U.S. patent application number 14/774962 was filed with the patent office on 2016-01-21 for newcastle disease viruses and uses thereof.
This patent application is currently assigned to ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, MEMORIAL SLOAN KETTERING CANCER CENTER. Invention is credited to James Allison, Adolfo Garcia-Sastre, Peter Palese, Jedd D. Wolchok, Dmitriy Zamarin.
Application Number | 20160015760 14/774962 |
Document ID | / |
Family ID | 51527979 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160015760 |
Kind Code |
A1 |
Palese; Peter ; et
al. |
January 21, 2016 |
NEWCASTLE DISEASE VIRUSES AND USES THEREOF
Abstract
Described herein are chimeric Newcastle disease viruses
engineered to express an agonist of a co-stimulatory signal of an
immune cell and compositions comprising such viruses. Also
described herein are chimeric Newcastle disease viruses engineered
to express an antagonist of an inhibitory signal of an immune cell
and compositions comprising such viruses. The chimeric Newcastle
disease viruses and compositions are useful in the treatment of
cancer. In addition, described herein are methods for treating
cancer comprising administering Newcastle disease viruses in
combination with an agonist of a co-stimulatory signal of an immune
and/or an antagonist of an inhibitory signal of an immune cell.
Inventors: |
Palese; Peter; (New York,
NY) ; Garcia-Sastre; Adolfo; (New York, NY) ;
Zamarin; Dmitriy; (New York, NY) ; Allison;
James; (Houston, TX) ; Wolchok; Jedd D.; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
MEMORIAL SLOAN KETTERING CANCER CENTER |
New York
New York |
NY
NY |
US
US |
|
|
Assignee: |
ICAHN SCHOOL OF MEDICINE AT MOUNT
SINAI
New York
NY
Memorial Sloan Kettering Cancer Center
New York
NY
|
Family ID: |
51527979 |
Appl. No.: |
14/774962 |
Filed: |
March 4, 2014 |
PCT Filed: |
March 4, 2014 |
PCT NO: |
PCT/US14/20299 |
371 Date: |
September 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61782994 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
424/135.1 ;
424/153.1; 424/173.1; 424/93.6; 435/235.1; 435/325; 435/349;
435/367 |
Current CPC
Class: |
C12N 7/00 20130101; A61P
35/00 20180101; C12N 2760/18132 20130101; A61K 39/3955 20130101;
A61K 2039/505 20130101; C12N 2760/18133 20130101; C07K 2317/76
20130101; C07K 16/2827 20130101; C07K 16/2818 20130101; A61K 35/768
20130101; C07K 16/2878 20130101; C12N 2760/18121 20130101; C12N
2760/18143 20130101; C12N 2760/18122 20130101 |
International
Class: |
A61K 35/768 20060101
A61K035/768; C12N 7/00 20060101 C12N007/00; C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395 |
Goverment Interests
[0002] This invention was made, in part, with Government support
under award numbers 5T32CA009207-35 and HHSN26620070010C from the
National Institutes of Health. The Government has certain rights in
this invention.
Claims
1. A chimeric Newcastle disease virus (NDV), comprising a packaged
genome which encodes an agonist of a co-stimulatory receptor of an
immune cell, wherein the agonist is expressed by the virus.
2. A chimeric NDV, comprising a packaged genome which encodes an
antagonist of an inhibitory receptor of an immune cell, wherein the
antagonist is expressed by the virus.
3. The chimeric NDV of claim 1 or 2, wherein the packaged genome
encodes a mutated F protein and the mutated F protein is expressed
by the virus.
4. The chimeric NDV of claim 1 or 2, wherein the immune cell is a T
lymphocyte or natural killer (NK) cell.
5. The chimeric NDV of claim 1, wherein the co-stimulatory receptor
is glucocorticoid-induced tumor necrosis factor receptor (GITR),
OX40, CD27, CD28, 4-1BB or CD40.
6. The chimeric NDV of claim 2, wherein the inhibitory receptor is
cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed
cell death protein 1 (PD1), B and T-lymphocyte attenuator (BTLA),
killer cell immunoglobulin-like receptor (KIR), lymphocyte
activation gene 3 (LAG3), or T-cell membrane protein 3 (TIM3).
7. The chimeric NDV of claim 1, wherein the agonist is an antibody
that specifically binds to the co-stimulatory receptor.
8. The chimeric NDV of claim 1, wherein the agonist is a ligand
that specifically binds to the co-stimulatory receptor.
9. The chimeric NDV of claim 1, wherein the agonist is an antibody
specifically binds to GITR, OX40, CD27, CD28, 4-1BB or CD40.
10. The chimeric NDV of claim 7 or 9, wherein the antibody is a
monoclonal antibody or single-chain Fv.
11. The chimeric NDV of claim 8, wherein the ligand is GITRL,
CD40L, CD137L, OX40L, CD70, or ICOSL.
12. The chimeric NDV of claim 2, wherein the antagonist is an
antibody that specifically binds to the inhibitory receptor.
13. The chimeric NDV of claim 2, wherein the antagonist is an
antibody that specifically binds to CTLA-4, PD-1, BTLA, KIR, LAG3,
or TIM3.
14. The chimeric NDV of claim 2, wherein the antagonist is a
soluble receptor of a ligand of the inhibitory receptor.
15. The chimeric NDV of claim 2, wherein the antagonist is an
antibody that specifically binds to a ligand of the inhibitory
receptor.
16. The chimeric NDV of claim 2, wherein the antagonist is an
antibody that specifically binds to PDL1, PDL2, B7-H3, B7-H4, HVEM,
or Gal9.
17. The chimeric NDV of claim 14, wherein the soluble receptor is
the extracellular domain of PD1, BTLA, KIR, LAG3 or TIM3.
18. The chimeric NDV of claim 12, 13, 15 or 16, wherein the
antibody is a monoclonal antibody or sc-Fv.
19. A pharmaceutical composition comprising the chimeric NDV of
claim 1, 5, 7, 8, 9 or 11 and a pharmaceutically acceptable
carrier.
20. A pharmaceutical composition comprising the chimeric NDV of
claim 2, 6, 12, 13, 14, 15, 16 or 17 and a pharmaceutically
acceptable carrier.
21. A method for producing a pharmaceutical composition, the method
comprising: a. propagating the chimeric NDV of any one of claims 1,
2, 5 to 9 or 11 to 17 in a cell line that is susceptible to a NDV
infection; and b. collecting the progeny virus, wherein the virus
is grown to sufficient quantities and under sufficient conditions
that the virus is free from contamination, such that the progeny
virus is suitable for formulation into a pharmaceutical
composition.
22. A method for producing a pharmaceutical composition, the method
comprising: a. propagating the chimeric NDV of any one of claims 1,
2, 5 to 9 or 11 to 17 in an embryonated egg; and b. collecting the
progeny virus, wherein the virus is grown to sufficient quantities
and under sufficient conditions that the virus is free from
contamination, such that the progeny virus is suitable for
formulation into a pharmaceutical composition.
23. A cell line comprising the chimeric NDV of any one of claims 1,
2, 5 to 9 or 11 to 17.
24. An embryonated egg comprising the chimeric NDV of any one of
claims 1, 2, 5 to 9 or 11 to 17.
25. A method for treating cancer, comprising administering to a
subject in need thereof a pharmaceutical composition comprising the
chimeric NDV of any one of claims 1, 5, 7, 8, 9, or 11.
26. A method for treating cancer, comprising administering to a
subject in need thereof a pharmaceutical composition comprising the
chimeric NDV of any one of claims 2, 6, 12, 13, 14, 15, 16 or
17.
27. The method of claim 25, wherein the packaged genome of the
chimeric NDV encodes a mutated F protein with a mutated cleavage
site, so that the mutated F protein is expressed by the virus.
28. The method of claim 26, wherein the packaged genome of the
chimeric NDV encodes a mutated F protein with a mutated cleavage
site, so that the mutated F protein is expressed by the virus.
29. The method of claim 25 further comprising administering to the
subject a second agonist of a co-stimulatory receptor of an immune
cell.
30. The method of claim 26, further comprising administering to the
subject an agonist of a co-stimulatory receptor of an immune
cell.
31. The method of claim 26 further comprising administering to the
subject a second antagonist of an inhibitory receptor of an immune
cell.
32. The method of claim 25 further comprising administering to the
subject an antagonist of an inhibitory receptor of an immune
cell.
33. A method for treating cancer, comprising administering to a
subject in need thereof an NDV and an agonist of a co-stimulatory
receptor of an immune cell.
34. A method for treating cancer, comprising administering to a
subject in need thereof an NDV and an antagonist of an inhibitory
receptor of an immune cell.
35. The method of claim 33, wherein the NDV is a chimeric NDV and
wherein the chimeric NDV comprises a packaged genome encoding a
cytokine which is expressed by the virus.
36. The method of claim 34, wherein the NDV is a chimeric NDV,
which comprises a packaged genome encoding a cytokine, wherein the
cytokine is expressed by the virus.
37. The method of claim 33, wherein the NDV is a chimeric NDV,
which comprises a packaged genome encoding a second agonist of a
co-stimulatory receptor of an immune cell or an antagonist of an
inhibitory receptor of an immune cell, wherein the second agonist
or antagonist is expressed by the virus.
38. The method of claim 34, wherein the NDV is a chimeric NDV,
which comprises a packaged genome encoding an agonist of a
co-stimulatory receptor of an immune cell or a second antagonist of
an inhibitory receptor of an immune cell, wherein the agonist or
second antagonist is expressed by the virus.
39. The method of claim 35 or 36, wherein the cytokine is IL-2,
IL-7, IL-15 or IL-21.
40. The method of claim 33, wherein the co-stimulatory receptor is
GITR, OX40, CD27, CD28, 4-1BB or CD40.
41. The method of claim 34, wherein the inhibitory receptor is
CTLA-4, PD1, BTLA, KIR, LAG3, or TIM3.
42. The method of claim 33, wherein the agonist is an antibody that
specifically binds to the co-stimulatory receptor.
43. The method of claim 33, wherein the agonist is a ligand that
specifically binds to the co-stimulatory receptor.
44. The method of claim 33, wherein the agonist is an antibody
specifically binds to GITR, OX40, CD27, CD28, 4-1BB or CD40.
45. The method of claim 42 or 44, wherein the antibody is a
monoclonal antibody or single-chain Fv.
46. The method of claim 43, wherein the ligand is CD137L, OX40L,
CD40L, GITRL, CD70, or ICOSL.
47. The method of claim 34, wherein the antagonist is an antibody
that specifically binds to the inhibitory receptor.
48. The method of claim 34, wherein the antagonist is an antibody
that specifically binds to CTLA-4, PD1, BTLA, KIR, LAG3, or
TIM3.
49. The method of claim 34, wherein the antagonist is a soluble
receptor of a ligand of the inhibitory receptor.
50. The method of claim 34, wherein the antagonist is an antibody
that specifically binds to a ligand of the inhibitory receptor.
51. The method of claim 34, wherein the antagonist is an antibody
that specifically binds to PDL1, PDL2, B7-H3, B7-H4, HVEM, or
Gal9.
52. The method of claim 51, wherein the soluble receptor is the
extracellular domain of PD1, BTLA, KIR, LAG3 or TIM3.
53. The method of claim 47, 48, 50 or 51, wherein the antibody is a
monoclonal antibody or scFv.
54. The method of claim 33 or 34 further comprising administering
adoptive T lymphocytes.
55. The method of any one of claims 33 to 38, 40 to 44 or 46 to 52,
wherein the cancer is melanoma, colorectal cancer, breast cancer,
ovarian cancer or renal cell cancer.
56. The method of any one of claims 33 to 38, 40 to 44 or 46 to 52,
wherein the cancer is malignant melanoma, malignant glioma, renal
cell carcinoma, pancreatic adenocarcinoma, malignant mesothelioma,
lung adenocarcinoma, lung small cell carcinoma, lung squamous cell
carcinoma, anaplastic thyroid cancer or head and neck squamous cell
carcinoma.
57. The method of any one of claims 33 to 38, 40 to 44 or 46 to 52,
wherein the subject is a human.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 61/782,994, filed on Mar. 14, 2013, which is
incorporated by reference herein in its entirety.
1. INTRODUCTION
[0003] Described herein are chimeric Newcastle disease viruses
engineered to express an agonist of a co-stimulatory signal of an
immune cell and compositions comprising such viruses. Also
described herein are chimeric Newcastle disease viruses engineered
to express an antagonist of an inhibitory signal of an immune cell
and compositions comprising such viruses. The chimeric Newcastle
disease viruses and compositions are useful in the treatment of
cancer. In addition, described herein are methods for treating
cancer comprising administering Newcastle disease viruses in
combination with an agonist of a co-stimulatory signal of an immune
cell and/or an antagonist of an inhibitory signal of an immune
cell.
2. BACKGROUND
[0004] Newcastle Disease Virus (NDV) is a member of the Avulavirus
genus in the Paramyxoviridae family, which has been shown to infect
a number of avian species (Alexander, D J (1988). Newcastle
disease, Newcastle disease virus--an avian paramyxovirus. Kluwer
Academic Publishers: Dordrecht, The Netherlands. pp 1-22). NDV
possesses a single-stranded RNA genome in negative sense and does
not undergo recombination with the host genome or with other
viruses (Alexander, D J (1988). Newcastle disease, Newcastle
disease virus--an avian paramyxovirus. Kluwer Academic Publishers:
Dordrecht, The Netherlands. pp 1-22). The genomic RNA contains
genes in the order of 3'-NP-P-M-F-HN-L-5', described in further
detail below. Two additional proteins, V and W, are produced by NDV
from the P gene by alternative mRNAs that are generated by RNA
editing. The genomic RNA also contains a leader sequence at the 3'
end.
[0005] The structural elements of the virion include the virus
envelope which is a lipid bilayer derived from the cell plasma
membrane. The glycoprotein, hemagglutinin-neuraminidase (HN)
protrudes from the envelope allowing the virus to contain both
hemagglutinin (e.g., receptor binding/fusogenic) and neuraminidase
activities. The fusion glycoprotein (F), which also interacts with
the viral membrane, is first produced as an inactive precursor,
then cleaved post-translationally to produce two disulfide linked
polypeptides. The active F protein is involved in penetration of
NDV into host cells by facilitating fusion of the viral envelope
with the host cell plasma membrane. The matrix protein (M), is
involved with viral assembly, and interacts with both the viral
membrane as well as the nucleocapsid proteins.
[0006] The main protein subunit of the nucleocapsid is the
nucleocapsid protein (NP) which confers helical symmetry on the
capsid. In association with the nucleocapsid are the P and L
proteins. The phosphoprotein (P), which is subject to
phosphorylation, is thought to play a regulatory role in
transcription, and may also be involved in methylation,
phosphorylation and polyadenylation. The L gene, which encodes an
RNA-dependent RNA polymerase, is required for viral RNA synthesis
together with the P protein. The L protein, which takes up nearly
half of the coding capacity of the viral genome is the largest of
the viral proteins, and plays an important role in both
transcription and replication. The V protein has been shown to
inhibit interferon-alpha and to contribute to the virulence of NDV
(Huang et al. (2003). Newcastle disease virus V protein is
associated with viral pathogenesis and functions as an Alpha
Interferon Antagonist. Journal of Virology 77: 8676-8685).
[0007] Naturally-occurring NDV has been reported to be an effective
oncolytic agent in a variety of animal tumor models (Sinkovics, J
G, and Horvath, J C (2000). Newcastle disease virus (NDV): brief
history of its oncolytic strains. J Clin Virol 16: 1-15; Zamarin et
al., 2009; Mol Ther 17: 697; Elankumaran et al., 2010; J Virol 84:
3835; Schirrmacher et al., 2009; Methods Mol Biol 542: 565; Bart et
al., 1973; Nat New Biol 245: 229). Naturally-occurring strains of
NDV have been used in multiple clinical trials against advanced
human cancers (Sinkovics, J G, and Horvath, J C (2000). Newcastle
disease virus (NDV): brief history of its oncolytic strains. J Clin
Virol 16: 1-15; Lorence et al. (2007). Phase 1 clinical experience
using intravenous administration of PV701, an oncolytic Newcastle
disease virus. Curr Cancer Drug Targets 7: 157-167; Hotte et al.
(2007). An optimized clinical regimen for the oncolytic virus
PV701. Clin Cancer Res 13: 977-985; Freeman et al. (2006). Phase
I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent
glioblastoma multiforme. Mol Ther 13: 221-228; Pecora et al.
(2002). Phase I trial of intravenous administration of PV701, an
oncolytic virus, in patients with advanced solid cancers. J Clin
Oncol 20: 2251-2266; Csatary et al. (2004). MTH-68/H oncolytic
viral treatment in human high-grade gliomas. J Neurooncol 67:
83-93). However, the success of naturally-occurring strains of NDV
in these clinical trials for advanced human cancers was only
marginal (Hotte et al. (2007). An optimized clinical regimen for
the oncolytic virus PV701. Clin Cancer Res 13: 977-985; Freeman et
al. (2006). Phase I/II trial of intravenous NDV-HUJ oncolytic virus
in recurrent glioblastoma multiforme. Mol Ther 13: 221-228; Pecora
et al. (2002). Phase I trial of intravenous administration of
PV701, an oncolytic virus, in patients with advanced solid cancers.
J Clin Oncol 20: 2251-2266). As such, there remains a need for
NDV-based therapies useful in the treatment of cancer, especially
advanced cancer.
3. SUMMARY
[0008] In one aspect, presented herein are chimeric Newcastle
disease viruses (NDVs) engineered to express an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell. In a specific embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes an agonist of a co-stimulatory signal of an immune
cell, wherein the agonist is expressed. In a specific embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes an antagonist of an inhibitory signal of an immune
cell, wherein the antagonist is expressed.
[0009] In another embodiment, presented herein are chimeric NDVs,
comprising a packaged genome which encodes an agonist of a
co-stimulatory signal of an immune cell and a mutated F protein
that causes the NDV to be highly fusogenic, wherein the agonist and
the mutated F protein are expressed. In another embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes an agonist of a co-stimulatory signal of an immune
cell and a mutated F protein with a mutated cleavage site, wherein
the agonist and the mutated F protein are expressed. In a specific
embodiment, the chimeric NDVs expressing the mutated F protein have
increased fusogenic activity relative to the corresponding virus
expressing the counterpart F protein without the mutations to the
cleavage site. In another specific embodiment, the modified F
protein is incorporated into the virion.
[0010] In another embodiment, presented herein are chimeric NDVs,
comprising a packaged genome which encodes an antagonist of an
inhibitory signal of an immune cell and a mutated F protein that
causes the NDV to be highly fusogenic, wherein the antagonist and
the mutated F protein are expressed. In another embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes antagonist of an inhibitory signal of an immune cell
and a mutated F protein with a mutated cleavage site, wherein the
antagonist and the mutated F protein are expressed. In a specific
embodiment, the chimeric NDVs expressing the mutated F protein have
increased fusogenic activity relative to the corresponding virus
expressing the counterpart F protein without the mutations to the
cleavage site. In another specific embodiment, the modified F
protein is incorporated into the virion.
[0011] In another embodiment, presented herein are chimeric NDVs,
comprising a packaged genome which encodes an agonist of a
co-stimulatory signal of an immune cell and a cytokine (e.g.,
interleukin (IL)-2), wherein the agonist and the cytokine are
expressed. In another embodiment, presented herein are chimeric
NDVs, comprising a packaged genome which encodes an agonist of a
co-stimulatory signal of an immune cell, a cytokine (e.g., IL-2)
and a mutated F protein that causes the NDV to be highly fusogenic,
wherein the agonist, the cytokine and the mutated F protein are
expressed. In another embodiment, presented herein are chimeric
NDVs, comprising a packaged genome which encodes an agonist of a
co-stimulatory signal of an immune cell, a cytokine (e.g., IL-2)
and a mutated F protein with a mutated cleavage site, wherein the
agonist, the cytokine and the mutated F protein are expressed. In a
specific embodiment, the chimeric NDVs expressing the mutated F
protein with the mutated cleavage site are highly fusogenic. In
another specific embodiment, the mutated F protein is incorporated
into the virion.
[0012] In another embodiment, presented herein are chimeric NDVs,
comprising a packaged genome which encodes an antagonist of an
inhibitory signal of an immune cell of an immune cell and a
cytokine (e.g., IL-2), wherein the antagonist and the cytokine are
expressed. In another embodiment, presented herein are chimeric
NDVs, comprising a packaged genome which encodes an antagonist of
an inhibitory signal of an immune cell, a cytokine (e.g., IL-2) and
a mutated F protein that causes the NDV to be highly fusogenic,
wherein the antagonist, the cytokine and the mutated F protein are
expressed. In another embodiment, presented herein are chimeric
NDVs, comprising a packaged genome which encodes an antagonist of
an inhibitory signal of an immune cell, a cytokine (e.g., IL-2) and
a mutated F protein with a mutated cleavage site, wherein the
antagonist, the cytokine and the mutated F protein are expressed.
In a specific embodiment, the chimeric NDVs expressing the mutated
F protein with the mutated cleavage site are highly fusogenic. In
another specific embodiment, the mutated F protein is incorporated
into the virion.
[0013] In a specific embodiment, the agonist of a co-stimulatory
signal of an immune cell is an agonist of a co-stimulatory receptor
expressed by an immune cell. Specific examples of co-stimulatory
receptors include glucocorticoid-induced tumor necrosis factor
receptor (GITR), Inducible T-cell costimulator (ICOS or CD278),
OX40 (CD134), CD27, CD28, 4-1BB (CD137), CD40, CD226, cytotoxic and
regulatory T cell molecule (CRTAM), death receptor 3 (DR3),
lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML
interactor (TACI), B cell-activating factor receptor (BAFFR), and B
cell maturation protein (BCMA). In a specific embodiment, the
agonist of a co-stimulatory receptor expressed by an immune cell is
an antibody (or an antigen-binding fragment thereof) or ligand that
specifically binds to the co-stimulatory receptor. In one
embodiment, the antibody is a monoclonal antibody. In another
embodiment, the antibody is an sc-Fv. In a specific embodiment, the
antibody is a bispecific antibody that binds to two receptors on an
immune cell. In one embodiment, the bispecific antibody binds to a
receptor on an immune cell and to another receptor on a cancer
cell. In specific embodiments, the antibody is a human or humanized
antibody. In certain embodiments, the ligand or antibody is a
chimeric protein comprising a NDV F protein or fragment thereof, or
NDV HN protein or fragment thereof. Methods for generating such
chimeric proteins are known in the art. See, e.g., U.S. Patent
Application Publication No. 2012-0122185, the disclosure of which
is herein incorporated by reference in its entirety. Also see Park
et al., PNAS 2006; 103:8203-8 and Murawski et al., J Virol 2010;
84:1110-23, the disclosures of which is herein incorporated by
reference in their entireties. In certain embodiments, the ligand
or antibody is expressed as a chimeric F protein or NDV F-fusion
protein, wherein the chimeric F protein or NDV F-fusion protein
comprises the cytoplasmic and transmembrane domains or fragments
thereof of the NDV F glycoprotein and the extracellular domain
comprises the ligand or antibody. In some embodiments, the ligand
is expressed as a chimeric HN protein or NDV HN-fusion protein,
wherein the chimeric HN protein or NDV HN-fusion protein comprises
the transmembrane and extracellular domains or fragments thereof of
the NDV HN glycoprotein and the extracellular domain comprises the
ligand or antibody. In a specific embodiment, the ligand or
antibody is expressed as a chimeric protein, such as described in
Section 7, Example 2, infra.
[0014] In a specific embodiment, the antagonist of an inhibitory
signal of an immune cell is an antagonist of an inhibitory receptor
expressed by an immune cell. Specific examples of inhibitory
receptors include cytotoxic T-lymphocyte-associated antigen 4
(CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B
and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like
receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell
membrane protein 3 (TIM3), adenosine A2a receptor (A2aR), T cell
immunoreceptor with immunoglobulin and ITIM domains (TIGIT),
leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and
CD160. In a specific embodiment, the antagonist of an inhibitory
receptor expressed by an immune cell is an antibody (or an
antigen-binding fragment thereof) that specifically binds to the
co-stimulatory receptor. In one embodiment, the antibody is a
monoclonal antibody. In another embodiment, the antibody is an
sc-Fv. In specific embodiments, the antibody is a human or
humanized antibody. In another specific embodiment, the antagonist
of an inhibitory receptor is a soluble receptor or antibody (or an
antigen-binding fragment thereof) that specifically binds to a
ligand of the inhibitory receptor. In certain embodiments, the
antibody is a chimeric protein comprising a NDV F protein or
fragment thereof, or NDV HN protein or fragment thereof. See, e.g.,
U.S. Patent Application Publication No. 2012-0122185, Park et al.,
PNAS 2006; 103: 8203-8, and Murawski et al., J. Virol 2010;
84:1110-23, which are each incorporated herein by reference in
their entirety. In certain embodiments, the antibody is expressed
as a chimeric F protein or NDV F-fusion protein, wherein the
chimeric F protein or NDV-F-fusion protein comprises the
cytoplasmic and transmembrane domains or fragments thereof of the
NDV F glycoprotein and the extracellular domain comprises the
antibody. In some embodiments, the antibody is expressed as a
chimeric HN protein or NDV HN-fusion protein, wherein the chimeric
HN protein or NDV HN-fusion protein comprises the transmembrane and
intracellular domains or fragments thereof of the NDV HN
glycoprotein and the extracellular domain comprises the
antibody.
[0015] In another aspect, presented herein are methods for
propagating the NDVs described herein (e.g., chimeric NDVs
described herein). The NDVs described herein (e.g., chimeric NDVs
described herein) can be propagated in any cell, subject, tissue or
organ susceptible to a NDV infection. In one embodiment, the NDVs
described herein (e.g., chimeric NDVs described herein) may be
propagated in a cell line. In another embodiment, the NDVs
described herein (e.g., chimeric NDVs described herein) may be
propagated in cancer cells. In another embodiment, the NDVs
described herein (e.g., chimeric NDVs described herein) may be
propagated in an embryonated egg. In certain embodiments, presented
herein are isolated cells, tissues or organs infected with an NDV
described herein (e.g., a chimeric NDV described herein). See,
e.g., Section 5.4, infra, for examples of cells, animals and eggs
to infect with an NDV described herein (e.g., a chimeric NDV
described herein). In specific embodiments, presented herein are
isolated cancer cells infected with an NDV described herein (e.g.,
a chimeric NDV described herein). In certain embodiments, presented
herein are cell lines infected with an NDV described herein (e.g.,
a chimeric NDV described herein). In other embodiments, presented
herein are embryonated eggs infected with an NDV described herein
(e.g., a chimeric NDV described herein).
[0016] In another aspect, presented herein are compositions
comprising an NDV described herein (e.g., a chimeric NDV described
herein). In a specific embodiment, presented herein are
pharmaceutical compositions comprising an NDV described herein
(e.g., a chimeric NDV described herein) and a pharmaceutically
acceptable carrier. In another embodiment, presented herein are
pharmaceutical compositions comprising cancer cells infected with
an NDV described herein (e.g., a chimeric NDV described herein),
and a pharmaceutically acceptable carrier. In specific embodiments,
the cancer cells have been treated with gamma radiation prior to
incorporation into the pharmaceutical composition. In specific
embodiments, the cancer cells have been treated with gamma
radiation before infection with the NDV (e.g., chimeric NDV). In
other specific embodiments, the cancer cells have been treated with
gamma radiation after infection with the NDV (e.g., chimeric NDV).
In another embodiment, presented herein are pharmaceutical
compositions comprising protein concentrate from lysed NDV-infected
cancer cells (e.g., chimeric-NDV infected cancer cells), and a
pharmaceutically acceptable carrier.
[0017] In another aspect, presented herein are methods for
producing pharmaceutical compositions comprising an NDV described
herein (e.g., a chimeric NDV described herein). In one embodiment,
a method for producing a pharmaceutical composition comprises: (a)
propagating an NDV described herein (e.g., a chimeric NDV described
herein) in a cell line that is susceptible to an NDV infection; and
(b) collecting the progeny virus, wherein the virus is grown to
sufficient quantities and under sufficient conditions that the
virus is free from contamination, such that the progeny virus is
suitable for formulation into a pharmaceutical composition. In
another embodiment, a method for producing a pharmaceutical
composition comprises: (a) propagating an NDV described herein
(e.g., a chimeric NDV described herein) in an embryonated egg; and
(b) collecting the progeny virus, wherein the virus is grown to
sufficient quantities and under sufficient conditions that the
virus is free from contamination, such that the progeny virus is
suitable for formulation into a pharmaceutical composition.
[0018] In another aspect, presented herein are methods for treating
cancer utilizing a chimeric NDV described herein (e.g., a chimeric
NDV described in Section 5.2, infra) or a composition comprising
such a chimeric NDV. In a specific embodiment, a method for
treating cancer comprises infecting a cancer cell in a subject with
a chimeric NDV described herein (e.g., a chimeric NDV described in
Section 5.2, infra) or a composition thereof. In another
embodiment, a method for treating cancer comprises administering to
a subject in need thereof a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, infra) or a composition
thereof. In specific embodiments, an effective amount of a chimeric
NDV described herein (e.g., a chimeric NDV described in Section
5.2, infra) or a composition comprising an effective amount of a
chimeric NDV described herein is administered to a subject to treat
cancer. In specific embodiments, the chimeric NDV comprises a
genome, the genome comprising an agonist of a co-stimulatory signal
of an immune cell (e.g., an agonist of a co-stimulatory receptor of
an immune cell) and/or an antagonist of an inhibitory signal of an
immune cell (e.g., an antagonist of an inhibitory receptor of an
immune cell). In certain embodiments, the genome of the NDV also
comprises a mutated F protein. In certain embodiments, two or more
chimeric NDVs are administered to a subject to treat cancer.
[0019] In another embodiment, a method for treating cancer
comprises administering to a subject in need thereof cancer cells
infected with a chimeric NDV described herein (e.g., a chimeric NDV
described in Section 5.2, infra) or composition thereof. In
specific embodiments, the cancer cells have been treated with gamma
radiation prior to administration to the subject or incorporation
into the composition. In another embodiment, a method for treating
cancer comprises administering to a subject in need thereof a
protein concentrate or plasma membrane fragments from cancer cells
infected with a chimeric NDV (e.g., a chimeric NDV described in
Section 5.2, infra) or a composition thereof. In specific
embodiments, the chimeric NDV comprises a genome, the genome
comprising an agonist of a co-stimulatory signal of an immune cell
(e.g., an agonist of a co-stimulatory receptor of an immune cell)
and/or an antagonist of an inhibitory signal of an immune cell
(e.g., an antagonist of an inhibitory receptor of an immune cell).
In certain embodiments, the genome of the NDV also comprises a
mutated F protein.
[0020] In another aspect, presented herein are methods for treating
cancer utilizing an NDV described herein (e.g., a chimeric NDV such
as described in Section 5.2, infra) or a composition comprising
such the NDV in combination with one or more other therapies. In
one embodiment, presented herein are methods for treating cancer
comprising administering to a subject an NDV described herein
(e.g., a chimeric NDV, such as described in Section 5.2.1, infra)
and one or more other therapies. In another embodiment, presented
herein are methods for treating cancer comprising administering to
a subject an effective amount of an NDV described herein or a
composition comprising an effective amount of an NDV described
herein, and one or more other therapies. The NDV and one or more
other therapies can be administered concurrently or sequentially to
the subject. In certain embodiments, the NDV and one or more other
therapies are administered in the same composition. In other
embodiments, the NDV and one or more other therapies are
administered in different compositions. The NDV and one or more
other therapies can be administered by the same or different routes
of administration to the subject.
[0021] Any NDV type or strain may be used in a combination therapy
disclosed herein, including, but not limited to,
naturally-occurring strains, variants or mutants, mutagenized
viruses, reassortants and/or genetically engineered viruses. In a
specific embodiment, the NDV used in a combination with one or more
other therapies is a naturally-occurring strain. In another
embodiment, the NDV used in combination with one or more other
therapies is a chimeric NDV. In a specific embodiment, the chimeric
NDV comprises a packaged genome, the genome comprising a cytokine
(e.g., IL-2, IL-7, IL-15, IL-17, or IL-21). In specific
embodiments, the cytokine is expressed by cells infected with the
chimeric NDV. In a specific embodiment, the chimeric NDV comprises
a packaged genome, the genome comprising a tumor antigen. In
specific embodiments, the tumor antigen is expressed by cells
infected with the chimeric NDV. In a specific embodiment, the
chimeric NDV comprises a packaged genome, the genome comprising a
pro-apoptotic molecule or an anti-apoptotic molecule. In specific
embodiments, the pro-apoptotic molecule or anti-apoptotic molecule
is expressed by cells infected with the chimeric NDV.
