U.S. patent application number 15/550567 was filed with the patent office on 2018-02-01 for immunotherapeutic vaccine and antibody combination therapy.
This patent application is currently assigned to Transgene SA. The applicant listed for this patent is Transgene SA. Invention is credited to Julie Hortelano, Xavier Preville, Karola Rittner, Philippe Slos.
Application Number | 20180028626 15/550567 |
Document ID | / |
Family ID | 55353205 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180028626 |
Kind Code |
A1 |
Slos; Philippe ; et
al. |
February 1, 2018 |
IMMUNOTHERAPEUTIC VACCINE AND ANTIBODY COMBINATION THERAPY
Abstract
The present invention relates to a combination product,
composition(s) and kit of parts comprising at least (i) a
therapeutic vaccine and (ii) one or more immune checkpoint
modulator(s). The present invention also concerns a method for
treating a proliferative or an infectious disease as well as a
method for eliciting or stimulating and/or re-orienting an immune
response, wherein said methods comprise administering to a subject
in need thereof said combination product or said
composition(s).
Inventors: |
Slos; Philippe; (Ingwiller,
FR) ; Hortelano; Julie; (Illkirch, FR) ;
Rittner; Karola; (Strasbourg, FR) ; Preville;
Xavier; (Saint Louis, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Transgene SA |
Illkirch Graffenstaden |
|
FR |
|
|
Assignee: |
Transgene SA
Illkirch Graffenstaden
FR
|
Family ID: |
55353205 |
Appl. No.: |
15/550567 |
Filed: |
February 12, 2016 |
PCT Filed: |
February 12, 2016 |
PCT NO: |
PCT/EP2016/052991 |
371 Date: |
August 11, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/2013 20130101;
A61K 39/00114 20180801; C07K 16/2818 20130101; A61K 2039/505
20130101; A61K 35/761 20130101; A61K 39/001139 20180801; A61K 39/12
20130101; A61K 38/1709 20130101; A61K 39/0011 20130101; C12N 7/00
20130101; C07K 2317/73 20130101; A61K 39/00 20130101; A61K 39/00117
20180801; A61K 2039/55533 20130101; A61K 39/39558 20130101; C12N
2710/24171 20130101; A61K 2039/545 20130101; C12N 2710/24132
20130101; A61K 38/193 20130101; C12N 2710/24143 20130101; C07K
16/3046 20130101; A61K 2039/5256 20130101; C07K 2317/76 20130101;
A61K 2039/575 20130101; A61P 35/00 20180101; A61K 39/3955 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 16/30 20060101 C07K016/30; C12N 7/00 20060101
C12N007/00; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 13, 2015 |
EP |
15305215.4 |
Apr 16, 2015 |
EP |
15305570.2 |
Claims
1. A combination product comprising at least (i) a therapeutic
vaccine and (ii) one or more immune checkpoint modulator(s).
2. The combination product of claim 1, wherein said therapeutic
vaccine comprises a recombinant plasmid or a viral vector.
3. The combination product of claim 2, wherein said therapeutic
vaccine comprises a poxvirus.
4. The combination product of claim 2, wherein said therapeutic
vaccine comprises a human or an animal adenovirus.
5. The combination product of claim 4, wherein said adenovirus is
replication-defective.
6. The combination product of claim 1, wherein said therapeutic
vaccine comprises or encodes one or more polypeptide(s) selected
from the group consisting of suicide gene products,
immunostimulatory polypeptides, and antigenic polypeptides.
7. The combination product of claim 6, wherein said therapeutic
vaccine comprises or encodes one or more polypeptide(s) selected
from the group consisting of the MUC1 antigen, HPV E6 and E7
antigens, the human IL-2, the human GM-CSF, the FCU1 suicide gene,
HBV antigens, mycobacteria antigens, and the HCV non structural
antigens.
8. The combination product of claim 7, wherein said therapeutic
vaccine comprises a MVA virus encoding the MUC-1 antigen and IL-2;
a MVA virus encoding a fusion of NS3 and NS4 HCV antigens and NS5b
antigen; a MVA virus encoding membrane anchored HPV-16
non-oncogenic E6 and E7 antigens and IL-2; a MVA virus encoding the
FCU1 suicide gene; a MVA virus encoding a combination of TB
antigens or an Ad vector encoding a combination of HBV
antigens.
9. (canceled)
10. The combination product of claim 1, wherein said one or more
immune checkpoint modulator(s) is a human or humanized monoclonal
antibody that specifically binds to any of PD-1, PD-L1, PD-L2,
LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and CTLA4.
11. The combination product of claim 10, wherein at least one of
said one or more immune checkpoint modulator(s) is selected from
the group consisting of (a) an antibody that specifically binds to
human PD-1; (b) an antibody that specifically binds to human PD-L1;
and (c) an antibody that specifically binds to human CTLA-4.
12. The combination product of claim 1, wherein said one or more
immune checkpoint modulator(s) is/are expressed by said therapeutic
vaccine.
13. The combination product of claim 1, wherein said combination
product is a composition comprising a therapeutically effective
amount of at least said therapeutic vaccine and said one or more
immune checkpoint modulator and a pharmaceutically acceptable
vehicle.
14. The combination product of claim 1, comprising from about 0.5
mg/kg to about 25 mg/kg of the immune checkpoint modulator(s).
15. The combination product of claim 1, wherein said combination
comprises from approximately 10.sup.8 vp to approximately
5.times.10.sup.11 vp of an adenoviral vector or from approximately
10.sup.6 pfu to approximately 10.sup.10 pfu of a MVA vector.
16. The combination product of claim 15, wherein the immune
checkpoint modulator(s) comprised in said combination is formulated
for intravenous or intratumoral administration and wherein the
therapeutic vaccine comprised in said combination is formulated for
intravenous, intramuscular, subcutaneous or intratumoral
administration.
17. The combination product of claim 1, wherein the administrations
of said therapeutic vaccine and said one or more immune checkpoint
inhibitor is concomitant, sequential, or interspersed.
18. The combination product of claim 17, wherein the
administration(s) of said therapeutic vaccine starts before the
administration(s) of said immune checkpoint modulator(s).
19. The combination product according to claim 1 comprising from 4
to 15 administrations of 10.sup.7 or 10.sup.9 pfu of a MVA-based
therapeutic vaccine at approximately 1 to 3 week interval
interspersed with 2 to 6 administrations of 3 to 10 mg/kg of
anti-immune checkpoint antibody/antibodies every 2 or 3 weeks.
20. A composition comprising effective amounts of: (a) a
therapeutic vaccine that comprises a recombinant plasmid or viral
vector; and (b) one or more immune checkpoint modulator(s) that
is/are a human or humanized monoclonal antibody/antibodies that
specifically bind(s) to any of PD-1, PD-L1, PD-L2, LAG3, Tim3,
BTLA, SLAM, 2B4, CD160, KLRG-1, and CTLA4.
21. A method for treating a proliferative or an infectious disease
comprising administering: (a) a combination product comprising at
least (i) a therapeutic vaccine and (ii) one or more immune
checkpoint modulator(s); or (b) a composition comprising effective
amounts of: (1) a therapeutic vaccine that comprises a recombinant
plasmid or viral vector; and (2) one or more immune checkpoint
modulator(s) that is/are a human or humanized monoclonal
antibody/antibodies that specifically bind(s) to any of PD-1,
PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and
CTLA4.
22. The method of claim 21, wherein said proliferative disease is a
cancer and wherein said infectious disease results from infection
with a virus selected from the group consisting of herpes virus
papillomavirus, poxvirus, retrovirus, HCV, HBV, and influenza
virus.
23. A method for eliciting or stimulating and/or re-orienting an
immune response comprising administering: (a) a combination product
comprising at least (i) a therapeutic vaccine and (ii) one or more
immune checkpoint modulator(s); or (b) a composition comprising
effective amounts of: (1) a therapeutic vaccine that comprises a
recombinant plasmid or viral vector; and (2) one or more immune
checkpoint modulator(s) that is/are a human or humanized monoclonal
antibody/antibodies that specifically bind(s) to any of PD-1,
PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and CTLA4
to a subject in need thereof so as to activate the patient's
immunity.
24. The method of claim 21 which is used or carried out in
association with one or more conventional therapeutic
modalities.
25. A kit comprising: (a) a container comprising a therapeutic
vaccine that comprises a recombinant plasmid or viral vector; and
(b) a different container comprising one or more immune checkpoint
modulator(s) that is/are a human or humanized monoclonal
antibody/antibodies that specifically bind(s) to any of PD-1,
PD-L1, PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1, and
CTLA4.
26. The combination product of claim 3, wherein said poxvirus is a
vaccinia virus.
27. The method of claim 23 which is used or carried out in
association with one or more conventional therapeutic modalities
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention generally relates to novel
combinations comprising of one or more immune checkpoint
modulator(s) and at least a therapeutic vaccine (more specifically
a vectorized vaccine encoding antigen(s)). Embodiments also include
compositions and kits comprising such components as well as methods
for treating, preventing or inhibiting proliferative and infectious
diseases. The invention is of very special interest in the field of
immunotherapy, specifically for enhancing host's immune response
and in particular for disrupting immune tolerance.
BACKGROUND ART
[0002] Using the host's immune system to eradicate persistent
infectious organisms and malignant cells is a promising approach.
This specific type of vaccine strategy is generally referred to as
immunotherapy. Widely used in traditional vaccination,
immunotherapy has shown some promise results in therapy for
treating severe, chronic and life-threatening diseases.
[0003] Numerous research groups have investigated immunotherapy as
a potential modality for treating cancer in an attempt to stimulate
the immune system and thus reject and destroy tumors. A vast number
of immunotherapeutic treatments have been described in the
literature for decades using various approaches, for example cancer
cells, parts of cells, purified antigens and vectorized antigens.
Cell vaccines are generally made up of cancer cells that have been
removed from the patient during surgery and altered in lab to make
them more amenable to be attacked by the patient's immune system
before being reintroduced in the patient. Alternatively, one may
use immune cells obtained from the patient's blood that have been
exposed to cancer cells or associated antigens, cultured in the
presence of chemokines that turn them into dendritic cells before
being given back to the patient by intravenous infusion in order to
help other immune system cells to attack the cancer cells. The
dendritic cell-based vaccine Provenge.RTM. (sipuleucel-T), was
tested in advanced clinical trials to treat advanced prostate
cancer and received FDA approval in 2010. However, such cell-based
vaccines require to be made individually for each patient and the
process used to produce them is thus complex and expensive.
[0004] Antigen vaccines are made up of only one or a few protein or
peptide antigen(s) that are specific for a certain type of cancers
or pathogens. Once administered, they will be able to induce
specific immunological responses against these antigens and boost
the patient's immune system. Several candidate peptide vaccines
reached clinical development. For example, the liposomal vaccine
Stimuvax.RTM. incorporates lipopeptides generated from the mucin 1
(MUC1) glycoprotein that is widely expressed by common cancers.
Although it did not provide in clinical trials a significant
improvement in overall survival in patients with advanced non-small
cell lung cancer (NSCLC), effects were nevertheless seen in some
subgroups of patients.
[0005] Vector-based vaccines have shown great promise and play an
important role in the development of new therapeutic strategies.
Vectors are used to deliver the targeted antigen into the body.
Typically, vectors originate from virus, bacteria, yeast cells, or
other structures that have been altered to make them no longer
harmful for the patient (e.g. inactivated, attenuated, etc). The
ideal viral vector should be safe and enable efficient antigen
presentation of the encoded antigens to the immune system.
Furthermore, the vector system must meet criteria that enable its
production on a large-scale basis. Live viral vectors are
attractive for their ability to both express antigens from a
variety of pathogens and tumoral tissue and to facilitate antigen
presentation through the endogenous pathway which has been shown to
be important for efficient induction of cellular immune responses
(Reyes-Sandoval, 2007, Immunology 121(2): 158-65). Several viral
vectors have thus emerged to date, all of them having relative
advantages and limits depending on the proposed application (see
for example Harrop and Carroll, 2006, Front Biosci., 11, 804-817;
Inchauspe et al., 2009, Int Rev Immunol 28(1): 7-19; Torresi et
al., 2011, J. Hepatol. 54(6): 1273-85). Significant research
efforts have also been undertaken to develop antigen-coding DNA
plasmids and associated delivery device to stimulate protective
immune responses (e.g. see Reyes-Sandoval and Ertl, 2001, Curr Mol
Med 1(2): 217-43).
[0006] For example, replication-defective adenovirus (Ad) vectors
have been extensively used because Ad infects replicating and
non-replicating cells, has a broad tissue tropism, propagates
efficiently in suitable packaging cell lines and production process
is scalable and affordable (Boukhebza et al., 2014, Vaccine 32(26):
3256-63). The attenuated non-replicative vaccinia virus Ankara
strain (MVA) is also an attractive candidate since it has been
shown to induce robust cellular immune responses with an excellent
safety profile both in the cancer and infectious diseases fields
(Boukhebza et al., 2012, Hum Vaccin Immunother 8(12): 1746-57;
Habersetzer et al., 2011, Gastroenterology 141(3): 890-99;
Fournillier et al., 2007, Vaccine 25(42): 7339-53; Drexler et al.,
2004, Curr Opin Biotechnol 15(6): 506-12). MVA has been attenuated
by more than 570 passages in chicken embryo fibroblasts resulting
in the loss of 15% of its genome. Consequently, MVA is unable to
produce mature virions in most mammalian cells that result in a
reduced risk of dissemination and an increased immunogenicity due
to the loss of several anti-immune defense genes (Sutter et al.,
1994, Vaccine 12(11): 1032-40).
[0007] However, there are limits on the immune system's ability to
fight chronic infectious diseases and cancers. Sometimes the immune
system doesn't detect the cancer or infected cells as foreign
because the cells are not different enough from normal cells. In
other cases, the response might not be strong enough to destroy the
diseased cells, especially in immuno-compromised patients. Finally,
the immune system might be ineffective due to the fact that
diseased cells have evolved different ways of eluding the immune
system.
[0008] One of the major mechanisms of immune suppression is a
process known as "T-cell exhaustion", which results from chronic
exposure to antigens and is characterized by the upregulation of
inhibitory receptors. These inhibitory receptors serve as immune
checkpoints in order to prevent uncontrolled immune reactions.
Various immune checkpoints acting at different levels of T cell
immunity have been described in the literature, including
programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte
associated protein-4 (CTLA-4), Lymphocyte-activation gene 3 (LAG3),
B and T lymphocyte attenuator, T-cell immunoglobulin, mucin
domain-containing protein 3 (TIM-3), and V-domain immunoglobulin
suppressor of T cell activation (VISTA). It has also been reported
that the interaction of PD-1 with its ligands PDL-1 and PDL-2 plays
a critical role in T cell exhaustion (Maier et al., 2007, J.
Immunol. 178: 2714-20; Tzeng et al., 2012, PLoS One 7: e39179).
[0009] Whatever the mechanism of action, these immune checkpoints
can inhibit the development of an efficient immune response. There
is an increasing interest of blocking such immune checkpoints as a
means of inhibiting immune system tolerance and thus rescue
exhausted T cells (Leach et al., 1996, Science 271: 1734-6). A vast
number of antagonistic antibodies have been developed during the
last decade (e.g. anti LAG3, -PD-L1, -CTLA-4, -PD1, etc) and three
are already marketed. The first to reach the market was the
monoclonal-CTLA-4-specific antibody ipilimumab (Yervoy trade name,
Bristol-Myers Squibb (BMS)) that has been approved for unresectable
or metastatic melanoma. BMS reported that from 1861 melanoma
patients treated with ipilimumab 22% and 17% are still alive 3 and
7 years later, respectively. Anti-PD1 nivolumab antibody (Opdivo
trade name, BMS) was approved in Japan in July 2014 for malignant
melanoma. Interim phase II data showed a 32% response rate in
pretreated metastatic melanoma with lower high grade adverse events
than Ipilimumab. Anti-PD-1 pembrolizumab (Keytruda trade name,
Merck), gained accelerated FDA approval for the treatment of
unresectable or metastatic melanoma. The marketed antibodies are
also in clinical trials for other indications including NSCLC
(non-small cell lung cancer) as well as a variety of other immune
checkpoint inhibitors targeting PD-1 (e.g. pidilizumab, CureTech),
CTLA-4 (e.g. Tremelimumab (AstraZeneca), PD-L1 (e.g. MPDL3280A,
Roche), KIR (lirilumab, BMS), IDO1 (e.g. indoximod, NewLink
genetics) and others.
[0010] Preclinical studies with antagonist antibodies are also
pursuing in infectious disease field (see e.g. Barber et al., 2006,
Nature 439: 682-7; Cecchinato et al., 2008, J. Immunol 180:
5439-47) and combinations with different vector platforms (DNA,
MVA, lentivirus, vaccinia, etc.) were envisaged. In particular, the
combination of PD-L1 blockade with a vaccinia virus expressing LMCV
(Lymphocytic Chronomeningitis Virus) epitope was shown to improve
the function of epitope-specific CD8+ T cells during persistent
viral infection (Ha et al., 2008, JEM 205: 543-55). Administration
of anti-PD-1 antibodies together with a SIV gag adenovirus vector
in naive macaques caused increased in Gag-specific T cells
(Finnefrock et al., 2009, J. Immunol. 182: 980-7). WO2004/058801
relates to the treatment of cancer using a recombinant MVA vector
encoding p53 oncogenic polypeptide in combination with anti-CTLA4
antibodies and CpG oligodeoxynucleotide immunomodulators.
[0011] One may expect that cancer and infectious diseases will
continue to be a serious global health threat for many years.
Although availability of antibiotics and vaccines, infectious
diseases cause 100.000 deaths per year throughout the world (data
WHO 2002). On the other hand, malignant and especially metastatic
tumors are often resistant to conventional therapies explaining the
significant morbidity of some cancers. The above-description
clearly illustrate that designing effective therapies is a
difficult task due to the numerous mechanisms set up by the host's
body to escape immune effector cells.
SUMMARY OF THE INVENTION
[0012] In the context of the invention, the inventors identified a
combination product able to potentiate the patient's immune
responses and/or restore exhausted T cell-mediated immunity.
Essential elements of such a combination product are a therapeutic
vaccine and an immune checkpoint inhibitor. The inventors
surprisingly found that administrations of a MVA vector encoding a
model antigen (.beta.Gal) in combination with anti-CTLA4 or
anti-PD-1 antibody are surprisingly effective to reduce the volume
of tumors implanted in a human cancer animal model and increase the
survival rate of those animals. The ability of such combinations to
provide antitumor effects is a good indication that the present
invention can be useful for treating human subjects against a
variety of diseases, and especially infectious and proliferative
diseases.
[0013] In a first aspect the invention provides a combination
product comprising at least a therapeutic vaccine and one or more
immune checkpoint modulator(s). Preferably, the therapeutic vaccine
comprises a viral vector and more preferably a recombinant viral
vector encoding an antigenic polypeptide. Preferably the immune
checkpoint modulator is a monoclonal antibody capable of
antagonizing at least partially the activity of immune checkpoint
such as CTLA-4 or PD-1.
[0014] The present invention also provides a composition comprising
the therapeutic vaccine and the one or more immune checkpoint
modulator(s) as well as the use or method of treatment using such a
composition or combination, especially for treating infectious and
proliferative diseases such as cancer.
[0015] Other and further aspects, features and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0016] As described above, the inventors identified a combination
product comprising at least (i) a therapeutic vaccine and (ii) one
or more immune checkpoint modulator(s).
[0017] As used throughout the entire application, the terms "a" and
"an" are used in the sense that they mean "at least one", "at least
a first", "one or more" or "a plurality" of the referenced
components or steps, unless the context clearly dictates otherwise.
For example, the term "a therapeutic vaccine" includes a plurality
of therapeutic vaccines, including mixtures thereof.
[0018] The term "one or more" refers to either one or a number
above one (e.g. 2, 3, 4, 5, etc).
[0019] The term "and/or" wherever used herein includes the meaning
of "and", "or" and "all or any other combination of the elements
connected by said term".
[0020] The term "about" or "approximately" as used herein means
within 10%, preferably within 8%, and more preferably within 5% of
a given value or range.
[0021] As used herein, when used to define products, compositions
and methods, the term "comprising" (and any form of comprising,
such as "comprise" and "comprises"), "having" (and any form of
having, such as "have" and "has"), "including" (and any form of
including, such as "includes" and "include") or "containing" (and
any form of containing, such as "contains" and "contain") are
open-ended and do not exclude additional, unrecited elements or
method steps. Thus, a polypeptide "comprises" an amino acid
sequence when the amino acid sequence might be part of the final
amino acid sequence of the polypeptide. "Consisting essentially of"
means excluding other components or steps of any essential
significance. Thus, a composition consisting essentially of the
recited components would not exclude trace contaminants and
pharmaceutically acceptable carriers. A polypeptide "consists
essentially of" an amino acid sequence when such an amino acid
sequence is present with optionally only a few additional amino
acid residues. "Consisting of" means excluding more than trace
elements of other components or steps. For example, a polypeptide
"consists of" an amino acid sequence when the polypeptide does not
contain any amino acids but the recited amino acid sequence.
[0022] The terms "polypeptide", "peptide" and "protein" refer to
polymers of amino acid residues which comprise at least nine or
more amino acids bonded via peptide bonds. The polymer can be
linear, branched or cyclic and may comprise naturally occurring
and/or amino acid analogs and it may be interrupted by non-amino
acids. As a general indication, if the amino acid polymer is more
than 50 amino acid residues, it is preferably referred to as a
polypeptide or a protein whereas if it is 50 amino acids long or
less, it is referred to as a "peptide".
[0023] Within the context of the present invention, the terms
"nucleic acid", "nucleic acid molecule", "polynucleotide" and
"nucleotide sequence" are used interchangeably and define a polymer
of any length of either polydeoxyribonucleotides (DNA) (e.g. cDNA,
genomic DNA, plasmids, vectors, viral genomes, isolated DNA,
probes, primers and any mixture thereof) or polyribonucleotides
(RNA) (e.g. mRNA, antisense RNA, SiRNA) or mixed
polyribo-polydeoxyribonucleotides. They encompass single or
double-stranded, linear or circular, natural or synthetic, modified
or unmodified polynucleotides. Moreover, a polynucleotide may
comprise non-naturally occurring nucleotides and may be interrupted
by non-nucleotide components.
