrantes alone or in combination
(FIGS. 14A-D) CTL responses were only seen in mice in which EL4
tumor regressed after direct delivery of the HSVB7.1 and/or
HSVrantes amplicons into pre-established tumor. Levels of CTL
activity were greater in mice which received both the HSVB7.1 and
HSVrantes vectors. The highest levels of CTL activity were observed
in mice which had been rechallenged with the parental EL4
cells.
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[0145]
1TABLE 1 Efficiency of IL-2 Secretion from Human Tumor Cells
Transduced with HSVil2 Diagnosis MOI Patient Clinical Histologic
Radiation 0 0.5 1 2 1 Met Colorectal Ca Moderately differentiated
No 0 580 .+-. 40 6400 .+-. 200 10700 .+-. 70 Adenocarcinoma Yes 0
334 .+-. 4 5500 .+-. 100 10500 .+-. 50 2 Hepatoma Clear cell No 0
2100 .+-. 10 2600 .+-. 20 5900 .+-. 70 adenocarcinoma Yes 0 580
.+-. 40 2490 .+-. 40 6450 .+-. 70 3 Gallbladder Ca Moderately
differentiated No 0 ND 12500 .+-. 700 ND Adenocarcinoma Yes 0 ND
4800 .+-. 100 ND 4 Hepatoma Poorly differentiated No 0 ND 17500
.+-. 500 ND Adenocarcinoma Yes 0 ND 19300 .+-. 600 ND ND, not
determined. Values are mean levels of samples transduced in
quadruplicate .+-. SEM. Levels are pg/10.sup.6 cells/24 hours.
[0146]
2TABLE 2 Effect of timing of irradiation and HSV exposure on cell
viability. Hepatoma cells were either exposed to radiation (10,000
rads) followed by a 20 minute exposure to HSV (Rad/HSV), or exposed
to HSV for 20 minutes followed by irradiation (10,000 rads)
(HSV/Rad). Cells (5 .times. 10.sup.5 cells were then plated and
left in culture for 48 hours. Non-viable cells were washed off
before harvesting cells for counting. In addition, harvested cells
were verified to be viable by trypan blue exclusion. Comparisons
were by student's t-test. Rad/HSV HSV/Rad MOI (.times.10.sup.5
cells) (.times.10.sup.5 cells) P 0 2.1 .+-. 0.1 1.8 .+-. 0.2 0.1
0.5 2.0 .+-. 0.1 1.8 .+-. 0.2 0.2 1.0 1.8 .+-. 0.1 1.5 .+-. 0.1
0.2
[0147]
3TABLE 3 Tumor growth of EL4 cells infected ex vivo with HSV
amplicons. EL4 cells were infected in vitro with HSV amplicon virus
and maintained in culture for 8 hours. 10.sup.6 viable HSV amplicon
infected EL4 cells were inoculated s.c. in mice and tumor presence
at one month recorded. # of mice with tumor/ HSV amplicon # of mice
inoculated HSV-B7.1 3/6 HSV-RANTES 4/6 HSV-B7.1 & HSV-RANTES
1/6 HSV-LacZ 6/6
[0148]
4TABLE 4 Intratumoral delivery of HSV amplicons into
pre-established EL4 tumors. EL4 cells were inoculated s.c. in mice
and tumors allowed to develop to a 5-6 mm diameter. HSV amplicon
virus was inoculated in two doses, on days 7 and 14, and tumor
growth monitored and recorded after one month. The values reported
correspond to the number of mice with tumor,/total number of mice.
Primary Tumor Tumor Growth HSV amplicon Growth Following
Rechallenge Experiment # 1 HSVB7.1 1/4 0/3 HSVB7.1 + HSVrantes 0/4
0/4 HSVlac 4/4 Experiment # 2 HSVB7.1 4/10 0/6 HSVrantes 5/10 0/5
HSVB7.1 + HSVrantes 1/10 0/9 HSVlac 5/5 Experiment # 3 HSVB7.1 4/12
0/4 HSVrantes 6/12 0/4 HSVB7.1 + HSVrantes 2/12 0/6 HSVlac 5/5
* * * * *