[0022] In another specific embodiment, the chimeric NDV comprises a
packaged genome, the genome comprising an agonist of a
co-stimulatory signal of an immune cell (e.g., an agonist of a
co-stimulatory receptor of an immune cell) and/or an antagonist of
an inhibitory signal of an immune cell (e.g., an antagonist of an
inhibitory receptor of an immune cell). In specific embodiments,
the agonist and/or antagonist are expressed by cells infected with
the chimeric NDV. In certain embodiments, the genome of the NDV
also comprises a mutated F protein. In certain embodiments, the one
or more therapies used in combination with an NDV described herein
is one or more other therapies described in Section 5.6.4, infra.
In particular embodiments, the one or more therapies used in
combination with an NDV described herein is an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell (see, e.g., Section 5.6.4.1,
infra). See, e.g., Section 5.2.1, infra, for examples of agonists
of a co-stimulatory signal of an immune cell and antagonists of an
inhibitory signal of an immune cell. In a specific embodiment, the
antagonist of an inhibitory signal of an immune cell is the
anti-CTLA-4 antibody described in Sections 6 and 7, infra. In
another specific embodiment, the antagonist of an inhibitory signal
of an immune cell is anti-PD-1 antibody or an anti-PD-L1 antibody
described in Section 7, infra. In another specific embodiment, the
agonist of a co-stimulatory signal of an immune cell is the ICOS
ligand described in Sections 6 and 7, infra.
[0023] 3.1 Terminology
[0024] As used herein, the term "about" or "approximately" when
used in conjunction with a number refers to any number within 1, 5
or 10% of the referenced number.
[0025] As used herein, the term "agonist(s)" refers to a
molecule(s) that binds to another molecule and induces a biological
reaction. In a specific embodiment, an agonist is a molecule that
binds to a receptor on a cell and triggers one or more signal
transduction pathways. For example, an agonist includes an antibody
or ligand that binds to a receptor on a cell and induces one or
more signal transduction pathways. In certain embodiments, the
antibody or ligand binds to a receptor on a cell and induces one or
more signal transduction pathways. In other embodiments, the
agonist facilitates the interaction of the native ligand with the
native receptor.
[0026] As used herein, the term "antagonist(s)" refers to a
molecule(s) that inhibits the action of another molecule without
provoking a biological response itself. In a specific embodiment,
an antagonist is a molecule that binds to a receptor on a cell and
blocks or dampens the biological activity of an agonist. For
example, an antagonist includes an antibody or ligand that binds to
a receptor on a cell and blocks or dampens binding of the native
ligand to the cell without inducing one or more signal transduction
pathways. Another example of an antagonist includes an antibody or
soluble receptor that competes with the native receptor on cells
for binding to the native ligand, and thus, blocks or dampens one
or more signal transduction pathways induced when the native
receptor binds to the native ligand.
[0027] As used herein, the terms "antibody" and "antibodies" refer
to molecules that contain an antigen binding site, e.g.,
immunoglobulins. Antibodies include, but are not limited to,
monoclonal antibodies, bispecific antibodies, multispecific
antibodies, human antibodies, humanized antibodies, synthetic
antibodies, chimeric antibodies, polyclonal antibodies, single
domain antibodies, camelized antibodies, single-chain Fvs (scFv),
single chain antibodies, Fab fragments, F(ab') fragments,
disulfide-linked bispecific Fvs (sdFv), intrabodies, and
antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id and
anti-anti-Id antibodies to antibodies), and epitope-binding
fragments of any of the above. In particular, antibodies include
immunoglobulin molecules and immunologically active fragments of
immunoglobulin molecules. Immunoglobulin molecules can be of any
type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In a specific
embodiment, an antibody is a human or humanized antibody. In
another specific embodiment, an antibody is a monoclonal antibody
or scFv. In certain embodiments, an antibody is a human or
humanized monoclonal antibody or scFv. In other specific
embodiments, the antibody is a bispecific antibody. In certain
embodiments, the bispecific antibody specifically binds to a
co-stimulatory receptor of an immune cell or an inhibitory receptor
of an immune, and a receptor on a cancer cell. In some embodiments,
the bispecific antibody specifically binds to two receptors immune
cells, e.g., two co-stimulatory receptors on immune cells, two
inhibitory receptors on immune cells, or one co-stimulatory
receptor on immune cells and one inhibitory receptor on immune
cells.
[0028] As used herein, the term "derivative" in the context of
proteins or polypeptides refers to: (a) a polypeptide that is at
least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, or 99% or is 40% to 65%, 50% to 90%, 65% to 90%, 70% to 90%,
75% to 95%, 80% to 95%, or 85% to 99% identical to a native
polypeptide; (b) a polypeptide encoded by a nucleic acid sequence
that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% or is 40% to 65%, 50% to 90%, 65% to 90%, 70%
to 90%, 75% to 95%, 80% to 95%, or 85% to 99% identical a nucleic
acid sequence encoding a native polypeptide; (c) a polypeptide that
contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more, or 2 to 5, 2 to 10, 5 to 10, 5 to 15, 5 to 20,
10 to 15, or 15 to 20 amino acid mutations (i.e., additions,
deletions and/or substitutions) relative to a native polypeptide;
(d) a polypeptide encoded by nucleic acid sequence that can
hybridize under high, moderate or typical stringency hybridization
conditions to a nucleic acid sequence encoding a native
polypeptide; (e) a polypeptide encoded by a nucleic acid sequence
that can hybridize under high, moderate or typical stringency
hybridization conditions to a nucleic acid sequence encoding a
fragment of a native polypeptide of at least 10 contiguous amino
acids, at least 12 contiguous amino acids, at least 15 contiguous
amino acids, at least 20 contiguous amino acids, at least 30
contiguous amino acids, at least 40 contiguous amino acids, at
least 50 contiguous amino acids, at least 75 contiguous amino
acids, at least 100 contiguous amino acids, at least 125 contiguous
amino acids, at least 150 contiguous amino acids, or 10 to 20, 20
to 50, 25 to 75, 25 to 100, 25 to 150, 50 to 75, 50 to 100, 75 to
100, 50 to 150, 75 to 150, 100 to 150, or 100 to 200 contiguous
amino acids; or (f) a fragment of a native polypeptide. Derivatives
also include a polypeptide that comprises the amino acid sequence
of a naturally occurring mature form of a mammalian polypeptide and
a heterologous signal peptide amino acid sequence. In addition,
derivatives include polypeptides that have been chemically modified
by, e.g., glycosylation, acetylation, pegylation, phosphorylation,
amidation, derivitization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other protein
moiety, etc. Further, derivatives include polypeptides comprising
one or more non-classical amino acids. In one embodiment, a
derivative is isolated. In specific embodiments, a derivative
retains one or more functions of the native polypeptide from which
it was derived.
[0029] Percent identity can be determined using any method known to
one of skill in the art. In a specific embodiment, the percent
identity is determined using the "Best Fit" or "Gap" program of the
Sequence Analysis Software Package (Version 10; Genetics Computer
Group, Inc., University of Wisconsin Biotechnology Center, Madison,
Wis.). Information regarding hybridization conditions (e.g., high,
moderate, and typical stringency conditions) have been described,
see, e.g., U.S. Patent Application Publication No. US 2005/0048549
(e.g., paragraphs 72-73).
[0030] As used herein, the term "fragment" is the context of a
fragment of a proteinaceous agent (e.g., a protein) refers to a
fragment that is 8 or more contiguous amino acids, 10 or more
contiguous amino acids, 15 or more contiguous amino acids, 20 or
more contiguous amino acids, 25 or more contiguous amino acids, 50
or more contiguous amino acids, 75 or more contiguous amino acids,
100 or more contiguous amino acids, 150 or more contiguous amino
acids, 200 or more contiguous amino acids, or in the range of
between 10 to 300 contiguous amino acids, 10 to 200 contiguous
amino acids, 10 to 250 contiguous amino acids, 10 to 150 contiguous
amino acids, 10 to 100 contiguous amino acids, 10 to 50 contiguous
amino acids, 50 to 100 contiguous amino acids, 50 to 150 contiguous
amino acids, 50 to 200 contiguous amino acids, 50 to 250 contiguous
amino acids, 50 to 300 contiguous amino acids, 25 to 50 contiguous
amino acids, 25 to 75 contiguous amino acids, 25 to 100 contiguous
amino acids, or 75 to 100 contiguous amino acids of a proteinaceous
agent. In a specific embodiment, a fragment of a proteinaceous
agent retains one or more functions of the proteinaceous agent--in
other words, it is a functional fragment. For example, a fragment
of a proteinaceous agent retains the ability to interact with
another protein and/or to induce, enhance or activate one or more
signal transduction pathways.
[0031] As used herein, the term "functional fragment," in the
context of a proteinaceous agent, refers to a portion of a
proteinaceous agent that retains one or more activities or
functions of the proteinaceous agent. For example, a functional
fragment of an inhibitory receptor may retain the ability to bind
one or more of its ligands. A functional fragment of a ligand of a
co-stimulatory receptor may retain the ability to bind to the
receptor and/or induce, enhance or activate one or more signal
transduction pathways mediated by the ligand binding to its
co-stimulatory receptor.
[0032] As used herein, the term "heterologous" refers an entity not
found in nature to be associated with (e.g., encoded by and/or
expressed by the genome of) a naturally occurring NDV.
[0033] As used herein, the term "elderly human" refers to a human
65 years or older.
[0034] As used herein, the term "human adult" refers to a human
that is 18 years or older.
[0035] As used herein, the term "human child" refers to a human
that is 1 year to 18 years old.
[0036] As used herein, the term "human toddler" refers to a human
that is 1 year to 3 years old.
[0037] As used herein, the term "human infant" refers to a newborn
to 1 year old year human.
[0038] In certain embodiments, the terms "highly fusogenic" and
"increased fusogenic activity", and the like, as used herein,
refers to an increase in the ability of the NDV to form syncytia
involving a large number of cells. In a specific embodiment, cells
infected with an NDV described herein that is engineered to express
a mutated F protein have an increased ability to form syncytia
relative to cells infected with the parental virus from which the
virus is derived, which parental virus has an unmutated F protein.
In another specific embodiment, about 10% to about 25%, about 25%
to about 50%, about 25% to about 75%, about 50% to about 75%, about
50% to about 95%, or about 75% to about 99% or about 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99% more cells infected with an NDV described herein
that is engineered to express a mutated F protein form syncytia
relative to the number of cells forming syncytia that are infected
with the parental virus from the chimeric virus is derived which
has an unmutated F protein. In certain embodiments, the syncytia
are quantitated microscopically by counting the number of nuclei
per syncytium after a certain period of time (e.g., about 8 hours
to about 12 hours, about 12 hours to about 24 hours, about 24 hours
to about 36 hours, or about 36 hours to about 48 hours).
[0039] As used herein, the term "interferon antagonist" refers to
an agent that reduces or inhibits the cellular interferon immune
response. In one embodiment, an interferon antagonist is a
proteinaceous agent that reduces or inhibits the cellular
interferon immune response. In a specific embodiment, an interferon
antagonist is a viral protein or polypeptide that reduces or
inhibits the cellular interferon response.
[0040] In a specific embodiment, an interferon antagonist is an
agent that reduces or inhibits interferon expression and/or
activity. In one embodiment, the interferon antagonist reduces or
inhibits the expression and/or activity of type I IFN. In another
embodiment, the interferon antagonist reduces or inhibits the
expression and/or activity of type II IFN. In another embodiment,
the interferon antagonist reduces or inhibits the expression and/or
activity of type III IFN. In a specific embodiment, the interferon
antagonist reduces or inhibits the expression and/or activity of
either IFN-.alpha., IFN-.beta. or both. In another specific
embodiment, the interferon antagonist reduces or inhibits the
expression and/or activity of IFN-.gamma.. In another embodiment,
the interferon antagonist reduces or inhibits the expression and/or
activity of one, two or all of IFN-.alpha., IFN-.beta., and
IFN-.gamma..
[0041] In certain embodiments, the expression and/or activity of
IFN-.alpha., IFN-.beta. and/or IFN-.gamma. in an embryonated egg or
cell is reduced approximately 1 to approximately 100 fold,
approximately 5 to approximately 80 fold, approximately 20 to
approximately 80 fold, approximately 1 to approximately 10 fold,
approximately 1 to approximately 5 fold, approximately 40 to
approximately 80 fold, or 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold by an
interferon antagonist relative to the expression and/or activity of
IFN-.alpha., IFN-.beta., and/or IFN-.gamma. in a control
embryonated egg or a cell not expressing or not contacted with such
an interferon antagonist as measured by the techniques described
herein or known to one skilled in the art. In other embodiments,
the expression and/or activity of IFN-.alpha., IFN-.beta. and/or
IFN-.gamma. in an embryonated egg or cell is reduced by at least
20% to 25%, at least 25% to 30%, at least 30% to 35%, at least 35%
to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to
55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%,
at least 70% to 75%, at least 75% to 80%, at least 80% to 85%, at
least 85% to 90%, at least 90% to 95%, at least 95% to 99% or by
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 99% by an interferon antagonist relative to the
expression and/or activity of IFN-.alpha., IFN-.beta., and/or
IFN-.gamma. in a control embryonated egg or a cell not expressing
or not contacted with such an interferon antagonist as measured by
the techniques described herein or known to one skilled in the
art.
[0042] As used herein, the phrases "IFN deficient systems" or
"IFN-deficient substrates" refer to systems, e.g., cells, cell
lines and animals, such as mice, chickens, turkeys, rabbits, rats,
horses etc., which do not produce one, two or more types of IFN, or
do not produce any type of IFN, or produce low levels of one, two
or more types of IFN, or produce low levels of any IFN (i.e., a
reduction in any IFN expression of 5-10%, 10-20%, 20-30%, 30-40%,
40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to
IFN-competent systems under the same conditions), do not respond or
respond less efficiently to one, two or more types of IFN, or do
not respond to any type of IFN, have a delayed response to one, two
or more types of IFN, and/or are deficient in the activity of
antiviral genes induced by one, two or more types of IFN, or
induced by any type of IFN.
[0043] As used herein, the terms "immunospecifically binds,"
"immunospecifically recognizes," "specifically binds," and
"specifically recognizes" are analogous terms in the context of
antibodies and refer to molecules that specifically bind to an
antigen (e.g., epitope or immune complex) as understood by one
skilled in the art. A molecule that specifically binds to an
antigen may bind to other peptides or polypeptides with lower
affinity as determined by, e.g., immunoassays (e.g., ELISA),
surface plasmon resonance (e.g., BIAcore.RTM.), a KinEx assay
(using, e.g., a KinExA 3000 instrument (Sapidyne Instruments,
Boise, Id.)), or other assays known in the art. In a specific
embodiment, molecules that specifically bind to an antigen bind to
the antigen with a dissociation constant (i.e., Ka) that is at
least 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs or greater than
the Ka when the molecules bind to another antigen. In a another
specific embodiment, molecules that specifically bind to an antigen
do not cross react with other proteins.
[0044] As used herein, the term "monoclonal antibody" is a term of
the art and generally refers to an antibody obtained from a
population of homogenous or substantially homogeneous antibodies,
and each monoclonal antibody will typically recognize a single
epitope (e.g., single conformation epitope) on the antigen.
[0045] As used herein, the phrase "multiplicity of infection" or
"MOI" is the average number of virus per infected cell. The MOI is
determined by dividing the number of virus added (ml
added.times.Pfu) by the number of cells added (ml
added.times.cells/ml).
[0046] As used herein, the term "native ligand" refers to any
naturally occurring ligand that binds to a naturally occurring
receptor. In a specific embodiment, the ligand is a mammalian
ligand. In another specific embodiment, the ligand is a human
ligand.
[0047] As used herein, the term "native polypeptide(s)" in the
context of proteins or polypeptides refers to any naturally
occurring amino acid sequence, including immature or precursor and
mature forms of a protein. In a specific embodiment, the native
polypeptide is a human protein or polypeptide.
[0048] As used herein, the term "native receptor" refers to any
naturally occurring receptor that binds to a naturally occurring
ligand. In a specific embodiment, the receptor is a mammalian
receptor. In another specific embodiment, the receptor is a human
receptor.
[0049] As used herein, the terms "subject" or "patient" are used
interchangeably. As used herein, the terms "subject" and "subjects"
refers to an animal. In some embodiments, the subject is a mammal
including a non-primate (e.g., a camel, donkey, zebra, cow, horse,
horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey,
chimpanzee, and a human). In some embodiments, the subject is a
non-human mammal. In certain embodiments, the subject is a pet
(e.g., dog or cat) or farm animal (e.g., a horse, pig or cow). In
other embodiments, the subject is a human. In certain embodiments,
the mammal (e.g., human) is 0 to 6 months old, 6 to 12 months old,
1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20
years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years
old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50
to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70
years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years
old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.
In specific embodiments, the subject is an animal that is not
avian.
[0050] As used herein, the terms "treat" and "treating" in the
context of the administration of a therapy refers to a
treatment/therapy from which a subject receives a beneficial
effect, such as the reduction, decrease, attenuation, diminishment,
stabilization, remission, suppression, inhibition or arrest of the
development or progression of cancer, or a symptom thereof. In
certain embodiments, the treatment/therapy that a subject receives
results in at least one or more of the following effects: (i) the
reduction or amelioration of the severity of cancer and/or a
symptom associated therewith; (ii) the reduction in the duration of
a symptom associated with cancer; (iii) the prevention in the
recurrence of a symptom associated with cancer; (iv) the regression
of cancer and/or a symptom associated therewith; (v) the reduction
in hospitalization of a subject; (vi) the reduction in
hospitalization length; (vii) the increase in the survival of a
subject; (viii) the inhibition of the progression of cancer and/or
a symptom associated therewith; (ix) the enhancement or improvement
the therapeutic effect of another therapy; (x) a reduction or
elimination in the cancer cell population; (xi) a reduction in the
growth of a tumor or neoplasm; (xii) a decrease in tumor size;
(xiii) a reduction in the formation of a tumor; (xiv) eradication,
removal, or control of primary, regional and/or metastatic cancer;
(xv) a decrease in the number or size of metastases; (xvi) a
reduction in mortality; (xvii) an increase in cancer-free survival
rate of patients; (xviii) an increase in relapse-free survival;
(xix) an increase in the number of patients in remission; (xx) a
decrease in hospitalization rate; (xxi) the size of the tumor is
maintained and does not increase in size or increases the size of
the tumor by less 5% or 10% after administration of a therapy as
measured by conventional methods available to one of skill in the
art, such as MRI, X-ray, and CAT Scan; (xxii) the prevention of the
development or onset of cancer and/or a symptom associated
therewith; (xxiii) an increase in the length of remission in
patients; (xxiv) the reduction in the number of symptoms associated
with cancer; (xxv) an increase in symptom-free survival of cancer
patients; and/or (xxvi) limitation of or reduction in metastasis.
In some embodiments, the treatment/therapy that a subject receives
does not cure cancer, but prevents the progression or worsening of
the disease. In certain embodiments, the treatment/therapy that a
subject receives does not prevent the onset/development of cancer,
but may prevent the onset of cancer symptoms.
[0051] As used herein, the term "in combination" in the context of
the administration of (a) therapy(ies) to a subject, refers to the
use of more than one therapy. The use of the term "in combination"
does not restrict the order in which therapies are administered to
a subject. A first therapy can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second therapy to a
subject.
[0052] As used herein, the terms "therapies" and "therapy" can
refer to any protocol(s), method(s), and/or agent(s) that can be
used in the treatment of cancer. In certain embodiments, the terms
"therapies" and "therapy" refer to biological therapy, supportive
therapy, hormonal therapy, chemotherapy, immunotherapy and/or other
therapies useful in the treatment of cancer. In a specific
embodiment, a therapy includes adjuvant therapy. For example, using
a therapy in conjunction with a drug therapy, biological therapy,
surgery, and/or supportive therapy. In certain embodiments, the
term "therapy" refers to a chimeric NDV described herein. In other
embodiments, the term "therapy" refers to an agent that is not a
chimeric NDV.
4. BRIEF DESCRIPTION OF THE FIGURES
[0053] FIG. 1. NDV infection upregulates the expression of MHC I,
MHC II, and ICAM-1 on the surface of in vitro infected B16-F10
cells (24 hours post-infection).
[0054] FIGS. 2A-2E. Intratumoral NDV treatment leads to
infiltration with macrophages, NK cells, CD8 and CD4 effector cells
and decreases the frequency of Tregs. A) Overall study scheme. B)
Total CD45+ infiltrates. C) Total immune cell infiltrates. D)
Representative flow cytometry dot plots of relative CD4 FoxP3+ and
FoxP3- subsets. E) Teff/Treg and CD8/Treg ratios.
[0055] FIGS. 3A-3C. Therapy with NDV exhibits favorable effects on
tumor microenvironment of distant tumors. A) Representative flow
cytometry dot plots of relative CD4 FoxP3+ and FoxP3- subsets. B)
Absolute numbers of CD4 effector, Treg, and CD8 cells per gram of
tumor. C) Teff/Treg and CD8/Treg ratios.
[0056] FIGS. 4A-4C. Lymphocytes infiltrating distant tumors
upregulate activation, lytic, and proliferation markers.
Representative expression plots on CD4 effector cells (left) and
the corresponding percentages in the CD4 effector, CD8, Tregs
(right) are shown for A) CD44, B) Granzyme B, and C) Ki-67.
[0057] FIGS. 5A-5D. NDV Monotherapy delays the growth of distant
tumors and provides some protection against tumor rechallenge.
Bilateral flank tumors were established as described in FIG. 2A and
the animals were treated and followed for survival. A) Growth of
right flank (treated) tumors. B) Growth of left flank (non-treated)
tumors. C) Overall survival. Numbers in boxes indicate percent of
animals free of tumors. D) Survival in animals cured of B16-F10
melanoma by NDV re-challenged on day 75 with B16-F10 melanoma
cells. Representative results of two different experiments with 10
mice per group.
[0058] FIGS. 6A-6B. Tumor-infiltrating lymphocytes from both
treated and non-treated tumors upregulate CTLA-4 in response to NDV
therapy. A) Representative dot plots of CTLA-4 expression in CD8,
CD4 effector, and Tregs in right (treated) tumors. B)
Representative dot plots of CTLA-4 expression in CD8, CD4 effector,
and Tregs in left (non-treated) tumors.
[0059] FIG. 7A-7C. Combination therapy with NDV and CTLA-4 blockade
enhances anti-tumor effect in the injected and distant tumors.
Bilateral B16 flank tumors were established and the animals were
treated as described in FIG. 2A with or without anti-CTLA-4
antibody 9H10. A) Growth of treated tumors. B) Growth of distant
tumors. Numbers in boxes represent percentage of mice free of
tumors. C) Long-term survival. Representative results of 2
different experiments with 10 mice per group.
[0060] FIG. 8. Combination therapy with NDV and anti-CTLA-4 is
effective systemically against non-virus-permissive prostate TRAMP
tumors. Right (day 12) and left (day 3) flank TRAMP tumors were
established and the animals were treated with NDV as described in
FIG. 2A with or without systemic anti-CTLA-4 antibody. Growth of
left flank (non-injected) tumors is shown. Numbers in boxes
indicate percent of animals free of tumors.
[0061] FIG. 9A-9C. NDV infection upregulates expression of PD-L1 in
B16-F10 tumors. A) Surface PD-L1 expression on B16-F10 cells
infected with NDV for 24 hours. B) Surface PD-L1 expression on
B16-F10 cells treated with UV-inactivated supernatant from infected
B16-F10 cells. C) Upregulation of PD-L1 on the surface of tumor
cells isolated from injected and distant tumors from the animals
treated as in FIG. 2A (2 left panels--representative flow cytometry
plots, right panel--calculated averages of 5 mice per group).
[0062] FIGS. 10A-10F. Combination therapy with NDV and anti-PD-1 is
effective systemically against B16 melanoma and results in
increased T cell infiltration with upregulation of activation
markers. A) Overall survival. Animals were treated as described in
FIG. 2A with or without anti-PD-1 antibody. B) Absolute numbers of
CD45, CD3, CD8, and CD4 effector cells in tumors. C) Relative
percentage of regulatory T cells in tumor-infiltrating lymphocytes.
D-E) Tumor-infiltrating lymphocytes from distant tumors were
isolated and stained for expression of ICOS (D) and Granzyme B (E).
F) Tumor infiltrating lymphocytes were restimulated with dendritic
cells loaded with tumor lysates and assessed for expression of IFN
gamma by intracellular cytokine staining.
[0063] FIG. 11. Combination therapy with NDV and CTLA-4 induces
upregulation of ICOS and CD4 effector cells in distant tumors and
tumor-draining lymph nodes (TDLN).
[0064] FIGS. 12A-12D. Generation and in vitro evaluation of
NDV-ICOSL virus. A) Viral genomic construct scheme. B) Expression
of ICOSL on the surface of B16-F10 cells infected for 24 hours
(representative histogram, left and average of 3 samples per group,
right). C) Cytolytic activity of NDV in the infected B16-F10 cells
determined by LDH assay. D) Replication of recombinant NDV in the
B16-F10 cells.
[0065] FIGS. 13A-13C. Combination therapy with NDV-mICOSL and
anti-CTLA-4 protects mice from contralateral tumor challenge and
results in long-term animal survival. Animals were challenged with
a larger tumor dose and treated with NDV as described in FIG. 2A
with or without systemic anti-CTLA-4 antibody. Growth of left flank
(non-injected) tumors is shown. B) Long-term survival. Numbers in
boxes indicate percent of animals protected from tumors. Pooled
data of 3 different experiments of 5-10 mice per group. C) Mice
treated with combination therapy develop vitiligo at the former
tumor sites, but not systemically.
[0066] FIG. 14A-14B. Combination therapy with NDV-mICOSL and
anti-CTLA-4 protects mice from contralateral tumor challenge and
results in long-term animal survival in the CT26 colon carcinoma
model. Animals were challenged with a larger tumor dose and treated
with NDV as described in FIG. 2A with or without systemic
anti-CTLA-4 antibody. Growth of left flank (non-injected) tumors is
shown. Numbers in boxes indicate percent of animals protected from
tumors. B) Long-term survival. Representative experiment with 5-10
mice per group (A) and pooled data of 2 different experiments of
5-10 mice per group (B).
[0067] FIGS. 15A-15C. NDV treatment leads to distant B16 tumor
infiltration with macrophages, NK cells, CD8 and CD4 effector cells
and decreases the frequency of Tregs. A) Total CD45+, CD3+, CD8+,
CD4+FoxP3- (Teff), and CD4+FoxP3+ (Treg) infiltrates. B) Teff/Treg
and CD8/Treg ratios. C) Total macrophage, NK, and NKT cell
infiltrates.
[0068] FIG. 16A-16B. Lymphocytes infiltrating distant B16 tumors
upregulate activation, lytic, and proliferation markers.
Representative Ki-67, Granzyme B (GrB) and ICOS expression plots
(A) and the corresponding percentages in the CD4 effector and CD8
cells (B).
[0069] FIG. 17. Tumor infiltrating lymphocytes from treated animals
secrete IFN-gamma in response to stimulation with DC's loaded with
B16-F10 lysates. Representative dot plots are shown.
[0070] FIGS. 18A-18B. Animals cured by combination therapy are
protected from further tumor challenge. A) B16-F10 melanoma, day
120 re-challenge with 1.times.10.sup.5 cells. B) CT26 colon
carcinoma, day 90 re-challenge with 1.times.10.sup.6 cells.
Representative results of two different experiments with 10 mice
per group.
[0071] FIG. 19A-19B. Recombinant ICOSL-F chimeric protein is
efficiently expressed on surface. A) Schematic diagram of the
chimeric protein. B) Expression of the chimeric ICOSL-Ftm fusion
protein on the surface of transfected cells.
[0072] FIG. 20A-20D. NDV infection is restricted to the injected
tumor. A) Recombinant NDV-Fluc was administered intratumorally (IT)
or intravenously (IV) into Balb/C animals bearing CT26 tumors and
images were acquired over the next 72 hours. B) NDV-Fluc was
administered to C57BL/6 mice bearing bilateral B16-F10 melanoma
tumors and animals were monitored for 120 hours. Representative
luminescence images are shown. C) Quantification of luminescence
from the tumor site normalized to background luminescence. D) Area
under the curve (AUC) calculated from the data in panel (C). Data
show representative results from 1 of 3 independent experiments
with 3-5 mice/group. ***p<0.001.
[0073] FIG. 21A-21F. NDV infection increases tumor leukocyte
infiltration in the virus-injected tumors. Animals were treated
according to the scheme described in FIG. 22A. Tumors were excised
on day 15, and TILs were labeled and analyzed by flow cytometry. A)
Representative flow cytometry plots of percentages of
tumor-infiltrating CD45+ and CD3+ cells. B) Absolute numbers of
CD45+ cells/g tumor. C) Absolute numbers of innate immune cells/g
tumor. D) Representative plots of percentages of CD4+FoxP3+ (Treg)
and CD4+FoxP3- (T cony) cells. E) Absolute numbers of conventional
and regulatory CD4+ cells and CD8+ cells/g tumor. F) Calculated
Tconv/Treg and CD8+/Treg ratios from the tumors. Data represent
cumulative results from 3 independent experiments with 3-5
mice/group. Mean+/-SEM is shown. *p<0.05, **p<0.01,
***p<0.001, ****p<0.0001.
[0074] FIG. 22A-22M. NDV increases distant tumor lymphocyte
infiltration and delays tumor growth. A) Treatment scheme. B)
Representative flow cytometry plots of percentages of
tumor-infiltrating CD45+ and CD3+ cells. C) Absolute numbers of
CD45+ cells/g tumor. D) Absolute numbers of innate immune cells/g
tumor. E) Tumor sections from distant tumors were stained with
H&E (upper panels) or labeled for CD3 and FoxP3 (bottom panels)
and analyzed by microscopy. Areas denoted by arrows indicate areas
of necrosis and inflammatory infiltrates. Scale bars represent 200
.mu.m. F) Representative flow cytometry plots of percentages of
CD4+FoxP3+ (Treg) and CD4+FoxP3- (Tconv) cells. G) Absolute numbers
of conventional and regulatory CD4+ cells and CD8+ cells/g tumor
calculated from flow cytometry. H) Relative percentages of Tregs
out of CD45+ cells. I) Calculated Tconv/Treg and CD8+/Treg ratios.
(J, K) Upregulation of ICOS, Granzyme B, and Ki-67 on
tumor-infiltrating Tconv (J) and CD8+ cells (K). L) Growth of
NDV-injected and distant tumors. M) Overall animal survival. Data
represent cumulative results from 3 (B-K) or 2 (L-M) independent
experiments with n=3-5 per group. Mean+/-SEM is shown. *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001.
[0075] FIG. 23A-23E. NDV therapy increases distant tumor lymphocyte
infiltration in bilateral footpad melanoma model. Animals bearing
bilateral footpad melanoma tumors were treated according to the
schedule described in FIG. 22A. Distant tumors were excised on day
15 and TILs were labeled and analyzed by flow cytometry. A)
Representative flow cytometry plots of percentages of
tumor-infiltrating CD45+ and CD3+ cells. B) Representative flow
cytometry plots of percentages of CD4+FoxP3+ and CD4+FoxP3- cells.
C) Absolute numbers of conventional and regulatory CD4+ cells and
CD8+ cells/g tumor. D, E) Upregulation of ICOS, Granzyme B, and
Ki-67 on tumor-infiltrating CD8+ (D) and Tconv (E) lymphocytes.
Data show representative results from 1 of 2 independent
experiments with 5 mice/group. *p<0.05, **p<0.01,
***p<0.001, ****p<0.0001.
[0076] FIG. 24A-24I. NDV induces infiltration of
adoptively-transferred tumor-specific lymphocytes and facilitates
tumor inflammation. A) Treatment scheme. B) Representative
luminescence images from animals treated with NDV and
adoptively-transferred Trp1-Fluc lymphocytes. C) Quantification of
average luminescence from the tumor sites. D) The area under the
curve (AUC) calculated from the data in panel (C). E) Absolute
number of Pmel lymphocytes from distant tumors calculated from flow
cytometry. F) Representative flow cytometry plots of percentages of
CD45+ and CD3+ cells infiltrating distant tumors of animals treated
per treatment scheme in panel (A). G) Experimental scheme for serum
transfer from animals treated intratumorally with single injection
of NDV or PBS. H) Representative flow cytometry plots of
percentages of CD45+ and CD3+ cells infiltrating serum-injected
tumors. I) Absolute numbers of the indicated cell subsets in
serum-injected tumors calculated from flow cytometry. Data for
panels B-E represent 1 of 3 experiments with n=4-5 per group. Data
for panels G-I represent pooled data from 2 independent experiments
with n=5 per group. Mean+/- SEM is shown. *p<0.05, **p<0.01,
***p<0.001, ****p<0.0001.
[0077] FIG. 25. Intratumoral NDV provides protection from tumor
rechallenge. Animals cured of B16-F10 melanoma by NDV were injected
on day 75 with 1.times.10.sup.5 B16-F10 melanoma cells, monitored
for tumor growth, and euthanized when the tumors reached 1000
mm.sup.3. Overall animal survival is shown. Data show cumulative
results from 1 of 2 independent experiments with 10 mice/group.