[0024] The term "analog", "mutant" or "variant" as used herein
refers to a component (polypeptide or nucleic acid) exhibiting one
or more modification(s) with respect to its native counterpart. Any
modification(s) can be envisaged, including substitution, insertion
and/or deletion of one or more nucleotide/amino acid residue(s).
When several mutations are contemplated, they can concern
consecutive residues and/or non-consecutive residues. Mutation(s)
can be generated by a number of ways known to those skilled in the
art, such as site-directed mutagenesis (e.g. using the Sculptor.TM.
in vitro mutagenesis system of Amersham, Les Ullis, France), PCR
mutagenesis, DNA shuffling and by chemical synthetic techniques
(e.g. resulting in a synthetic nucleic acid molecule). Preferred
are analogs that retain a degree of sequence identity of at least
80%, preferably at least 85%, more preferably at least 90%, and
even more preferably at least 98% identity with the sequence of the
native counterpart.
[0025] In a general manner, the term "identity" refers to an amino
acid to amino acid or nucleotide to nucleotide correspondence
between two polypeptide or nucleic acid sequences. The percentage
of identity between two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps which need to be introduced for optimal global
alignment and the length of each gap. Various computer programs and
mathematical algorithms are available in the art to determine the
percentage of identity between amino acid sequences, such as for
example the algorithm of Needleman et Wunsch. J. Mol. Biol. 48,
443-453, 1970, the Blast program available at NCBI or ALIGN in
Atlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl.,
3: 482-9) or the needle software available from ebi.ac.uk world
wide under the name <<Align>>. Programs for determining
identity between nucleotide sequences are also available in
specialized data base (e.g. Genbank, the Wisconsin Sequence
Analysis Package, BESTFIT, FASTA and GAP programs). For
illustrative purposes, "at least 80% identity" means 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100%.
[0026] As used herein, the term "isolated" refers to a component
(e.g. a polypeptide, peptide, polynucleotide, vector, etc.), that
is removed from its natural environment (i.e. separated from at
least one other component(s) with which it is naturally associated
or found in nature). An isolated component refers to a component
that is maintained in a heterologous context or purified (partially
or substantially). For example, a nucleic acid molecule is isolated
when it is separated of sequences normally associated with it in
nature (e.g. dissociated from a chromosome or a genome) but it can
be associated with heterologous sequences (e.g. within a
recombinant vector).
[0027] The term "obtained from", "originating" or "originate" is
used to identify the original source of a component (e.g. a
polypeptide, peptide, polynucleotide, vector, etc.) but is not
meant to limit the method by which the component is made which can
be, for example, by chemical synthesis or recombinant means.
[0028] The term "combination" as used herein refers to any
arrangement possible of two or more entities (e.g. at least the
therapeutic vaccine and the one or more immune checkpoint
modulator(s) described herein).
[0029] A "therapeutic vaccine" as used herein refers to any
substance (e.g. polypeptide, polysaccharide, nucleic acid, etc.),
including complex substance (e.g. cells, cell mixtures, live or
dead organisms such as bacteria, viruses, and other microorganisms,
etc. . . . ), part thereof (e.g. immunogenic fragment, epitope,
cell wall, flagella, etc) or analogs thereof, that is capable of
being the target of an immune response. The term encompasses
"antigens" (i.e. native antigens as well as fragments and analogs
thereof).
[0030] The term "immune checkpoint modulator" refers to a molecule
capable of modulating the function of an immune checkpoint protein
in a positive or negative way (in particular the interaction
between an antigen presenting cell (APC) and an immune T effector
cell).
[0031] The term "subject" generally refers to an organism for whom
any product and method of the invention is needed or may be
beneficial. Typically, the organism is a mammal, particularly a
mammal selected from the group consisting of domestic animals, farm
animals, sport animals, and primates. Preferably, the subject is a
human who have been diagnosed as being or at risk of having a
pathological condition such as an infectious disease caused by or
associated with a pathogenic organism or a proliferative disease
such as cancer. The terms "subject" and "patients" may be used
interchangeably when referring to a human organism and encompasses
male and female. The subject to be treated may be a newborn, an
infant, a young adult or an adult.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In a first aspect the present invention provides a
combination product comprising at least (i) a therapeutic vaccine
and (ii) one or more immune checkpoint modulator(s).
[0033] Combination Product
[0034] In the context of the present invention, such an arrangement
includes a mixture of the individual entities (e.g. a single
composition) meaning that the individual entities making up the
combination are placed together in a common container before
administration to the subject. By contrast, distinct combinations
refer to the case where the individual entities are not mixed
together meaning that they are into separate containers (e.g.
distinct compositions) for administration in conjunction with one
another either concomitantly, sequentially or in an interspersed
manner.
[0035] Exemplary combinations include, but are not limited to,
combination of polypeptides (e.g. peptide or protein-based
therapeutic vaccine and one or more immune checkpoint modulator(s)
in the form of recombinant polypeptide) or combination of nucleic
acid molecule(s) (e.g. a vectorized therapeutic vaccine and one or
more vector(s) engineered for encoding and expressing immune
checkpoint modulator(s)) as well as combination of both
polypeptide(s) and nucleic acid molecule(s) (e.g. a vectorized
therapeutic vaccine and one or more recombinant immune checkpoint
modulator polypeptide(s)). For illustrative purposes, a single
composition can be in the form of a) a mixture of the therapeutic
vaccine with one or more immune checkpoint modulator
polypeptide(s); b) a mixture of the therapeutic vaccine with
vector(s) encoding of the one or more immune checkpoint
modulator(s) or c) a specific design (e.g. the therapeutic vaccine
encodes both polypeptide(s) of therapeutic interest and the one or
more immune checkpoint modulator(s)). The present invention
encompasses combinations comprising equal molar concentrations of
each entity as well as combinations with very different
concentrations of the different entities. It is appreciated that
optimal concentration of each entity of the combination can be
determined by the artisan skilled in the art. Preferably, the
combination is synergistic providing higher efficacy (e.g. an
increased survival) than each entity alone.
[0036] In the context of the invention, the combination product of
the present invention can be used for prophylaxis (e.g. to reduce
the risk of having a given disease or pathological condition)
and/or therapy (e.g. in a subject diagnosed as having a given
disease or pathological condition). Therapeutic use is
preferred.
[0037] Therapeutic Vaccine
[0038] In one embodiment, the "therapeutic vaccine" as used herein
is a biological product designed to elicit or increase immunity to
a particular target (e.g. cancerous or infected cells, etc.)
through the presence or expression of a polypeptide of interest
which is expected to cause a beneficial effect on the course or a
symptom of the disease or pathological condition when administered
appropriately to a subject.
[0039] Several types of therapeutic vaccines can be used in the
context of the invention including, but not limited to, cell-based
vaccines, peptide or polypeptide-based vaccines and vector-based
vaccines.
[0040] Representative examples of cell based vaccines can be
obtained for example [0041] From stem cells (such as SL-701
developed by Stemline Therapeutics for treating glioblastoma);
[0042] From specialized cells such as immune cells that are
reprogrammed in vitro to attack cancer cells (e.g. the Dendritic
cell vaccine developed by Immunocellular Therapeutics targeting six
tumor-associated antigens (TAA) involved in glioblastoma, and
Provenge vaccine approved for treating advanced prostate cancer);
[0043] From patient's cancer cells altered in lab to make them more
amenable to be attacked by the patient's immune system; or [0044]
From microorganisms that have been engineered for being avirulent
or attenuated by disabling their virulent properties and optionally
for expressing polypeptides of interest. Well-known examples of
such microorganisms include without limitation bacterium (e.g.
Mycobacterium; Lactobacillus (e.g. Lactococcus lactis); Listeria
(e.g. Listeria monocytogenes) Salmonella and Pseudomona), and yeast
(e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia
pastoris). Representative examples of bacterium and yeast
therapeutic vaccines include Mycobacterium bovis BCG and
Tarmogens.sup.R developed by GlobeImmune made from
genetically-modified yeast that express one or more
disease-associated antigens.
[0045] Typically, peptide or polypeptide vaccine includes antigenic
peptide(s)/polypeptide(s) optionally mixed to an adjuvant. One may
cite for illustrative purpose Newax E75 developed by Galena and
Genentech for breast cancer.
[0046] Vector-based vaccines are preferred in the context of the
invention. The term "vector" as used herein refers to a vehicle,
preferably a nucleic acid molecule or a viral particle that
contains the elements necessary to allow delivery, propagation
and/or expression of biological molecules within a host cell or
subject. For the purpose of the invention, the vectors may be of
naturally occurring genetic sources, synthetic or artificial, or
some combination of natural and artificial genetic elements. This
term encompasses extrachromosomal vectors (e.g. that remain in the
cell cytosol or nucleus) and integration vectors (e.g. designed to
integrate into the cell genome) as well as cloning and expression
vectors.
[0047] In one embodiment, the therapeutic vaccine comprises a
recombinant plasmid or viral vector. A "plasmid vector" as used
herein refers to a replicable DNA construct. Usually plasmid
vectors contain selectable marker genes that allow host cells
carrying the plasmid vector to be selected for or against in the
presence of a corresponding selective drug. A variety of positive
and negative selectable marker genes are known in the art. By way
of illustration, an antibiotic resistance gene can be used as a
positive selectable marker gene that allows selection of the
plasmid-containing cells in the presence of the corresponding
antibiotic. Suitable plasmid vectors include, without limitation,
pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 (Seed, 1987,
Nature 329: 840), pMT2PC (Kaufman et al., 1987, EMBO J. 6: 187-95),
pVAX (Invitrogen) and pgWiz (Gene Therapy System Inc; Himoudi et
al., 2002, J. Virol. 76: 12735-46).
[0048] The term "viral vector" or "virus" or "virions" or
"therapeutic virus" as used herein refers to a nucleic acid vector
that includes at least one element of a virus genome allowing
packaging into a viral particle. In the context of the present
invention, these terms have to be understood broadly as including
nucleic acid vector (e.g. vector DNA) as well as viral particles
generated thereof, and especially infectious viral particles. The
term "infectious" refers to the ability of a viral vector to infect
and enter into a host cell or subject.
[0049] Viral vectors can be replication-competent or selective
(e.g. engineered to replicate better or selectively in specific
host cells), or can be genetically disabled so as to be
replication-defective or replication-impaired. In a preferred
embodiment, the therapeutic vaccine comprised in the combination of
the invention is a replication-defective or replication-impaired
viral vector which means that it cannot replicate to any
significant extent in normal cells, especially in normal human
cells. The impairment or defectiveness of replication functions can
be evaluated by conventional means, such as by measuring DNA
synthesis and/or viral titer in non-permissive cells. The viral
vector can be rendered replication-defective by partial or total
deletion or inactivation of regions critical to viral replication.
Such replication-defective or impaired viral vectors typically
require for propagation, permissive cell lines which bring up or
complement the missing/impaired functions.
[0050] Viral vectors can be engineered from a variety of viruses
and in particular from the group of viruses consisting of
adenovirus, adenovirus-associated virus (AAV), poxvirus, herpes
virus, measles virus, foamy virus, alphavirus, vesicular stomatis
virus, Newcastle disease virus, picorna virus, Sindi virus, etc).
One may use either parental strains as well as derivatives thereof
(i.e. a virus that is modified compared to a parental strain of
said virus, e.g. by truncation, deletion, substitution, and/or
insertion of one or more nucleotide(s) contiguous or not within the
viral genome). Modification(s) can be within endogenous viral genes
(e.g. coding and/or regulatory sequences) and/or within intergenic
regions. Moreover, modification(s) can be silent or not (e.g.
resulting in a modified viral gene product). Modification(s) can be
made in a number of ways known to those skilled in the art using
conventional molecular biology techniques.
[0051] Preferably, the modifications encompassed by the present
invention affect, for example, virulence, toxicity, or
pathogenicity of the virus compared to a virus without such
modification, but do not completely inhibit infection and
production of new virus at least in permissive cells. Said
modification(s) preferably lead(s) to the synthesis of a defective
protein (or lack of synthesis) so as to be unable to ensure the
activity of the protein produced under normal conditions by the
unmodified gene. Exemplary modifications are disclosed in the
literature with a specific preference for those altering viral
genes involved in DNA metabolism, host virulence and IFN pathway
(see e.g. Guse et al., 2011, Expert Opinion Biol. Ther.
11(5):595-608). Other suitable modifications include the insertion
of exogenous gene(s) (e.g. nucleic acid molecule(s) of interest) as
described hereinafter.
[0052] A particularly suitable viral vector to be comprised in the
therapeutic vaccine in use herein is obtained from a poxvirus. As
used herein the term "poxvirus" refers to a virus belonging to the
Poxviridae family with a preference for the Chordopoxvirinae
subfamily directed to vertebrate host which includes several genus
such as Orthopoxvirus, Capripoxvirus, Avipoxvirus, Parapoxvirus,
Leporipoxvirus and Suipoxvirus. Orthopoxviruses are preferred in
the context of the present invention as well as the Avipoxviruses
including Canarypoxvirus (e.g. ALVAC) and Fowlpoxvirus (e.g. the
FP9 vector).
[0053] In a preferred embodiment, the therapeutic vaccine comprises
a poxviral vector belonging to the Orthopoxvirus genus and even
more preferably to the vaccinia virus (VV) species. Vaccinia
viruses are large, complex, enveloped viruses with a linear,
double-stranded DNA genome of approximately 200 kb in length which
encodes numerous viral enzymes and factors that enable the virus to
replicate independently from the host cell machinery. Two distinct
infectious viral particles exist, the intracellular IMV (for
intracellular mature virion) surrounded by a single lipid envelop
that remains in the cytosol of infected cells until lysis and the
double enveloped EEV (for extracellular enveloped virion) that buds
out from the infected cell. Any vaccinia virus strain can be used
in the context of the present invention including, without
limitation, Western Reserve (WR), Copenhagen (Cop), Lister, LIVP,
Wyeth, Tashkent, Tian Tan, Brighton, Ankara, MVA (Modified vaccinia
virus Ankara), LC16M8, LC16M0 strains, etc. and any derivative
thereof.
[0054] Engineered poxviruses can be used with modifications aimed
at improving safety (e.g. increased attenuation) and/or efficacy
(e.g. improved selectivity for cancer cells and/or decreasing
toxicity in healthy cells) of the resulting virus. One may cite
more particularly defective modifications within the thymidine
kinase (J2R; see Weir and Moss, 1983, Genbank accession number
AAA48082), the deoxyuridine triphosphatase (F2L), the viral
hemagglutinin (A56R); the small (F4L) and/or the large (I4L)
subunit of the ribonucleotide reductase, the serine protease
inhibitor (B13R/B14R) and the complement 4b binding protein (C3L).
Representative examples of suitable VV for use in this invention
include NYVAC (U.S. Pat. No. 5,494,807) as well as TK-defective,
TK- and F2L-defective (WO2009/065547) and TK- and I4L-defective VV
(WO2009/065546). The gene nomenclature used herein is that of
Copenhagen Vaccinia strain. It is also used herein for the
homologous genes of other poxviridae unless otherwise indicated.
However, gene nomenclature may be different according to the pox
strain but correspondence between Copenhagen and other vaccinia
strains are generally available in the literature.
[0055] Sequences of the genome of various Poxviridae, are available
in the art in specialized databanks such as Genebank. For example,
the vaccinia virus strains Western Reserve, Copenhagen, Cowpoxvirus
and Canarypoxvirus genomes are available in Genbank under accession
numbers NC_006998, M35027, NC_003663, NC_005309, respectively. A
particularly appropriate viral vector in the context of the present
invention is MVA due to its highly attenuated phenotype (Mayr et
al., 1975, Infection 3: 6-14; Sutter and Moss, 1992, Proc. Natl.
Acad. Sci. USA 89: 10847-51), a more pronounced IFN-type 1 response
generated upon infection compared to non-attenuated vectors and
availability of the sequence of its genome in the literature
(Antoine et al., 1998, Virol. 244: 365-96) and in Genbank (under
accession number U94848).
[0056] Other viral vectors appropriate in the context of the
invention are morbillivirus which can be obtained from the
paramyxoviridae family, with a specific preference for measles
virus. Various attenuated strains are available in the art
(Brandler et al, 2008, CIMID, 31: 271; Singh et al., 1999, J.
Virol. 73(6): 4823), such as and without limitation, the Edmonston
A and B strains (Griffin et al., 2001, Field's in Virology,
1401-1441), the Schwarz strain (Schwarz A, 1962, Am J Dis Child,
103: 216), the S-191 or C-47 strains (Zhang et al., 2009, J Med
Virol. 81 (8): 1477). One may also use recombinant Newcastle
Disease Virus (NDV) (Bukreyev and Collins, 2008, Curr Opin Mol Ther
10: 46-55) with a specific preference for an attenuated strain
thereof such as MTH-68 that was already used in cancer patients
(Csatary et al., 1999, Anti Cancer Res 19: 635-8) and NDV-HUJ,
which showed promising results in glioblastoma patients
(isracast.com Mar. 1, 2006).
[0057] Still another suitable viral vector for use in the present
invention is an adenoviral vector. It can be derived from a variety
of human or animal adenoviruses (e.g. canine, ovine, simian, etc)
and any serotype can be employed. It can also be a chimeric
adenovirus (WO2005/001103). One of skill will recognize that
elements derived from multiple serotypes can be combined in a
single adenovirus.
[0058] Desirably, the adenoviral vector originates from a human Ad,
including those of rare serotypes, or from a primate (e.g.
chimpanzee, gorilla). Representative examples of human adenoviruses
include subgenus C (e.g. Ad2 Ad5 and Ad6), subgenus B (e.g. Ad3,
Ad7, Ad11, Ad14, Ad34, Ad35 and Ad50), subgenus D (e.g. Ad19, Ad24,
Ad26, Ad48 and Ad49) and subgenus E (Ad4). Representative examples
of chimp Ad include without limitation AdCh3 (Peruzzi et al., 2009,
Vaccine 27: 1293-300) and AdCh63 (Dudareva et al, 2009, Vaccine 27:
3501-4) and any of those described in the art (see for example,
WO2010/086189; WO2009/105084; WO2009/073104; WO2009/073103;
WO2005/071093; and WO03/046124). A number of adenoviruses are now
well characterized genetically and biochemically (Hoffmann et al.,
2007, Human Gene Ther. 18: 51-62). An exemplary genome sequence of
human adenovirus type 5 (Ad5) is found in GenBank Accession M73260
and in Chroboczek et al. (1992, Virol. 186: 280-5).
[0059] Preferably, the adenovirus employed in this invention is
replication-defective, e.g. by total or partial deletion of E1
region. An appropriate E1 deletion extends from approximately
positions 459 to 3510 by reference to the sequence of the Ad5
disclosed in the GenBank under the accession number M 73260.
Preferably, the virus retains a functional viral pIX gene. The
adenoviral genome may comprise additional modification(s) (e.g.
deletion of all or part of other essential E2 and/or E4 regions as
described in WO94/28152; Lusky et al, 1998, J. Virol 72: 2022). In
addition, the non-essential E3 region can also be mutated or
deleted.
[0060] More preferably, the adenovirus comprised in the therapeutic
vaccine of the invention is a human adenovirus of serotype 5 (Ad5),
defective for E1 and/or E3 function and comprising a nucleic acid
molecule of interest inserted in the E1 region.
[0061] The present invention also encompasses vectors or viral
particles complexed to lipids or polymers (e.g. polyethylene
glycol) to form particulate structures such as liposomes,
lipoplexes or nanoparticles as well as vectors or viral particles
modified to allow preferential targeting to a specific host cell. A
characteristic feature of targeted vectors is the presence at their
surface of a ligand capable of recognizing and binding to a
cellular and surface-exposed component. Examples of suitable
ligands include antibodies or fragments thereof directed to
cell-specific, tissue-specific and pathogen-associated markers.
Targeting can be carried out by genetically inserting the ligand
into a polypeptide present on the surface of the virus (e.g. in the
adenoviral fiber or in the poxviral p14 IMV exposed polypeptide,
etc.) or by chemically modifying the viral surface envelope.
Furthermore, when using a virus-based therapeutic vaccine, said
virus can be live, attenuated, inactivated or killed.
Polypeptides of Interest
[0062] The therapeutic vaccine comprised in the combination product
of the present invention preferably contains or encodes one or more
polypeptides of therapeutic interest that can compensate for
pathological symptoms, e.g. by acting through toxic effects to
limit or remove harmful cells from the body, by improving immunity
or by reversing immune exhaustion mechanisms. Such polypeptides may
be encoded by native genes or genes obtained from the latter
following suitable sequence modification(s). In the context of the
invention, the polypeptide of interest can be of mammal origin
(e.g. human) or not (e.g. of bacterial or viral origin).
[0063] Advantageously, the therapeutic vaccine in use in the
present invention comprises or encodes one or more polypeptide(s)
selected from the group consisting of suicide gene products,
immunostimulatory polypeptides and antigenic polypeptides.
Preferred antigenic polypeptides for use herein are
tumor-associated antigens and antigens of pathogenic organisms.
Preferably, the polypeptide of interest is not an oncogenic
transcription factor such as p53.
Suicide Genes
[0064] The term "suicide gene" refers to a nucleic acid molecule
coding for a protein (e.g. enzyme) able to convert a precursor of a
drug into a cytotoxic compound. Appropriate suicide genes for use
in this invention are disclosed in the following table with the
corresponding prodrug (or drug precursor) and the active
(cytotoxic) drug.
