Note: Descriptions are shown in the official language in which they were submitted.
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RNA viruses expressing IL-12 for immunovirotherapy
The present invention relates to a recombinant virus of the family
Paramyxoviridae,
comprising at least one expressible polynucleotide encoding an IL-12
polypeptide, wherein
said IL-12 polypeptide is an IL-12 fusion polypeptide comprising a p35 subunit
of an IL-12
and a p40 subunit of an IL-12; to a polynueleotide encoding the same, and to a
kit comprising
the same. Moreover, the present invention relates to a method for treating
cancer in a subject
afflicted with cancer, comprising contacting said subject with a recombinant
virus of the
family Paramyxoviridae of the invention, and, thereby, treating cancer in a
subject afflicted
with cancer.
Interleukin 12 (IL-12) is a heterodimeric polypeptide interleukin consisting
of two subunits,
p35 and p40, encoded by two separate genes, IL-12A and IL-12B, respectively.
IL-12 is
produced in response to immune stimuli by dendritic cells, macrophages,
neutrophils, and by
human B-lymphoblastoid cells, and has been known as an important stimulator of
immune
cell activity, in particular of T cells and natural killer cells, which are,
among other effects,
stimulated to secrete IFN-y by IL-12. 1FN- y, in turn, is known to stimulate
expression of
immune checkpoint blockade proteins on non-immune cells, e.g. of PD-L1, a
mechanism used
by cancer cells to evade the immune system (Abiko et al. (2015), British
journal of cancer
112(9): 1501; Quetglas et al. (2015) Cancer Research 75(15 Supplement): 281).
Due to the
known immunostimulatory effects of IL-12, it was attempted to use a
recombinant measles
virus expressing both subunits of IL-12 as a vaccine to improve immune
response against
measles virus. However, it was found that the transgene had a detrimental
effect on the
neutralizing antibody response and that lymphoproliferative responses were not
improved
(Hoffman et al. (2003), J Infect Dis 188:1553).
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Oncolytic viruses (0V) which replicate selectively in tumor cells are an
emerging modality of
cancer treatment. Aside from direct cytopathic effects and lysis of tumor
cells, interactions of
OV with the immune system can trigger systemic anti-tumor immunity. OV have
been
modified to express immunomodulatory transgenes to further enhance these
effects (Melcher
et at., Mol Then 2011, 19: 1008-1016). The vaccinia virus JX-594 and
herpesvirus
talimogene laherpavec (TVEC), both harboring GM-CSF, have shown promising
results in
clinical phase II and III trials (Heo et at., Nat Med. 2013,19: 329-336 and
Andtbacka et at. J
Clin Oncol. 2013, 31, suppl; abstr LBA9008).
RNA viruses, in particular members of the family Paramyxoviridae like, e.g.
measles virus
(MV), have also shown potential use in oncolysis. Viruses of the family
Paramyxoviridae are
negative-sense single-stranded RNA viruses and include human pathogens like,
e.g. human
parainfluenza viruses, mumps virus, human respiratory syncytial virus, and
measles virus.
From wild type measles virus, several non-pathogenic strains, including a
vaccine strain, have
been derived, which have been shown to remain oncolytic. The measles virus
vaccine strain
has been developed as a vector platform to target multiple tumor entities and
several clinical
trials are ongoing (Russell et al., Nat Biotechnol. 2012, 30: 658-670).
Recently, the capacity
of oncolytic MV encoding GM-CSF to support the induction of a specific anti-
tumor immune
response in terms of a tumor vaccination effect was demonstrated (Grossardt et
al. Hum Gene
Ther. 2013, 24: 644-654.).
There is, however, still a need in the art for improved cancer therapies, in
particular for
improved oncolytic virus therapies. It is therefore an objective of the
present invention to
provide an improved oncolytic virus, which fully or partially avoids the short-
comings of
known oncolytic viruses.
Accordingly, the present invention relates to a recombinant virus of the
family
Paramyxoviridae, comprising an expressible polynucleotide encoding an IL-12
polypeptide,
wherein said IL-12 polypeptide is an IL-12 fusion polypeptide comprising a p35
subunit of an
IL-12 and a p40 subunit of an IL-12.
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As used in the following, the terms "have", "comprise" or "include" or any
arbitrary
grammatical variations thereof are used in a non-exclusive way. Thus, these
terms may both
refer to a situation in which, besides the feature introduced by these terms,
no further features
are present in the entity described in this context and to a situation in
which one or more
further features are present. As an example, the expressions "A has B", "A
comprises B" and
"A includes B" may both refer to a situation in which, besides B, no other
element is present
in A (i.e. a situation in which a solely and exclusively .consists of B) and
to a situation in
which, besides B, one or more further elements are present in entity A, such
as element C,
elements C and D or even further elements.
Further, as used in the following, the terms "preferably", "more preferably",
"most
preferably", "particularly", "more particularly", "specifically", "more
specifically" or similar
temis are used in conjunction with optional features, without restricting
alternative
possibilities. Thus, features introduced by these terms are optional features
and are not
intended to restrict the scope of the claims in any way. The invention may, as
the skilled
person will recognize, be performed by using alternative features. Similarly,
features
introduced by "in an embodiment of the invention" or similar expressions are
intended to be
optional features, without any restriction regarding alternative embodiments
of the invention,
without any restrictions regarding the scope of the invention and without any
restriction
regarding the possibility of combining the features introduced in such way
with other optional
or non-optional features of the invention. Moreover, if not otherwise
indicated, the term
"about" relates to the indicated value with the commonly accepted technical
precision in the
relevant field, preferably relates to the indicated value 20%.
The terms "virus" and "virus of the family Paramyxoviridae" are known to the
skilled person.
Preferably, the virus of the family Paramyxoviridae is a member of the genus
Morbillivirus.
More preferably, the virus of the family Paramyxoviridae is a measles virus
(MV), still more
preferably an MV of strain Edmonston A or B, preferably B. Most preferably,
the virus of the
family Paramyxoviridae is an MV of vaccine strain Schwarz/Moraten.
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The term "recombinant virus", as used herein, relates to a virus comprising a
genome
modified by biotechnological means as compared to known, naturally occurring,
virus
genomes. Preferably, the recombinant virus is a virus comprising a genome
modified as
compared to naturally occurring virus genomes. Preferred biotechnological
means for
.. modifying a viral genome are known to the skilled person and include any of
the methods of
molecular cloning, in particular recombinant DNA techniques including, without
limitation,
cleavage of DNA by restriction enzymes, ligation of DNA, polymerase chain
reaction (PCR),
cloning of viral genomes, and the like. It is understood by the skilled person
that viruses of the
family Paramyxoviridae have a single-stranded (-)-RNA as a genome.
Accordingly, the
genome of the recombinant virus of the present invention, preferably, is
obtained by cloning
an expression vector as described herein below comprising an expressible
nucleotide
sequence encoding said recombinant virus genome, followed by expressing said
expressible
nucleotide sequence encoding said recombinant virus in a permissive host cell.
Alternatively,
the recombinant virus genome may also be expressed in non-permissive host
cells, e.g.,
preferably, from rodents or other higher eukaryotes. Preferably, the
recombinant virus of the
present invention is a recombinant virus of the family Paramyxoviridae, more
preferably a
recombinant Morbillivirus, most preferably, a recombinant measles virus (MV).
As will be
understood by the skilled person, the recombinant virus of he present
invention may
comprises further modifications as compared to a naturally occurring virus.
Preferably, the
recombinant virus comprises a polypeptide mediating a modified tropism and/or
a
polynucleotide encoding the same. More preferably, said polypeptide mediating
a modified
tropism is a fusion polypeptide of a viral membrane integral polypeptide or of
a viral
membrane associated polypeptide with a polypeptide mediating binding to a
target, e.g. a cell,
preferably a specific kind of cell, more preferably a cancer cell. Preferably,
said fusion
polypeptide comprises a viral hemagglutinin or a fragment thereof, preferably
a membrane
integral fragment thereof. Preferably, said fusion polypeptide comprises a
single-chain
antibody specifically binding to a target molecule, e.g. to Carcinoembryonic
antigen (CEA) or
CD20. Most preferably, said fusion polypeptide is a fusion polypeptide of a
truncated viral
hemagglutinin with an anti-CD20 single-chain antibody or with an anti-CEA
single-chain
antibody. Preferably, the recombinant virus comprises a polynucleotide
comprising the
nucleic acid sequence of any one of SEQ ID NOs: 4 to 7, 14, and 15. SEQ ID NO:
4 is an
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artificial MV genome encoding an IL-12 fusion polypeptide comprising the mouse
p40
subunit of IL-12 and the mouse p35 subunit of IL-12 as specified elsewhere
herein. SEQ ID
NO: 5 is an artificial MV genome encoding an IL-12 fusion polypeptide
comprising the
human p40 subunit of IL-12 and the human p35 subunit of IL-12 as specified
elsewhere
.. herein. SEQ ID NO: 6 is an artificial MV genome encoding an 1L-12 fusion
polypeptide
comprising the mouse p40 subunit of IL-12 and the mouse p35 subunit of IL-12
as specified
elsewhere herein, and a fusion polypeptide comprising a viral hemagglutinin
and an anti-
human-CEA single-chain antibody. SEQ ID NO: 7 is an artificial MV genome
encoding an
IL-12 fusion polypeptide comprising the mouse p40 subunit = of IL-12 and the
mouse p35
.. subunit of IL-12 as specified elsewhere herein, and a fusion polypeptide
comprising a viral
hemagglutinin and an anti-human-CD20 single-chain antibody. SEQ ID NO: 14 is
an artificial
MV genome derived from strain Edmonston B encoding an IL-12 fusion polypeptide
comprising the mouse p40 subunit of IL-12 and the mouse p35 subunit of IL-12
as specified
elsewhere herein. SEQ ID NO: 15 is an artificial MV genome encoding an IL-12
fusion
.. polypeptide comprising the human p40 subunit of IL-12 and the human p35
subunit of IL-12
as specified elsewhere herein.
As used herein, the term "IL-12" relates to an interleukin 12 which is, in
principle, known to
the skilled person. Preferably, IL-12 is the heterodimeric IL-12 having the
activity of
stimulating the immune response of a subject. Preferably, the IL-12 is an IL-
12 of a vertebrate
species, more preferably of a mammal, even more preferably of a rat, a mouse,
or a human,
most preferably of a human. Preferably, the IL-12 has the subunits of rat IL-
12, i.e. p35
comprising the amino acid sequence of Genbank Acc. No: NP 445842.1
GI:16758120, and
p40 comprising the amino acid sequence of Genbank Ace. No: NP_072133.1
GI:12018288,
.. Preferably, the subunits of the rat IL-12 are encoded by a polynucleotide
comprising the
nucleic acid sequence of Genbank Ace. No: NM_053390.1 GI:16758119 (rat mRNA
expressed from the rat IL-12A gene) and/or of Genbank Ace. No: NM_022611.1
GI:12018287 (rat mRNA expressed from the rat IL-12B gene). More preferably,
the IL-12 has
the subunits of mouse IL-12, i.e. p35 comprising the amino acid sequence of
Genbank Ace.
No: NP 001152896.1 GI:226874945, and p40 comprising the amino acid sequence of
Genbank Ace. No: NP 001290173.1 GI:735997434. Preferably, the subunits of the
mouse
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IL-12 are encoded by a polynucleotide comprising the nucleic acid sequence of
Genbank Ace.