****p<0.0001.
[0078] FIG. 26A-26B. Tumor-infiltrating CD8+ lymphocytes upregulate
CTLA-4 in response to NDV therapy. Representative dot plots (left)
and cumulative results (right) of CTLA-4 expression in CD8+ cells
in NDV-treated (A), and distant (B) tumors. Representative results
from 1 of 3 experiments with 3 mice per group. *p<0.05.
[0079] FIG. 27A-27K. NDV and CTLA-4 blockade synergize to reject
local and distant tumors. A) Treatment scheme. B) Growth of
virus-treated (right flank) B16-F10 tumors. C) Growth of distant
(left flank) B16-F10 tumors. D) Long-term survival in the B16-F10
model. E) Surviving animals were injected with 1.times.10.sup.5
B16-F10 cells in right flank on day 90 and followed for survival.
Data represent cumulative results from 3 experiments with n=6-11
per group. F) Growth of virus-treated (right flank) and distant
(left flank) TRAMP C2 tumors. G) Long-term survival in the TRAMP C2
model. H) In vitro sensitivity of B16-F10 and TRAMP C2 cells to
NDV-mediated lysis at different multiplicities of infection
(MOI's). I-K) Upregulation of MHC I, MHC II, CD80, and CD86 in
B16-F10 and TRAMP C2 cells infected with NDV. Representative flow
cytometry plots from B16-F10 cells (I) and calculated average
median fluorescent intensities (MFI) for B16-F10 (J) and TRAMP C2
(K) cells are shown. Mean+/- SEM is shown. Data represent results
from 1 of 3 (B-E), or 1 of 2 (F, G) independent experiments with
n=5-10 per group. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001.
[0080] FIG. 28A-28E. Systemic anti-tumor effect is restricted to
the injected tumor type. A) Animals were injected i.d. in right
flank with B16-F10 melanoma, MC38 colon carcinoma, or PBS, and in
the left flank with B16-F10 cells and treated as outlined in the
scheme. B, C) Growth of distant tumors (B) and overall survival (C)
of animals that received right B16-F10 or no right flank tumors.
Data show representative results from 1 out of 2 independent
experiments with 5-10 mice/group. D, E) Growth of distant tumors
(D) and overall survival (E) of animals that received right B16-F10
or MC38 tumors. Data represent results from 1 out of 2 independent
experiments with n=10 per group. **p<0.01, ****p<0.0001.
[0081] FIG. 29A-29E. Combination therapy with NDV and anti-CTLA-4
enhances tumor infiltration with innate and adaptive immune cells.
Animals were treated with combination therapy as described in FIG.
27A. Tumors were harvested on day 15 and analyzed for infiltrating
immune cells by flow cytometry. A) Absolute numbers of CD45+
cells/g tumor. B) Absolute numbers of CD11b+ and NK 1.1+ cells/g
tumor. C) Absolute numbers of conventional and regulatory CD4+
cells and CD8+ cells/g tumor. D) Relative percentages of Tregs out
of CD45+ cells. E) Calculated Tconv/Treg and CD8+/Treg ratios. Data
represent cumulative results from 4 independent experiments with
3-5 mice/group. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001.
[0082] FIG. 30A-30J. Combination therapy with NDV and CTLA-4
blockade induces inflammatory changes in distant tumors. Animals
were treated per schema in FIG. 27A. Tumors were harvested on day
15 and analyzed for infiltrating immune cells. A) Tumor sections
from distant tumors were stained with H&E (upper panels) or for
CD3 and FoxP3 (lower panels) and analyzed by light and fluorescence
microscopy, respectively. Areas denoted by arrows indicate necrosis
and inflammatory infiltrates. Scale bars represent 200 .mu.m. B)
Absolute number of tumor-infiltrating CD45+ and CD3+ cells/g tumor
calculated from flow cytometry. C) Representative flow cytometry
plots of percent of tumor-infiltrating CD4+ and CD8+ cells gated on
CD45+ population. D) Absolute numbers of Tconv, Treg, and CD8+
cells per gram of tumor. E) Relative percentages of
tumor-infiltrating Tregs out of CD45+ cells. F) Calculated
Tconv/Treg and CD8+/Treg ratios. G-I) Upregulation of ICOS,
Granzyme B, and Ki-67 on tumor-infiltrating CD8+ and Tconv
lymphocytes. Representative flow cytometry plots (upper panels) and
cumulative results (bottom panels) are shown. J) TILs were
restimulated with DC's pulsed with B16-F10 tumor lysates, and
IFN.gamma. production was determined by intracellular cytokine
staining Representative flow cytometry plots (left panel) and
cumulative results (right panel) are shown. Data represent
cumulative results from 5 (A-I) or 2 (J) independent experiments
with n=3-5 per group. Mean+/-SEM is shown. *p<0.05, **p<0.01,
***p<0.001, ****p<0.0001.
[0083] FIG. 31. Antibodies to CD8, CD4, and NK1.1 deplete the cells
of interest in vivo. Depleting antibodies were injected as
discussed in Materials and Methods in Section 7.1, infra. Blood
samples were collected on day 5 and processed by flow cytometry for
CD4+, CD8+, and NK cells with non-crossreactive antibodies.
Positive staining is represented by the horizontal bars.
Representative plots from 1 of 2 independent experiments with 5
mice per group are shown.
[0084] FIG. 32A-32F. Anti-tumor activity of NDV combination therapy
depends on CD8+ and NK cells and type I and type II interferons.
A-C) Animals were treated as described in FIG. 27A with or without
depleting antibodies for CD4+, CD8+, NK cells, or IFN.gamma.. A)
Growth of injected tumors. B) Growth of distant tumors. C)
Long-term survival. D-F) IFNAR-/- or age-matched C57BL/6 mice
(BL/6) were treated as described in FIG. 3A and monitored for tumor
growth. D) Growth of injected tumors. E) Growth of distant tumors.
F) Long-term survival. Data for all panels represent cumulative
results from 2 independent experiments with n=3-10 per group.
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0085] FIG. 33A-33B. NDV therapy leads to upregulation of PD-L1 on
tumors and tumor-infiltrating leukocytes. A). PD-L1 expression on
B16-F10 cells infected in vitro (left panel), and in vivo in virus
injected and distant tumors. Left, representative flow cytometry
histograms, right, average median fluorescence intensity (MFI) of
PD-L1 expression on B16-F10 cells from tumors. B) PD-L1 expression
on the surface of tumor-infiltrating leukocytes isolated from
distant tumors. Left: representative flow cytometry histograms,
right: calculated average MFI for each cell subset.
[0086] FIG. 34A-34D. Combination therapy of NDV with antibodies
blocking PD-1 leads to enhanced anti-tumor efficacy in bilateral
flank B16 melanoma model. A) Treatment scheme. B) Right flank
(NDV-injected) tumor growth. C) Left flank (distant) tumor growth.
D) Overall survival.
[0087] FIG. 35A-35D. Combination therapy of NDV with antibodies
blocking PD-L1 leads to enhanced anti-tumor efficacy in bilateral
flank B16 melanoma model. A) Treatment scheme. B) Right flank
(NDV-injected) tumor growth. C) Left flank (distant) tumor growth.
D) Overall survival.
[0088] FIG. 36A-36E. Combination therapy with NDV and anti-PD-1
therapy results in increased distant tumor infiltration with
effector but not regulatory T cells. A) Representative flow
cytometry plots of percentages of CD4+ and CD8+ cells in tumors. B)
Representative flow cytometry plots of percentages of Tconv
(CD4+FoxP3-) and Treg (CD4+FoxP3+) cells. C) Absolute numbers of T
cell subsets per gram of tumor, calculated from flow cytometry. D)
Relative percentages of Tregs from CD4+ T cells. E) Calculated
Tconv/Treg and CD8/Treg ratios.
[0089] FIG. 37A-37B. TILs from distant tumors in animals treated
with combination NDV and anti-PD-1 therapy upregulate lytic and
proliferation markers. A) Representative flow cytometry plots of
percentages of Tconv and CD8 lymphocytes positive for Granzyme B
and Ki67. B) Percentages of Tconv and CD8+ T cells positive for
Granzyme B and Ki67.
[0090] FIG. 38A-38C. NDV induces tumor immune infiltration and
upregulation of ICOS on CD4 and CD8 cells in the virus-injected and
distant tumors. A) Treatment scheme. B) Expression of ICOS on
tumor-infiltrating CD4+FoxP3- and CD8+ cells isolated from
NDV-injected (right flank) tumors. Representative flow cytometry
plots (top) and median fluorescence intensities (MFI) (bottom) are
shown. C) Expression of ICOS on tumor-infiltrating CD4+FoxP3- and
CD8+ cells isolated from distant (left flank) tumors.
Representative flow cytometry plots (top) and median fluorescence
intensities (MFI) (bottom) are shown.
[0091] FIG. 39A-39D. Generation and in vitro evaluation of
NDV-ICOSL virus. A) Viral genomic construct scheme. B) Expression
of ICOSL on the surface of B16-F10 cells infected for 24 hours
(representative histogram, left and average of 3 samples per group,
right). C) Cytolytic activity of NDV in the infected B16-F10 cells
determined by LDH assay. D) Replication of recombinant NDV in the
B16-F10 cells.
[0092] FIG. 40A-40F. NDV-ICOSL causes growth delay of distant
tumors and induces enhanced tumor lymphocyte infiltration.
Bilateral flank B16-F10 tumors were established as previously and
the animals were treated with 4 intratumoral injections of the
indicated virus to the right tumor. A) Growth of virus-injected
tumors. B) Growth of distant tumors. C) Overall survival. D)
Absolute numbers of tumor-infiltrating leukocytes in the right
(virus-injected tumors). E) Absolute numbers of tumor-infiltrating
leukocytes in the left (distant tumors). F) Relative percentage of
Tregs in the distant tumors.
[0093] FIG. 41A-41E. Combination therapy of NDV-ICOSL and CTLA-4
blockade results in rejection of the injected and distant tumors in
the B16-F10 model and protects against tumor rechallenge. A)
Treatment schema. B) Growth of virus-injected (right) tumors. C)
Growth of distant (left) tumors. D) Overall survival. E) Surviving
animals on day 90 were re-challenged in right flank with
2.times.10.sup.5 B16-F10 cells and followed for survival.
[0094] FIG. 42A-42E. Combination therapy of NDV-ICOSL and CTLA-4
blockade results in rejection of the injected and distant tumors in
the CT26 model. A) Treatment schema. B) Growth of virus-injected
(right) tumors. C) Growth of distant (left) tumors. D) Overall
survival. E) Surviving animals on day 90 were re-challenged in
right flank with 1.times.10.sup.6 CT26 cells and followed for
survival.
[0095] FIG. 43A-43J. Combination therapy of NDV-ICOSL and
anti-CTLA-4 leads to enhanced tumor infiltration with innate and
adaptive immune cells. Animals bearing bilateral flank B16-F10
tumors were treated according to the schedule described in FIG.
41A. On day 15 the animals were sacrificed and distant tumors were
processed for analysis of TIL's. A) Representative flow cytometry
plots of CD45+ and CD3+ cells gating on the entire tumor cell
population. Absolute number of tumor-infiltrating CD45+ (B), CD11b+
(C), and NK1.1+ cells (D) per gram of tumor was calculated from
flow cytometry. E) Absolute numbers of tumor-infiltrating, CD3+,
CD8+, CD4+FoxP3- (CD4eff), and CD4+FoxP3+ (Treg) per gram of tumor.
F) Relative percentage of Tregs of all CD45+ cells. G) Calculated
effector/Treg ratios. H, I, J) relative percentages of
tumor-infiltrating CD8+ and CD4+ effector cells positive for ICOS,
granzyme B, and Ki67, respectively.
[0096] FIG. 44A-44C. Schematic diagram for additional generated
recombinant NDV viruses expressing chimeric and native
immunostimulatory proteins. A) Diagram of chimeric proteins of TNF
receptor superfamily (GITRL, OX40L, 4-1BBL, CD40L) fused to the NDV
HN intracellular and transmembrane region of HN at the N terminus
(upper panel). Lower panel demonstrates the diagram of chimeric
proteins of immunoglobulin receptor superfamily, with anti-CD28scfv
and ICOSL extracellular domains fused to the intracellular and
transmembrane region of F at the C terminus. B) Length of
intracellular-transmembrane (HN and F) and extracellular domains of
each of the described chimeric proteins. C) Schematic diagram of
the site of insertion of transgene and list of all recombinant NDVs
expressing immunostimulatory ligands generated by this
strategy.
[0097] FIG. 45A-45C. Confirmation of rescue of recombinant NDV's.
A) Hemagglutination assay demonstrating positive hemagglutinating
activity in the wells for NDV-HN-GITRL, NDV-aCD28scfv-F,
NDV-HN-OX40L, NDV-HN-CD40L, NDV-IL15, and NDV-IL21. B) Primer
locations for confirmation of gene insert in the rescued viruses by
RT-PCR. C) RT-PCR on RNA isolated from rescued viruses.
[0098] FIG. 46. B16-F10 cells infected with recombinant NDVs
express the ligands on the surface. B16-F10 cells were infected
with the indicated recombinant NDV's at MOI of 2 and were analyzed
for surface ligand expression by flow cytometry 18 hours later.
Representative flow cytometry plots are shown.
[0099] FIG. 47. NDV-HN-4-1BBL induces increased distant tumor
immune infiltration. Animals bearing bilateral flank B16 melanoma
tumors were treated intratumorally into single flank with the
indicated virus as previously. After 3 treatments, animals were
euthanized and tumor-infiltrating lymphocytes from distant tumors
were analyzed by flow cytometry. Total number of tumor-infiltrating
CD3, CD4+FoxP3+ (Treg), CD4+FoxP3- (Tconv), CD8, NK, and CD11b+
cells per gram of tumor is shown.
5. DETAILED DESCRIPTION
[0100] In one aspect, presented herein are chimeric Newcastle
disease viruses (NDVs) engineered to express an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell. In a specific embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes an agonist of a co-stimulatory signal of an immune
cell, wherein the agonist is expressed. In a specific embodiment,
presented herein are chimeric NDVs, comprising a packaged genome
which encodes an antagonist of an inhibitory signal of an immune
cell, wherein the antagonist is expressed.
[0101] In another aspect, presented herein are methods for
propagating the NDVs described herein (e.g., chimeric NDVs
described herein). The NDVs described herein (e.g., chimeric NDVs
described herein) can be propagated in any cell, subject, tissue,
organ or animal susceptible to a NDV infection.
[0102] In another aspect, presented herein are compositions
comprising an NDV described herein (e.g., a chimeric NDV described
herein). In a specific embodiment, presented herein are
pharmaceutical compositions comprising an NDV described herein
(e.g., a chimeric NDV described herein) and a pharmaceutically
acceptable carrier. In another embodiment, presented herein are
pharmaceutical compositions comprising cancer cells infected with
an NDV described herein (e.g., a chimeric NDV described herein),
and a pharmaceutically acceptable carrier. In another embodiment,
presented herein are pharmaceutical compositions comprising protein
concentrate from lysed NDV-infected cancer cells (e.g.,
chimeric-NDV infected cancer cells), and a pharmaceutically
acceptable carrier.
[0103] In another aspect, presented herein are methods for
producing pharmaceutical compositions comprising an NDV described
herein (e.g., a chimeric NDV described herein). In one embodiment,
a method for producing a pharmaceutical composition comprises: (a)
propagating an NDV described herein (e.g., a chimeric NDV described
herein) in a cell line that is susceptible to an NDV infection; and
(b) collecting the progeny virus, wherein the virus is grown to
sufficient quantities and under sufficient conditions that the
virus is free from contamination, such that the progeny virus is
suitable for formulation into a pharmaceutical composition. In
another embodiment, a method for producing a pharmaceutical
composition comprises: (a) propagating an NDV described herein
(e.g., a chimeric NDV described herein) in an embryonated egg; and
(b) collecting the progeny virus, wherein the virus is grown to
sufficient quantities and under sufficient conditions that the
virus is free from contamination, such that the progeny virus is
suitable for formulation into a pharmaceutical composition.
[0104] In another aspect, presented herein are methods for treating
cancer utilizing a chimeric NDV described herein (e.g., a chimeric
NDV described in Section 5.2, infra) or a composition comprising
such a chimeric NDV. In a specific embodiment, a method for
treating cancer comprises infecting a cancer cell in a subject with
a chimeric NDV described herein (e.g., a chimeric NDV described in
Section 5.2, infra) or a composition thereof. In another
embodiment, a method for treating cancer comprises administering to
a subject in need thereof a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, infra) or a composition
thereof. In specific embodiments, an effective amount of a chimeric
NDV described herein (e.g., a chimeric NDV described in Section
5.2, infra) or a composition comprising an effective amount of a
chimeric NDV described herein is administered to a subject to treat
cancer. In specific embodiments, the chimeric NDV comprises a
packaged genome, the genome comprising an agonist of a
co-stimulatory signal of an immune cell (e.g., an agonist of a
co-stimulatory receptor of an immune cell) and/or an antagonist of
an inhibitory signal of an immune cell (e.g., an antagonist of an
inhibitory receptor of an immune cell), wherein the agonist and/or
antagonist are expressed by the NDV. In certain embodiments, the
genome of the NDV also comprises a mutated F protein. In certain
embodiments, two or more chimeric NDVs are administered to a
subject to treat cancer.
[0105] In another embodiment, a method for treating cancer
comprises administering to a subject in need thereof cancer cells
infected with a chimeric NDV described herein (e.g., a chimeric NDV
described in Section 5.2, infra) or composition thereof. In
specific embodiments, the cancer cells have been treated with gamma
radiation prior to administration to the subject or incorporation
into the composition. In another embodiment, a method for treating
cancer comprises administering to a subject in need thereof a
protein concentrate or plasma membrane fragments from cancer cells
infected with a chimeric NDV (e.g., a chimeric NDV described in
Section 5.2, infra) or a composition thereof. In specific
embodiments, the chimeric NDV comprises a packaged genome, the
genome comprising an agonist of a co-stimulatory signal of an
immune cell (e.g., an agonist of a co-stimulatory receptor of an
immune cell) and/or an antagonist of an inhibitory signal of an
immune cell (e.g., an antagonist of an inhibitory receptor of an
immune cell), wherein the agonist and/or antagonist are expressed
by the NDV. In certain embodiments, the genome of the NDV also
comprises a mutated F protein, which is expressed by the NDV.
[0106] In another aspect, presented herein are methods for treating
cancer utilizing an NDV described herein (e.g., a chimeric NDV such
as described in Section 5.2, infra) or a composition comprising
such the NDV in combination with one or more other therapies. In
one embodiment, presented herein are methods for treating cancer
comprising administering to a subject an NDV described herein
(e.g., a chimeric NDV, such as described in Section 5.2, infra) and
one or more other therapies. In another embodiment, presented
herein are methods for treating cancer comprising administering to
a subject an effective amount of an NDV described herein or a
composition comprising an effective amount of an NDV described
herein, and one or more other therapies. The NDV and one or more
other therapies can be administered concurrently or sequentially to
the subject. In certain embodiments, the NDV and one or more other
therapies are administered in the same composition. In other
embodiments, the NDV and one or more other therapies are
administered in different compositions. The NDV and one or more
other therapies can be administered by the same or different routes
of administration to the subject.
[0107] Any NDV type or strain may be used in a combination therapy
disclosed herein, including, but not limited to,
naturally-occurring strains, variants or mutants, mutagenized
viruses, reassortants and/or genetically engineered viruses. In a
specific embodiment, the NDV used in a combination with one or more
other therapies is a naturally-occurring strain. In another
embodiment, the NDV used in combination with one or more other
therapies is a chimeric NDV. In a specific embodiment, the chimeric
NDV comprises a packaged genome, the genome comprising a cytokine
(e.g., IL-2, IL-7, IL-15, IL-17 or IL-21). In specific embodiments,
the chimeric NDV comprises a packaged genome, the genome comprising
a tumor antigen. In specific embodiments, the tumor antigen is
expressed by cells infected with the chimeric NDV. In another
specific embodiment, the chimeric NDV comprises a packaged genome,
the genome comprising a pro-apoptotic molecule (e.g., Bax, Bak,
Bad, BID, Bcl-xS, Bim, Noxa, Puma, AIF, FasL, and TRAIL) or an
anti-apoptotic molecule (e.g., Bcl-2, Bcl-xL, Mcl-1, and XIAP). In
specific embodiments, the pro-apoptotic molecule or anti-apoptotic
molecule is expressed by cells infected with the chimeric NDV. In
another specific embodiment, the chimeric NDV comprises a packaged
genome, the genome comprising an agonist of a co-stimulatory signal
of an immune cell (e.g., an agonist of a co-stimulatory receptor of
an immune cell) and/or an antagonist of an inhibitory signal of an
immune cell (e.g., an antagonist of an inhibitory receptor of an
immune cell). In specific embodiments, the agonist and/or
antagonist are expressed by cells infected with the chimeric NDV.
In certain embodiments, the genome of the NDV also comprises a
mutated F protein, a tumor antigen, a heterologous interferon
antagonist, a pro-apoptotic molecule and/or an anti-apoptotic
molecule. In certain embodiments, the one or more therapies used in
combination with an NDV described herein is one or more other
therapies described in Section 5.6.4, infra. In particular
embodiments, the one or more therapies used in combination with an
NDV described herein are an agonist of a co-stimulatory signal of
an immune cell and/or an antagonist of an inhibitory signal of an
immune cell. See, e.g., Section 5.2.1, infra, for examples of
agonists of a co-stimulatory signal of an immune cell and
antagonists of an inhibitory signal of an immune cell. In a
specific embodiment, the antagonist of an inhibitory signal of an
immune cell is the anti-CTLA-4 antibody described in Section 6,
infra. In another specific embodiment, the agonist of a
co-stimulatory signal of an immune cell is the ICOS ligand
described in Section 6, infra.
[0108] 5.1 Newcastle Disease Virus
[0109] Any NDV type or strain may be used in a combination therapy
disclosed herein, including, but not limited to,
naturally-occurring strains, variants or mutants, mutagenized
viruses, reassortants and/or genetically engineered viruses. In a
specific embodiment, the NDV used in a combination therapy
disclosed herein is a naturally-occurring strain. In certain
embodiments, the NDV is a lytic strain. In other embodiments, the
NDV used in a combination therapy disclosed herein is a non-lytic
strain. In certain embodiments, the NDV used in a combination
therapy disclosed herein is lentogenic strain. In some embodiments,
the NDV is a mesogenic strain. In other embodiments, the NDV used
in a combination therapy disclosed herein is a velogenic strain.
Specific examples of NDV strains include, but are not limited to,
the 73-T strain, NDV HUJ strain, Ulster strain, MTH-68 strain,
Italien strain, Hickman strain, PV701 strain, Hitchner B1 strain
(see, e.g., Genbank No. AF309418 or NC.sub.--002617), La Sota
strain (see, e.g., Genbank No. AY845400), YG97 strain, MET95
strain, Roakin strain, and F48E9 strain. In a specific embodiment,
the NDV used in a combination therapy disclosed herein that is the
Hitchner B1 strain. In another specific embodiment, the NDV used in
a combination therapy disclosed herein is a B1 strain as identified
by Genbank No. AF309418 or NC.sub.--002617. In another specific
embodiment, the NDV used in a combination therapy disclosed herein
is the NDV identified by ATCC No. VR2239. In another specific
embodiment, the NDV used in a combination therapy disclosed herein
is the La Sota strain.
[0110] In specific embodiments, the NDV used in a combination
therapy disclosed herein is not pathogenic birds as assessed by a
technique known to one of skill. In certain specific embodiments,
the NDV used in a combination therapy is not pathogenic as assessed
by intracranial injection of 1-day-old chicks with the virus, and
disease development and death as scored for 8 days. In some
embodiments, the NDV used in a combination therapy disclosed herein
has an intracranial pathogenicity index of less than 0.7, less than
0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 or
less than 0.1. In certain embodiments, the NDV used in a
combination therapy disclosed herein has an intracranial
pathogenicity index of zero.
[0111] In certain embodiments, the NDV used in a combination
therapy disclosed herein is a mesogenic strain that has been
genetically engineered so as not be a considered pathogenic in
birds as assessed by techniques known to one skilled in the art. In
certain embodiments, the NDV used in a combination therapy
disclosed herein is a velogenic strain that has been genetically
engineered so as not be a considered pathogenic in birds as
assessed by techniques known to one skilled in the art.
[0112] In certain embodiments, the NDV used in a combination
therapy disclosed herein expresses a mutated F protein. In a
specific embodiment, the NDV used in a combination therapy
expresses a mutated F protein is highly fusogenic and able to form
syncytia. In another specific embodiment, the mutated F protein is
incorporated into the virion.
[0113] In one embodiment, a genome of a NDV used in a combination
therapy disclosed herein is engineered to express a mutated F
protein with a mutated cleavage site. In a specific embodiment, the
NDV used in a combination therapy disclosed herein is engineered to
express a mutated F protein in which the cleavage site of the F
protein is mutated to produce a polybasic amino acid sequence,
which allows the protein to be cleaved by intracellular proteases,
which makes the virus more effective in entering cells and forming
syncytia. In another specific embodiment, the NDV used in a
combination therapy disclosed herein is engineered to express a
mutated F protein in which the cleavage site of the F protein is
replaced with one containing one or two extra arginine residues,
allowing the mutant cleavage site to be activated by ubiquitously
expressed proteases of the furin family. Specific examples of NDVs
that express such a mutated F protein include, but are not limited
to, rNDV/F2aa and rNDV/F3aa. For a description of mutations
introduced into a NDV F protein to produce a mutated F protein with
a mutated cleavage site, see, e.g., Park et al. (2006) Engineered
viral vaccine constructs with dual specificity: avian influenza and
Newcastle disease. PNAS USA 103: 8203-2808, which is incorporated
herein by reference in its entirety. In some embodiments, the NDV
used in a combination therapy disclosed herein is engineered to
express a mutated F protein with the amino acid mutation L289A. In
specific embodiments the L289A mutated F protein possesses one, two
or three arginine residues in the cleavage site. In certain
embodiments, the mutated F protein is from a different type or
strain of NDV than the backbone NDV. In some embodiments, the
mutated F protein is in addition to the backbone NDV F protein. In
specific embodiments, the mutated F protein replaces the backbone
NDV F protein.
[0114] In certain embodiments, the NDV used in a combination
therapy disclosed herein is attenuated such that the NDV remains,
at least partially, infectious and can replicate in vivo, but only
generate low titers resulting in subclinical levels of infection
that are non-pathogenic (see, e.g., Khattar et al., 2009, J. Virol.
83:7779-7782). In a specific embodiment, the NDV is attenuated by
deletion of the V protein. Such attenuated NDVs may be especially
suited for embodiments wherein the virus is administered to a
subject in order to act as an immunogen, e.g., a live vaccine. The
viruses may be attenuated by any method known in the art.
[0115] In certain embodiments, the NDV used in a combination
therapy disclosed herein does not comprise an NDV V protein
encoding sequence. In other embodiments, the NDV used in a
combination therapy disclosed herein expresses a mutated V protein.
See, e.g., Elankumaran et al., 2010, J. Virol. 84(8): 3835-3844,
which is incorporated herein by reference, for examples of mutated
V proteins. In certain embodiments, a mesogenic or velogenic NDV
strain used in a combination therapy disclosed herein expresses a
mutated V protein, such as disclosed by Elankumaran et al., 2010,
J. Virol. 84(8): 3835-3844.
[0116] In certain embodiments, the NDV used in a combination
therapy disclosed herein is an NDV disclosed in U.S. Pat. No.
7,442,379, U.S. Pat. No. 6,451,323, or U.S. Pat. No. 6,146,642,
which is incorporated herein by reference in its entirety. In
specific embodiments, the NDV used in a combination therapy
disclosed herein is genetically engineered to encode and express a
heterologous peptide or protein. In certain embodiments, the NDV
used in a combination therapy disclosed herein is a chimeric NDV
known to one of skill in the art, or a chimeric NDV disclosed
herein (see, e.g., Section 5.2, infra). In some embodiments, the
NDV used in a combination therapy disclosed herein is a chimeric
NDV comprising a genome engineered to express a tumor antigen (see
below for examples of tumor antigens). In certain embodiments, the
NDV used in a combination therapy disclosed herein is a chimeric
NDV comprising a genome engineered to express a heterologous
interferon antagonist (see below for examples of heterologous
interferon antagonists). In some embodiments, the NDV used in a
combination therapy disclosed herein is a chimeric NDV disclosed in
U.S. patent application publication No. 2012/0058141, which is
incorporated herein by reference in its entirety. In certain
embodiments, the NDV used in a combination therapy disclosed herein
is a chimeric NDV disclosed in U.S. patent application publication
No. 2012/0122185, which is incorporated herein by reference in its
entirety. In some embodiments, the NDV used in a combination
therapy disclosed herein is a chimeric NDV comprising a genome
engineered to express a cytokine, such as, e.g., IL-2, IL-7, IL-9,
IL-15, IL-17, IL-21, IL-22, IFN-gamma, GM-CSF, and TNF-alpha. In
some embodiments, the NDV used in a combination therapy disclosed
herein is a chimeric NDV comprising a genome engineered to express
IL-2, IL-15, or IL-21. In a specific embodiment, the NDV used in a
combination therapy disclosed herein is a chimeric NDV comprising a
genome engineered to express a cytokine as described in Section 7,
Example 2, infra.
[0117] 5.2 Chimeric Newcastle Disease Virus
[0118] In one aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or Natural Killer
(NK) cell. In some embodiments, the agonist and/or antagonist is
incorporated into the virion. In a specific embodiment, a genome of
a NDV is engineered to express an agonist of a co-stimulatory
signal of an immune cell, such as, e.g., a T-lymphocyte or NK cell.
In another specific embodiment, a genome of a NDV is engineered to
express an antagonist of an inhibitory signal of an immune cell,
such as, e.g., a T-lymphocyte or NK cell. In other words, the NDV
serves as the "backbone" that is engineered to express an agonist
of a co-stimulatory signal and/or an antagonist of an inhibitory
signal of an immune cell, such as, e.g., a T-lymphocyte or Natural
Killer (NK) cell. Specific examples of agonists of co-stimulatory
signals as well as specific examples of antagonists of inhibitory
signal are provided below.
[0119] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
mutated F protein. In one embodiment, a genome of a NDV is
engineered to express an agonist of a co-stimulatory signal of an
immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
mutated F protein. In another embodiment, a genome of a NDV is
engineered to express an antagonist of an inhibitory signal of an
immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
mutated F protein. In a specific embodiment, the mutated F protein
is highly fusogenic and able to form syncytia. In another specific
embodiment, the mutated F protein is incorporated into the virion.
In certain embodiments, the genome of a NDV engineered to express
an agonist of a co-stimulatory signal and/or an antagonist of an
inhibitory signal of an immune cell, such as, e.g., a T-lymphocyte
or NK cell, comprises an NDV V protein encoding sequence.
[0120] In one embodiment, a genome of a NDV is engineered to
express an agonist of a co-stimulatory signal and/or an antagonist
of an inhibitory signal of an immune cell, such as, e.g., a
T-lymphocyte or NK cell, and a mutated F protein with a mutated
cleavage site. In a specific embodiment, the NDV is engineered to
express a mutated F protein in which the cleavage site of the F
protein is mutated to produce a polybasic amino acid sequence,
which allows the protein to be cleaved by intracellular proteases,
which makes the virus more effective in entering cells and forming
syncytia. In another specific embodiment, the NDV is engineered to
express a mutated F protein in which the cleavage site of the F
protein is replaced with one containing one or two extra arginine
residues, allowing the mutant cleavage site to be activated by
ubiquitously expressed proteases of the furin family. Specific
examples of NDVs that express such a mutated F protein include, but
are not limited to, rNDV/F2aa and rNDV/F3aa. For a description of
mutations introduced into a NDV F protein to produce a mutated F
protein with a mutated cleavage site, see, e.g., Park et al. (2006)
Engineered viral vaccine constructs with dual specificity: avian
influenza and Newcastle disease. PNAS USA 103: 8203-2808, which is
incorporated herein by reference in its entirety. In some
embodiments, the chimeric NDV is engineered to express a mutated F
protein with the amino acid mutation L289A. In certain embodiments,
the mutated F protein is from a different type or strain of NDV
than the backbone NDV. In specific embodiments the L289A mutated F
protein possesses one, two or three arginine residues in the
cleavage site. In some embodiments, the mutated F protein is in
addition to the backbone NDV F protein. In specific embodiments,
the mutated F protein replaces the backbone NDV F protein. In
specific embodiments, the mutated F protein is incorporated into
the virion.