TABLE-US-00001 TABLE 1 Enzyme Prodrug Active Drug Thymidine
phosphorylase 5-FU 5-FdUMP 5'-DFUR 5-FU Deoxycitidine kinase
Gemcitabine Gemcitabine monophosphate Cytidine deaminase 5'-DFCR
5'-DFUR Cytosine deaminase 5-FC 5-FU Uracil 5-FU 5-FUMP
phosphoribosyltransferase Thymidine phosphorylase 5-FU 5-FdUMP
Thymidine kinase (HSV) Ganciclovir Ganciclovir-triphosphate
nucleotide Nitroreductase CB1954 5-(Aziridin-1-yl)-4-hydroxyl-
amino-2-nitro-benzamide Cytochrome P450 Ifosfamide Isophosphoramide
mustard Cyclophosmamide Phosphoramide mustard Purine-nucleoside
Fludarabine 2-Fluoroadenine phosphorylase Alkaline phosphatase
Etoposide phosphate Etoposide Mitomycin C phosphate Mitomycin C
N-(4-phophonooxy- Doxorubicin phenylacetyl)doxorubicin
Carboxypeptidase Methotrexate-amino acids Methotrexate Penicillin
amidase N-(phenylacetyl) doxorubicin Doxorubicin .beta.-Lactamase
C-DOX Doxorubicin
[0065] Desirably, the therapeutic vaccine comprises or encodes a
polypeptide having at least cytosine deaminase (CDase) activity.
CDase encoding nucleic acid molecule can be obtained from any
prokaryotes and lower eukaryotes such as Saccharomyces cerevisiae
(FCY1 gene), Candida Albicans (FCA1 gene) and Escherichia coli
(codA gene).
[0066] Alternatively or in combination, the therapeutic vaccine
comprises or encodes a polypeptide having uracil phosphoribosyl
transferase (UPRTase) activity. UPRTase-encoding nucleic acid
molecule can be obtained from E. coli (Andersen et al., 1992,
European J. Biochem. 204: 51-56), Lactococcus lactis (Martinussen
et al., 1994, J. Bacteriol. 176: 6457-63), Mycobacterium bovis (Kim
et al., 1997, Biochem. Mol. Biol. Internat. 41: 1117-24), Bacillus
subtilis (Martinussen et al., 1995, J. Bacteriol. 177: 271-4) and
yeast (e.g. S. cerevisiae FUR1 disclosed by Kern et al., 1990, Gene
88: 149-57).
[0067] The nucleotide sequence of such CDase and UPRTase-encoding
nucleic acid molecules and amino acids of the encoded enzyme are
available in specialized data banks (SWISSPROT EMBL, Genbank,
Medline and the like). Functional analogues may also be used. Such
analogues preferably have an amino acid sequence having a degree of
identity of at least 70%, advantageously of at least 80%,
preferably of at least 90%, and most preferably of at least 95%
with the native polypeptide. It is within the reach of the skilled
person to engineer analogs from the published data, and test the
enzymatic activity in an acellular or cellular system according to
conventional techniques (see e.g. EP998568). For illustrative
purposes, suitable functional analogues comprise the N-terminally
truncated FUR1 mutant described in EP998568 (with a deletion of the
35 first residues up to the second Met residue present at position
36 in the native protein) which exhibits a higher UPRTase activity
than that of the native enzyme as well as the FCY1::FUR1 fusions
named FCU1 (amino acid sequence represented in the sequence
identifier SEQ ID NO: 1 of WO2009/065546) and FCU1-8 described in
WO96/16183, EP998568 and WO2005/07857.
Immunostimulatory Polypeptides
[0068] As used herein, the term "immunostimulatory polypeptide"
refers to a polypeptide which has the ability to stimulate the
immune system, in a specific or non-specific way. A vast number of
proteins are known in the art for their ability to exert an
immunostimulatory effect. Examples of suitable immunostimulatory
proteins in the context of the invention include without limitation
cytokines, with a specific preference for interleukins (e.g. IL-2,
IL-6, IL-12, IL-15, IL-24), chemokines (e.g. CXCL10, CXCL9,
CXCL11), interferons (e.g. IFN.alpha., IFN.beta., IFN.gamma.),
tumor necrosis factor (TNF), colony-stimulating factors (e.g.
GM-CSF, C-CSF, M-CSF . . . ), APC (for Antigen Presenting
Cell)-exposed proteins (e.g. B7.1, B7.2 and the like), growth
factors (Transforming Growth Factor TGF, Fibroblast Growth Factor
FGF, Vascular Endothelial Growth Factors VEGF, and the like), major
histocompatibility complex (MHC) antigens of class I or II,
apoptosis inducers or inhibitors (e.g. Bax, Bcl2, BclX . . . ) and
immunotoxins. Preferably, the immunostimulatory protein is an
interleukin or a colony-stimulating factor (e.g. GM-CSF).
Antigenic Polypeptides
[0069] In one embodiment, the therapeutic vaccine contains or
encodes an antigen in connection with the disease to treat. The
term "antigenic" refers to the capacity of eliciting or stimulating
an immune response (e.g. a cell-mediated and/or humoral immunity).
The antigen stimulates the body's immune system to recognize the
target as foreign so that the immune system can more easily
recognize and destroy it when later encounters. The present
invention encompasses native antigenic polypeptides (present in/on
live or dead organisms or cells) as well as modified version
thereof (analogs, fragments) and combination thereof as described
herein.
[0070] In the context of the invention, preferred antigens
contained in or encoded by the therapeutic vaccine are
tumour-specific or tumour-related antigens (i.e. tumor-associated
antigens) as well as antigens form pathogenic organisms such as
virus, bacteria, parasite and the like as well as allergens.
[0071] Viral antigenic polypeptides include for example antigens
from hepatitis viruses A, B, C, D and E, immunodeficiency viruses
(e.g. HIV), herpes viruses, cytomegalovirus, varicella zoster,
papilloma viruses, Epstein Barr virus, influenza viruses,
para-influenza viruses, adenoviruses, coxsakie viruses, picorna
viruses, rotaviruses, respiratory syncytial viruses, pox viruses,
rhinoviruses, rubella virus, papovirus, mumps virus, measles virus.
Some non-limiting examples of HIV antigens include gp120 gp40,
gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef tat, nef.
Some non-limiting examples of human herpes viruses antigens include
gH, gL gM gB gC gK gE or gD or Immediate Early protein such as
ICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2. Some non-limiting
examples of cytomegalovirus antigens include gB. Some non-limiting
examples of derived from Epstein Barr virus (EBV) include gp350.
Some non-limiting examples of Varicella Zoster Virus antigens
include gp1, 11, 111 and IE63. Some non-limiting examples of
hepatitis C virus antigens includes env E1 or E2 protein, core
protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7. Some non-limiting
examples of human papilloma viruses (HPV) antigens include L1, L2,
E1, E2, E3, E4, E5, E6, E7. Antigens derived from other viral
pathogens, such as Respiratory Syncytial virus (e.g. F and G
proteins), parainfluenza virus, measles virus, mumps virus,
flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne
encephalitis virus, Japanese Encephalitis Virus) and Influenza
virus cells (e.g. HA, NP, NA, or M proteins) can also be used in
accordance with the present invention.
[0072] Bacterial antigenic polypeptides include for example
antigens from Mycobacteria causing TB and leprosy, pneumocci,
aerobic gram negative bacilli, mycoplasma, staphyloccocus,
streptococcus, salmonellae, chlamydiae, neisseriae and the
like.
[0073] Parasitic antigenic polypeptides include for example
antigens from malaria, leishmaniasis, trypanosomiasis,
toxoplasmosis, schistosomiasis and filariasis.
[0074] Allergenic polypeptides refer to any substance that can
induce an allergic or asthmatic response in a susceptible subject.
Allergens include pollens, insect venoms, animal dander dust,
fungal spores and drugs (e.g. penicillin).
[0075] Tumor-associated antigenic polypeptides (TAA) include
various categories of antigens, e.g. those which are normally
silent (i.e. not expressed) in normal cells, those that are
expressed only at low levels or at certain stages of
differentiation and those that are temporally expressed such as
embryonic and foetal antigens as well as those resulting from
mutation of cellular genes, such as oncogenes (e.g. activated ras
oncogene), proto-oncogenes (e.g. ErbB family), or proteins
resulting from chromosomal translocations. Such tumor-associated
antigens also encompass antigens encoded by pathogenic organisms
that are capable to induce a malignant condition in a subject
(especially chronically infected subject) such as RNA and DNA tumor
viruses (e.g. HPV, HCV, EBV, etc) and bacteria (e.g. Helicobacter
pilori). Some non-limiting examples of tumor-associated antigens
include, without limitation, MART-1/Melan-A, gp100, Dipeptidyl
peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp),
cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/GA733,
Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1
and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its
immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific
membrane antigen (PSMA), T-cell receptor/CD3-zeta chain,
MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,
MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,
MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),
MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),
GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE,
LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family (e.g.
MUC1, MUC16, etc; see e.g. U.S. Pat. No. 6,054,438; WO98/04727; or
WO98/37095), HER2/neu, p21ras, RCAS1, alpha-fetoprotein,
E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, p120ctn,
gp100.sup.Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis
coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75,
GM2 and GD2 gangliosides, Smad family of tumor antigens brain
glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4,
SSX-5, SCP-1 and CT-7, and c-erbB-2 and viral antigens such as the
HPV-16 and HPV-18 E6 and E7 antigens and the EBV-encoded nuclear
antigen (EBNA)-1 as well as markers (beta-galactosidase, luciferase
. . . ).
[0076] The present invention also encompasses therapeutic vaccine
comprising/expressing two or more polypeptides of interest as
described above, e.g. at least two antigens, at least one antigen
and one immunostimulatory polypeptide, at least two antigens and
one immunostimulatory polypeptide, etc.
[0077] A preferred therapeutic vaccine comprised in the combination
product of the invention comprises or encodes one or more
polypeptides of interest selected from the group consisting of:
[0078] The MUC-1 antigen [0079] HPV E6 and E7 antigens, in
particular non-oncogenic variants thereof; [0080] The human IL-2
[0081] The human GM-CSF; [0082] The FCU-1 suicide gene; [0083] HBV
antigens, in particular HBV pol, HBsAg and/or core; [0084]
Mycobacteria (e.g. MtB or M bovis) antigens in particular one or
more selected from the group consisting of RpfB, RpfD, Ag85A,
Ag85B, ESAT6, CFP10, TB10.4, Rv0111, Rv0287, Rv0569, Rv1733,
Rv1813, Rv2029, Rv2626, Rv3407, Rv3477, Rv3478, and [0085] The HCV
NS antigens (e.g. NS3, NS4a, NS4b, NS5a, NS5b).
[0086] The present invention encompasses the use/expression of
native polypeptide(s) of interest as well as analogs thereof (e.g.
fragments thereof such as peptides; and modified ones), especially
when the native polypeptide exerts undesired properties (e.g.
oncogenic or transforming properties, cytotoxicity, etc). For
example, to circumvent oncogenicity of HPV E6 and E7 polypeptides,
one may use or express non oncogenic analogs displaying reduced
capacity to bind p53 and Rb, respectively. Such non oncogenic
analogs are described e.g. in Munger et al. (1989, EMBO J. 8:
4099-105); Crook et al. (1991, Cell 67: 547-56); Heck et al. (1992,
Proc. Natl. Acad. Sci. USA 89: 4442-6); Munger et al. (1991, J.
Virol. 65: 3943-8); Phelps et al. (1992, J. Virol. 66, 2418-27) and
WO99/03885. A non-oncogenic HPV-16 E6 variant which is suitable for
the purpose of the present invention is deleted of one or more
amino acid residues located from approximately position 118 to
approximately position 122 (+1 representing the first methionine
residue of the native HPV-16 E6 polypeptide), with a special
preference for the complete deletion of residues 118 to 122
(CPEEK). A non-oncogenic HPV-16 E7 variant which is suitable for
the purpose of the present invention is deleted of one or more
amino acid residues located from approximately position 21 to
approximately position 26 (+1 representing the first amino acid of
the native HPV-16 E7 polypeptide, with a special preference for the
complete deletion of residues 21 to 26 (DLYCYE). In one embodiment,
it might be advantageous to modify or include additional features
to the polypeptide of interest so as to improve its immunogenic
activity and/or therapeutic activity. For example, it can be useful
to associate (e.g. by mixture, fusion or independent expression) in
a same therapeutic vaccine (in particular a vector-based
therapeutic vaccine): [0087] A nucleotide sequence encoding an
antigen (e.g. a TAA, or an antigen from a pathogenic organism as
previously described), and [0088] A nucleotide sequence encoding an
immunostimulatory polypeptide such a cytokine or an interleukin
(for instance IL-2; tumour necrosis factor (TNF); interferon (IFN);
colony stimulating factor (CSF), or granulocyte-macrophage
colony-stimulating factor (GMCSF).
[0089] One may also use or express with the polypeptide of interest
one or more peptides or polypeptides capable of enhancing
immunogenicity. Such peptides or polypeptides have been described
in the literature and include, without limitation, calreticulin
(Cheng et al., 2001, J. Clin. Invest. 108: 669-78), Mycobacterium
tuberculosis heat shock protein 70 (HSP70) (Chen et al., 2000,
Cancer Res. 60: 1035-42), ubiquitin (Rodriguez et al., 1997, J.
Virol. 71: 8497-503), bacterial toxin such as the translocation
domain of Pseudomonas aeruginosa exotoxin A (ETA(dIII)) (Hung et
al., 2001 Cancer Res. 61: 3698-703) as well as T.sub.H Pan-Dr
epitope (Sidney et al., 1994, Immunity 1: 751), pstS1 GCG epitope
(Vordermeier et al., 1992, Eur. J. Immunol. 22: 2631), tetanus
toxoid P2TT (Panina-Bordignon et al., 1989, Eur. J. Immunol. 19:
2237) and P30TT (Demotz et al., 1993, Eur. J. Immunol. 23: 425)
peptides, and influenza epitope (Lamb et al., 1982, Nature 300: 66;
Rothbard et al., 1989, Int. Immunol. 1: 479).
[0090] Other suitable structural features are those which are
beneficial to the synthesis, processing, stability and solubility
of the polypeptide of interest that is used in or expressed by the
therapeutic vaccine of the invention; e.g. those aimed to modify
potential cleavage sites, potential glycosylation sites and/or
membrane anchorage so as to improve MHC class I and/or MHC class II
presentation. Membrane presentation can be achieved by
incorporating in the polypeptide of interest a membrane-anchoring
sequence and a secretory sequence (i.e. a signal peptide) if the
native polypeptide lacks it. Briefly, signal peptides usually
comprise 15 to 35 essentially hydrophobic amino acids which are
then removed by a specific ER (endoplasmic reticulum)-located
endopeptidase to give the mature polypeptide. Trans-membrane
peptides are also highly hydrophobic in nature and serve to anchor
the polypeptides within cell membrane. The choice of the
trans-membrane and/or signal peptides which can be used in the
context of the present invention is vast. They may be obtained from
cellular or viral polypeptides such as those of immunoglobulins,
tissue plasminogen activator, insulin, rabies glycoprotein, the HIV
virus envelope glycoprotein or the measles virus F protein or may
be synthetic. Preferably, the secretory sequence is inserted at the
N-terminus of the polypeptide downstream of the codon for
initiation of translation and the membrane-anchoring sequence at
the C-terminus, preferably immediately upstream of the stop
codon.
Recombinant Vector
[0091] In a preferred embodiment, the therapeutic vaccine comprises
a recombinant vector engineered to express at least one nucleic
acid molecule encoding a polypeptide of interest as described
herein. It may be easily generated by a number of ways known to
those skilled in the art (e.g. cloning, PCR amplification, DNA
shuffling). For example, such a nucleic acid molecule can be
isolated independently from any available source (e.g. biologic
materials described in the art, cDNA and genomic libraries, viral
genomes or any prior art vector known to include it) using sequence
data available to the skilled person and the sequence information
provided herein, and then suitably cloned by conventional molecular
biology techniques. Alternatively, they can also be generated by
chemical synthesis in automatized process (e.g. assembled from
overlapping synthetic oligonucleotides or synthetic gene).
Preferably, such a nucleic acid molecule of interest is obtained
from cDNA and does not comprise intronic sequences. Modification(s)
can be generated by a number of ways known to those skilled in the
art, such as chemical synthesis, site-directed mutagenesis, PCR
mutagenesis, etc.
[0092] In addition, the nucleic acid molecule for use in this
invention can be optimized for providing high level expression in a
particular host cell or subject. It has been indeed observed that,
the codon usage patterns of organisms are highly non-random and the
use of codons may be markedly different between different hosts. As
the therapeutic gene might be from prokaryote (e.g. bacterial or
viral antigen) or lower eukaryote (e.g. the suicide gene) origin,
it may have an inappropriate codon usage pattern for efficient
expression in higher eukaryotic cells (e.g. human). Typically,
codon optimization is performed by replacing one or more "native"
codon corresponding to a codon infrequently used by one or more
codon encoding the same amino acid which is more frequently used in
the subject to treat. It is not necessary to replace all native
codons corresponding to infrequently used codons since increased
expression can be achieved even with partial replacement.
[0093] Further to optimization of the codon usage, expression can
also be improved through additional modifications of the nucleotide
sequence. For example, the nucleic acid sequence can be modified so
as to prevent clustering of rare, non-optimal codons being present
in concentrated areas and/or to suppress or modify "negative"
sequence elements which are expected to negatively influence
expression levels. Such negative sequence elements include without
limitation the regions having very high (>80%) or very low
(<30%) GC content; AT-rich or GC-rich sequence stretches;
unstable direct or inverted repeat sequences; R A secondary
structures; and/or internal cryptic regulatory elements such as
internal TATA-boxes, chi-sites, ribosome entry sites, and/or
splicing donor/acceptor sites.
[0094] Moreover, when homologous nucleic acid molecules are to be
expressed, such homologous sequences can be degenerated over the
full length nucleic acid molecule or portion(s) thereof so as to
reduce sequence homology. It is indeed advisable to degenerate the
portions of nucleic acid sequences that show a high degree of
sequence identity (e.g. the same antigen obtained from various
serotypes of a given virus such as HPV-16 and HPV-18 E6 and/or E7
antigens; overlapping antigens such as HBV antigens) so as to avoid
homologous recombination problems during production process and the
skilled person is capable of identifying such portions by sequence
alignment.
[0095] The nucleic acid molecule(s) encoding the polypeptide(s) of
interest may be inserted in the vector, e.g. within a viral gene,
an intergenic region, in a non-essential gene or region or in place
of viral sequences. The general conditions for constructing and
producing recombinant poxviruses are well known in the art (see for
example WO2010/130753; WO03/008533; U.S. Pat. No. 6,998,252; U.S.
Pat. No. 5,972,597 and U.S. Pat. No. 6,440,422). The nucleic acid
molecule(s) of interest is/are preferably inserted within the
poxviral genome in a non-essential locus. Thymidine kinase gene is
particularly appropriate for insertion in Copenhagen vaccinia
vectors and deletion II or III for insertion in MVA vector
(WO97/02355; Meyer et al., 1991, J. Gen. Virol. 72: 1031-8). The
general conditions for constructing and producing recombinant
measles viruses are well known in the art. Insertion of the nucleic
acid molecule(s) of interest between P and M genes or between H and
L genes is particularly appropriate. The general conditions for
constructing and producing recombinant adenoviruses are well known
in the art (see e.g. Chartier et al., 1996, J. Virol. 70: 4805-10
and WO96/17070). E1 or E3 region is the preferred site of insertion
for the nucleic acid molecule(s) to be expressed which can be
positioned in sense or antisense orientation relative to the
natural transcriptional direction of the region in question.
[0096] In a particularly preferred embodiment, the therapeutic
vaccine is selected from the group consisting of: [0097] A MVA
virus encoding the MUC-1 TAA and human IL-2 as represented by
TG4010 described in WO92/07000, U.S. Pat. No. 5,861,381 and
Limacher and Quoix (2012, Oncolmmunology 1(5): 791-2); [0098] A MVA
virus encoding a fusion of NS3 and NS4 HCV antigens and NS5b
antigen (as represented by TG4040 described in WO2004/111082);
[0099] A MVA virus encoding membrane anchored HPV-16 non-oncogenic
E6 and E7 antigens and human IL-2 as represented by TG4001
described in WO99/03885; [0100] A MVA virus encoding the FCU1 gene
as represented by TG4023 (WO99/54481); and [0101] A MVA virus
encoding a combination of TB antigens (as described in
WO2014/009438).
Expression of the Nucleic Acid Molecule(s) Encoding the
Polypeptide(s) of Interest
[0102] In accordance with the present invention, the nucleic acid
molecule(s) expressed by the therapeutic vaccine for use in the
invention is/are operably linked to suitable regulatory elements
for expression in a desired host cell or subject.
[0103] As used herein, the term "regulatory elements" or
"regulatory sequence" refers to any element that allows,
contributes or modulates the expression of the nucleic acid
molecule(s) in a given host cell or subject, including replication,
duplication, transcription, splicing, translation, stability and/or
transport of the nucleic acid(s) or its derivative (i.e. m RNA). As
used herein, "operably linked" means that the elements being linked
are arranged so that they function in concert for their intended
purposes. For example, a promoter is operably linked to a nucleic
acid molecule if the promoter effects transcription from the
transcription initiation to the terminator of said nucleic acid
molecule in a permissive host cell.
[0104] It will be appreciated by those skilled in the art that the
choice of the regulatory sequences can depend on factors such as
the nucleic acid molecule(s) itself, the vector from which it is
expressed, the level of expression desired, etc. The promoter is of
special importance. In the context of the invention, it can be
constitutive directing expression of the nucleic acid molecule(s)
in many types of cells or specific to certain types of cells or
tissues or regulated in response to specific events or exogenous
factors (e.g. by temperature, nutrient additive, hormone, etc) or
according to the phase of a viral cycle (e.g. late or early). One
may also use promoters that are repressed during the production
step in response to specific events or exogenous factors, in order
to optimize production of the therapeutic vaccine and circumvent
potential toxicity of the expressed polypeptide(s).