No: NM 001159424.2 GI:746816821 (mouse mRNA expressed from the mouse IL-12A
gene) and/or of Genbank Acc. No: NM_001303244.1 GI:735997433 (mouse mRNA
expressed from the mouse IL-12B gene). Most preferably, the IL-12 has the
subunits of
human IL-12, i.e. p35 comprising the amino acid sequence of Genbank Ace. No:
NP 000873.2 GI:24430219, and p40 comprising the amino acid sequence of Genbank
Ace.
No: NP 002178.2 GI:24497438. Preferably, the subunits of the human IL-12 are
encoded by
a polynucleotide comprising the nucleic acid sequence of Genbank Ace. No:
NM_000882.3
GI:325974478 (human mRNA expressed from the human IL-12A gene) and/or of
Genbank
Ace. No: NM 002187.2 GI:24497437 (human mRNA expressed from the human IL-12B
gene). In its natural form, IL-12 is a secreted interleukin, i.e. it is
processed and transported
from the interior of the producing cell to the exterior of the producing cell
by said producing
cell. Accordingly, IL-12 preferably is a secreted IL-12.
More preferably, the IL-12 according to the present invention is an IL-12
fusion polypeptide
comprising a p40 subunit of an IL-12 and a p35 subunit of an IL-12, preferably
comprising
subunits as specified herein above. More preferably, the p40 subunit and the
p35 subunit of
said IL-12 fusion polypeptide are from the same species; i.e. preferably, the
p40 subunit and
the p35 subunit of said IL-12 fusion polypeptide are a rat p40 subunit and a
rat p35 subunit,
more preferably are a mouse p40 subunit and a mouse p35 subunit, most
preferably are a
human p40 subunit and a human p35 subunit. Preferably, said p40 subunit and
said p35
subunit are comprised in the order N-terminus- p40 subunit- p35 subunit - C-
terminus in said
fusion polypeptide. Preferably, said p40 subunit and said p35 subunit are
separated by a
linker, i.e., the fusion polypeptide comprises the structure p40-linker-35.
The term "linker" is known to the skilled person and, preferably, relates to a
short sequence of
amino acids separating two domains of a polypeptide or two components of a
fusion
polypeptide. The skilled person knows how to select appropriate linker
sequences in order to
construct functional fusion polypeptides, e.g. from Xue et al. (2004), NAR 32
(Web server
issue):W562. Preferably, said linker comprises of from 1 to 50, more
preferably of from 2 to
25, most preferably of from 10 to 20 amino acids. Preferably, the amino acids
of the linker are
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small amino acids and/or amino acids promoting turns in protein structure;
accordingly, the
amino acids comprised in the linker, preferably, are glycinc, alanine, serine,
and/or proline.
Preferably, the linker has a repetitive structure; thus, preferably, the
linker comprises of from
1 to 10, more preferably of from 2 to 5, most preferably 3 repetitions of an
amino acid
sequence comprising 3 or 4, preferably 5 amino acids. Preferably, the
repetitive sequence of
the linker comprises the sequence (glycinex-serine), with x= 3 to 6,
preferably 4 to 6, more
preferably 4 or 6. More preferably, the repetitive sequence of the linker
comprises the
sequence (glycine4-serine), i.e. gly-gly-gly-gly-ser (SEQ ID NO: I),
preferably repeated as
specified above. Thus, preferably the linker comprises or consists of the
amino acid sequence
-(glycine4-serine)n-, with n=1 to 10, preferably n=2 to 5, more preferably
n=3. Most
preferably, the linker has the amino acid sequence of SEQ ID NO:8. Also more
preferably,
the repetitive sequence of the linker comprises the sequence (g1ycine6-
serine), i.e. gly-gly-gly-
gly-gly-gly-ser (SEQ ID NO 9).
As used herein, the term "fusion polypeptide" relates to a polypeptide wherein
all
components, e.g. p35 subunit, linker, and p40 subunit, are covalently linked
and, preferably,
are produced as a contiguous polypeptide chain. Thus, preferably, the fusion
polypeptide of
the present invention, preferably, is expressed from a single gene. Thus, the
IL-12 of the
present invention preferably is fused mouse IL-12 (FmIL-12), preferably
comprising the
amino acid sequence of SEQ ID NO:10, preferably encoded by a polynucleotide
comprising
the nucleic acid sequence of SEQ ID NO:11. More preferably, the IL-12 of the
present
invention preferably is fused human IL-12 (FhIL-12), preferably comprising the
amino acid
sequence of SEQ ID NO:12, preferably encoded by a polynucleotide comprising
the nucleic
acid sequence of SEQ ID NO:13. The terms "polypeptide" and "fusion
polypeptide", as used
herein, preferably encompass variants of said polypeptides and fusion
polypeptides as
specified elsewhere herein.
Preferably, the recombinant virus of the family Paramyxoviridae of the present
invention
further comprises at least one expressible polynucleotide encoding a further
activator of the
immune response. preferably an immunoglobulin or part thereof, preferably a
secreted
immunoglobul in.
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As used herein, the term "further activator of the immune response" relates to
a compound
which, when contacted with a mixture of immune cells and immune-response
inducing cells,
e.g. cancer cells, causes at least one type of immune cell to be more active
as compared to an
immune cell of the same type comprised in the same mixture but lacking said
compound. As
used herein, the term IL-12 as specified elsewhere herein relates to an
activator of the immune
response, but not to a further activator of the immune response. Preferably,
the immune cell
activated is a cell mediating a response increasing a subject's resistance to
an antigen, i.e.
preferably, said immune cell is not a tolerance-mediating immune cell.
Preferably, the
immune cell activated by the further activator of the immune response is a T-
cell, more
preferably a helper T-cell or a cytotoxic T-cell. Most preferably, the immune
cell activated by
the further activator of the immune response is a cytotoxic T-cell expressing
PD-1. Measures
of immune cell activity are known to the skilled person and include,
preferably, expression of
activation markers, production of antibodies, excretion of cytokines, and
release of cytotoxins,
e.g. perforin, granzymes, and/or granolysin.
Preferably, the further activator of the immune response is an antagonist of a
signaling
pathway causing at least one type of immune cell to become inhibited.
Accordingly,
preferably, the further activator of the immune response is a ligand for an
immune checkpoint
blockade protein. More preferably, the further activator of the immune
response is a ligand for
an immune checkpoint blockade protein. Still more preferably, the activator of
the immune
response is an inhibitor of PD-1 receptor signaling. It is understood by the
skilled person that
signaling through a receptor signaling pathway can be inhibited by either
preventing the
receptor from being activated, or by preventing the signal generated by the
activated receptor
from being further transmitted. Accordingly, preferably, the further activator
of the immune
response is a PD-L1 antagonist, the term "antagonist" relating to a compound
binding to the
molecule the effect of which is antagonized and through said binding
preventing said
molecule from interacting with its native binding partner in a productive,
i.e. signaling-
inducing, way. Preferred assays for said activity are described e.g. in WO
2015/128313 Al.
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Preferably, the further activator of the immune response is an antagonist as
described above
selected from the list of molecule types consisting of a peptide aptamer, an
anticalin, a
Designed Ankyrin Repeat Protein (DARPin), an inhibitory peptide, and,
preferably, an
immunoglobulin, more preferably, an antibody.
In the context of this invention, a "peptide aptamer" is a peptide
specifically binding its
interaction partner and having the activity of activating the immune response
as specified
herein above, preferably, the activity of being an antagonist of PD-Li as
specified herein
above. Peptide aptamers, preferably, are peptides comprising 8-80 amino acids,
more
preferably 10-50 amino acids, and most preferably 15-30 amino acids. They can
e.g. be
isolated from randomized peptide expression libraries in a suitable host
system like baker's
yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-
1998). A peptide
aptamer, preferably, is a free peptide; it is, however, also contemplated by
the present
invention that a peptide aptamer is fused to a polypeptide serving as
"scaffold", meaning that
the covalent linking to said polypeptide serves to fix the three-dimensional
structure of said
peptide aptamer to one specific conformation. More preferably, the peptide
aptamer is fused
to a transport signal, in particular a peptide export signal.
As used herein, the term "anticalin" relates to an artificial polypeptide
derived from a
lipocalin specifically binding its interaction partner. Similarly, a "Designed
Ankyrin Repeat
Protein" or "DARPin", as used herein, is an artificial polypeptide comprising
several ankyrin
repeat motifs and specifically binding its interaction partner. The anticalins
and the DARPins
of the present invention have the activity of activating the immune response
as specified
herein above, preferably, the activity of being an antagonist of PD-Li as
specified herein
above.
As used herein, the term "inhibitory peptide" relates to any chemical molecule
comprising at
least one peptide having the activity of activating the immune response as
specified herein
above, preferably, the activity of being an antagonist PD-L1 as specified
herein above.
Preferably, the inhibitory peptide comprises a peptide having an amino acid
sequence
corresponding to an amino acid sequence of at least five, at least six, at
least seven, at least
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eight, at least nine, at least ten, at least eleven, at least twelve, at least
13, at least 14, or at
least 15 consecutive amino acids comprised in a PD-Ll polypeptide. Preferably,
the inhibitory
peptide comprises a peptide having an amino acid sequence corresponding to an
amino acid
sequence of 5 to 200, more preferably 6 to 100, even more preferably 7 to 50,
or, most
preferably, 8 to 30 consecutive amino acids comprised in a PD-L I polypeptide.
Moreover,
also encompassed are variants of the aforementioned inhibitory peptides. Such
variants have
at least the same essential biological activity as the specific inhibitory
peptides.
As used herein, the term "immunoglobulin" relates to a polypeptide being a
soluble
immunoglobulin, preferably an antibody from any of the classes IgA, IgD, IgE,
IgG, or IgM,
preferably having the activity of binding, more preferably specifically
binding, a molecule of
interest. Immunoglobulins against antigens of interest can be prepared by well
known
methods using, e.g., a purified molecule of interest or a suitable fragment
derived therefrom
as an antigen. A fragment which is suitable as an antigen may be identified by
antigenicity
determining algorithms well known in the art. Such fragments may be obtained
either from
one of the molecules of interest by proteolytic digestion, may be a synthetic
peptide, or may
be obtained by recombinant expression. Preferably, a peptide of a molecule of
interest used as
an antigen is located at the exterior of a cell expressing the molecule of
interest; i.e.
preferably, the epitope the binding domain interacts with, preferably, is an
extracellular
domain. Preferably, the immunoglobulin of the present invention is a
monoclonal antibody, a
human or humanized antibody or primatized, chimerized antibody or a fragment
thereof, so
long as they exhibit the desired binding activity as specified elsewhere
herein. Also comprised
as antibodies of the present invention are a bispecific antibody, a synthetic
antibody, or a
chemically modified derivative of any of these. Preferably, the antibody of
the present
invention shall specifically bind (i.e. does only to a negligible extent or,
preferably, not cross
react with other polypeptides or peptides) to a molecule of interest as
specified above.