[0121] In some embodiments, the genome of a NDV engineered to
express an agonist of a co-stimulatory signal and/or an antagonist
of an inhibitory signal of an immune cell, such as, e.g., a
T-lymphocyte or NK cell, comprises a mutated NDV V protein encoding
sequence, such as disclosed by Elankumaran et al., 2010, J. Virol.
84(8): 3835-3844. In other embodiments, the genome of a NDV
engineered to express an agonist of a co-stimulatory signal and/or
an antagonist of an inhibitory signal of an immune cell, such as,
e.g., a T-lymphocyte or NK cell does not comprise an NDV V protein
encoding sequence. In certain embodiments, parental backbone of the
chimeric NDV is a mesogenic or velogenic NDV strain that is
engineered to express a mutated V protein, such as disclosed by
Elankumaran et al., 2010, J. Virol. 84(8): 3835-3844.
[0122] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
cytokine. In a specific embodiment, a genome of a NDV is engineered
to express an agonist of a co-stimulatory signal of an immune cell,
such as, e.g., a T-lymphocyte or NK cell, and a cytokine. In a
specific embodiment, a genome of a NDV is engineered to express an
antagonist of an inhibitory signal of an immune cell, such as,
e.g., a T-lymphocyte or NK cell, and a cytokine Specific examples
of cytokines include, but are not limited to, interleukin (IL)-2,
IL-7, IL-9, IL-15, IL-17, IL-21, IL-22, interferon (IFN) gamma,
GM-CSF, and tumor necrosis factor (TNF)-alpha.
[0123] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, a
mutated F protein, and a cytokine (e.g., IL-2, IL-7, IL-9, IL-15,
IL-17, IL-21, IL-22, IFN-gamma, GM-CSF, and TNF-alpha). In a
specific embodiment, the mutated F protein are highly fusogenic. In
a specific embodiment, the mutated F protein has a mutant cleavage
site (such as described herein). In some embodiments, the mutated F
protein comprises the amino acid mutation L289A. In some
embodiments, the chimeric NDV is engineered to express a mutated F
protein with the amino acid mutation L289A. In certain embodiments,
the mutated F protein is from a different type or strain of NDV
than the backbone NDV. In specific embodiments the L289A mutated F
protein possesses one, two or three arginine residues in the
cleavage site. In some embodiments, the mutated F protein is in
addition to the backbone NDV F protein. In specific embodiments,
the mutated F protein replaces the backbone NDV F protein. In
specific embodiments, the mutated F protein is incorporated into
the virion.
[0124] In certain aspects, provided herein are chimeric NDV
comprising a genome engineered to express a cytokine such as, e.g.,
IL-7, IL-15, IL-21 or another cytokine described herein or known to
one of skill in the art. See, e.g., Section 7 for examples of
chimeric NDVs engineered to express cytokines as well as methods of
producing such chimeric NDVs.
[0125] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express (i) an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, and (ii) a tumor antigen. In a specific
embodiment, a genome of a NDV is engineered to express an agonist
of a co-stimulatory signal of an immune cell, such as, e.g., a
T-lymphocyte or NK cell, and a tumor antigen. In a specific
embodiment, a genome of a NDV is engineered to express an
antagonist of an inhibitory signal of an immune cell, such as,
e.g., a T-lymphocyte or NK cell, and a tumor antigen.
[0126] Tumor antigens include tumor-associated antigens and
tumor-specific antigens. Specific examples of tumor antigens
include, but are not limited to, MAGE-1, MAGE-3, BAGE, GAGE-1,
GAGE-2, N-acetylglucosaminyltransferase-V, p-15, gp100,
MART-1/MelanA, TRP-1 (gp75), Tyrosinase, cyclin-dependent kinase 4,
.beta.-catenin, MUM-1, CDK4, HER-2/neu, human papillomavirus-E6,
human papillomavirus E7, CD20, carcinoembryonic antigen (CEA),
epidermal growth factor receptor, MUC-1, caspase-8, CD5, mucin-1,
Lewisx, CA-125, p185HER2, IL-2R, Fap-.alpha., tenascin, antigens
associated with a metalloproteinase, and CAMPATH-1. Other examples
include, but are not limited to, KS 1/4 pan-carcinoma antigen,
ovarian carcinoma antigen (CA125), prostatic acid phosphate,
prostate specific antigen, melanoma-associated antigen p97,
melanoma antigen gp75, high molecular weight melanoma antigen
(HMW-MAA), prostate specific membrane antigen, CEA, polymorphic
epithelial mucin antigen, milk fat globule antigen, colorectal
tumor-associated antigens (such as: CEA, TAG-72, CO17-1A, GICA
19-9, CTA-1 and LEA), Burkitt's lymphoma antigen-38.13, CD19,
B-lymphoma antigen-CD20, CD33, melanoma specific antigens (such as
ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside
GM3), tumor-specific transplantation type of cell-surface antigen
(TSTA) (such as virally-induced tumor antigens including T-antigen
DNA tumor viruses and Envelope antigens of RNA tumor viruses),
oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder
tumor oncofetal antigen, differentiation antigen (such as human
lung carcinoma antigen L6 and L20), antigens of fibrosarcoma,
leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids,
breast cancer antigens (such as EGFR (Epidermal growth factor
receptor), HER2 antigen (p185.sup.HER2) and HER2 neu epitope),
polymorphic epithelial mucin (PEM), malignant human lymphocyte
antigen-APO-1, differentiation antigen (such as I antigen found in
fetal erythrocytes, primary endoderm, I antigen found in adult
erythrocytes, preimplantation embryos, I(Ma) found in gastric
adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1 found
in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D.sub.156-22 found in
colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic
adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in
gastric cancer, Y hapten, Le.sup.y found in embryonal carcinoma
cells, TL5 (blood group A), EGF receptor found in A431 cells,
E.sub.1 series (blood group B) found in pancreatic cancer, FC10.2
found in embryonal carcinoma cells, gastric adenocarcinoma antigen,
CO-514 (blood group Le.sup.a) found in Adenocarcinoma, NS-10 found
in adenocarcinomas, CO-43 (blood group Le.sup.b), G49 found in EGF
receptor of A431 cells, MH2 (blood group ALe.sup.b/Le.sup.y) found
in colonic adenocarcinoma, 19.9 found in colon cancer, gastric
cancer mucins, T.sub.5A.sub.7 found in myeloid cells, R.sub.24
found in melanoma, 4.2, G.sub.D3, D1.1, OFA-1, G.sub.M2, OFA-2,
G.sub.D2, and M1:22:25:8 found in embryonal carcinoma cells, and
SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos), T cell
receptor derived peptide from a Cutaneous T cell Lymphoma,
C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer antigen
15-3 (CA15-3) associated with breast cancer, cancer antigen-19
(CA-19) and cancer antigen-242 associated with gastrointestinal
cancers, carcinoma associated antigen (CAA), chromogranin A,
epithelial mucin antigen (MC5), human epithelium specific antigen
(E1A), Lewis(a)antigen, melanoma antigen, melanoma associated
antigens 100, 25, and 150, mucin-like carcinoma-associated antigen,
multidrug resistance related protein (MRPm6), multidrug resistance
related protein (MRP41), Neu oncogene protein (C-erbB-2), neuron
specific enolase (NSE), P-glycoprotein (mdr1 gene product),
multidrug-resistance-related antigen, p170,
multidrug-resistance-related antigen, prostate specific antigen
(PSA), CD56, and NCAM.
[0127] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, a
mutated F protein, and a tumor antigen. In a specific embodiment,
the mutated F protein are highly fusogenic. In a specific
embodiment, the mutated F protein has a mutant cleavage site (such
as described herein). In some embodiments, the mutated F protein
comprises the amino acid mutation L289A. In some embodiments, the
chimeric NDV is engineered to express a mutated F protein with the
amino acid mutation L289A. In certain embodiments, the mutated F
protein is from a different type or strain of NDV than the backbone
NDV. In specific embodiments the L289A mutated F protein possesses
one, two or three arginine residues in the cleavage site. In some
embodiments, the mutated F protein is in addition to the backbone
NDV F protein. In specific embodiments, the mutated F protein
replaces the backbone NDV F protein. In specific embodiments, the
mutated F protein is incorporated into the virion.
[0128] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express (i) an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, and (ii) a heterologous interferon antagonist.
In a specific embodiment, a genome of a NDV is engineered to
express an agonist of a co-stimulatory signal of an immune cell,
such as, e.g., a T-lymphocyte or NK cell, and a heterologous
interferon antagonist. In a specific embodiment, a genome of a NDV
is engineered to express an antagonist of an inhibitory signal of
an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
heterologous interferon antagonist. See, e.g., U.S. patent
application publication No. 2012-0058141, which is incorporated
herein by reference, for examples of chimeric NDV engineered to
express heterologous interferon antagonists.
[0129] Interferon antagonists may be identified using any technique
known to one of skill in the art, including, e.g., the techniques
described in U.S. Pat. Nos. 6,635,416; 7,060,430; and 7,442,527;
which are incorporated herein by reference in their entirety. In a
specific embodiment, the heterologous interferon antagonist is a
viral protein. Such viral proteins may be obtained or derived from
any virus and the virus may infect any species (e.g., the virus may
infect humans or non-human mammals). Exemplary heterologous
interferon antagonists include, without limitation, Nipah virus W
protein, Nipah V protein, Ebola virus VP35 protein, vaccinia virus
E3L protein, influenza virus NS1 protein, respiratory syncytial
virus (RSV) NS2 protein, herpes simplex virus (HSV) type 1 ICP34.5
protein, Hepatitis C virus NS3-4 protease, dominant-negative
cellular proteins that block the induction or response to innate
immunity (e.g., STAT1, MyD88, IKK and TBK), and cellular regulators
of the innate immune response (e.g., SOCS proteins, PIAS proteins,
CYLD proteins, IkB protein, AtgS protein, Pin1 protein, IRAK-M
protein, and UBP43). See, e.g., U.S. patent application publication
No. 2012-0058141, which is incorporated herein by reference in its
entirety, for additional information regarding heterologous
interferon antagonist.
[0130] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, a
mutated F protein, and a heterologous interferon antagonist. In a
specific embodiment, the mutated F protein are highly fusogenic. In
a specific embodiment, the mutated F protein has a mutant cleavage
site (such as described herein). In some embodiments, the mutated F
protein comprises the amino acid mutation L289A. In some
embodiments, the chimeric NDV is engineered to express a mutated F
protein with the amino acid mutation L289A. In certain embodiments,
the mutated F protein is from a different type or strain of NDV
than the backbone NDV. In specific embodiments the L289A mutated F
protein possesses one, two or three arginine residues in the
cleavage site. In some embodiments, the mutated F protein is in
addition to the backbone NDV F protein. In specific embodiments,
the mutated F protein replaces the backbone NDV F protein. In
specific embodiments, the mutated F protein is incorporated into
the virion.
[0131] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
pro-apoptotic molecule. In a specific embodiment, a genome of a NDV
is engineered to express an agonist of a co-stimulatory signal of
an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
pro-apoptotic molecule. In a specific embodiment, a genome of a NDV
is engineered to express an antagonist of an inhibitory signal of
an immune cell, such as, e.g., a T-lymphocyte or NK cell, and a
pro-apoptotic molecule. Specific examples of pro-apoptotic
molecules include, but are not limited to, Bax, Bak, Bad, BID,
Bcl-xS, Bim, Noxa, Puma, AIF, FasL, and TRAIL.
[0132] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, a
mutated F protein, and a pro-apoptotic molecule. In a specific
embodiment, the mutated F protein are highly fusogenic. In a
specific embodiment, the mutated F protein has a mutant cleavage
site (such as described herein). In some embodiments, the mutated F
protein comprises the amino acid mutation L289A. In some
embodiments, the chimeric NDV is engineered to express a mutated F
protein with the amino acid mutation L289A. In certain embodiments,
the mutated F protein is from a different type or strain of NDV
than the backbone NDV. In specific embodiments the L289A mutated F
protein possesses one, two or three arginine residues in the
cleavage site. In some embodiments, the mutated F protein is in
addition to the backbone NDV F protein. In specific embodiments,
the mutated F protein replaces the backbone NDV F protein. In
specific embodiments, the mutated F protein is incorporated into
the virion.
[0133] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and an
anti-apoptotic molecule. In a specific embodiment, a genome of a
NDV is engineered to express an agonist of a co-stimulatory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and an
anti-apoptotic molecule. In a specific embodiment, a genome of a
NDV is engineered to express an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, and an
anti-apoptotic molecule. Specific examples of anti-apoptotic
molecules include, but are not limited to, Bcl-2, Bcl-xL, Mcl-1,
and XIAP.
[0134] In another aspect, described herein are chimeric NDVs,
comprising a genome engineered to express an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell, such as, e.g., a T-lymphocyte or NK cell, a
mutated F protein, and an anti-apoptotic molecule. In a specific
embodiment, the mutated F protein are highly fusogenic. In a
specific embodiment, the mutated F protein has a mutant cleavage
site (such as described herein). In some embodiments, the mutated F
protein comprises the amino acid mutation L289A. In some
embodiments, the chimeric NDV is engineered to express a mutated F
protein with the amino acid mutation L289A. In certain embodiments,
the mutated F protein is from a different type or strain of NDV
than the backbone NDV. In specific embodiments the L289A mutated F
protein possesses one, two or three arginine residues in the
cleavage site. In some embodiments, the mutated F protein is in
addition to the backbone NDV F protein. In specific embodiments,
the mutated F protein replaces the backbone NDV F protein. In
specific embodiments, the mutated F protein is incorporated into
the virion.
[0135] In certain aspects, provided herein are chimeric NDVs
comprising a genome engineered express a pro-apoptotic molecule. In
certain aspects, provided herein are chimeric NDVs comprising a
genome engineered to express an anti-apoptotic molecule. Examples
of pro-apoptotic molecules and anti-apoptotic molecules are
provided herein.
[0136] Any NDV type or strain may be used as a backbone that is
engineered to express an agonist of a co-stimulatory signal of an
immune cell and/or an antagonist of an inhibitory signal of an
immune cell, such as, e.g., a T-lymphocyte or NK cell, and in
certain embodiments, engineered to express a cytokine, tumor
antigen, heterologous interferon antagonist, pro-apoptotic
molecule, anti-apoptotic molecule and/or mutated F protein,
including, but not limited to, naturally-occurring strains,
variants or mutants, mutagenized viruses, reassortants and/or
genetically engineered viruses. In a specific embodiment, the NDV
used in a combination therapy disclosed herein is a
naturally-occurring strain. In certain embodiments, the NDV that
serves as the backbone for genetic engineering is a lytic strain.
In other embodiments, the NDV that serves as the backbone for
genetic engineering is a non-lytic strain. In certain embodiments,
the NDV that serves as the backbone for genetic engineering is
lentogenic strain. In some embodiments, the NDV that serves as the
backbone for genetic engineering is mesogenic strain. In other
embodiments, the NDV that serves as the backbone for genetic
engineering is a velogenic strain. Specific examples of NDV strains
include, but are not limited to, the 73-T strain, NDV HUJ strain,
Ulster strain, MTH-68 strain, Italien strain, Hickman strain, PV701
strain, Hitchner B1 strain, La Sota strain (see, e.g., Genbank No.
AY845400), YG97 strain, MET95 strain, Roakin strain, and F48E9
strain. In a specific embodiment, the NDV that serves as the
backbone for genetic engineering is the Hitchner B1 strain. In
another specific embodiment, the NDV that serves as the backbone
for genetic engineering is a B1 strain as identified by Genbank No.
AF309418 or NC.sub.--002617. In another specific embodiment, the
NDV that serves as the backbone for genetic engineering is the NDV
identified by ATCC No. VR2239. In another specific embodiment, the
NDV that serves as the backbone for genetic engineering is the La
Sota strain.
[0137] In certain embodiments, attenuation, or further attenuation,
of the chimeric NDV is desired such that the chimeric NDV remains,
at least partially, infectious and can replicate in vivo, but only
generate low titers resulting in subclinical levels of infection
that are non-pathogenic (see, e.g., Khattar et al., 2009, J. Virol.
83:7779-7782). In a specific embodiment, the NDV is attenuated by
deletion of the V protein. Such attenuated chimeric NDVs may be
especially suited for embodiments wherein the virus is administered
to a subject in order to act as an immunogen, e.g., a live vaccine.
The viruses may be attenuated by any method known in the art.
[0138] In certain embodiments, a chimeric NDV described herein
expresses one, two, three, or more, or all of the following, and a
suicide gene: (1) an agonist of a co-stimulatory signal of an
immune cell; (2) an antagonist of an inhibitory signal of an immune
cell; (3) a cytokine; (4) a tumor antigen; (5) a heterologous
interferon antagonist; (6) a pro-apoptotic molecule; (7) an
anti-apoptotic molecule; and/or (8) a mutated F protein. In
specific embodiments, in addition to expressing an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell, such as, e.g., a T-lymphocyte
or NK cell, and in certain embodiments, a mutated F protein and a
cytokine, a chimeric NDV is engineered to express a suicide gene
(e.g., thymidine kinase) or another molecule that inhibits NDV
replication or function (a gene that makes NDV sensitive to an
antibiotic or an anti-viral agent). In some embodiments, in
addition to expressing an agonist of a co-stimulatory signal of an
immune cell and/or an antagonist of an inhibitory signal of an
immune cell, such as, e.g., a T-lymphocyte or NK cell, and in
certain embodiments, a mutated F protein and a cytokine, a chimeric
NDV is engineered to encode tissue-specific microRNA (miRNA) target
sites (e.g., sites targeted by miR-21, miR-184, miR-133a/133b,
miR-137, and/or miR-193a microRNAs).
[0139] In certain embodiments, the tropism of the chimeric NDV is
altered. In a specific embodiment, the tropism of the virus is
altered by modification of the F protein cleavage site to be
recognized by tissue-specific or tumor-specific proteases such as
matrix metalloproteases (MMP) and urokinase. In other embodiments,
tropism of the virus is altered by introduction of tissue-specific
miRNA target sites. In certain embodiments, NDV HN protein is
mutated to recognize tumor-specific receptor.
[0140] In certain embodiments, one or more of the following are
expressed by a chimeric NDV as a chimeric protein or fusion
protein: (1) an agonist of a co-stimulatory signal of an immune
cell; (2) an antagonist of an inhibitory signal of an immune cell;
(3) a cytokine; (4) a tumor antigen; (5) a heterologous interferon
antagonist; (6) a pro-apoptotic molecule; (7) an anti-apoptotic
molecule; and/or (8) a mutated F protein. In specific embodiments,
the chimeric protein or fusion protein comprises the transmembrane
and cytoplasmic domains or fragments thereof of the NDV F or NDV HN
protein and an extracellular domain that comprises one of the
molecules referenced in the previous sentence. See U.S. Patent
Application No. 2012-0122185 for a description of such chimeric
proteins or fusion proteins, and International Application
Publication No. WO 2007/064802, which are incorporated herein by
reference.
[0141] In embodiments herein, the agonist of a co-stimulatory
signal and/or the antagonist of an inhibitory signal of an immune
cell may be inserted into the genome of the backbone NDV between
two transcription units. In a specific embodiment, the agonist of a
co-stimulatory signal and/or the antagonist of an inhibitory signal
of an immune cell is inserted into the genome of the backbone NDV
between the M and P transcription units or between the HN and L
transcription units. In accordance with other embodiments herein,
the cytokine, tumor antigen, heterologous interferon antagonist,
pro-apoptotic molecule, anti-apoptotic molecule and/or mutated F
protein are inserted into the genome of the backbone NDV between
two or more transcription units (e.g., between the M and P
transcription units or between the HN and L transcription
units).
[0142] 5.2.1. Immune Cell Stimulatory Agents
[0143] The chimeric NDVs described herein may be engineered to
express any agonist of a co-stimulatory signal and/or any
antagonist of an inhibitory signal of an immune cell, such as,
e.g., a T-lymphocyte, NK cell or antigen-presenting cell (e.g., a
dendritic cell or macrophage), known to one of skill in the art. In
specific embodiments, the agonist and/or antagonist is an agonist
of a human co-stimulatory signal of an immune cell and/or
antagonist of a human inhibitory signal of an immune cell. In
certain embodiments, the agonist of a co-stimulatory signal is an
agonist of a co-stimulatory molecule (e.g., co-stimulatory
receptor) found on immune cells, such as, e.g., T-lymphocytes
(e.g., CD4+ or CD8+ T-lymphocytes), NK cells and/or
antigen-presenting cells (e.g., dendritic cells or macrophages).
Specific examples of co-stimulatory molecules include
glucocorticoid-induced tumor necrosis factor receptor (GITR),
Inducible T-cell costimulator (ICOS or CD278), OX40 (CD134), CD27,
CD28, 4-1BB (CD137), CD40, lymphotoxin alpha (LT alpha), LIGHT
(lymphotoxin-like, exhibits inducible expression, and competes with
herpes simplex virus glycoprotein D for HVEM, a receptor expressed
by T lymphocytes), CD226, cytotoxic and regulatory T cell molecule
(CRTAM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR),
transmembrane activator and CAML interactor (TACI), B
cell-activating factor receptor (BAFFR), and B cell maturation
protein (BCMA). In specific embodiments, the agonist is an agonist
of a human co-stimulatory receptor of an immune cell. In certain
embodiments, the agonist of a co-stimulatory receptor is not an
agonist of ICOS. In some embodiments, the antagonist is an
antagonist of an inhibitory molecule (e.g., inhibitory receptor)
found on immune cells, such as, e.g., T-lymphocytes (e.g., CD4+ or
CD8+ T-lymphocytes), NK cells and/or antigen-presenting cells
(e.g., dendritic cells or macrophages). Specific examples of
inhibitory molecules include cytotoxic T-lymphocyte-associated
antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or
CD279), B and T-lymphocyte attenuator (BTLA), killer cell
immunoglobulin-like receptor (KIR), lymphocyte activation gene 3
(LAG3), T-cell membrane protein 3 (TIM3), CD160, adenosine A2a
receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM
domains (TIGIT), leukocyte-associated immunoglobulin-like receptor
1 (LAIR1), and CD160. In specific embodiments, the antagonist is an
antagonist of a human inhibitory receptor of an immune cell.
[0144] In a specific embodiment, the agonist of a co-stimulatory
receptor is an antibody or antigen-binding fragment thereof that
specifically binds to the co-stimulatory receptor. Specific
examples of co-stimulatory receptors include GITR, ICOS, OX40,
CD27, CD28, 4-1BB, CD40, LT alpha, LIGHT, CD226, CRTAM, DR3, LTBR,
TACI, BAFFR, and BCMA. In certain specific embodiments, the
antibody is a monoclonal antibody. In other specific embodiments,
the antibody is an sc-Fv. In a specific embodiment, the antibody is
a bispecific antibody that binds to two receptors on an immune
cell. In other embodiments, the bispecific antibody binds to a
receptor on an immune cell and to another receptor on a cancer
cell. In specific embodiments, the antibody is a human or humanized
antibody. In some embodiments, the antibody is expressed as a
chimeric protein with NDV F protein or fragment thereof, or NDV HN
protein or fragment thereof. See, e.g., U.S. patent application
Publication No. 2012/0122185, which is incorporated herein by
reference for a description regarding generation of chimeric F or
chimeric HN proteins. In a specific embodiment, the chimeric
protein is the chimeric F protein described in Sections 6 and/or 7,
infra. The techniques described below for generating the chimeric
ICOSL-F protein and the chimeric CD28-F protein can be used to
generate other chimeric F proteins or chimeric HN proteins.
[0145] In another embodiment, the agonist of a co-stimulatory
receptor is a ligand of the co-stimulatory receptor. In certain
embodiments, the ligand is fragment of a native ligand. Specific
examples of native ligands include ICOSL, B7RP1, CD137L, OX40L,
CD70, herpes virus entry mediator (HVEM), CD80, and CD86. The
nucleotide sequences encoding native ligands as well as the amino
acid sequences of native ligands are known in the art. For example,
the nucleotide and amino acid sequences of B7RP1 (otherwise known
as ICOSL; GenBank human: NM.sub.--015259.4, NP.sub.--056074.1
murine: NM.sub.--015790.3, NP.sub.--056605.1), CD137L (GenBank
human: NM.sub.--003811.3, NP.sub.--003802.1, murine:
NM.sub.--009404.3, NP.sub.--033430.1), OX40L (GenBank human:
NM.sub.--003326.3, NP.sub.--003317.1, murine: NM.sub.--009452.2,
NP.sub.--033478.1), CD70 (GenBank human: NM.sub.--001252.3,
NP.sub.--001243.1, murine: NM.sub.--011617.2, AAD00274.1), CD80
(GenBank human: NM.sub.--005191.3, NP.sub.--005182.1, murine:
NM.sub.--009855.2, NP.sub.--033985.3), and CD86 (GenBank human:
NM.sub.--005191.3, CAG46642.1, murine: NM.sub.--019388.3,
NP.sub.--062261.3) can be found in GenBank. In other embodiments,
the ligand is a derivative of a native ligand. In some embodiments,
the ligand is a fusion protein comprising at least a portion of the
native ligand or a derivative of the native ligand that
specifically binds to the co-stimulatory receptor, and a
heterologous amino acid sequence. In specific embodiments, the
fusion protein comprises at least a portion of the native ligand or
a derivative of the native ligand that specifically binds to the
co-stimulatory receptor, and the Fc portion of an immunoglobulin or
a fragment thereof. An example of a ligand fusion protein is a
4-1BB ligand fused to Fc portion of immunoglobulin (described by
Meseck M et al., J Immunother. 2011 34:175-82). In some
embodiments, the ligand is expressed as a chimeric protein with the
NDV F protein or fragment thereof, or NDV HN protein or fragment
thereof. In a specific embodiment, the protein is the chimeric HN
protein described in Section 7, infra. The techniques described
below for generating the chimeric HN-GITRL, chimeric HN-OX40-L,
chimeric HN-4-1BBL, and/or chimeric HN-CD40L can be used to
generate other chimeric F proteins or chimeric HN proteins.
[0146] In another embodiment, the antagonist of an inhibitory
receptor is an antibody (or an antigen-binding fragment) or a
soluble receptor that specifically binds to the native ligand for
the inhibitory receptor and blocks the native ligand from binding
to the inhibitory receptor and transducing an inhibitory signal(s).
Specific examples of native ligands for inhibitory receptors
include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, Gal9 and adenosine.
Specific examples of inhibitory receptors that bind to a native
ligand include CTLA-4, PD-1, BTLA, KIR, LAG3, TIM3, and A2aR.
[0147] In specific embodiments, the antagonist of an inhibitory
receptor is a soluble receptor that specifically binds to the
native ligand for the inhibitory receptor and blocks the native
ligand from binding to the inhibitory receptor and transducing an
inhibitory signal(s). In certain embodiments, the soluble receptor
is a fragment of a native inhibitory receptor or a fragment of a
derivative of a native inhibitory receptor that specifically binds
to native ligand (e.g., the extracellular domain of a native
inhibitory receptor or a derivative of an inhibitory receptor). In
some embodiments, the soluble receptor is a fusion protein
comprising at least a portion of the native inhibitory receptor or
a derivative of the native inhibitory receptor (e.g., the
extracellular domain of the native inhibitory receptor or a
derivative of the native inhibitory receptor), and a heterologous
amino acid sequence. In specific embodiments, the fusion protein
comprises at least a portion of the native inhibitory receptor or a
derivative of the native inhibitory receptor, and the Fc portion of
an immunoglobulin or a fragment thereof. An example of a soluble
receptor fusion protein is a LAG3-Ig fusion protein (described by
Huard B et al., Eur J Immunol. 1995 25:2718-21).
[0148] In specific embodiments, the antagonist of an inhibitory
receptor is an antibody (or an antigen-binding fragment) that
specifically binds to the native ligand for the inhibitory receptor
and blocks the native ligand from binding to the inhibitory
receptor and transducing an inhibitory signal(s). In certain
specific embodiments, the antibody is a monoclonal antibody. In
other specific embodiments, the antibody is an scFv. In particular
embodiments, the antibody is a human or humanized antibody. A
specific example of an antibody to inhibitory ligand is anti-PD-L1
antibody (Iwai Y, et al. PNAS 2002; 99:12293-12297).
[0149] In another embodiment, the antagonist of an inhibitory
receptor is an antibody (or an antigen-binding fragment) or ligand
that binds to the inhibitory receptor, but does not transduce an
inhibitory signal(s). Specific examples of inhibitory receptors
include CTLA-4, PD1, BTLA, KIR, LAG3, TIM3, and A2aR. In certain
specific embodiments, the antibody is a monoclonal antibody. In
other specific embodiments, the antibody is an scFv. In particular
embodiments, the antibody is a human or humanized antibody. A
specific example of an antibody to inhibitory receptor is
anti-CTLA-4 antibody (Leach D R, et al. Science 1996; 271:
1734-1736). Another example of an antibody to inhibitory receptor
is anti-PD-1 antibody (Topalian S L, NEJM 2012; 28:3167-75).
[0150] In certain embodiments, a chimeric NDV described herein is
engineered to an antagonist of CTLA-4, such as, e.g., Ipilimumab or
Tremelimumab. In certain embodiments, a chimeric NDV described
herein is engineered to an antagonist of PD1, such as, e.g.,
MDX-1106 (BMS-936558), MK3475, CT-011, AMP-224, or MDX-1105. In
certain embodiments, a chimeric NDV described herein is engineered
to express an antagonist of LAG3, such as, e.g., IMP321. In certain
embodiments, a chimeric NDV described herein is engineered to
express an antibody (e.g., a monoclonal antibody or an
antigen-binding fragment thereof, or scFv) that binds to B7-H3,
such as, e.g., MGA271. In specific embodiments, a chimeric NDV
described herein is engineered to express an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell described in Section 6 and/or
Section 7, infra. In specific embodiments, NDV described herein is
engineered to express anti-CD28 scvFv, ICOSL, CD40L, OX40L, CD137L,
GITRL, and/or CD70.
[0151] In certain embodiments, an agonist of a co-stimulatory
signal of an immune cell induces (e.g., selectively) induces one or
more of the signal transduction pathways induced by the binding of
a co-stimulatory receptor to its ligand. In specific embodiments,
an agonist of a co-stimulatory receptor induces one or more of the
signal transduction pathways induced by the binding of the
co-stimulatory receptor to one or more of its ligands by at least
25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in
the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to
95%, 75% to 95%, or 75% to 100% relative to the one or more signal
transduction pathways induced by the binding of the co-stimulatory
receptor to one or more of its ligands in the absence of the
agonist. In specific embodiments, an agonist of a co-stimulatory
receptor: (i) induces one or more of the signal transduction
pathways induced by the binding of the co-stimulatory receptor to
one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%,
80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to
50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100%
relative to the one or more signal transduction pathways induced by
the binding of the co-stimulatory receptor to the particular ligand
in the absence of the agonist; and (ii) does not induce, or induces
one or more of the signal transduction pathways induced by the
binding of the co-stimulatory receptor to one or more other ligands
by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between
2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%,
or 15% to 20% relative to the one or more signal transduction
pathways induced by the binding of the co-stimulatory receptor to
such one or more other ligands in the absence of the agonist.
[0152] In certain embodiments, an agonist of a co-stimulatory
signal of an immune cell activates or enhances (e.g., selectively
activates or enhances) one or more of the signal transduction
pathways induced by the binding of a co-stimulatory receptor to its
ligand. In specific embodiments, an agonist of a co-stimulatory
receptor activates or enhances one or more of the signal
transduction pathways induced by the binding of the co-stimulatory
receptor to one or more of its ligands by at least 25%, 30%, 40%,
50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of
between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%,
or 75% to 100% relative to the one or more signal transduction
pathways induced by the binding of co-stimulatory receptor to one
or more of its ligands in the absence of the agonist. In specific
embodiments, an agonist of a co-stimulatory receptor: (i) an
agonist of a co-stimulatory signal activates or enhances one or
more of the signal transduction pathways induced by the binding of
the co-stimulatory receptor to one particular ligand by at least
25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in
the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to
95%, 75% to 95%, or 75% to 100% relative to the one or more signal
transduction pathways induced by the binding of the co-stimulatory
receptor to the particular ligand in the absence of the agonist;
and (ii) does not activate or enhance, or activates or enhances one
or more of the signal transduction pathways induced by the binding
of the co-stimulatory receptor to one or more other ligands by less
than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%,
2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to
20% relative to the one or more signal transduction pathways
induced by the binding of the co-stimulatory receptor to such one
or more other ligands in the absence of the agonist.