[0105] Suitable constitutive promoters for expression in
recombinant adenovirus and plasmid vectors include, but are not
limited to, the cytomegalovirus (CMV) immediate early promoter
(U.S. Pat. No. 5,168,062), the RSV promoter, the adenovirus major
late promoter, the phosphoglycero kinase (PGK) promoter (Adra et
al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of
herpes simplex virus (HSV)-1 and the T7 polymerase promoter
(WO98/10088). Vaccinia virus promoters are particularly adapted for
expression in recombinant poxviruses. Representative examples
include without limitation the vaccinia 7.5K, HSR, 11K7.5 (Erbs et
al., 2008, Cancer Gene Ther. 15(1): 18-28), TK, pB2R, p28, p11 and
K1L promoter, as well as synthetic promoters such as those
described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7;
Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and
Boyle, 1990, Virology 179: 151-8) as well as early/late chimeric
promoters. Promoters suitable for measles viruses include without
limitation any promoter directing expression of measles
transcription units (Brandler and Tangy, 2008, CIMID 31: 271).
[0106] Those skilled in the art will appreciate that the regulatory
elements controlling the expression of the nucleic acid molecule(s)
of interest may further comprise additional elements for proper
initiation, regulation and/or termination of transcription (e.g.
polyA transcription termination sequences), mRNA transport (e.g.
nuclear localization signal sequences), processing (e.g. splicing
signals), and stability (e.g. introns and non-coding 5' and 3'
sequences), translation (e.g. an initiator Met, tripartite leader
sequences, IRES ribosome binding sites, signal peptides, etc.) and
purification steps (e.g. a tag). In a preferred embodiment, the
therapeutic vaccine for use in the invention comprises a MVA vector
which contains inserted into its genome (preferably in deletion II)
a nucleic acid molecule encoding a tumor-associated antigen such as
MUC-1 (preferably under the transcriptional control of the
early/late vaccinia pH5R promoter) and a nucleic acid molecule
encoding an immunostimulatory polypeptide such as the human IL-2
(preferably under the transcriptional control of the early/late
vaccinia p7.5 promoter). More preferably, the encoded MUC1 antigen
comprises an amino acid sequence that is at least 90% identical to
SEQ ID NO: 1. Even more preferably, the MUC1 antigen is encoded by
a nucleotide sequence that is at least 80% identical to SEQ ID NO:
2.
Production of Virus-Based Therapeutic Vaccine
[0107] In a preferred embodiment, the therapeutic vaccine present
in the combination product of the present invention is a viral
vector. Typically, viral vectors are produced into a suitable host
cell line using conventional techniques including a) preparing a
producer (e.g. permissive) host cell, b) transfecting or infecting
the prepared producer host cells, c) culturing the transfected or
infected host cell under suitable conditions so as to allow the
production of the vector (e.g. infectious viral particles), d)
recovering the produced vector from the culture of said cell and
optionally e) purifying said recovered vector.
[0108] As used herein, the term "host cell" should be understood
broadly without any limitation concerning particular organization
in tissue, organ, or isolated cells. Such cells may be of a unique
type of cells or a group of different types of cells such as
cultured cell lines, primary cells and dividing cells. In the
context of the invention, the term "host cells" include prokaryotic
cells, lower eukaryotic cells such as yeast, and other eukaryotic
cells such as insect cells, plant and mammalian (e.g. human or
non-human) cells as well as producer cells capable of producing the
plasmid or virus-based therapeutic vaccine and/or the immune
checkpoint modulator(s) for use in the invention. This term also
includes cells which can be or has been the recipient of the
combination product described herein as well as progeny of such
cells.
[0109] In step a), suitable producer cells depend on the type of
viral vector to be amplified. Replication-defective recombinant
adenoviruses are typically propagated and produced in a cell that
supplies in trans the adenoviral protein(s) encoded by those genes
that have been deleted or inactivated in the replication-defective
adenovirus, thus allowing the virus to replicate in the cell.
Suitable cell lines for complementing E1-deleted adenoviruses
include the HEK-293 cells (Graham et al., 1997, J. Gen. Virol. 36:
59-72) as well as the HER-96 and PER-C6 cells (e.g. Fallaux et al.,
1998, Human Gene Ther. 9: 1909-1917; WO97/00326) and E1 A549 (Imler
et al., 1996, Gene Ther. 3: 75-84) or any derivative of these cell
lines. But any other cell line described in the art can also be
used in the context of the present invention, especially cell lines
approved for producing products for human use. The infectious
adenoviral particles may be recovered from the culture supernatant
and/or from the cells after lysis. They can be further purified
according to standard techniques (ultracentrifugation in a cesium
chloride gradient, chromatography, etc. as described for example in
WO96/27677, WO98/00524, WO98/22588, WO98/26048, WO00/40702,
EP1016711 and WO00/50573).
[0110] MVA is strictly host-restricted and is typically amplified
on avian cells, either primary avian cells (such as chicken embryo
fibroblasts (CEF) prepared from chicken embryos obtained from
fertilized eggs) or immortalized avian cell lines, and in
particular a Cairina moschata cell line immortalized with a duck
TERT gene (see e.g. WO2010/130756 and WO2012/001075); avian cell
line produced according to the process described in WO2007/077256
or WO2009/004016; avian cell line immortalized with a combination
of viral and/or cellular genes (see e.g. WO2005/042728); a
spontaneously immortalized cell (e.g. the chicken DF1 cell line
disclosed in U.S. Pat. No. 5,879,924); or immortalized cells which
derive from embryonic cells by progressive severance from growth
factors and feeder layer (e.g. Ebx chicken cell lines disclosed in
WO2005/007840 and WO2008/129058).
[0111] For other vaccinia virus or other poxvirus strains, in
addition to avian primary cells (such as CEF) and avian cell lines,
many other non-avian cell lines are available for production,
including human cell lines such as HeLa (ATCC-CRM-CCL-2.TM. or
ATCC-CCL-2.2.TM.), MRC-5, HEK-293; hamster cell lines such as
BHK-21 (ATCC CCL-10), and Vero cells. In a preferred embodiment,
vaccinia virus other than MVA is amplified in HeLa cells (see e.g.
WO2010/130753).
[0112] Producer cells are preferably cultivated in a medium free
from animal- or human-derived products, using a chemically defined
medium with no product of animal or human origin. In particular,
while growth factors may be present, they are preferably
recombinantly produced and not purified from animal material. An
appropriate animal-free medium may be easily selected by those
skilled in the art depending on selected producer cells. Such media
are commercially available. In particular, when CEFs are used as
producer cells, they may be cultivated in VP-SFM cell culture
medium (Invitrogen). Producer cells are preferably cultivated at a
temperature comprised between +30.degree. C. and +38.degree. C.
(more preferably at about +37.degree. C.) for between 1 and 8 days
(preferably for 1 to 5 days for CEF and 2 to 7 days for
immortalized cells) before infection. If needed, several passages
of 1 to 8 days may be made in order to increase the total number of
cells.
[0113] In step b), producer cells are infected by the viral vector
under appropriate conditions (in particular using an appropriate
multiplicity of infection (MOI) to permit productive infection of
producer cells. In particular, when the therapeutic vaccine is
based on MVA and is amplified using CEF, it may be seeded in the
cell culture vessel containing CEFs at a MOI which is preferably
comprised between 0.001 and 0.1 (more preferably about 0.05).
Infection step is also preferably performed in a medium (which may
be the same as or different from the medium used for culture of
producer cells) free from animal- or human-derived products, using
a chemically defined medium with no product of animal or human
origin.
[0114] In step c), infected producer cells are then cultured under
appropriate conditions well known to those skilled in the art until
progeny viral vector (e.g. infectious virus particles) is produced.
Culture of infected producer cells is also preferably performed in
a medium (which may be the same as or different from the medium
used for culture of producer cells and/or for infection step) free
from animal- or human-derived products (using a chemically defined
medium with no product of animal or human origin) at a temperature
between +30.degree. C. and +37.degree. C., for 1 to 5 days.
[0115] In step d), the viral vector produced in step c) is
collected from the culture supernatant and/or the producer cells.
Recovery from producer cells (and optionally also from culture
supernatant), may require a step allowing the disruption of the
producer cell membrane to allow the liberation of the vector from
producer cells. The disruption of the producer cell membrane can be
induced by various techniques well known to those skilled in the
art, including but not limited to: freeze/thaw, hypotonic lysis,
sonication, microfluidization, or high speed homogenization.
[0116] Viral vectors may then be further purified, using
purification steps well known in the art. Various purification
steps can be envisaged, including clarification, enzymatic
treatment (e.g. endonuclease, protease, etc), chromatographic and
filtration steps. Appropriate methods are described in the art
(e.g. WO2007/147528; WO2008/138533, WO2009/100521, WO2010/130753,
WO2013/022764).
Immune Checkpoint Modulator(s)
[0117] Immune checkpoints and modulators thereof as well as methods
of using such compounds are described in the literature. "Immune
checkpoint" proteins are directly or indirectly involved in an
immune pathway that under normal physiological conditions is
crucial for preventing uncontrolled immune reactions and thus for
the maintenance of self-tolerance and/or tissue protection. But
under pathological conditions, they play a critical role in T cell
exhaustion.
[0118] The one or more immune checkpoint modulator(s) in use herein
may independently act at any step of the T cell-mediated immunity
including clonal selection of antigen-specific cells, T cell
activation, proliferation, trafficking to sites of antigen and
inflammation, execution of direct effector function and signaling
through cytokines and membrane ligands. Each of these steps is
regulated by counterbalancing stimulatory and inhibitory signals
that in fine tune the response. In the context of the present
invention, the term encompasses (i) immune checkpoint modulator(s)
capable of down-regulating at least partially the function of an
inhibitory immune checkpoint (e.g. by direct binding or inhibition
of a ligand binding to said targeted immune checkpoint) so as to
exert an antagonist function and, thus antagonize an immune
checkpoint-mediated inhibitory signal as well as (ii) immune
checkpoint modulator(s) capable of up-regulating at least partially
the function of a stimulatory immune checkpoint so as to exert an
agonist function and, thus, amplify an immune checkpoint-mediated
stimulatory signal.
[0119] The one or more immune checkpoint modulator(s) in use herein
may independently be a polypeptide or a nucleic acid molecule; with
a specific preference for peptide ligands, soluble domains of
natural receptors, RNAi, antisense molecules, antibodies and
protein scaffolds.
[0120] In a preferred embodiment, the immune checkpoint modulator
is an antibody. In the context of the invention, "antibody" ("Ab")
is used in the broadest sense and encompasses naturally occurring
and engineered by man as well as full length antibodies or
functional fragments or analogs thereof that are capable of binding
the target immune checkpoint or epitope (thus retaining the
target-binding portion). The antibody in use in the invention can
be of any origin, e.g. human, humanized, animal (e.g. rodent or
camelid antibody) or chimeric. It may be of any isotype (e.g. IgG1,
IgG2, IgG3, IgG4, IgM, etc.). In addition, it may be glycosylated
or non-glycosylated. The term antibody also includes bispecific or
multispecific antibodies so long as they exhibit the binding
specificity described herein.
[0121] For illustrative purposes, full length antibodies are
glycoproteins comprising at least two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. Each heavy
chain is comprised of a heavy chain variable region (VH) and a
heavy chain constant region which is made of three CH1, CH2 and CH3
domains (optionally with a hinge between CH1 and CH2). Each light
chain is comprised of a light chain variable region (VL) and a
light chain constant region which comprises one CL domain. The VH
and VL regions comprise hypervariable regions, named
complementarity determining regions (CDR), and interspersed with
more conserved regions named framework regions (FR). Each VH and VL
is composed of three CDRs and four FRs in the following order:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and
light chains are determinant for the binding specificity.
[0122] As used herein, a "humanized antibody" refers to a non-human
(e.g. murine, camel, rat, etc) antibody whose protein sequence has
been modified to increase its similarity to a human antibody (i.e.
produced naturally in humans). The process of humanization is well
known in the art (see e.g. Pestra et al., 1997, Cancer Res. 57(20):
4593-9; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; U.S. Pat.
No. 6,180,370; WO2012/110360). For example, a monoclonal antibody
developed for human use can be humanized by substituting one or
more residue of the FR regions to look like human immunoglobulin
sequence whereas the vast majority of the residues of the variable
regions (especially the CDRs) are not modified and correspond to
those of a non-human immunoglobulin. For general guidance, the
number of these amino acid substitutions in the FR regions is
typically no more than 20 in each variable region VH or VL.
[0123] As used herein, a "chimeric antibody" refers to an antibody
comprising one or more element(s) of one species and one or more
element(s) of another species, for example, a non-human antibody
comprising at least a portion of a constant region (Fc) of a human
immunoglobulin.
[0124] Antibody fragments can be engineered for use in the
combination of the invention. Representative examples include
without limitation Fab, Fab', F(ab')2, dAb, Fd, Fv, scFv, di-scFv,
diabody and any other artificial antibody. More specifically:
[0125] (i) a Fab fragment is represented by a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; [0126] (ii) a F(ab')2
fragment is represented by a bivalent fragment comprising two Fab
fragments linked by at least one disulfide bridge at the hinge
region; [0127] (iii) a Fd fragment consists of the VH and CH1
domains; [0128] (iv) a Fv fragment consists of the VL and VH
domains of a single arm of an antibody, [0129] (v) a dAb fragment
consists of a single variable domain fragment (VH or VL domain);
[0130] (vi) a single chain Fv (scFv) comprises the two domains of a
Fv fragment, VL and VH, that are fused together, optionally with a
linker to make a single protein chain (see e.g. Bird et al., 1988,
Science 242: 423-6; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85: 5879-83; U.S. Pat. No. 4,946,778; U.S. Pat. No. 5,258,498); and
[0131] (vii) any other artificial antibody.
[0132] Methods for preparing antibodies, fragments and analogs
thereof are known in the art (see e.g. Harlow and Lane, 1988,
Antibodies--A laboratory manual; Cold Spring Harbor Laboratory,
Cold Spring Harbor N.Y.). In one embodiment, such an antibody can
be generated a host animal with the targeted immune checkpoint
modulator. Alternatively, it can be produced from hybridomas (see
e.g. Kohler and Milstein, 1975, Nature 256: 495-7; Cote et al.,
1983, Proc. Natl. Acad. Sci. USA 80: 2026-30; Cole et al. in
Monoclonal antibodies and Cancer Therapy; Alan Liss pp77-96),
recombinant techniques (e.g. using phage display methods), peptide
synthesis and enzymatic cleavage. Antibody fragments can be
produced by recombinant technique as described herein. They may
also be produced by proteolytic cleavage with enzymes such as
papain to produce Fab fragments or pepsin to produce F(ab')2
fragments as described in the literature (see e.g. Wahl et al.,
1983, J. Nucl. Med. 24: 316-25). Analogs (or fragment thereof) can
be generated by conventional molecular biology methods (PCR,
mutagenesis techniques). If needed, such fragments and analogs may
be screened for functionality in the same manner as intact
antibodies (e.g. by standard ELISA assay).
[0133] In a preferred embodiment, at least one of the one or more
immune checkpoint modulator(s) for use in the present invention is
a monoclonal antibody, with a specific preference for a human (in
which both the framework regions are derived from human germline
immunoglobin sequences) or a humanized antibody according to
well-known humanization process.
[0134] Desirably, the one or more immune checkpoint modulator(s) in
use in the present invention antagonizes at least partially (e.g.
more than 50%) the activity of inhibitory immune checkpoint(s), in
particular those mediated by any of the following PD-1, PD-L1,
PD-L2, LAG3, Tim3, BTLA, SLAM, 2B4, CD160, KLRG-1 and CTLA4, with a
specific preference for a human or humanized monoclonal antibody
that specifically binds to any of such target proteins. The term
"specifically binds to" refers to the capacity to a binding
specificity and affinity for a particular target or epitope even in
the presence of a heterogeneous population of other proteins and
biologics. Thus, under designated assay conditions, the antibody in
use in the invention binds preferentially to its target and does
not bind in a significant amount to other components present in a
test sample or subject. Preferably, such an antibody shows high
affinity binding to its target with an equilibrium dissociation
constant equal or below 1.times.10.sup.-6M (e.g. at least
0.5.times.10.sup.-6, 1.times.10.sup.-7, 1.times.10.sup.-8,
1.times.10.sup.-9, 1.times.10.sup.-10, etc). Alternatively, the one
or more immune checkpoint modulator(s) in use in the present
invention exerts an agonist function in the sense that it is
capable of stimulating or reinforcing stimulatory signals, in
particular those mediated by CD28 with a specific preference for
any of ICOS, CD137 (4-1BB), OX40, CD27, CD40 and GITR immune
checkpoints. Standard assays to evaluate the binding ability of the
antibodies toward immune checkpoints are known in the art,
including for example, ELISAs, Western blots, RIAs and flow
cytometry. The binding kinetics (e.g., binding affinity) of the
antibodies also can be assessed by standard assays known in the
art, such as by Biacore analysis.
[0135] In a preferred embodiment, at least one of the one or more
checkpoint modulator(s) for use in this invention is an antibody
capable of antagonizing at least partially the protein Programmed
Death 1 (PD-1), and especially an antibody that specifically binds
to human PD-1. PD-1 is part of the immunoglobulin (Ig) gene
superfamily and a member of the CD28 family. It is a 55 kDa type 1
transmembrane protein expressed on antigen-experienced cells (e.g.
activated B cells, T cells, and myeloid cells) (Agata et al., 1996,
Int. Immunol. 8: 765-72; Okazaki et al., 2002, Curr. Opin. Immunol.
14: 391779-82; Bennett et al., 2003, J. Immunol 170: 711-8). In
normal context, it acts by limiting the activity of T cells at the
time of inflammatory response, thereby protecting normal tissues
from destruction (Topalian, 2012, Curr. Opin. Immunol. 24: 207-12).
Two ligands have been identified for PD-1, respectively PD-L1
(programmed death ligand 1) and PD-L2 (programmed death ligand 2)
(Freeman et al., 2000, J. Exp. Med. 192: 1027-34; Carter et al.,
2002, Eur. J. Immunol. 32: 634-43). PD-L1 was identified in 20-50%
of human cancers (Dong et al., 2002, Nat. Med. 8: 787-9). The
interaction between PD-1 and PD-L1 resulted in a decrease in tumor
infiltrating lymphocytes, a decrease in T-cell receptor mediated
proliferation, and immune evasion by the cancerous cells (Dong et
al., 2003, J. Mol. Med. 81: 281-7; Blank et al., 2005, Cancer
Immunol. Immunother. 54: 307-314). The complete nucleotide and
amino acid PD-1 sequences can be found under GenBank Accession No
U64863 and NP_005009.2. A number of anti PD1 antibodies are
available in the art (see e.g. those described in WO2004/004771;
WO2004/056875; WO2006/121168; WO2008/156712; WO2009/014708;
WO2009/114335; WO2013/043569; and WO2014/047350). Preferred anti
PD-1 antibodies in the context of this invention are FDA approved
or under advanced clinical development and one may use in
particular an anti-PD-1 antibody selected from the group consisting
of Nivolumab (also termed BMS-936558 under development by Bristol
Myer Squibb), Lanbrolizumab (also termed MK-3475 under development
by Merck), and Pidilizumab (also termed CT-011 under development by
CureTech).
[0136] Another preferred example of immune checkpoint modulator is
represented by a modulator capable of antagonizing at least
partially the PD-1 ligand termed PD-L1, and especially an antibody
that recognizes human PD-L1. A number of anti PD-L1 antibodies are
available in the art (see e.g. those described in EP1907000).
Preferred anti PD-L1 antibodies are FDA approved or under advanced
clinical development (e.g. MPDL3280A under development by
Genentech/Roche and BMS-936559 under development by Bristol Myer
Squibb).
[0137] Still another preferred example of immune checkpoint
modulator is represented by a modulator capable of antagonizing at
least partially the CTLA-4 protein, and especially an antibody that
recognizes human CTLA-4. CTLA4 (for cytotoxic
T-lymphocyte-associated antigen 4) also known as CD152 was
identified in 1987 (Brunet et al., 1987, Nature 328: 267-70) and is
encoded by the CTLA4 gene (Dariavach et al., Eur. J. Immunol. 18:
1901-5). It is expressed on the surface of T cells where it
primarily regulates the amplitude of the early stages of T cell
activation. Recent work has suggested that CTLA-4 may function in
vivo by capturing and removing B7-1 and B7-2 from the membranes of
antigen-presenting cells, thus making these unavailable for
triggering of CD28 (Qureshi et al., Science, 2011, 332: 600-3). The
complete CTLA-4 nucleic acid sequence can be found under GenBank
Accession No LI 5006. A number of anti CTLA-4 antibodies are
available in the art (see e.g. those described in U.S. Pat. No.
8,491,895). Preferred anti CTLA-4 antibodies in the context of this
invention are FDA approved or under advanced clinical development.
One may cite more particularly ipilimumab marketed by Bristol Myer
Squibb as Yervoy (see e.g. U.S. Pat. No. 6,984,720; U.S. Pat. No.
8,017,114), tremelimumab under development by Pfizer (see e.g. U.S.
Pat. No. 7,109,003 and U.S. Pat. No. 8,143,379) and single chain
anti-CTLA4 antibodies (see e.g. WO97/20574 and WO2007/123737).
[0138] Immune checkpoint modulator for antagonizing the TIM3
receptor may also be used in the combination product of the present
invention (see e.g. Ngiow et al., 2011, Cancer Res. 71: 3540-51;
US2012-0189617).
[0139] Another Immune checkpoint modulator for antagonizing the
LAG3 receptor may also be used in the combination of the present
invention (see e.g. Toni-Jun et al., 2011, 2014, ACCR poster LB266;
Woo et al., 2012, Cancer Res. 72: 917-27).
[0140] Still another example of immune checkpoint modulator is
represented by an OX40 agonist such as agonist ligand of OX40
(OX40L) (see e.g. U.S. Pat. No. 5,457,035, U.S. Pat. No. 7,622,444;
WO03/082919) or an antibody directed to the OX40 receptor (see e.g.
U.S. Pat. No. 7,291,331 and WO03/106498).