Specific binding can be tested by various well known techniques. Antibodies or
fragments
thereof can be obtained by using methods which are described, e.g., in Harlow
and Lane
"Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
Monoclonal
antibodies can be prepared by the techniques originally described in Kohler
and Milstein,
Nature. 1975. 256: 495; and Galfr6, Meth. Enzymol. 1981, 73: 3, which comprise
the fusion
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of mouse myeloma cells to spleen cells derived from immunized mammals. As will
be
understood by the skilled person, a molecule of interest, bound by an
immunoglobulin of the
present invention, may also be an Fe receptor or a complement protein binding
an Fe part of
an antibody; accordingly, the immunoglobulin preferably is an Fe domain of an
antibody,
more preferably a soluble Fe domain of an antibody, most preferably a secreted
soluble Fc
domain of an antibody. Preferably, said antibody the Fe domain is derived from
is an IgG,
more preferably an IgG1 , most preferably a human IgG1 . Preferably, the
secreted soluble Fe
domain comprises the amino acid sequence of SEQ ID NO: 16 or a variant
thereof, preferably
encoded by the nucleic acid sequence of SEQ ID NO: 17. More preferably, the
immunoglobulin is an antagonistic anti-PD-L1 antibody, still more preferably
comprising the
amino acid sequence of SEQ ID NO:2 or a variant thereof, preferably encoded by
a
polynucleotide comprising the nucleic acid sequence of SEQ ID NO:3 or a
variant thereof.
More preferably, the immunoglobulin is an antagonistic anti-PD-Ll antibody,
more
preferably comprising the amino acid sequence of SEQ ID NO:2, preferably
encoded by a
polynucleotide comprising the nucleic acid sequence of SEQ ID NO:3.
"Immunoglobulin fragments" comprise a portion of an intact immunoglobulin,
preferably of
an antibody, in an embodiment, comprise the antigen-binding region thereof
Examples of
antibody fragments and fusion proteins of variable regions include Fab, Fab',
F(ab')2, and Fv
fragments; diabodies; linear antibodies; single-chain antibody molecules;
single-domain-
antibodies (VHH), also known as nanobodies, and multispecific antibodies
formed from
antibody fragments. Papain digestion of antibodies produces two identical
antigen-binding
fragments, called "Fab" fragments, each with a single antigen-binding site,
and a residual
"Fe" fragment, whose name reflects its ability to crystallize readily. Pepsin
treatment yields
an F(ab')2 fragment that has two antigen-combining sites and is still capable
of cross-linking
antigen. "Fv" is the minimum antibody fragment which contains a complete
antigen-binding
site. Preferably, a two-chain Fv species consists of a dimer of one heavy- and
one light-chain
variable domain in tight, non-covalent association. In a single-chain Fv
(scFv) species, one
heavy- and one light-chain variable domain can be covalently linked by a
flexible peptide
linker such that the light and heavy chains can associate in a "dimeric"
structure analogous to
that in a two-chain Fv species. It is in this configuration that the three
hypervariable regions
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(HVRs, also referred to as complementarity determining regions (CDRs)) of each
variable
domain interact to define an antigen-binding site. Collectively, the six HVRs
of one scFv
confer antigen-binding specificity to the antibody. However, even a single
variable domain
(or half of an Fv comprising only three 1 IVRs specific for an antigen) has
the ability to
recognize and bind antigen, although at a lower affinity than the entire
binding site. The term
"diabodies" refers to antibody fragments with two antigen-binding sites, which
fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that is too
short to allow
pairing between the two domains on the same chain, the domains are forced to
pair with the
complementary domains of another chain and create two antigen-binding sites.
Diabodies
may be bivalent or bispecific. Diabodies are described more fully in, for
example, EP 0 404
097; WO 1993/01161; Hudson et al., Nat. Med. 9 (2003) 129-134; and Hollinger
et al., PNAS
USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in
Hudson et al.,
Nat. Med. 9 (2003) 129-134.
The term "secreted", as used herein, relates to a compound being transferred
from the interior
of a host cell to the exterior of said host cell by a mechanism intrinsic to
said host cell.
Preferably, secretion of a polypeptide or fusion polypeptide is mediated by a,
preferably
eukaryotic, signal peptide mediating import of said peptide or polypeptide
into the lumen of
the endoplasmic reticulum and, more preferably, by the absence of retention
signals. Signal
peptides causing secretion of peptides or polypeptides are known in the art.
Preferably, the
signal peptide is an IL-12 signal peptide. Also preferably, the signal peptide
is or comprises
an Ig leader sequence. More preferably, the signal peptide is or comprises a
human Ig leader
sequence. Still more preferably, the signal peptide is or comprises a matching
leader
sequence, i.e. a leader sequence selected from the same Ig kappa subgroup as
the variable
light chain of the antibody, preferably, of the single-chain antibody.
As used herein, the terms "polypeptide variant" relates to any chemical
molecule comprising
at least one polypeptide or fusion polypeptide as specified elsewhere herein,
having the
indicated activity, but differing in primary structure from said polypeptide
or fusion
polypeptide indicated above. Thus, the polypeptide variant, preferably, is a
mutein having the
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indicated activity. Preferably, the polypeptide variant comprises a peptide
having an amino
acid sequence corresponding to an amino acid sequence of 5 to 200, more
preferably 6 to 100,
even more preferably 7 to 50, or, most preferably, 8 to 30 consecutive amino
acids comprised
in a polypeptide as specified above. Moreover, also encompassed are further
polypeptide
variants of the aforementioned polypeptides. Such polypeptide variants have at
least
essentially the same biological activity as the specific polypeptides.
Moreover, it is to be
understood that a polypeptide variant as referred to in accordance with the
present invention
shall have an amino acid sequence which differs due to at least one amino acid
substitution,
deletion and/or addition, wherein the amino acid sequence of the variant is
still, preferably, at
least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with
the
amino acid sequence of the specific polypeptide. The degree of identity
between two amino
acid sequences can be determined by algorithms well known in the art.
Preferably, the degree
of identity is to be determined by comparing two optimally aligned sequences
over a
comparison window, where the fragment of amino acid sequence in the comparison
window
may comprise additions or deletions (e.g., gaps or overhangs) as compared to
the sequence it
is compared to for optimal alignment. The percentage is calculated by
determining, preferably
over the whole length of the polypeptide, the number of positions at which the
identical amino
acid residue occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the window of
comparison
and multiplying the result by 100 to yield the percentage of sequence
identity. Optimal
alignment of sequences for comparison may be conducted by the local homology
algorithm of
Smith and Waterman (1981), by the homology alignment algorithm of Needleman
and
Wunsch (1970), by the search for similarity method of Pearson and Lipman
(1988), by
computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA,
and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group
(GCG),
575 Science Dr., Madison, WI), or by visual inspection. Given that two
sequences have been
identified for comparison, GAP and BESTFIT are preferably employed to
determine their
optimal alignment and, thus, the degree of identity. Preferably, the default
values of 5.00 for
gap weight and 0.30 for gap weight length are used. Polypeptide variants
referred to herein
may be allelic variants or any other species specific homologs, paralogs, or
orthologs.
Moreover, the polypeptide variants referred to herein include fragments of the
specific
CA 02921864 2016-02-25
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polypeptides or the aforementioned types of polypeptide variants as long as
these fragments
and/or variants have the biological activity as referred to above. Such
fragments may be or be
derived from, e.g., degradation products or splice variants of the
polypeptides. Further
included are variants which differ due to posttranslational modifications such
as
phosphorylation, glycosylation, ubiquitinylation, sumoylation, or
myristylation, by including
non-natural amino acids, and/or by being peptidomimetics.
The term "expressible polynucleotide", as used herein, relates to a
polynucleotide operatively
linked to at least one expression control sequence causing transcription of
the nucleic acid
sequence comprised in said polynucleotide to occur, preferably in eukaryotic
cells or isolated
fractions thereof, preferably into a translatable mRNA or into a viral genome.
Regulatory
elements ensuring expression in eukaryotic cells, preferably mammalian cells,
are well known
in the art. They, preferably, comprise regulatory sequences ensuring
initiation of transcription
and, optionally, poly-A signals ensuring termination of transcription and
stabilization of the
transcript. Additional regulatory elements may include transcriptional as well
as translational
enhancers. Preferably, the aforesaid at least one expression control sequence
is an expression
control sequence of a (-)strand RNA virus, more preferably of a Paramyxovirus
as described
herein above, most preferably of an MV. Thus, preferably, at least one
expression control
sequence comprises a (-)strand RNA viral regulatory sequence ensuring
initiation of
transcription (consensus "gene start signal", preferably consensus MV "gene
start signal") and
termination signals (consensus "gene stop signal", preferably, consensus MV
"gene stop
signal") ensuring termination of transcription and stabilization of the
transcript. It is known in
the art that production of viral particles in permissive host cells can be
initiated by
transfecting into said permissive host cells one or more expressible DNA
constructs encoding
(i) a recombinant viral anti-genome, (ii) the viral L gene, (iii) the viral P
gene, and (iv) the
viral N gene. It is also understood by the skilled person that, once a viral
genome and the
aforesaid viral genes were expressed in said host cell, replication and
assembly of viral
particles occurs in the cytoplasm of the host cell and is, therefore, solely
dependent on viral
regulatory signals. The term polynucleotide, as used herein, preferably
encompasses
polynucleotide variants as specified elsewhere herein. Preferably, the
expressible
polynucleotide encoding an IL-12 is comprised in the genome of the recombinant
virus of the
CA 02921864 2016-02-25
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family Paramyxoviridae in a region corresponding to the region intervening the
P and the M
gene of measles virus.
The term "polynucleotide encoding a recombinant virus", as used herein,
relates to a
polynucleotide comprising a nucleic acid sequence or nucleic acid sequences
required for
generating a virus particle or a virus-like particle in a host cell. It is
understood by the skilled
person that a virus is constituted by a polynucleotide genome and at least one
kind of capsid
polypeptide. Accordingly, the polynucleotide encoding a recombinant virus of
the present
invention, preferably, comprises a recombinant virus genome. As will be
understood by the
skilled person, in case the polynucleotide encoding a recombinant virus is
comprised in a
virus according to the present invention, i.e. a virus of the family
Paramyxoviridae, the
polynucleotide is (-)strand RNA. It is also understood by the skilled person
that in case the
polynucleotide is DNA comprised in a host cell, at least an RNA-dependent RNA
polymerase
activity will additionally be required to produce viral particles from said
DNA polynucleotide.
Preferably, the polynucleotide encoding a recombinant virus comprises or
consists of the
nucleic acid sequence as specified elsewhere herein. As annotated herein, the
sequence of the
DNA copy of negative-strand (-)RNA viruses is annotated in the usual 5'-3'-
orientation; this
corresponds to the viral sequence in antigenomic (+)RNA orientation with
respect to the
natural 3'¨+5'-orientation of negative-strand (-)RNA viruses.
The term "polynucleotide variant", as used herein, relates to a variant of a
polynucleotide
related to herein comprising a nucleic acid sequence characterized in that the
sequence can be
derived from the aforementioned specific nucleic acid sequence by at least one
nucleotide
substitution, addition and/or deletion, wherein the polynucleotide variant
shall have the
activity as specified for the specific polynucleotide. Preferably, said
polynucleotide variant is
an ortholog, a paralog or another homolog of the specific polynucleotide. Also
preferably,
said polynucleotide variant is a naturally occurring allele of the specific
polynucleotide.
Polynucleotide variants also encompass polynucleotides comprising a nucleic
acid sequence
which is capable of hybridizing to the aforementioned specific
polynucleotides, preferably,
under stringent hybridization conditions. These stringent conditions are known
to the skilled
worker and can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.