[0153] In some embodiments, an antagonist of an inhibitory signal
of an immune cell (e.g., selectively) inhibits or reduces one or
more of the signal transduction pathways induced by the binding of
an inhibitory receptor to its ligand. In specific embodiments, an
antagonist of an inhibitory receptor inhibits or reduces one or
more of the signal transduction pathways induced by the binding of
the inhibitory receptor to one or more of its ligands by at least
25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in
the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to
95%, 75% to 95%, or 75% to 100% relative to the one or more signal
transduction pathways induced by the binding of the inhibitory
receptor to one or more of its ligands in the absence of the
antagonist. In specific embodiments, an antagonist of an inhibitory
receptor: (i) inhibits or reduces one or more of the signal
transduction pathways induced by the binding of the inhibitory
receptor to one particular ligand by at least 25%, 30%, 40%, 50%,
60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of
between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%,
or 75% to 100% relative to the one or more signal transduction
pathways induced by the binding of the inhibitory receptor to the
one particular ligand in the absence of the antagonist; and (ii)
does not inhibit or reduce, or inhibits or reduces one or more of
the signal transduction pathways induced by the binding of the
inhibitory receptor to one or more other ligands by less than 20%,
15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to
10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20%
relative to the one or more signal transduction pathways induced by
the binding of inhibitory receptor to such one or more other
ligands in the absence of the antagonist.
[0154] In specific embodiments, an agonist of a co-stimulatory
signal of an immune cell and/or an antagonist of an inhibitory
signal of an immune cell induces, activates and/or enhances one or
more immune activities, functions or responses. The one or more
immune activities, functions or responses can be in the form of,
e.g., an antibody response (humoral response) or a cellular immune
response, e.g., cytokine secretion (e.g., interferon-gamma), helper
activity or cellular cytotoxicity. In one embodiment, expression of
an activation marker on immune cells (e.g., CD44, Granzyme, or
Ki-67), expression of a co-stimulatory receptor on immune cells
(e.g., ICOS, CD28, OX40, or CD27), expression of a ligand for a
co-stimulatory receptor (e.g., B7HRP1, CD80, CD86, OX40L, or CD70),
cytokine secretion, infiltration of immune cells (e.g.,
T-lymphocytes, B lymphocytes and/or NK cells) to a tumor, antibody
production, effector function, T cell activation, T cell
differentiation, T cell proliferation, B cell differentiation, B
cell proliferation, and/or NK cell proliferation is induced,
activated and/or enhanced following contact with an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune cell. In another embodiment,
myeloid-derived suppressor cell (MDSC) tumor infiltration and
proliferation, Treg tumor infiltration, activation and
proliferation, peripheral blood MDSC and Treg counts are inhibited
following contact with an agonist of a co-stimulatory signal of an
immune cell and/or an antagonist of an inhibitory signal of an
immune cell.
[0155] 5.3 Construction of NDVS
[0156] The NDVs described herein can be generated using the reverse
genetics technique. The reverse genetics technique involves the
preparation of synthetic recombinant viral RNAs that contain the
non-coding regions of the negative-strand, viral RNA which are
essential for the recognition by viral polymerases and for
packaging signals necessary to generate a mature virion. The
recombinant RNAs are synthesized from a recombinant DNA template
and reconstituted in vitro with purified viral polymerase complex
to form recombinant ribonucleoproteins (RNPs) which can be used to
transfect cells. A more efficient transfection is achieved if the
viral polymerase proteins are present during transcription of the
synthetic RNAs either in vitro or in vivo. The synthetic
recombinant RNPs can be rescued into infectious virus particles.
The foregoing techniques are described in U.S. Pat. No. 5,166,057
issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec. 29,
1998; in U.S. Pat. No. 6,146,642 issued Nov. 14, 2000; in European
Patent Publication EP 0702085A1, published Feb. 20, 1996; in U.S.
patent application Ser. No. 09/152,845; in International Patent
Publications PCT WO97/12032 published Apr. 3, 1997; WO96/34625
published Nov. 7, 1996; in European Patent Publication EP A780475;
WO 99/02657 published Jan. 21, 1999; WO 98/53078 published Nov. 26,
1998; WO 98/02530 published Jan. 22, 1998; WO 99/15672 published
Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998; WO 97/06270
published Feb. 20, 1997; and EPO 780 475A1 published Jun. 25, 1997,
each of which is incorporated by reference herein in its
entirety.
[0157] The helper-free plasmid technology can also be utilized to
engineer a NDV described herein. Briefly, a complete cDNA of a NDV
(e.g., the Hitchner B1 strain) is constructed, inserted into a
plasmid vector and engineered to contain a unique restriction site
between two transcription units (e.g., the NDV P and M genes; or
the NDV HN and L genes). A nucleotide sequence encoding a
heterologous amino acid sequence (e.g., a nucleotide sequence
encoding an agonist of a co-stimulatory signal and/or an antagonist
of an inhibitory signal of an immune cell) may be inserted into the
viral genome at the unique restriction site. Alternatively, a
nucleotide sequence encoding a heterologous amino acid sequence
(e.g., a nucleotide sequence encoding an agonist of a
co-stimulatory signal and/or an antagonist of an inhibitory signal
of an immune cell) may be engineered into a NDV transcription unit
so long as the insertion does not affect the ability of the virus
to infect and replicate. The single segment is positioned between a
T7 promoter and the hepatitis delta virus ribozyme to produce an
exact negative transcript from the T7 polymerase. The plasmid
vector and expression vectors comprising the necessary viral
proteins are transfected into cells leading to production of
recombinant viral particles (see, e.g., International Publication
No. WO 01/04333; U.S. Pat. Nos. 7,442,379, 6,146,642, 6,649,372,
6,544,785 and 7,384,774; Swayne et al. (2003). Avian Dis.
47:1047-1050; and Swayne et al. (2001). J. Virol. 11868-11873, each
of which is incorporated by reference in its entirety).
[0158] Techniques for the production of a chimeric NDV that express
an antibody are known in the art. See, e.g., Puhler et al., Gene
Ther. 15(5): 371-283 (2008) for the generation of a recombinant NDV
expressing a full IgG from two transgenes.
[0159] Bicistronic techniques to produce multiple proteins from a
single mRNA are known to one of skill in the art. Bicistronic
techniques allow the engineering of coding sequences of multiple
proteins into a single mRNA through the use of IRES sequences. IRES
sequences direct the internal recruitment of ribozomes to the RNA
molecule and allow downstream translation in a cap independent
manner. Briefly, a coding region of one protein is inserted into
the ORF of a second protein. The insertion is flanked by an IRES
and any untranslated signal sequences necessary for proper
expression and/or function. The insertion must not disrupt the open
reading frame, polyadenylation or transcriptional promoters of the
second protein (see e.g., Garcia-Sastre et al., 1994, J. Virol.
68:6254-6261 and Garcia-Sastre et al., 1994 Dev. Biol. Stand.
82:237-246, each of which are incorporated by reference herein in
their entirety).
[0160] 5.4 Propagation of NDVS
[0161] The NDVs described herein (e.g., the chimeric NDVs) can be
propagated in any substrate that allows the virus to grow to titers
that permit the uses of the viruses described herein. In one
embodiment, the substrate allows the NDVs described herein (e.g.,
the chimeric NDVs) to grow to titers comparable to those determined
for the corresponding wild-type viruses.
[0162] The NDVs described herein (e.g., the chimeric NDVs) may be
grown in cells (e.g., avian cells, chicken cells, etc.) that are
susceptible to infection by the viruses, embryonated eggs (e.g.,
chicken eggs or quail eggs) or animals (e.g., birds). Such methods
are well-known to those skilled in the art. In a specific
embodiment, the NDVs described herein (e.g., the chimeric NDVs) may
be propagated in cancer cells, e.g., carcinoma cells (e.g., breast
cancer cells and prostate cancer cells), sarcoma cells, leukemia
cells, lymphoma cells, and germ cell tumor cells (e.g., testicular
cancer cells and ovarian cancer cells). In another specific
embodiment, the NDVs described herein (e.g., the chimeric NDVs) may
be propagated in cell lines, e.g., cancer cell lines such as HeLa
cells, MCF7 cells, THP-1 cells, U87 cells, DU145 cells, Lncap
cells, and T47D cells. In certain embodiments, the cells or cell
lines (e.g., cancer cells or cancer cell lines) are obtained and/or
derived from a human(s). In another embodiment, the NDVs described
herein (e.g., the chimeric NDVs) are propagated in chicken cells or
embryonated eggs. Representative chicken cells include, but are not
limited to, chicken embryo fibroblasts and chicken embryo kidney
cells. In a specific embodiment, the NDVs described herein (e.g.,
the chimeric NDVs) are propagated in Vero cells. In another
specific embodiment, the NDVs described herein (e.g., the chimeric
NDVs) are propagated in cancer cells in accordance with the methods
described in Section 6 and/or Section 7, infra. In another specific
embodiment, the NDVs described herein (e.g., the chimeric NDVs) are
propagated in chicken eggs or quail eggs. In certain embodiments, a
NDV virus described herein (e.g., a chimeric NDV) is first
propagated in embryonated eggs and then propagated in cells (e.g.,
a cell line).
[0163] The NDVs described herein (e.g., the chimeric NDVs) may be
propagated in embryonated eggs, e.g., from 6 to 14 days old, 6 to
12 days old, 6 to 10 days old, 6 to 9 days old, 6 to 8 days old, or
10 to 12 days old. Young or immature embryonated eggs can be used
to propagate the NDVs described herein (e.g., the chimeric NDVs).
Immature embryonated eggs encompass eggs which are less than ten
day old eggs, e.g., eggs 6 to 9 days old or 6 to 8 days old that
are IFN-deficient. Immature embryonated eggs also encompass eggs
which artificially mimic immature eggs up to, but less than ten day
old, as a result of alterations to the growth conditions, e.g.,
changes in incubation temperatures; treating with drugs; or any
other alteration which results in an egg with a retarded
development, such that the IFN system is not fully developed as
compared with ten to twelve day old eggs. The NDVs described herein
(e.g., the chimeric NDVs) can be propagated in different locations
of the embryonated egg, e.g., the allantoic cavity. For a detailed
discussion on the growth and propagation viruses, see, e.g., U.S.
Pat. No. 6,852,522 and U.S. Pat. No. 7,494,808, both of which are
hereby incorporated by reference in their entireties.
[0164] For virus isolation, the NDVs described herein (e.g., the
chimeric NDVs) can be removed from cell culture and separated from
cellular components, typically by well known clarification
procedures, e.g., such as gradient centrifugation and column
chromatography, and may be further purified as desired using
procedures well known to those skilled in the art, e.g., plaque
assays.
[0165] 5.5 Compositions & Routes of Administration
[0166] Encompassed herein is the use of a NDV described herein
(e.g., the chimeric NDVs) in compositions. Also encompassed herein
is the use of plasma membrane fragments from NDV infected cells or
whole cancer cells infected with NDV in compositions. In a specific
embodiment, the compositions are pharmaceutical compositions, such
as immunogenic formulations (e.g., vaccine formulations). The
compositions may be used in methods of treating cancer.
[0167] In one embodiment, a pharmaceutical composition comprises a
NDV described herein (e.g., the chimeric NDVs), in an admixture
with a pharmaceutically acceptable carrier. In some embodiments,
the pharmaceutical composition further comprises one or more
additional prophylactic or therapeutic agents, such as described in
Section 5.6.4, infra. In a specific embodiment, a pharmaceutical
composition comprises an effective amount of a NDV described herein
(e.g., the chimeric NDVs), and optionally one or more additional
prophylactic of therapeutic agents, in a pharmaceutically
acceptable carrier. In some embodiments, the NDV (e.g., a chimeric
NDV) is the only active ingredient included in the pharmaceutical
composition.
[0168] In another embodiment, a pharmaceutical composition (e.g.,
an oncolysate vaccine) comprises a protein concentrate or a
preparation of plasma membrane fragments from NDV infected cancer
cells, in an admixture with a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition further
comprises one or more additional prophylactic or therapeutic
agents, such as described in Section 5.6.4, infra. In another
embodiment, a pharmaceutical composition (e.g., a whole cell
vaccine) comprises cancer cells infected with NDV, in an admixture
with a pharmaceutically acceptable carrier. In some embodiments,
the pharmaceutical composition further comprises one or more
additional prophylactic or therapeutic agents, such as described in
Section 5.6.4, infra.
[0169] The pharmaceutical compositions provided herein can be in
any form that allows for the composition to be administered to a
subject. In a specific embodiment, the pharmaceutical compositions
are suitable for veterinary and/or human administration. As used
herein, the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeiae
for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the pharmaceutical composition is administered. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable excipients include starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like. Examples
of suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. The formulation should
suit the mode of administration.
[0170] In a specific embodiment, the pharmaceutical compositions
are formulated to be suitable for the intended route of
administration to a subject. For example, the pharmaceutical
composition may be formulated to be suitable for parenteral,
intravenous, intraarterial, intrapleural, inhalation,
intraperitoneal, oral, intradermal, colorectal, intraperitoneal,
intracranial, and intratumoral administration. In a specific
embodiment, the pharmaceutical composition may be formulated for
intravenous, intraarterial, oral, intraperitoneal, intranasal,
intratracheal, intrapleural, intracranial, subcutaneous,
intramuscular, topical, pulmonary, or intratumoral
administration.
[0171] 5.6 Anti-Cancer Uses and Other Uses
[0172] In one aspect, a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra) may be used in the
treatment of cancer. In one embodiment, provided herein are methods
for treating cancer, comprising administering to a subject in need
thereof a chimeric NDV described herein (e.g., a chimeric NDV
described in Section 5.2, supra) or a composition thereof. In a
specific embodiment, provided herein is a method for treating
cancer, comprising administering to a subject in need thereof an
effective amount of a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra) or a composition
thereof.
[0173] In specific embodiments, a chimeric NDV engineered to
express an agonist of a co-stimulatory signal of an immune cell, or
a composition thereof is administered to a subject to treat cancer.
In another specific embodiments, a chimeric NDV engineered to
express an antagonist of an inhibitory signal of an immune cell, or
a composition thereof is administered to a subject to treat cancer.
In certain embodiments, a chimeric NDV engineered to express an
agonist of a co-stimulatory signal of an immune cell and a mutated
F protein or a composition thereof is administered to a subject to
treat cancer. In certain embodiments, a chimeric NDV engineered to
express an antagonist of an inhibitory signal of an immune cell and
a mutated F protein or a composition thereof is administered to a
subject to treat cancer.
[0174] A chimeric NDV (e.g., a chimeric NDV described in Section
5.2, supra) described herein or a composition thereof, an
oncolysate vaccine, or a whole cell cancer vaccine used in a method
for treating cancer may be used as any line of therapy (e.g., a
first, second, third, fourth or fifth line therapy).
[0175] In certain embodiments, a chimeric NDV described herein
(e.g., a chimeric NDV described in Section 5.2, supra) is the only
active ingredient administered to treat cancer. In specific
embodiments, a chimeric NDV described herein (e.g., a chimeric NDV
described in Section 5.2, supra) is the only active ingredient in a
composition administered to treat cancer.
[0176] The chimeric NDV (e.g., a chimeric NDV described in Section
5.2, supra) or a composition thereof may be administered locally or
systemically to a subject. For example, the chimeric NDV (e.g., a
chimeric NDV described in Section 5.2, supra) or a composition
thereof may be administered parenterally (e.g., intravenously,
intraarterially, or subcutaneously), intratumorally,
intrapleurally, intranasally, intraperitoneally, intracranially,
orally, rectally, by inhalation, intramuscularly, topically or
intradermally to a subject. In a specific embodiment, the chimeric
NDV is administered via the hepatic artery, by, e.g., hepatic
artery injection, which can be performed by interventional
radiology or through placement of an arterial infusion pump. In
another specific embodiment, the chimeric NDV is administered
intraoperatively, laparoscopically, or endoscopically. In a
specific embodiment, intraperitoneal administration of the chimeric
NDV is performed by direct injection, infusion via catheter, or
injection during laparoscopy.
[0177] In certain embodiments, the methods described herein include
the treatment of cancer for which no treatment is available. In
some embodiments, a chimeric NDV described herein (e.g., a chimeric
NDV described in Section 5.2, supra) or a composition thereof is
administered to a subject to treat cancer as an alternative to
other conventional therapies.
[0178] In one embodiment, provided herein is a method for treating
cancer, comprising administering to a subject in need thereof a
chimeric NDV described herein (e.g., a chimeric NDV described in
Section 5.2, supra) or a composition thereof and one or more
additional therapies, such as described in Section 5.6.4, infra. In
a particular embodiment, one or more therapies are administered to
a subject in combination with a chimeric NDV described herein
(e.g., a chimeric NDV described in Section 5.2, supra) or a
composition thereof to treat cancer. In a specific embodiment, the
additional therapies are currently being used, have been used or
are known to be useful in treating cancer. In another embodiment, a
chimeric NDV described herein (e.g., a chimeric NDV described in
Section 5.2, supra) or a composition thereof is administered to a
subject in combination with a supportive therapy, a pain relief
therapy, or other therapy that does not have a therapeutic effect
on cancer. In a specific embodiment, the one or more additional
therapies administered in combination with a chimeric NDV described
herein (e.g., a chimeric NDV described in Section 5.2, supra) is
one or more of the therapies described in Section 5.6.4.1, infra.
In certain embodiments, a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra) and one or more
additional therapies are administered in the same composition. In
other embodiments, a chimeric NDV and one or more additional
therapies are administered in different compositions.
[0179] In certain embodiments, two, three or multiple NDVs
(including one, two or more chimeric NDVs described herein, such as
one, two or more of the chimeric NDVs described in Section 5.2,
supra) are administered to a subject to treat cancer. The second or
more chimeric NDVs used in accordance with methods described herein
that comprise administration of two, three or multiple NDVs to a
subject to treat cancer may be naturally occurring chimeric NDVs or
engineered chimeric NDVs that have been engineered to express
heterologous amino acid sequence (e.g., a cytokine) The first and
second chimeric NDVs may be part of the same pharmaceutical
composition or different pharmaceutical compositions. In certain
embodiments, the first chimeric NDV and the second chimeric NDV are
administered by the same route of administration (e.g., both are
administered intratumorally or intravenously). In other
embodiments, the first chimeric NDV and the second chimeric NDV are
administered by different routes of administration (e.g., one is
administered intratumorally and the other is administered
intravenously).
[0180] In specific embodiments, a first chimeric NDV engineered to
express an agonist of a co-stimulatory signal of an immune cell is
administered to a patient to treat cancer in combination with a
second chimeric NDV engineered to express an antagonist of an
inhibitory signal of an immune cell. In other specific embodiments,
a first chimeric NDV engineered to express an agonist of a
co-stimulatory signal of an immune cell and/or an antagonist of an
inhibitory signal of an immune is administered in combination with
a second chimeric NDV engineered to express one, two or more of the
following: a cytokine (e.g., IL-2), a heterologous interferon
antagonist, a tumor antigen, a pro-apoptotic molecule, and/or
anti-apoptotic molecule. In a specific embodiment, the first
chimeric NDV, the second chimeric NDV, or both express a mutated F
protein that increases the fusogenic activity of the chimeric NDV.
In another specific embodiment, the first chimeric NDV, the second
chimeric NDV or both express a mutated F protein with a mutation in
the cleavage site (such as described herein).
[0181] In specific embodiments, a first composition (e.g., a
pharmaceutical composition) comprising a first chimeric NDV
engineered to express an agonist of a co-stimulatory signal of an
immune cell is administered to a patient to treat cancer in
combination with a second composition (e.g., a pharmaceutical
composition) comprising a second chimeric NDV engineered to express
an antagonist of an inhibitory signal of an immune cell. In other
specific embodiments, a first composition (e.g., a pharmaceutical
composition) comprising a first chimeric NDV engineered to express
an agonist of a co-stimulatory signal of an immune cell and/or an
antagonist of an inhibitory signal of an immune is administered in
combination with a second composition (e.g., a pharmaceutical
composition) comprising a second chimeric NDV engineered to express
one, two or more of the following: a cytokine (e.g., IL-2), a
heterologous interferon antagonist, a tumor antigen, a
pro-apoptotic molecule, and/or anti-apoptotic molecule. In a
specific embodiment, the first chimeric NDV, the second chimeric
NDV, or both express a mutated F protein that increases the
fusogenic activity of the chimeric NDV. In another specific
embodiment, the first chimeric NDV, the second chimeric NDV or both
express a mutated F protein with a mutation in the cleavage site
(such as described herein).
[0182] In another aspect, an NDV described herein (e.g., an NDV
described in Section 5.1, supra) may be used in combination with
one or more additional therapies, such as described herein in
Section 5.6.4, infra (e.g., Section 5.6.4.1, infra), in the
treatment of cancer. In one embodiment, provided herein are methods
for treating cancer, comprising administering to a subject in need
thereof an NDV described herein (e.g., an NDV described in Section
5.1, supra) or a composition thereof and one or more additional
therapies, such as described herein in Section 5.6.4, infra. (e.g.,
Section 5.6.4.1). In a specific embodiment, provided herein is a
method for treating cancer, comprising administering to a subject
in need thereof an effective amount of an NDV described herein
(e.g., an NDV described in Section 5.1, supra) or a composition
thereof and an effective amount of one or more additional
therapies, such as described in Section 5.6.4, infra. (e.g.,
Section 5.6.4.1). In certain embodiments, an NDV described herein
(e.g., an NDV described in Section 5.1, supra) and one or more
additional therapies, such as described in Section 5.6.4, infra
(e.g., Section 5.6.4.1), are administered in the same composition.
In other embodiments, an NDV (e.g., an NDV described in Section
5.1, supra) and one or more additional therapies are administered
in different compositions.
[0183] The NDV used in combination with one ore more additional
therapies can be administered systemically or locally. For example,
the NDV or composition thereof may be administered parenterally
(e.g., intravenously, intraarterially, or subcutaneously),
intratumorally, intrapleurally, intranasally, intraperitoneally,
intracranially, orally, rectally, by inhalation, intramuscularly,
topically or intradermally to a subject. In a specific embodiment,
the NDV is administered via the hepatic artery, by, e.g., hepatic
artery injection, which can be performed by interventional
radiology or through placement of an arterial infusion pump. In
another specific embodiment, the NDV is administered
intraoperatively, laparoscopically, or endoscopically. In a
specific embodiment, intraperitoneal administration of the NDV is
performed by direct injection, infusion via catheter, or injection
during laparoscopy.
[0184] An NDV (e.g., an NDV described in Section 5.1, supra)
described herein or a composition thereof, an oncolysate vaccine,
or a whole cell cancer vaccine in combination with one or more
additional therapies, such as described herein in Section 5.6.4,
infra, may be used as any line of therapy (e.g., a first, second,
third, fourth or fifth line therapy) for treating cancer in
accordance with a method described herein.
[0185] In another aspect, whole cancer cells infected with a
chimeric NDV described herein (e.g., a chimeric NDV described in
Section 5.2, supra) can be used to treat cancer. In a specific
embodiment, a chimeric NDV described herein (e.g., a chimeric NDV
described in Section 5.2, supra) may be contacted with a cancer
cell or a population of cancer cells and the infected cancer cell
or population of cancer cells may be administered to a subject to
treat cancer. In one embodiment, the cancer cells are subjected to
gamma radiation prior to infection with a chimeric NDV described
herein (e.g., a chimeric NDV described in Section 5.2, supra). In
another embodiment, the cancer cells are subjected to gamma
radiation after infection with a chimeric NDV described herein
(e.g., a chimeric NDV described in Section 5.2, supra). In a
particular embodiment, the cancer cells are treated prior to
administration to a subject so that the cancer cells cannot
multiply in the subject. In a specific embodiment, the cancer cells
cannot multiply in the subject and the virus cannot infect the
subject. In one embodiment, the cancer cells are subjected to gamma
radiation prior to administration to subject. In another
embodiment, the cancer cells are sonicated prior to administration
to a subject. In another embodiment, the cancer cells are treated
with mitomycin C prior to administration to a subject. In another
embodiment, the cancer cells are treated by freezing and thawing
prior to administration to a subject. In another embodiment, the
cancer cells are treated with heat treatment prior to
administration to a subject. The cancer cells may be administered
locally or systemically to a subject. For example, the cancer cells
may be administered parenterally (e.g., intravenously or
subcutaneously), intratumorally, intranasally, orally, by
inhalation, intrapleurally, topically or intradermally to a
subject. In a specific embodiment, the cancer cells are
administered intratumorally or to the skin (e.g., intradermally) of
a subject. The cancer cells used may be autologous or allogeneic.
In a specific embodiment, the backbone of the chimeric NDV is a
non-lytic strain. The cancer cells may be administered to a subject
alone or in combination with an additional therapy. The cancer
cells are preferably in a pharmaceutical composition. In certain
embodiments, the cancer cells are administered in combination with
one or more additional therapies, such as described in Section
5.6.4, infra. In certain embodiments, the cancer cells and one or
more additional therapies are administered in the same composition.
In other embodiments, the cancer cells and one or more additional
therapies are administered in different compositions.
[0186] In another aspect, whole cancer cells infected with an NDV
described herein (e.g., an NDV described in Section 5.1, supra) may
be used in combination with one or more additional therapies
described herein in Section 5.6.4, infra, in the treatment of
cancer. In one embodiment, provided herein are methods for treating
cancer, comprising administering to a subject in need thereof whole
cancer cells infected with an NDV described herein (e.g., an NDV
described in Section 5.1, supra) in combination with one or more
additional therapies described herein in Section 5.6.4, infra. In a
specific embodiment, provided herein is a method for treating
cancer, comprising administering to a subject in need thereof an
effective amount of whole cancer cells infected with an NDV
described herein (e.g., an NDV described in Section 5.1, supra) in
combination with an effective amount of one or more additional
therapies described in Section 5.6.4, infra. In certain
embodiments, whole cancer cells infected with an NDV described
herein (e.g., an NDV described in Section 5.1, supra) and one or
more additional therapies described in Section 5.6.4, infra, are
administered in the same composition. In other embodiments, whole
cancer cells infected with an NDV described herein (e.g., an NDV
described in Section 5.1, supra) and one or more additional
therapies are administered in different compositions.
[0187] In another aspect, a protein concentrate or plasma membrane
preparation from lysed cancer cells infected with a chimeric NDV
(e.g., a chimeric NDV described in Section 5.2, supra) can be used
to treat cancer. In one embodiment, a plasma membrane preparation
comprising fragments from cancer cells infected with a chimeric NDV
described herein can be used to treat cancer. In another
embodiment, a protein concentrate from cancer cells infected with a
chimeric NDV described herein can be used to treat cancer.
Techniques known to one of skill in the art may be used to produce
the protein concentrate or plasma membrane preparation. In a
specific embodiment, a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra) may be contacted with
a cancer cell or a population of cancer cells and the infected
cancer cell or population of cancer cells may be lysed using
techniques known to one of skill in the art to obtain protein
concentrate or plasma membrane fragments of the NDV-infected cancer
cells, and the protein concentrate or plasma membrane fragments of
the NDV-infected cancer cells may be administered to a subject to
treat cancer. The protein concentrate or plasma membrane fragments
may be administered locally or systemically to a subject. For
example, the protein concentrate or plasma membrane fragments may
be administered parenterally, intratumorally, intranasally,
intrapleurally, orally, by inhalation, topically or intradermally
to a subject. In a specific embodiment, such a protein concentrate
or plasma membrane preparation is administered intratumorally or to
the skin (e.g., intradermally) of a subject. The cancer cells used
to produce the protein concentrate or plasma membrane preparation
may be autologous or allogeneic. In a specific embodiment, the
backbone of the chimeric NDV is a lytic strain. The protein
concentrate or plasma membrane preparation may be administered to a
subject alone or in combination with an additional therapy. The
protein concentrate or plasma membrane preparation is preferably in
a pharmaceutical composition. In certain embodiments, the protein
concentrate or plasma membrane preparation is administered in
combination with one or more additional therapies, such as
described in Section 5.6.4, infra (e.g., Section 5.6.4.1) In
certain embodiments, the protein concentrate or plasma membrane
preparation and one or more additional therapies are administered
in the same composition. In other embodiments, the protein
concentrate or plasma membrane preparation and one or more
additional therapies are administered in different
compositions.
[0188] In another aspect, a protein concentrate or plasma membrane
preparation from lysed cancer cells infected with an NDV (e.g., an
NDV described in Section 5.1, supra) may be used in combination
with one or more additional therapies, such as described herein in
Section 5.6.4, infra (e.g., Section 5.6.4.1), in the treatment of
cancer. In one embodiment, provided herein are methods for treating
cancer, comprising administering to a subject in need thereof a
protein concentrate or plasma membrane preparation from lysed
cancer cells infected with an NDV (e.g., an NDV described in
Section 5.1, supra) in combination with one or more additional
therapies, such as described herein in Section 5.6.4, infra. (e.g.,
Section 5.6.4.1). In a specific embodiment, provided herein is a
method for treating cancer, comprising administering to a subject
in need thereof an effective amount of a protein concentrate or
plasma membrane preparation from lysed cancer cells infected with
an NDV (e.g., an NDV described in Section 5.1, supra) in
combination with an effective amount of one or more additional
therapies, such as described in Section 5.6.4, infra. (e.g.,
Section 5.6.4.1). In certain embodiments, the protein concentrate
or plasma membrane preparation and one or more additional
therapies, such as described in Section 5.6.4, infra, are
administered in the same composition. In other embodiments, the
protein concentrate or plasma membrane preparation and one or more
additional therapies are administered in different
compositions.
[0189] In another aspect, the chimeric NDVs described herein (e.g.,
a chimeric NDV described in Section 5.2, supra) can be used to
produce antibodies which can be used in diagnostic immunoassays,
passive immunotherapy, and the generation of antiidiotypic
antibodies. For example, a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra) can be administered
to a subject (e.g., a mouse, rat, pig, horse, donkey, bird or
human) to generate antibodies which can then be isolated and used,
e.g., in diagnostic assays, passive immunotherapy and generation of
antiidiotypic antibodies. In certain embodiments, an NDV described
herein (e.g., an NDV described in Section 5.1 or 5.2, supra) is
administered to a subject (e.g., a mouse, rat, pig, horse, donkey,
bird, or human) in combination with one or more additional
therapies, such as described in Section 5.6.4, infra, to generated
antibodies which can then be isolated and used, e.g., in diagnostic
assays, passive immunotherapy and generation of antiidiotypic
antibodies. The generated antibodies may be isolated by standard
techniques known in the art (e.g., immunoaffinity chromatography,
centrifugation, precipitation, etc.) and used in diagnostic
immunoassays, passive immunotherapy and generation of antiidiotypic
antibodies.
[0190] In certain embodiments, the antibodies isolated from
subjects administered a chimeric NDV described herein (e.g., a
chimeric NDV described in Section 5.2, supra), or isolated from
subjects administered an NDV described herein (e.g., an NDV
described in Section 5.1 or 5.2, supra) in combination with one or
more additional therapies, such as described in Section 5.6.4,
infra, are used to assess the expression of NDV proteins, a
heterologous peptide or protein expressed by a chimeric NDV, or
both. Any immunoassay system known in the art may be used for this
purpose including but not limited to competitive and noncompetitive
assay systems using techniques such as radioimmunoassays, ELISA
(enzyme linked immunosorbent assays), "sandwich" immunoassays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays and immunoelectrophoresis assays, to name but a
few.
[0191] 5.6.1. Patient Population
[0192] In some embodiments, an NDV (e.g., a chimeric NDV) described
herein or a composition thereof, an oncolysate vaccine described
herein, or a whole cell vaccine described herein, or a combination
therapy described herein is administered to a subject suffering
from cancer. In other embodiments, an NDV (e.g., a chimeric NDV)
described herein or a composition thereof, an oncolysate vaccine
described herein, or a whole cell vaccine described herein, or a
combination therapy described herein is administered to a subject
predisposed or susceptible to cancer. In some embodiments, an NDV
(e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein is administered to a
subject diagnosed with cancer. Specific examples of the types of
cancer are described herein. In an embodiment, the subject has
metastatic cancer. In another embodiment, the subject has stage 1,
stage 2, stage 3, or stage 4 cancer. In another embodiment, the
subject is in remission. In yet another embodiment, the subject has
a recurrence of cancer.
[0193] In certain embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof, an oncolysate vaccine described herein, or a
whole cell vaccine described herein, or a combination therapy
described herein is administered to a human that is 0 to 6 months
old, 6 to 12 months old, 6 to 18 months old, 18 to 36 months old, 1
to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20
years old, 20 to 25 years old, 25 to 30 years old, 30 to 35 years
old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50
to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70
years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years
old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.