[0141] Other examples of immune checkpoint modulators are
represented by anti-KIR or anti-CD96 antibody targeting the
inhibitory receptors harboured by CD8+ T cells and NK cells.
[0142] The present invention encompasses a combination comprising
more than one immune checkpoint modulator(s). A preferred example
includes without limitation using an anti-CTLA-4 antibody with an
anti-PD-1 or an anti-PD-L1 antibody in combination with the
therapeutic vaccine described herein.
[0143] Nucleic acid molecules encoding the relevant portion(s) of
the desired immune checkpoint modulator can be obtained by standard
molecular biology techniques using sequence data accessible in the
art and the information provided herein. For example, cDNAs
encoding the light and heavy chains of the antibody or their CDRs
can be isolated from the producing hybridoma, immunoglobulin gene
libraries or any available source.
[0144] In one embodiment, the one or more immune checkpoint
modulator(s) for use in this invention can be comprised in the
therapeutic vaccine described herein. For example, the encoding
nucleic acid molecule can be inserted in a vector-based therapeutic
vaccine (e.g. in an antigen-encoding viral vector). In this
context, the nucleic acid molecules encoding the polypeptide(s) of
interest and the immune checkpoint modulator(s) are preferably
expressed independently using distinct regulatory elements.
Alternatively, the one or more immune checkpoint modulator(s) for
use in this invention can be expressed from an independent vector
system such as one of those described herein in connection with the
therapeutic vaccine for separate or concomitant administration to
the subject in need thereof.
[0145] Still alternatively, the one or more immune checkpoint
modulator(s) for use in this invention can be produced by
recombinant means using suitable expression vectors and host cells
for administration as recombinant polypeptide to the subject in
need thereof.
Production of Immune Checkpoint Modulator
[0146] Insertion into the expression vector can be performed by
routine molecular biology, e.g. as described in Sambrook et al.
(2001, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor
Laboratory). Insertion into a virus-based therapeutic vaccine can
be performed through homologous recombination as described in
Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al.
(2002, Cancer Gene Ther. 9: 470-7).
[0147] A variety of host-vector systems may be used or constructed
to express the one or more immune checkpoint modulator(s) for use
in the present invention, including prokaryotic organisms such as
bacteria (e.g. E. coli or Bacillus subtilis); yeast (e.g.
Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris);
insect cell systems (e.g. Sf 9 cells and baculovirus); plant cell
systems (e.g. cauliflower mosaic virus CaMV; tobacco mosaic virus
TMV) and mammalian cell systems (e.g. cultured cells). Typically,
such vectors are commercially available (e.g. in Invitrogen,
Stratagene, Amersham Biosciences, Promega, etc.) or available from
depositary institutions such as the American Type Culture
Collection (ATCC, Rockville, Md.) or have been the subject of
numerous publications describing their sequence, organization and
methods of producing, allowing the artisan to apply them. For
general purposes, such vectors usually comprise one or more
element(s) enabling maintenance, propagation or expression of the
nucleic acid molecule in the host cell. Representative elements
include without limitation marker gene(s) in order to facilitate
identification and isolation of the recombinant host cells (e.g. by
complementation of a cell auxotrophy or by antibiotic resistance),
stabilizing elements (e.g. DAP system as described in U.S. Pat. No.
5,198,343), and integrative elements (e.g. LTR viral sequences and
transposons).
[0148] Suitable plasmid vectors for use in prokaryotic systems
include without limitation pBR322 (Gibco BRL), pUC (Gibco BRL),
pbluescript (Stratagene), p Poly (Lathe et al., 1987, Gene 57:
193-201), pTrc (Amann et al., 1988, Gene 69: 301-15); pET lid
(Studier et al., 1990, Gene Expression Technology: Methods in
Enzymology 185: 60-89); pIN (Inouye et al., 1985, Nucleic Acids
Res. 13: 3101-9; Van Heeke et al., 1989, J. Biol. Chem. 264:
5503-9); and pGEX vectors where the nucleic acid molecule can be
expressed in fusion with glutathione S-transferase (GST) (Amersham
Biosciences Product). Suitable vectors for expression in yeast
(e.g. S. cerevisiae) include, but are not limited to pYepSec1
(Baldari et al., 1987, EMBO J. 6: 229-34), pMFa (Kujan et al.,
1982, Cell 30: 933-43), pJRY88 (Schultz et al., 1987, Gene 54:
113-23), pYES2 (Invitrogen Corporation) and pTEF-MF (Dualsystems
Biotech Product). Plasmid and viral vectors such as those described
herein in connection with the therapeutic vaccine may also be used
to produce the immune checkpoint modulator(s) by recombinant
means.
[0149] Recombinant DNA technologies can also be used to improve
expression of the nucleic acid molecule encoding the immune
checkpoint modulator in the host cell, e.g. by using high-copy
number vectors, substituting or modifying one or more
transcriptional regulatory sequences (e.g. promoter, enhancer and
the like), optimizing the codon usage and suppressing negative
sequences that may destabilize the transcript as described herein
in connection with the nucleic acid molecule(s) encoding the
polypeptide(s) of interest).
[0150] As before, the nucleic acid molecule encoding the immune
checkpoint modulator is in a form suitable for its expression in a
host cell, which means that the nucleic acid molecule is placed
under the control of one or more regulatory sequences, appropriate
to the vector, the host cell and/or the level of expression desired
as described above. Constitutive promoters (e.g. PGK, CMV
promoters, etc), inducible eukaryotic promoters regulated by
exogenously supplied compounds (e.g. TRP and IPTG-inducible pTAC
promoters, zinc-inducible metallothionein (MT) promoter,
dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV)
promoter, tetracycline-repressible and rapamycin-inducible
promoter, etc) can be used as well as any of the promoters
described hereinafter for expression of nucleic acid molecule
encoding the polypeptide of interest.
[0151] The methods for the recombinant production of the immune
checkpoint modulator are conventional in the art. Typically such
methods comprise (a) introducing the expression vector described
herein into a suitable producer cell to produce a transfected or
infected producer cell, (b) culturing in-vitro said transfected or
infected producer cell under conditions suitable for its growth,
(c) recovering the immune checkpoint modulator from the cell
culture, and (d) optionally, purifying the recovered immune
checkpoint modulator.
[0152] In the context of the invention, producer cells include
prokaryotic cells, lower eukaryotic cells such as yeast, and other
eukaryotic cells such as insect cells, plant and mammalian (e.g.
human or non-human) cells. Preferred E. coli cells include without
limitation E. coli BL21 (Amersham Biosciences). Preferred yeast
producer cells include without limitation S. cerevisiae, S. pombe,
Pichia pastoris. Preferred mammalian producer cells include without
limitation BHK-21 (baby hamster kidney), CV-1 (African monkey
kidney cell line), COS (e.g. COS-7) cells, Chinese hamster ovary
(CHO) cells, mouse NIH/3T3 cells, HeLa cells, Vero cells, HEK293
cells and PERC.6 cells as well as the corresponding hybridoma
cells.
[0153] Transfection/infection of producer host cells is
conventional and may use additional compounds so as to improve the
transfection efficiency and/or stability of the vector. These
compounds are widely documented in the literature such as
polycationic polymers (e.g. chitosan, polymethacrylate, PEI, etc),
cationic lipids (e.g. DC-Chol/DOPE, transfectam lipofectin now
available from Promega) and liposomes.
[0154] The producer cells can be cultured in conventional
fermentation bioreactors, flasks, and petri plates. Culturing can
be carried out at a temperature, pH and oxygen content appropriate
for a given host cell. No attempts to describe in detail the
various methods known for the production of proteins in prokaryote
and eukaryote cells will be made here. Production of the immune
checkpoint modulator can be periplasmic, intracellular or
preferably secreted outside the producer cell (e.g. in the culture
medium). If necessary, especially when the immune checkpoint
modulator is not secreted outside the producer cell or where it is
not secreted completely, it can be recovered by standard lysis
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. If secreted, it can
be recovered directly from the culture medium.
[0155] Optionally, the immune checkpoint modulator can then be
purified by well-known purification methods including ammonium
sulfate precipitation, acid extraction, gel electrophoresis,
filtration and chromatographic methods (e.g. reverse phase, size
exclusion, ion exchange, affinity, phosphocellulose,
hydrophobic-interaction or hydroxylapatite chromatography, etc).
The conditions and technology used to purify a particular protein
will depend on factors such as net charge, molecular weight,
hydrophobicity, hydrophilicity and will be apparent to those having
skill in the art. Moreover, the level of purification will depend
on the intended use. It is also understood that depending upon the
producer cell, the immune checkpoint modulator proteins can have
various glycosylation patterns, or may be non-glycosylated (e.g.
when produced in bacteria) as described herein.
[0156] Desirably, the immune checkpoint modulator in use in the
present invention is at least partially purified in the sense that
it is substantially free of other antibodies having different
antigenic specificities and/or other cellular material. Further,
the immune checkpoint modulator may be formulated according to the
conditions conventionally used in the art (e.g. WO2009/073569).
[0157] In accordance with the present invention, a variety of
modifications can be introduced in the immune checkpoint inhibitor
so as to increase its biological half-life, its affinity, its
stability and/or its production. For example, a signal peptide may
be included for facilitating secretion of the immune checkpoint
modulator in a cell culture as described herein. As an additional
example, a tag peptide (typically a short peptide sequence able to
be recognized by available antisera or compounds) may also be added
for facilitating purification of the recombinant immune checkpoint
modulator. A vast variety of tag peptides can be used in the
context of the invention including, without limitation, PK tag,
FLAG octapeptide, MYC tag, HIS tag (usually a stretch of 4 to 10
histidine residues) and e-tag (U.S. Pat. No. 6,686,152). The tag
peptide(s) may be independently positioned at the N-terminus of the
protein or alternatively at its C-terminus or alternatively
internally or at any of these positions when several tags are
employed. Tag peptides can be detected by immunodetection assays
using anti-tag antibodies.
[0158] Another approach that may be pursued in the context of the
present invention is coupling of the immune checkpoint modulator to
an external agent such as a radiosensitizer agent, a cytotoxic
agent and/or a labelling agent. The coupling can be covalent or
not. As used herein, the term "radiosensitizer" refers to a
molecule that makes cells more sensitive to radiation therapy.
Radiosensitizer includes, but are not limited to, metronidazole,
misonidazole, desmethylmisonidazole, pimonidazole, etanidazole,
nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145,
nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuhdine (IUdR),
bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea and
cisplatin. As used herein, the term "cytotoxic agent" refers to a
compound that is directly toxic to cells, preventing their
reproduction or growth such as toxins (e.g. an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof). As used herein, "a labeling agent" refers to a detectable
compound. The labeling agent may be detectable by itself (e.g.,
radioactive isotope labels or fluorescent labels) or, in the case
of an enzymatic label, may catalyze chemical modification of a
substrate compound which is detectable.
[0159] Another modification is pegylation for example to increase
the biological half-life of the antibody. Methods for pegylating
proteins are known in the art (see e.g. EP154316; EP401384;
WO98/15293, WO01/23001, etc).
Combination Product and Therapy
[0160] The term "combination therapy" and any variation such as
"combined use" refers to the action of administering in the same
subject at least the two entities being an object of the invention
and described herein.
[0161] In one embodiment, the present invention relates to a
combination product in the form of a composition comprising a
therapeutically effective amount of at least the therapeutic
vaccine and one or more immune checkpoint modulator entities
described herein and a pharmaceutically acceptable vehicle. In
another embodiment, the present invention relates to distinct
compositions for combined use, one comprising at least a
therapeutically effective amount of the therapeutic vaccine and a
pharmaceutically acceptable vehicle and another comprising a
therapeutically effective amount of the one or more immune
checkpoint modulator and a pharmaceutically acceptable vehicle. One
may proceed with one or more administration(s) of each entity (or
composition(s) thereof) which can be concomitant, sequential or
interspersed via the same or different routes.
[0162] A "therapeutically effective amount" corresponds to the
amount of each of the active entities (therapeutic vaccine and the
one or more immune check point modulator(s)) comprised in the
combination or composition(s) of the invention that is sufficient
for producing one or more beneficial results. Such a
therapeutically effective amount may vary as a function of various
parameters such as the mode of administration; the age and weight
of the subject; the nature and extent of symptoms; the ability of
the subject to respond to the treatment, kind of concurrent
treatment; the frequency of treatment and/or the need for
prevention or therapy, etc.
[0163] When "prophylactic" use is concerned, the combination is
administered at a dose sufficient to prevent or to delay the onset
and/or establishment and/or relapse of a pathologic condition,
especially in a subject at risk. For "therapeutic" use, the
therapeutic vaccine and the immune checkpoint modulator(s) are both
administered to a subject diagnosed as having a disease or
pathological condition with the goal of treating it, optionally in
association with one or more conventional therapeutic
modalities.
[0164] The term "pharmaceutically acceptable vehicle" is intended
to include any and all carriers, solvents, diluents, excipients,
adjuvants, dispersion media, coatings, antibacterial and antifungal
agents, absorption agents and the like compatible with
administration in mammals and in particular human subjects.
[0165] Each of the therapeutic vaccine and the one or more immune
check point modulator(s) or composition(s) thereof can
independently be placed in a solvent or diluent appropriate for
human or animal use. In particular, each or both may be formulated
so as to ensure its stability in particular under the conditions of
manufacture and long-term storage (i.e. for at least 6 months, with
a preference for at least two years) at freezing (e.g. -70.degree.
C., -20.degree. C.), refrigerated (e.g. 4.degree. C.) or ambient
(e.g. 20-25.degree. C.) temperature. Such formulations generally
include a liquid carrier such as aqueous solutions. Physiological
saline solution, Ringer's solution, Hank's solution, saccharide
solution (e.g. glucose, trehalose, saccharose, dextrose, etc) and
other aqueous physiologically balanced salt solutions may be used
(see for example the most current edition of Remington: The Science
and Practice of Pharmacy, A. Gennaro, Lippincott,
Williams&Wilkins). Animal or vegetable oils, mineral or
synthetic oils are also suitable. Advantageously, the formulation
appropriate for the therapeutic vaccine and the one or more immune
check point modulator(s) or composition(s) thereof is suitably
buffered for human use, preferably at physiological or slightly
basic pH (e.g. from approximately pH 7 to approximately pH 9 with a
specific preference for a pH comprised between 7 and 8 and more
particularly close to 7.5). Suitable buffers include without
limitation TRIS (tris(hydroxymethyl)methylamine), TRIS-HCl
(tris(hydroxymethyl)methylamine-HCl), HEPES
(4-2-hydroxyethyl-1-piperazineethanesulfonic acid), phosphate
buffer (e.g. PBS), ACES (N-(2-Acetamido)-aminoethanesulfonic acid),
PIPES (Piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO
(3-(N-Morpholino)-2-hydroxypropanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), TES
(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), DIPSO
(3-[bis(2-hydroxyethyl)amino]-2-hydroxypropane-1-sulfonic acid),
MOBS (4-(N-morpholino)butanesulfonic acid), TAPSO
(3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic
Acid), HEPPSO
(4-(2-Hydroxyethyl)-piperazine-1-(2-hydroxy)-propanesulfonic acid),
POPSO
(2-hydroxy-3-[4-(2-hydroxy-3-sulfopropyl)piperazin-1-yl]propane-1-sulfoni-
c acid), TEA (triethanolamine), EPPS
(N-(2-Hydroxyethyl)-piperazine-N'-3-propanesulfonic acid), and
TRICINE (N-[Tris(hydroxymethyl)-methyl]-glycine). Preferably, said
buffer is selected from TRIS-HCl, TRIS, Tricine, HEPES and
phosphate buffer comprising a mixture of Na.sub.2HPO.sub.4 and
KH.sub.2PO.sub.4 or a mixture of Na.sub.2HPO.sub.4 and
NaH.sub.2PO.sub.4. Said buffer (in particular those mentioned above
and notably TRIS-HCl) is preferably present in a concentration of
10 to 50 mM. It might be beneficial to also include in such
formulations a monovalent salt so as to ensure an appropriate
osmotic pressure. Said monovalent salt may notably be selected from
NaCl and KCl, preferably said monovalent salt is NaCl, preferably
in a concentration of 10 to 500 mM.
[0166] The formulation appropriate for use in the context of the
present invention, and especially liquid or frozen formulation, may
also include a cryoprotectant so as to protect the therapeutic
vaccine and/or the one or more immune check point modulator(s) (in
particular virus-based composition) at low storage temperature,
such as at about +5.degree. C. and lower. Suitable cryoprotectants
include without limitation sucrose (or saccharose), trehalose,
maltose, lactose, mannitol, sorbitol and glycerol, preferably in a
concentration of 0.5 to 20% (weight in g/volume in L, referred to
as w/v). For example, sucrose is preferably present in a
concentration of 5 to 15% (w/v), with a specific preference for
about 10%.
[0167] The formulation appropriate for use in the present invention
and especially liquid formulation may further comprise a
pharmaceutically acceptable chelating agent, and in particular an
agent chelating dications for improving stability. The
pharmaceutically acceptable chelating agent may notably be selected
from ethylenediaminetetraacetic acid (EDTA),
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid
(DMSA), diethylene triamine pentaacetic acid (DTPA), and
2,3-Dimercapto-1-propanesulfonic acid (DMPS). The pharmaceutically
acceptable chelating agent is preferably present in a concentration
of at least 50 .mu.M with a specific preference for a concentration
of 50 to 1000 .mu.M. Preferably, said pharmaceutically acceptable
chelating agent is EDTA present in a concentration close to 150
.mu.M.
[0168] Additional compounds may further be present to increase
stability of the formulated therapeutic vaccine and/or immune check
point modulator(s) or composition(s) thereof. Such additional
compounds include, without limitation, C.sub.2-C.sub.3 alcohol
(desirably in a concentration of 0.05 to 5% (volume/volume or
v/v)), sodium glutamate (desirably in a concentration lower than 10
mM), non-ionic surfactant (Evans et al. 2004, J Pharm Sci.
93:2458-75, Shi et al., 2005, J Pharm Sci. 94:1538-51, U.S. Pat.
No. 7,456,009, US2007/0161085) such as Tween 80 (also known as
polysorbate 80) at low concentration below 0.1%. Divalent salts
such as MgCl.sub.2 or CaCl.sub.2 have been found to induce
stabilization of various biological products in the liquid state
(see Evans et al. 2004, J Pharm Sci. 93:2458-75 and U.S. Pat. No.
7,456,009). Amino acids, and in particular histidine, arginine or
methionine, have been found to induce stabilization of various
viruses in the liquid state (see Evans et al., 2004, J Pharm Sci.
93:2458-75, U.S. Pat. No. 7,456,009, US2007/0161085, U.S. Pat. No.
7,914,979, WO2014/029702 and WO2014/053571).
[0169] The presence of high molecular weight polymers such as
dextran or polyvinylpyrrolidone (PVP) is particularly suited for
freeze-dried formulations. Lyophilized formulations are generally
obtained by a process involving vacuum drying and freeze-drying
(see e.g. WO03/053463; WO2006/0850082; WO2007/056847;
WO2008/114021) and the presence of these polymers assists in the
formation of the cake during freeze-drying (see EP1418942 and
WO2014/053571).
[0170] Various formulations available in the art either in frozen,
liquid or freeze-dried form can be independently used to preserve
the therapeutic vaccine and/or immune check point modulator(s) or
composition(s) thereof (e.g. WO98/02522, WO00/29024, WO00/34444,
WO01/66137, WO03/053463, WO2006/0850082, WO2007/056847 and
WO2008/114021, etc). For illustrative purposes, sterile histidine,
acetate citrate or phosphate buffers saline containing surfactant
such as polysorbate 80 and protectants such as sucrose or mannitol
are adapted to the preservation of recombinant antibodies and
buffered formulations including NaCl and/or sugar are particularly
adapted to the preservation of vectorised therapeutic vaccine (e.g.
Tris-HCl 10 mM pH 8 with saccharose 5% (w/v), Sodium glutamate 10
mM, and NaCl 50 mM or phosphate-buffered saline with glycerol (10%)
and NaCl).
[0171] Formulation can be adapted according to the mode of
administration to ensure proper distribution or delayed release in
vivo. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polyethylene glycol.
Many methods for the preparation of such formulations are described
in the art (e.g. J. R. Robinson in "Sustained and Controlled
Release Drug Delivery Systems", ed., Marcel Dekker, Inc., New York,
1978; WO01/23001; WO2006/93924; WO2009/53937). Gastro-resistant
capsules and granules are particularly appropriate for oral
administration, suppositories for rectal or vaginal administration,
optionally in combination with absorption enhancers useful to
increase the pore size of the mucosal membranes. Such absorption
enhancers are typically substances having structural similarities
to the phospholipid domains of the mucosal membranes (such as
sodium deoxycholate, sodium glycocholate,
dimethyl-beta-cyclodextrin, lauryl-1-lysophosphatidylcholine).
[0172] Each of the therapeutic vaccine and/or the immune check
point modulator(s) or composition(s) thereof may also contain
pharmaceutically acceptable excipients for providing desirable
pharmaceutical or pharmacodynamic properties, including for example
osmolarity, viscosity, clarity, colour, sterility, stability,
dissolution, release or absorption into the subject, or delivery to
a particular organ.
[0173] The appropriate dosage of the therapeutic vaccine and the
immune checkpoint modulator(s) as well as the optimal ratios of
each entity may be determined by techniques well known in the art.
Further refinement of the calculations necessary to adapt the
appropriate dosage for a subject or a group of subjects may be
routinely made by a practitioner, in the light of the relevant
circumstances.
[0174] Suitable dosage of the immune checkpoint modulator(s) varies
from about 0.01 mg/kg to about 50 mg/kg, advantageously from about
0.1 mg/kg to about 30 mg/kg, desirably from about 0.5 mg/kg to
about 25 mg/kg, preferably from about 1 mg/kg to about 20 mg/kg,
more preferably from about 2 mg/kg to about 15 mg/kg, with a
specific preference for doses from about 3 mg/kg to about 10 mg/kg
when used by parenteral injection. However, doses may be adapted to
the administration route and the subject to be treated by a factor
of variation comprised between 1.5 and 100. In some embodiments,
two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the
dosage of each antibody administered falls within the ranges
indicated.
[0175] Suitable dosage for a virus-based therapeutic vaccine varies
from approximately 10.sup.5 to approximately 10.sup.13 vp (viral
particles), iu (infectious unit) or pfu (plaque-forming units)
depending on the quantitative technique used. As a general
guidance, adenovirus doses from approximately 10.sup.6 to
approximately 5.times.10.sup.12 vp are suitable, preferably from
approximately 10.sup.7 vp to approximately 10.sup.12 vp, more
preferably from approximately 10.sup.8 vp to approximately
5.times.10.sup.11 vp; doses of approximately 5.times.10.sup.8 vp to
approximately 10.sup.11 vp being particularly preferred especially
for human use. Individual doses which are suitable for MVA-based
therapeutic vaccine comprise from approximately 10.sup.4 to
approximately 10.sup.12 pfu, preferably from approximately 10.sup.5
pfu to approximately 10.sup.11 pfu, more preferably from
approximately 10.sup.6 pfu to approximately 10.sup.10 pfu; doses of
approximately 10.sup.7 pfu to approximately 10.sup.8 pfu being
particularly preferred especially for human use. Individual doses
which are suitable for Vaccinia-based therapeutic vaccine comprise
from approximately 10.sup.5 to approximately 10.sup.13 pfu,
preferably from approximately 10.sup.6 pfu to approximately
10.sup.11 pfu, more preferably from approximately 10.sup.7 pfu to
approximately 10.sup.10 pfu; doses of approximately 10.sup.8 pfu to
approximately 5.times.10.sup.9 vp being particularly preferred
especially for human use. The quantity of virus present in a sample
can be determined by routine titration techniques, e.g. by counting
the number of plaques following infection of permissive cells (e.g.
293 or PER6C6 for Ad, BHK-21 or CEF for MVA, HeLa for VV), by
measuring the A260 absorbance (vp titers), or still by quantitative
immunofluorescence, e.g. using anti-virus antibodies (iu titers).
Suitable dosage for a plasmid-based therapeutic vaccine varies from
10 .mu.g to 20 mg, advantageously from 100 .mu.g to 10 mg and
preferably from approximately 0.5 mg to approximately 5 mg.
Administration
[0176] The combination product of the invention is suitable for
single administration or a series of administrations. In particular
when distinct compositions are contemplated, the therapeutic
vaccine and the immune check point modulator(s) may be administered
together or separately to the subject and in a single dose or
multiple doses. Administrations may be concomitant (e.g. mixed in
the same composition or in different compositions administered at
approximately the same time), sequential (therapeutic vaccine
followed by immune checkpoint modulator or vice versa) or
interspersed (intermixed administrations at various intervals) and
performed by the same or different routes at the same site or at
alternative sites.
[0177] Any of the conventional administration routes is applicable
in the context of the invention including parenteral, topical or
mucosal routes, for the combination product or composition(s) of
the invention. Parenteral routes are intended for administration as
an injection or infusion and encompass systemic as well as local
routes. Parenteral injection types that may be used to administer
the combination product of the invention are intravenous (into a
vein, such as the portal vein feeding liver), intravascular (into a
blood vessel), intra-arterial (into an artery such as hepatic
artery), intradermal (into the dermis), subcutaneous (under the
skin), intramuscular (into muscle), intraperitoneal (into the
peritoneum) and intratumoral (into a tumor or its close vicinity)
or still by scarification. Infusions typically are given by
intravenous route. Mucosal administrations include without
limitation oral/alimentary, intranasal, intratracheal,
intrapulmonary, intravaginal or intra-rectal route. Topical
administration can also be performed using transdermal means (e.g.
patch and the like). Preferred routes of administration for the
immune checkpoint modulator(s) include intravenous (e.g.
intravenous injection or infusion), and intratumoral. Preferred
routes of administration for the therapeutic vaccine include
intravenous, intramuscular, subcutaneous and intratumoral. For
example, intratumoral inoculations of the therapeutic vaccine could
be advantageously combined with intravenous injections of the
immune checkpoint modulator(s).
[0178] Administrations may use conventional syringes and needles
(e.g. Quadrafuse injection needles) or any compound or device
available in the art capable of facilitating or improving delivery
of the active agent(s) in the subject (e.g. electroporation for
facilitating intramuscular administration). An alternative is the
use of a needleless injection device to administer at least one of
the active entities comprised in the combination product of the
invention (e.g. Biojector.TM. device). Transdermal patches may also
be envisaged.
[0179] In one embodiment, the therapeutic vaccine and the one or
more immune checkpoint modulator(s) or composition(s) thereof are
administered sequentially, such as the vaccines being administered
first and the immune checkpoint modulator(s) second, or vise-versa
(immune checkpoint modulator(s) being administered first and the
therapeutic vaccine second). The sequence may vary. For example,
the order of the administrations can be reversed or kept in the
same order at each time point of administration.
[0180] One may also proceed by interspersed administrations of the
therapeutic vaccine and the immune checkpoint modulator(s). The
period of time between the first administration of the therapeutic
vaccine and the first administration of the immune check point
modulator(s) may vary from approximately several minutes to several
week(s). It is also possible to proceed for each entity via
sequential cycles of administrations that are repeated after a rest
period. Intervals between each administration can be from one hour
to one year (e.g. 24 h, 48 h, 72 h, weekly, every two weeks,
monthly or yearly). Intervals can also be irregular (e.g. following
the measurement of monoclonal antibodies in the patient blood
levels). The doses can vary for each administration within the
range described above. Preferably, the time interval between each
therapeutic vaccine administration can vary from approximately 1
day to approximately 8 weeks, advantageously from approximately 2
days to approximately 6 weeks, preferably from approximately 3 days
to approximately 4 weeks and even more preferably from
approximately 1 week to approximately 3 weeks with a specific
preference for about one week. In combination, the time interval
between each administration of immune check point modulator(s) can
vary from approximately 2 days to approximately 8 weeks,
advantageously from approximately 1 week to approximately 6 weeks,
preferably every 3 weeks.
[0181] A preferred therapeutic scheme involves from 4 to 15 (4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) administrations of 10.sup.7
or 10.sup.9 pfu of a MVA-based therapeutic vaccine at approximately
1 to 3 week interval interspersed with 2 to 6 administrations of 3
to 10 mg/kg of anti-immune checkpoint antibody(ies)(s) every 2 or 3
weeks. For illustrative purposes, a preferred administration
schedule comprises subcutaneous administrations of a
MUC1-expressing MVA vector (such as TG4010) at a dose of
approximately 10.sup.8 pfu weekly for 6 weeks and then every three
weeks interspersed with intravenous administrations of an
anti-CTLA4 antibody such as ipilimumab at a dose of approximately 3
mg/kg every 3 weeks for a total of four doses.
[0182] The combination product or composition of the invention is
for use for treating or preventing diseases or pathologic
condition, especially those caused by a pathogenic organism or an
unwanted cell division according to the modalities described
herein. A "disease" (and any form of disease such as "disorder" or
"pathological condition") is typically characterized by
identifiable symptoms. Exemplary diseases include, but are not
limited to, infectious diseases that result from an infection with
a pathogenic organism (e.g. bacteria, parasite, virus, fungus, etc)
and proliferative diseases involving abnormal proliferation of
cells such as neoplastic diseases (e.g. cancer), rheumatoid
arthritis and restenosis.
[0183] The present invention also relates to a method of treatment
comprising administering a combination product or composition(s) of
therapeutic vaccine and/or one or more immune checkpoint
modulator(s) in an amount sufficient to treat or prevent a disease
or a pathologic condition in a subject in need thereof or alleviate
one or more symptoms related to or associated with the diseases and
pathologic condition, according to the modalities described herein.
In a preferred embodiment, the disease or pathologic condition to
be treated is a proliferative or an infectious disease (e.g.
especially a chronic infection). Accordingly, the present invention
also relates to a method for inhibiting tumor cell growth
comprising administering a combination product or composition(s) of
therapeutic vaccine and/or one or more immune checkpoint
modulator(s) to a subject in need thereof. In the context of the
invention, the methods and use according to the invention aim at
slowing down, curing, ameliorating or controlling the occurrence or
the progression of the targeted disease.
[0184] As used herein, the term "cancer" includes, but is not
limited to, solid tumors and blood borne tumors. The term "cancer"
encompasses both primary and metastatic cancers. Representative
examples of cancers that may be treated using the combination and
methods of the invention include, without limitation, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia and more particularly
bone cancer, gastrointestinal cancer, liver cancer, pancreatic
cancer, gastric cancer, colorectal cancer, esophageal cancer,
oro-pharyngeal cancer, laryngeal cancer, salivary gland carcinoma,
thyroid cancer, lung cancer, cancer of the head or neck, skin
cancer, squamous cell cancer, melanoma, uterine cancer, cervical
cancer, endometrial carcinoma, vulvar cancer, ovarian cancer,
breast cancer, prostate cancer, cancer of the endocrine system,
sarcoma of soft tissue, bladder cancer, kidney cancer, glioblastoma
and various types of the central nervous system (CNS), etc. The
present invention is particularly useful for treatment of cancers
that express PD-L1 (Iwai et al., 2005, Int. Immunol. 17: 133-44),
especially metastatic ones and those that overexpress MUC1
(especially hypoglycosylated form thereof) such as renal cancer
(e.g. clear cell carcinoma), prostate cancer (e.g. hormone
refractory prostate adenocarcinoma), breast cancer (e.g. metastatic
breast cancer), colorectal cancer, lung cancer (e.g. non-small cell
lung cancer) liver cancer (e.g. hepatocarcinoma), gastric cancer,
bile duct carcinoma, endometrial cancer, pancreatic cancer and
ovarian cancer. Preferably said cancer is non small cell lung
cancer (NSCL).
[0185] Representative examples of infectious diseases that may be
treated using the combination and methods of the invention include,
without limitation, a) viral diseases such as those resulting from
infection by an herpes virus (HSV1, HSV2, or VZV), a papillomavirus
(HPV), a poxvirus causing variola or chicken pox, an enterovirus, a
retrovirus such as HIV causing AIDS, a cytomegalovirus, a
flavivirus (e.g. causing Japanese encephalitis, hepatitis C, dengue
and yellow fever), an Hepadnavirus (e.g. HBV), an orthomyxovirus
(e.g. influenza virus), a paramyxovirus (e.g. parainfluenzavirus,
mumps virus, measles virus and respiratory syncytial virus (RSV)),
a coronavirus (e.g. SARS), rhabdovirus and rotavirus; b) diseases
resulting from infection by bacteria, for example, Escherichia,
Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria,
Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas,
Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria,
Clostridium, Bacillus, Corynebacterium, Mycobacterium,
Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium,
Brucella, Yersinia, Haemophilus, or Bordetella; and (c) fungal
diseases including but not limited to candidiasis, aspergillosis,
histoplasmosis, cryptococcal meningitis; and d) parasitic diseases
including but not limited to malaria, Pneumocystis carnii
pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and
trypanosome infection.
[0186] Typically, upon administration according to the modalities
described herein, the combination product of the invention provides
a therapeutic benefit to the treated subject over the baseline
status or over the expected status if not treated, which can be
evidenced by an observable improvement of the clinical status over
the baseline status or over the expected status if not treated in
combination as described herein. An improvement of the clinical
status can be easily assessed by any relevant clinical measurement
typically used by physicians or other skilled healthcare staff. In
the context of the invention, the therapeutic benefit can be
transient (for one or a couple of months after cessation of
administration) or sustained (for several months or years). As the
natural course of clinical status which may vary considerably from
a subject to another, it is not required that the therapeutic
benefit be observed in each subject treated but in a significant
number of subjects (e.g. statistically significant differences
between two groups can be determined by any statistical test known
in the art, such as a Tukey parametric test, the Kruskal-Wallis
test the U test according to Mann and Whitney, the Student's
t-test, the Wilcoxon test, etc).
[0187] When the method is aimed at treating a proliferative
disease, in particular cancer, a therapeutic benefit can be
evidenced by for instance a reduction in the tumor number; a
reduction of the tumor size, a reduction in the number or extent of
metastases, an increase in the length of remission, a stabilization
(i.e. not worsening) of the state of disease, a delay or slowing of
disease progression or severity, a prolonged survival, a better
response to the standard treatment, an improvement of quality of
life, a reduced mortality, etc.
[0188] When the method is aimed at treating an infectious disease,
a therapeutic benefit can be evidenced by for instance, a decrease
of the amount of the infecting pathogenic organism quantified in
blood, plasma, or sera of a treated subject, and/or a stabilized
(not worsening) state of the infectious disease (e.g. stabilization
of conditions typically associated with the infectious disease such
as inflammatory status), and/or the reduction of the level of
specific seric markers (e.g. decrease of alanine aminotransferase
(ALT) and/or aspartate aminotransferase (AST) associated with liver
poor condition usually observed in chronic hepatitis C), decrease
in the level of any antigen associated with the occurrence of an
infectious disease and/or the appearance or the modification of the
level of antibodies to the pathogenic organism and/or an improved
response of the treated subject to conventional therapies (e.g.
antibiotics) and/or a survival extension as compared to expected
survival if not receiving the combination treatment.
[0189] The appropriate measurements such as blood tests, analysis
of biological fluids and biopsies as well as medical imaging
techniques can be used to assess a clinical benefit. They can be
performed before the administration (baseline) and at various time
points during treatment and after cessation of the treatment. For
general guidance, such measurements are evaluated routinely in
medical laboratories and hospitals and a large number of kits are
available commercially (e.g. immunoassays, quantitative PCR
assays).
[0190] Preferably, the combination product of the invention is used
or administered for eliciting or stimulating and/or redirecting an
immune response in the treated subject. Accordingly, the present
invention also encompasses a method for eliciting or stimulating
and/or re-orienting an immune response (e.g. to tumor or infected
cells) comprising administering a combination product or
composition(s) of therapeutic vaccine and/or one or more immune
checkpoint modulator(s) to a subject in need thereof, in an amount
sufficient according to the modalities described herein so as to
activate the patient's immunity.
[0191] In a particular embodiment, the combination product and
method(s) of the invention may be employed according to the
modalities described herein to break immune tolerance usually
encountered in chronically infected and cancerous subjects.
[0192] The elicited, stimulated or redirected immune response can
be specific (i.e. directed to epitopes/antigens) and/or
non-specific (innate), humoral and/or cellular. In the context of
the invention, the immune response is preferably a T cell response
CD4+ or CD8+-mediated or both, directed to
polypeptide(s)/epitope(s), in particular associated with a tumor or
an infecting pathogenic organism.
[0193] The ability of the combination product described herein to
elicit, stimulate or redirect an immune response can be evaluated
either in vitro (e.g. using biological samples collected from the
subject) or in vivo using a variety of direct or indirect assays
which are standard in the art. For a general description of
techniques available to evaluate the onset and activation of an
immune response, see for example Coligan et al. (1992 and 1994,
Current Protocols in Immunology; ed J Wiley & Sons Inc,
National Institute of Health or subsequent editions). The ability
to stimulate a humoral response may be determined by antibody
binding and/or competition in binding (see for example Harlow,
1989, Antibodies, Cold Spring Harbor Press). Evaluation of
non-specific immunity can be performed by for example measurement
of the NK/NKT-cells (e.g. representatively and level of
activation), as well as, IFN-related cytokine and/or chemokine
producing cascades, activation of TLRs and other markers of innate
immunity (Scott-Algara et al., 2010 PLOS One 5(1), e8761; Zhou et
al., 2006, Blood 107, 2461-2469; Chan, 2008, Eur. J. Immunol. 38,
2964-2968). Evaluation of cellular immunity can be performed for
example by quantification of cytokine(s) produced by activated T
cells including those derived from CD4+ and CD8+ T-cells using
routine bioassays (e.g. characterization and/or quantification of T
cells by ELISpot, by multiparameters flow cytometry or ICS, by
cytokine profile analysis using multiplex technologies or ELISA),
by determination of the proliferative capacity of T cells (e.g. T
cell proliferation assays by [.sup.3H] thymidine incorporation
assay), by assaying cytotoxic capacity for antigen-specific T
lymphocytes in a sensitized subject or by immunization of
appropriate animal models. For example, techniques routinely used
in laboratories (e.g. flow cytometry, histology) may be used to
perform tumor surveillance. One may also use various available
antibodies so as to identify different immune cell populations
involved in anti-tumor response that are present in the treated
subjects, such as cytotoxic T cells, activated cytotoxic T cells,
natural killer cells and activated natural killer cells.
[0194] If desired, the combination product, composition(s) or
methods of the invention can be used or carried out in association
with one or more conventional therapeutic modalities which are
available for treating or preventing the disease or pathological
condition to be treated or prevented (e.g. chemotherapy, radiation,
and/or surgery). Such conventional therapy may be administered to
the subject sequentially or concomitantly with the combination or
method according to the invention.
[0195] Representative examples of conventional therapeutic drugs
that may be useful in association with the combination product,
composition or method of the invention include among others
nitrosoureas, antibiotics, antimetabolites, antimitotics, antiviral
drugs (e.g. interferon alpha), monoclonal antibodies, signaling
inhibitors as well as chemotherapeutic drugs routinely used in
cancer therapy. One may cite more specifically: [0196] alkylating
agents such as e.g. mitomycin C, cyclophosphamide, busulfan,
ifosfamide, isosfamide, melphalan, hexamethylmelamine, thiotepa,
chlorambucil, or dacarbazine; [0197] antimetabolites such as, e.g.
gemcitabine, capecitabine, 5-fluorouracil, cytarabine,
2-fluorodeoxy cytidine, methotrexate, idatrexate, tomudex or
trimetrexate; [0198] topoisomerase II inhibitors such as, e.g.
doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone;
[0199] topoisomerase I inhibitors such as, e.g. irinotecan
(CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan;
[0200] antimitotic drugs such as, e.g., paclitaxel, docetaxel,
vinblastine, vincristine or vinorelbine; [0201] platinum
derivatives such as, e.g., cisplatin, oxaliplatin, spiroplatinum or
carboplatinum; [0202] inhibitors of tyrosine kinase receptors such
as sunitinib (Pfizer), sorafenib (Bayer), gefitinib, erlotinib and
lapatinib; and [0203] antibodies (in particular anti-neoplastic
cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab) or
inhibitors of Human Epidermal Growth Factor Receptor-2 (in
particular trastuzumab); and agents that affect angiogenesis such
as, e.g. inhibitor of Vascular Endothelial Growth Factor (in
particular bevacizumab or ranibizumab); [0204] antibiotics
conventionally used against infectious pathogenic organisms such as
Aminoglycosides, Ansamycins, Carbapenems, Cephalosporins,
Glycopeptides, Macrolides, Penicillins, Qionolones and tetracyclins
among others; One may cite in particular antibiotics currently used
in first line therapy to treat a Mtb infection such as isoniazid,
rifamycins (e.g. rifampin, rifapentine and rifabutin), ethambutol,
streptomycin, pyrazinamide and fluoroquinolones as well as those
used in "second-line" therapy in Mtb-infected subjects that has
demonstrated drug resistance such as ofloxacin, ciprofloxacin,
ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin
and capreomycin; [0205] Antiviral treatment such as
interferon-alpha (IFNa and pegylated derivative) and
nucleoside/nucleotide analogues (NUCs). For example, lamivudine,
entecavir, telbivudine, adefovir and tenofovir are currently used
for treating HBV. Other antivirals include protease inhibitors
(e.g. serine protease inhibitors such as VX950 of Vertex),
polymerase inhibitors and helicase inhibitors that are suitable for
treating hepatitis C; [0206] TLR agonists and N-glycosylation
inhibitors; [0207] Interleukins (e.g. IL-2, IL-6, IL-15, IL-24,
etc), interferons (e.g. IFN.alpha., IFN.beta. or IFN.gamma.), tumor
necrosis factor (TNF), colony-stimulating factors (e.g. GM-CSF,
C-CSF, M-CSF, etc) and chemokines (e.g. CXCL10, CXCL9, CXCL11,
etc); [0208] siRNA and antisense oligonucleotides that target genes
of infectious pathogenic organism or cellular gene associated with
the targeted disease.
[0209] According to an advantageous embodiment, especially when the
therapeutic vaccine is armed with a suicide gene, the combination
product or methods according to the present invention may be used
in association with the corresponding prodrug (see Table 1). The
prodrug is administered in accordance with standard practice (e.g.
per os, systematically, etc). Preferably, the prodrug
administration takes place subsequent to the administration of the
therapeutic vaccine, (e.g. at least 3 days after the administration
of the suicide-gene encoding therapeutic vaccine). The oral route
is preferred. It is possible to administer a single dose of prodrug
or doses which are repeated for a time which is sufficiently long
to enable the toxic metabolite to be produced within the subject.
By way of illustration, a dose of 50 to 500 mg/kg/day,
advantageously, a dose of 200 mg/kg/day or, preferably, a dose of
100 mg/kg/day is appropriate.
[0210] The combination product and method of the invention may also
be used in combination with one or more adjuvant(s) or "immune
stimulant" to enhance immunity (especially a T cell-mediated
immunity), e.g. through toll-like receptors (TLR) such as TLR-7,
TLR-8 and TLR-9. For illustrative purposes, such adjuvants include,
without limitation, alum, mineral oil emulsion such as, Freunds
complete and incomplete (IFA), lipopolysaccharides (Ribi et al.,
1986, Immunology and Immunopharmacology of Bacterial Endotoxins,
Plenum Publ. Corp., NY, p407-419), saponins such as ISCOMATRIX,
AbISCO, QS21 (Sumino et al., 1998, J. Virol. 72: 4931; WO98/56415),
imidazo-quinoline compounds such as Imiquimod (Suader, 2000, J. Am
Acad Dermatol. 43:S6), S-27609 (Smorlesi, 2005, Gene Ther. 12:
1324) and related compounds such as those described in
WO2007/147529; cationic peptides such as IC-31 (Kritsch et al.,
2005, J. Chromatogr Anal. Technol. Biomed. Life Sci. 822: 263-70),
polysaccharides such as Adjuvax and squalenes such as MF59. Other
suitable adjuvants include ds RNA like NAB2 with Lipofectin
(Claudepierre et al., 2014, J. Virol 88: 5242-55) or 3pRNA (Hornung
et al., 2006, Science 314: 994-7), both stimulating IFN.alpha.
responses via activation of cytoplasmic helicase MDA-5 and
RIG-1.
[0211] Alternatively or in combination, the combination or method
according to the invention can also be used in association with
radiotherapy. Those skilled in the art can readily formulate
appropriate radiation therapy protocols and parameters (see for
example Perez and Brady, 1992, Principles and Practice of Radiation
Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations
and modifications as will be readily apparent to those skilled in
the field). The types of radiation that may be used in cancer
treatment are well known in the art and include electron beams,
high-energy photons from a linear accelerator or from radioactive
sources such as cobalt or cesium, protons, and neutrons.
[0212] The present invention also provides kits including the
active entities of the combination product of the invention in kit
form. A kit is a packaged combination, optionally, including
instructions for use optionally with other components. In one
embodiment, a kit includes at least the therapeutic vaccine as
discussed herein in one container and one or more immune checkpoint
modulator(s) as described herein in another container. Such
containers are preferably sterile glass or plastic vial. A
preferred kit comprises a MVA-based therapeutic vaccine (e.g. a MVA
virus expressing the tumor-associated MUC1 antigen and the human
IL-2) and an immune checkpoint modulator(s) which specifically
binds CTLA-4 (e.g. an anti-CTLA-4 antibody, such as ipilimumab).
Another preferred kit comprises a MVA-based therapeutic vaccine
(e.g. a MVA virus expressing the tumor-associated MUC1 antigen and
the human IL-2) and an immune checkpoint modulator(s) which
specifically binds PD-1 (e.g. an anti-PD-1 antibody, such as
nivolumab or lanbrolizumab). Another preferred kit comprises a
MVA-based therapeutic vaccine (e.g. a MVA virus expressing the
tumor-associated MUC1 antigen and the human IL-2) and an immune
checkpoint modulator(s) which specifically binds PD-L1 (e.g. an
anti-PD-L1 antibody, such as MPDL3280A or BMS936559). Optionally,
the kit can include suitable devices for performing the
administration of the active agents. The kit can also include a
package insert including information concerning the compositions or
individual component and dosage forms in the kit.
[0213] All of the above cited disclosures of patents, publications
and database entries are specifically incorporated herein by
reference in their entirety to the same extent as if each such
individual patent, publication or entry were specifically and
individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0214] FIG. 1 illustrates the effects on tumor volume (FIG. 1A) and
mice survival (FIG. 1B) in a subcutaneous CT26-CL25 tumor model of
the administration of the formulation buffer (vehicle), MVATG18124
alone, the anti PD-1 clone RMP1.14 alone, a combination of both
MVATG18124 and anti PD-1 clone RMP1.14 and a combination of both
MVATG18124 and the rat IgG2a isotype control.
[0215] FIG. 2 illustrates the effects on mice survival in
metastatic CT26-CL25 tumor model of the administration of an empty
MVA (MVATGN33.1), MVATG18124 alone, an anti CTLA-4 antibody alone,
its IgG2b isotype control, a combination of both MVATG18124 and
anti CTLA-4 and a combination of both MVATG18124 and the IgG2b
isotype control.
[0216] FIG. 3A illustrates the percentage of gated
CD8.sup.+CD3.sup.dim cells (referred as CD8.sup.dimCD3.sup.dim
herein after) in dissociated lungs obtained from untreated (i.e.
naive) mice or mice treated with MVATG18124, anti-CTLA-4 and both
MVATG18124 and anti-CTLA-4. FIG. 3B illustrates an example of
CD8.sup.dimCD3.sup.dim (referred as CD8.sup.+CD3.sup.dim in the
Figure) population in CD3/CD8 dot blot lung cells from mice treated
with anti CTLA-4 and MVATG18124.
[0217] FIG. 4 represents the IFN-.gamma. positive
CD8.sup.dimCD3.sup.dim cell population obtained following ConA
induction in lung samples obtained from untreated (i.e. naive) mice
or mice treated with MVATG18124, anti-CTLA-4 and both. FIG. 4A:
ConA-induced fold induction of percentage of IFN.gamma..sup.+
CD8.sup.dimCD3.sup.dim cells upon incubation of lung cells with
bmDCs to facilitate activation. FIG. 4B: ConA-induced fold
induction of percentage of IFN.gamma..sup.+ CD8.sup.dimCD3.sup.dim
cells upon incubation of lung cells with anti-CD28 to facilitate
activation.
[0218] FIG. 5 illustrates ELIspot analysis of splenic lymphocytes
stimulated with the .beta.-gal-specific peptide T9L3 showed
.beta.-gal specific response in splenic lymphocytes treated with
MVATG18124. The raw data was transformed into histogram graph.
Results are expressed as number of spot forming units (sfu) per
1.times.10.sup.6 splenocytes (mean) for each triplicate or
quadriplicate.
[0219] FIG. 6 represents the induction of IFN-.gamma., CD107a and
KLRG1 positive cells in CD8.sup.dimCD3.sup.dim cell population
obtained in lung samples stimulated with T9L-3 peptide (hatched) or
irrelevant T8G peptide (dotted) following treatment with an empty
vector MVATGN33.1 alone or in combination with anti-CTLA-4,
MVATG18124 alone or in combination with anti-CTLA-4 or anti-CTLA-4
alone.
[0220] FIG. 7 illustrates the effect provided by TG4010 treatment
on mice survival and tumor volume in a CT26-MUC1 tumor model. FIG.
7A: CT26-MUC1 lung tumor model. CT26-MUC1 cells were injected iv.
Day 2 and 9, 5.10.sup.7 pfu TG4010, empty MVA vector (MVATGN33.1)
or buffer (SO8) were injected intravenously. Mice were weighed
twice per week and sacrificed when loosing 10% of weight. Percent
survival was monitored. FIG. 7B: CT26-MUC1 s.c. tumor model.
CT26-MUC1 cells were injected s.c. Day 2 and 9, 1.10.sup.7 pfu
TG4010, empty MVA vector (MVATGN33.1) or buffer (SO8) were injected
s.c. in the same flank. Mice were sacrificed when the tumors
reached the size of 2000 mm.sup.3. Mean tumor volumes with SEM are
shown.
[0221] FIG. 8 illustrates the effect on mice survival (FIG. 8A) and
tumor volume (FIG. 8B) provided by TG4010 treatment in combination
with anti PD-1 antibody in the CT26-MUC1 s.c. tumor model. BALB/c
mice were injected s.c. with 2.10.sup.5 CT26-MUC1 cells. On days 2
and 9 after tumor implantation, mice were treated sc with TG4010
(also called MVATG9931) or an empty control vector at the
suboptimal dose of 1.10.sup.6 pfu followed by i.p. administration
of 250 .mu.g anti PD-1 (RMP1.14, IgG2a, BioXCell) on days 10, 13,
15 and 17. Mice were sacrificed when the tumors reached the size of
2000 mm.sup.3. Percent survival and mean tumor volumes were
monitored over time.
[0222] FIG. 9 illustrates the survival of BALB/C mice injected s.c.
with CT26-MUC1 tumor cells and immunized with two injections of
1.times.10.sup.7 PFU of MVATGN33.1 or MVATG9931 on days D2 and D9
followed by i.p. administrations of 250 .mu.g of anti-PD-1 Ab on
days D10, D13, D15 and D17. (*: p<0.05; **:: p<0.01; ***:
p<0.001)
EXAMPLES
[0223] We set out to combine immune checkpoint blocking approaches
with therapeutic MVA vectors with the goal of inducing
antigen-specific T cell immune response with MVA and release the
brakes from T cell generation with immune checkpoint antibodies.
Preclinical evidence for synergistic effects of immune checkpoint
blockers combined with viral vectors was to be demonstrated in
mouse tumor models. This implies the use of i) murine-specific
anti-immune check point antibodies and ii) an antigen-expressing
MVA vector.
[0224] The MVA vector chosen for these studies (MVATG18124)
contains the bacterial LacZ gene encoding the beta-galactosidase Ag
model (SEQ ID NO: 3) under the control of poxvirus promoter pH5R
(SEQ ID NO: 4). pH5R promoter was isolated by PCR amplification
from Vaccinia virus Copenhagen strain using appropriate primers.
The E. Coli LacZ gene was obtained by PCR amplification using
primers otg19678 (SEQ ID NO: 5) and otg19679 (SEQ ID NO: 6) with
pCMVBeta (Clontech) as DNA template. The pH5R and LacZ genes were
cloned into a shuttle plasmid between MVA sequences extending from
positions 142006 to 142987 and positions 142992 to 143992 according
to GenBank sequence EF675191.1. MVATG18124 was generated into
chicken embryo fibroblast (CEF) cells by transfection of shuttle
plasmid into previously MVA-infected CEF, resulting in homologous
recombination between shuttle plasmid DNA and MVA genome and
insertion of the pH5R-LacZ cassette into deletion III. Recombinant
MVA clones were isolated using conventional technology (Lullo et
al., 2010, J Virol Methods 163: 195-204) and the selected clones
were controlled by PCR, then amplified in CEF cells. Virus stocks
were titrated on DF1 cells by plaque assay. Absence of mutation
into the inserted DNA and the surrounding region was checked by DNA
sequencing.
[0225] It was first chosen to target the immune checkpoint blocker
murine PD-1 (mPD-1) with an appropriate antibody. The rat anti
mPD-1 antibody RMP1-14 (BioXcell) was chosen. This antibody was
shown to block the interaction of mPD1 with its ligands (Yamazaki
et al., 2005, J. Immunol. 175(3): 1586-92).
[0226] The combination of mPD-1 inhibitors with the
antigen-expressing MVATG18124 was tested in vivo in two mice
models, respectively metastatic and subcutaneous tumor models. The
colon carcinoma cell line CT26.CL25 (ATCC CRL-2639), transduced
with the LacZ gene and thus stably expressing beta-galactosidase,
was either injected subcutaneously to generated palpable tumors
(subcutaneous model) or intravenously to generate lung metastasis.
Mice were then treated with MVATG18124 expressing
beta-galactosidase and murine-specific immune checkpoint blockers
like anti PD-1 or anti CTLA-4 antibodies.
Example 1: Combination of MVATG18124 with Anti-PD-1 Mab
[0227] The combination of mPD-1 inhibitor (commercial clone
RMP1-14; BioXCell) with beta-gal-expressing MVATG18124 was tested
in vivo in a subcutaneous tumor model. Balb/c mice were
subcutaneously injected with 2.times.10.sup.5 CT26.CL25 cells. Day
2 and 9 after cell implantation, mice were then intravenously
immunized with either 1.times.10.sup.4 pfu of MVATG18124 or
formulation vehicle as negative control in combination with 4
intraperitoneal (ip) administrations at days 10, 13, 15 and 17 of
250 .mu.g of either murine anti PD-1 antibody RMP1.14 (BioXcell) or
its isotype control IgG2a (clone 2A3). In other terms, 5 groups of
10 mice were tested; a first group treated with MVATG18124
receiving 2 iv injections (group 1); a second group treated with 4
ip injections of anti-PD-1 antibody (group 2); a third group
treated with both MVATG18124 and anti-PD-1 antibody receiving 2 iv
injections of the therapeutic MVA and 4 ip injections of anti-PD-1
antibody (group 3); a fourth group receiving 2 iv injections of the
therapeutic MVATG18124 and 4 ip injections of isotype antibody
(group 4) and a control group receiving the formulation buffer
(group 5). Tumor growth and survival were measured over time as
illustrated in FIG. 1.
[0228] As expected, tumor volume increased very rapidly in control
group receiving formulation buffer. Rapid tumour growth was also
observed in the group receiving mPD-1 antibody. A slight delay in
tumor volume was seen in groups receiving MVATG18124 either alone
or in combination with the isotype control. Tumor growth was
greatly reduced in the "combination" group injected with both
MVATG18124 and mPD-1 antibodies (FIG. 1A). Effects on mice survival
are more pronounced since about 70% of mice treated with both
MVATG18124 and mPD-1 antibodies are still alive more than 100 days
following tumor implantation versus 10% of MVATG18124-treated
animal group (significant differences). In contrast, control,
antibody-treated and isotype-treated animals died within less than
50 days (FIG. 1B).
Example 2: Combination of MVATG18124 with Anti-CTLA-4 Mab
[0229] The combination of CTLA-4 inhibitor (commercial clone 9D9;
BioXCell) with antigen-expressing MVATG18124 was tested in vivo in
a metastatic CT26-CL25 model. 2.times.10.sup.5 CT26.CL25 cells were
injected intravenously (iv) in Balb/c mice. Days 2 and 9,
MVATG18124 encoding beta-galactosidase or its empty vector control
MVAN33.1 was administered iv at the dose of 1.10.sup.4 pfu. 250
.mu.g of the murine anti-CTLA4 antibody 9D9 (BioXCell) or its IgG2b
isotype control (clone MPC-11 BioXCell) were injected
intraperitoneally (ip) at days 3 and 10. The survival of mice was
followed for more than 60 days. The viral dose of 1.10.sup.4 pfu
was identified as optimal dose to increase survival rates in this
tumor model (data not shown).
[0230] As illustrated in FIG. 2, treatment with anti CTLA4
antibodies or its isotype control alone showed weak effects
comparable to those observed with the empty MVA vector MVATGN33.1
(35% survival at day 20 for all three groups). In contrast,
treatment with MVATG18124 alone or in combination with the isotype
control increased mice survival (35% survival at day 35 for both
groups). The group of mice treated with the combination of
MVATG18124 and anti-CTLA4 antibodies greatly increased the
percentage of 35% survival to more than 60 days.
[0231] Thus, we have clearly demonstrated a clear anti-tumor effect
of treating MVA-based immunotherapeutic vaccine and the immune
checkpoint blocker anti CTLA4.
Example 3: Lymphoid Cell Population Studies in Dissociated Lungs of
Treated Mice
Determination of IFN.gamma. Positive CD8dim CD3dim Cells
[0232] Cellular response was examined in mice treated with either
the anti-CTLA-4 antibody the antigen-expressing MVA or both as well
as in untreated (i.e. naive) mice. Five BALB/c mice per group were
injected iv with MVATG18124 (1.10.sup.4 pfu) day 1 and 8 or ip with
250 .mu.g anti CTLA-4 (clone 9D9, BioXCell) day 2 and 9 or both.
Mice were sacrificed day 15, and lungs were isolated. Lungs from
all 5 mice per group were pooled, cut into small pieces in C-tubes
(Miltenyi) and enzymatically dissociated using a tissue
dissociation kit (Miltenyi, 130-096-730) using the Gentle OctoMACS
(Miltenyi) according to the manufacturer's recommendations.
[0233] Lung-derived cells were plated at 2.10.sup.6 cells/well (96
well plate) in T cell-specific medium (TexMACS, Miltenyi). Cells
were activated by co-cultivation with peptide-loaded bone
marrow-derived murine dendritic cells (bmDCs): bmDCs from BALB/c
mice were generated from bone marrow cells matured in the presence
of murine GM-CSF (Peprotech, 100 .mu.g/ml) for 10 days.
Alternatively, activation was facilitated by incubation with 1
.mu.g of anti CD28 (clone PV-1). Concanavalin A (ConA, 5 .mu.g/ml)
served as non-specific activator. Anti CD107a antibody (clone
eBio1D4B) was added to label degranulating cells. Secretion of
cytokines was blocked after one 20 hour of incubation by adding
GolgiPlug/Brefeldin (1:1000, BD Biosciences). After 5 hours total
incubation time, cells were washed and stained for viability
(LiveDead, Fixable violet dead cell staining kit) and surface
markers CD8a (clone 53-6.7) and CD3c (clone 145-2C11). Cells were
stained intracellularly for IFN-.gamma. (clone XMG1.2) using the BD
Cytofix/Cytoperm kit (BD Biosciences). Cells were fixed and
analyzed by flow cytometry (Navios, Beckman Coulter).
[0234] Combinatorial treatment of MVATG18124 and anti CTLA-4 in
naive BALB/c mice leads to the appearance of a sub population of
lymphocyte cells named CD8.sup.dimCD3.sup.dim in the lung. FIG. 3B
represents an example of CD8.sup.dimCD3.sup.dim population in
CD3/CD8 dot blot lung cells from mice treated with anti CTLA-4 and
MVATG18124. Gating living lymphocytes, the percentages of gated
CD8.sup.dimCD3.sup.dim cells in two independent experiments are
illustrated in FIG. 3A. More specifically, the population of
CD8.sup.dimCD3.sup.dim was clearly increased after treatment with
MVATG18124 and even more after combinatorial treatment with
MVATG18124+anti CTLA-4.
[0235] Next, the percentage of intracellular IFN .gamma..sup.+
cells was assessed in the CD8.sup.dimCD3.sup.dim population. Within
this CD8.sup.dimCD3.sup.dim population, highest induction of
IFN-.gamma.+ cells was observed in mice treated with
MVATG18124+anti CTLA-4 (FIGS. 4A and 4B).
[0236] In summary, combinatorial treatment of MVATG18124 and anti
CTLA-4 in naive BALB/c mice leads to the appearance of a
CD8.sup.dimCD3.sup.dim lymphocyte cell population in the lung. Upon
stimulation with ConA and a high percentage of
CD8.sup.dimCD3.sup.dim from mice treated with MVATG18124+anti
CTLA-4 can be induced to secrete IFN-.gamma..
[0237] The CD3.sup.dimCD8.sup.dim cell population was analyzed in
greater detail. [0238] In the course of our analyses, we observed
that the CD3.sup.dimCD8.sup.dim population was positive for the
killer cell lectin like receptor G1 (KLRG1') and negative or low
for CD127 (IL-7R.alpha.) (CD127.sup.-/low). This phenotype is
associated with antigen experienced short lived effector cells
(SLECs) (Obar et al., 2011, J Immunol., doi:
10.4049/jimmunol.1102335; Sarkar et al., 2008, J Exp Med 205(3):
625-40). [0239] The CD3.sup.dimCD8.sup.dim KLRG1.sup.+ population
infiltrating/present in the lung of mice treated with MVATG18124
and anti CTLA-4 responds to an antigen-specific stimulus with
IFN-.gamma. secretion and degranulation (CD107a). Determination of
IFN-.gamma., CD107a and KLRG1 Positive Cells in
CD8.sup.dimCD3.sup.dim Cell Population
[0240] As described above, BALB/c mice were injected i.v. with
MVA-.beta.-gal or an empty vector MVATGN33.1 at 1.10.sup.4 pfu. On
days 3 and 10, mice received 250 .mu.g anti CTLA-4 i.p. Lungs were
taken day 14 and enzymatically dissociated. Lung derived cells were
plated at 2.10.sup.6 cells/well (96 well plate) in T cell-specific
medium (TexMACS, Miltenyi), activated by incubation with 1 .mu.g of
anti CD28 (clone PV-1) and stimulated with a .beta.-gal specific
peptide (T9L-3) or a control peptide (T8G) in the presence of anti
CD107a antibody (clone eBio1D4B) to label degranulating cells.
Secretion of cytokines was blocked after one hour of incubation by
adding GolgiPlug/Brefeldin (1:1000, BD Biosciences). After 5h total
incubation time, cells were washed and stained for viability
(LiveDead, Fixable violet dead cell staining kit) and for the
surface markers CD8a (clone 53-6.7), CD3.epsilon. (clone 145-2C11)
and KLRG1 (clone 2F1/KLRG1). Cells were stained intracellularly for
IFN-.gamma. (clone XMG1.2) using the BD Cytofix/Cytoperm kit (BD
Biosciences). Cells were fixed and analyzed by flow cytometry
(Navios, Beckman Coulter). After 5 hours, cells and stained
CD8a.
[0241] Treatment with MVATG18124 or an empty control vector, and
even more the combination of MVATG18124 and anti CTLA-4 in naive
BALB/c mice lead to the appearance of a
CD8.sup.dimCD3.sup.dimKLRG1.sup.+ lymphocyte cell population in the
lung. Upon ex vivo stimulation with the .beta.-gal peptide T9L-3,
this population secreted IFN.gamma. and degranulated (CD107a) and,
as illustrated in FIG. 6, the percentage was higher after treatment
with MVATG18124 and anti CTLA-4 than in the other groups treated
with the control vector (MVATGN33.1 alone or with anti CTLA-4),
with MVATG18124 alone or with anti CTLA-4 alone. As expected,
stimulation with an irrelevant peptide (T8G peptide) did not yield
such induction.
[0242] In conclusion, the treatment with MVATG18124 and anti CTLA-4
increases the b-gal specific response in a
CD3.sup.dimCD8.sup.dimKLRG1.sup.+ cell population in the lung.
Example 4: Secretion of IFN-.gamma.
[0243] Further, the number of IFN.gamma. secreting splenic
lymphocytes was investigated following BALB/c mice treatment with
MVATG18124 or MVAN33.1 at 1.10.sup.4 pfu (D1 and 8) and anti CTLA-4
or its isotype control (D2 and D9, 250 .mu.g ip). Measurement was
performed at day 14 by ELISpot (Enzyme-linked immunospot)
assay.
Plate Preparation
[0244] The day preceeding the experiments, membrane ELISpot plates
(Millipore, ref. MSIPS4W10) were prewetted with 15 .mu.L of 35%
ethanol per well with maximum incubation time of 2 min. Plates were
washed five times with 200 .mu.L per well of sterile water.