CA 02921864 2016-02-25
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Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization
conditions are
hybridization conditions in 6x sodium chloride/sodium citrate (= SSC) at
approximately 45 C,
followed by one or more wash steps in 0.2x SSC, 0.1% SDS at 50 to 65 C. The
skilled worker
knows that these hybridization conditions differ depending on the type of
nucleic acid and, for
example when organic solvents are present. with regard to the temperature and
concentration
of the buffer. For example, under "standard hybridization conditions" the
temperature differs
depending on the type of nucleic acid between 42 C and 58 C in aqueous buffer
with a
concentration of 0.1x to 5x SSC (pH 7.2). If organic solvent is present in the
abovementioned
buffer, for example 50% formamide, the temperature under standard conditions
is
approximately 42 C. The hybridization conditions for DNA:DNA hybrids are
preferably for
example 0.1x SSC and 20 C to 45 C, preferably between 30 C and 45 C. The
hybridization
conditions for DNA:RNA hybrids are preferably, for example, 0.1x SSC and 30 C
to 55 C,
preferably between 45 C and 55 C. The abovementioned hybridization
temperatures are
determined for example for a nucleic acid with approximately 100 bp (= base
pairs) in length
and a G + C content of 50% in the absence of formamide. The skilled worker
knows how to
determine the hybridization conditions required by referring to textbooks such
as the textbook
mentioned above, or the following textbooks: Sambrook et al., ''Molecular
Cloning", Cold
Spring Harbor Laboratory, 1989; Harries and 'Higgins (Ed.) 1985, "Nucleic
Acids
Hybridization: A Practical Approach", IRL Press at Oxford University Press,
Oxford; Brown
(Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at
Oxford
University Press, Oxford. Alternatively, polynucleotide variants are
obtainable by PCR-based
techniques such as mixed oligonucleotide primer-based amplification of DNA,
i.e. using
degenerated primers against conserved domains of a polypeptide of the present
invention.
Conserved domains of a polypeptide may be identified by a sequence comparison
of the
nucleic acid sequence of the polynucleotide or the amino acid sequence of the
polypeptide of
the present invention with sequences of other organisms. As a template, DNA or
cDNA from
bacteria, fungi, or plants preferably, from animals may be used. Further,
variants include
polynucleotides comprising nucleic acid sequences which are at least 70%, at
least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least
99% identical to
the specifically indicated nucleic acid sequences. Moreover, also encompassed
are
polynueleotides which comprise nucleic acid sequences encoding amino acid
sequences
CA 02921864 2016-02-25
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which are at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at
least 98% or at least 99% identical to the amino acid sequences specifically
indicated. The
percent identity values are, preferably, calculated over the entire amino acid
or nucleic acid
sequence region. A series of programs based on a variety of algorithms is
available to the
skilled worker for comparing different sequences. In this context, the
algorithms of
Needleman and Wunsch or Smith and Waterman give particularly reliable results.
To carry
out the sequence alignments, the program PileUp (J. Mol. Evolution., 25, 351-
360, 1987,
Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit
(Needleman and
Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl.
Math. 2;
482-489 (1981))), which are part of the GCG software packet (Genetics Computer
Group, 575
Science Drive, Madison, Wisconsin, USA 53711 (1991)), are to be used. The
sequence
identity values recited above in percent (%) are to be determined, preferably,
using the
program GAP over the entire sequence region with the following settings: Gap
Weight: 50,
Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which,
unless
otherwise specified, shall always be used as standard settings for sequence
alignments.
A polynucleotide comprising a fragment of any of the specifically indicated
nucleic acid
sequences is also encompassed as a variant polynucleotide of the present
invention. The
fragment shall still encode a polypeptide or fusion polypeptide which still
has the activity as
specified. Accordingly, the polypeptide encoded may comprise or consist of the
domains of
the polypeptide of the present invention conferring the said biological
activity. A fragment as
meant herein, preferably, comprises at least 50, at least 100, at least 250 or
at least 500
consecutive nucleotides of any one of the specific nucleic acid sequences or
encodes an amino
acid sequence comprising at least 20, at least 30, at least 50, at least 80,
at least 100 or at least
150 consecutive amino acids of any one of the specific amino acid sequences.
The polynucleotides of the present invention either consist of, essentially
consist of, or
comprise the aforementioned nucleic acid sequences. Thus, they may contain
further nucleic
acid sequences as well. Specifically, the polynucleotides of the present
invention may encode
fusion proteins wherein one partner of the fusion protein is a polypeptide
being encoded by a
nucleic acid sequence recited above. Such fusion proteins may comprise as
additional part
CA 02921864 2016-02-25
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polypcptides for monitoring expression (e.g., green, yellow, blue or red
fluorescent proteins,
alkaline phosphatase and the like) or so called "tags" which may serve as a
detectable marker
or as an auxiliary measure for purification purposes. Tags for the different
purposes are well
known in the art and are described elsewhere herein.
The polynucleotide of the present invention shall be provided, preferably,
either as an isolated
polynucleotide (i.e. isolated from its natural context) or in genetically
modified form. The
polynucleotide, preferably, is DNA, including cDNA, or RNA. The term
encompasses single
as well as double stranded polynucleotides. Moreover, preferably, comprised
are also
chemically modified polynucleotides including naturally occurring modified
polynucleotides
such as glycosylated or methylated polynucleotides or artificial modified one
such as
biotinylated polynucleotides.
As used herein, the term "host cell" relates to a vertebrate cell. Preferably,
the cell is a
mammalian cell, more preferably, a mouse, rat, cat, dog, hamster, guinea pig,
sheep, goat, pig,
cattle, or horse cell. Still more preferably, the host cell is a primate cell.
Most preferably, the
host cell is a human cell. Preferably, the host cell is a tumor cell, more
preferably a cancer
cell.
Advantageously, it was found in the work underlying the present invention that
oncolytic
measles virus can be engineered to express IL-12, in particular an IL-12
fusion polypeptide,
while infecting cancer cells and that IL-12 expression strongly enhances the
immune response
induced by the measles virus against said cancer cells. Moreover, it was found
that by further
expressing immunoglobulins, in particular an anti-PD-Li antibody, measles
virus can further
augment the immunological response to cancer cells, thus further contributing
to their
elimination..
The definitions made above apply mutatis mutandis to the following. Additional
definitions
and explanations made further below also apply for all embodiments described
in this
specification mutatis mutandis.
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The present invention further relates to a polynucleotide encoding the
recombinant virus of
the family Paramyxoviridae according to the present invention.
The present invention further relates to a host cell comprising the
recombinant virus of the
family Paramyxoviridae of the present invention and/or the polynucleotide
encoding the
recombinant virus of the family Paramyxoviridae of the present invention.
As used herein, the term "host cell" relates to a host cell as specified
herein above. Moreover,
the host cell comprising the polynucleotide encoding the recombinant virus of
the family
.. Paramyxoviridae of the present invention may also be a bacterial, yeast, or
insect cell,
preferably a bacterial cell of the genus Escherichia, more preferably an
Escherichia coli cell.
The present invention also relates to a medicament comprising (a) (i) a
recombinant virus of
the family Paramyxoviridae comprising an expressible polynucleotide encoding
an IL-12 and
not comprising an expressible polynucleotide encoding a CTLA-4 antagonist, a
PD-1
antagonist, a CD80 antagonist, a CD86 antagonist, or a PD-L1 antagonist; (ii)
a recombinant
virus of the family Paramyxoviridae comprising an expressible polynucleotide
encoding an
IL-12 fusion polypeptide of the present invention; (iii) a polynucleotide
encoding the
recombinant virus of the family Paramyxoviridae of (i) and/or (ii), (iv) a
host cell comprising
the recombinant virus of the family Paramyxoviridae and/or the polynucleotide
encoding the
recombinant virus of the family Paramyxoviridae according to; or (v) any
combination of (i)
to (iv); and (b) at least one pharmacologically acceptable excipient.
The terms "medicament" and "pharmaceutical composition", as used herein,
relate to the
compounds of the present invention and optionally one or more pharmaceutically
acceptable
carrier, i.e. excipient. The compounds of the present invention can be
formulated as
pharmaceutically acceptable salts. Acceptable salts comprise acetate, methyl
ester, HC1,
sulfate, chloride and the like. The pharmaceutical compositions are,
preferably, administered
locally, topically or systemically. Suitable routes of administration
conventionally used for
drug administration are oral, intravenous, or parenteral administration as
well as inhalation. A
preferred route of administration is intra-tumoral administration. However,
depending on the
CA 02921864 2016-02-25
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nature and mode of action of a compound, the pharmaceutical compositions may
be
administered by other routes as well. For example, polynucleotide compounds
may be
administered in a gene therapy approach by using viral vectors or viruses or
liposomes.
Moreover, the compounds can be administered in combination with other drugs
either in a
common pharmaceutical composition or as separated pharmaceutical compositions
wherein
said separated pharmaceutical compositions may be provided in form of a kit of
parts. The
compounds are, preferably, administered in conventional dosage forms prepared
by
combining the drugs with standard pharmaceutical carriers according to
conventional
procedures. These procedures may involve mixing, granulating and compressing
or dissolving
the ingredients as appropriate to the desired preparation. It will be
appreciated that the form
and character of the pharmaceutically acceptable carrier or diluent is
dictated by the amount
of active ingredient with which it is to be combined, the route of
administration and other
well-known variables.
The excipient(s) must be acceptable in the sense of being compatible with the
other
ingredients of the formulation and being not deleterious to the recipient
thereof. The excipient
employed may be, for example, a solid, a gel or a liquid carrier. Exemplary of
solid carriers
are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,
magnesium stearate, stearic
acid and the like. Exemplary of liquid carriers are phosphate buffered saline
solution, syrup,
oil such as peanut oil and olive oil, water, emulsions, various types of
wetting agents, sterile
solutions and the like. Similarly, the carrier or diluent may include time
delay material well
known to the art, such as glyceryl mono-stearate or glyceryl distearate alone
or with a wax.
Said suitable carriers comprise those mentioned above and others well known in
the art, see,
e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,
Pennsylvania. The diluent(s) is/are selected so as not to affect the
biological activity of the
combination. Examples of such diluents are distilled water, physiological
saline, Ringer's
solutions, dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition
or formulation may also include other carriers, adjuvants, or nontoxic,
nontherapeutic, non-
immunogenic stabilizers and the like.
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A therapeutically effective dose refers to an amount of the compounds to be
used in a
pharmaceutical composition of the present invention which prevents,
ameliorates or treats the
symptoms accompanying a disease or condition referred to in this
specification. Therapeutic
efficacy and toxicity of such compounds can be determined by standard
pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50 (the dose
therapeutically
effective in 50% of the population) and LD50 (the dose lethal to 50% of the
population). The
dose ratio between therapeutic and toxic effects is the therapeutic index, and
it can be
expressed as the ratio, LD50/ED50.
The dosage regimen will be determined by the attending physician and other
clinical factors;
preferably in accordance with any one of the above described methods. As is
well known in
the medical arts, dosages for any one patient depends upon many factors,
including the
patient's size, body surface area, age, the particular compound to be
administered, sex, time
and route of administration, general health, and other drugs being
administered concurrently.
Progress can be monitored by periodic assessment. A typical dose can be, for
example, in the
range of 1 to 1000 l_tg for a polypeptide or polynucleotide, or 104-108 viral
particles for a virus
or a virus-like particle; however, doses below or above this exemplary range
are envisioned,
especially considering the aforementioned factors. Progress can be monitored
by periodic
assessment. The pharmaceutical compositions and formulations referred to
herein are
administered at least once in order to treat or ameliorate or prevent a
disease or condition
recited in this specification. However, the said pharmaceutical compositions
may be
administered more than one time, for example from one to four times daily up
to a non-
limited number of days. Specific pharmaceutical compositions are prepared in a
manner well
known in the pharmaceutical art and comprise at least one active compound
referred to herein
above in admixture or otherwise associated with a pharmaceutically acceptable
carrier or
diluent. For making those specific pharmaceutical compositions, the active
compound(s) will
usually be mixed with a carrier or the diluent, or enclosed or encapsulated in
a capsule, sachet,
cachet, paper or other suitable containers or vehicles. The resulting
formulations are to be
adapted to the mode of administration, i.e. in the forms of tablets, capsules,
suppositories,
solutions, suspensions or the like. Dosage recommendations shall be indicated
in the
CA 02921864 2016-02-25
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prescribers or users instructions in order to anticipate dose adjustments
depending on the
considered recipient.