In some embodiments, an NDV (e.g., a chimeric NDV) or a composition
thereof, an oncolysate vaccine described herein, or a whole cell
vaccine described herein, or a combination therapy described herein
is administered to a human infant. In other embodiments, an NDV
(e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein is administered to a
human toddler. In other embodiments, an NDV (e.g., a chimeric NDV)
or a composition thereof, an oncolysate vaccine described herein,
or a whole cell vaccine described herein, or a combination therapy
described herein is administered to a human child. In other
embodiments, an NDV (e.g., a chimeric NDV) or a composition
thereof, an oncolysate vaccine described herein, or a whole cell
vaccine described herein, or a combination therapy described herein
is administered to a human adult. In yet other embodiments, an NDV
(e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein is administered to an
elderly human.
[0194] In certain embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof, an oncolysate vaccine described herein, or a
whole cell vaccine described herein, or a combination therapy
described herein is administered to a subject in an
immunocompromised state or immunosuppressed state or at risk for
becoming immunocompromised or immunosuppressed. In certain
embodiments, an NDV (e.g., a chimeric NDV) or a composition
thereof, an oncolysate vaccine described herein, or a whole cell
vaccine described herein, or a combination therapy described herein
is administered to a subject receiving or recovering from
immunosuppressive therapy. In certain embodiments, an NDV (e.g., a
chimeric NDV) or a composition thereof, an oncolysate vaccine
described herein, or a whole cell vaccine described herein, or a
combination therapy described herein is administered to a subject
that has or is at risk of getting cancer. In certain embodiments,
the subject is, will or has undergone surgery, chemotherapy and/or
radiation therapy. In certain embodiments, the patient has
undergone surgery to remove the tumor or neoplasm. In specific
embodiments, the patient is administered an NDV (e.g., a chimeric
NDV) or a composition thereof, an oncolysate vaccine described
herein, or a whole cell vaccine described herein, or a combination
therapy described herein following surgery to remove a tumor or
neoplasm. In other embodiment, the patient is administered an NDV
(e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein prior to undergoing
surgery to remove a tumor or neoplasm. In certain embodiments, an
NDV (e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein is administered to a
subject that has, will have or had a tissue transplant, organ
transplant or transfusion.
[0195] In some embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof, an oncolysate vaccine described herein, or a
whole cell vaccine described herein, or a combination therapy
described herein is administered to a patient who has proven
refractory to therapies other than the chimeric NDV or composition
thereof, oncolysate, whole cell vaccine, or a combination therapy
but are no longer on these therapies. In a specific embodiment, an
NDV (e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein is administered to a
patient who has proven refractory to chemotherapy. In one
embodiment, that a cancer is refractory to a therapy means that at
least some significant portion of the cancer cells are not killed
or their cell division arrested. The determination of whether the
cancer cells are refractory can be made either in vivo or in vitro
by any method known in the art for assaying the effect of a therapy
on cancer cells, using the art-accepted meanings of "refractory" in
such a context. In a certain embodiment, refractory patient is a
patient refractory to a standard therapy. In certain embodiments, a
patient with cancer, is refractory to a therapy when the tumor or
neoplasm has not significantly been eradicated and/or the symptoms
have not been significantly alleviated. The determination of
whether a patient is refractory can be made either in vivo or in
vitro by any method known in the art for assaying the effectiveness
of a treatment of cancer, using art-accepted meanings of
"refractory" in such a context.
[0196] In certain embodiments, the patient to be treated in
accordance with the methods described herein is a patient already
being treated with antibiotics, anti-virals, anti-fungals, or other
biological therapy/immunotherapy or anti-cancer therapy. Among
these patients are refractory patients, and patients who are too
young for conventional therapies. In some embodiments, the subject
being administered an NDV (e.g., a chimeric NDV), an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein has not received therapy
prior to the administration of the chimeric NDV or composition, the
oncolysate vaccine, or the whole cell vaccine, or the combination
therapy.
[0197] In some embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof, an oncolysate vaccine described herein, or a
whole cell vaccine described herein, or a combination therapy
described herein is administered to a patient to prevent the onset
of cancer in a patient at risk of developing cancer. In some
embodiments, compounds are administered to a patient who are
susceptible to adverse reactions to conventional therapies.
[0198] In some embodiments, the subject being administered an NDV
(e.g., a chimeric NDV) or a composition thereof, an oncolysate
vaccine described herein, or a whole cell vaccine described herein,
or a combination therapy described herein has not received prior
therapy. In other embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof, an oncolysate vaccine described herein, or a
whole cell vaccine described herein, or a combination therapy
described herein is administered to a subject who has received a
therapy prior to administration of the NDV (e.g., a chimeric NDV)
or composition, the oncolysate vaccine, the whole cell vaccine, or
the combination therapy. In some embodiments, the subject
administered an NDV (e.g., a chimeric NDV) or a composition
thereof, an oncolysate vaccine described herein, or a whole cell
vaccine described herein, or a combination therapy described herein
experienced adverse side effects to a prior therapy or a prior
therapy was discontinued due to unacceptable levels of toxicity to
the subject.
[0199] 5.6.2. Dosage & Frequency
[0200] The amount of an NDV or a composition thereof, an oncolysate
vaccine, or a whole cell vaccine which will be effective in the
treatment of cancer will depend on the nature of the cancer, the
route of administration, the general health of the subject, etc.
and should be decided according to the judgment of a medical
practitioner. Standard clinical techniques, such as in vitro
assays, may optionally be employed to help identify optimal dosage
ranges. However, suitable dosage ranges of an NDV for
administration are generally about 10.sup.2, 5.times.10.sup.2,
10.sup.3, 5.times.10.sup.3, 10.sup.4, 5.times.10.sup.4, 10.sup.5,
5.times.10.sup.5, 10.sup.6, 5.times.10.sup.6, 10.sup.7,
5.times.10.sup.7, 10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.1.degree., 5.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11 or 10.sup.12 pfu, and most
preferably about 10.sup.4 to about 10.sup.12, 10.sup.6 to
10.sup.12, 10.sup.8 to 10.sup.12, 10.sup.9 to 10.sup.12 or 10.sup.9
to 10.sup.11, and can be administered to a subject once, twice,
three, four or more times with intervals as often as needed. Dosage
ranges of oncolysate vaccines for administration may include 0.001
mg, 0.005 mg, 0.01 mg, 0.05 mg. 0.1 mg. 0.5 mg, 1.0 mg, 2.0 mg. 3.0
mg, 4.0 mg, 5.0 mg, 10.0 mg, 0.001 mg to 10.0 mg, 0.01 mg to 1.0
mg, 0.1 mg to 1 mg, and 0.1 mg to 5.0 mg, and can be administered
to a subject once, twice, three or more times with intervals as
often as needed. Dosage ranges of whole cell vaccines for
administration may include 10.sup.2, 5.times.10.sup.2, 10.sup.3,
5.times.10.sup.3, 10.sup.4, 5.times.10.sup.4, 10.sup.5,
5.times.10.sup.5, 10.sup.6, 5.times.10.sup.6, 10.sup.7,
5.times.10.sup.7, 10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11 or 10.sup.12 cells, and can be
administered to a subject once, twice, three or more times with
intervals as often as needed. In certain embodiments, dosages
similar to those currently being used in clinical trials for NDV,
oncolysate vaccines or whole cell vaccines are administered to a
subject. Effective doses may be extrapolated from dose response
curves derived from in vitro or animal model test systems.
[0201] In certain embodiments, an NDV (e.g., a chimeric NDV) or a
composition thereof is administered to a subject as a single dose
followed by a second dose 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks,
1 to 3 weeks, 1 to 2 weeks later. In accordance with these
embodiments, booster inoculations may be administered to the
subject at 6 to 12 month intervals following the second
inoculation. In certain embodiments, an oncolysate vaccine or a
whole cell vaccine is administered to a subject as a single dose
followed by a second dose 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks,
1 to 3 weeks, 1 to 2 weeks later.
[0202] In certain embodiments, administration of the same NDV
(e.g., chimeric NDV) or a composition thereof, oncolysate vaccine,
or whole cell vaccine may be repeated and the administrations may
be separated by at least 1 day, 2 days, 3 days, 5 days, 6 says, 7
days, 10 days, 14 days, 15 days, 21 days, 28 days, 30 days, 45
days, 2 months, 75 days, 3 months, or at least 6 months. In other
embodiments, administration of the same NDV (e.g., a NDV) or a
composition thereof, oncolysate vaccine, or whole cell vaccine may
be repeated and the administrations may be separated by 1 to 14
days, 1 to 7 days, 7 to 14 days, 1 to 30 days, 15 to 30 days, 15 to
45 days, 15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6
months, 3 to 12 months, or 6 to 12 months. In some embodiments, a
first NDV (e.g., a first chimeric NDV) or a composition thereof is
administered to a subject followed by the administration of a
second NDV (e.g., a second chimeric NDV) or a composition thereof.
In certain embodiments, the first and second NDVs (e.g., the first
and second chimeric NDVs) or compositions thereof may be separated
by at least 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days,
14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75
days, 3 months, or at least 6 months. In other embodiments, the
first and second NDVs (e.g., the first and second chimeric NDVs) or
compositions thereof may be separated by 1 to 14 days, 1 to 7 days,
7 to 14 days, 1 to 30 days, 15 to 30 days, 15 to 45 days, 15 to 75
days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months,
or 6 to 12 months.
[0203] In certain embodiments, an NDV or composition thereof, or
oncolysate vaccine or whole cell vaccine is administered to a
subject in combination with one or more additional therapies, such
as a therapy described in Section 5.6.4, infra. The dosage of the
other one or more additional therapies will depend upon various
factors including, e.g., the therapy, the nature of the cancer, the
route of administration, the general health of the subject, etc.
and should be decided according to the judgment of a medical
practitioner. In specific embodiments, the dose of the other
therapy is the dose and/or frequency of administration of the
therapy recommended for the therapy for use as a single agent is
used in accordance with the methods disclosed herein. In other
embodiments, the dose of the other therapy is a lower dose and/or
less frequent administration of the therapy than recommended for
the therapy for use as a single agent is used in accordance with
the methods disclosed herein. Recommended doses for approved
therapies can be found in the Physician's Desk Reference.
[0204] In certain embodiments, an NDV or composition thereof, or
oncolysate vaccine or whole cell vaccine is administered to a
subject concurrently with the administration of one or more
additional therapies. In other embodiments, an NDV or composition
thereof, or oncolysate vaccine or whole cell vaccine is
administered to a subject every 3 to 7 days, 1 to 6 weeks, 1 to 5
weeks, 1 to 4 weeks, 2 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks
and one or more additional therapies (such as described in Section
5.6.4, infra) is administered every 3 to 7 days, 1 to 6 weeks, 1 to
5 weeks, 1 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks. In certain
embodiments, an NDV or composition thereof, or oncolysate vaccine
or whole cell vaccine is administered to a subject every 1 to 2
weeks and one or more additional therapies (such as described in
Section 5.6.4, infra) is administered every 2 to 4 weeks. In some
embodiments, an NDV or composition thereof, or oncolysate vaccine
or whole cell vaccine is administered to a subject every week and
one or more additional therapies (such as described in Section
5.6.4, infra) is administered every 2 weeks.
[0205] 5.6.3. Types of Cancer
[0206] Specific examples of cancers that can be treated in
accordance with the methods described herein include, but are not
limited to: leukemias, such as but not limited to, acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemias, such as,
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia leukemias and myelodysplastic syndrome; chronic
leukemias, such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, placancer cell leukemia, solitary
placancercytoma and extramedullary placancercytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, glioblastoma multiforme, acoustic neurinoma,
craniopharyngioma, medulloblastoma, meningioma, pineocytoma,
pineoblastoma, primary brain lymphoma; breast cancer including but
not limited to ductal carcinoma, adenocarcinoma, lobular (cancer
cell) carcinoma, intraductal carcinoma, medullary breast cancer,
mucinous breast cancer, tubular breast cancer, papillary breast
cancer, Paget's disease, and inflammatory breast cancer; adrenal
cancer such as but not limited to pheochromocytom and
adrenocortical carcinoma; thyroid cancer such as but not limited to
papillary or follicular thyroid cancer, medullary thyroid cancer
and anaplastic thyroid cancer; pancreatic cancer such as but not
limited to, insulinoma, gastrinoma, glucagonoma, vipoma,
somatostatin-secreting tumor, and carcinoid or islet cell tumor;
pituitary cancers such as but limited to Cushing's disease,
prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye
cancers such as but not limited to ocular melanoma such as iris
melanoma, choroidal melanoma, and cilliary body melanoma, and
retinoblastoma; vaginal cancers such as squamous cell carcinoma,
adenocarcinoma, and melanoma; vulvar cancer such as squamous cell
carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,
and Paget's disease; cervical cancers such as but not limited to,
squamous cell carcinoma, and adenocarcinoma; uterine cancers such
as but not limited to endometrial carcinoma and uterine sarcoma;
ovarian cancers such as but not limited to, ovarian epithelial
carcinoma, borderline tumor, germ cell tumor, and stromal tumor;
esophageal cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, placancercytoma,
verrucous carcinoma, and oat cell (cancer cell) carcinoma; stomach
cancers such as but not limited to, adenocarcinoma, fungating
(polypoid), ulcerating, superficial spreading, diffusely spreading,
malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma;
colon cancers; rectal cancers; liver cancers such as but not
limited to hepatocellular carcinoma and hepatoblastoma; gallbladder
cancers such as adenocarcinoma; cholangiocarcinomas such as but not
limited to papillary, nodular, and diffuse; lung cancers such as
non-cancer cell lung cancer, squamous cell carcinoma (epidermoid
carcinoma), adenocarcinoma, large-cell carcinoma and cancer-cell
lung cancer; testicular cancers such as but not limited to germinal
tumor, seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, prostatic intraepithelial neoplasia, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
such as but not limited to squamous cell carcinoma; basal cancers;
salivary gland cancers such as but not limited to adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx
cancers such as but not limited to squamous cell cancer, and
verrucous; skin cancers such as but not limited to, basal cell
carcinoma, squamous cell carcinoma and melanoma, superficial
spreading melanoma, nodular melanoma, lentigo malignant melanoma,
acral lentiginous melanoma; kidney cancers such as but not limited
to renal cell carcinoma, adenocarcinoma, hypernephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/or
uterer); Wilms' tumor; bladder cancers such as but not limited to
transitional cell carcinoma, squamous cell cancer, adenocarcinoma,
carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma,
mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma and papillary
adenocarcinomas (for a review of such disorders, see Fishman et
al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and
Murphy et al., 1997, Informed Decisions: The Complete Book of
Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin
Books U.S.A., Inc., United States of America).
[0207] In a specific embodiment, the chimeric NDVs described herein
or compositions thereof, an oncolysate vaccine described herein, a
whole cell vaccine herein, or a combination therapy described
herein are useful in the treatment of a variety of cancers and
abnormal proliferative diseases, including (but not limited to) the
following: carcinoma, including that of the bladder, breast, colon,
kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and
skin; including squamous cell carcinoma; hematopoietic tumors of
lymphoid lineage, including leukemia, acute lymphocytic leukemia,
acute lymphoblastic leukemia, B-cell lymphoma, T cell lymphoma,
Burkitt's lymphoma; hematopoietic tumors of myeloid lineage,
including acute and chronic myelogenous leukemias and promyelocytic
leukemia; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma; other tumors, including melanoma, seminoma,
teratocarcinoma, neuroblastoma and glioma; tumors of the central
and peripheral nervous system, including astrocytoma,
neuroblastoma, glioma, and schwannomas; tumors of mesenchymal
origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma;
and other tumors, including melanoma, xeroderma pigmentosum,
keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma.
[0208] In some embodiments, cancers associated with aberrations in
apoptosis are treated in accordance with the methods described
herein. Such cancers may include, but are not limited to,
follicular lymphomas, carcinomas with p53 mutations, hormone
dependent tumors of the breast, prostate and ovary, and
precancerous lesions such as familial adenomatous polyposis, and
myelodysplastic syndromes. In specific embodiments, malignancy or
dysproliferative changes (such as metaplasias and dysplasias), or
hyperproliferative disorders of the skin, lung, liver, bone, brain,
stomach, colon, breast, prostate, bladder, kidney, pancreas, ovary,
and/or uterus are treated in accordance with the methods described
herein. In other specific embodiments, a sarcoma or melanoma is
treated in accordance with the methods described herein.
[0209] In a specific embodiment, the cancer being treated in
accordance with the methods described herein is leukemia, lymphoma
or myeloma (e.g., multiple myeloma). Specific examples of leukemias
and other blood-borne cancers that can be treated in accordance
with the methods described herein include, but are not limited to,
acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell
leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic
leukemia "AML", acute promyelocytic leukemia "APL", acute
monoblastic leukemia, acute erythroleukemic leukemia, acute
megakaryoblastic leukemia, acute myelomonocytic leukemia, acute
nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic
myelocytic leukemia "CML", chronic lymphocytic leukemia "CLL", and
hairy cell leukemia.
[0210] Specific examples of lymphomas that can be treated in
accordance with the methods described herein include, but are not
limited to, Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple
myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera.
[0211] In another embodiment, the cancer being treated in
accordance with the methods described herein is a solid tumor.
Examples of solid tumors that can be treated in accordance with the
methods described herein include, but are not limited to
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon cancer, colorectal cancer, kidney cancer, pancreatic cancer,
bone cancer, breast cancer, ovarian cancer, prostate cancer,
esophageal cancer, stomach cancer, oral cancer, nasal cancer,
throat cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, uterine cancer,
testicular cancer, cancer cell lung carcinoma, bladder carcinoma,
lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, skin cancer, melanoma, neuroblastoma, and
retinoblastoma. In another embodiment, the cancer being treated in
accordance with the methods described herein is a metastatic. In
another embodiment, the cancer being treated in accordance with the
methods described herein is malignant.
[0212] In a specific embodiment, the cancer being treated in
accordance with the methods described herein is a cancer that has a
poor prognosis and/or has a poor response to conventional
therapies, such as chemotherapy and radiation. In another specific
embodiment, the cancer being treated in accordance with the methods
described herein is malignant melanoma, malignant glioma, renal
cell carcinoma, pancreatic adenocarcinoma, malignant pleural
mesothelioma, lung adenocarcinoma, lung small cell carcinoma, lung
squamous cell carcinoma, anaplastic thyroid cancer, and head and
neck squamous cell carcinoma. In another specific embodiment, the
cancer being treated in accordance with the methods described
herein is a type of cancer described in Section 6 and/or Section 7,
infra.
[0213] 5.6.4. Additional Therapies
[0214] Additional therapies that can be used in a combination with
an NDV described herein or a composition thereof, an oncolysate
vaccine, or a whole cell vaccine for the treatment of cancer
include, but are not limited to, small molecules, synthetic drugs,
peptides (including cyclic peptides), polypeptides, proteins,
nucleic acids (e.g., DNA and RNA nucleotides including, but not
limited to, antisense nucleotide sequences, triple helices, RNAi,
and nucleotide sequences encoding biologically active proteins,
polypeptides or peptides), antibodies, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules. In a specific embodiment, the additional therapy
is a chemotherapeutic agent.
[0215] In some embodiments, an NDV described herein or a
composition thereof, an oncolysate vaccine, or a whole cell vaccine
is used in combination with radiation therapy comprising the use of
x-rays, gamma rays and other sources of radiation to destroy cancer
cells. In specific embodiments, the radiation therapy is
administered as external beam radiation or teletherapy, wherein the
radiation is directed from a remote source. In other embodiments,
the radiation therapy is administered as internal therapy or
brachytherapy wherein a radioactive source is placed inside the
body close to cancer cells and/or a tumor mass.
[0216] In certain embodiments, an NDV described herein or a
composition thereof, an oncolysate vaccine, or a whole cell cancer
vaccine is used in combination with adoptive T cell therapy. In a
specific embodiment, the T cells utilized in the adoptive T cell
therapy are tumor infiltrating lymphocytes that have been isolated
from a subject and a particular T cell or clone has been expanded
for use thereof. In some embodiments, the T cells utilized in the
adoptive T cell therapy are T cells taken from a patient's blood
after they have received a cancer vaccine and expanded in vitro
before use. In another specific embodiment, the T cells utilized in
the adoptive T cell therapy are T cells that have been influenced
to potently recognize and attack tumors. In another specific
embodiment, the T cells utilized in the adoptive T cell therapy
have been genetically modified to express tumor-antigen specific T
cell receptor or a chimeric antigen receptor (CAR). In a specific
embodiment, the adoptive T cell therapy utilized is analogous to
that described in Section 7, infra.
[0217] In certain embodiments, an NDV described herein or a
composition thereof, an oncolysate vaccine, or a whole cell cancer
vaccine is used in combination with a cytokine. In a specific
embodiment, an NDV described herein or a composition thereof, an
oncolysate vaccine, or a whole cell cancer vaccine is used in
combination with interferon (e.g., IFN-.gamma.).
[0218] Currently available cancer therapies and their dosages,
routes of administration and recommended usage are known in the art
and have been described in such literature as the Physician's Desk
Reference (67th ed., 2013).
[0219] Specific examples of anti-cancer agents that may be used in
combination with an NDV described herein or a composition thereof
include: hormonal agents (e.g., aromatase inhibitor, selective
estrogen receptor modulator (SERM), and estrogen receptor
antagonist), chemotherapeutic agents (e.g., microtubule disassembly
blocker, antimetabolite, topoisomerase inhibitor, and DNA
crosslinker or damaging agent), anti-angiogenic agents (e.g., VEGF
antagonist, receptor antagonist, integrin antagonist, vascular
targeting agent (VTA)/vascular disrupting agent (VDA)), radiation
therapy, and conventional surgery.
[0220] Non-limiting examples of hormonal agents that may be used in
combination with an NDV described herein or a composition thereof
include aromatase inhibitors, SERMs, and estrogen receptor
antagonists. Hormonal agents that are aromatase inhibitors may be
steroidal or nonsteroidal. Non-limiting examples of nonsteroidal
hormonal agents include letrozole, anastrozole, aminoglutethimide,
fadrozole, and vorozole. Non-limiting examples of steroidal
hormonal agents include aromasin (exemestane), formestane, and
testolactone. Non-limiting examples of hormonal agents that are
SERMs include tamoxifen (branded/marketed as Nolvadex.RTM.),
afimoxifene, arzoxifene, bazedoxifene, clomifene, femarelle,
lasofoxifene, ormeloxifene, raloxifene, and toremifene.
Non-limiting examples of hormonal agents that are estrogen receptor
antagonists include fulvestrant. Other hormonal agents include but
are not limited to abiraterone and lonaprisan.
[0221] Non-limiting examples of chemotherapeutic agents that may be
used in combination with an NDV described herein or a composition
thereof, an oncolysate vaccine, or a whole cell vaccine include
microtubule disasssembly blocker, antimetabolite, topoisomerase
inhibitor, and DNA crosslinker or damaging agent. Chemotherapeutic
agents that are microtubule disassembly blockers include, but are
not limited to, taxenes (e.g., paclitaxel (branded/marketed as
TAXOL.RTM.), docetaxel, abraxane, larotaxel, ortataxel, and
tesetaxel); epothilones (e.g., ixabepilone); and vinca alkaloids
(e.g., vinorelbine, vinblastine, vindesine, and vincristine
(branded/marketed as)) ONCOVIN.RTM..
[0222] Chemotherapeutic agents that are antimetabolites include,
but are not limited to, folate antimetabolites (e.g., methotrexate,
aminopterin, pemetrexed, raltitrexed); purine antimetabolites
(e.g., cladribine, clofarabine, fludarabine, mercaptopurine,
pentostatin, thioguanine); pyrimidine antimetabolites (e.g.,
5-fluorouracil, capecitabine, gemcitabine (GEMZAR.RTM.),
cytarabine, decitabine, floxuridine, tegafur); and
deoxyribonucleotide antimetabolites (e.g., hydroxyurea).
[0223] Chemotherapeutic agents that are topoisomerase inhibitors
include, but are not limited to, class I (camptotheca)
topoisomerase inhibitors (e.g., topotecan (branded/marketed as
HYCAMTIN.RTM.) irinotecan, rubitecan, and belotecan); class II
(podophyllum) topoisomerase inhibitors (e.g., etoposide or VP-16,
and teniposide); anthracyclines (e.g., doxorubicin, epirubicin,
Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin,
pirarubicin, valrubicin, and zorubicin); and anthracenediones
(e.g., mitoxantrone, and pixantrone).
[0224] Chemotherapeutic agents that are DNA crosslinkers (or DNA
damaging agents) include, but are not limited to, alkylating agents
(e.g., cyclophosphamide, mechlorethamine, ifosfamide
(branded/marketed as IFEX.RTM.), trofosfamide, chlorambucil,
melphalan, prednimustine, bendamustine, uramustine, estramustine,
carmustine (branded/marketed as BiCNU.RTM.), lomustine, semustine,
fotemustine, nimustine, ranimustine, streptozocin, busulfan,
mannosulfan, treosulfan, carboquone,
N,N'N'-triethylenethiophosphoramide, triaziquone,
triethylenemelamine); alkylating-like agents (e.g., carboplatin
(branded/marketed as PARAPLATIN.RTM.), cisplatin, oxaliplatin,
nedaplatin, triplatin tetranitrate, satraplatin, picoplatin);
nonclassical DNA crosslinkers (e.g., procarbazine, dacarbazine,
temozolomide (branded/marketed as TEMODAR.RTM.), altretamine,
mitobronitol); and intercalating agents (e.g., actinomycin,
bleomycin, mitomycin, and plicamycin).
[0225] 5.6.4.1 Immune Modulators
[0226] In specific embodiments, an NDV described herein (e.g., a
chimeric NDV) or a composition thereof, an oncolysate vaccine, or a
whole cell vaccine are administered to a subject in combination
with one or more of the following: any agonist of a co-stimulatory
signal of an immune cell (such as, e.g., a T-lymphocyte, NK cell or
antigen-presenting cell (e.g., a dendritic cell or macrophage)
and/or any antagonist of an inhibitory signal of an immune cell
(such as, e.g., a T-lymphocyte, NK cell or antigen-presenting cell
(e.g., a dendritic cell or macrophage), known to one of skill in
the art. In particular embodiments, an NDV described herein (e.g.,
a chimeric NDV) or a composition thereof, an oncolysate vaccine, or
a whole cell vaccine are administered to a subject in combination
with one or more of the agonists of a co-stimulatory signal of an
immune cell described in Section 5.2.1, supra. In some embodiments,
an NDV described herein (e.g., a chimeric NDV) or a composition
thereof, an oncolysate vaccine, or a whole cell vaccine are
administered to a subject in combination with one or more of the
antagonists of an inhibitory signal of an immune cell described in
Section 5.2.1, supra. In certain embodiments, an NDV described
herein (e.g., a chimeric NDV) or a composition thereof, an
oncolysate vaccine, or a whole cell vaccine are administered to a
subject in combination with one or more of the agonists of a
co-stimulatory signal of an immune cell and/or one or more of the
antagonists of an inhibitory signal of an immune cell described in
Section 6 and/or Section 7, infra (e.g., an anti-CTLA-4 antibody,
an ICOS-L, an anti-PD-1 antibody, or an anti-PD-L1 antibody)
[0227] 5.7 Biological Assays
In Vitro Viral Assays
[0228] Viral assays include those that measure altered viral
replication (as determined, e.g., by plaque formation) or the
production of viral proteins (as determined, e.g., by western blot
analysis) or viral RNAs (as determined, e.g., by RT-PCR or northern
blot analysis) in cultured cells in vitro using methods which are
well known in the art.
[0229] Growth of the NDVs described herein can be assessed by any
method known in the art or described herein (e.g., in cell culture
(e.g., cultures of chicken embryonic kidney cells or cultures of
chicken embryonic fibroblasts (CEF)). Viral titer may be determined
by inoculating serial dilutions of a NDV described herein into cell
cultures (e.g., CEF, MDCK, EFK-2 cells, Vero cells, primary human
umbilical vein endothelial cells (HUVEC), H292 human epithelial
cell line or HeLa cells), chick embryos, or live animals (e.g.,
avians). After incubation of the virus for a specified time, the
virus is isolated using standard methods. Physical quantitation of
the virus titer can be performed using PCR applied to viral
supernatants (Quinn & Trevor, 1997; Morgan et al., 1990),
hemagglutination assays, tissue culture infectious doses (TCID50)
or egg infectious doses (EID50). An exemplary method of assessing
viral titer is described in Section 6 and Section 7, below.
[0230] Incorporation of nucleotide sequences encoding a
heterologous peptide or protein (e.g., a cytokine, a mutated F
protein, a mutated V protein, or miRNA target site into the genome
of a chimeric NDV described herein can be assessed by any method
known in the art or described herein (e.g., in cell culture, an
animal model or viral culture in embryonated eggs). For example,
viral particles from cell culture of the allantoic fluid of
embryonated eggs can be purified by centrifugation through a
sucrose cushion and subsequently analyzed for fusion protein
expression by Western blotting using methods well known in the
art.
[0231] Immunofluorescence-based approaches may also be used to
detect virus and assess viral growth. Such approaches are well
known to those of skill in the art, e.g., fluorescence microscopy
and flow cytometry (see Section 6 and Section 7, infra).
[0232] Antibody Assays
[0233] Antibodies generated by the NDVs described herein may be
characterized in a variety of ways well-known to one of skill in
the art (e.g., ELISA, Surface Plasmon resonance display (BIAcore),
Western blot, immunofluorescence, immunostaining and/or
microneutralization assays). In particular, antibodies generated by
the chimeric NDVs described herein may be assayed for the ability
to specifically bind to an antigen of the virus or a heterologous
peptide or protein. Such an assay may be performed in solution
(e.g., Houghten, 1992, Bio/Techniques 13:412 421), on beads (Lam,
1991, Nature 354:82 84), on chips (Fodor, 1993, Nature 364:555
556), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et
al., 1992, Proc. Natl. Acad. Sci. USA 89:1865 1869) or on phage
(Scott and Smith, 1990, Science 249:386 390; Cwirla et al., 1990,
Proc. Natl. Acad. Sci. USA 87:6378 6382; and Felici, 1991, J. Mol.
Biol. 222:301 310) (each of these references is incorporated herein
in its entirety by reference).
[0234] Antibodies generated by the chimeric NDVs described herein
that have been identified to specifically bind to an antigen of the
virus or a heterologous peptide or protein can be assayed for their
specificity to said antigen of the virus or heterologous peptide or
protein. The antibodies may be assayed for specific binding to an
antigen of the virus or a heterologous peptide or protein and for
their cross-reactivity with other antigens by any method known in
the art. Immunoassays which can be used to analyze specific binding
and cross-reactivity include, but are not limited to, competitive
and non-competitive assay systems using techniques such as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds., 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety).
[0235] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. Alternatively, a surface plasmon
resonance assay (e.g., BIAcore kinetic analysis) or KinExA assay
(Blake, et al., Analytical Biochem., 1999, 272:123-134) may be used
to determine the binding on and off rates of antibodies to an
antigen of the chimeric NDVs described herein.
IFN Assays
[0236] IFN induction and release by an NDV described herein may be
determined using techniques known to one of skill in the art or
described herein. For example, the amount of IFN induced in cells
following infection with an NDV described herein may be determined
using an immunoassay (e.g., an ELISA or Western blot assay) to
measure IFN expression or to measure the expression of a protein
whose expression is induced by IFN. Alternatively, the amount of
IFN induced may be measured at the RNA level by assays, such as
Northern blots and quantitative RT-PCR, known to one of skill in
the art. In specific embodiments, the amount of IFN released may be
measured using an ELISPOT assay. (See, e.g., the methods described
in Section 6 and Section 7, below). Further, the induction and
release of cytokines may be determined by, e.g., an immunoassay or
ELISPOT assay at the protein level and/or quantitative RT-PCR or
northern blots at the RNA level. See Section 6 and/or Section 7,
infra, regarding assays to measure cytokine induction and
release.
Activation Marker Assays
[0237] Techniques for assessing the expression of activation
marker, co-stimulatory molecule, ligand, or inhibitory molecule by
immune cells are known to one of skill in the art. For example, the
expression of an activation marker, co-stimulatory molecule,
ligand, or inhibitory molecule by an immune cell (e.g., T
lymphocyte or NK cell) can be assessed by flow cytometry. In a
specific embodiment, techniques described in Section 6 and/or
Section 7, infra, are used to assess the expression of an
activation marker, co-stimulatory molecule, ligand, or inhibitory
molecule by an immune cell.
Immune Cell Infiltration Assays
[0238] Techniques for assessing immune cell infiltration are known
to one of skill in the art. In a specific embodiment, techniques
described in Section 6 and/or Section 7, infra, are used to assess
immune cell infiltration.
Toxicity Studies
[0239] In some embodiments, the NDVs described herein or
compositions thereof, oncolysate vaccines described herein, whole
cell vaccines described herein, or combination therapies described
herein are tested for cytotoxicity in mammalian, preferably human,
cell lines (see, e.g., the cytotoxicity assay described in Section
6 and/or Section 7, infra). In certain embodiments, cytotoxicity is
assessed in one or more of the following non-limiting examples of
cell lines: U937, a human monocyte cell line; primary peripheral
blood mononuclear cells (PBMC); Huh7, a human hepatoblastoma cell
line; HL60 cells, HT1080, HEK 293T and 293H, MLPC cells, human
embryonic kidney cell lines; human melanoma cell lines, such as
SkMel2, SkMel-119 and SkMel-197; THP-1, monocytic cells; a HeLa
cell line; and neuroblastoma cells lines, such as MC-IXC, SK-N-MC,
SK-N-MC, SK-N-DZ, SH-SY5Y, and BE(2)-C. In certain embodiments,
cytotoxicity is assessed in various cancer cells. In some
embodiments, the ToxLite assay is used to assess cytotoxicity.
[0240] Many assays well-known in the art can be used to assess
viability of cells or cell lines following infection with an NDV
described herein or composition thereof, or treatment with an
oncolysate vaccine described herein, a whole cell vaccine described
herein, or a combination therapy described herein and, thus,
determine the cytotoxicity of the NDV or composition thereof,
oncolysate vaccine, whole cell vaccine, or combination therapy. For
example, cell proliferation can be assayed by measuring
Bromodeoxyuridine (BrdU) incorporation, (.sup.3H) thymidine
incorporation, by direct cell count, or by detecting changes in
transcription, translation or activity of known genes such as
proto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2,
cyclin A, D1, D2, D3, E, etc). The levels of such protein and mRNA
and activity can be determined by any method well known in the art.
For example, protein can be quantitated by known immunodiagnostic
methods such as ELISA, Western blotting or immunoprecipitation
using antibodies, including commercially available antibodies. mRNA
can be quantitated using methods that are well known and routine in
the art, for example, using northern analysis, RNase protection, or
polymerase chain reaction in connection with reverse transcription.
Cell viability can be assessed by using trypan-blue staining or
other cell death or viability markers known in the art. In a
specific embodiment, the level of cellular ATP is measured to
determined cell viability. In preferred embodiments, an NDV
described herein or composition thereof, oncolysate vaccine, whole
cell vaccine, or combination therapy kills cancer cells but does
not kill healthy (i.e., non-cancerous) cells. In one embodiment, an
NDV described herein or composition thereof, oncolysate vaccine,
whole cell vaccine, or combination therapy preferentially kills
cancer cells but does not kill healthy (i.e., non-cancerous)
cells.
[0241] In specific embodiments, cell viability is measured in
three-day and seven-day periods using an assay standard in the art,
such as the CellTiter-Glo Assay Kit (Promega) which measures levels
of intracellular ATP. A reduction in cellular ATP is indicative of
a cytotoxic effect. In another specific embodiment, cell viability
can be measured in the neutral red uptake assay. In other
embodiments, visual observation for morphological changes may
include enlargement, granularity, cells with ragged edges, a filmy
appearance, rounding, detachment from the surface of the well, or
other changes.
[0242] The NDVs described herein or compositions thereof,
oncolysate vaccines, whole cell vaccines or combination therapies
can be tested for in vivo toxicity in animal models (see, e.g., the
animal models described in Section 6 and/or Section 7, below). For
example, animal models, described herein and/or others known in the
art, used to test the effects of compounds on cancer can also be
used to determine the in vivo toxicity of the NDVs described herein
or compositions thereof, oncolysate vaccines, whole cell vaccines,
or combination therapies. For example, animals are administered a
range of pfu of an NDV described herein (e.g., a chimeric NDV
described in Section 5.2, infra). Subsequently, the animals are
monitored over time for lethality, weight loss or failure to gain
weight, and/or levels of serum markers that may be indicative of
tissue damage (e.g., creatine phosphokinase level as an indicator
of general tissue damage, level of glutamic oxalic acid
transaminase or pyruvic acid transaminase as indicators for
possible liver damage). These in vivo assays may also be adapted to
test the toxicity of various administration mode and/or regimen in
addition to dosages.
[0243] The toxicity and/or efficacy of an NDV described herein or a
composition thereof, an oncolysate vaccine described herein, a
whole cell vaccine described herein, or a combination therapy
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Therapies that exhibits large therapeutic indices are
preferred. While therapies that exhibits toxic side effects may be
used, care should be taken to design a delivery system that targets
such therapies to the site of affected tissue in order to minimize
potential damage to noncancerous cells and, thereby, reduce side
effects.
[0244] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of the
therapies for use in subjects. The dosage of such agents lies
preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized. For any therapy described herein,
the therapeutically effective dose can be estimated initially from
cell culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the chimeric NDV that achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in subjects. Levels in plasma may be measured, for example,
by high performance liquid chromatography.
Anti-Cancer Studies
[0245] The NDVs described herein or compositions thereof,
oncolysate vaccines described herein, whole cell vaccines described
herein, or combination therapies described herein can be tested for
biological activity using animal models for cancer. Such animal
model systems include, but are not limited to, rats, mice, chicken,
cows, monkeys, pigs, dogs, rabbits, etc. In a specific embodiment,
the anti-cancer activity of an NDV described herein or combination
therapy is tested in a mouse model system. Such model systems are
widely used and well-known to the skilled artisan such as the SCID
mouse model or transgenic mice.
[0246] The anti-cancer activity of an NDV described herein or a
composition thereof, oncolysate vaccine described herein, whole
cell vaccine described herein, or a combination therapy described
herein can be determined by administering the NDV or composition
thereof, oncolysate vaccine, whole cell vaccine, or combination
therapy to an animal model and verifying that the NDV or
composition thereof, oncolysate vaccine, whole cell vaccine, or
combination therapy is effective in reducing the severity of
cancer, reducing the symptoms of cancer, reducing cancer
metastasis, and/or reducing the size of a tumor in said animal
model (see, e.g., Section 6 and/or Section 7, below). Examples of
animal models for cancer in general include, include, but are not
limited to, spontaneously occurring tumors of companion animals
(see, e.g., Vail & MacEwen, 2000, Cancer Invest 18(8):781-92).
Examples of animal models for lung cancer include, but are not
limited to, lung cancer animal models described by Zhang & Roth
(1994, In-vivo 8(5):755-69) and a transgenic mouse model with
disrupted p53 function (see, e.g. Morris et al., 1998, J La State
Med Soc 150(4): 179-85). An example of an animal model for breast
cancer includes, but is not limited to, a transgenic mouse that
over expresses cyclin D1 (see, e.g., Hosokawa et al., 2001,
Transgenic Res 10(5):471-8). An example of an animal model for
colon cancer includes, but is not limited to, a TCR b and p53
double knockout mouse (see, e.g., Kado et al., 2001, Cancer Res.
61(6):2395-8). Examples of animal models for pancreatic cancer
include, but are not limited to, a metastatic model of PancO2
murine pancreatic adenocarcinoma (see, e.g., Wang et al., 2001,
Int. J. Pancreatol. 29(1):37-46) and nu-nu mice generated in
subcutaneous pancreatic tumors (see, e.g., Ghaneh et al., 2001,
Gene Ther. 8(3):199-208). Examples of animal models for
non-Hodgkin's lymphoma include, but are not limited to, a severe
combined immunodeficiency ("SCID") mouse (see, e.g., Bryant et al.,
2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenic mouse
(see, e.g., Hough et al., 1998, Proc. Natl. Acad. Sci. USA
95(23):13853-8). An example of an animal model for esophageal
cancer includes, but is not limited to, a mouse transgenic for the
human papillomavirus type 16 E7 oncogene (see, e.g., Herber et al.,
1996, J. Virol. 70(3):1873-81). Examples of animal models for
colorectal carcinomas include, but are not limited to, Apc mouse
models (see, e.g., Fodde & Smits, 2001, Trends Mol Med 7(8):369
73 and Kuraguchi et al., 2000). In a specific embodiment, the
animal models for cancer described in Section 6 and/or Section 7,
infra, are used to assess efficacy of an NDV or composition
thereof, an oncolysate, a whole cell vaccine, or a combination
therapy.
6. EXAMPLE 1
[0247] This example demonstrates the therapeutic efficacy of NDV
therapy in combination with immune checkpoint modulators that are
immunostimulatory in the treatment of cancer.
[0248] 6.1 Materials & Methods
Mice
[0249] BALB/c mice (6-8 weeks old), and WT C57BL/6 mice were
purchased from Jackson Laboratory. All mice were maintained in
microisolator cages and treated in accordance with the NIH and
American Association of Laboratory Animal Care regulations. All
mouse procedures and experiments for this study were approved by
the Memorial Sloan-Kettering Cancer Center Institutional Animal
Care and Use Committee.
Cell Lines
[0250] The murine cancer cell lines for melanoma (B16-F10), and
colon carcinoma (CT26 and MC38) were maintained in RPMI medium
supplemented with 10% fetal calf serum and penicillin with
streptomycin. The murine prostate cancer cell line TRAMP-C2 was
maintained in DMEM medium supplemented with 5% fetal calf serum
(FCS; Mediatech, Inc.), 5% Nu Serum IV (BD Biosciences) HEPES,
2-ME, pen/strep, L-glut, 5 .mu.g/mL insulin (Sigma), and 10 nmol/L
DHT (Sigma).
Antibodies
[0251] Therapeutic anti-CTLA-4 (clone 9H10), anti-PD-1 (clone
RMP1-14), and anti-PD-L1 monoclonal antibodies were produced by
BioXcell. Antibodies used for flow cytometry were purchased from
eBioscience, Biolegend, Invitrogen, and BD Pharmingen.
Viruses and Cloning
[0252] Recombinant lentogenic NDV LaSota strain was used for all
experiments. To generate NDV virus expressing murine ICOSL, a DNA
fragment encoding the murine ICOSL flanked by the appropriate
NDV-specific RNA transcriptional signals was inserted into the
SacII site created between the P and M genes of pT7NDV/LS. Viruses
were rescued from cDNA using methods described previously and
sequenced by reverse transcription PCR for insert fidelity. Virus
titers were determined by serial dilution and immunofluorescence in
Vero cells. Recombinant ICOSL-F fusion construct was generated by
PCR amplification of the ICOSL DNA encoding the extracellular
domain (amino acids 1-277) with flanking EcoRI and MluI restriction
sites, and the NDV F DNA encoding the F transmembrane and
intracellular domains (amino acids 501-554) with flanking MluI and
XhoI restriction sites. The resultant DNA fragments were assembled
in pCAGGS vector utilizing 3-part ligation.
In Vitro Infection Experiments
[0253] For evaluation of upregulation of surface MHC-I, MHC-II, and
ICAM-1 by NDV, and for evaluation of surface expression of the
ICOSL transgene from the NDV-ICOSL virus, B16-F10 cells were
infected in 6-well dishes at MOI 2 in triplicate. Twenty-four hours
later, the cells were harvested by mechanical scraping and
processed for surface labeling and quantification by flow
cytometry. For virus growth curve experiments, B16-F10 cells were
incubated at room temperature with the virus in 6-well culture
dishes at the indicated MOIs in a total volume of 100 .mu.l. One
hour after the incubation, the infection media was aspirated and
the cells were incubated at 37.degree. C. in 1 ml of DMEM with 10%
chick allantoic fluid. After 24, 48, and 72 hours, the supernatants
were collected and virus titers were determined as above. For in
vitro cytotoxicity experiments, the infections were carried out in
a similar fashion. At 24, 48, 72, and 96 hours post infection the
cells were washed and incubated with 1% Triton X-100 at 37.degree.
C. for 30 minutes. LDH activity in the lysates was determined using
the Promega CytoTox 96 assay kit, according to the manufacturer's
instructions.
Tumor Challenge Survival Experiments.
[0254] Bilateral flank tumor models were established to monitor for
therapeutic efficacy in both injected and systemic tumors.
Treatment schedules and cell doses were established for each tumor
model to achieve 10-20% tumor clearance by NDV or
anti-CTLA-4/anti-PD-1 as single agents. For experiments evaluating
combination therapy of wild-type NDV (NDV-WT) with immune
checkpoint blockade, B16F10 tumors were implanted by injection of
2.times.10.sup.5 B16F10 cells in the right flank i.d. on day 0 and
5.times.10.sup.4 cells in the left flank on day 4. On days 7, 10,
13, and 16 the mice were treated with 4 intratumoral injections of
2.times.10.sup.7 pfu of NDV in PBS in a total volume of 100 .mu.l.
Concurrently, on days 7, 10, 13, and 16 the mice received 4 i.p.
injections of anti-CTLA-4 antibody (100 .mu.g) or anti-PD-1
antibody (250 .mu.g). Control groups received a corresponding dose
of isotype antibody i.p. and intratumoral injection of PBS. Tumor
size and incidence were monitored over time by measurement with a
caliper.
[0255] For the TRAMP-C2 model, 5.times.10.sup.5 cells were
implanted in right flank on day 0 and 5.times.10.sup.5 cells were
implanted in the left flank on day 8. Treatment was performed on
days 11, 14, 17, and 20 in the similar fashion to above.
[0256] For experiments evaluating recombinant NDV expressing ICOSL
(NDV-ICOSL), B16F10 tumors were implanted by injection of
2.times.10.sup.5 B16F10 cells in the right flank i.d. on day 0 and
1.times.10.sup.5 cells in the left flank on day 4. Treatment was
carried out as above.
[0257] For the CT26 model, tumors were implanted by injection of
1.times.10.sup.6 CT26 cells in the right flank i.d. on day 0 and
1.times.10.sup.6 cells in the left flank on day 2. Treatment was
carried out as above on days 6, 9, and 12.
Isolation of Tumor-Infiltrating Lymphocytes
[0258] B16F10 tumors were implanted by injection of
2.times.10.sup.5 B16F10 cells in the right flank i.d. on day 0 and
2.times.10.sup.5 cells in the left flank on day 4. On days 7, 10,
and 13 the mice were treated with 3 intratumoral injections of
2.times.10.sup.7 pfu of NDV, and 100 .mu.g of i.p. anti-CTLA-4
antibody or 250 .mu.g of i.p. anti-PD-1 antibody, where specified.
On day 15, mice were sacrificed by CO.sub.2 inhalation. Tumors and
tumor-draining lymph nodes were removed using forceps and surgical
scissors and weighed. Tumors from each group were minced with
scissors prior to incubation with 1.67 Wunsch U/mL Liberase and 0.2
mg/mL DNase for 30 minutes at 37.degree. C. Tumors were homogenized
by repeated pipetting and filtered through a 70-.mu.m nylon filter.
Cell suspensions were washed once with complete RPMI and purified
on a Ficoll gradient to eliminate dead cells. Cells from tumor
draining lymph nodes were isolated by grinding the lymph nodes
through a 70-.mu.m nylon filter.
Flow Cytometry
[0259] Cells isolated from tumors or tumor-draining lymph nodes
were processed for surface labeling with several antibody panels
staining CD45, CD3, CD4, CD8, CD44, PD-1, ICOS, CD11c, CD19, NK1.1,
CD11b, F4/80, Ly6C and Ly6G. Fixable viability dye eFluor780
(eBioscience) was used to distinguish the live cells. Cells were
further permeabilized using FoxP3 fixation and permeabilization kit
(eBioscience) and stained for Ki-67, FoxP3, Granzyme B, CTLA-4, and
IFN gamma. Data was acquired using the LSRII Flow cytometer (BD
Biosciences) and analyzed using FlowJo software (Treestar).
DC Purification and Loading
[0260] Spleens from naive mice were isolated and digested with 1.67
Wunsch U/mL Liberase and 0.2 mg/mL DNase for 30 minutes at
37.degree. C. The resulting cell suspensions were filtered through
70 um nylon filter and washed once with complete RPMI. CD11c+
dendritic cells were purified by positive selection using Miltenyi
magnetic beads. Isolated dendritic cells were cultured overnight
with recombinant GM-CSF and B16-F10 tumor lysates and were purified
on Ficoll gradient.
Analysis of Cytokine Production
[0261] Cell suspensions from tumors or tumor-draining lymph nodes
were pooled and enriched for T cells using a Miltenyi T-cell
purification kit. Isolated T cells were counted and co-cultured for
8 hours with dendritic cells loaded with B16-F10 tumor cell lysates
in the presence of 20 U/ml IL-2 (R and D) plus Brefeldin A (BD
Bioscience). After restimulation, lymphocytes were processed for
flow cytometry as above.
Statistics.
[0262] Data were analyzed by 2-tailed Student's t test, and
P<0.05 was considered statistically significant.
[0263] 6.2 Results
[0264] In order to characterize the anti-tumor immune response
induced by Newcastle disease virus (NDV) infection, the expression
of MHC I and MHC II molecules as well as ICAM-1 on the surface of
in vitro infected cells was assessed. As shown in FIG. 1, NDV
infection in B16 melanoma cells induces upregulation of MHC class I
and II molecules as well as adhesion molecule ICAM-1, all of which
are thought to be important for recruitment of tumor-specific
lymphocytes and activation of anti-tumor immune response. Next, the
anti-tumor immune response induced by NDV infection in vivo was
assessed in a murine melanoma model and an established 2-flank
model that allowed for monitoring of responses both in the
virus-injected tumors as well as distant tumors which do not
receive the virus. As shown in FIG. 2, the virus-infected tumors
show dramatic infiltration with immune cells such as NK cells,
macrophages, and CD8 and CD4 cells, but not regulatory T cells.
Since part of this immune response could be a response to virus,
rather than tumor, the immune response with respect to
contralateral tumors was assessed (FIG. 3). Interestingly, these
tumors demonstrated a similar degree of increased CD8 and CD4
effector, but not T reg infiltrate. Analysis of these cells
revealed that they upregulate activation, proliferation, and lytic
markers (FIG. 4). NDV monotherapy was effective in controlling the
treated tumors (FIG. 5A), but only marginally slowed down the
growth of the contralateral tumors (FIG. 5B). Mice that cleared the
tumors, however, demonstrated some degree of protection against
further tumor challenge (FIG. 5D), suggesting that NDV therapy can
induce a lasting immunity.
[0265] Next, it was assessed whether additional mechanisms could be
targeted to enhance the anti-tumor effect generated by NDV.
Characterization of tumor-infiltrating lymphocytes from both
NDV-injected and non-injected tumors revealed upregulation of the
inhibitory receptor CTLA-4 on lymphocytes (FIG. 6). It was then
assessed whether inhibition of the CTLA-4 receptor could result in
a better therapeutic efficacy of NDV. Strikingly, combination
therapy resulted in rejection in bilateral tumors in the majority
of the animals, an effect that was not seen with either treatment
alone (FIG. 7). This effect was present even when the prostate
adenocarcinoma TRAMP model was used, which is not susceptible to
viral infection (FIG. 8), suggesting that the minimal viral
replication and the resultant inflammatory response were sufficient
for generation of protective anti-tumor immunity.
[0266] To determine whether targeting other immune checkpoints in
combination with NDV therapy could be beneficial, the effect on the
PD-1-PD-L1 pathway following NDV infection was assessed. As shown
in FIG. 9, NDV infected tumor cells both in vitro and in vivo had
upregulated the expression of the inhibitory PD-L1 ligand on the
surface of the cells. This effect was not just a result of a direct
virus infection, but was also seen when non-infected cells were
treated with UV-inactivated supernatants from the virus infected
cells (FIG. 9B) and in contralateral, noninfected, tumors (FIG.
9C). This prompted testing combination therapy with NDV and
anti-PD-1 antibody. Similar to CTLA-4 blockade, NDV therapy in
combination with anti-PD-1 in the aggressive B16 melanoma model
resulted in cures in the majority of animals, an effect that was
associated with increased tumor infiltration with activated
effector lymphocytes (FIG. 10).
[0267] Throughout the studies conducted, the therapeutic efficacy
of a combination therapy decreased when larger tumor challenge was
used. Next, activation markers that could predict a better response
and could be targeted for further improvement in therapeutic
efficacy were assessed. Analysis of lymphocytes isolated from the
tumors and tumor-draining lymph nodes identified upregulation of
the co-stimulatory molecule ICOS as one of the activation markers
in the treated animals (FIG. 11). ICOS upregulation has been
previously been shown to be associated with more durable
therapeutic responses and increased survival in patients treated
with anti-CTLA-4 therapy for malignant melanoma. It was assessed
whether intratumoral expression of the ICOS ligand (ICOSL) could
further boost the therapeutic response of combination therapy.
Using reverse-genetics system for NDV, NDV expressing murine ICOSL
(NDV-ICOSL) were generated. In vitro characterization of the virus
revealed that it had similar replicative and lytic properties to
the parental NDV strain (FIG. 12). When tested in vivo, however,
with a larger B16 tumor challenge, NDV-ICOSL demonstrated
significant advantage over the parental NDV virus when used in
combination with CTLA-4 blockade, with long-term survival in the
majority of treated animals (FIG. 13). This effect was not limited
to B16 melanoma and was demonstrated for CT26 colon carcinoma in
the Balb/C mouse strain, suggesting that this therapeutic strategy
could be translatable to different tumor types (FIG. 14). Analysis
of B16 tumors from the treated animals demonstrated significant
infiltration with different immune cell subtypes with upregulation
of the activation markers (FIGS. 15 and 16). These lymphocytes were
tumor-specific and demonstrated secretion of IFN gamma in response
to stimulation with dendritic cells loaded with tumor lysates (FIG.
17). Finally, animals that were cured of their B16 or CT26 tumors
were re-challenged with tumor cells and demonstrated complete
protection against tumor re-challenge (FIG. 18).
[0268] To further improve the expression of the ICOSL in the tumor
and to incorporate the ligand into the virion, a chimeric protein
consisting of the extracellular domain of the ICOSL (amino acids
1-277) and the transmembrane and intracellular domains of the NDV F
protein (amino acids 501-554) was generated (FIG. 19A).
Transfection of the resultant construct into B16-F10 cells resulted
in increased expression of the chimeric ICOSL-F ligand on the
surface of the transfected cells, when compared to the transfected
native ICOSL, suggesting that the regulatory mechanisms governing
the transport of NDV F protein to the surface can be utilized to
increase the surface expression of immune stimulatory ligands (FIG.
19B).
[0269] Overall, these studies demonstrate that 1) combination of
NDV with immune checkpoint regulatory antibodies can be used as a
strategy to circumvent the limitation of both oncolytic virus
therapy and antibody therapy; and 2) expression of
immunostimulatory ligands by NDV can further improve the
therapeutic efficacy of the virus, especially when used in
combination with immunoregulatory antibodies. These findings have
clinical application.
7. EXAMPLE 2
[0270] This example demonstrates the anti-tumor immune responses
induced by oncolytic NDV and the anti-tumor responses induced by
NDV in combination with CTLA-4 blockade.
[0271] 7.1 Materials & Methods
Mice
[0272] C57BL/6J and Balb/C mice were purchased from Jackson
Laboratory. IFNAR-/- mice on C57BL/6J background were a kind gift
of Dr. Eric Pamer. Pmel-1 and Tip-1 TCR transgenic mice have been
reported (Overwijk et al., 2003, J. Exp. Med, 198:568, Muransky et
al., 2008, Blood 112:362) and were provided by N. Restifo (National
Cancer Institute, Bethesda, Md.). Trp1 mice were crossed to
CD2:luciferase mice provided by Patrick Hwu at MD Anderson Cancer
Center (Houston, Tex.) to create Trp1 Luciferase.sup.+ (Trp1-Fluc)
mice. All mice were maintained in microisolator cages and treated
in accordance with the NIH and American Association of Laboratory
Animal Care regulations. All mouse procedures and experiments for
this study were approved by the Memorial Sloan-Kettering Cancer
Center Institutional Animal Care and Use Committee.
Cell Lines
[0273] The murine cancer cell lines for melanoma (B16-F10), and
colon carcinoma (CT26 and MC38) were maintained in RPMI medium
supplemented with 10% fetal calf serum and penicillin with
streptomycin. The murine prostate cancer cell line TRAMP-C2 was
maintained in DMEM medium supplemented with 5% fetal calf serum
(FCS; Mediatech, Inc.), 5% Nu Serum IV (BD Biosciences) HEPES,
2-ME, pen/strep, L-glut, 5 .mu.g/mL insulin (Sigma), and 10 nmol/L
DHT (Sigma).
Antibodies
[0274] Therapeutic anti-CTLA-4 (clone 9H10), anti-PD-1 (clone
RMP1-14), anti-PD-L1 (clone 9G2), anti-CD8 (clone 2.43), anti-CD4
(clone GK1.5), anti-IFN-gamma (clone XMG1.2), and anti-NK1.1 (clone
PK136) monoclonal antibodies were produced by BioXcell. Antibodies
used for flow cytometry were purchased from eBioscience, Biolegend,
Invitrogen, and BD Pharmingen.
Viruses and Cloning
[0275] Recombinant lentogenic NDV LaSota strain was used for all
experiments. To generate NDV virus expressing murine ICOSL, a DNA
fragment encoding the murine ICOSL flanked by the appropriate
NDV-specific RNA transcriptional signals was inserted into the
SacII site created between the P and M genes of pT7NDV/LS. Viruses
were rescued from cDNA using methods described previously and
sequenced by reverse transcription PCR for insert fidelity. Virus
titers were determined by serial dilution and immunofluorescence in
Vero cells. Recombinant ICOSL-F fusion construct was generated by
PCR amplification of the ICOSL DNA encoding the extracellular
domain (amino acids 1-277) with flanking EcoRI and MluI restriction
sites, and the NDV F DNA encoding the F transmembrane and
intracellular domains (amino acids 501-554) with flanking MluI and
XhoI restriction sites. The resultant DNA fragments were assembled
in pCAGGS vector utilizing 3-part ligation. Recombinant anti-mouse
CD28scfv-F fusion construct was generated by PCR amplification of
the cDNA encoding hamster anti-CD28scfv with flanking EcoRI and
MluI restriction sites, and the NDV F DNA encoding the F
transmembrane and intracellular domains (amino acids 501-554) with
flanking MluI and XhoI restriction sites. The resultant DNA
fragments were assembled in pCAGGS vector utilizing 3-part ligation
and then subcloned into pNDV vector between the P and M genes. To
generate recombinant viruses expressing other chimeric proteins
(HN-GITRL, HN-4-1BBL, HN-CD40L, HN-OX40L), cDNA encoding
extracellular domain of each gene (FIG. 44) was amplified with
gene-specific primers with flanking EcoRI and MluI restriction
sites, and the transmembrane and intracellular domain of HN protein
was amplified with specific primers with flanking MluI and XhoI
restriction sites. The full chimeric genes were assembled in pCAGGS
vector using 3-part ligation and then subcloned into NDV vector
between the P and M genes. The details of each chimeric construct
are demonstrated in FIG. 44. To generate recombinant NDV encoding
murine IL-2, IL-15, and IL-21, the cDNA for each gene was amplified
with gene-specific primers with flanking SacII restriction sites
and then cloned into pNDV between the P and M genes. Viruses were
rescued from cDNA using methods described previously and sequenced
by reverse transcription PCR for insert fidelity. Virus titers were
determined by serial dilution and immunofluorescence in Vero
cells.
In Vitro Infection Experiments
[0276] For cell surface labeling, cells were infected in 6-well
dishes at MOI 2 (B16-F10) or MOI 5 (TRAMP C2) in triplicate.
Twenty-four hours later, the cells were harvested by scraping and
processed for surface labeling and quantification by flow
cytometry. For in vitro cytotoxicity experiments, cells were
infected at the indicated MOI's and incubated at 37.degree. C. in
serum-free media in presence of 250 ng/ml TPCK trypsin. At 24, 48,
72, and 96 hours post infection the cells were washed and incubated
with 1% Triton X-100 at 37.degree. C. for 30 minutes. LDH activity
in the lysates was determined using the Promega CytoTox 96 assay
kit, according to the manufacturer's instructions.
Tumor Challenge Survival Experiments
[0277] Bilateral flank tumor models were established to monitor for
therapeutic efficacy in both injected and systemic tumors.
Treatment schedules and cell doses were established for each tumor
model to achieve 10-20% tumor clearance by NDV or anti-CTLA-4 as
single agents. For experiments evaluating combination therapy of
NDV with anti-CTLA-4 antibody, B16-F10 tumors were implanted by
injection of 2.times.10.sup.5 B16-F10F10 cells in the right flank
intradermally (i.d.) on day 0 and 5.times.10.sup.4 cells in the
left flank on day 4. On days 7, 9, 11, and 13 the mice were treated
with intratumoral injections of 2.times.10.sup.7 pfu of NDV in PBS
in a total volume of 100 .mu.l. Concurrently, on days 7, 9, 11, and
13 the mice received intraperitoneal (i.p.) injections of
anti-CTLA-4 antibody (100 .mu.g), anti-PD-1 antibody (250 .mu.g),
or anti-PD-L1 antibody (250 .mu.g). Control groups received a
corresponding dose of isotype antibody i.p. and intratumoral
injection of PBS. The animals were euthanized for signs of distress
or when the total tumor volume reached 1000 mm.sup.3. For depletion
of immune cells, mice were injected i.p. with 500 .mu.g of
monoclonal antibodies to CD8+, CD4+, NK1.1 or IFN.gamma. one day
before and two days after tumor challenge, followed by injection of
250 .mu.g every 5 days throughout the experiment. For the TRAMP-C2
model, 1.times.10.sup.6 cells were implanted in the right flank on
day 0 and 5.times.10.sup.5 cells were implanted in the left flank
on day 4. Treatment was performed on days 7, 10, 13, and 16 in the
similar fashion to above. For the CT26 model, tumors were implanted
by injection of 1.times.10.sup.6 CT26 cells in the right flank i.d.
on day 0 and 1.times.10.sup.6 cells in the left flank on day 2.
Treatment was carried out as above on days 6, 9, and 12. For
experiments evaluating recombinant NDV expressing ICOSL, 4-1BBL,
OX40L, CD40L, GITRL, anti-CD28scfv, IL-2, IL-15, and IL-21
(NDV-transgene), B16F10 tumors are implanted by injection of
2.times.10.sup.5 B16F10 cells in the right flank i.d. on day 0 and
1.times.10.sup.5 cells in the left flank on day 4. On days 7, 9,
11, and 13 the mice are treated with intratumoral injections of
2.times.10.sup.7 pfu of NDV in PBS in a total volume of 100 .mu.l.
Concurrently, on days 7, 9, 11, and 13 the mice receive
intraperitoneal (i.p.) injections of anti-CTLA-4 antibody (100
.mu.g), anti-PD-1 antibody (250 .mu.g), or anti-PD-L1 antibody (250
.mu.g).
Isolation of Trp1 and Pmel Lymphocytes and Adoptive Transfer
[0278] Spleens and lymph nodes from transgenic mice were isolated
and grinded through 70-um nylon filters. CD4+ and CD8+ cells were
purified by positive selection using Miltenyi magnetic beads.
[0279] The isolated Trp1 or Pmel cells were injected into recipient
animals via the tail vein at the indicated schedule at
2.5.times.10.sup.4 cells per mouse and 1.times.10.sup.6 cells per
mouse, respectively.
Serum Transfer Experiments
[0280] Groups of tumor-bearing mice were treated intratumorally
with single injection of NDV or PBS. On day 4, blood was collected
by terminal bleeding and serum was isolated by centrifugation. Sera
were pooled from each group and UV-treated in Stratalinker 1800
with six pulses of 300 mJ/cm.sup.2 UV light to inactivate any virus
that could be potentially present. Undiluted 100 .mu.l of serum was
injected intratumorally into naive B16-F10 tumor-bearing mice for a
total of 3 injections given every other day. Tumors were removed 3
days after the last injection and processed for isolation of
tumor-infiltrating lymphocytes as described below.
Bioluminescence Imaging
[0281] Mice were imaged every 2-3 days starting on day 6. Mice were
injected retro-orbitally with 50 .mu.l of 40 mg/ml D-luciferin
(Caliper Life Sciences) in PBS and imaged immediately using the
IVIS Imaging System (Caliper Life Sciences). Gray-scale
photographic images and bioluminescence color images were
superimposed using The Living Image, version 4.0 (Caliper Life
Sciences) software overlay. A region of interest (ROI) was manually
selected over the tumor and the area of the ROI was kept
constant.
Isolation of Tumor-Infiltrating Lymphocytes
[0282] B16-F10 tumors were implanted by injection of
2.times.10.sup.5 B16-F10 cells in the right flank i.d. on day 0 and
2.times.10.sup.5 cells in the left flank on day 4. On days 7, 9,
and 11 the mice were treated with intratumoral injections of
2.times.10.sup.7 pfu of NDV, and i.p. anti-CTLA-4 or anti-PD-1
antibody where specified. Rare animals that died from tumor burden
(always in untreated control groups) or animals that completely
cleared the tumors (always in treatment groups) were not used for
the analysis. On day 15, mice were sacrificed and tumors and
tumor-draining lymph nodes were removed using forceps and surgical
scissors and weighed. Tumors from each group were minced with
scissors prior to incubation with 1.67 Wunsch U/mL Liberase and 0.2
mg/mL DNase for 30 minutes at 37.degree. C. Tumors were homogenized
by repeated pipetting and filtered through a 70-.mu.m nylon filter.
Cell suspensions were washed once with complete RPMI and purified
on a Ficoll gradient to eliminate dead cells. Cells from tumor
draining lymph nodes were isolated by grinding the lymph nodes
through a 70-.mu.m nylon filter.
Flow Cytometry
[0283] Cells isolated from tumors or tumor-draining lymph nodes
were processed for surface labeling with several antibody panels
staining for CD45, CD3, CD4, CD8, CD44, ICOS, CD11c, CD19, NK1.1,
CD11b, F4/80, Ly6C and Ly6G. Fixable viability dye eFluor506
(eBioscience) was used to distinguish the live cells. Cells were
further permeabilized using FoxP3 fixation and permeabilization kit
(eBioscience) and stained for Ki-67, FoxP3, Granzyme B, CTLA-4, and
IFN.gamma.. Data was acquired using the LSRII Flow cytometer (BD
Biosciences) and analyzed using FlowJo software (Treestar).
DC Purification and Loading
[0284] Spleens from naive mice were isolated and digested with 1.67
Wunsch U/mL Liberase and 0.2 mg/mL DNase for 30 minutes at
37.degree. C. The resulting cell suspensions were filtered through
70 um nylon filter and washed once with complete RPMI. CD11c+DC's
were purified by positive selection using Miltenyi magnetic beads.
Isolated DC's were cultured overnight with recombinant GM-CSF and
B16-F10 tumor lysates and were purified on Ficoll gradient.
Analysis of Cytokine Production
[0285] Cell suspensions from tumors or tumor-draining lymph nodes
were pooled and enriched for T cells using a Miltenyi T-cell
purification kit. Isolated T cells were counted and co-cultured for
8 hours with DC's loaded with B16-F10 tumor cell lysates in the
presence of 20 U/ml IL-2 (R and D) plus Brefeldin A (BD
Bioscience). After restimulation, lymphocytes were processed for
flow cytometry as above.
Immunofluorescence and Microscopy
[0286] Tumors were dissected from the mice, washed in PBS, fixed in
4% paraformaldehyde, and processed for paraffin embedding according
to protocols described previously. Sections were cut using a
microtome, mounted on slides, and processed for staining with
hematoxylin and eosin (H&E) or with anti-CD3 and anti-FoxP3
antibody. Slides were analyzed on Zeiss Axio 2 wide-field
microscope using 10.times. and 20.times. objectives.
Statistics
[0287] Data were analyzed by 2-tailed Student's t test (for
comparisons of 2 groups) and ANOVA where appropriate. Data for
survival were analyzed by Log-Rank (Mantel-Cox) Test. Two-sided
p<0.05 was considered statistically significant (P.ltoreq.0.05
(*), P.ltoreq.0.01 (**), P.ltoreq.0.001 (***), P<0.0001
(****)).
[0288] 7.2 Results
NDV Replication is Restricted to the Injected Tumor Site
[0289] The viral distribution kinetics with intratumoral and
systemic administration of NDV were characterized. Intratumoral
injection of recombinant NDV expressing firefly luciferase reporter
(NDV-Fluc) resulted in sustained luciferase signal in the injected
flank tumor, while systemic administration of the virus resulted in
no detectable luciferase signal in the tumor (FIG. 20A). As limited
systemic virus delivery was unlikely to induce sufficient tumor
lysis and immune response, the intratumoral NDV injection was
explored as a means to elicit an anti-tumor immune response that
could potentially overcome the limitations of systemic OV therapy.
As such, for further studies modeled metastatic disease was modeled
by using the bilateral flank B16-F10 tumor model (FIG. 22A).
NDV-Fluc administration into the right flank tumor resulted in
viral replication within the injected tumor, with the luciferase
signal detectable for up to 96 hours (FIG. 20B-D). No virus was
detected in the contralateral (left flank) tumor by luminescent
imaging (FIG. 20B-D), by passage in embryonated eggs, or RT-PCR.
This system thus allowed for the characterization of the immune
responses in both virus-injected and distant tumors, which were not
directly affected by NDV.
NDV Therapy Increases Local and Distant Tumor Lymphocyte
Infiltration and Delays Tumor Growth
[0290] Analysis of the virus-injected tumors revealed an
inflammatory response as evidenced by increased infiltration with
cells expressing leukocyte common antigen CD45 (FIGS. 21A-B). The
immune infiltrates were characterized by increase in innate immune
compartment, including myeloid cells, NK cells, and NKT cells (FIG.
21C), and the adaptive compartment, including CD8+ and conventional
CD4+FoxP3- (Tconv) T cells, leading to significant increase of CD8
and Tconv to regulatory (Treg) T cell ratios (p=0.0131 and
p=0.0006, respectively) (FIGS. 21D-21F). Remarkably, analysis of
the contralateral tumors revealed a similar increase in the
inflammatory infiltrates (FIG. 22B,C), characterized by increased
numbers of both innate immune cells (FIG. 22D) and effector T cells
(FIG. 22E,G). Notably, although there were no major changes in the
absolute number of Tregs (FIG. 22G), there was a substantial
decrease in their relative percentages (FIG. 22E,F,H), with
significant enhancement of the CD8 and Tconv to Treg ratios
(p=0.002 and p=0.0021, respectively) (FIG. 22I). Effector T cells
isolated from the distal tumors expressed increased activation,
proliferation, and lytic markers ICOS, Ki-67, and Granzyme B,
respectively (FIG. 1J,K). As previously, virus or viral RNA was
unable to be isolated from the distant tumors, suggesting that the
observed changes in the distant tumor microenvironment were not due
to direct viral infection. In order to further exclude the
possibility of undetectable local viral spread, tumors were
implanted at other distant sites, such as bilateral posterior
footpads, which generated similar findings (FIG. 23).
[0291] Consistent with the observed inflammatory effect,
intratumoral administration of NDV resulted in growth delay not
only of the injected, but also of the contralateral tumors,
resulting in prolonged animal survival (FIG. 1L,M). To determine
whether this effect was transient and whether durable anti-tumor
protection was possible, single-flank B16-F10 tumor-bearing mice
were intratumorally treated with NDV, and long-term survivors were
injected with B16-F10 cells on the opposite flank. The majority of
the animals demonstrated tumor growth delay, and 30% of the animals
completely rejected rechallenged cells, suggesting that
intratumoral therapy with NDV can indeed induce protective
anti-tumor memory responses (FIG. 25).
NDV Induces Tumor Infiltration and Expansion of Tumor-Specific
Lymphocytes
[0292] To determine whether the anti-tumor immune response was
dependent on the NDV-injected tumor type or a result of nonspecific
inflammation generated by NDV infection, the experiment was
performed with heterologous tumors (MC38 colon carcinoma and
B16-F10 melanoma) implanted at the opposite flanks (FIG. 24A). To
track tumor-specific lymphocytes, T cell receptor-transgenic
congenitally-marked CD8+ (Pmel) cells or luciferase-marked CD4+
(Trp1) cells recognizing the melanoma differentiation antigens
gp100 (Pmel) and Trp1 (Trp1) were adoptively transferred (Muranski
et al., 2008, Blood, 112: 362; Overwijk et al., 2003, J Exp Med,
198: 569). Bioluminescent imaging was used to measure the
distribution and expansion kinetics of the adoptively transferred
Trp1 cells. Transfer of Trp1 cells into PBS-treated tumor-bearing
animals failed to result in Trp1 accumulation in the tumors,
highlighting the highly immunosuppressive nature of the tumor
microenvironment in this model (FIG. 24B-D). NDV injection into
B16-F10 tumors resulted in significant increase in the luciferase
signal within the injected tumors (FIG. 24B-D), indicating Trp1 T
cell expansion (area under the curve (AUC) p=0.0084). Remarkably,
similar expansion was seen in the contralateral tumor, albeit at a
delay (p=0.0009) (FIG. 24B-D). In contrast, NDV injection into MC38
tumors failed to induce substantial Trp1 infiltration into the
injected MC38 tumors or distant B16-F10 tumors (FIG. 24B-D),
suggesting that the distant tumor-specific lymphocyte infiltration
is likely dependent on the antigen identity of the injected tumor.
Similarly, intratumoral injection of NDV resulted in increased
infiltration of Pmel cells in distant tumors, which was more
pronounced when the injected tumor was B16-F10 rather than MC38
(FIG. 24E).
[0293] Interestingly, although infiltration of distant B16-F10
tumors with adoptively-transferred lymphocytes was dependent on the
injected tumor identity, distant tumors did demonstrate increased
immune infiltration even when the primary injected tumor was MC38
(FIG. 24F), suggesting that a nonspecific inflammatory response
component may also play a role. Indeed, serum from NDV-treated
animals, treated with UV irradiation to inactivate any potential
virus, induced tumor leukocyte infiltration when injected
intratumorally into naive B16-F10 tumor-bearing mice (FIG. 24G,H),
with the majority of the increase seen in the NK and CD8+
compartments (p=0.0089 and p=0.0443, respectively) (FIG. 24I).
NDV and CTLA-4 Blockade Synergize to Reject Local and Distant
Tumors
[0294] Despite the prominent inflammatory response and growth delay
seen in distant tumors, complete contralateral tumor rejection with
long-term survival was only seen in approximately 10% of animals
(FIG. 22M), suggestive of active immunosuppressive mechanisms in
the tumor microenvironment. Characterization of NDV-injected and
distant tumors revealed upregulation of CTLA-4 on
tumor-infiltrating T cells (FIG. 26), suggesting that NDV-induced
tumor inflammation would make the tumors sensitive to systemic
therapy with CTLA-4 blockade. Remarkably, combination therapy of
NDV with anti-CTLA-4 antibody (FIG. 27A) resulted in rejection of
bilateral tumors and long-term survival in the majority of the
animals, an effect that was not seen with either treatment alone
(FIG. 27B-D). To determine the durability of the observed
protection, the surviving animals were injected in the right flank
on day 90 with B16-F10 cells without any further therapy. Animals
treated with NDV and anti-CTLA-4 combination therapy demonstrated
over 80% protection against tumor re-challenge, compared with 40%
protection in the animals treated with single agent anti-CTLA-4
antibody (FIG. 27E).
Combination Therapy with NDV and CTLA-4 Blockade is Effective
Against Virus Non-Permissive Tumors
[0295] To determine whether this treatment strategy could be
extended to other tumor types, the strategy was evaluated in the
poorly-immunogenic TRAMP C2 prostate adenocarcinoma model.
Similarly to the B16-F10 model, combination therapy caused
regression of the injected tumors (FIG. 27F), and either delayed
the outgrowth of distant tumors or led to complete distant tumor
regression with prolonged long-term survival (FIG. 27F,G).
Interestingly, whereas B16-F10 cells were susceptible to
NDV-mediated lysis in vitro, TRAMP C2 cells were strongly
resistant, with low cytotoxicity observed at a multiplicity of
infection (MOI) of up to 10 (FIG. 27H). In both cell lines, NDV
infection in vitro resulted in surface upregulation of MHC and
co-stimulatory molecules (FIG. 27I-K). MHC class I was upregulated
uniformly in all cells, even though not all cells get infected with
NDV at the MOI of 1. Previous studies demonstrated that NDV induces
type I IFN expression in B16-F10 cells (Zamarin et al., 2009, Mol
Ther 17:697). Both type I IFN (Dezfouli et al., 2003, Immunol.
Cell. Biol., 81:459, Seliger et al., 2001, Cancer Res., 61:1095)
are known to upregulate MHC class Ion B16-F10 cells, suggesting
that within the context of the infected tumors these mechanisms may
play an additional role in enhancement of tumor immunogenicity.
These results thus suggest that in vitro sensitivity to
virus-mediated lysis is not necessary for sensitivity to NDV
therapy in vivo and further highlight the importance of a
virus-generated inflammatory response, rather than direct
oncolysis, in the observed anti-tumor efficacy.
Systemic Anti-Tumor Effect is Antigen-Restricted to the Injected
Tumor Type
[0296] To determine whether the observed anti-tumor effect in the
distant tumor was specific to the injected tumor type, the
combination therapy in animals bearing a unilateral distant B16-F10
tumor and in animals with heterologous tumor types (MC38 colon
carcinoma and B16-F10 melanoma) implanted at the opposite flanks
was evaluated (FIG. 28A). Although administration of the virus
intradermally into the non-tumor-bearing right flank resulted in
delayed left flank tumor outgrowth, it failed to result in
long-term protection and tumor rejection seen in the animals
bearing bilateral B16-F10 tumors (FIG. 28B,C). Similarly, injection
of NDV into the right flank MC38 tumors of the animals bearing left
flank B16-F10 tumors failed to induce B16-F10 tumor rejection (FIG.
28D,E), suggesting that the NDV-induced anti-tumor immune response
is likely antigen-restricted to the injected tumor.
Combination Therapy with NDV and Anti-CTLA-4 Induces Tumor
Infiltration with Activated Lymphocytes
[0297] To examine the B16-F10 tumor microenvironment in the treated
animals, bilateral tumors were collected and processed for analysis
of infiltrating cells. Analysis of the injected and distant tumors
from the treated animals revealed prominent inflammatory
infiltrates and large areas of tumor necrosis in the animals
treated with combination therapy (FIG. 30A, FIG. 29). This
correlated with increased numbers of CD45+ cells and T cells in the
combination therapy group (FIG. 30A-C, FIG. 29A-C). As previously,
the observed increase in TILs was primarily due to infiltration of
CD8+ and Tconv, but not Treg cells, leading to enhanced effector to
Treg ratios (FIG. 30D-F, FIG. 29C-E). Phenotypic characterization
of CD4+ and CD8+ TILs from animals receiving the combination
treatment demonstrated upregulation of ICOS, Granzyme B, and Ki-67
over the untreated and anti-CTLA-4 treated animals (FIG. 30G-I) and
a larger percentage of IFNgamma-expressing CD8+ cells in response
to re-stimulation with dendritic cells (DCs) pulsed with B16-F10
tumor lysates (FIG. 30J).
Anti-Tumor Activity of NDV Combination Therapy Depends on
CD8+Cells, NK Cells and Type I and II Interferons
[0298] To determine which components of cellular immunity were
responsible for the observed therapeutic effect, the treatment was
repeated in the presence of depleting antibodies for CD4+, CD8+, or
NK cells. Adequate cell depletion of each cell subset was confirmed
by flow cytometry of peripheral blood (FIG. 31). Depletion of
either CD8+ or NK cells resulted in abrogation of therapeutic
effect in both virus-injected and distant tumors (FIG. 32A,B), with
significant reduction in long-term survival (p<0.0001 for CD8
and p=0.0011 for NK depletion) (FIG. 32C). Consistent with these
findings, treatment of the animals with an anti-IFN.gamma.
neutralizing antibody also decreased therapeutic efficacy. In
contrast, depletion of CD4+ cells did not result in appreciable
change in anti-tumor effect, though these results must be
interpreted with caution since anti-CD4+ depletion also results in
concurrent depletion of Tregs.
[0299] Type I IFN has been previously demonstrated to play an
important role in priming of CD8+ cells for anti-tumor immune
response (Fuertes et al., 2011, J Exp Med, 208: 2005; Diamond et
al, 2011, J Exp Med, 208: 1989). To investigate the role of type I
IFN in tumor rejection by NDV, the experiments were repeated in the
type I IFN receptor knockout (IFNAR-/-) mice. The IFNAR-/- mice
demonstrated rapid progression of both injected and contralateral
tumors and were completely resistant to the combination therapy
(FIG. 32D-F). Overall, these findings highlight the important role
of both innate and adaptive immune responses for the systemic
therapeutic efficacy of the virus observed in this study.
NDV Therapy Leads to Upregulation of PD-L1 on Tumor Cells and on
Tumor Infiltrating Leukocytes
[0300] To determine whether targeting other immune checkpoints in
combination with NDV therapy could be beneficial, the effect on the
PD-1-PD-L1 pathway following NDV infection was assessed. As shown
in FIG. 33, NDV infected tumor cells both in vitro and in vivo had
upregulated the expression of the inhibitory PD-L1 ligand on the
surface of the cells (FIG. 33A), which was also seen in the
distant, noninfected, tumors. The upregulation of PD-L1 was not
just restricted to tumor cells, but was also seen on tumor
infiltrating leukocytes of both innate and adaptive immune lineages
(FIG. 33B).
Combination Therapy of NDV with PD-1 and PD-L1-Blocking Antibodies
Leads to Improved Anti-Tumor Immunity and Long-Term Animal
Survival
[0301] The combination of NDV with antibody blocking PD-1 and the
combination of NDV with antibody blocking PD-L1 were evaluated in
the bilateral flank melanoma model described above. Remarkably,
similar to CTLA-4 blockade, NDV therapy in combination with either
anti-PD-1 or anti-PD-L1 antibody led to improved animal survival
(FIGS. 34 and 35). Distant tumors from animals treated with
combination of NDV and anti-PD-1 antibody were characterized. As
can be seen from FIG. 36, combination of intratumoral NDV with
systemic PD-1 blockade led to marked distant tumor infiltration
with immune cells, with the increase in tumor-infiltrating CD8
cells being the most pronounced finding. The infiltrating cells
upregulated proliferation and lytic markers Ki67 and granzyme B,
respectively (FIG. 37).
NDV Induces Tumor Immune Infiltration Upregulation of ICOS on CD4
and CD8 Cells in the Virus-Injected and Distant Tumors and Tumor
Draining Lymph Nodes (TDLN)
[0302] The findings above demonstrated that combination of
intratumoral NDV with systemic immune checkpoint blockade results
in significant synergy between the two therapeutic approaches. To
further build on these findings, enhancement of T cell effector
function within the tumor microenvironment through a relevant
co-stimulatory pathway may drive a better anti-tumor immune
response was investigated. Previous studies identified the
sustained upregulation of inducible costimulator (ICOS) on T cells
as a strong indicator of response to CTLA-4 blockade in patients
(Carthon et al., 2010, Clin. Canc. Res., 16:2861). ICOS is a CD28
homologue upregulated on the surface of activated T cells that has
been shown to be critical for T cell-dependent B lymphocyte
responses and development of all T helper subsets (Simpson et al.,
2010 Curr Opin Immunol. 22:326). The role of ICOS in anti-tumor
tumor efficacy of CTLA-4 blockade was recently confirmed by mouse
studies, where ICOS-deficient mice were severely compromised in
development of anti-tumor response with CTLA-4 blockade (Fu et al.,
2011, Cancer Res., 71:5445).
[0303] The expression of ICOS in bilateral flank tumor models
treated with NDV were characterized to determine whether the
receptor could serve as a target in this therapeutic approach. To
characterize the local and abscopal effects of intratumoral NDV
therapy, bilateral flank B16-F10 melanoma models were utilized,
with the virus administered to a unilateral tumor (FIG. 38A).
Activation markers that could predict a better response and could
be targeted for further improvement in therapeutic efficacy were
assessed. The example focused on ICOS, as sustained ICOS
upregulation has been previously been shown to be associated with
more durable therapeutic responses and increased survival in
patients treated with anti-CTLA-4 therapy for malignant melanoma.
Analysis of lymphocytes isolated from the tumors and tumor-draining
lymph nodes identified upregulation of the co-stimulatory molecule
ICOS as one of the activation markers in the treated animals (FIG.
38B, C).
Generation and In Vitro Evaluation of NDV-ICOSL Virus.
[0304] Using reverse-genetics system for NDV, NDV expressing murine
ICOSL (NDV-ICOSL) was generated (FIG. 39A). The expression of ICOSL
on the surface of infected B16-F10 cells was confirmed by flow
cytometry after 24 hour infection (FIG. 39B). In vitro
characterization of the virus revealed that it had similar
replicative (FIG. 39D) and lytic (FIG. 39C) properties to the
parental NDV strain.
NDV-ICOSL Growth Delay of Distant Tumors and Induces Enhanced Tumor
Lymphocyte Infiltration
[0305] To evaluate NDV-ICOSL for therapeutic efficacy in the
virus-injected and distant tumors, animals bearing bilateral
B16-F10 tumors were treated with 4 intratumoral injections of the
virus given to a unilateral flank tumor. Both NDV-ICOSL and
wild-type NDV were comparable in their ability to cause tumor
regressions within the tumors directly injected with the virus
(FIG. 40A). However, when compared to the wild-type NDV, NDV-ICOSL
resulted in significant tumor growth delay of the distant tumors
with several animals remaining tumor-free long-term (FIG. 40B-C).
Analysis of virus-injected tumors revealed enhanced tumor
infiltration with CD4 and CD8 effector cells in the animals treated
with wild-type NDV and NDV-ICOSL, although the differences between
the two viruses were not statistically significant, mirroring the
similar activity of the two viruses against the right flank tumors
(FIGS. 40A and 40D). In contrast, analysis of the left flank tumors
revealed more prominent increase in tumor-infiltrating CD8 and
Tconv cells in the NDV-ICOSL-treated group (FIG. 40E).
Interestingly, there was also an increase in absolute number of
regulatory T cells, with the highest increase seen in the NDV-ICOSL
group (FIG. 40E), although the relative percentage of regulatory T
cells was significantly lower in the NDV-treated animals (FIG.
40F).
Combination Therapy of NDV-ICOSL and CTLA-4 Blockade Results in
Rejection of the Injected and Distant Tumors.
[0306] Overall, the findings above demonstrated that despite the
significant inflammatory response seen in distant tumors with
intratumoral administration of NDV-ICOSL, the majority of the
animals still succumbed to tumors, suggesting that the inhibitory
mechanisms active within the tumor microenvironment prevent tumor
rejection by the infiltrating immune cells. Thus the efficacy of
combination therapy of localized NDV-ICOSL with systemic CTLA-4
blockade was evaluated. For these experiments, the tumor challenge
doses were increased to the levels where no significant therapeutic
efficacy with NDV or anti-CTLA-4 as single agents was observed. As
previously, the animals were treated with 4 doses of NDV
administered to a unilateral tumor, concomitantly given with
systemic anti-CTLA-4 antibody (FIG. 41A). In the B16-F10 model,
combination therapy with NDV-ICOSL and anti-CTLA-4 led to
regression of the majority of the injected and distant tumors with
long-term animal survival, which was significantly superior to the
combination of NDV-WT with anti-CTLA-4 (FIG. 41B-D). To determine
whether these findings could be extended to other tumor models, the
same experiment was performed in the bilateral flank CT26 colon
carcinoma model. Despite the poor sensitivity of CT26 cells to
NDV-mediated lysis in vitro, significant therapeutic efficacy of
combination therapy of NDV and anti-CTLA-4 against both
virus-injected and distant tumors was observed, with superior
efficacy again seen in the group utilizing the combination of
NDV-ICOSL with anti-CTLA-4 (FIG. 42A-D). In both tumor models,
animals that completely cleared the tumors were re-challenged with
lethal dose of tumor cells on day 90 without further therapy and
demonstrated protection against re-challenge in the majority of the
animals (FIG. 41E and FIG. 42E). Interestingly, while in the CT26
model all of the cured animals were protected from re-challenge, in
the B16-F10 model the animals treated with combination therapy
demonstrated superior protection, when compared to the animals that
were cured by anti-CTLA-4 alone (FIG. 41E), indicating that the
combination approach leads to a more effective protective memory
response.
Combination Therapy Leads to Enhanced Tumor Infiltration with
Innate and Adaptive Immune Cells
[0307] Analysis of distant B16 tumors from the animals treated with
combination of NDV and anti-CTLA-4 therapy demonstrated significant
tumor infiltration with different immune cell subtypes (FIG.
43A,B). The increased infiltration was evident in both the innate
(FIG. 43C,D) and the adaptive (FIG. 43E) immune compartments, with
the highest increase seen in the group treated with combination of
NDV-ICOSL and anti-CTLA-4. Interestingly, while this group
demonstrated the highest number of infiltrating CD8+ lymphocytes,
there was also a statistically-significant increase in regulatory T
cells seen in this group (FIG. 43E), though the overall percentage
of Tregs was significantly decreased, when compared to the
untreated animals or animals treated with single-agent anti-CTLA-4
(FIG. 43F), with resultant increase of the effector to Treg ratios
(FIG. 43G). A detailed analysis of the TILs demonstrated that the
TILs isolated from the animals treated with NDV-ICOSL and
anti-CTLA-4 combination expressed the highest levels of activation,
lytic, and proliferation makers ICOS, granzyme B, and Ki67
respectively (FIG. 43H-J).
Generation of Recombinant NDV Expressing Other Co-Stimulatory
Molecules
[0308] This example thus demonstrates that expression of a
co-stimulatory ligand by NDV can result in activation of stronger
immune responses, which can lead to more effective anti-tumor
immunity, especially in the setting of combination therapies with
immune checkpoint blockade. To evaluate additional co-stimulatory
molecules, ligands targeting the immunoglobulin superfamily of
receptors (ICOS and CD28) and the TNF receptor superfamily (GITR,
4-1BB, OX40, and CD40) were studied. For targeting CD28 an
artificial ligand composed of a chimeric protein with the
cytoplasmic and transmembrane domains of the NDV F glycoprotein and
the extracellular domain composed of a single-chain antibody
against CD28 (aCD28-scfv) was engineered (FIG. 44A,B). For the
ligands targeting the TNF receptor superfamily, the extracellular
domain of each ligand was fused to the transmembrane and
intracellular domains of the NDV HN glycoprotein, in order to
ensure enhanced expression of the ligands on the surface of the
infected cells (FIG. 44A,B). In addition, recombinant viruses
expressing cytokines of the common gamma chain receptor family
(IL-2, IL-15, and IL-21) were generated. The resultant constructs
are illustrated in the diagram in FIG. 44C. Recombinant viruses
were generated by reverse genetics and presence of the viruses was
confirmed by hemagglutination assays (FIG. 45A). To ensure the
fidelity of the inserted genes, RNA was isolated from each virus
and RT-PCR was performed with primers annealing outside of the
cloned gene region (FIG. 45B,C). Sequence of each gene was further
confirmed by Sanger sequencing. To confirm the expression of
co-stimulatory ligands on the surface of infected cells, cultured
B16-F10 cells were infected at MOI of 2 and analyzed 24 hours later
by flow cytometry with antibodies specific to each gene (FIG.
46).
NDV-4-1BBL Induces Increased Tumor Infiltration with Lymphocytes in
the Distant Tumors
[0309] The ability of the engineered viruses to demonstrate any
evidence of enhanced immune response was evaluated, using
NDV-4-1BBL as an example. Mice bearing bilateral flank B16-F10
melanomas were treated with intratumoral injection to right tumor
of control NDV or NDV-4-1BBL, as described previously and distant
tumors were collected on day 15. As can be seen in FIG. 47, therapy
with NDV-4-1BBL demonstrated enhanced infiltration of both innate
and adaptive immune cells into the contralateral tumors, consistent
with previous findings demonstrating similar results with NDV
expressing ICOSL (FIG. 40). Overall, these findings suggest that
expression of immunostimulatory molecules by NDV within the context
of tumor microenvironment can lead to enhanced anti-tumor
immunity.
[0310] The generated viruses NDV-4-1BBL, NDV-GITRL, NDV-OX40L,
NDV-CD40L, NDV-IL-2, NDV-IL-15, NDV-IL-21 are evaluated for the
ability to induce tumor immune infiltration using similar assays as
described in this Section 7. In addition, for therapeutic
evaluation, each of the viruses is evaluated in bilateral flank
tumor models with the virus being administered to a single-flank
tumor in combination with systemic antibodies targeting the
inhibitory checkpoints PD-1, PD-L1, and/or CTLA-4.
CONCLUSION
[0311] To trigger immunogenic tumor cell death and an inflammatory
response, nonpathogenic NDV was employed, which, despite its
relatively weak lytic activity, has been demonstrated to be a
potent inducer of type I IFN and DC maturation (Wilden et al.,
2009, Int J Oncol 34: 971; Kato et al., 2005, Immunity 23: 19). A
bilateral flank melanoma model with staggered implantation of
tumors at a schedule that was previously demonstrated not to be
affected by concomitant immunity was utilized (Turk et al., 2004, J
Exp Med 200: 771). This example demonstrates that intratumoral
injection of NDV results in distant tumor immune infiltration in
the absence of distant virus spread. Notably, this effect was
associated with relative reduction in the number of Tregs and
marked enhancement of CD4 and CD8 effector to Treg ratios, which
has been previously demonstrated to be a marker of a favorable
immunological response to immunotherapy (Quezada et al., 2006, J
Clin Invest 116: 1935; Curran et al., 2010, Proc Natl Acad Sci USA
107: 4275).
[0312] The data in this example demonstrates that NDV enhances
tumor infiltration with tumor-specific lymphocytes, an effect that
was dependent on the identity of the virus-injected tumor. The
enhanced tumor infiltration and expansion of adoptively-transferred
lymphocytes further suggest the synergy between oncolytic virus
therapy and therapeutic approaches utilizing adoptive T cell
transfer. It is plausible that the tumor-specific lymphocytes
undergo activation and expansion at the site of the initial viral
infection, followed by their migration to other tumor sites, which
is likely dependent on chemokines and lymphocyte homing receptors
(Franciszkiewicz et al., 2012, Cancer Res 72: 6325). The data in
this example also demonstrates that distant tumor immune
infiltration was in part non-specific and could be induced by NDV
infection of a heterologous tumor or by transfer of serum from
treated animals to naive tumor-bearing mice. Increased vascular
permeability induced by inflammatory cytokines such as IL-6 may
strongly contribute to activation of tumor vasculature and
lymphocyte recruitment into the tumors (Fisher et al., 2011, The
Journal of clinical investigation 121: 3846).
[0313] Despite the pronounced increase in TILs, therapeutic effect
in distant tumors was rather modest with NDV monotherapy,
highlighting the immunosuppressive nature of the microenvironment
of these tumors (Spranger et al., 2013, Sci Transl Med 5).
Remarkably, combination of systemic anti-CTLA-4 antibody with
intratumoral NDV led to rejection of distant B16-F10 tumors with
long-term animal survival. The animals were also protected against
further tumor rechallenge, suggestive of establishment of long-term
memory. Interestingly, therapeutic efficacy was also seen with
TRAMP C2 and CT26 tumor models, which exhibit poor sensitivity to
NDV-mediated cell lysis in vitro. These findings highlight the
importance of the NDV-induced anti-tumor immune/inflammatory
response, rather than direct lysis, as the primary mechanism
driving the anti-tumor efficacy in this model. Indeed, analysis of
NDV-injected and distant tumors treated with combination therapy
demonstrated prominent infiltration with innate immune cells and
activated CD8+ and CD4+ effector cells, while depletion of CD8+ and
NK cells abrogated the therapeutic efficacy. Furthermore, the
combination strategy was completely ineffective in IFNAR-/- mice,
which support the role of the type I IFN pathway in the induction
of anti-tumor immunity in this system (Fuertes et al., 2011, J Exp
Med 208, 2005; Diamond et al., 2011, J Exp Med 208: 1989; Swann et
al., 2007, J Immunol 178: 7540).
[0314] In summary, this example demonstrates localized intratumoral
therapy of B16 melanoma with NDV induces inflammatory responses
leading to lymphocytic infiltrates and anti-tumor effect in distant
(non-virally injected) tumors without distant virus spread. The
inflammatory effect coincided with distant tumor infiltration with
tumor-specific CD4+ and CD8+ T cells, which was dependent on the
identity of the virus-injected tumor. Combination therapy with
localized NDV and systemic CTLA-4 blockade led to rejection of
pre-established distant tumors and protection from tumor
re-challenge in poorly-immunogenic tumor models, irrespective of
tumor cell line sensitivity to NDV-mediated lysis. Therapeutic
effect was associated with marked distant tumor infiltration with
activated CD8+ and CD4+ effector but not regulatory T cells, and
was dependent on CD8+ cells, NK cells and type I interferon. This
example demonstrates that localized therapy with oncolytic NDV
induces inflammatory immune infiltrates in distant tumors, making
them susceptible to systemic therapy with immunomodulatory
antibodies.
[0315] The invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described will become apparent to
those skilled in the art from the foregoing description and
accompanying Figures. Such modifications are intended to fall
within the scope of the appended claims.
[0316] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
* * * * *