[0245] ELISpot plates were coated with a rat anti-mouse IFN-.gamma.
monoclonal antibody (AN18 Mabtech, ref. 3321-3-1000) diluted at 15
.mu.g/mL in sterile DPBS (Sigma, ref. D8357) (100 .mu.L/well). The
plates were covered and incubated overnight at 4.degree. C. The
next day, the plates were washed 3 times with sterile PBS (200
.mu.L/well) and were saturated for 1 h at 37.degree. C. with 200
.mu.L/well of complete RPMI 1640 medium (RPM11640 medium, (Sigma
R0883); L-Glutamine 2 mM, (Sigma G6392); Gentamycin 0.01 g/L
(Schering Plough U570036); Fetal Calf Serum 10% (JRH 12003-1000M)
550 .mu.l of a solution 5.times.10.sup.-2 M bmercaptoethanol).
Sample Preparation
[0246] For ex vivo evaluation of the frequency of the specific CD8+
T cells induced by immunization, euthanized animals were
splenectomized 7 days after last immunization. Spleens from the
same group were pooled in a cell strainer in a well of a 6-wells
culture plate containing 5 mL of complete medium. Spleens were
crushed with a syringe piston and the cell strainer discarded.
Splenocytes were collected with 8 mL of complete medium and
transferred in a 15 mL falcon tube. The splenocytes suspension was
laid over 4 mL of Lympholyte.RTM.-M separation cell media
(Cedarlane, ref. CL5035) and centrifuged (20 min, 1500.times.g,
room temperature). The interphase containing lymphocytes was
collected and rinsed three times with 10 mL of PBS. Between each
rinse step, cells were centrifuged (4 min at 400 xg) and the
supernatants were discarded. The remaining red blood cells were
lysed by addition on the lymphocyte pellet of 2 mL of RBC lysis
buffer 1.times. (10.times. solution: BD Pharm Lyse.TM. lysing
solution, ref. 555899) diluted in sterile water. Each tube was
gently vortexed immediately after adding the lysis solution and
incubated at room temperature for 15 minutes. Lysis was topped by
the addition of 10 mL DPBS followed by centrifugation 4 min at
400.times.g., the cells were resuspended in 10 mL of complete RPMI
1640 medium. The cells were counted with a Z2 Cell Counter (Beckman
Coulter) and the cell concentration was adjusted at
1.times.10.sup.7 cells per mL in complete RPMI 1640 medium.
Assay
[0247] To perform the ELISpot assay, 100 .mu.L of lymphocyte
suspension from each group (1.times.10.sup.6 cells) were added to
each wells of a coated 96-wells plate. One given condition was
tested in triplicates or in quadriplicates. One hundred microliter
of different indicated peptides (2 .mu.g/mL in complete RPMI 1640
medium) was added to the cell suspension. ConA (Sigma, ref. C5275)
was used as positive control (5 .mu.g/mL final concentration)
MVA-specific peptide (S9L-8) was used for immunization control. The
plates were then incubated at 37.degree. C. in 5% CO.sub.2 for 16
to 20 hours. Then, plates were washed three times with DPBS (200
.mu.L). Biotinylated rat anti-mouse IFN-.gamma. monoclonal antibody
(Mabtech, ref. 3321-6-1000) was diluted at 1 .mu.g/mL in antibody
mix buffer (PBS, 0.5% SVF) and distributed at 100 .mu.L/well.
Plates were incubated 2 hours at room temperature in darkness, and
then washed three times in DPBS (200 .mu.L). One hundred microliter
of Extravidin-Phosphatase alkaline (SIGMA, ref. E2636) (Diluted
1/5000 in antibody mix buffer) was added to each well and the
plates were incubated for 1 hour at room temperature in darkness.
Plates were finally washed three times in DPBS (200 .mu.L). One
hundred microliter of BCIP/NBT (Sigma, ref. B5655, one caps in 10
mL MilliQ water) was added to each well until blue spots develop
and then plates were washed thoroughly in tap water and dried.
Data Acquisition
[0248] Spots were counted with an ELISpot reader (CTL Immunospot
reader, S5 UV). A visual quality control (comparing machine scans
and plates) was performed on each well to ensure that the counts
provided by the ELISpot reader match the reality of the picture.
Results were expressed as number of spot forming units (sfu) per
1.times.10.sup.6 splenic lymphocytes (mean) for each triplicate or
quadriplicate. Specific ELISpot response was determined either with
the DFR(eq) method (Moodie et al., Cancer Immunol Immunother. 2010
October; 59(10):1489-501) or with an empirical cut-off calculated
as the mean number of spots from blank wells plus two times the
standard deviation of this mean number of spots.
[0249] As shown in FIG. 5, ELIspot analysis of splenic lymphocytes
stimulated with the .beta.-gal-specific peptide T9L3 showed
.beta.-gal specific response in splenic lymphocytes treated with
MVATG18124. The specific responses increased further after
combinatorial treatment with anti CTLA-4 but not with its isotype
control. Stimulation with the non-specific peptide T8G showed no
increase of IFN-.beta. secreting cells.
Example 5: Combinatorial Effect of TG4010 (MVA-MUC1-IL-2) and Anti
PD-1 (RMP1.14) in a MU1-Positive CT26-Based Tumor Model
[0250] TG4010 is a MVA vector encoding the full cDNA sequence of
human MUC1 and human IL-2. Anti-tumoral efficacy provided by this
vector was tested in a CT26-based MUC1-positive cell line which
gives rise to MUC1-positive tumors after s.c. injection, as well as
lung tumors after i.v. injection.
Generation of CT26-MUC1 Cell Line
[0251] The murine colon carcinoma cell line CT26 WT (ATCC CRL-2638)
was stably transfected with the plasmid pTG5077 encoding the full
cDNA sequence of human MUC1 under the control of the CMV promoter
as well as a G418-resistance gene under the control of the SV40
promoter. CT26 cells were transfected by means of Lipofectamine LTX
with pTG5077, and cultivated in the presence of 0.4 mg/ml G418 to
select for stable transfectants. After 14 days, living cells were
labeled with a monoclonal antibody against MUC1 (H23+second
antibody Goat anti mouse-FITC). Positive cells were sorted (FACS
ARIA), transferred in 96 well plates at 1 cell/well. Outgrowing
clones were analyzed for stable MUC1 expression by flow cytometry
up to day 60 after transfection. Four stably MUC1-expressing clones
were then tested for their ability to induce tumor growth in BALB/c
mice after sc injection and after iv injection. One clone was
retained after verification that s.c.-implanted tumors and lung
tumors obtained after iv injection were MUC1-positive.
Therapeutic Efficacy of TG4010 in the CT26-MUC1 Tumor Model
[0252] 2.10.sup.5 CT26-MUC1 cells were injected s.c. or i.v. in
BALB/c mice to generate sc tumors or lung tumors, respectively. On
day 2 and 9 after tumor challenge, mice were treated s.c. or i.v.,
respectively, with 1.10.sup.7 TG4010 or the empty control vector
MVATGN33.1. Mean tumor volume or percent survival were monitored
over time. TG4010 showed significant improvement of survival in the
iv/iv lung tumor model (p=0.00642) and significant reduction of
tumor growth in the sc/sc tumor model (see FIGS. 7A and 7B,
respectively).
Therapeutic Efficacy of TG4010 in Combination with Anti-PD1 in the
CT26-MUC1 Tumor Model
[0253] BALB/c mice were injected s.c. with 2.10.sup.6 CT26.MUC1
cells. On days 2 and 9 after tumor implantation, mice were treated
sc with TG4010 (also designated MVATG9931) or an empty control
vector (MVATGN33.1) at the suboptimal dose of 1.10.sup.6 pfu. On
days 10, 13, 15 and 17, mice received 250 .mu.g anti PD-1 (RMP1.14,
IgG2a, BioXCell). Mice were sacrificed when the tumors reached the
size of 2,000 mm.sup.3. Tumor volume and animal survival were
followed over time.
[0254] As illustrated in FIG. 8B, tumors started to grow at day 16
and tumor volume increased rapidly and regularly over time in
control group receiving formulation buffer, as expected. A delay in
tumour growth was observed in the groups receiving mPD-1 antibody,
TG4010 alone and empty MVATGN33.1 alone or with anti-PD-1. In
contrast, tumor growth was greatly reduced in the "combination"
group injected with both TG4010 and mPD-1 antibody. Effects on mice
survival are also observed as shown in FIG. 8A. Indeed, about 70%
of mice treated with both TG4010 and mPD-1 antibody are still alive
more than 60 days following tumor implantation while approximately
10% and 20% of animals treated with the empty control (without or
with the anti-PD-1 antibody) remained alive. Only 30 and 40% of
animals respectively treated with TG4010 alone or with anti-PD-1
antibody survived 2 months after tumor implantation. In contrast,
control group treated with formulation buffer died within less than
45 days. The increased survival provided by the combinatorial
treatment was maintained overtime (120 days after tumor
implantation) (data not shown).
[0255] The same experiment as above was also conducted except that
mice were treated with 2 injections of 1.10.sup.7 pfu of MVATG9931
(i.e. TG4010) or the negative MVATGN33.1 control vector, optionally
followed by four i.p. administrations of 250 .mu.g of anti-PD1. As
illustrated in FIG. 9, a substantial increase in median survival of
70 days for the combinatorial treatment is however noticeable in
comparison to 46 and 49.5 days for the mice treated with MVATG9931
and anti-PD-1, respectively. Note also that the survival increase
is significant (p=0.008) when the combination of MVATG9931 and
anti-PD-1 treatments is compared to the control empty virus
MVATGN33.1 and anti-PD-1 modalities, suggesting a MUC-1 specific
interaction at this dose of virus. At the highest dose of
1.times.10.sup.7 PFU for MVATG9931, the best therapeutic activity
among all the conditions tested is achieved for the combination
with significance reached against each treatment modality alone
(p=0.014 against MVATG9931 and p<0.001 against anti-PD-1; FIG.
9). In conclusion, the combination of MVATG9931 with an anti-PD-1
antibody allowed to obtain the best therapeutic index in the
ectopic CT26-MUC1 model in comparison to each treatment alone which
paves the way to the clinical evaluation of this combination
therapy approach.
[0256] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific method and reagents described herein,
including alternatives, variants, additions, deletions,
modifications and substitutions. Such equivalents are considered to
be within the scope of this invention and are covered by the
following claims.
Sequence CWU 1
1
61557PRTHomo sapiens 1Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu
Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly
His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala
Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala
Val Ser Met Thr Ser Ser Val Leu Ser Ser His 50 55 60 Ser Pro Gly
Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu 65 70 75 80 Ala
Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln 85 90
95 Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr
100 105 110 Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro
Ala Pro 115 120 125 Ser Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
Thr Ser Ala Pro 130 135 140 Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
Pro Pro Ala His Gly Val 145 150 155 160 Thr Ser Ala Pro Asp Thr Arg
Pro Ala Pro Gly Ser Thr Ala Pro Pro 165 170 175 Ala His Gly Val Thr
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser 180 185 190 Thr Ala Pro
Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro 195 200 205 Ala
Pro Gly Ser Thr Ala Pro Pro Gly His Gly Val Thr Ser Ala Pro 210 215
220 Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
225 230 235 240 Thr Ser Ala Pro Asp Asn Arg Pro Ala Leu Gly Ser Thr
Ala Pro Pro 245 250 255 Val His Asn Val Thr Ser Ala Ser Gly Ser Ala
Ser Gly Ser Ala Ser 260 265 270 Thr Leu Val His Asn Gly Thr Ser Ala
Arg Ala Thr Thr Thr Pro Ala 275 280 285 Ser Lys Ser Thr Pro Phe Ser
Ile Pro Ser His His Ser Asp Thr Pro 290 295 300 Thr Thr Leu Ala Ser
His Ser Thr Lys Thr Asp Ala Ser Ser Thr His 305 310 315 320 His Ser
Thr Val Pro Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro 325 330 335
Gln Leu Ser Thr Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser 340
345 350 Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr
Tyr 355 360 365 Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln
Ile Tyr Lys 370 375 380 Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys
Phe Arg Pro Gly Ser 385 390 395 400 Val Val Val Gln Leu Thr Leu Ala
Phe Arg Glu Gly Thr Ile Asn Val 405 410 415 His Asp Val Glu Thr Gln
Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser 420 425 430 Arg Tyr Asn Leu
Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe 435 440 445 Pro Phe
Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala 450 455 460
Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu 465
470 475 480 Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly
Gln Leu 485 490 495 Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met
Ser Glu Tyr Pro 500 505 510 Thr Tyr His Thr His Gly Arg Tyr Val Pro
Pro Ser Ser Thr Asp Arg 515 520 525 Ser Pro Tyr Glu Lys Val Ser Ala
Gly Asn Gly Gly Ser Ser Leu Ser 530 535 540 Tyr Thr Asn Pro Ala Val
Ala Ala Thr Ser Ala Asn Leu 545 550 555 21674DNAHomo sapiens
2atgacaccgg gcacccagtc tcctttcttc ctgctgctgc tcctcacagt gcttacagtt
60gttacaggtt ctggtcatgc aagctctacc ccaggtggag aaaaggagac ttcggctacc
120cagagaagtt cagtgcccag ctctactgag aagaatgctg tgagtatgac
cagcagcgta 180ctctccagcc acagccccgg ttcaggctcc tccaccactc
agggacagga tgtcactctg 240gccccggcca cggaaccagc ttcaggttca
gctgccacct ggggacagga tgtcacctcg 300gtcccagtca ccaggccagc
cctgggctcc accaccccgc cagcccacga tgtcacctca 360gccccggaca
acaagccagc cccatctcca ggctccaccg cccccccagc ccacggtgtc
420acctcggccc cggacaccag gccggcacca ggaagcacag caccacccgc
gcatggtgta 480acatctgccc ctgatacacg tcctgctccc ggtagtactg
cgccgcctgc acacggggtg 540acgtctgctc ccgacactcg gccggcgcct
gggtcaacgg ctcctccggc acatggagtt 600acttctgcgc cagacacgcg
acccgctcca ggtagtacgg cgccaccggg gcatggagtg 660acatctgcgc
ccgatactag gccggccccg ggctccaccg cccccccagc ccatggtgtc
720acctcggccc cggacaacag gcccgccttg ggctccaccg cccctccagt
ccacaatgtc 780acctcggcct caggctctgc atcaggctca gcttctactc
tggtgcacaa cggcacctct 840gccagggcta ccacaacccc agccagcaag
agcactccat tctcaattcc cagccaccac 900tctgatactc ctaccaccct
tgccagccat agcaccaaga ctgatgccag tagcactcac 960catagcacgg
tacctcctct cacctcctcc aatcacagca cttctcccca gttgtctact
1020ggggtctctt tctttttcct gtcttttcac atttcaaacc tccagtttaa
ttcctctctg 1080gaagatccca gcaccgacta ctaccaagag ctgcagagag
acatttctga aatgtttttg 1140cagatttata aacaaggggg ttttctgggc
ctctccaata ttaagttcag gccaggatct 1200gtggtggtac aattgactct
ggccttccga gaaggtacca tcaatgtcca cgacgtggag 1260acacagttca
atcagtataa aacggaagca gcctctcgat ataacctgac gatctcagac
1320gtcagcgtga gtgatgtgcc atttcctttc tctgcccagt ctggggctgg
ggtgccaggc 1380tggggcatcg cgctgctggt gctggtctgt gttctggttg
cgctggccat tgtctatctc 1440attgccttgg ctgtctgtca gtgccgccga
aagaactacg ggcagctgga catctttcca 1500gcccgggata cctaccatcc
tatgagcgag taccccacct accacaccca tgggcgctat 1560gtgcccccta
gcagtaccga tcgtagcccc tatgagaagg tttctgcagg taatggtggc
1620agcagcctct cttacacaaa cccagcagtg gcagccactt ctgccaactt gtag
167431047PRTEscherichia coli 3Met Ser Phe Thr Leu Thr Asn Lys Asn
Val Ile Phe Val Ala Gly Leu 1 5 10 15 Gly Gly Ile Gly Leu Asp Thr
Ser Lys Glu Leu Leu Lys Arg Asp Pro 20 25 30 Val Val Leu Gln Arg
Arg Asp Trp Glu Asn Pro Gly Val Thr Gln Leu 35 40 45 Asn Arg Leu
Ala Ala His Pro Pro Phe Ala Ser Trp Arg Asn Ser Glu 50 55 60 Glu
Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly 65 70
75 80 Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val Pro Glu
Ser 85 90 95 Trp Leu Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val
Val Pro Ser 100 105 110 Asn Trp Gln Met His Gly Tyr Asp Ala Pro Ile
Tyr Thr Asn Val Thr 115 120 125 Tyr Pro Ile Thr Val Asn Pro Pro Phe
Val Pro Thr Glu Asn Pro Thr 130 135 140 Gly Cys Tyr Ser Leu Thr Phe
Asn Val Asp Glu Ser Trp Leu Gln Glu 145 150 155 160 Gly Gln Thr Arg
Ile Ile Phe Asp Gly Val Asn Ser Ala Phe His Leu 165 170 175 Trp Cys
Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro 180 185 190
Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu 195
200 205 Ala Val Met Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp
Gln 210 215 220 Asp Met Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser
Leu Leu His 225 230 235 240 Lys Pro Thr Thr Gln Ile Ser Asp Phe His
Val Ala Thr Arg Phe Asn 245 250 255 Asp Asp Phe Ser Arg Ala Val Leu
Glu Ala Glu Val Gln Met Cys Gly 260 265 270 Glu Leu Arg Asp Tyr Leu
Arg Val Thr Val Ser Leu Trp Gln Gly Glu 275 280 285 Thr Gln Val Ala
Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp 290 295 300 Glu Arg
Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu Asn Val Glu 305 310 315
320 Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val
325 330 335 Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala Glu
Ala Cys 340 345 350 Asp Val Gly Phe Arg Glu Val Arg Ile Glu Asn Gly
Leu Leu Leu Leu 355 360 365 Asn Gly Lys Pro Leu Leu Ile Arg Gly Val
Asn Arg His Glu His His 370 375 380 Pro Leu His Gly Gln Val Met Asp
Glu Gln Thr Met Val Gln Asp Ile 385 390 395 400 Leu Leu Met Lys Gln
Asn Asn Phe Asn Ala Val Arg Cys Ser His Tyr 405 410 415 Pro Asn His
Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr 420 425 430 Val
Val Asp Glu Ala Asn Ile Glu Thr His Gly Met Val Pro Met Asn 435 440
445 Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val
450 455 460 Thr Arg Met Val Gln Arg Asp Arg Asn His Pro Ser Val Ile
Ile Trp 465 470 475 480 Ser Leu Gly Asn Glu Ser Gly His Gly Ala Asn
His Asp Ala Leu Tyr 485 490 495 Arg Trp Ile Lys Ser Val Asp Pro Ser
Arg Pro Val Gln Tyr Glu Gly 500 505 510 Gly Gly Ala Asp Thr Thr Ala
Thr Asp Ile Ile Cys Pro Met Tyr Ala 515 520 525 Arg Val Asp Glu Asp
Gln Pro Phe Pro Ala Val Pro Lys Trp Ser Ile 530 535 540 Lys Lys Trp
Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys 545 550 555 560
Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr 565
570 575 Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly Phe Val
Trp 580 585 590 Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn
Gly Asn Pro 595 600 605 Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr
Pro Asn Asp Arg Gln 610 615 620 Phe Cys Met Asn Gly Leu Val Phe Ala
Asp Arg Thr Pro His Pro Ala 625 630 635 640 Leu Thr Glu Ala Lys His
Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser 645 650 655 Gly Gln Thr Ile
Glu Val Thr Ser Glu Tyr Leu Phe Arg His Ser Asp 660 665 670 Asn Glu
Leu Leu His Trp Met Val Ala Leu Asp Gly Lys Pro Leu Ala 675 680 685
Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu Ile 690
695 700 Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln Leu Trp
Leu 705 710 715 720 Thr Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp
Ser Glu Ala Gly 725 730 735 His Ile Ser Ala Trp Gln Gln Trp Arg Leu
Ala Glu Asn Leu Ser Val 740 745 750 Thr Leu Pro Ala Ala Ser His Ala
Ile Pro His Leu Thr Thr Ser Glu 755 760 765 Met Asp Phe Cys Ile Glu
Leu Gly Asn Lys Arg Trp Gln Phe Asn Arg 770 775 780 Gln Ser Gly Phe
Leu Ser Gln Met Trp Ile Gly Asp Lys Lys Gln Leu 785 790 795 800 Leu
Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu Asp Asn Asp 805 810
815 Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala Trp Val Glu
820 825 830 Arg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala Leu
Leu Gln 835 840 845 Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile
Thr Thr Ala His 850 855 860 Ala Trp Gln His Gln Gly Lys Thr Leu Phe
Ile Ser Arg Lys Thr Tyr 865 870 875 880 Arg Ile Asp Gly Ser Gly Gln
Met Ala Ile Thr Val Asp Val Glu Val 885 890 895 Ala Ser Asp Thr Pro
His Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu 900 905 910 Ala Gln Val
Ala Glu Arg Val Asn Trp Leu Gly Leu Gly Pro Gln Glu 915 920 925 Asn
Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg Trp Asp Leu 930 935
940 Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser Glu Asn Gly
945 950 955 960 Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His
Gln Trp Arg 965 970 975 Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser
Gln Gln Gln Leu Met 980 985 990 Glu Thr Ser His Arg His Leu Leu His
Ala Glu Glu Gly Thr Trp Leu 995 1000 1005 Asn Ile Asp Gly Phe His
Met Gly Ile Gly Gly Asp Asp Ser Trp 1010 1015 1020 Ser Pro Ser Val
Ser Ala Glu Leu Gln Leu Ser Ala Gly Arg Tyr 1025 1030 1035 His Tyr
Gln Leu Val Trp Cys Gln Lys 1040 1045 4114DNAVaccinia virus
4tttattctat acttaaaaaa tgaaaataaa tacaaaggtt cttgagggtt gtgttaaatt
60gaaagcgaga aataatcata aattatttca ttatcgcgat atccgttaag tttg
114546DNAartificial sequencePCR primer for LacZ cloning 5ccaccttaat
taagccgcca ccatgtcgtt tactttgacc aacaag 46643DNAartificial
sequencePCR primer for LacZ cloning 6accaactcga gagaaaaatt
atttttgaca ccagaccaac tgg 43
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