Accordingly, the present invention also relates to a method for treating
cancer in a subject
afflicted with cancer, comprising
a) contacting said subject with
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 fusion polypeptide of the present invention;
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae of (i)
and/or (ii) and/or the polynucleotide encoding the recombinant virus of the
family
Paramyxoviridae according to (iii); or
(v) any combination of (i) to (iv), and thereby,
b) treating cancer in a subject afflicted with cancer.
The methods of treatment of the present invention, preferably, may comprise
steps in addition
to those explicitly mentioned above. For example, further steps may relate,
e.g., to localizing
a tumor and/or diagnosing cancer for step a), or administration of additional
medication for
step b). Moreover, one or more of said steps may be performed by automated
equipment. The
method of the present invention, preferably, is an in vivo method of
treatment.
The term "treatment" refers to an amelioration of the diseases or disorders
referred to herein
or the symptoms accompanied therewith to a significant extent. Said treating
as used herein
also includes an entire restoration of the health with respect to the diseases
or disorders
referred to herein. It is to be understood that treating as used in accordance
with the present
invention may not be effective in all subjects to be treated. However, the
term shall require
CA 02921864 2016-02-25
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that, preferably, a statistically significant portion of subjects suffering
from a disease or
disorder referred to herein can be successfully treated. Whether a portion is
statistically
significant can be determined without further ado by the person skilled in the
art using various
well known statistic evaluation tools, e.g., determination of confidence
intervals, p-value
determination, Student's t-test, Mann-Whitney test etc.. Preferred confidence
intervals are at
least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-
values are,
preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall
be effective for at
least 10%, at least 20% at least 50%at least 60%, at least 70%, at least 80%,
or at least 90% of
the subjects of a given cohort or population. Preferably, treating cancer is
reducing tumor
burden in a subject. As will be understood by the skilled person,
effectiveness of treatment of
e.g. cancer is dependent on a variety of factors including, e.g. cancer stage
and cancer type.
As used herein, the term "subject" relates to a vertebrate. Preferably, the
subject is a mammal,
more preferably, a mouse, rat, cat, dog, hamster, guinea pig, sheep, goat,
pig, cattle, or horse.
Still more preferably, the subject is a primate. Most preferably, the subject
is a human.
Preferably, the subject is afflicted with a disease caused or aggravated by an
insufficient
response of the immune response of said subject, more preferably, the subject
is afflicted with
cancer.
The term "cancer", as used herein, relates to a disease of an animal,
including man,
characterized by uncontrolled growth by a group of body cells ("cancer
cells"). This
uncontrolled growth may be accompanied by intrusion into and destruction of
surrounding
tissue and possibly spread of cancer cells to other locations in the body.
Preferably, also
included by the term cancer is a relapse. Thus, preferably, the cancer is a
solid cancer, a
metastasis, or a relapse thereof.
Preferably, the cancer is selected from the list consisting of acute
lymphoblastic leukemia,
acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, anal
cancer,
appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile
duct cancer,
bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid
tumor,
cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic
leukemia, chronic
CA 02921864 2016-02-25
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myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma,
endometrial
cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ
cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma,
gallbladder cancer,
gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic
tumor, hairy cell
leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma,
hypopharyngeal
cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi
sarcoma,
laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell
carcinoma,
mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple
myeloma,
mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal
cancer,
neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer,
oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer,
ovarian germ
cell tumor, ovarian low malignant potential tumor, pancreatic cancer,
papillomatosis,
paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer,
pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central
nervous
system lymphoma, prostate cancer, rectal cancer, renal cell cancer,
retinoblastoma,
rhabdomyosareoma, salivary gland cancer, sezary syndrome, small cell lung
cancer, small
intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck
cancer,
testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer,
urethral cancer,
uterine sarcoma, vaginal cancer, vulvar cancer, waldenstrom macroglobulinemia,
and wilms
tumor. More preferably, the cancer is a solid cancer, a metastasis, or a
relapse thereof. Most
preferably, the cancer is a tumor derived from malignant melanoma, head and
neck cancer,
hepatocellular carcinoma, pancreatic carcinoma, prostate cancer, renal cell
carcinoma, gastric
carcinoma, colorectal carcinoma, lymphomas or leukemias.
Preferably, the method of treatment of the present invention comprises
contacting a subject
with a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding
a CTLA-4 antagonist, a PD-1 antagonist, a CD80 antagonist, a CD86 antagonist,
or a PD-L1
antagonist. Thus, preferably, the method comprises contacting a subject with a
recombinant
virus of the family Paramyxoviridae comprising an expressible polynucleotide
encoding an
IL-12, wherein said recombinant virus of the family Paramyxoviridae is not a
virus disclosed
CA 02921864 2016-02-25
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in WO 2015/128313 Al. Preferably, said recombinant virus of the family
Paramyxoviridae of
(i) is a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 polypeptide and not comprising an expressible
polynucleotide encoding a ligand for an immune checkpoint blockade protein.
More
preferably, the recombinant virus of the family Paramyxoviridae of (i) is a
recombinant virus
of the family Paramyxoviridae comprising an expressible polynucleotide
encoding an IL-12
polypeptide as the only expressible polynucleotide encoding an activator of
the immune
response, i. e. preferably, the recombinant virus of the family
Paramyxoviridae comprising an
expressible polynucleotide encoding an IL-12 does not comprise an expressible
.. polynucleotide encoding a further activator of the immune response.
The present invention further relates to an in vitro method for activating
immune cells with
antitumor activity in a sample comprising cancer cells and immune cells,
comprising
a) contacting said sample comprising cancer cells and immune cells with
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 fusion polypeptide of the present invention;
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae of (i)
and/or (ii) and/or the polynucleotide encoding the recombinant virus of the
family
Paramyxoviridae according to (iii); or
(v) any combination of (i) to (iv), and thereby,
b) activating immune cells with antitumor activity comprised in said
sample..
The method for activating immune cells with antitumor activity may comprise
steps in
addition to those explicitly mentioned above. For example, further steps may
relate, e.g., to
providing the recombinant virus of the family Paramyxoviridae for step a),
administering
CA 02921864 2016-02-25
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further activating compounds, e.g. cytokines, to the immune cells in step b),
or separating
immune cells from cancer cells after step b). Moreover, one or more of said
steps may be
performed by automated equipment.
Moreover, the present invention relates to a recombinant virus of the family
Paramyxoviridae
of the present invention for use in treatment of inappropriate cell
proliferation.
The term "inappropriate cell proliferation" relates to any proliferation of
cells of a subject
which is not appropriate to the physiological state of said subject and/or to
the tissue context
of said cells. Preferably, inappropriate cell proliferation is caused or
aggravated by an
inhibition or insufficient activation of the immune system, more preferably
inhibition or
insufficient activation of T cells. Also preferably, inappropriate cell
proliferation is cancer.
The present invention further relates to a kit comprising at least
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide
encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80 antagonist, a CD86
antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 fusion polypcptide of the present invention;
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae of (i)
and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae of (i) and/or
(ii) and/or the polynucleotide encoding the recombinant virus of the family
Paramyxoviridae according to (iii); or
(v) any combination of (i) to (iv),
housed in a container.
The term "kit", as used herein, refers to a collection of the aforementioned
components.
Preferably, said components are combined with additional components,
preferably within an
outer container. The outer container, also preferably, comprises instructions
for carrying out a
CA 02921864 2016-02-25
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method of the present invention. Examples for such the components of the kit
as well as
methods for their use have been given in this specification. The kit,
preferably, contains the
aforementioned components in a ready-to-use formulation. Preferably, the kit
may
additionally comprise instructions, e.g., a user's manual for applying the
recombinant virus of
the family Paramyxoviridae with respect to the applications provided by the
methods of the
present invention. Details are to be found elsewhere in this specification.
Additionally, such
user's manual may provide instructions about correctly using the components of
the kit. A
user's manual may be provided in paper or electronic form, e.g., stored on CD
or CD ROM.
The present invention also relates to the use of said kit in any of the
methods according to the
present invention.
Moreover, the present invention relates to a use of
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide
encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80 antagonist, a CD86
antagonist, or a PD-Ll antagonist;
(ii) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 fusion polypeptide of the present invention;
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae of (i)
and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae of (i) and/or
(ii) and/or the polynucleotide encoding the recombinant virus of the family
Paramyxoviridae according to (iii); or
(v) any combination of (i) to (iv),
for the manufacture of a medicament for treating inappropriate cell
proliferation, preferably
for treating cancer.
Summarizing the findings of the present invention, the following embodiments
are preferred:
CA 02921864 2016-02-25
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I. A
recombinant virus of the family Paramyxoviridae, comprising an expressible
polynucleotide encoding an IL-12 polypeptide, wherein said IL-12 polypeptide
is an IL-12
fusion polypeptide comprising a p40 subunit of an IL-12 and a p35 subunit of
an IL-12.
2. The
recombinant virus of the family Paramyxoviridae of embodiment I, wherein said
p40 subunit and said p35 subunit of said IL-12 fusion polypeptide are from the
same species.
3. The
recombinant virus of the family Paramyxoviridae of embodiment 1 or 2, wherein
said p40 subunit and said p35 subunit of said IL-12 fusion polypeptide are a
mouse p40
subunit and a mouse p35 subunit or a variant thereof, preferably are a human
p40 subunit and
a human p35 subunit or a variant thereof.
4. The
recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to
3, wherein said p40 subunit and said p35 subunit of said IL-12 fusion
polypeptide are a
mouse p40 subunit and a mouse p35 subunit, preferably are a human p40 subunit
and a human
p35 subunit.
5. The
recombinant virus of the family Paramyxoviridae of any one of embodiments I to
4, wherein said IL-12 fusion polypeptide comprises the structure p40-linker-
p35.
6. The
recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to
5, wherein said linker is -(glycine4-serine),-, with n=1 to 10, preferably n=2
to 5, more
preferably n=3.
7. The
recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to
5, wherein said linker is -(glycine6-serine)-.
8. The
recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to
7, wherein said expressible polynuelcotide encoding an IL-12 is comprised in
the genome of
the recombinant
virus of the family Paramyxoviridae in a region corresponding to the region
intervening the P and the M gene of measles virus.
CA 02921864 2016-02-25
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9. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 1 to
8, further comprising at least one expressible polynucleotide encoding a
further activator of
the immune response.
10. The recombinant virus of the family Paramyxoviridae of embodiment 9,
wherein said
further activator of the immune response is an immunoglobulin or fragment
thereof.
11. The recombinant virus of the family Paramyxoviridae of embodiments 9 or
10,
wherein said further activator of the immune response is a secreted
immunoglobulin.
12. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 9 to
11, wherein said further activator of the immune response is an Pc domain of
an antibody.
13. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 9 to
12, wherein said further activator of the immune response is a secreted
soluble Fe domain of a
human IgG1 antibody.
14. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 9 to
13, wherein said further activator of the immune response is a secreted
soluble activator of the
immune response.
15. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 9 to
14, wherein said further activator of the immune response is a single-chain
antibody or a
nanobody.
16. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 9 to
15, wherein said further activator of the immune response is a secreted
soluble anti-PD-L1
antibody.
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17. The recombinant virus of the family Paramyxoviridae of embodiment
16, wherein said
secreted soluble anti-PD-Ll antibody comprises an amino acid sequence
according to SEQ ID
NO: 2.
18. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 1 to
17, wherein said recombinant virus is a recombinant Morbillivirus, preferably,
a recombinant
measles virus (MV).
19. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 1 to
18, wherein said recombinant MV is derived from MV strain Edmonston A or B,
preferably
B, more preferably from MV vaccine strain Schwarz/Moraten.
20. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 1 to
19, wherein the at least one expressible polynucleotide encoding an IL-12
polypeptide is
comprised in a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae.
21. The recombinant virus of the family Paramyxoviridae of any one of
embodiments 1 to
20, wherein said polynucleotide encoding the recombinant virus of the family
Paramyxoviridae comprises the nucleic acid sequence of any one of SEQ ID NOs:
4 to 7, 14,
and 15.
22. A polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
according to any one of embodiments 1 to 21.
23. The polynucleotide according to embodiment 22, wherein said
polynucleotide
comprises the nucleic acid sequence any one of SEQ ID NOs: 4 to 7, 14, and 15.
24. A host cell comprising the recombinant virus of the family
Paramyxoviridae according
to any one of embodiments 1 to 21 and/or the polynucleotide encoding the
recombinant virus
of the family Paramyxoviridae according to embodiment 21 or 22.
CA 02921864 2016-02-25
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25. A medicament comprising
(a) (i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-L1 antagonist;
(ii) a recombinant virus of the family Paramyxoviridae according of any one of
embodiments 1 to 21,
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae
according to (i) or (ii) and/or a polynucleotide encoding the recombinant
virus of
the family Paramyxoviridae of (iii); or
(v) any combination of (i) to (iv); and
(b) at least one pharmacologically acceptable excipient.
26. A method for treating cancer in a subject afflicted with cancer,
comprising
a) contacting said subject with
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae according of any one of
embodiments Ito 21,
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae
according to (i) or (ii) and/or a polynucleotide encoding the recombinant
virus of
the family Paramyxoviridae of (iii); or
(v) any combination of (i) to (iv); and, thereby,
b) treating cancer in a subject afflicted with cancer.
CA 02921864 2016-02-25
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27. The method of embodiment 26, wherein said recombinant virus of the family
Paramyxoviridae of (i) is a recombinant virus of the family Paramyxoviridae
comprising an
expressible polynucleotide encoding an IL-12 polypeptide and not comprising an
expressible
polynucleotide encoding a ligand for an immune checkpoint blockade protein.
28. The method of embodiment 26 or 27, wherein said recombinant virus of the
family
Paramyxoviridae of (i) is a recombinant virus of the family Paramyxoviridae
comprising an
expressible polynucleotide encoding an IL-12 polypeptide as the only
expressible
polynucleotide encoding an activator of the immune response.
29. The method of any one of embodiments 26 to 28, wherein said cancer is a
solid
cancer, a metastasis, or a relapse thereof.
30. The method of any one of embodiments 26 to 29, wherein treating cancer
is reducing
tumor burden.
31. The method of any one of embodiments 26 to 30, wherein said cancer is
malignant
melanoma, head and neck cancer, hepatocellular carcinoma, pancreatic
carcinoma, prostate
cancer, renal cell carcinoma, gastric carcinoma, colorectal carcinoma,
lymphomas or
leukemias.
32. An in vitro method for activating immune cells with antitumor activity
in a sample
comprising cancer cells and immune cells, comprising
a) contacting said sample comprising cancer cells and immune cells with
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae according of any one of
embodiments 1 to 21,
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
CA 02921864 2016-02-25
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(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae
according to (i) or (ii) and/or a polynucleotide encoding the recombinant
virus of
the family Paramyxoviridae of (iii); or
(v) any combination of (i) to (iv); and thereby,
b) activating immune cells with antitumor activity comprised in said
sample.
33. Use of
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-Li antagonist;
(ii) a recombinant virus of the family Paramyxoviridae according of any one of
embodiments 1 to 21,
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae
according to (i) or (ii) and/or a polynucleotide encoding the recombinant
virus of
the family Paramyxoviridae of (iii); or
(v) any combination of (i) to (iv);
for the manufacture of a medicament for treating cancer.
34. A recombinant virus of the family Paramyxoviridae according to any
one of
embodiments 1 to 21 and/or a polynucleotide according to embodiment 22 or 23
for use in
medical treatment.
'15
35. A recombinant virus of the family Paramyxoviridae
(i) comprising an expressible polynucleotide encoding an IL-12 and not
comprising an
expressible polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a
CD80
antagonist, a CD86 antagonist, or a PD-Ll antagonist; and/or
(ii) according of any one of embodiments 1 to 21,
for use in treatment of inappropriate cell proliferation.
- 34 -
36. The recombinant virus of the family Paramyxoviridae for use of
embodiment 35,
wherein treatment of inappropriate cell proliferation is cancer treatment.
37. A kit comprising
(i) a recombinant virus of the family Paramyxoviridae comprising an
expressible
polynucleotide encoding an IL-12 and not comprising an expressible
polynucleotide encoding a CTLA-4 antagonist, a PD-1 antagonist, a CD80
antagonist, a CD86 antagonist, or a PD-L1 antagonist;
(ii) a recombinant virus of the family Paramyxoviridae according of any one of
embodiments 1 to 21,
(iii) a polynucleotide encoding the recombinant virus of the family
Paramyxoviridae
of (i) and/or (ii),
(iv) a host cell comprising the recombinant virus of the family
Paramyxoviridae
according to (i) or (ii) and/or a polynucleotide encoding the recombinant
virus of
the family Paramyxoviridae of (iii); or
(v) any combination of (i) to (iv);
housed in a container.
Figure Legends
Fig.1: One step growth curves in Vero-allis (A) and MC38cea (B) cells: Cells
were
transduced with MeVac encoding the respective transgenes at MOI=3. Cell
suspensions were
collected by scraping in the culture medium and titre determined at the
depicted time points.
Fig. 2: Cytotoxic effect in the target MC8cea cells: Cells were transduced
with MeVac
encoding the respective transgenes at MOI=5 and cell viability was determined
by XTT assay
Date Recue/Date Received 2021-02-02
CA 02921864 2016-02-25
- 35 -
at the depicted time points. Mean results of triplicate infections per time
point with standard
errors of the mean (not visible for some data points) are shown.
Fig. 3: Expression of MeVac encoded immunomodulators in MC38cea cells. MC38cea
cells
were transduced with MeVac encoding the respective immunomodulators and eGFP
or IgGl-
Fc as control vectors at MOI=3. Supernatant samples were collected at the
depicted time
points and transgene expression detected by ELISA. Unspecific binding was
controlled by
IgG 1-Fe (upper panels) or eGFP (lower panels) supernatants and subtracted
from the specific
measurements. In case of mIP-10 an increase of the signal was observed in the
eGFP controls
which was not subtracted from the specific measurements and is depicted
accordingly.
Fig. 4: MeVac encoded anti-PD-Li binding to MC38cea cells. MC38cea cells were
incubated
with supernatant from Vero-uHis infected with MeVac encoding anti-PD-I,1 or
IgG 1 -Fc. For
detection of bound anti-PD-L1, cells were stained with primary Ab specific for
HA tag and
secondary Ab coupled to PE. DAPI staining was used to exclude dead cells and
samples were
analyzed by flow cytometry. (A) Overlay histogram for PE of DAPI- MC38cea
populations
from one of three independent experiments is shown on panel (B). (B)Average
median
fluorescence intensity (MFI) of PE for DAPI- populations with standard error
of the means
from the three independent experiments is shown on the left panel.
Fig. 5: Functionality of MeVac encoded immunomodulators. (A) MC38cea cells
were treated
with supernatants from Vero-aHis cells infected with MeVac encoding the
respective
immunomodulators and cocultured in ratio 2:1 with murine splenocytes in the
presence of
PMA and ionomycin in 96-well plate. After 24h the supernatants were collected
and IFN-y
concentration measured by ELISA. Relative activation corresponds to ratio of
the optical
density (absorbance at 450nm minus 570nm) of the respective samples to
activated
splenocytes. Data for one of three independent experiments are shown; (B)
splenocytes were
stimulated with recombinant murine IL-2 and cultivated in the presence of
medium from
Vero-al us infected with MeVac encoding FmIL-12 or eGFP. After 48h the
supernatants were
collected and IFN-y concentration measured by ELISA. Mean results with
standard error of
CA 02921864 2016-02-25
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the mean of triplicate splenocyte cultures per FmIL-12 concentration are
shown. IFN-y
concentration in the eGFP controls was close to background.
Fig. 6: Therapeutic efficacy of immunomodulatory MeVac in vivo: Breaking
immunosuppression: MC38cea cells were implanted subcutaneously (s.c.) into the
right flank
of C57BL/6J mice (6-9 animals per group). When tumors reached an average
volume of 50
mm3 mice received intratumoral injections with 1 x106 cell infectious units
(ciu) with the
respective viruses on four consecutive days in 100 IA. Tumor volume was
determined every
third day and mice were sacrificed when tumor volumes exceeded 1500mm3 or when
.. ulceration occurred.
Fig. 7: Therapeutic efficacy of immunomodulatory MeVac in vivo: Activating DCs
and
effector cells: Therapeutic. MC38cea cells were implanted subcutaneously
(s.c.) into the right
flank of C57BL/6J mice (6-9 animals per group). When tumors reached an average
volume of
50 mm3 mice received intratumoral injections with 5 x105 ciu with the
respective viruses on
five consecutive days in 100 1.d. Tumor volume was detemiined every third day
and mice
were sacrificed when tumor volumes exceeded 1500 mm3 or when ulceration
occurred.
Fig. 8: Rechallenge of long term survivors with MC38cea. Mice experiencing
complete tumor
remission in the experiments identifying the most effective MeVac vectors and
were
rechallenged with MC38cea cells 3 to 6 months after the initinal tumor cell
implantation.
Eight naive mice served as a control group. 1x105 MC38cea cells were implanted
subcutaneously (s.c.) in the left flank of the mice. Tumor engraftment rates
were monitored.
.. Fig. 9: Comparison of therapeutic efficacy of MeVac encoding FmIL-12 and
anti-PD-Li.
MC38cea cells were implanted subcutaneously (s.c.) into the right flank of
C57BL/6J mice
(10 animals per group). When tumors reached an average volume of 50 mm3 mice
received
intratumoral injections with lx106 ciu with the respective viruses on four
consecutive days in
100 [il. Tumor volume was determined every third day and mice were sacrificed
when tumor
volumes exceeded 1500 mm3 or when ulceration occurred.
CA 02921864 2016-02-25
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Fig. 10: Rechallenge of long term survivors from the experiment comparing
efficacy of
FmIL-12 and anti-PD-Li encoding vectors with MC38cea. Mice were rechallenged
with
MC38cea cells ca. 6 months after the initinal tumor cell implantation. Ten
naive mice served
as a control group. 1 x105 MC38cea cells were implanted subcutaneously (s.c.)
in the left
flank of the mice. Tumor engraftment rates were monitored.
Fig. 11: IFN-y memory recall in murine splenocytes from mice experiencing
complete tumor
remissions in MeVac FmIL-12 versus MeVac anti-PD-L1 efficacy experiment. Anti-
tumor:
Freshly isolated splenocytes from mice treated with MeVac encoding the
respective
immunomodulators or naïve mice were stimulated with recombinant murine IL-2
and
cocultivated with MC38cea (A) or MC38 (B) and B16 (B) tumor cells or
irrelevant human
cell lysate (DLD-1). After 48h of cultivation cell culture medium was
collected and IFN-y
concentration was measured by ELISA. IFN-y concentrations in the individual
cocultures
with median in the group (A) or average concentration from two replicate
measurements with
standard error of the mean (SEM) are shown (B).
Fig. 12: IFN-y memory recall in murine splenocytes from mice experiencing
complete tumor
remissions in MeVac FmIL-12 versus MeVac anti-PD-Ll efficacy experiment: Anti-
MeVac:
Freshly isolated splenocytes from mice treated with MeVac encoding the
respective
immunomodulators or naïve mice were stimulated with recombinant murine IL-2
and
cocultivated with MeVac (A) or as controls with an irrelevant human cell
lysate (DLD-1) (B)
or as a negative control splenocytes were cultivated alone. After 48h of
cultivation cell culture
medium was collected and IFN-y concentration was measured by ELISA. IFN-y
concentrations in the individual cocultures with median in the group (A) or
average
concentration from two replicate measurements with standard error of the mean
(SEM) are
shown (B).
Fig. 13: Comparison of therapeutic efficacy of combination of MeVac encoding
Fm-IL-12
and anti-PD-L1 with either vector in combination with a control vector
encoding IgGI-Fc.
MC38cea cells were implanted subcutaneously (s.c.) into the right flank of
C57BL/6J mice
CA 02921864 2016-02-25
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(8-10 animals per group). When tumors reached an average volume of 50 mm3 mice
received
intratumoral injections with 1x106 ciu with the respective viruses on four
consecutive days in
100 al. Tumor volume was determined every third day and mice were sacrificed
when tumor
volumes exceeded 1500 mm3 or when ulceration occurred.
Fig. 14: Rechallenge of long term survivors from the experiment comparing
efficacy of
combination of MeVac encoding Fm-IL-12 and anti-PD-Li with either vector in
combination
with a control vector encoding IgG 1-Fe. Mice were rechallenged with MC38cea
cells ca. 5
months after the initinal tumor cell implantation. Ten naive mice served as a
control group.
lx105 MC38cea cells were implanted subcutaneously (s.c.) in the left flank of
the mice.
Tumor engraftment rates were monitored.
Fig. 15: IFN-y memory recall in murine splenocytes from mice experiencing
complete tumor
remissions in MeVac FmIL-12 and MeVac anti-PD-Li combination experiment: Anti-
tumor:
Freshly isolated splenocytes from mice treated with MeVac encoding the
respective
immunomodulators or naïve mice were stimulated with recombinant murine IL-2
and
cocultivated with MC38cca (A), MC38 (B) or B16 (C) tumor cells. After 48h of
cultivation
cell culture medium was collected and IFN-y concentration was measured by
ELISA. IFN-y
concentrations in the individual cocultures with median in the group (A) or
average
concentration from two replicate measurements with standard error of the mean
(SEM) are
shown (B).
Fig. 16: IFN-y memory recall in murine splenocytes from mice experiencing
complete tumor
remissions in MeVac FmIL-12 and MeVac anti-PD-Li combination experiment: Anti-
MeVac: Freshly isolated splenocytes from mice treated with MeVac encoding the
respective
immunomodulators or naïve mice were stimulated with recombinant murine IL-2
and
cocultivated with MeVac (A) or Vero-aHis lysate (B). After 48h of cultivation
cell culture
medium was collected and IFN-y concentration was measured by ELISA. IFN-y
concentrations in the individual cocultures with median in the group (A) or
average
concentration from two replicate measurements with standard error of the mean
(SEM) are
shown (B).
-39-
Fig. 17: Comparison of therapeutic efficacy of combination of MeVac
encoding Fm-IL-12
and anti-PD-L1 with either vector in combination with a control vector
encoding IgGl-Fc. B16-
CD20 cells were implanted subcutaneously (s.c.) into the right flank of
C57BL/6J mice (8-10
animals per group). When tumors reached an average volume of 50 mm3 mice
received
intratumoral injections with 1 x106 ciu with the respective viruses on four
consecutive days in 100
[tl. Tumor volume was determined every third day and mice were sacrificed when
tumor volumes
exceeded 1500 mm3 or when ulceration occurred.
Fig. 18: Schemes of the constructed recombinant MeVac genomes. Transgenes
encoding different
immunomodulators as well as eGFP and IgGl-Fc as controls were inserted in
different positions
of MeVac genome. Murine IL-12 was inserted as a fusion protein consisting of
p40 and p35 protein
subunits linked by a (Gly4Ser)3 linker (FmIL-12). Murine CD80 was inserted as
a soluble form
of the protein consisting of the extracellular part of the protein fused to a
human IgGl-Fc (CD80-
Fc). The MeVac H gene in the novel constructs was fully retargeted to human
CEA (hCEA)
antigen by ablating attachment to the natural receptors, fusing the H protein
to a single chain
antibody (scAb) against the hCEA and including a six-histidine tag at the C
terminus to allow
specific transduction of murine MC38cea cells via human CEA antigen and Vero-
aHis cells via
anti-His scAb.
Fig. 19: Scheme of MeVac genomes encoding FmIL-12 or anti-PD-L1 or IgGl-Fc
retargeted to
human CD20. MeVac H gene was fully retargeted to human CD20 antigen by
ablating attachment
to the natural receptors, fusing the H protein to a single chain antibody
(scAb) against the CD20
and including a six-histidine tag at the C terminus to allow specific
transduction of murine
melanoma B16-CD20 cells via human CD20 antigen and Vero-aHis cells via anti-
His scAb.
The following Examples shall merely illustrate the invention. They shall not
be construed,
whatsoever, to limit the scope of the invention.
Examples
Example 1: Cell culture
Date Recue/Date Received 2021-03-19
CA 02921864 2016-02-25
- 40 -
Vero African green monkey kidney cells were obtained from the American Type
Culture Collection (Manassas, VA). Vero-aHis cell line stably expressing a
single chain
antibody (scAb) against His6 tag (Nakamura et al. 2005) was a kind gift of S.
J. Russel (Mayo
Clinic, Rochester, MN). Murine colon adenocarcinoma cells MC38cea (transduced
for stable
expression of human CEA antigen) and the parental MC38 cell line were a gift
of R. Cattaneo
(Mayo Clinic, Rochester, MN). 1116-CD20 have previously been generated by
transducing the
parental cell line with a lentiviral vector encoding human CD20 (Engeland et
al. 2014). All
cell lines were cultivated in either Dulbecco's modified Eagle's medium (DMEM;
Life
Technologies, Darmstadt, Germany) or Roswell Park Memorial Institute 1640
medium
(RPMI 1640; Life Technologies) supplemented with 10% Fetal Calf Serum (FCS) at
37 C in
a humidified atmosphere with 5% CO2 and routinely tested for mycoplasma
contamination.
Example 2: Cloning of recombinant MeVac genomes
The cDNA plasmids encoding recombinant MeV genomes were constructed on the
basis of the commercially used Schwarz/Moraten vaccine strain (MeVac)
(Combredet et al.
2003). Transgenes were inserted in additional transcription units (ATUs)
containing
additional gene-end gene-start signals and a unique cloning site. Transgenes
smaller than 1
kbp including murine GM-CSF (426 bp), murine IP-10 (312 bp) and eGFP (720 bp)
were
inserted into the leader position of pcMeVac as M/u/-Asc/ fragments via the
unique AscI
restriction site. The mGM-CSF and eGFP fragments were amplified from the MeV
Edmonston B (NSe) vaccine strain genomes encoding the respective transgenes
(Grossardt et
al. 2013). The mIP-10 (mCxci./ 0) gene was amplified with primers flanking the
novel
construct and adding a Al/u/ site and a Kozak sequence (GCCACC) in the 5'-end
and a two
nucleotide TA spacer and AscI site in the 3'-end using cDNA obtained from
murine
splenocytes.
Cassette encoding a murine IL-12 fusion protein (FmIL-12) consisting of murine
IL-
12 p40 and p35 subunits linked by a (Gly4Ser)3 for insertion into MeV genome
had previously
been constructed by C. Grossardt (Grossardt 2013) based on results of Lieschke
and
colleagues (Lieschke et al. 1997). FmII,-12 construct (1650 bp) was excised
from pCG
expression vector (constructed by C. Grossardt) as Paul-Mlul fragment and
inserted into the
MeVac genome downstream the P ORF via the unique MauBI cloning site.
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Antibodies against negative murine T cell regulators CTLA-4 and PD-Li as well
as
soluble form of murine CD80 T cell costimulatory molecule and human IgGl-Fc
fragment
for use as a control were inserted into the ATU downstream the H gene.
Cassettes encoding
antibodies against murine CTLA-4 and PD-Li and human IgG1 -Fc fragment
previously
designed by C. E. Engeland (Engeland et al. 2014) were used as templates. The
respective
constructs were excised from pCG expression vectors as Mlul-Paul fragments and
inserted
into the pcMeVac H-ATU via the unique MauBI cloning site.
Murine CD80 molecule was inserted for expression from MeVac in a soluble form.
CD8O-Fc was constructed by fusing the extracellular part of the murine CD80
with the same
human IgG1 -Fc region as used in both anti-CTLA-4 and anti-PD-L1 constructs
via fusion
PCR. The first PCR fragment consisting of the MluI restriction site followed
by Kozak
sequence (GCCACC), murine CD80 signal peptide, extracellular part (up to the
asparagine in
position 246) of murine CD80 and first 26 nucleotides of the hinge of IgGl-Fc
was
synthesized using pCG vector encoding murine CD80 as a template. The second
PCR
fragment consisting of the human IgGl-Fe region followed by myc tag, stop
codon and Ascl
restriction site was synthesized using pCG vector encoding human IgGl-Fc as a
template. The
obtained PCR products were fused with flanking primers in an overlap PCR
obtaining the
mCD80-Fc construct of 1614 bp. The mCD80-Fc was inserted into the pcMeVac H-
ATU as a
MluI-AscI fragment via the unique MauBI cloning site.
MeVac genomes encoding the previously described transgenes with a fully
retargeted
MeV H attachment gene were constructed to allow targeted transduction of
murine MC38cea
and B16-CD20 cells. MeVac H gene was exchanged for H gene with mutated
attachment sites
to the natural MeV receptors CD46 and CD150, fused to a single chain antibody
(scAb)
against human CEA or CD20 and containing a C-terminal His6 tag. The
retargeting system
allows a flexible change of the targeted antigen by exchanging the specific
scAb as a Sill-Noll
fragment.
Example 3: Virus propagation and titration
Recombinant MeVac particles were obtained from cDNA constructs according to
Radecke et al. (Radecke et al. 1995) and propagated on Vero-allis cells
according to
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Nakamura et al. (Nakamura et al. 2005). For propagation Vero-aHis cells were
infected at a
multiplicity of infection (M01) of 0.03 and cultivated at 37 C 5% CO2 until
syncytia had
spread across the whole cell layer (36 - 48 h post infection). Subsequently
culture medium
was completely removed, cells were scraped and collected and viral particles
released by one
freeze-thaw cycle. Cellular debris was removed by centrifugation at 6000xg for
5min. The
amount of viral particles was determined by 1:10 serial dilution titrations in
octuplicates on
1.5x104 Vero- His cells per well in 96-well cell culture plates. Individual
syncytia were
counted 72h post infection and titers calculated as cell infectious units per
ml (ciu/ml).
Example 4: Statistical analyses
Statistical analyses were performed using GraphPad Prism software (version
5.04; GraphPad
Software, La Jolla, CA). Tumor volumes and ELISA results in restimulation
experiments
were analysed by one-way ANOVA with Tukey's multiple comparison test. Survival
curves
were analyzed by log-rank (Mantel-Cox) test with Bonferroni-Holm correction
for multiple
comparisons. Result was considered statistically significant if p value was
lower than 0.05
after correcting for multiple comparisons.
Example 5: Characterization of virus replication
Vero-aHis and MC38cea cells were seeded in 12-well plates (1 x105 cells per
well). After 12h
the cell culture medium was removed and cells were infected with the
respective viruses at
MOI=3 in 300 I OptiMEM in triplicates for each time point and cultivated at
37 C 5% CO2.
After adsorption for ca. 2h the inoculum was removed and substituted with 1 ml
DMEM+10 A FCS per well. Cells were scraped in the culture medium at the
designated time
points, collected and snap frozen in liquid nitrogen. The amount of viral
particles was
determined by 1:10 serial dilution titrations in quadruplicates on 1.5 x104
Vero-otHis cells per
well in 96-well cell culture plates. Individual syncytia were counted 72h post
infection and
titers calculated as ciu/ml.
Example 6: Assessment of virus cytotoxic potential in vitro
MC38cea cells were seeded in 6-well plates (2x105 cells per well). After 12h
the cell culture
medium was removed and cells were infected with the respective viruses at
M01=5 in 800 1
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OptiMEM in triplicates for each time point and cultivated at 37 C 5% CO2.
After adsorption
for ca. 2h the inoculum was removed and substituted with 2 ml DMEM+10% FCS per
well.
At the designated time points cell viability was determined using Colorimetric
Cell Viability
Kit III (XTT) (PromoKine, Heidelberg, Germany) according to instructions of
the
manufacturer.
Example 7: Characterization of transgene expression
MC38cea cells were seeded in 12-well plates (1x105 cells per well). After 12h
the cell culture
medium was removed and cells were infected with the respective viruses at
MOI=3 in 300 [11
OptiMEM in triplicates for each time point and cultivated at 37 C 5% CO2.
After adsorption
for ca. 2h 700 1..t1 DMEM+10% FCS per well was added. Supernatants were
collected at the
designated time points. Time point Oh was represented by inoculum in OptiMEM
used for
infection. Expression of the respective immunomodulators was detected by
ELISA.
Commercially available ELISA kits were used for detection of mGM-CSF, FmIL-12,
mIP-10
(R&D Systems, Wiesbaden, Germany) and CD8O-Fc (Boster Biological Technology,
Offenbach, Germany) according to instructions of the manufacturer. Anti-CTLA-4
and anti-
PD-Li were detected by binding to their respective murine proteins. Ninety-six
well plates
(Nunc Maxisorp, Thermo Fisher Scientific, Schwerte, Germany) were coated with
100 ng
recombinant His-tagged murine CTLA-4 or PD-L1 (Life Technologies). Wells were
blocked
and 100 1.11 of the respective samples were added and incubated for 2h. After
washing the
antibodies were detected with anti-human IgG-Fc Biotin (clone HP-6071; Sigma-
Aldrich,
Tautkirchen, Germany), Peroxidase conjugated Streptavidin (Dianova, Hamburg,
Germany)
and 1-Step Ultra-TMB ELISA Substrate Solution (Thermo Scientific, Karlsruhe,
Germany).
Absorbance was measured using Infinite M200 Pro microplate reader and i-
control software
(Tecan, Mannedorf, Switzerland).
Example 8: Flow cytometry for detection of anti-PD-Li binding to MC38cea cells
Vero-aHis cells were seeded in 15 cm cell culture dishes and infected with
MeVac encoding
anti-PD-Li or IgGl-Fe with MOI=0.03. Supernatants were collected (15m1 per
plate) when
syncytia had spread over the whole cell layer (ca. 36h post infection).
lx106MC38cea cells
were incubated with anti-PD-L1 or IgG 1 -Fe containing supernatant previously
collected from
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one fully infected 15 cm dish for lh with rotation at room to. After washing
the bound anti-
PD-L1 was detected by staining with anti-HA (clone HA-7; Sigma-Aldrich) and
goat anti-
mouse IgG PE (polyclonal; BD Biosciences, Heidelberg, Germany). The stained
cells were
resuspended in DPBS with 0.2 vig/m1 DAPI (Sigma-Aldrich) and directly acquired
on LSRII
flow cytometer (BD Biosciences) collecting at least 10000 events per sample.
Example 9: Isolation of murine splenocytes
Spleens were aseptically isolated and maintained in RPMI 1640 (Life
Technologies,
Darmstadt, Germany) at 4 C until further processing. Spleen was passed through
a 100 vtin
nylon cell strainer (BD Biosciences, Heidelberg, Germany) into 10 ml RPMI 1640
and cells
were pelleted at 300xg for 5min. For red blood cell lysis pellet was
resuspended in 1 ml ACK
Lysing solution (Life Technologies), incubated 10min at room t and
centrifuged at 300xg for
5min. Cells were resuspended in DPBS (Life Technologies) and cell
concentration
determined using Neubauer hemocytometer and Trypan blue (Sigma-Aldrich)
staining for
dead cell exclusion.
Example 10: Functional assay for MeVac encoded anti-PD-L1. CD8O-Fc and anti-
CTLA-4
Vero-allis cells were seeded in 15 cm cell culture dishes and infected with
MeVac
encoding anti-PD-L I, anti-CTLA-4, CD8O-Fc or IgGl-Fc with MO1=0.03.
Supernatants were
collected (15m1 per plate) when syncytia had spread over the whole cell layer
(ca. 36h post
infection). 2x105 MC38cea cells were incubated with 2m1 medium collected from
the Vero-
aHis infected with the respective viruses for 5min with rotation at room to
and pelleted by
centrifugation 5min at 300xg. The procedure was repeated six times. The
treated cells were
resuspended in 100 vtl activation medium ¨ RPMI 1640 supplemented with 5% FCS,
1%
Penicillin-Streptomycin (Life Technologies), 500 1iM ionomycin (Cayman
Chemical
Company, Hamburg, Germany) and 5 1.11VI PMA (Cayman Chemical Company) and
seeded in
.. 96-well plate. 2x105 freshly isolated splenocytes from C57BL/6J mouse in
100 Ill activation
medium were added per each well with the treated MC38cea cells. Cells were
cocultivated
24h at 37 C 5% CO2 and supernatants collected subsequently. IFN-y
concentration was
determined using mouse IFN gamma ELISA Ready-SET-Go ! (eBioscience, Frankfurt
am
Main, Germany) according to the instructions of the manufacturer.
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Example 11: Functional assay for MeVac encoded FmIL-12
Vero-otHis cells were seeded in 15 cm cell culture dishes and infected with
MeVac
encoding FmIL-12 or eGFP. Supernatants were collected (15m1 per plate) when
syncytia had
spread over the whole cell layer (ca. 36h post infection). FmIL-12
concentration was assessed
using Mouse IL-12 p70 Quantikine ELISA Kit (R&D Systems). 2x106 freshly
isolated
splenocytes from a C57BL/6J mouse were resuspended in RPMI 1640 supplemented
with
10% FCS, 1% Penicillin-Streptomycin solution and 50 U/ml recombinant murine IL-
2
(Miltenyi, Bergisch Gladbach, Germany) with varying concentrations of MeVac
encoded
FmIL-12 or respective parts of supernatant from cells infected with eGFP
encoding MeVac.
Splenocytes were seeded in 12-well plates and incubated 48h at 37 C 5% CO2.
Supernatants
were collected and IFN-y concentration assessed using mouse IFN gamma ELISA
Ready-
SET-Go! (eBioscience) according to the instructions of the manufacturer.
Example 12: Assessment of therapeutic efficacy in vivo
MC38cea cells were subcutaneously (s.c.) implanted into six to eight weeks old
C57B1/6J
mice (Harlan Laboratories, Rossdorf Germany or DKFZ, Heidelberg, Germany).
When
average tumor volume reached 50 ¨ 100 mm3 (depending on experiment) treatment
was
initiated. Mice received intratumoral (i.t.) injections with the respective
viruses on four or five
consecutive days with 5x105 or 1x106 ciu in 100 I. Mice in the mock group
received
treatment with 100 .1 OptiMEM. Tumor volume was determined every third day
measuring
largest and smallest diameter with a caliper and calculating the volume using
a formula:
largest diameterx(smallest diameter)2x0.5. Mice were sacrificed when tumor
volume
exceeded 1500 mm3, ulceration occurred or signs of severe illness were
observed.
Example 13: Antigen specific IFN-y memory recall with murinc splenocytes
MC38cea, MC38 and 816 cells were treated with 20 ug/m1 mitomycin-C (Sigma-
Aldrich) for
2h with shaking at 37 C. After subsequent washing three times with DPBS cells
were
resuspended in activation medium containing RPMI 1640 supplemented with 10%
FCS, 1%
Penicillin-Streptomycin and 50 U/ml recombinant murine IL-2. Freshly isolated
murine
splenocytes were resuspended in the same activation medium. Cocultures were
prepared in
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24-well plates seeding 1x105 mitomycin-c treated tumor cells or lx106ciu MeVac
with 1x106
splenoeytes per well in 0.5 ml total volume of activation medium. As controls
1>106
splenocytes were cocultivated also with Vero-aHis or DLD-1 cell lysates
prepared by lysis of
l x106 cells per ml with one freeze-thaw cycle. Cells were cocultivated for
48h, supernatants
collected and IFN-7 concentration assessed using mouse IFN gamma ELISA Ready-
SET-
Go! (eBioscience) according to the instructions of the manufacturer.
References (Examples)
Combredet, C. et al., 2003. A Molecularly Cloned Schwarz Strain of Measles
Virus Vaccine
Induces Strong Immune Responses in Macaques and Transgenic Mice A Molecularly
Cloned Schwarz Strain of Measles Virus Vaccine Induces Strong Immune Responses
in
Macaques and Transgenic Mice.
Engeland, C.E. et al., 2014. CTLA-4 and PD-Li Checkpoint Blockade Enhances
Oncolytic
Measles Virus Therapy. Molecular Therapy, 22(11), pp.1949-1959.
Grossardt, C., 2013. Engineering Targeted and Cytokine-armed Oncolytic Measles
Viruses.
Ruperto-Carola University of Heidelberg.
Grossardt, C. et al., 2013. Granulocyte-Macrophage Colony-Stimulating Factor-
Armed
Oncolytic Measles Virus Is an Effective Therapeutic Cancer Vaccine. Human Gene
Therapy, 24(7), pp.644-654.
Lieschke, G.J. et al., 1997. Bioactive murine and human interleukin-12 fusion
proteins which
retain antitumor activity in vivo. Nature biotechnology, 15(1), pp.35-40.
Nakamura, T. et al., 2005. Rescue and propagation of fully retargeted
oncolytic measles
viruses. Nature biotechnology. 23(2), pp.209-14.
Radecke, F. et al., 1995. Rescue of measles viruses from cloned DNA. The EMBO
journal,
14(23), pp.5773-84. Available at:
