U.S. patent application number 15/091060 was filed with the patent office on 2016-07-21 for combination of vaccination and inhibition of the pd-1 pathway.
This patent application is currently assigned to CureVac AG. The applicant listed for this patent is CureVac AG. Invention is credited to Mariola FOTIN-MLECZEK, Karl-Josef KALLEN, Jochen PROBST.
Application Number | 20160206719 15/091060 |
Document ID | / |
Family ID | 47748572 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206719 |
Kind Code |
A1 |
FOTIN-MLECZEK; Mariola ; et
al. |
July 21, 2016 |
COMBINATION OF VACCINATION AND INHIBITION OF THE PD-1 PATHWAY
Abstract
The present invention relates to a vaccine/inhibitor combination
comprising an RNA vaccine comprising at least one RNA comprising at
least one open reading frame (ORF) coding for at least one antigen
and a composition comprising at least one PD-1 pathway inhibitor,
preferably directed against PD-1 receptor or its ligands PD-L1 and
PD-L2. The present invention furthermore relates to a
pharmaceutical composition and a kit of parts comprising the
components of such a vaccine/inhibitor combination. Additionally
the present invention relates to medical use of such a
vaccine/inhibitor combination, the pharmaceutical composition and
the kit of parts comprising such a vaccine/inhibitor combination,
particularly for the prevention or treatment of tumor or cancer
diseases or infectious diseases. Furthermore, the present invention
relates to the use of an RNA vaccine in therapy in combination with
a PD-1 pathway inhibitor and to the use of a PD-1 pathway inhibitor
in therapy in combination with an RNA vaccine.
Inventors: |
FOTIN-MLECZEK; Mariola;
(Sindelfingen, DE) ; KALLEN; Karl-Josef;
(Konigsdorf, DE) ; PROBST; Jochen; (Wolfschlugen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac AG |
Tubingen |
|
DE |
|
|
Assignee: |
CureVac AG
Tubingen
DE
|
Family ID: |
47748572 |
Appl. No.: |
15/091060 |
Filed: |
April 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14769720 |
Aug 21, 2015 |
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PCT/EP2014/000461 |
Feb 21, 2014 |
|
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15091060 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61P 33/00 20180101; A61P 43/00 20180101; A61K 39/39558 20130101;
A61P 35/00 20180101; C07K 2317/76 20130101; C07K 16/2803 20130101;
A61K 2039/55516 20130101; A61K 39/0005 20130101; A61K 39/0011
20130101; A61P 31/04 20180101; A61K 2039/505 20130101; A61P 31/10
20180101; C07K 16/2818 20130101; A61K 39/39 20130101; Y02A 50/30
20180101; A61P 31/12 20180101; A61K 2039/53 20130101; A61K 39/39558
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2013 |
EP |
PCT/EP2013/000526 |
Claims
1. A method of treating a subject comprising administering to the
subject an effective amount of: (i) an immunogenic composition
comprising at least one RNA encoding at least one antigen; and (ii)
an immune checkpoint inhibitor.
2. The method of claim 1, wherein the immune checkpoint inhibitor
comprises a CTLA-4 pathway inhibitor or a PD-1 pathway
inhibitor
3. The method of claim 2, wherein the CTLA-4 pathway inhibitor
comprises an antibody directed against the CTLA-4 receptor.
4. The method of claim 3, wherein the subject has a cancer and the
at least one RNA is a mRNA encoding a tumor antigen.
5. The method of claim 2, wherein the PD-1 pathway inhibitor
comprises an inhibitor directed against PD-1.
6. The method of claim 2, wherein the PD-1 pathway inhibitor
comprises an inhibitor directed against PD-L1 or PD-L2.
7. The method of claim 1, wherein the immune checkpoint inhibitor
is administered after said immunogenic composition.
8. The method of claim 1, wherein the immune checkpoint inhibitor
is comprises an antibody, a nucleic acid encoding an antibody or an
siRNA.
9. The method of claim 8, wherein the immune checkpoint inhibitor
is an antibody, or a nucleic-acid encoded antibody.
10. The method of claim 1, wherein the at least one RNA encodes an
infectious disease antigen.
11. The method of claim 1, wherein the at least one RNA encodes a
cancer or tumor cell antigen.
12. The method of claim 1, wherein the at least one RNA is an
mRNA.
13. The method of claim 1, wherein the RNA is a codon optimized
RNA.
14. The method of claim 1, wherein the at least one RNA comprises
an increased G/C content relative to a wild type RNA encoding the
antigen.
15. The method of claim 1, wherein the at least one RNA is
complexed with a carrier.
16. The method of claim 15, wherein the carrier is a cationic,
polycationic or polymeric carrier.
17. The method of claim 16, wherein the carrier comprises
protamine.
18. The method of claim 1, wherein the subject has a cancer or
tumor disease.
19. A kit of parts comprising: (i) an immunogenic composition
comprising at least one RNA encoding at least one antigen; and (ii)
a composition comprising an immune checkpoint inhibitor.
20. A pharmaceutical composition comprising an effective amount of:
(i) at least one RNA encoding at least one antigen and (ii) an
immune checkpoint inhibitor.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 14/769,720, filed Aug. 21, 2015, which is a national phase
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/EP2014/000461, filed Feb. 21, 2014, which claims priority
to International Application No. PCT/EP2013/000526, filed Feb. 22,
2013. The entire text of each of the above referenced disclosures
is specifically incorporated herein by reference.
[0002] The present invention relates to a vaccine/inhibitor
combination comprising an RNA vaccine comprising at least one RNA
comprising at least one open reading frame (ORF) coding for at
least one antigen and a composition comprising at least one PD-1
pathway inhibitor, preferably directed against PD-1 receptor or its
ligands PD-L1 and/or PD-L2. The present invention furthermore
relates to a pharmaceutical composition and a kit of parts
comprising such a vaccine/inhibitor combination. Additionally, the
present invention relates to the medical use of such a
vaccine/inhibitor combination, the pharmaceutical composition and
the kit of parts comprising such a vaccine/inhibitor combination,
particularly for the prevention or treatment of tumor or cancer
diseases or infectious diseases. Furthermore, the present invention
relates to the use of an RNA vaccine in therapy in combination with
a PD-1 pathway inhibitor and to the use of a PD-1 pathway inhibitor
in therapy in combination with an RNA vaccine.
[0003] Traditionally, cancer immunotherapy was focused on
stimulating the immune system through vaccination or adoptive
cellular immunotherapy to elicit an anti-tumor response. This
approach was based on the assumption that tumor cells express
antigenic targets but that anti-tumor T cells were not sufficiently
activated. Therefore, to circumvent this problem, it was mainly
tried to increase the recognition of these antigenic targets by
stimulating key positive co-stimulatory and innate immune pathways
(such as CD28, CD154 and TLR receptors).
[0004] More recently, it became clear that the immune system does
recognize tumor antigens but remains quiescent although tumor
antigens are present. This observation led to the hypothesis that
there are specific inhibitory mechanisms which limit or even shut
down the anti-tumor response. This hypothesis was confirmed when
negative regulatory T cell surface molecules were discovered which
are upregulated in activated T cells to dampen their activity,
resulting in less effective killing of tumor cells. These
inhibitory molecules were termed negative co-stimulatory molecules
due to their homology to the T cell co-stimulatory molecule CD28.
These proteins, also referred to as immune checkpoint proteins,
function in multiple pathways including the attenuation of early
activation signals, competition for positive co-stimulation and
direct inhibition of antigen presenting cells (Bour-Jordan et al.,
2011. Immunol Rev. 241(1):180-205; PMID: 21488898). One member of
this protein family is programmed death-1 (PD-1) and its ligands
B7-H1/PD-L1 (CD274) and B7-DC/PD-L2 (CD273).
[0005] PD-1 is expressed on activated T and B cells as well as on
monocytes involved in regulating the balance between immune
activation and tolerance. The major role of PD-1 is to limit the
activity of T cells in the periphery during an inflammatory
response to infection and to limit autoimmunity. The basis for this
regulation is that the PD-1 ligands B7-H1/PD-L1 and B7-DC/PD-L2 are
upregulated in response to various proinflammatory cytokines and
can bind to PD-1 on activated T cells in inflamed tissues, thereby
limiting the immune response. Deletion of the PD-1 gene leads to
autoimmune complications, for example lupus-like symptoms
(Nishimura et al., 1999. Immunity 11(2): 141-51; PMID:
10485649).
[0006] In addition, it was found that B7-H1/PD-L1 is frequently
upregulated on many different tumor types, where it inhibits local
anti-tumor T cell responses by binding to PD-1 on tumor
infiltrating lymphocytes. For example, it was demonstrated that
PD-L1 plays a role in tumor immune evasion in squamous cell
carcinomas of the head and neck (Dong et al., 2002. Nat Med.
8(8):793-800). The authors showed that forced expression of
B7-H1/PD-L1 in tumors that are normally B7-H1/PD-L1 negative
inhibits their immune elimination and that antibody blockade
restores anti-tumor responses. Furthermore, it was shown that many
human tumors, such as carcinomas of the lung, ovary and colon,
upregulate B7-H1/PD-L1 relative to their normal tissue
counterparts. In summary, these observations suggested to use
blockade of the PD-1 pathway for cancer immunotherapy (Zitvogel and
Kroemer, 2012. Oncoimmunology 1, 1223-1125).
[0007] In this context US2010/0055102 describes the use of PD-1
antagonists for increasing a T cell immune response in a
mammal.
[0008] Recently, the first clinical trials with immune checkpoint
inhibitors (ICIs) against the PD-1 receptor and its ligand PD-L1
have been reported. The first phase I trial with the anti-PD-1
monoclonal antibody BMS-936558 was conducted in patients with
treatment-refractory metastatic solid tumors. Clinical activity was
observed in patients with melanoma, renal carcinoma, colorectal
cancer and non-small cell lung cancer (NSCLC). It could be shown
that PD-L1 is overexpressed in many cancers and is often associated
with poor prognosis. Furthermore tumor cell surface expression of
PD-L1 in pretreatment biopsies emerged as a potential biomarker of
response, consistent with pathway biology (Brahmer et al., 2010. J
Clin Oncol. 28(19):3167-75; PMID: 20516446).
[0009] Another clinical study with BMS-936558 produced objective
responses in approximately one in four to one in five patients with
non-small-cell lung cancer, melanoma, or renal-cell cancer; the
adverse-event profile does not appear to preclude its use.
Preliminary data suggest a relationship between PD-L1 expression on
tumor cells and objective response (Topalian et al., 2012. N Engl J
Med. 366(26):2443-54; PMID: 22658127).
[0010] Furthermore, a phase I study with the anti-PD-L1 antibody
BMS-936559 induced durable tumor regression (objective response
rate of 6 to 17%) and prolonged stabilization of disease (rates of
12 to 41% at 24 weeks) in patients with advanced cancers, including
non-small-cell lung cancer, melanoma, and renal-cell cancer.
Specifically, an objective response (a complete or partial
response) was observed in 9 of 52 patients with melanoma, 2 of 17
with renal-cell cancer, 5 of 49 with non-small-cell lung cancer,
and 1 of 17 with ovarian cancer (Brahmer et al. 2012. N Engl J Med.
366(26):2455-65; PMID: 22658128).
[0011] Nevertheless, only relatively low responses of some cancers
could be demonstrated in these studies (e.g. lung and prostate
cancer) to PD-1 pathway checkpoint inhibitors. In more recent
studies combinations with PD-1 pathway checkpoint inhibitors have
been evaluated in preclinical models.
[0012] A study reported e.g. the generation of a dendritic cell
(DC) vaccine with a combination of a PD-1 ligand siRNA and target
antigen mRNA. These PD-L1-silenced DC's loaded with antigen mRNA
increased ex vivo antigen-specific CD8+ T cell responses from
transplanted cancer patients (Hobo et al. 2013. Cancer Immunol
Immunother. 62(2):285-97; PMID: 22903385)
[0013] Likewise, Dai et al. showed that a dendritic cell-directed
lentiviral vector (DCLV) system encoding the human immunodeficiency
virus (HIV)-1 Gag protein combined with blocking of the PD-1/PD-1
ligand (PD-L) inhibitory signal via an anti-PD-L1 antibody
generated an enhanced HIV-1 Gag-specific CD8+ immune response
following DCLV immunization (Dai et al. 2012. Mol Ther.
20(9):1800-9; PMID: 22588271).
[0014] Another study examined the combination of recombinant
lentivector (rLV) vaccination with PD-1 and PD-L1 blocking
antibodies. Combining blocking antibodies with rLV vaccination
delayed tumor growth but was not sufficient to induce the complete
rejection of established tumors (Sierro et al. 2011. Eur J
Immunol.; 41(8):2217-28; PMID: 21538347).
[0015] Additionally, WO2008/11344 discloses the modulation of an
immune response by combination of a DNA-based recombinant
adenoviral construct (rAAV) coding for an antigen or therapeutic
protein and a nucleic acid coding for a modulator of PD-1
signaling, particularly siRNA, antisense RNA or a ribozyme specific
for PD-L1 gene.
[0016] In this context also the fusion of soluble PD-1 protein and
antigenic protein fragments was studied (WO2012/062218).
[0017] A further preclinical study reported that combination
blockade of the CTLA-4- and PD-1-negative costimulatory receptors
leads to synergistic levels of tumor rejection in the context of a
suitable vaccine (Curran et al., 2010. Proc Natl Acad Sci USA
107(9):4275-80; PMID: 20160101). Mice preimplanted with B16
melanoma cells were vaccinated with irradiated B16 melanoma cells
expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with
antibody blockade of the negative T-cell costimulatory receptor
cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1
(PD-1) and its ligand PD-L1. In the context of Gvax, neither
blockade of any single co-inhibitory receptor nor of any
combination resulted in greater than 20% survival. Using Fvax,
blockade of either PD-L1 (8% survival), CTLA-4 (10% survival), or
PD-1 (25% survival) showed modest therapeutic benefit. However,
only combined blockade of CTLA-4 and PD-1 led to tumor rejection in
50% of mice and further addition of anti-PD-L1 antibody increased
the rate to 65%. In summary, only CTLA-4-blockade combined with
PD-1 blockade had a significant effect in promoting the rejection
of B16 melanomas in conjunction with the B16 melanoma cell vaccine.
Blocking of a single inhibitory receptor (PD-1 or CTLA-4) leads to
upregulation of the unblocked pathway. Therefore, this study
suggests that a simultaneous blockade of multiple negative
co-stimulatory receptors may be required to enable
tumor-infiltrating T cells to be effective. Furthermore, based on
their preclinical data in the B16 melanoma model, the authors
suggest that combining these agents may have synergistic effects in
driving tumor rejection but they remark that combining multiple
co-inhibitory blocking antibodies in the clinic should proceed with
great caution.
[0018] Mkrtichya et al. additionally reported synergistic
antigen-specific immune responses by using an anti-PD-1 antibody
(CT-011) with Treg-cell depletion by low-dose cyclophosphamide
(CPM), combined with a tumor vaccine (HPV16-E7 peptide vaccine).
But only the combination of these 3 treatments resulted in a
complete regression of tumors (Mkrtichya et al. 2011. Eur J
Immunol. 41(10):2977-86; PMID: 21710477)
[0019] A further report examining the effect of PD-L1 inhibition on
tumor growth showed that the systemic administration of anti-PD-L1
antibody plus melanoma peptide-pulsed dendritic cells (DCs)
resulted in a higher number of melanoma peptide-specific CD8+ T
cells, but that this combination was insufficient to delay the
growth of established B16 melanoma. Although the additional body
irradiation delayed tumor growth, further adoptive transfer of
antigen-specific CD8+ T cells was needed to achieve tumor
regression and long-term survival of the treated mice (Pilon-Thomas
et al. 2010. J Immunol. 1; 184(7):3442-9; PMID: 20194714).
[0020] In a preclinical study Fotin-Mleczek et al. showed the
beneficial combination of a tumour vaccine based on mRNA and an
antibody directed against the CTLA4 receptor which attenuates the
signaling of T cells (Fotin-Mleczek et al., 2012. J Gene Med.
14(6):428-39; PMID: 22262664).
[0021] In summary, the use of immune checkpoint inhibitors appears
to represent a promising approach for improved cancer
immunotherapy. However, the combination of vaccines with a single
ICI often did not lead to the hoped improvement of the
immunotherapy and the combined clinical use of ICIs targeting
multiple negative co-stimulatory receptors or the combination of
ICIs with other treatments may induce clinical complications as
e.g. induction of an autoimmune disease.
[0022] Therefore, it is the object of the present invention to
provide safe and effective means for a therapy based on ICIs,
particularly based on PD-1 pathway inhibitors, in particular for a
therapy of tumor, cancer and/or infectious diseases.
[0023] The object underlying the present invention is solved by the
claimed subject matter. In particular, the object of the invention
is solved by the provision of a vaccine/inhibitor combination
comprising as vaccine an RNA vaccine comprising at least one RNA
comprising at least one open reading frame coding for at least one
antigen and as inhibitor a PD-1 pathway inhibitor, preferably
directed against PD-1 receptor or its ligands PD-L1 and/or PD-L2.
Furthermore, the object is solved by a pharmaceutical composition
or a kit of parts comprising the vaccine/inhibitor combination or
the respective components thereof. Additionally, the object is
solved by a combination of an RNA vaccine with ICIs, particularly
PD-1 pathway inhibitors for use in a method of treatment of tumour
or cancer diseases or infection diseases.
[0024] For the sake of clarity and readability the following
definitions are provided. Any technical feature mentioned for these
definitions may be read on each and every embodiment of the
invention. Additional definitions and explanations may be
specifically provided in the context of these embodiments.
Immune response: An immune response may typically either be a
specific reaction of the adaptive immune system to a particular
antigen (so called specific or adaptive immune response) or an
unspecific reaction of the innate immune system (so called
unspecific or innate immune response). In essence, the invention is
associated with specific reactions (adaptive immune responses) of
the adaptive immune system. However, this specific response can be
supported by an additional unspecific reaction (innate immune
response). Therefore, the invention also relates to a compound,
composition or combination for simultaneous stimulation of the
innate and the adaptive immune system to evoke an efficient
adaptive immune response. Immune system: The immune system may
protect organisms from infection. If a pathogen succeeds in passing
a physical barrier of an organism and enters this organism, the
innate immune system provides an immediate, but non-specific
response. If pathogens evade this innate response, vertebrates
possess a second layer of protection, the adaptive immune system.
Here, the immune system adapts its response during an infection to
improve its recognition of the pathogen. This improved response is
then retained after the pathogen has been eliminated, in the form
of an immunological memory, and allows the adaptive immune system
to mount faster and stronger attacks each time this pathogen is
encountered. According to this, the immune system comprises the
innate and the adaptive immune system. Each of these two parts
typically contains so called humoral and cellular components.
Adaptive immune response: The adaptive immune response is typically
understood to be an antigen-specific response of the immune system.
Antigen specificity allows for the generation of responses that are
tailored to specific pathogens or pathogen-infected cells. The
ability to mount these tailored responses is usually maintained in
the body by "memory cells". Should a pathogen infect the body more
than once, these specific memory cells are used to quickly
eliminate it. In this context, the first step of an adaptive immune
response is the activation of naive antigen-specific T cells or
different immune cells able to induce an antigen-specific immune
response by antigen-presenting cells. This occurs in the lymphoid
tissues and organs through which naive T cells are constantly
passing. The three cell types that may serve as antigen-presenting
cells are dendritic cells, macrophages, and B cells. Each of these
cells has a distinct function in eliciting immune responses.
Dendritic cells may take up antigens by phagocytosis and
macropinocytosis and may become stimulated by contact with e.g. a
foreign antigen to migrate to the local lymphoid tissue, where they
differentiate into mature dendritic cells. Macrophages ingest
particulate antigens such as bacteria and are induced by infectious
agents or other appropriate stimuli to express MHC molecules. The
unique ability of B cells to bind and internalize soluble protein
antigens via their receptors may also be important to induce T
cells. MHC-molecules are, typically, responsible for presentation
of an antigen to T-cells. Therein, presenting the antigen on MHC
molecules leads to activation of T cells which induces their
proliferation and differentiation into armed effector T cells. The
most important function of effector T cells is the killing of
infected cells by CD8+ cytotoxic T cells and the activation of
macrophages by Th1 cells which together make up cell-mediated
immunity, and the activation of B cells by both Th2 and Th1 cells
to produce different classes of antibody, thus driving the humoral
immune response. T cells recognize an antigen by their T cell
receptors which do not recognize and bind the antigen directly, but
instead recognize short peptide fragments e.g. of pathogen-derived
protein antigens, e.g. so-called epitopes, which are bound to MHC
molecules on the surfaces of other cells. Adaptive immune system:
The adaptive immune system is essentially dedicated to eliminate or
prevent pathogenic growth. It typically regulates the adaptive
immune response by providing the vertebrate immune system with the
ability to recognize and remember specific pathogens (to generate
immunity), and to mount stronger attacks each time the pathogen is
encountered. The system is highly adaptable because of somatic
hypermutation (a process of accelerated somatic mutations), and
V(D)J recombination (an irreversible genetic recombination of
antigen receptor gene segments). This mechanism allows a small
number of genes to generate a vast number of different antigen
receptors, which are then uniquely expressed on each individual
lymphocyte. Because the gene rearrangement leads to an irreversible
change in the DNA of each cell, all of the progeny (offspring) of
such a cell will then inherit genes encoding the same receptor
specificity, including the Memory B cells and Memory T cells that
are the keys to long-lived specific immunity. Cellular
immunity/cellular immune response: Cellular immunity relates
typically to the activation of macrophages, natural killer cells
(NK), antigen-specific cytotoxic T-lymphocytes, and the release of
various cytokines in response to an antigen. In more general terms,
cellular immunity is not based on antibodies, but on the activation
of cells of the immune system. Typically, a cellular immune
response may be characterized e.g. by activating antigen-specific
cytotoxic T-lymphocytes that are able to induce apoptosis in cells,
e.g. specific immune cells like dendritic cells or other cells,
displaying epitopes of foreign antigens on their surface. Such
cells may be virus-infected or infected with intracellular
bacteria, or cancer cells displaying tumor antigens. Further
characteristics may be activation of macrophages and natural killer
cells, enabling them to destroy pathogens and stimulation of cells
to secrete a variety of cytokines that influence the function of
other cells involved in adaptive immune responses and innate immune
responses. Humoral immunity/humoral immune response: Humoral
immunity refers typically to antibody production and optionally to
accessory processes accompanying antibody production. An humoral
immune response may be typically characterized, e.g., by Th2
activation and cytokine production, germinal center formation and
isotype switching, affinity maturation and memory cell generation.
Humoral immunity also typically may refer to the effector functions
of antibodies, which include pathogen and toxin neutralization,
classical complement activation, and opsonin promotion of
phagocytosis and pathogen elimination. Innate immune system: The
innate immune system, also known as non-specific (or unspecific)
immune system, typically comprises the cells and mechanisms that
defend the host from infection by other organisms in a non-specific
manner. This means that the cells of the innate system may
recognize and respond to pathogens in a generic way, but unlike the
adaptive immune system, it does not confer long-lasting or
protective immunity to the host. The innate immune system may be,
e.g., activated by ligands of Toll-like receptors (TLRs) or other
auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40
ligand, or cytokines, monokines, lymphokines, interleukins or
chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,
IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth
factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2,
TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine
Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like
receptor, a ligand of a RIG-I like receptor, an immunostimulatory
nucleic acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an
antibacterial agent, or an anti-viral agent. The vaccine/inhibitor
combination, the pharmaceutical composition or the kit of parts
according to the present invention may comprise one or more such
substances. Typically, a response of the innate immune system
includes recruiting immune cells to sites of infection, through the
production of chemical factors, including specialized chemical
mediators, called cytokines; activation of the complement cascade;
identification and removal of foreign substances present in organs,
tissues, the blood and lymph, by specialized white blood cells;
activation of the adaptive immune system; and/or acting as a
physical and chemical barrier to infectious agents.
Adjuvant/adjuvant component: An adjuvant or an adjuvant component
in the broadest sense is typically a pharmacological and/or
immunological agent that may modify, e.g. enhance, the effect of
other agents, such as a drug or vaccine. It is to be interpreted in
a broad sense and refers to a broad spectrum of substances.
Typically, these substances are able to increase the immunogenicity
of antigens. For example, adjuvants may be recognized by the innate
immune systems and, e.g., may elicit an innate immune response.
"Adjuvants" typically do not elicit an adaptive immune response.
Insofar, "adjuvants" do not qualify as antigens. Their mode of
action is distinct from the effects triggered by antigens resulting
in an adaptive immune response. Antigen: In the context of the
present invention "antigen" refers typically to a substance which
may be recognized by the immune system, preferably by the adaptive
immune system, and is capable of triggering an antigen-specific
immune response, e.g. by formation of antibodies and/or
antigen-specific T cells as part of an adaptive immune response.
Typically, an antigen may be or may comprise a peptide or protein
which comprises at least one epitope and which may be presented by
the MHC to T cells. In the sense of the present invention an
antigen may be the product of translation of a provided RNA,
preferably an mRNA as defined herein. In this context, also
fragments, variants and derivatives of peptides and proteins
comprising at least one epitope are understood as antigens. In the
context of the present invention, tumour antigens and pathogenic
antigens as defined herein are particularly preferred. Epitope:
Epitopes (also called `antigen determinant`) can be distinguished
in T cell epitopes and B cell epitopes. T cell epitopes or parts of
the proteins in the context of the present invention may comprise
fragments preferably having a length of about 6 to about 20 or even
more amino acids, e.g. fragments as processed and presented by MHC
class I molecules, preferably having a length of about 8 to about
10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids),
or fragments as processed and presented by MHC class II molecules,
preferably having a length of about 13 or more amino acids, e.g.
13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein
these fragments may be selected from any part of the amino acid
sequence. These fragments are typically recognized by T cells in
form of a complex consisting of the peptide fragment and an MHC
molecule, i.e. the fragments are typically not recognized in their
native form. B cell epitopes are typically fragments located on the
outer surface of (native) protein or peptide antigens as defined
herein, preferably having 5 to 15 amino acids, more preferably
having 5 to 12 amino acids, even more preferably having 6 to 9
amino acids, which may be recognized by antibodies, i.e. in their
native form.
[0025] Such epitopes of proteins or peptides may furthermore be
selected from any of the herein mentioned variants of such proteins
or peptides. In this context antigenic determinants can be
conformational or discontinuous epitopes which are composed of
segments of the proteins or peptides as defined herein that are
discontinuous in the amino acid sequence of the proteins or
peptides as defined herein but are brought together in the
three-dimensional structure or continuous or linear epitopes which
are composed of a single polypeptide chain.
Vaccine: A vaccine is typically understood to be a prophylactic or
therapeutic material providing at least one antigen, preferably an
immunogen. "Providing at least on antigen" means, for example, that
the vaccine comprises the antigen or that the vaccine comprises a
molecule that, e.g., codes for the antigen or a molecule comprising
the antigen. For example, the vaccine may comprise a nucleic acid,
such as an RNA (e.g. RNA vaccine), which codes for a peptide or
protein that comprises the antigen. The antigen or immunogen may be
derived from any material that is suitable for vaccination. For
example, the antigen or immunogen may be derived from a pathogen,
such as from bacteria or virus particles etc., or from a tumor or
cancerous tissue. The antigen or immunogen stimulates the body's
adaptive immune system to provide an adaptive immune response. In
the context, of the present invention, the vaccine preferably does
not comprise cells, such as dendritic cells or cancer cells, e.g.,
B16 melanoma cells. Furthermore, in the context of the present
invention the vaccine preferably does not consist of peptide
antigens, such as peptide tumor antigens. In the context of the
present invention, the vaccine is preferably an RNA vaccine. RNA
vaccine: An RNA vaccine is defined herein as a vaccine comprising
at least one RNA molecule comprising at least one open reading
frame (ORF) coding for at least one antigen. In the context of the
present invention, the at least one RNA molecule comprised by the
vaccine is preferably an isolated RNA molecule. This at least one
RNA is preferably viral RNA, self-replicating RNA (replicon) or
most preferably mRNA. Also included herein are RNA/DNA hybrids
which means that the at least one RNA molecule of the RNA vaccine
consists partially of ribonucleotides and partially of
deoxyribonucleotides. In this context, the at least one RNA of the
RNA vaccine consists to at least 50% of ribonucleotides, more
preferably to at least 60%, 70%, 80%, 90% and most preferably to
100%. In this context, the at least one RNA of the RNA vaccine can
also be provided as complexed RNA or mRNA, as virus particle and as
replicon particle as defined herein. In the context of the present
invention, the at least one RNA comprised by the RNA vaccine is
preferably not present in cells, such as in dendritic cell or
cancer cells, e.g. B16 melanoma cells. Furthermore it is
particularly preferred that the RNA vaccine of the invention does
not comprise or does not consist of a lentiviral vector, in
particular not a dendritic cell--directed lentiviral vector, or an
(recombinant) adenoviral/adeno-associated vector ((r)AAV vector).
Preferably, the RNA vaccine of the invention is not an HIV vaccine.
In particular, it is preferred that the RNA vaccine of the
invention does not comprise an AAV or lentiviral vector encoding
one or more antigens. Also, the RNA vaccine of the invention may
preferably not encode HIV-specific antigens, e.g. Gag protein, in
particular not a lentiviral vector encoding HIV specific antigens,
like Gag. Preferably, the RNA vaccine of the invention does not
comprise a fusion protein of a PD1 protein and an antigenic protein
or a fusion protein of a PD1 protein and an immunoglobulin or a
portion thereof and does not encode such a PD1 fusion protein.
Genetic vaccination: Genetic vaccination may typically be
understood to be vaccination by administration of a nucleic acid
molecule encoding an antigen or an immunogen or fragments thereof.
The nucleic acid molecule may be administered to a subject's body
or to isolated cells of a subject. Upon transfection of certain
cells of the body or upon transfection of the isolated cells, the
antigen or immunogen may be expressed by those cells and
subsequently presented to the immune system, eliciting an adaptive,
i.e. antigen-specific immune response. Accordingly, genetic
vaccination typically comprises at least one of the steps of a)
administration of a nucleic acid, preferably an (isolated) RNA as
defined herein, to a subject, preferably a patient, or to isolated
cells of a subject, preferably a patient, which usually results in
transfection of the subject's cells either in vivo or in vitro; b)
transcription and/or translation of the introduced nucleic acid
molecule; and optionally c) re-administration of isolated,
transfected cells to the subject, preferably the patient, if the
nucleic acid has not been administered directly to the patient.
Nucleic acid: The term nucleic acid means any DNA- or RNA-molecule
and is used synonymous with polynucleotide. Furthermore,
modifications or derivatives of the nucleic acid as defined herein
are explicitly included in the general term "nucleic acid". For
example, PNA is also included in the term "nucleic acid".
Monocistronic RNA: A monocistronic RNA may typically be an RNA,
preferably an mRNA, that comprises only one open reading frame. An
open reading frame in this context is a sequence of several
nucleotide triplets (codons) that can be translated into a peptide
or protein. Bi-/multicistronic RNA: RNA, preferably mRNA, that
typically may have two (bicistronic) or more (multicistronic) open
reading frames (ORF). An open reading frame in this context is a
sequence of several nucleotide triplets (codons) that can be
translated into a peptide or protein. 5'-Cap structure: A 5' Cap is
typically a modified nucleotide, particularly a guanine nucleotide,
added to the 5' end of an RNA molecule. Preferably, the 5'-Cap is
added using a 5'-5'-triphosphate linkage. Poly(C) sequence: A
poly(C) sequence is typically a long sequence of cytosine
nucleotides, typically about 10 to about 200 cytidine nucleotides,
preferably about 10 to about 100 cytidine nucleotides, more
preferably about 10 to about 70 cytidine nucleotides or even more
preferably about 20 to about 50 or even about 20 to about 30
cytidine nucleotides. A poly(C) sequence may preferably be located
3' of the coding region comprised by a nucleic acid. Poly(A) tail:
A poly(A) tail also called "3'-poly(A) tail" is typically a long
sequence of adenine nucleotides of up to about 400 adenosine
nucleotides, e.g. from about 25 to about 400, preferably from about
50 to about 400, more preferably from about 50 to about 300, even
more preferably from about 50 to about 250, most preferably from
about 60 to about 250 adenosine nucleotides, added to the 3' end of
a nucleic acid sequence, preferably an mRNA. A poly(A) tail may
preferably be located 3' of the coding region comprised by a
nucleic acid, e.g. an mRNA. Stabilized nucleic acid: A stabilized
nucleic acid, typically, may be essentially resistant to in vivo
degradation (e.g. degradation by an exo- or endo-nuclease) and/or
ex vivo degradation (e.g. by the manufacturing process prior to
vaccine administration, e.g. in the course of the preparation of
the RNA vaccine solution to be administered). Stabilization of RNA,
particularly mRNA can, e.g., be achieved by providing a 5'-Cap
structure, a Poly(A) tail, a poly (C) tail, and/or any other UTR
modification. It can also be achieved by backbone modification,
sugar modification, base modification, and/or modification of the
G/C-content of the nucleic acid. Various other methods are
conceivable in the context of the invention. Modification of a
nucleic acid (modified nucleic acid): Modification of a nucleic
acid molecule, particularly of RNA or mRNA, may contain backbone
modifications, sugar modifications or base modifications. A
backbone modification in connection with the present invention is a
modification in which phosphates of the backbone of the nucleotides
contained in the nucleic acid molecule are chemically modified. A
sugar modification in connection with the present invention is a
chemical modification of the sugar of the nucleotides of the
nucleic acid. Furthermore, a base modification in connection with
the present invention is a chemical modification of the base moiety
of the nucleotides of the nucleic acid molecule. Therefore a
modified nucleic acid is also defined herein as a nucleic acid
molecule which may include nucleotide analogues. Furthermore a
modification of a nucleic acid molecule can contain a lipid
modification. Such a lipid-modified nucleic acid typically
comprises a nucleic acid as defined herein. Such a lipid-modified
nucleic acid molecule typically further comprises at least one
linker covalently linked with that nucleic acid molecule, and at
least one lipid covalently linked with the respective linker.
Alternatively, the lipid-modified nucleic acid molecule comprises
at least one nucleic acid molecule as defined herein and at least
one (bifunctional) lipid covalently linked (without a linker) with
that nucleic acid molecule. According to a third alternative, the
lipid-modified nucleic acid molecule comprises a nucleic acid
molecule as defined herein, at least one linker covalently linked
with that nucleic acid molecule, and at least one lipid covalently
linked with the respective linker, and also at least one
(bifunctional) lipid covalently linked (without a linker) with that
nucleic acid molecule. A modification of a nucleic acid may also
comprise the modification of the G/C content of the coding region
of a nucleic acid molecule, especially of the at least one RNA of
the RNA vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination. In this context it is particularly
preferred that the G/C content of the coding region of the nucleic
acid molecule is increased, compared to the G/C content of the
coding region of its particular wild type coding sequence, i.e. the
unmodified RNA. The encoded amino acid sequence of the nucleic acid
sequence is preferably not modified compared to the coded amino
acid sequence of the particular wild type mRNA. The modification of
the G/C-content of the nucleic acid molecule, especially if the
nucleic acid molecule is in the form of an mRNA or codes for an
mRNA, is based on the fact that the sequence of any mRNA region to
be translated is important for efficient translation of that mRNA.
Thus, the composition and the sequence of various nucleotides are
important. In particular, sequences having an increased G
(guanosine)/C (cytosine) content are more stable than sequences
having an increased A (adenosine)/U (uracil) content. Therefore,
the codons of the coding sequence or mRNA are therefore varied
compared to its wild type coding sequence or mRNA, while retaining
the translated amino acid sequence, such that they include an
increased amount of G/C nucleotides. In respect to the fact that
several codons code for one and the same amino acid (so-called
degeneration of the genetic code), the most favourable codons for
the stability can be determined (so-called alternative codon
usage). Preferably, the G/C content of the coding region of the
nucleic acid molecule, especially of the at least one RNA of the
RNA vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination, is increased by at least 7%, more
preferably by at least 15%, particularly preferably by at least
20%, compared to the G/C content of the coded region of the wild
type mRNA. According to a specific embodiment at least 5%, 10%,
20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more
preferably at least 80% and most preferably at least 90%, 95% or
even 100% of the substitutable codons in the region coding for a
protein or peptide as defined herein or its fragment, variant
and/or derivative thereof or the whole sequence of the wild type
mRNA sequence or coding sequence are substituted, thereby
increasing the G/C content of said sequence. In this context, it is
particularly preferable to increase the G/C content of the nucleic
acid molecule, especially of the at least one RNA of the RNA
vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination, to the maximum (i.e. 100% of the
substitutable codons), in particular in the region coding for a
protein, compared to the wild type sequence. Furthermore a
modification of the nucleic acid, especially of the at least one
RNA of the RNA vaccine encoding at least one antigen in the
inventive vaccine/inhibitor combination, is based on the finding
that the translation efficiency is also determined by a different
frequency in the occurrence of tRNAs in cells. The frequency in the
occurrence of tRNAs in a cell, and thus the codon usage in said
cell, is dependent on the species the cell is derived from.
Accordingly, a yeast cell generally exhibits a different codon
usage than a mammalian cell, such as a human cell. Thus, if
so-called "rare codons" are present in the nucleic acid molecule
(with respect to the respective expression system), especially if
the nucleic acid is in the form of an mRNA or codes for an mRNA, to
an increased extent, the corresponding modified nucleic acid
molecule is translated to a significantly poorer degree than in the
case where codons coding for relatively "frequent" tRNAs are
present. Therefore, the coding region of the modified nucleic acid,
particularly the at least one RNA of the RNA vaccine encoding at
least one antigen in the inventive vaccine/inhibitor combination is
preferably modified compared to the corresponding region of the
wild type mRNA or coding sequence such that at least one codon of
the wild type sequence which codes for a tRNA which is relatively
rare in the cell is exchanged for a codon which codes for a tRNA
which is relatively frequent in the cell and carries the same amino
acid as the relatively rare tRNA. By this modification, the
sequences of the nucleic acid molecule, particularly of the at
least one RNA of the RNA vaccine encoding at least one antigen in
the inventive vaccine/inhibitor combination, is modified such that
codons for which frequently occurring tRNAs are available are
inserted. In other words, by this modification all codons of the
wild type sequence which code for a tRNA which is relatively rare
in the cell can in each case be exchanged for a codon which codes
for a tRNA which is relatively frequent in the cell and which, in
each case, carries the same amino acid as the relatively rare tRNA.
Such a modified nucleic acid, preferably is termed herein as
"codon-optimized nucleic acid or RNA". Which tRNAs occur relatively
frequently in the cell and which, in contrast, occur relatively
rarely is known to a person skilled in the art; cf. e.g. Akashi,
Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. It is particularly
preferred that a nucleic acid sequence coding for a protein,
particularly the at least one RNA coding for at least one antigen
comprised by the RNA vaccine, used in the present invention is
codon optimized for the human codon usage. The codons which use for
the particular amino acid the tRNA which occurs the most
frequently, e.g. the Gly codon, which uses the tRNA which occurs
the most frequently in the (human) cell, are particularly
preferred. In this context, it is particularly preferable to link
the sequential G/C content which is increased, in particular
maximized, in the modified nucleic acid molecule, particularly of
the at least one RNA of the RNA vaccine encoding at least one
antigen in the inventive vaccine/inhibitor combination, with
the
"frequent" codons without modifying the amino acid sequence of the
protein encoded by the coding region of the nucleic acid molecule.
This preferred embodiment allows provision of a particularly
efficiently translated and stabilized (modified) nucleic acid,
particularly of the at least one RNA of the RNA vaccine encoding at
least one antigen in the inventive vaccine/inhibitor combination.
Derivative of a nucleic acid molecule: A derivative of a nucleic
acid molecule is defined herein in the same manner as a modified
nucleic acid, as defined above. Nucleotide analogues: Nucleotide
analogues are nucleotides structurally similar (analogue) to
naturally occurring nucleotides which include phosphate backbone
modifications, sugar modifications, or modifications of the
nucleobase. UTR modification: A nucleic acid molecule, especially
if the nucleic acid is in the form of a coding nucleic acid
molecule, particularly the at least one RNA of the RNA vaccine
comprising at least one open reading frame coding for at least one
antigen according to the invention, preferably has at least one
modified 5' and/or 3' UTR sequence (UTR modification). These in the
5' and/or 3' untranslated regions (UTR) included sequences may have
the effect of increasing the half-life of the nucleic acid in the
cytosol or may increase the translation of the encoded protein or
peptide. These UTR sequences can have 100% sequence identity to
naturally occurring sequences which occur in viruses, bacteria and
eukaryotes, but can also be partly or completely synthetic. The
untranslated sequences (UTR) of the (alpha-) globin gene, e.g. from
Homo sapiens or Xenopus laevis may be mentioned as an example of
stabilizing sequences which can be used for a stabilized nucleic
acid. Another example of a stabilizing sequence has the general
formula (C/U)CCANxCCC(U/A)PyxUC(C/U)CC which is contained in the
3'UTR of the very stable RNA which codes for (alpha-)globin,
type(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf.
Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
Particularly preferred in the context of the present invention is
the mutated UTR of (alpha-)globin comprising the following sequence
GCCCGaTGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG (SEQ ID NO. 1)
(the underlined nucleotide shows the mutation compared to the wild
type sequence), which is also termed herein as muag. Such
introduced UTR sequences can of course be used individually or in
combination with one another and also in combination with other
sequence modifications known to a person skilled in the art.
Histone stem-loop: In the context of the present invention, a
histone stem-loop sequence is preferably selected from at least one
of the following formulae (I) or (II):
##STR00001##
wherein: [0026] stem1 or stem2 bordering elements N.sub.1-6 is a
consecutive sequence of 1 to 6, preferably of 2 to 6, more
preferably of 2 to 5, even more preferably of 3 to 5, most
preferably of 4 to 5 or 5 N, wherein each N is independently from
another selected from a nucleotide selected from A, U, T, G and C,
or a nucleotide analogue thereof; [0027] stem1
[N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse
complementary with element stem2, and is a consecutive sequence
between of 5 to 7 nucleotides; [0028] wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; [0029] wherein N.sub.3-5 is a
consecutive sequence of 3 to 5, preferably of 4 to 5, more
preferably of 4 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof, and [0030] wherein G is guanosine or
an analogue thereof, and may be optionally replaced by a cytidine
or an analogue thereof, provided that its complementary nucleotide
cytidine in stem2 is replaced by guanosine; [0031] loop sequence
[N.sub.0-4(U/T)N.sub.0-4] is located between elements stem1 and
stem2, and is a consecutive sequence of 3 to 5 nucleotides, more
preferably of 4 nucleotides; [0032] wherein each N.sub.0-4 is
independent from another a consecutive sequence of 0 to 4,
preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N
is independently from another selected from a nucleotide selected
from A, U, T, G and C or a nucleotide analogue thereof; and [0033]
wherein U/T represents uridine, or optionally thymidine; [0034]
stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially
reverse complementary with element stem1, and is a consecutive
sequence between of 5 to 7 nucleotides; [0035] wherein N.sub.3-5 is
a consecutive sequence of 3 to 5, preferably of 4 to 5, more
preferably of 4 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G and C or a
nucleotide analogue thereof; [0036] wherein N.sub.0-2 is a
consecutive sequence of 0 to 2, preferably of 0 to 1, more
preferably of 1 N, wherein each N is independently from another
selected from a nucleotide selected from A, U, T, G or C or a
nucleotide analogue thereof; and [0037] wherein C is cytidine or an
analogue thereof, and may be optionally replaced by a guanosine or
an analogue thereof provided that its complementary nucleoside
guanosine in stem1 is replaced by cytidine; [0038] wherein [0039]
stem1 and stem2 are capable of base pairing with each other forming
a reverse complementary sequence, wherein base pairing may occur
between stem1 and stem2, e.g. by Watson-Crick base pairing of
nucleotides A and U/T or G and C or by non-Watson-Crick base
pairing e.g. wobble base pairing, reverse Watson-Crick base
pairing, Hoogsteen base pairing, reverse Hoogsteen base pairing or
are capable of base pairing with each other forming a partially
reverse complementary sequence, wherein an incomplete base pairing
may occur between stem1 and stem2, on the basis that one or more
bases in one stem do not have a complementary base in the reverse
complementary sequence of the other stem. Nucleic acid synthesis:
Nucleic acid molecules used according to the invention as defined
herein may be prepared using any method known in the art, including
synthetic methods such as e.g. solid phase synthesis, in vivo
propagation (e.g. in vivo propagation of viruses), as well as in
vitro methods, such as in vitro transcription reactions.
[0040] For preparation of a nucleic acid molecule, especially if
the nucleic acid is in the form of an RNA or mRNA, a corresponding
DNA molecule may e.g. be transcribed in vitro. This DNA matrix
preferably comprises a suitable promoter, e.g. a T7 or SP6
promoter, for in vitro transcription, which is followed by the
desired nucleotide sequence coding for the nucleic acid molecule,
e.g. mRNA, to be prepared and a termination signal for in vitro
transcription. The DNA molecule, which forms the matrix of the at
least one RNA of interest, may be prepared by fermentative
proliferation and subsequent isolation as part of a plasmid which
can be replicated in bacteria. Plasmids which may be mentioned as
suitable for the present invention are e.g. the plasmids pT7Ts
(GenBank accession number U26404; Lai et al., Development 1995,
121: 2349 to 2360), pGEM.RTM. series, e.g. pGEM.RTM.-1 (GenBank
accession number X65300; from Promega) and pSP64 (GenBank accession
number X65327); cf. also Mezei and Storts, Purification of PCR
Products, in: Griffin and Griffin (ed.), PCR Technology: Current
Innovation, CRC Press, Boca Raton, Fla., 2001.
RNA: RNA is the usual abbreviation for ribonucleic-acid. It is a
nucleic acid molecule, i.e. a polymer consisting of nucleotides.
These nucleotides are usually adenosine-monophosphate,
uridine-monophosphate, guanosine-monophosphate and
cytidine-monophosphate monomers which are connected to each other
along a so-called backbone. The backbone is formed by
phosphodiester bonds between the sugar, i.e. ribose, of a first and
a phosphate moiety of a second, adjacent monomer. The specific
succession of the monomers is called the RNA-sequence. Messenger
RNA (mRNA): In eukaryotic cells, transcription is typically
performed inside the nucleus or the mitochondria. In vivo,
transcription of DNA usually results in the so-called premature RNA
which has to be processed into so-called messenger RNA, usually
abbreviated as mRNA. Processing of the premature RNA, e.g. in
eukaryotic organisms, comprises a variety of different
posttranscriptional-modifications such as splicing, 5'-capping,
polyadenylation, export from the nucleus or the mitochondria and
the like. The sum of these processes is also called maturation of
RNA. The mature messenger RNA usually provides the nucleotide
sequence that may be translated into an amino acid sequence of a
particular peptide or protein. Typically, a mature mRNA comprises a
5'-cap, a 5'UTR, an open reading frame, a 3'UTR and a poly(A)
sequence. In the context of the present invention, an mRNA may also
be an artificial molecule, i.e. a molecule not occurring in nature.
This means that the mRNA in the context of the present invention
may, e.g., comprise a combination of a 5'UTR, open reading frame,
3'UTR and poly(A) sequence, which does not occur in this
combination in nature. Retrovirus: A retrovirus is an RNA virus
that is duplicated in a host cell using the reverse transcriptase
enzyme to produce DNA from its RNA genome. The DNA is then
incorporated into the host's genome by an integrase enzyme. The
virus thereafter replicates as part of the host cell's DNA and then
undergoes the usual transcription and translational processes to
express the genes carried by the virus. Often lentiviruses were
used for gene therapy purposes. For safety reasons lentiviral
vectors normally do not carry the genes required for their
replication. To produce a lentivirus, several plasmids are
transfected into a so-called packaging cell line, commonly HEK 293.
One or more plasmids, generally referred to as packaging plasmids,
encode the virion proteins, such as the capsid and the reverse
transcriptase. Another plasmid contains the genetic material to be
delivered by the vector. It is transcribed to produce the
single-stranded RNA viral genome which is packaged into the virion,
which is used for infection of cells for gene therapy purposes or
genetic vaccination. Virion: Virus particles (known as virions)
consist of two or three parts: i) the genetic material (comprising
viral genes and optional substituted heterologous genes) made from
either DNA or RNA; ii) a protein coat that protects these genes;
and in some cases iii) an envelope of lipids that surrounds the
protein coat when they are outside a cell. Self-replicating RNA
(Replicons): Self-replicating RNA are delivery vectors based on
alphaviruses which have been developed from Semliki Forest virus
(SFV), Sindbis (SIN) virus, and Venezuelan equine encephalitis
(VEE) virus. Alphaviruses are single stranded RNA viruses in which
heterologous genes of interest may substitute for the alphavirus'
structural genes. By providing the structural genes in trans, the
replicon RNA is packaged into replicon particles (RP) which may be
used for gene therapy purposes or genetic vaccination (see for
example Vander Veen et al., 2012. Alphavirus replicon vaccines.
Animal Health Research Reviews 13(1):1-9). After entry into the
host cell, the genomic viral RNA initially serves as an mRNA for
translation of the viral nonstructural proteins (nsPs) required for
initiation of viral RNA amplification. RNA replication occurs via
synthesis of a full-length minus-strand intermediate that is used
as the template for synthesis of additional genome-length RNAs and
for transcription of a plus-strand subgenomic RNA from an internal
promoter. Such RNA may then be considered as self-replicating RNA,
since the non-structural proteins responsible for replication (and
transcription of the heterologous genes) are still present in such
replicon. Such alphavirus vectors are referred to as "replicons."
Replicon particle: A replicon particle consist of two or three
parts: i) the genetic material (=the replicon) (comprising viral
genes and optional substituted heterologous genes) made from either
DNA or RNA; ii) a protein coat that protects these genes; and in
some cases iii) an envelope of lipids that surrounds the protein
coat when they are outside a cell. Isolated RNA: Isolated RNA is
defined herein as RNA which is not part of a cell, an irradiated
cell or a cell lysate. An isolated RNA may be produced by isolation
and/or purification from cells or cell lysates, or from in vitro
transcription systems.
[0041] The term isolated RNA in the context of the present
invention is defined herein as RNA which is not part of cells,
irradiated cells or cell lysates including the RNA of the RNA
vaccine or which is not part of cells transfected with the RNA of
the RNA vaccine. In other words, the term "isolated RNA" excludes
(ex vivo) transfected or modulated cells used as RNA vaccine, in
particular excludes ex vivo transfected or modulated immune cells,
such as dendritic cells (DC), e.g. DC transfected/transduced with
an RNA used as RNA vaccine. Furthermore, the term isolated RNA
excludes RNA comprised in cells, irradiated cells or cell lysate
comprising naturally the at least one RNA coding for an antigen
used as RNA vaccine. Accordingly, the RNA vaccine of the inventive
combination does preferably not comprise modulated or transfected
cells, in particular no transfected or modulated immune cells (e.g.
antigen presenting cells), more particularly no transfected or
modulated DC, irradiated cells or cell lysates comprising naturally
the at least one RNA of the RNA vaccine. By that embodiment, it
becomes clear that the RNA vaccine used in the present invention
does preferably not correspond to a cell-based vaccine or a
DC-based vaccine, or, more generally, does preferably not
correspond to a cell-based vaccine or, more generally, the RNA
vaccine used in the present invention is preferably free of cells
and the inventive combination to be administered provides the RNA
vaccine as RNA, more preferably as mRNA, optionally in association
with a carrier and/or adjuvant component. The term isolated RNA
also includes RNA that is complexed with further components e.g.
peptides, proteins, carriers etc., RNA packaged in particles like
e.g. replicon particles or virus particles (virions) and RNA
contained in solution which may additional to the RNA comprise
further components e.g. buffer, stabilization reagents, RNAse
inhibitors, etc.
Sequence of a nucleic acid molecule: The sequence of a nucleic acid
molecule is typically understood to be the particular and
individual order, i.e. the succession of its nucleotides. Sequence
of a protein or peptide: The sequence of a protein or peptide is
typically understood to be the order, i.e. the succession of its
amino acids. Sequence identity: Two or more sequences are identical
if they exhibit the same length and order of nucleotides or amino
acids. The percentage of identity typically describes the extent to
which two sequences are identical, i.e. it typically describes the
percentage of nucleotides that correspond in their sequence
position with identical nucleotides of a reference-sequence. For
determination of the degree of identity, the sequences to be
compared are considered to exhibit the same length, i.e. the length
of the longest sequence of the sequences to be compared. This means
that a first sequence consisting of 8 nucleotides is 80% identical
to a second sequence consisting of 10 nucleotides comprising the
first sequence. In other words, in the context of the present
invention, identity of sequences preferably relates to the
percentage of nucleotides of a sequence which have the same
position in two or more sequences having the same length. Gaps are
usually regarded as non-identical positions, irrespective of their
actual position in an alignment. Fragment of a sequence: a fragment
of a sequence is typically a shorter portion of a full-length
sequence of e.g. a nucleic acid sequence or an amino acid sequence.
Accordingly, a fragment of a sequence, typically, consists of a
sequence that is identical to the corresponding stretch or
corresponding stretches within the full-length sequence. A
preferred fragment of a sequence in the context of the present
invention, consists of a continuous stretch of entities, such as
nucleotides or amino acids, corresponding to a continuous stretch
of entities in the molecule the fragment is derived from, which
represents at least 5%, preferably at least 20%, preferably at
least 30%, more preferably at least 40%, more preferably at least
50%, even more preferably at least 60%, even more preferably at
least 70%, and most preferably at least 80% of the total (i.e.
full-length) molecule from which the fragment is derived. Thus, for
example, a fragment of a protein or peptide antigen preferably
corresponds to a continuous stretch of entities in the protein or
peptide antigen the fragment is derived from, which represents at
least 5%, preferably at least 20%, preferably at least 30%, more
preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, even more preferably at least 70%, and
most preferably at least 80% of the total (i.e. full-length)
protein or peptide antigen. It is particularly preferred that the
fragment of a sequence is a functional fragment, i.e. that the
fragment fulfils one or more of the functions fulfilled by the
sequence the fragment is derived from. For example, a fragment of a
protein or peptide antigen preferably exhibits at least one
antigenic function (e.g. is capable of eliciting a specific immune
reaction against at least one antigen determinant in said protein
or peptide antigen) of the protein or peptide antigen the fragment
is derived from. Fragments of proteins: "Fragments" of proteins or
peptides, i.e., fragments of amino acid sequences, in the context
of the present invention may, typically, comprise a sequence of a
protein or peptide as defined herein, which is, with regard to its
amino acid sequence (or its encoding nucleic acid molecule),
N-terminally, C-terminally and/or intrasequentially truncated
compared to the amino acid sequence of the original (native)
protein (or its encoded nucleic acid molecule). Such truncation may
thus occur either on the amino acid level or correspondingly on the
nucleic acid level. A sequence identity with respect to such a
fragment as defined herein may therefore preferably refer to the
entire protein or peptide as defined herein or to the entire
(coding) nucleic acid molecule of such a protein or peptide.
[0042] Likewise, "fragments" of nucleic acid sequences in the
context of the present invention may comprise a sequence of a
nucleic acid as defined herein, which is, with regard to its
nucleic acid molecule 5'-, 3'- and/or intrasequentially truncated
compared to the nucleic acid molecule of the original (native)
nucleic acid molecule. A sequence identity with respect to such a
fragment as defined herein may therefore preferably refer to the
entire nucleic acid as defined herein.
Transfection: The term `transfection` refers to the introduction of
nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules,
into cells, preferably into eukaryotic cells. In the context of the
present invention, the term `transfection` encompasses any method
known to the skilled person for introducing nucleic acid molecules,
preferably RNA molecules into cells, preferably into eukaryotic
cells, such as into mammalian cells. Such methods encompass, for
example, electroporation, lipofection, e.g. based on cationic
lipids and/or liposomes, calcium phosphate precipitation,
nanoparticle based transfection, virus based transfection, or
transfection based on cationic polymers, such as DEAE-dextran or
polyethylenimine etc. Carrier: A carrier in the context of the
invention may typically be a compound that facilitates transport
and/or complexation of another compound (cargo). A carrier may be
associated to its cargo by covalent or non-covalent interaction. A
carrier may transport nucleic acids, e.g. RNA or DNA, to the target
cells. The carrier may--for some embodiments--be a cationic or
polycationic compound or a polymeric carrier as defined herein. A
carrier, in the context of the present invention, is preferably
suitable as carrier for nucleic acid molecules, e.g. for mediating
dissolution in physiological acceptable liquids, transport and
cellular uptake of the nucleic acid molecules or a vector.
Accordingly, a carrier, in the context of the present invention,
may be a component which may be suitable for depot and delivery of
a nucleic acid molecule or vector. Such carriers may be, for
example, cationic or polycationic carriers or compounds which may
serve as transfection or complexation agent. Particularly preferred
carriers or polymeric carriers in this context are cationic or
polycationic compounds. Cationic or polycationic
compound/component:
[0043] The term "cationic or polycationic compound/component"
typically refers to a charged molecule, which is positively charged
(cation) at a pH value typically from 1 to 9, preferably at a pH
value of or below 9 (e.g. from 5 to 9), of or below 8 (e.g. from 5
to 8), of or below 7 (e.g. from 5 to 7), most preferably at a
physiological pH, e.g. from 7.3 to 7.4. Accordingly, a cationic or
polycationic compound/component may be any positively charged
compound or polymer, preferably a cationic or polycationic peptide
or protein which is positively charged under physiological
conditions, particularly under physiological conditions in vivo. A
`cationic peptide or protein` may contain at least one positively
charged amino acid, or more than one positively charged amino acid,
e.g. selected from Arg, His, Lys or Orn. Accordingly,
`polycationic` compounds are also within the scope exhibiting more
than one positive charge under the conditions given. In this
context a cationic peptide or protein contains a larger number of
cationic amino acids, e.g. a larger number of Arg, His, Lys or Orn,
than negatively charged amino acids. In a preferred embodiment, a
cationic peptide or protein in the context of the present invention
contains a larger number of cationic amino acids, e.g. a larger
number of Arg, His, Lys or Orn, than other residues.
[0044] The term "cationic or polycationic compound" in the context
of the present invention preferably refers to compounds which can
be used as transfection or complexation agent, particularly of
nucleic acids, used according to the invention.
[0045] Cationic or polycationic compounds according to the
invention, being particularly preferred agents in this context
include protamine, nucleoline, spermine or spermidine, or other
cationic peptides or proteins, such as poly-L-lysine (PLL),
poly-arginine, basic polypeptides, cell penetrating peptides
(CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),
Tat-derived peptides, Penetratin, VP22 derived or analog peptides,
HSV VP22 (Herpes simplex), MAP, KALA or protein transduction
domains (PTDs), PpT620, prolin-rich peptides, arginine-rich
peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,
Calcitonin peptide(s), Antennapedia-derived peptides (particularly
from Drosophila antennapedia), pAntp, pIsl, FGF, Lactoferrin,
Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived
peptides, SAP, or histones. Protamine is particularly
preferred.
[0046] Additionally, preferred cationic or polycationic proteins or
peptides used as transfection or complexation agent may be selected
from the following proteins or peptides having the following total
formula (III):
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x,
(formula (III))
wherein l+m+n+o+x=8-15, and l, m, n or o independently of each
other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or 15, provided that the overall content of Arg,
Lys, His and Orn represents at least 50% of all amino acids of the
oligopeptide; and Xaa may be any amino acid selected from native
(=naturally occurring) or non-native amino acids except of Arg,
Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3
or 4, provided, that the overall content of Xaa does not exceed 50%
of all amino acids of the oligopeptide. Particularly preferred
cationic peptides in this context are e.g. Arg.sub.7, Arg.sub.8,
Arg.sub.9, H.sub.3R.sub.9, R.sub.9H.sub.3, H.sub.3R.sub.9H.sub.3,
YSSR.sub.9SSY, (RKH).sub.4, Y(RKH).sub.2R, etc.
[0047] Further preferred cationic or polycationic compounds, which
can be used as transfection or complexation agent may include
cationic polysaccharides, for example chitosan, polybrene, cationic
polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium
chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,
DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,
DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:
Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:
dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:
O,O-ditetradecanoyl-N-(.alpha.-trimethylammonioacetyl)diethanolamine
chloride, CLIP1:
rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,
CLIPS:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylamm-
onium, oligofectamine, Lipofectamine.RTM. or cationic or
polycationic polymers, e.g. modified polyaminoacids, such as
.beta.-aminoacid-polymers or reversed polyamides, etc., modified
polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium
bromide)), etc., modified acrylates, such as pDMAEMA
(poly(dimethylaminoethyl methylacrylate)), etc., modified
amidoamines such as pAMAM (poly(amidoamine)), etc., modified
polybetaaminoester (PBAE), such as diamine end modified 1,4
butanediol diacrylate-co-5-amino-1-pentanol polymers, etc.,
dendrimers, such as polypropylamine dendrimers or pAMAM based
dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine),
poly(propyleneimine), etc., polyallylamine, sugar backbone based
polymers, such as cyclodextrin based polymers, dextran based
polymers, chitosan, etc., silan backbone based polymers, such as
PMOXA-PDMS copolymers, etc., blockpolymers consisting of a
combination of one or more cationic blocks (e.g. selected from a
cationic polymer as mentioned above) and of one or more hydrophilic
or hydrophobic blocks (e.g. polyethyleneglycole); etc.
Polymeric carrier: A polymeric carrier is typically a carrier that
is formed of a polymer. A polymeric carrier in the context of the
present invention used as transfection or complexation agent might
be a polymeric carrier formed by disulfide-crosslinked cationic
components. The disulfide-crosslinked cationic components may be
the same or different from each other. The polymeric carrier can
also contain further components. It is also particularly preferred
that the polymeric carrier comprises mixtures of cationic peptides,
proteins or polymers and optionally further components as defined
herein, which are crosslinked by disulfide bonds as described
herein.
[0048] In this context the cationic components, which form basis
for the polymeric carrier by disulfide-crosslinkage, are typically
selected from any suitable cationic or polycationic peptide,
protein or polymer suitable for this purpose, particular any
cationic or polycationic peptide, protein or polymer capable to
complex a nucleic acid as defined according to the present
invention, and thereby preferably condensing the nucleic acid. The
cationic or polycationic peptide, protein or polymer, is preferably
a linear molecule, however, branched cationic or polycationic
peptides, proteins or polymers may also be used.
[0049] Every disulfide-crosslinking cationic or polycationic
protein, peptide or polymer of the polymeric carrier, which may be
used to complex e.g. the at least one RNA of the RNA vaccine
encoding at least one antigen or an adjuvant nucleic acid in the
inventive vaccine/inhibitor combination contains at least one --SH
moiety, most preferably at least one cysteine residue or any
further chemical group exhibiting an --SH moiety, capable to form a
disulfide linkage upon condensation with at least one further
cationic or polycationic protein, peptide or polymer as cationic
component of the polymeric carrier as mentioned herein.
[0050] As defined above, the polymeric carrier, which may be used
to complex e.g. the at least one RNA of the RNA vaccine encoding at
least one antigen or an adjuvant nucleic acid in the inventive
vaccine/inhibitor combination may be formed by
disulfide-crosslinked cationic (or polycationic) components.
[0051] According to one first alternative, at least one cationic
(or polycationic) component of the polymeric carrier, which may be
used in this context may be selected from cationic or polycationic
peptides or proteins. Such cationic or polycationic peptides or
proteins preferably exhibit a length of about 3 to 100 amino acids,
preferably a length of about 3 to 50 amino acids, more preferably a
length of about 3 to 25 amino acids, e.g. a length of about 3 to
10, 5 to 15, 10 to 20 or 15 to 25 amino acids. Alternatively or
additionally, such cationic or polycationic peptides or proteins
may exhibit a molecular weight of about 0.01 kDa to about 100 kDa,
including a molecular weight of about 0.5 kDa to about 100 kDa,
preferably of about 10 kDa to about 50 kDa, even more preferably of
about 10 kDa to about 30 kDa.
[0052] In the specific case that the cationic component of the
polymeric carrier, which may be used to complex e.g. the at least
one RNA of the RNA vaccine encoding at least one antigen or an
adjuvant nucleic acid in the inventive vaccine/inhibitor
combination comprises a cationic or polycationic peptide or
protein, the cationic properties of the cationic or polycationic
peptide or protein or of the entire polymeric carrier, if the
polymeric carrier is entirely composed of cationic or polycationic
peptides or proteins, may be determined upon its content of
cationic amino acids. Preferably, the content of cationic amino
acids in the cationic or polycationic peptide or protein and/or the
polymeric carrier is at least 10%, 20%, or 30%, preferably at least
40%, more preferably at least 50%, 60% or 70%, but also preferably
at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most
preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,
more preferably in the range of about 15% to 75%, even more
preferably in the range of about 20% to 50%, e.g. 20, 30, 40 or
50%, or in a range formed by any two of the afore mentioned values,
provided, that the content of all amino acids, e.g. cationic,
lipophilic, hydrophilic, aromatic and further amino acids, in the
cationic or polycationic peptide or protein, or in the entire
polymeric carrier, if the polymeric carrier is entirely composed of
cationic or polycationic peptides or proteins, is 100%.
[0053] Preferably, such cationic or polycationic peptides or
proteins of the polymeric carrier, which comprise or are
additionally modified to comprise at least one --SH moiety, are
selected from, without being restricted thereto, cationic peptides
or proteins such as protamine, nucleoline, spermine or spermidine,
oligo- or poly-L-lysine (PLL), basic polypeptides, oligo or
poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs,
such as Transportan, or MPG peptides, HIV-binding peptides, Tat,
HIV-1 Tat (HIV), Tat-derived peptides, members of the penetratin
family, e.g. Penetratin, Antennapedia-derived peptides
(particularly from Drosophila antennapedia), pAntp, pIsl, etc.,
antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB,
SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Loligomere,
FGF, Lactoferrin, histones, VP22 derived or analog peptides,
Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALA or protein
transduction domains (PTDs, PpT620, prolin-rich peptides,
arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers,
Calcitonin peptide(s), etc.
[0054] Alternatively or additionally, such cationic or polycationic
peptides or proteins of the polymeric carrier, which comprise or
are additionally modified to comprise at least one --SH moiety, are
selected from, without being restricted thereto, following cationic
peptides having the following sum formula (IV):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x};
formula (IV)
wherein l+m+n+o+x=3-100, and l, m, n or o independently of each
other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60,
61-70, 71-80, 81-90 and 91-100 provided that the overall content of
Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine)
represents at least 10% of all amino acids of the oligopeptide; and
Xaa is any amino acid selected from native (=naturally occurring)
or non-native amino acids except of Arg, Lys, His or Orn; and x is
any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,
71-80, 81-90, provided, that the overall content of Xaa does not
exceed 90% of all amino acids of the oligopeptide. Any of amino
acids Arg, Lys, His, Orn and Xaa may be positioned at any place of
the peptide. In this context cationic peptides or proteins in the
range of 7-30 amino acids are particular preferred. Even more
preferred peptides of this formula are oligoarginines such as e.g.
Arg.sub.7, Arg.sub.8, Arg.sub.9, Arg.sub.12, His.sub.3Arg.sub.9,
Arg.sub.9His.sub.3, His.sub.3Arg.sub.9His.sub.3,
His.sub.6Arg.sub.9His.sub.6, His.sub.3Arg.sub.4His.sub.3,
His.sub.6Arg.sub.4His.sub.6, TyrSer.sub.2Arg.sub.9Ser.sub.2Tyr,
(ArgLysHis).sub.4, Tyr(ArgLysHis).sub.2Arg, etc.
[0055] According to a one further particular preferred embodiment,
the cationic or polycationic peptide or protein of the polymeric
carrier, when defined according to formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (IV)) as shown above and which comprises or is
additionally modified to comprise at least one --SH moiety, may be,
without being restricted thereto, selected from subformula
(IVa):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa').sub.x(Cys).sub.y-
} formula (IVa)
wherein (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o; and x are
as defined herein, Xaa' is any amino acid selected from native
(=naturally occurring) or non-native amino acids except of Arg,
Lys, His, Orn or Cys and y is any number selected from 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and 81-90, provided that
the overall content of Arg (Arginine), Lys (Lysine), His
(Histidine) and Orn (Ornithine) represents at least 10% of all
amino acids of the oligopeptide.
[0056] This embodiment may apply to situations, wherein the
cationic or polycationic peptide or protein of the polymeric
carrier, e.g. when defined according to empirical formula
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula (IV)) as shown above, comprises or has been modified with
at least one cysteine as --SH moiety in the above meaning such that
the cationic or polycationic peptide as cationic component carries
at least one cysteine, which is capable to form a disulfide bond
with other components of the polymeric carrier.
[0057] According to another particular preferred embodiment, the
cationic or polycationic peptide or protein of the polymeric
carrier, when defined according to formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (IV)) as shown above, may be, without being restricted
thereto, selected from subformula (IVb):
Cys.sup.1{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cy-
s.sup.2 formula (IVb)
wherein empirical formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (IV)) is as defined herein and forms a core of an amino
acid sequence according to (semiempirical) formula (IV) and wherein
Cys.sup.1 and Cys.sup.2 are Cysteines proximal to, or terminal to
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x. This
embodiment may apply to situations, wherein the cationic or
polycationic peptide or protein of the polymeric carrier, which may
be used to complex the at least one RNA of the RNA vaccine encoding
at least one antigen or an adjuvant nucleic acid in the inventive
vaccine/inhibitor combination, e.g. when defined according to
empirical formula
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula (IV)) as shown above, has been modified with at least two
cysteines as --SH moieties in the above meaning such that the
cationic or polycationic peptide of the inventive polymeric carrier
carries at least two (terminal) cysteines, which are capable to
form a disulfide bond with other components of the polymeric
carrier.
[0058] According to a second alternative, at least one cationic (or
polycationic) component of the polymeric carrier may be selected
from e.g. any (non-peptidic) cationic or polycationic polymer
suitable in this context, provided that this (non-peptidic)
cationic or polycationic polymer exhibits or is modified to exhibit
at least one --SH-moiety, which provide for a disulfide bond
linking the cationic or polycationic polymer with another component
of the polymeric carrier as defined herein. Thus, likewise as
defined herein, the polymeric carrier may comprise the same or
different cationic or polycationic polymers.
[0059] In the specific case that the cationic component of the
polymeric carrier comprises a (non-peptidic) cationic or
polycationic polymer the cationic properties of the (non-peptidic)
cationic or polycationic polymer may be determined upon its content
of cationic charges when compared to the overall charges of the
components of the cationic polymer. Preferably, the content of
cationic charges in the cationic polymer at a (physiological) pH as
defined herein is at least 10%, 20%, or 30%, preferably at least
40%, more preferably at least 50%, 60% or 70%, but also preferably
at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most
preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,
more preferably in the range of about 30% to 100%, even preferably
in the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or
100%, or in a range formed by any two of the afore mentioned
values, provided, that the content of all charges, e.g. positive
and negative charges at a (physiological) pH as defined herein, in
the entire cationic polymer is 100%.
[0060] Preferably, the (non-peptidic) cationic component of the
polymeric carrier represents a cationic or polycationic polymer,
typically exhibiting a molecular weight of about 0.1 or 0.5 kDa to
about 100 kDa, preferably of about 1 kDa to about 75 kDa, more
preferably of about 5 kDa to about 50 kDa, even more preferably of
about 5 kDa to about 30 kDa, or a molecular weight of about 10 kDa
to about 50 kDa, even more preferably of about 10 kDa to about 30
kDa. Additionally, the (non-peptidic) cationic or polycationic
polymer typically exhibits at least one --SH-moiety, which is
capable to form a disulfide linkage upon condensation with either
other cationic components or other components of the polymeric
carrier as defined herein.
[0061] In the above context, the (non-peptidic) cationic component
of the polymeric carrier, which may be used to complex e.g. the at
least one RNA of the RNA vaccine encoding at least one antigen or
an adjuvant nucleic acid in the inventive vaccine/inhibitor
combination may be selected from acrylates, modified acrylates,
such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)),
chitosanes, aziridines or 2-ethyl-2-oxazoline (forming oligo
ethylenimines or modified oligoethylenimines), polymers obtained by
reaction of bisacrylates with amines forming oligo beta aminoesters
or poly amido amines, or other polymers like polyesters,
polycarbonates, etc. Each molecule of these (non-peptidic) cationic
or polycationic polymers typically exhibits at least one
--SH-moiety, wherein these at least one --SH-moiety may be
introduced into the (non-peptidic) cationic or polycationic polymer
by chemical modifications, e.g. using imonothiolan, 3-thio
propionic acid or introduction of --SH-moieties containing amino
acids, such as cysteine or any further (modified) amino acid. Such
--SH-moieties are preferably as already defined above.
[0062] According to a particularly preferred embodiment, the
further component, which may be contained in the polymeric carrier,
which may be used to modify the different (short) cationic or
polycationic peptides or (non-peptidic) polymers forming basis for
the polymeric carrier or the biophysical/biochemical properties of
the polymeric carrier as defined herein, is an amino acid component
(AA). According to the present invention, the amino acid component
(AA) comprises a number of amino acids preferably in a range of
about 1 to 100, preferably in a range of about 1 to 50, more
preferably selected from a number comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14 or 15-20, or may be selected from a range
formed by any two of the afore mentioned values. In this context
the amino acids of amino acid component (AA) can be chosen
independently from each other. For example if in the polymeric
carrier two or more (AA) components are present they can be the
same or can be different from each other.
[0063] The amino acid component (AA) may contain or may be flanked
(e.g. terminally) by a --SH containing moiety, which allows
introducing this component (AA) via a disulfide bond into the
polymeric carrier as defined herein. In the specific case that the
--SH containing moiety represents a cysteine, the amino acid
component (AA) may also be read as -Cys-(AA)-Cys- wherein Cys
represents cysteine and provides for the necessary --SH-moiety for
a disulfide bond. The --SH containing moiety may be also introduced
into amino acid component (AA) using any of modifications or
reactions as shown above for the cationic component or any of its
components.
[0064] Furthermore, the amino acid component (AA) may be provided
with two --SH-moieties (or even more), e.g. in a form represented
by formula HS-(AA)-SH to allow binding to two functionalities via
disulfide bonds, e.g. if the amino acid component (AA) is used as a
linker between two further components (e.g. as a linker between two
cationic polymers).
[0065] Alternatively, the amino acid component (AA) may be provided
with other functionalities as already described above for the other
components of the polymeric carrier, which allow binding of the
amino acid component (AA) to any of components of the polymeric
carrier.
[0066] Thus, according to the present invention, the amino acid
component (AA) of the polymeric carrier may be bound to further
components of the polymeric carrier, which may be used to complex
e.g. the at least one RNA of the RNA vaccine encoding at least one
antigen or an adjuvant nucleic acid in the inventive
vaccine/inhibitor with or without using a disulfide linkage.
[0067] According to a further and particularly preferred
alternative, the amino acid component (AA), may be used to modify
the polymeric carrier, particularly the content of cationic
components in the polymeric carrier as defined above.
[0068] In the context of the present invention, the amino acid
component (AA) may be selected from the following alternatives: an
aromatic amino acid component, a hydrophilic (and preferably non
charged polar) amino acid component, a lipophilic amino acid
component, or a weak basic amino acid component.
[0069] According to a further alternative, the amino acid component
(AA) may be a signal peptide or signal sequence, a localisation
signal or sequence, a nuclear localisation signal or sequence
(NLS), an antibody, a cell penetrating peptide (e.g. TAT), etc.
Additionally, according to another alternative, the amino acid
component (AA) may be a functional peptide or protein, which may
modulate the functionality of the polymeric carrier accordingly.
Such functional peptides or proteins as the amino acid component
(AA) preferably comprise any peptides or proteins as defined
herein, e.g. as defined herein as antigens. According to one
alternative, such further functional peptides or proteins may
comprise so called cell penetrating peptides (CPPs) or cationic
peptides for transportation.
[0070] According to a last alternative, the amino acid component
(AA) may consist of or may comprise any peptide or protein which
can execute any favourable function in the cell. Particularly
preferred are peptides or proteins selected from therapeutically
active proteins or peptides, from antigens, e.g. tumour antigens,
pathogenic antigens (e.g. animal antigens, viral antigens,
protozoan antigens, bacterial antigens), from antibodies, from
immunostimulatory proteins or peptides, from antigen-specific T
cell receptors, or from any other protein or peptide suitable for a
specific (therapeutic) application. Particularly preferred are
peptide epitopes from those antigen(s) encoded by the at least one
RNA of the RNA vaccine encoding at least one antigen in the
inventive vaccine/inhibitor combination.
[0071] The polymeric carrier, which may be used to complex e.g. the
at least one RNA of the RNA vaccine encoding at least one antigen
or an adjuvant nucleic acid in the inventive vaccine/inhibitor
combination may comprise at least one of the above mentioned
cationic or polycationic peptides, proteins or polymers or further
components, e.g. (AA), wherein any of the above alternatives may be
combined with each other, and may be formed by polymerizing same in
a polymerization condensation reaction via their --SH-moieties.
[0072] Further, the polymeric carrier may be selected from a
polymeric carrier molecule according to generic formula (V):
L-P.sup.1--S--[S--P.sup.2--S].sub.n--S--P.sup.3-L formula (V)
wherein, [0073] P.sup.1 and P.sup.3 are different or identical to
each other and represent a linear or branched hydrophilic polymer
chain, each P.sup.1 and P.sup.3 exhibiting at least one
--SH-moiety, capable to form a disulfide linkage upon condensation
with component P.sup.2, or alternatively with (AA), (AA).sub.x, or
[(AA).sub.x].sub.z if such components are used as a linker between
P.sup.1 and P.sup.2 or P.sup.3 and P.sup.2) and/or with further
components (e.g. (AA), (AA).sub.x, [(AA).sub.x].sub.z or L), the
linear or branched hydrophilic polymer chain selected independent
from each other from polyethylene glycol (PEG),
poly-N-(2-hydroxypropyl)methacrylamide,
poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl
L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),
hydroxyethyl starch or poly(hydroxyalkyl L-glutamine), wherein the
hydrophilic polymer chain exhibits a molecular weight of about 1
kDa to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or
more preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to
about 25 kDa or 5 kDa to about 10 kDa; [0074] P.sup.2 is a cationic
or polycationic peptide or protein, e.g. as defined above for the
polymeric carrier formed by disulfide-crosslinked cationic
components, and preferably having a length of about 3 to about 100
amino acids, more preferably having a length of about 3 to about 50
amino acids, even more preferably having a length of about 3 to
about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15, 10
to 20 or 15 to 25 amino acids, more preferably a length of about 5
to about 20 and even more preferably a length of about 10 to about
20; or [0075] is a cationic or polycationic polymer, e.g. as
defined above for the polymeric carrier formed by
disulfide-crosslinked cationic components, typically having a
molecular weight of about 0.5 kDa to about 30 kDa, including a
molecular weight of about 1 kDa to about 20 kDa, even more
preferably of about 1.5 kDa to about 10 kDa, or having a molecular
weight of about 0.5 kDa to about 100 kDa, including a molecular
weight of about 10 kDa to about 50 kDa, even more preferably of
about 10 kDa to about 30 kDa; [0076] each P.sup.2 exhibiting at
least two --SH-moieties, capable to form a disulfide linkage upon
condensation with further components P.sup.2 or component(s)
P.sup.1 and/or P.sup.3 or alternatively with further components
(e.g. (AA), (AA).sub.x, or [(AA).sub.x].sub.z); [0077] --S--S-- is
a (reversible) disulfide bond (the brackets are omitted for better
readability), wherein S preferably represents sulphur or a --SH
carrying moiety, which has formed a (reversible) disulfide bond.
The (reversible) disulfide bond is preferably formed by
condensation of --SH-moieties of either components P' and P.sup.2,
P.sup.2 and P.sup.2, or P.sup.2 and P.sup.3, or optionally of
further components as defined herein (e.g. L, (AA), (AA).sub.x,
[(AA).sub.x].sub.z, etc); The --SH-moiety may be part of the
structure of these components or added by a modification as defined
below; [0078] L is an optional ligand, which may be present or not,
and may be selected independent from the other from RGD,
Transferrin, Folate, a signal peptide or signal sequence, a
localization signal or sequence, a nuclear localization signal or
sequence (NLS), an antibody, a cell penetrating peptide, (e.g. TAT
or KALA), a ligand of a receptor (e.g. cytokines, hormones, growth
factors etc), small molecules (e.g. carbohydrates like mannose or
galactose or synthetic ligands), small molecule agonists,
inhibitors or antagonists of receptors (e.g. RGD peptidomimetic
analogues), or any further protein as defined herein, etc.; [0079]
n is an integer, typically selected from a range of about 1 to 50,
preferably from a range of about 1, 2 or 3 to 30, more preferably
from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about
1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15,
or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range
of about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20,
or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a
range of about 6 to 11 or 7 to 10. Most preferably, n is in a range
of about 1, 2, 3, 4, or 5 to 10, more preferably in a range of
about 1, 2, 3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8,
or in a range of about 1, 2, or 3 to 7.
[0080] Each of hydrophilic polymers P.sup.1 and P.sup.3 typically
exhibits at least one --SH-moiety, wherein the at least one
--SH-moiety is capable to form a disulfide linkage upon reaction
with component P.sup.2 or with component (AA) or (AA).sub.x, if
used as linker between P.sup.1 and P.sup.2 or P.sup.3 and P.sup.2
as defined below and optionally with a further component, e.g. L
and/or (AA) or (AA).sub.x, e.g. if two or more --SH-moieties are
contained. The following subformulae "P.sup.1--S--S--P.sup.2" and
"P.sup.2--S--S--P.sup.3" within generic formula (V) above (the
brackets are omitted for better readability), wherein any of S,
P.sup.1 and P.sup.3 are as defined herein, typically represent a
situation, wherein one-SH-moiety of hydrophilic polymers P.sup.1
and P.sup.3 was condensed with one --SH-moiety of component P.sup.2
of generic formula (V) above, wherein both sulphurs of these
--SH-moieties form a disulfide bond --S--S-- as defined herein in
formula (V). These --SH-moieties are typically provided by each of
the hydrophilic polymers P.sup.1 and P.sup.3, e.g. via an internal
cysteine or any further (modified) amino acid or compound which
carries a --SH moiety. Accordingly, the subformulae
"P.sup.1--S--S--P.sup.2" and "P.sup.2--S--S--P.sup.3" may also be
written as "P.sup.1-Cys-Cys-P.sup.2" and "P.sup.2-Cys-Cys-P.sup.3",
if the --SH-- moiety is provided by a cysteine, wherein the term
Cys-Cys represents two cysteines coupled via a disulfide bond, not
via a peptide bond. In this case, the term "--S--S--" in these
formulae may also be written as "--S-Cys", as "-Cys-S" or as
"-Cys-Cys-". In this context, the term "-Cys-Cys-" does not
represent a peptide bond but a linkage of two cysteines via their
--SH-moieties to form a disulfide bond. Accordingly, the term
"-Cys-Cys-" also may be understood generally as
"-(Cys-S)--(S-Cys)-", wherein in this specific case S indicates the
sulphur of the --SH-moiety of cysteine. Likewise, the terms
"--S-Cys" and "--Cys-S" indicate a disulfide bond between a --SH
containing moiety and a cysteine, which may also be written as
"--S--(S-Cys)" and "-(Cys-S)--S". Alternatively, the hydrophilic
polymers P.sup.1 and P.sup.3 may be modified with a --SH moiety,
preferably via a chemical reaction with a compound carrying a --SH
moiety, such that each of the hydrophilic polymers P' and P.sup.3
carries at least one such --SH moiety. Such a compound carrying a
--SH moiety may be e.g. an (additional) cysteine or any further
(modified) amino acid, which carries a --SH moiety. Such a compound
may also be any non-amino compound or moiety, which contains or
allows to introduce a --SH moiety into hydrophilic polymers P.sup.1
and P.sup.3 as defined herein. Such non-amino compounds may be
attached to the hydrophilic polymers P.sup.1 and P.sup.3 of formula
(VI) of the polymeric carrier according to the present invention
via chemical reactions or binding of compounds, e.g. by binding of
a 3-thio propionic acid or thioimolane, by amide formation (e.g.
carboxylic acids, sulphonic acids, amines, etc), by Michael
addition (e.g maleinimide moieties, .quadrature., .quadrature.
unsatured carbonyls, etc), by click chemistry (e.g. azides or
alkines), by alkene/alkine methatesis (e.g. alkenes or alkines),
imine or hydrozone formation (aldehydes or ketons, hydrazins,
hydroxylamins, amines), complexation reactions (avidin, biotin,
protein G) or components which allow Sn-type substitution reactions
(e.g halogenalkans, thiols, alcohols, amines, hydrazines,
hydrazides, sulphonic acid esters, oxyphosphonium salts) or other
chemical moieties which can be utilized in the attachment of
further components. A particularly preferred PEG derivate in this
context is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In
each case, the SH-moiety, e.g. of a cysteine or of any further
(modified) amino acid or compound, may be present at the terminal
ends or internally at any position of hydrophilic polymers P.sup.1
and P.sup.3. As defined herein, each of hydrophilic polymers
P.sup.1 and P.sup.3 typically exhibits at least one --SH-moiety
preferably at one terminal end, but may also contain two or even
more --SH-moieties, which may be used to additionally attach
further components as defined herein, preferably further functional
peptides or proteins e.g. a ligand, an amino acid component (AA) or
(AA).sub.x, antibodies, cell penetrating peptides or enhancer
peptides (e.g. TAT, KALA), etc.
In the context of the entire formula (V) of the inventive polymeric
carrier may be preferably defined as follows:
L-P.sup.1--S-[Cys-P.sup.2-Cys].sub.n-S--P.sup.3-L formula (VI)
wherein L, P.sup.1, P.sup.2, P.sup.3 and n are as defined herein, S
is sulphur and each Cys provides for one --SH-moiety for the
disulfide bond.
[0081] The amino acid component (AA) or (AA).sub.x in the polymeric
carrier of formula (V or VI), e.g. as defined above for the
polymeric carrier formed by disulfide-crosslinked cationic
components may also occur as a mixed repetitive amino acid
component [(AA).sub.x].sub.z, wherein the number of amino acid
components (AA) or (AA).sub.x is further defined by integer z. In
this context, z may be selected from a range of about 1 to 30,
preferably from a range of about 1 to 15, more preferably 1 to 10
or 1 to 5 and even more preferably selected from a number selected
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be
selected from a range formed by any two of the afore mentioned
values.
[0082] According to a specific and particularly preferred
alternative, the amino acid component (AA) or (AA).sub.x,
preferably written as S-(AA).sub.x-S or [S-(AA).sub.x-S] may be
used to modify component P.sup.2, particularly the content of
component S--P.sup.2--S in repetitive component [S--P.sup.2--S]n of
the polymeric carrier of formula (V) above. This may be represented
in the context of the entire polymeric carrier according to formula
(VI) e.g. by following formula (VIa):
L-P.sup.1--S--{[S--P.sup.2--S].sub.a[S-(AA).sub.x-S].sub.b}--S--P.sup.3--
L, formula (VIa)
wherein x, S, L, AA, P.sup.1, P.sup.2 and P.sup.3 are preferably as
defined herein. In formula (VIa) above, any of the single
components [S--P.sup.2--S] and [S-(AA).sub.x-S] may occur in any
order in the subformula {[S--P.sup.2--S].sub.a[S-(AA).sub.x-S]b}.
The numbers of single components [S--P.sup.2--S] and
[S-(AA).sub.x-S] in the subformula
{[S--P.sup.2--S].sub.a[S-(AA).sub.x-S].sub.b} are determined by
integers a and b, wherein a+b=n. n is an integer and is defined as
above for formula (V).
[0083] According to another embodiment, the polymeric carrier,
which may be used to complex e.g. the at least one RNA of the RNA
vaccine encoding at least one antigen or an adjuvant nucleic acid
in the inventive vaccine/inhibitor combination or single components
thereof, e.g. of the above mentioned cationic or polycationic
peptides, proteins or polymers or further components, e.g. (AA),
may be further modified with a ligand, preferably a carbohydrate,
more preferably a sugar, even more preferably mannose.
[0084] According to one specific embodiment, the entire polymeric
carrier may be formed by a polymerization condensation (of at least
one) of the above mentioned cationic or polycationic peptides,
proteins or polymers or further components, e.g. (AA), via their
--SH-moieties in a first step and complexing e.g. the at least one
RNA of the RNA vaccine encoding at least one antigen or an adjuvant
nucleic acid in the inventive vaccine/inhibitor combination to such
a polymeric carrier in a second step. The polymeric carrier may
thus contain a number of at least one or even more of the same or
different of the above defined cationic or polycationic peptides,
proteins or polymers or further components, e.g. (AA), the number
preferably determined by the above range.
[0085] According to one alternative specific embodiment, the
polymeric carrier, which may be used to complex e.g. the at least
one RNA of the RNA vaccine encoding at least one antigen or an
adjuvant nucleic acid in the inventive vaccine/inhibitor
combination is formed by carrying out the polymerization
condensation of at least one of the above mentioned cationic or
polycationic peptides, proteins or polymers or further components,
e.g. (AA), via their --SH-moieties simultaneously to complexing
e.g. the at least one RNA of the RNA vaccine encoding at least one
antigen or an adjuvant nucleic acid in the inventive
vaccine/inhibitor combination to the (in situ prepared) polymeric
carrier. Likewise, the polymeric carrier may thus also here contain
a number of at least one or even more of the same or different of
the above defined cationic or polycationic peptides, proteins or
polymers or further components, e.g. (AA), the number preferably
determined by the above range.
[0086] N/P ratio: The N/P ratio is a measure of the ionic charge of
the cationic (side chain) component of the cationic or polycationic
compound or of the polymeric carrier used as carrier or
complexation agent as defined herein. In particular, if the
cationic properties of the cationic component are generated by
nitrogens (e.g. of the amino acid side chains), the N/P ratio
expresses the ratio of basic nitrogen atoms to phosphate residues
in the nucleotide backbone, considering that (side chain) nitrogen
atoms in the cationic component of the cationic or polycationic
compound or of the polymeric carrier contribute to positive charges
and phosphate of the phosphate backbone of the nucleic acid cargo
e.g. the at least one RNA coding for at least one antigen comprised
in the RNA vaccine or an adjuvant nucleic acid contribute to the
negative charge. Generally, one phosphate provides one negative
charge, e.g. one nucleotide in the cargo nucleic acid molecule
provides one negative charge. It may be calculated on the basis
that, for example, 1 .mu.g RNA typically contains about 3 nmol
phosphate residues, provided that RNA exhibits a statistical
distribution of bases. Additionally, 1 nmol peptide typically
contains about x nmol nitrogen residues, dependent on the molecular
weight and the number of its (cationic) amino acids.
Zetapotential: The "zetapotential" is a widely used parameter for
the electrical surface charge of a particle. It is typically
determined by moving the charged particle through an electrical
field. In the context of the present invention, the zetapotential
is the preferred parameter for characterizing the charge of a
particle, e.g. of a complex comprising as carrier or complexation
agent a cationic or polycationic compound and/or a polymeric
carrier and as nucleic acid cargo the at least one RNA coding for
at least one antigen of the RNA vaccine or an adjuvant nucleic
acid. Thus, in the context of the present invention, the charge of
a particle is preferably determined by determining the
zetapotential by the laser Doppler electrophoresis method using a
Zetasizer Nano instrument (Malvern Instruments, Malvern, UK) at
25.degree. C. and a scattering angle of 173.degree.. The surface
charge of a given particle also depends on the ionic strength of
the utilized matrix (e.g. salt containing buffer) and the pH of the
solution. Therefore, the actual zetapotential of a given complex at
a charge ratio (N/P) may differ slightly between different buffers
used for injection. For the measurement, the particles, such as
complexes comprising as carrier or complexation agent a cationic or
polycationic compound and/or a polymeric carrier and as nucleic
acid cargo the at least one RNA coding for at least one antigen of
the RNA vaccine or an adjuvant nucleic acid according to the
invention are preferably suspended in Ringer Lactate solution. In a
specific embodiment the present invention refers to the use of a
negatively charged complex under the conditions of a given
injection buffer, preferably under the conditions of a Ringer
lactate solution, assessed by its Zetapotential. A Ringer lactate
solution according to the present invention preferably contains 130
mmol/L sodium ions, 109 mmol/L chloride ions, 28 mmol/L lactate, 4
mmol/L potassium ions and 1.5 mmol/L calcium ion. The sodium,
chloride, potassium and lactate typically come from NaCl (sodium
chloride), NaC.sub.3H.sub.5O.sub.3 (sodium lactate), CaCl.sub.2
(calcium chloride), and KCl (potassium chloride). The osmolarity of
the Ringer lactate solution is 273 mOsm/L and the pH is adjusted to
6.5. Immunostimulatory composition: In the context of the
invention, an immunostimulatory composition may be typically
understood to be a composition containing at least one component
which is able to induce an immune response or from which a
component which is able to induce an immune response is derivable.
Such immune response may be preferably an innate immune response or
a combination of an adaptive and an innate immune response.
Preferably, an immunostimulatory composition in the context of the
invention contains at least one immunostimulating/adjuvant nucleic
acid molecule, more preferably an RNA, for example an mRNA
molecule. The immunostimulatory component, such as the mRNA may be
complexed with a suitable carrier. Thus, the immunostimulatory
composition may comprise an mRNA/carrier-complex. Furthermore, the
immunostimulatory composition may comprise an adjuvant and/or a
suitable vehicle for the immunostimulatory component, such as the
mRNA. Adjuvant nucleic acid: An adjuvant nucleic acid, as used
herein, is preferably selected from nucleic acids which are known
to bind to TLR receptors. Such an adjuvant nucleic acid can be in
the form of a(n) (immunostimulatory) CpG nucleic acid, in
particular CpG-RNA or CpG-DNA, which preferably induces an innate
immune response. A CpG-RNA or CpG-DNA used according to the
invention can be a single-stranded CpG-DNA (ss CpG-DNA), a
double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss
CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic
acid used according to the invention is preferably in the form of
CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss
CpG-RNA). Also preferably, such CpG nucleic acids have a length as
described above. Preferably, the CpG motifs are unmethylated.
Furthermore, an adjuvant nucleic acid, as used herein, can be an
immunostimulatory RNA (isRNA), which preferably elicits an innate
immune response.
[0087] Preferably, an adjuvant nucleic acid, preferably an
immunostimulatory RNA (isRNA), as used herein, may comprise any
nucleic acid sequence known to be immunostimulatory, including,
e.g., nucleic acid sequences representing and/or encoding ligands
of TLRs, preferably selected from human family members TLR1-TLR10
or murine family members TLR1-TLR13, more preferably selected from
(human) family members TLR1-TLR10, even more preferably from TLR7
and TLR8, ligands for intracellular receptors for RNA (such as
RIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006).
Toll-like receptors and RNA helicases: two parallel ways to trigger
antiviral responses. Mol. Cell 22, 561-569), or any other
immunostimulatory RNA sequence. Such an adjuvant nucleic acid may
comprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or
of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5
to 30 nucleotides.
[0088] According to a particularly preferred embodiment, an
adjuvant nucleic acid sequence, particularly an isRNA, as used
herein, may consist of or comprise a nucleic acid of formula (VII)
or (VIII):
G.sub.lX.sub.mG.sub.n, (formula (VII))
wherein: [0089] G is guanosine, uracil or an analogue of guanosine
or uracil; [0090] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0091]
l is an integer from 1 to 40, [0092] wherein [0093] when l=1 G is
guanosine or an analogue thereof, [0094] when l>1 at least 50%
of the nucleotides are guanosine or an analogue thereof; [0095] m
is an integer and is at least 3; [0096] wherein [0097] when m=3 X
is uracil or an analogue thereof, [0098] when m>3 at least 3
successive uracils or analogues of uracil occur; [0099] n is an
integer from 1 to 40, [0100] wherein [0101] when n=1 G is guanosine
or an analogue thereof, [0102] when n>1 at least 50% of the
nucleotides are guanosine or an analogue thereof.
[0102] C.sub.lX.sub.mC.sub.n, (formula (VIII))
wherein: [0103] C is cytosine, uracil or an analogue of cytosine or
uracil; [0104] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0105]
l is an integer from 1 to 40, [0106] wherein [0107] when l=1 C is
cytosine or an analogue thereof, [0108] when l>1 at least 50% of
the nucleotides are cytosine or an analogue thereof; [0109] m is an
integer and is at least 3; [0110] wherein [0111] when m=3 X is
uracil or an analogue thereof, [0112] when m>3 at least 3
successive uracils or analogues of uracil occur; [0113] n is an
integer from 1 to 40, [0114] wherein [0115] when n=1 C is cytosine
or an analogue thereof, [0116] when n>1 at least 50% of the
nucleotides are cytosine or an analogue thereof.
[0117] The nucleic acids of formula (VII) or (VIII), which may be
used as an adjuvant nucleic acid sequence, particularly an isRNA,
may be relatively short nucleic acid molecules with a typical
length of approximately from 5 to 100 (but may also be longer than
100 nucleotides for specific embodiments, e.g. up to 200
nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably
a length of approximately from 5 to 70, more preferably a length of
approximately from 8 to 60 and, more preferably a length of
approximately from 15 to 60 nucleotides, more preferably from 20 to
60, most preferably from 30 to 60 nucleotides. If the nucleic acid
of formula (VII) or (VIII) has a maximum length of e.g. 100
nucleotides, m will typically be <=98. The number of nucleotides
G in the nucleic acid of formula (I) is determined by l or n. l and
n, independently of one another, are each an integer from 1 to 40,
wherein when l or n=1 G is guanosine or an analogue thereof, and
when l or n>1 at least 50% of the nucleotides are guanosine or
an analogue thereof. For example, without implying any limitation,
when l or n=4 G.sub.1 or G.sub.n can be, for example, a GUGU, GGUU,
UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.; when l or
n=5 G.sub.1 or G.sub.n can be, for example, a GGGUU, GGUGU, GUGGU,
UGGGU, UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG,
UGGGG, or GGGGG, etc.; etc. A nucleotide adjacent to X.sub.m in the
nucleic acid of formula (VII) according to the invention is
preferably not a uracil. Similarly, the number of nucleotides C in
the nucleic acid of formula (VIII) according to the invention is
determined by l or n. l and n, independently of one another, are
each an integer from 1 to 40, wherein when l or n=1 C is cytosine
or an analogue thereof, and when l or n>1 at least 50% of the
nucleotides are cytosine or an analogue thereof. For example,
without implying any limitation, when l or n=4, C.sub.1 or C.sub.n
can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC,
CUCC, UCCC or CCCC, etc.; when l or n=5 C.sub.1 or C.sub.n can be,
for example, a CCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC,
CUCUC, CCCCU, CCCUC, CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A
nucleotide adjacent to X.sub.m in the nucleic acid of formula
(VIII) according to the invention is preferably not a uracil.
Preferably, for formula (VII), when l or n>1, at least 60%, 70%,
80%, 90% or even 100% of the nucleotides are guanosine or an
analogue thereof, as defined above. The remaining nucleotides to
100% (when guanosine constitutes less than 100% of the nucleotides)
in the flanking sequences G.sub.1 and/or G.sub.n are uracil or an
analogue thereof, as defined hereinbefore. Also preferably, l and
n, independently of one another, are each an integer from 2 to 30,
more preferably an integer from 2 to 20 and yet more preferably an
integer from 2 to 15. The lower limit of l or n can be varied if
necessary and is at least 1, preferably at least 2, more preferably
at least 3, 4, 5, 6, 7, 8, 9 or 10. This definition applies
correspondingly to formula (VIII).
[0118] According to a further particularly preferred embodiment, an
adjuvant nucleic acid sequence, particularly an isRNA, as used
herein, may consist of or comprise a nucleic acid of formula (IX)
or (X):
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a, (formula (IX))
wherein: [0119] G is guanosine (guanine), uridine (uracil) or an
analogue of guanosine (guanine) or uridine (uracil), preferably
guanosine (guanine) or an analogue thereof; [0120] X is guanosine
(guanine), uridine (uracil), adenosine (adenine), thymidine
(thymine), cytidine (cytosine), or an analogue of these nucleotides
(nucleosides), preferably uridine (uracil) or an analogue thereof;
[0121] N is a nucleic acid sequence having a length of about 4 to
50, preferably of about 4 to 40, more preferably of about 4 to 30
or 4 to 20 nucleic acids, each N independently being selected from
guanosine (guanine), uridine (uracil), adenosine (adenine),
thymidine (thymine), cytidine (cytosine) or an analogue of these
nucleotides (nucleosides); [0122] a is an integer from 1 to 20,
preferably from 1 to 15, most preferably from 1 to 10; [0123] l is
an integer from 1 to 40, [0124] wherein [0125] when l=1, G is
guanosine (guanine) or an analogue thereof, [0126] when l>1, at
least 50% of these nucleotides (nucleosides) are guanosine
(guanine) or an analogue thereof; [0127] m is an integer and is at
least 3; [0128] wherein [0129] when m=3, X is uridine (uracil) or
an analogue thereof, and [0130] when m>3, at least 3 successive
uridines (uracils) or analogues of uridine (uracil) occur; [0131] n
is an integer from 1 to 40, [0132] wherein [0133] when n=1, G is
guanosine (guanine) or an analogue thereof, [0134] when n>1, at
least 50% of these nucleotides (nucleosides) are guanosine
(guanine) or [0135] an analogue thereof; [0136] u, v may be
independently from each other an integer from 0 to 50, [0137]
preferably wherein when u=0, v 1, or [0138] when v=0, u.gtoreq.1;
wherein the nucleic acid molecule of formula (IX) has a length of
at least 50 nucleotides, preferably of at least 100 nucleotides,
more preferably of at least 150 nucleotides, even more preferably
of at least 200 nucleotides and most preferably of at least 250
nucleotides.
[0138] (N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a (formula
(X))
wherein: [0139] C is cytidine (cytosine), uridine (uracil) or an
analogue of cytidine (cytosine) or uridine (uracil), preferably
cytidine (cytosine) or an analogue thereof; [0140] X is guanosine
(guanine), uridine (uracil), adenosine (adenine), thymidine
(thymine), cytidine (cytosine) or an analogue of the
above-mentioned nucleotides (nucleosides), preferably uridine
(uracil) or an analogue thereof; [0141] N is each a nucleic acid
sequence having independent from each other a length of about 4 to
50, preferably of about 4 to 40, more preferably of about 4 to 30
or 4 to 20 nucleic acids, each N independently being selected from
guanosine (guanine), uridine (uracil), adenosine (adenine),
thymidine (thymine), cytidine (cytosine) or an analogue of these
nucleotides (nucleosides); [0142] a is an integer from 1 to 20,
preferably from 1 to 15, most preferably from 1 to 10; [0143] l is
an integer from 1 to 40, [0144] wherein [0145] when l=1, C is
cytidine (cytosine) or an analogue thereof, [0146] when l>1, at
least 50% of these nucleotides (nucleosides) are cytidine
(cytosine) or an analogue thereof; [0147] m is an integer and is at
least 3; [0148] wherein [0149] when m=3, X is uridine (uracil) or
an analogue thereof, [0150] when m>3, at least 3 successive
uridines (uracils) or analogues of uridine (uracil) occur; [0151] n
is an integer from 1 to 40, [0152] wherein [0153] when n=1, C is
cytidine (cytosine) or an analogue thereof, [0154] when n>1, at
least 50% of these nucleotides (nucleosides) are cytidine
(cytosine) or [0155] an analogue thereof. [0156] u, v may be
independently from each other an integer from 0 to 50, [0157]
preferably wherein when u=0, v.gtoreq.1, or [0158] when v=0,
u.gtoreq.1; wherein the nucleic acid molecule of formula (X)
according to the invention has a length of at least 50 nucleotides,
preferably of at least 100 nucleotides, more preferably of at least
150 nucleotides, even more preferably of at least 200 nucleotides
and most preferably of at least 250 nucleotides.
[0159] Any of the definitions given above in formulae (VII) and
(VIII), e.g. for elements N (i.e. N.sub.u and N.sub.v) and X
(X.sub.m), particularly the core structure as defined above, as
well as for integers a, l, m, n, u and v, similarly apply to
elements of formula (IX) and (X) correspondingly. The definition of
bordering elements N.sub.u and N.sub.v in formula (X) is identical
to the definitions given above for N.sub.u and N.sub.v in formula
(IX).
Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the
context of the invention may typically be an RNA that is able to
induce an innate immune response. It usually does not have an open
reading frame and thus does not provide a peptide-antigen or
immunogen but elicits an innate immune response e.g. by binding to
a specific kind of Toll-like-receptor (TLR) or other suitable
receptors. However, of course also mRNAs having an open reading
frame and coding for a peptide/protein may induce an innate immune
response and, thus, may be immunostimulatory RNAs. Preferably, the
immunostimulatory RNA may be a single-stranded, a double-stranded
or a partially double-stranded RNA, more preferably a
single-stranded RNA, and/or a circular or linear RNA, more
preferably a linear RNA. More preferably, the immunostimulatory RNA
may be a (linear) single-stranded RNA. Even more preferably, the
immunostimulatory RNA may be a (long) (linear) (single-stranded)
non-coding RNA. In this context it is particular preferred that the
isRNA carries a triphosphate at its 5'-end which is the case for in
vitro transcribed RNA. An immunostimulatory RNA may also occur as a
short RNA oligonucleotide as defined herein. An immunostimulatory
RNA as used herein may furthermore be selected from any class of
RNA molecules, found in nature or being prepared synthetically, and
which can induce an innate immune response and may support an
adaptive immune response induced by an antigen. Furthermore,
(classes of) immunostimulatory RNA molecules, used as a further
compound of the inventive vaccine/inhibitor combination, may
include any other RNA capable of eliciting an innate immune
response. E.g., such an immunostimulatory RNA may include ribosomal
RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral
RNA (vRNA). Such an immunostimulatory RNA may comprise a length of
1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to
500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.
siRNA: A small interfering RNA (siRNA), is of interest particularly
in connection with the phenomenon of RNA interference. The in vitro
technique of RNA interference (RNAi) is based on double-stranded
RNA molecules (dsRNA), which trigger the sequence-specific
suppression of gene expression (Zamore (2001) Nat. Struct. Biol. 9:
746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon (2002) Nature
41: 244-251). In the transfection of mammalian cells with long
dsRNA, the activation of protein kinase R and RnaseL brings about
unspecific effects, such as, for example, an interferon response
(Stark et al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze
(2002) Viral Immunol. 15: 95-119). These unspecific effects are
avoided when shorter, for example 21- to 23-mer, so-called siRNA
(small interfering RNA), is used, because unspecific effects are
not triggered by siRNA that is shorter than 30 bp (Elbashir et al.
(2001) Nature 411: 494-498).
[0160] In the context of the present invention, siRNAs directed
against PD-1, PD-L1 or PD-L2 may thus be double-stranded RNAs
(dsRNAs) having a length of from 17 to 29, preferably from 19 to
25, and preferably is at least 90%, more preferably 95% and
especially 100% (of the nucleotides of a dsRNA) complementary to a
section of the naturally occurring mRNA sequence coding for PD-1,
PD-L1 or PD-L2 either to a coding or a non-coding section,
preferably a coding section. Such a section of the naturally
occurring mRNA sequence may be termed herein a "target sequence"
and may be any section of the naturally occurring mRNA coding for
PD-1, PD-L1 or PD-L2. 90% complementary means that with a length of
a dsRNA described herein of, for example, 20 nucleotides, the dsRNA
contain not more than 2 nucleotides showing no complementarity with
the corresponding section of the target sequence. The sequence of
the double-stranded siRNA used according to the invention is,
however, preferably wholly complementary in its general structure
with a section of the target sequence. In this context the nucleic
acid molecule of the complex may be a dsRNA having the general
structure 5'-(N.sub.17-29)-3', preferably having the general
structure 5'-(N.sub.19-25)-3', more preferably having the general
structure 5'-(N.sub.19-24)-3', or yet more preferably having the
general structure 5'-(N.sub.21-23)-3', wherein for each general
structure each N is a (preferably different) nucleotide of a
section of the target sequence, preferably being selected from a
continuous number of 17 to 29 nucleotides of a section of the
target sequence, and being present in the general structure
5'-(N.sub.17-29)-3' in their natural order. In principle, all the
sections having a length of from 17 to 29, preferably from 19 to
25, base pairs that occur in the target sequence can serve for
preparation of a dsRNA as defined herein. Equally, dsRNAs used as
siRNAs can also be directed against mRNA sequences that do not lie
in the coding region, in particular in the 5' non-coding region of
the target sequence, for example, therefore, against non-coding
regions of the target sequence having a regulatory function. The
target sequence of the dsRNA used as siRNA can therefore lie in the
translated and untranslated region of the target sequence and/or in
the region of the control elements of the mRNA sequence. The target
sequence for a dsRNA used as siRNA directed against PD-1, PD-L1 or
PD-L2 can also lie in the overlapping region of untranslated and
translated sequence; in particular, the target sequence can
comprise at least one nucleotide upstream of the start triplet of
the coding region of the mRNA sequence.
Open reading frame: An open reading frame (ORF) in the context of
the invention may typically be a sequence of several nucleotide
triplets which may be translated into a peptide or protein. An open
reading frame preferably contains a start codon, i.e. a combination
of three subsequent nucleotides coding usually for the amino acid
methionine (ATG or AUG), at its 5'-end and a subsequent region
which usually exhibits a length which is a multiple of 3
nucleotides. An ORF is preferably terminated by a stop-codon (e.g.,
TAA, TAG, TGA). Typically, this is the only stop-codon of the open
reading frame. Thus, an open reading frame in the context of the
present invention is preferably a nucleotide sequence, consisting
of a number of nucleotides that may be divided by three, which
starts with a start codon (e.g. ATG or AUG) and which preferably
terminates with a stop codon (e.g., TAA, TGA, or TAG or UAA, UAG,
UGA, respectively). The open reading frame may be isolated or it
may be incorporated in a longer nucleic acid sequence, for example
in a vector or an mRNA. An open reading frame may also be termed
"protein coding region" or "coding region". IRES (internal
ribosomal entry site) sequence: An IRES can function as a sole
ribosome binding site, but it can also serve to provide a bi- or
even multicistronic RNA as defined herein which codes for several
proteins or peptides, which are to be translated by the ribosomes
independently of one another. Examples of IRES sequences which can
be used according to the invention are those from picornaviruses
(e.g. FMDV), pestiviruses (CFFV), polioviruses (PV),
encephalomyocarditis viruses (ECMV), foot and mouth disease viruses
(FMDV), hepatitis C viruses (HCV), classical swine fever viruses
(CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses
(SIV) or cricket paralysis viruses (CrPV). Fragment or part of an
(protein/peptide) antigen: Fragments or parts of a
(protein/peptide) antigen in the context of the present invention
are typically understood to be peptides corresponding to a
continuous part of the amino acid sequence of a (protein/peptide)
antigen, preferably having a length of about 6 to about 20 or even
more amino acids, e.g. parts as processed and presented by MHC
class I molecules, preferably having a length of about 8 to about
10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids),
or fragments as processed and presented by MHC class II molecules,
preferably having a length of about 13 or more amino acids, e.g.
13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein
these fragments may be selected from any part of the amino acid
sequence. These fragments are typically recognized by T cells in
form of a complex consisting of the peptide fragment and an MHC
molecule, i.e. the fragments are typically not recognized in their
native form. Fragments or parts of the (protein/peptide) antigens
as defined herein may also comprise epitopes or functional sites of
those (protein/peptide) antigens. Preferably, fragments or parts of
a (protein/peptide) antigen in the context of the invention are or
comprise epitopes, or do have antigenic characteristics, eliciting
an adaptive immune response. Therefore, fragments of
(protein/peptide) antigens may comprise at least one epitope of
those (protein/peptide) antigens. Furthermore, also domains of a
(protein/peptide) antigen, like the extracellular domain, the
intracellular domain or the transmembrane domain and shortened or
truncated versions of a (protein/peptide) antigen may be understood
to comprise a fragment of a (protein/peptide) antigen. Variants of
proteins: "Variants" of proteins or peptides as defined in the
context of the present invention may be generated, having an amino
acid sequence which differs from the original sequence in one or
more mutation(s), such as one or more substituted, inserted and/or
deleted amino acid(s). Preferably, these variants have the same
biological function or specific activity compared to the
full-length native protein, e.g. its specific antigenic property.
"Variants" of proteins or peptides as defined in the context of the
present invention may comprise conservative amino acid
substitution(s) compared to their native, i.e. non-mutated
physiological, sequence. Those amino acid sequences as well as
their encoding nucleotide sequences in particular fall under the
term variants as defined herein. Substitutions in which amino
acids, which originate from the same class, are exchanged for one
another are called conservative substitutions. In particular, these
are amino acids having aliphatic side chains, positively or
negatively charged side chains, aromatic groups in the side chains
or amino acids, the side chains of which can enter into hydrogen
bridges, e.g. side chains which have a hydroxyl function. This
means that e.g. an amino acid having a polar side chain is replaced
by another amino acid having a likewise polar side chain, or, for
example, an amino acid characterized by a hydrophobic side chain is
substituted by another amino acid having a likewise hydrophobic
side chain (e.g. serine (threonine) by threonine (serine) or
leucine (isoleucine) by isoleucine (leucine)). Insertions and
substitutions are possible, in particular, at those sequence
positions which cause no modification to the three-dimensional
structure or do not affect the binding region. Modifications to a
three-dimensional structure by insertion(s) or deletion(s) can
easily be determined e.g. using CD spectra (circular dichroism
spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of
Polypeptides, in: Modern Physical Methods in Biochemistry,
Neuberger et al. (ed.), Elsevier, Amsterdam).
[0161] Furthermore, variants of proteins or peptides as defined
herein, which may be encoded by a nucleic acid molecule, may also
comprise those sequences, wherein nucleotides of the nucleic acid
are exchanged according to the degeneration of the genetic code,
without leading to an alteration of the respective amino acid
sequence of the protein or peptide, i.e. the amino acid sequence or
at least part thereof may not differ from the original sequence in
one or more mutation(s) within the above meaning.
[0162] In order to determine the percentage to which two sequences
are identical, e.g. nucleic acid sequences or amino acid sequences
as defined herein, preferably the amino acid sequences encoded by a
nucleic acid sequence of the polymeric carrier as defined herein or
the amino acid sequences themselves, the sequences can be aligned
in order to be subsequently compared to one another. Therefore,
e.g. a position of a first sequence may be compared with the
corresponding position of the second sequence. If a position in the
first sequence is occupied by the same component (residue) as is
the case at a position in the second sequence, the two sequences
are identical at this position. If this is not the case, the
sequences differ at this position. If insertions occur in the
second sequence in comparison to the first sequence, gaps can be
inserted into the first sequence to allow a further alignment. If
deletions occur in the second sequence in comparison to the first
sequence, gaps can be inserted into the second sequence to allow a
further alignment. The percentage to which two sequences are
identical is then a function of the number of identical positions
divided by the total number of positions including those positions
which are only occupied in one sequence. The percentage to which
two sequences are identical can be determined using a mathematical
algorithm. A preferred, but not limiting, example of a mathematical
algorithm which can be used is the algorithm of Karlin et al.
(1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic
Acids Res., 25:3389-3402. Such an algorithm is integrated in the
BLAST program. Sequences which are identical to the sequences of
the present invention to a certain extent can be identified by this
program. A "variant" of a protein or peptide may have at least 70%,
75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a
stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or
peptide, preferably over the full-length sequence that variant is
derived from. Analogously, a "variant" of a nucleic acid sequence
may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%
nucleotide identity over a stretch of 10, 20, 30, 50, 75 or 100
nucleotide of such nucleic acid sequence, preferably over the
full-length sequence the variant is derived from.
Derivative of a protein or peptide: A derivative of a peptide or
protein is typically understood to be a molecule that is derived
from another molecule, such as said peptide or protein. A
"derivative" of a peptide or protein also encompasses fusions
comprising a peptide or protein used in the present invention. For
example, the fusion comprises a label, such as, for example, an
epitope, e.g., a FLAG epitope or a V5 epitope. For example, the
epitope is a FLAG epitope. Such a tag is useful for, for example,
purifying the fusion protein. Pharmaceutically effective amount: A
pharmaceutically effective amount in the context of the invention
is typically understood to be an amount that is sufficient to
induce a pharmaceutical effect, such as an immune response,
altering a pathological level of an expressed peptide or protein,
or substituting a lacking gene product, e.g., in case of a
pathological situation. Vehicle: A vehicle is typically understood
to be a material that is suitable for storing, transporting, and/or
administering a compound, such as a pharmaceutically active
compound. For example, it may be a physiologically acceptable
liquid which is suitable for storing, transporting, and/or
administering a pharmaceutically active compound. PD-1 pathway:
Members of the PD-1 pathway are all proteins which are associated
with PD-1 signaling. On the one hand these might be proteins which
induce PD-1 signaling upstream of PD-1 as e.g. the ligands of PD-1
PD-L1 and PD-L2 and the signal transduction receptor PD-1. On the
other hand these might be signal transduction proteins downstream
of PD-1 receptor. Particularly preferred as members of the PD-1
pathway in the context of the present invention are PD-1, PD-L1 and
PD-L2. PD-1 pathway inhibitor: In the context of the present
invention, a PD-1 pathway inhibitor is preferably defined herein as
a compound capable to impair the PD-1 pathway signaling, preferably
signaling mediated by the PD-1 receptor. Therefore, the PD-1
pathway inhibitor may be any inhibitor directed against any member
of the PD-1 pathway capable of antagonizing PD-1 pathway signaling.
In this context, the inhibitor may be an antagonistic antibody as
defined herein, targeting any member of the PD-1 pathway,
preferably directed against PD-1 receptor, PD-L1 or PD-L2. This
antagonistic antibody may also be encoded by a nucleic acid. Such
encoded antibodies are also called "intrabodies" as defined herein.
Also, the PD-1 pathway inhibitor may be a fragment of the PD-1
receptor or the PD1-receptor blocking the activity of PD1 ligands.
B7-1 or fragments thereof may act as PD1-inhibiting ligands as
well. Furthermore, the PD-1 pathway inhibitor may be siRNA (small
interfering RNA) or antisense RNA directed against a member of the
PD-1 pathway, preferably PD-1, PD-L1 or PD-L2. Additionally, a PD-1
pathway inhibitor may be a protein comprising (or a nucleic acid
coding for) an amino acid sequence capable of binding to PD-1 but
preventing PD-1 signaling, e.g. by inhibiting PD-1 and B7-H1 or
B7-DL interaction. Additionally, a PD-1 pathway inhibitor may be a
small molecule inhibitor capable of inhibiting PD-1 pathway
signaling, e.g. a PD-1 binding peptide or a small organic molecule.
Antibody: An antibody may be selected from any antibody, e.g. any
recombinantly produced or naturally occurring antibodies, known in
the art, in particular antibodies suitable for therapeutic,
diagnostic or scientific purposes, particularly directed against
PD-1, PD-L1 or PD-L2. Herein, the term "antibody" is used in its
broadest sense and specifically covers monoclonal and polyclonal
antibodies (including, antagonist, and blocking or neutralizing
antibodies) and antibody species with polyepitopic specificity.
According to the invention, "antibody" typically comprises any
antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgE
antibodies), such as naturally occurring antibodies, antibodies
generated by immunization in a host organism, antibodies which were
isolated and identified from naturally occurring antibodies or
antibodies generated by immunization in a host organism and
recombinantly produced by biomolecular methods known in the art, as
well as chimeric antibodies, human antibodies, humanized
antibodies, bispecific antibodies, intrabodies, i.e. antibodies
expressed in cells and optionally localized in specific cell
compartments, and fragments and variants of the aforementioned
antibodies. In general, an antibody consists of a light chain and a
heavy chain both having variable and constant domains. The light
chain consists of an N-terminal variable domain, VL, and a
C-terminal constant domain, CL. In contrast, the heavy chain of the
IgG antibody, for example, is comprised of an N-terminal variable
domain, VH, and three constant domains, CH1, CH2 and CH3. Single
chain antibodies may be used according to the present invention as
well.
[0163] Antibodies may preferably comprise full-length antibodies,
i.e. antibodies composed of the full heavy and full light chains,
as described above. However, derivatives of antibodies such as
antibody fragments, variants or adducts may also be used as PD-1
pathway inhibitor according to the invention. Antibody fragments
may be selected from Fab, Fab', F(ab')2, Fc, Facb, pFc', Fd and Fv
fragments of the aforementioned (full-length) antibodies. In
general, antibody fragments are known in the art. For example, a
Fab ("fragment, antigen binding") fragment is composed of one
constant and one variable domain of each of the heavy and the light
chain. The two variable domains bind the epitope on specific
antigens. The two chains are connected via a disulfide linkage. A
scFv ("single chain variable fragment") fragment, for example,
typically consists of the variable domains of the light and heavy
chains. The domains are linked by an artificial linkage, in general
a polypeptide linkage such as a peptide composed of 15-25 glycine,
proline and/or serine residues.
Polyclonal antibody: Polyclonal antibody typically means mixtures
of antibodies directed to specific antigens or immunogens or
epitopes of a protein which were generated by immunization of a
host organism, such as a mammal, e.g. including goat, cattle,
swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse,
hamster and rabbit. Polyclonal antibodies are generally not
identical, and thus usually recognize different epitopes or regions
from the same antigen. Thus, in such a case, typically a mixture (a
composition) of different antibodies will be used, each antibody
being directed to specific antigens or immunogens or epitopes of a
protein, particularly directed to PD-1, PD-L1 or PD-L2. Monoclonal
antibody: The term "monoclonal antibody" herein typically refers to
an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed to a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed to different determinants (epitopes),
each monoclonal antibody is directed to a single determinant on the
antigen. For example, monoclonal antibodies as defined above may be
made by the hybridoma method first described by Kohler and
Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA
methods, e.g. as described in U.S. Pat. No. 4,816,567. "Monoclonal
antibodies" may also be isolated from phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990), for example. According to Kohler and Milstein,
an immunogen (antigen) of interest is injected into a host such as
a mouse and B-cell lymphocytes produced in response to the
immunogen are harvested after a period of time. The B-cells are
combined with myeloma cells obtained from mouse and introduced into
a medium which permits the B-cells to fuse with the myeloma cells,
producing hybridomas. These fused cells (hybridomas) are then
placed into separate wells of microtiter plates and grown to
produce monoclonal antibodies. The monoclonal antibodies are tested
to determine which of them are suitable for detecting the antigen
of interest. After being selected, the monoclonal antibodies can be
grown in cell cultures or by injecting the hybridomas into mice. In
the context of the present invention particularly preferred are
monoclonal antibodies directed against PD-1, PD-L1 and PD-L2.
Chimeric antibodies: Chimeric antibodies, which may be used as PD-1
pathway inhibitor according to the invention are preferably
antibodies in which the constant domains of an antibody described
above are replaced by sequences of antibodies from other organisms,
preferably human sequences. Humanized antibodies: Humanized
(non-human) antibodies, which may be used as PD-1 pathway inhibitor
according to the invention are antibodies in which the constant and
variable domains (except for the hypervariable domains) of an
antibody are replaced by human sequences. Human antibodies: Human
antibodies can be isolated from human tissues or from immunized
non-human host organisms which are transgene for the human IgG gene
locus. Additionally, human antibodies can be provided by the use of
a phage display. Bispecific antibodies: Bispecific antibodies in
context of the invention are preferably antibodies which act as an
adaptor between an effector and a respective target by two
different F.sub.a/b-domains, e.g. for the purposes of recruiting
effector molecules such as toxins, drugs, cytokines etc., targeting
effector cells such as CTL, NK cells, makrophages, granulocytes,
etc. (see for review: Kontermann R. E., Acta Pharmacol. Sin, 2005,
26(1): 1-9). Bispecific antibodies as described herein are, in
general, configured to recognize by two different
F.sub.a/b-domains, e.g. two different antigens, immunogens,
epitopes, drugs, cells (or receptors on cells), or other molecules
(or structures) as described above. Bispecificity means herewith
that the antigen-binding regions of the antibodies are specific for
two different epitopes. Thus, different antigens, immunogens or
epitopes, etc. can be brought close together, what, optionally,
allows a direct interaction of the two components. For example,
different cells such as effector cells and target cells can be
connected via a bispecific antibody. Encompassed, but not limited,
by the present invention are antibodies or fragments thereof which
bind, on the one hand, a soluble antigen and, on the other hand, an
antigen or receptor e.g. PD-1 or its ligands PD-L1 and PD-L2 on the
surface of a cell, e.g. a tumor cell. Intrabodies: Intrabodies may
be antibodies as defined above. These antibodies are intracellular
expressed antibodies, and therefore these antibodies may be encoded
by nucleic acids to be used for expression of the encoded
antibodies. Therefore nucleic acids coding for an antibody,
preferably as defined above, particularly an antibody directed
against a member of the PD-1 pathway, e.g. PD-1, PD-L1 or PD-L2 may
be used as PD-1 pathway inhibitor according to the present
invention.
[0164] According to a first aspect, the object underlying the
present invention is solved by a vaccine/inhibitor combination
comprising: [0165] (i) as vaccine an RNA vaccine comprising at
least one RNA comprising at least one open reading frame (ORF)
coding for at least one antigen, and [0166] (ii) as an inhibitor a
composition comprising at least one PD-1 pathway inhibitor.
[0167] In the context of the present invention, the term
"vaccine/inhibitor combination" preferably means a combined
occurrence of an RNA vaccine comprising at least one RNA comprising
at least one open reading frame (ORF) coding for at least one
antigen and of a composition comprising at least one PD-1 pathway
inhibitor. Therefore, this vaccine/inhibitor combination may occur
either as one composition, comprising all these components in one
and the same mixture (e.g. in a pharmaceutical composition), or may
occur as a kit of parts, wherein the different components form
different parts of such a kit of parts. This inventive
vaccine/inhibitor combination preferably allows to elicit an
adaptive immune response (and optional an innate immune response)
in a patient to be treated, preferably a mammal, by using as a
first component an RNA vaccine, comprising at least one RNA
comprising at least one open reading frame encoding at least one
antigen, preferably encoding a tumour antigen or a pathogenic
antigen. The inhibitor of the inventive vaccine/inhibitor
combination, preferably a PD-1 pathway inhibitor may antagonize
PD-1 pathway signaling by preferably inhibiting or suppressing
signal transduction mediated by the PD-1 receptor. Thus, the
administration of the vaccine and the inhibitor may occur either
simultaneously or timely staggered, either at the same site of
administration or at different sites of administration, as further
outlined below. Such a vaccine/inhibitor combination may induce an
active immune response and thereby prevents e.g. tumour growth or
induces tumour regression. The inventive vaccine/inhibitor
combination is thus suitable to effectively stimulate
antigen-specific immune responses against cancer and pathogen
infected cells. More precisely, the inventive vaccine/inhibitor
combination is particularly suitable in the treatment of tumour
diseases and infectious diseases which may be associated with an
overexpression of PD-1, PD-L1 or PD-L2 and to further improve the
immune response against such tumour cells and infected cells.
[0168] The invention is therefore based on the surprising finding
that the combination of an RNA vaccine and a PD-1 pathway inhibitor
shows an extremely advantageous inhibition of tumour growth
resulting in enhanced survival which could not be expected from the
prior art. Thus, the combined treatment with an RNA vaccine, e.g.
coding for a specific antigen (active vaccination) such as a tumour
antigen, and with an inhibitor directed to a member of the PD-1
pathway, particularly PD-1 receptor or its ligands PD-L1 and PD-L2,
could strongly decrease the harmful impact of a disease to be
treated, e.g. the growth rate of a tumour. In this context, the
inventors surprisingly found that treatment with an RNA vaccine
comprising an RNA coding for a tumor antigen in combination with a
PD-1 pathway inhibitor unexpectedly inhibited tumor growth
resulting in an improved survival of tumor challenged mice in a
synergistic manner as evidenced by the occurrence of 50% complete
responses.
[0169] As a first component, the inventive vaccine/inhibitor
combination includes as a vaccine an RNA vaccine which comprises at
least one RNA comprising at least one open reading frame (ORF)
coding for at least one antigen, preferably a tumour antigen or a
pathogenic antigen.
[0170] According to the invention, the RNA vaccine of the inventive
vaccine/inhibitor combination preferably comprises at least one RNA
comprising at least one open reading frame encoding at least one
antigen as defined herein.
[0171] The at least one RNA of the RNA vaccine may be selected from
any RNA suitable to encode an amino acid sequence, preferably from
a messenger RNA (mRNA).
[0172] However other forms of RNA may likewise find its application
in carrying out the teaching of the present invention. For example,
the RNA may be a virus derived RNA such as RNA of a retrovirus or
an RNA replicon as defined herein e.g. derived from an
alphavirus.
[0173] In specific embodiments the at least one RNA of the RNA
vaccine does not comprise or does not consist of a lentiviral
vector or a adenoviral/adeno-associated vector.
[0174] In a specific embodiment, the RNA vaccine comprises or
consists of isolated RNA as defined herein.
[0175] Furthermore, the at least one RNA of the RNA vaccine of the
inventive vaccine/inhibitor combination may be a single- or a
double-stranded RNA (which may also be regarded as an RNA
(molecule) due to non-covalent association of two single-stranded
RNA (molecules)) or a partially double-stranded or partially single
stranded RNA, which are at least partially self-complementary (both
of these partially double-stranded or partially single stranded RNA
molecules are typically formed by a longer and a shorter
single-stranded RNA molecule or by two single stranded
RNA-molecules, which are about equal in length, wherein one
single-stranded RNA molecule is in part complementary to the other
single-stranded RNA molecule and both thus form a double-stranded
RNA molecule in this region, i.e. a partially double-stranded or
partially single stranded RNA). Preferably, the at least one RNA of
the RNA vaccine of the inventive vaccine/inhibitor combination may
be a single-stranded RNA. Furthermore, the at least one RNA of the
RNA vaccine of the inventive vaccine/inhibitor combination may be a
circular or linear RNA, preferably a linear RNA. More preferably,
the at least one RNA of the RNA vaccine of the inventive
vaccine/inhibitor combination may be a (linear) single-stranded
RNA.
[0176] Preferably, the at least one RNA of the RNA vaccine of the
inventive vaccine/inhibitor combination comprises a length of about
5 to about 20000, or 100 to about 20000 nucleotides, preferably of
about 250 to about 20000 nucleotides, more preferably of about 500
to about 10000, even more preferably of about 500 to about
5000.
[0177] In a particular preferred embodiment of the first aspect of
the invention, the at least one RNA of the RNA vaccine comprising
at least one open reading frame codes for at least one tumour
antigen. In this context tumour antigens are preferably located on
the surface of the (tumour) cell. Tumour antigens may also be
selected from proteins, which are overexpressed in tumour cells
compared to a normal cell (e.g. non-tumor cells). Furthermore,
tumour antigens also include antigens expressed in cells which are
(were) not themselves (or originally not themselves) degenerated
but are associated with the supposed tumour. Antigens which are
connected with tumour-supplying vessels or (re)formation thereof,
in particular those antigens which are associated with
neovascularization, e.g. growth factors, such as VEGF, bFGF etc.,
are also included herein. Antigens associated with a tumour
furthermore include antigens from cells or tissues, typically
embedding the tumour. Further, some substances (usually proteins or
peptides) are expressed in patients suffering (knowingly or
not-knowingly) from a cancer disease and they occur in increased
concentrations in the body fluids of said patients. These
substances are also referred to as "tumour antigens", however they
are not antigens in the stringent meaning of an immune response
inducing substance. The class of tumour antigens can be divided
further into tumour-specific antigens (TSAs) and
tumour-associated-antigens (TAAs). TSAs can only be presented by
tumour cells and never by normal "healthy" cells. They typically
result from a tumour specific mutation. TAAs, which are more
common, are usually presented by both tumour and healthy cells.
These antigens are recognized and the antigen-presenting cell can
be destroyed by cytotoxic T cells. Additionally, tumour antigens
can also occur on the surface of the tumour in the form of, e.g., a
mutated receptor. In this case, they can be recognized by
antibodies.
[0178] Further, tumour associated antigens may be classified as
tissue-specific antigens, also called melanocyte-specific antigens,
cancer-testis antigens and tumour-specific antigens. Cancer-testis
antigens are typically understood to be peptides or proteins of
germ-line associated genes which may be activated in a wide variety
of tumours. Human cancer-testis antigens may be further subdivided
into antigens which are encoded on the X chromosome, so-called CT-X
antigens, and those antigens which are not encoded on the X
chromosome, the so-called (non-X CT antigens). Cancer-testis
antigens which are encoded on the X-chromosome comprise, for
example, the family of melanoma antigen genes, the so-called
MAGE-family. The genes of the MAGE-family may be characterised by a
shared MAGE homology domain (MHD). Each of these antigens, i.e.
melanocyte-specific antigens, cancer-testis antigens and
tumour-specific antigens, may elicit autologous cellular and
humoral immune responses. Accordingly, the tumour antigen encoded
by the RNA comprised in the RNA vaccine used in the present
invention is preferably a melanocyte-specific antigen, a
cancer-testis antigen or a tumour-specific antigen, preferably it
may be a CT-X antigen, a non-X CT-antigen, a binding partner for a
CT-X antigen or a binding partner for a non-X CT-antigen or a
tumour-specific antigen, more preferably a CT-X antigen, a binding
partner for a non-X CT-antigen or a tumour-specific antigen.
[0179] Particular preferred tumour antigens according to the
present invention are selected from the list consisting of 5T4,
707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,
alpha-5-beta-6-integrin, alpha-actinin-4/m,
alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,
BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA
15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL,
CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30,
CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA,
CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage
XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1,
DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,
EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250,
GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8,
GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu,
HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox
NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,
HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin
receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,
KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3,
MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2,
MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17,
MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1,
MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A,
MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m,
mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1,
MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A,
N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,
NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1,
OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15,
p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1,
PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME,
PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA,
PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE,
SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1,
SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B,
SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta,
TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,
TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1. Such
tumour antigens preferably may be selected from the group
consisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1,
MAGE-A3, Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA,
PSMA, STEAP-1, VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more
preferably from PAP, MAGE-A3, WT1, and MUC-1. Such tumour antigens
preferably may be selected from the group consisting of MAGE-A1
(e.g. MAGE-A1 according to accession number M77481), MAGE-A2,
MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number
NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to
accession number NM_005511), GP100 (e.g. GP100 according to
accession number M77348), tyrosinase (e.g. tyrosinase according to
accession number NM_000372), surviving (e.g. survivin according to
accession number AF077350), CEA (e.g. CEA according to accession
number NM_004363), Her-2/neu (e.g. Her-2/neu according to accession
number M11730), WT1 (e.g. WT1 according to accession number
NM_000378), PRAME (e.g. PRAME according to accession number
NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI
(epidermal growth factor receptor 1) according to accession number
AF288738), MUC1, mucin-1 (e.g. mucin-1 according to accession
number NM_002456), SEC61G (e.g. SEC61G according to accession
number NM_014302), hTERT (e.g. hTERT accession number NM_198253),
5T4 (e.g. 5T4 according to accession number NM_006670), TRP-2 (e.g.
TRP-2 according to accession number NM_001922), STEAP1
(Six-transmembrane epithelial antigen of prostate 1), PSCA, PSA,
PSMA, etc.
[0180] In this context it is particularly preferred that the at
least one RNA of the RNA vaccine of the inventive vaccine/inhibitor
combination encodes the tumour antigens selected from PCA, PSA,
PSMA, STEAP and optional MUC-1, or fragments, variants or
derivatives thereof.
[0181] In a further particularly preferred embodiment the RNA
vaccine of the inventive vaccine/inhibitor combination comprises at
least one RNA coding for the tumour antigens selected from
NY-ESO-1, MAGE-C1, MAGE-C2, Survivin, optional 5T4 and optional
MUC-1, or fragments, variants or derivatives thereof.
[0182] Furthermore, tumour antigens also may encompass idiotypic
antigens associated with a cancer or tumour disease, particularly
lymphoma or a lymphoma associated disease, wherein said idiotypic
antigen is an immunoglobulin idiotype of a lymphoid blood cell or a
T cell receptor idiotype of a lymphoid blood cell.
[0183] In a further particularly preferred embodiment of the first
aspect of the invention, the at least one RNA of the RNA vaccine
comprises at least one open reading frame coding for at least one
pathogenic antigen. Pathogenic antigens are peptide or protein
antigens derived from a pathogen associated with infectious disease
which are preferably selected from antigens derived from the
pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma
phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale,
Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus
genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus
cereus, Bartonella henselae, BK virus, Blastocystis hominis,
Blastomyces dermatitidis, Bordetella pertussis, Borrelia
burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia
malayi, Bunyaviridae family, Burkholderia cepacia and other
Burkholderia species, Burkholderia mallei, Burkholderia
pseudomallei, Caliciviridae family, Campylobacter genus, Candida
albicans, Candida spp, Chlamydia trachomatis, Chlamydophila
pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis,
Clostridium botulinum, Clostridium difficile, Clostridium
perfringens, Clostridium perfringens, Clostridium spp, Clostridium
tetani, Coccidioides spp, coronaviruses, Corynebacterium
diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever
virus, Cryptococcus neoformans, Cryptosporidium genus,
Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and
DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus
genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus,
Entamoeba histolytica, Enterococcus genus, Enterovirus genus,
Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71),
Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli
0157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola
gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,
Francisella tularensis, Fusobacterium genus, Geotrichum candidum,
Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,
Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,
Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus,
Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D
Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and
HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus),
Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6
(HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus
(hMPV), Human papillomavirus (HPV), Human parainfluenza viruses
(HPIV), Japanese encephalitis virus, JC virus, Junin virus,
Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus,
Legionella pneumophila, Leishmania genus, Leptospira genus,
Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),
Machupo virus, Malassezia spp, Marburg virus, Measles virus,
Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum
virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium
lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans,
Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus,
Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides,
Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi,
Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis,
Paragonimus spp, Paragonimus westermani, Parvovirus B19,
Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii,
Poliovirus, Rabies virus, Respiratory syncytial virus (RSV),
Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus,
Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi,
Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus,
Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma
genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix
schenckii, Staphylococcus genus, Staphylococcus genus,
Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus
pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium,
Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara
cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis,
Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura,
Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum,
Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola
major or Variola minor, vCJD prion, Venezuelan equine encephalitis
virus, Vibrio cholerae, West Nile virus, Western equine
encephalitis virus, Wuchereria bancrofti, Yellow fever virus,
Yersinia enterocolitica, Yersinia pestis, and Yersinia
pseudotuberculosis.
[0184] In this context, particularly preferred are antigens from
the pathogens selected from Influenza virus, respiratory syncytial
virus (RSV), Herpes simplex virus (HSV), human Papilloma virus
(HPV), Human immunodeficiency virus (HIV), Plasmodium,
Staphylococcus aureus, Dengue virus, Chlamydia trachomatis,
Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium
tuberculosis, Rabies virus, and Yellow Fever Virus.
[0185] The at least one RNA of the RNA vaccine comprising at least
one open reading frame coding for at least one antigen according to
the first aspect of the present invention may occur as a mono-,
di-, or even multicistronic RNA, i.e. an RNA which contains the
open reading frame of one, two or more proteins or peptides. Such
open reading frames in di-, or even multicistronic RNAs may be
separated by at least one internal ribosome entry site (IRES)
sequence, e.g. as described herein or by signal peptides which
induce the cleavage of the resulting polypeptide which comprises
several proteins or peptides.
[0186] The at least one RNA of the RNA vaccine of the inventive
vaccine/inhibitor combination may be stabilized in order to prevent
instability and (fast) degradation of the RNA by various
approaches. This instability of RNA is typically due to
RNA-degrading enzymes, "RNases" (ribonucleases), wherein
contamination with such ribonucleases may sometimes completely
degrade RNA in solution. Accordingly, the natural degradation of
RNA in the cytoplasm of cells is very finely regulated and RNase
contaminations may be generally removed by special treatment prior
to use of said compositions, in particular with diethyl
pyrocarbonate (DEPC). A number of mechanisms of natural degradation
are known in this context in the prior art, which may be utilized
as well. E.g., the terminal structure is typically of critical
importance particularly for an mRNA. As an example, at the 5' end
of naturally occurring mRNAs there is usually a so-called cap
structure, which is a modified guanosine nucleotide also called 5
`Cap structure, and at the 3` end is typically a sequence of up to
200 adenosine nucleotides (the so-called poly-A tail). In a further
embodiment, the at least one RNA of the RNA vaccine of the
inventive vaccine/inhibitor combination comprises at least one of
the following structural elements: a 5' and/or 3'-UTR sequence,
preferably a 5' and/or 3'-UTR modification, a 5'Cap structure, a
poly(C) sequence, a poly-A tail and/or a polyadenylation signal,
preferably as defined herein.
[0187] In a further embodiment, the at least one RNA of the RNA
vaccine of the inventive vaccine/inhibitor combination preferably
comprises at least two of the following structural elements: a 5'
and/or 3'-UTR sequence, preferably a 5' and/or 3'-UTR modification
(e.g. the mutated sequence of the 3'-UTR of the (alpha)globin gene
(muag)); a histone-stem-loop structure, preferably a
histone-stem-loop in its 3' untranslated region; a 5'-Cap
structure; a poly(C) sequence; a poly-A tail; or a polyadenylation
signal, e.g. given a 5'-Cap structure and a histone-stem-loop and,
potentially a poly-A-tail.
[0188] In this context it is particularly preferred that the at
least one RNA of the RNA vaccine encoding at least one antigen
comprised in the inventive vaccine/inhibitor combination has the
following structure in 5' to 3'-direction: [0189] a) an optional
5'-UTR sequence comprising a UTR modification [0190] b) an open
reading frame encoding an antigen as defined above; [0191] c) a
3'-UTR sequence comprising a UTR modification [0192] d) at least
one histone stem-loop, optionally without a histone downstream
element 3' to the histone stem-loop [0193] e) a poly(A) sequence or
optional a polyadenylation signal; and [0194] f) a poly(C)
sequence.
[0195] In another particular preferred embodiment the at least one
RNA of the RNA vaccine encoding at least one antigen in the
inventive vaccine/inhibitor combination has the following structure
in 5' to 3'-direction: [0196] a) an optional 5'-UTR sequence
comprising a UTR modification [0197] b) an open reading frame
encoding an antigen as defined above; [0198] c) a 3'-UTR sequence
comprising a UTR modification [0199] d) a poly(A) sequence [0200]
e) a poly(C) sequence; and [0201] f) at least one histone
stem-loop.
[0202] For further improvement of the resistance to e.g. in vivo
degradation (e.g. by an exo- or endo-nuclease), the at least one
RNA of the RNA vaccine encoding at least one antigen in the
inventive vaccine/inhibitor combination may be provided as a
stabilized nucleic acid, e.g. in the form of a modified nucleic
acid as defined herein. According to a further embodiment of the
invention, it is therefore preferred that the at least one RNA of
the RNA vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination is stabilized, preferably by backbone
modifications, sugar modifications and/or base modifications, more
preferred stabilized by modification of the G/C-content as defined
herein. All of these modifications may be introduced into the at
least one RNA without impairing the RNA's function to be translated
into the antigen, to be reverse transcribed or to be
replicated.
[0203] According to another embodiment, the at least one RNA of the
RNA vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination may be modified and thus stabilized
by modifying the G (guanosine)/C (cytosine) content of the mRNA,
preferably of the open reading frame thereof.
[0204] Therein, the G/C content of the at least one RNA of the RNA
vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination is particularly increased compared to
the G/C content of the open reading frame of its particular wild
type open reading frame, i.e. the unmodified RNA as defined herein.
However, the encoded amino acid sequence of the open reading frame
of the at least one RNA of the RNA vaccine encoding at least one
antigen in the inventive vaccine/inhibitor combination RNA is
preferably not modified compared to the encoded amino acid sequence
of the particular wild type open reading frame.
[0205] According to a further preferred embodiment of the
invention, the at least one RNA of the RNA vaccine encoding at
least one antigen in the inventive vaccine/inhibitor combination is
optimized for translation (codon-optimized) as defined herein,
preferably optimized for translation by replacing codons for less
frequent tRNAs of a given amino acid by codons for more frequently
occurring tRNAs of the respective amino acid.
[0206] In this context, it is particularly preferred to link the
sequential G/C content which is increased, in particular maximized,
in the at least one RNA of the RNA vaccine encoding at least one
antigen in the inventive vaccine/inhibitor combination, with the
"frequent" codons without modifying the amino acid sequence of the
protein encoded by the open reading frame comprised in the at least
one RNA of the RNA vaccine.
[0207] In the context of the present invention, the at least one
RNA of the RNA vaccine encoding at least one antigen in the
inventive vaccine/inhibitor combination, may be prepared using any
method known in the art, including synthetic methods such as e.g.
solid phase synthesis, as well as in vivo propagation like e.g.
production of virus-like particles or replicon particles in cells
or in vitro methods, such as in vitro transcription reactions.
Replicons such as self-amplifying RNA based on an alphavirus genome
can be produced by constructing DNA plasmids encoding the
self-amplifying RNA using standard molecular techniques. Linearized
DNA is transcribed in vitro by, for example, T7 RNA polymerase and
the resulting RNA is introduced into cells, e.g. by
electroporation. Replicon particle production can be evaluated in
packaging assays in which in vitro transcribed replicon and
defective helper RNA are cotransfected into cells (Perri et al.,
2003. J. Virol. 77(19):10394-403; 12970424).
[0208] In a further embodiment the RNA vaccine of the inventive
vaccine/inhibitor combination comprises a plurality or more than
one, preferably 2 to 10, more preferably 2 to 5, most preferably 2
to 4 of RNA molecules as defined herein. These RNA vaccines
comprise more than one RNA molecules, preferably encoding different
peptides or proteins which comprise preferably different tumour
antigens or pathogenic antigens.
[0209] In this context it is particularly preferred that the RNA
vaccine of the inventive vaccine/inhibitor combination comprising a
plurality (which means typically more than 1, 2, 3, 4, 5, 6 or more
than 10 nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic
acids) of RNA molecules, particularly for use in the treatment of
prostate cancer (PCa) comprises at least: [0210] a) an RNA molecule
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen PSA, or a fragment,
variant or derivative thereof; and [0211] b) an RNA molecule
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen PSMA, or a
fragment, variant or derivative thereof; and [0212] c) an RNA
molecule encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen PSCA, or a
fragment, variant or derivative thereof; [0213] d) an RNA molecule
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen STEAP-1, or a
fragment, variant or derivative thereof; and optional [0214] e) an
RNA molecule encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen MUC-1, or a
fragment, variant or derivative thereof.
[0215] In a further preferred embodiment the RNA vaccine of the
inventive vaccine/inhibitor combination comprising a plurality
(which means typically more than 1, 2, 3, 4, 5, 6 or more than 10
nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) of
RNA molecules, particularly for use in the treatment of non-small
lung cancer (NSCLC) comprises at least: [0216] a) an RNA molecule
encoding at least one peptide or protein, wherein said encoded
peptide or protein comprises the tumour antigen NY-ESO-1, or a
fragment, variant or derivative thereof; and [0217] d) an RNA
molecule encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen MAGE-C1, or
a fragment, variant or derivative thereof; and [0218] e) an RNA
molecule encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen MAGE-C2, or
a fragment, variant or derivative thereof; [0219] f) an RNA
molecule encoding at least one peptide or protein, wherein said
encoded peptide or protein comprises the tumour antigen Survivin,
or a fragment, variant or derivative thereof; and optional [0220]
g) an RNA molecule encoding at least one peptide or protein,
wherein said encoded peptide or protein comprises the tumour
antigen 5T4, or a fragment, variant or derivative thereof; and
optional [0221] h) an RNA molecule encoding at least one peptide or
protein, wherein said encoded peptide or protein comprises the
tumour antigen MUC-1, or a fragment, variant or derivative
thereof.
[0222] According to one embodiment of the present invention, the at
least one RNA of the RNA vaccine encoding at least one antigen in
the inventive vaccine/inhibitor combination may be administered
naked without being associated with any further vehicle,
transfection or complexation agent for increasing the transfection
efficiency and/or the immunostimulatory properties of the at least
one RNA.
[0223] In a preferred embodiment, the at least one RNA of the RNA
vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination may be formulated together with a
cationic or polycationic compound and/or with a polymeric carrier
as defined herein. Accordingly, in a specific embodiment of the
invention it is preferred that the at least one RNA of the RNA
vaccine encoding at least one antigen in the inventive
vaccine/inhibitor combination is associated with or complexed with
a cationic or polycationic compound or a polymeric carrier as
defined herein, optionally in a weight ratio selected from a range
of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from
about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about
4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w) to about 1:1
(w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1
(w/w) of RNA to cationic or polycationic compound and/or with a
polymeric carrier; or optionally in an N/P-ratio of at least 0.1,
0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5 or 2. Preferably, the N/P-ratio
lies within a range of about 0.1, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5 or
2 to 20, preferably in a range of about 0.2 (0.5 or 0.75 or 1.0) to
12, more preferably in an N/P-ratio of about 0.4 (0.75 or 1.0) to
10, and even more preferably in an N/P ratio of about 0.4 (0.75 or
1.0) to 5. Most preferably the N/P ratio lies in a ratio between
0.1 and 0.9.
[0224] In this context it is preferable that the cationic or
polycationic compound or the polymeric carrier used as carrier or
complexation agent and the at least one RNA of the RNA vaccine
encoding at least one antigen in the inventive vaccine/inhibitor
combination as defined herein are provided in an N/P-ratio of at
least about 1 or, preferably, of a range of about 1 to 20 for in
vitro applications (e.g. in the case cells extracted from the
patient would be treated in vitro with the inventive pharmaceutical
composition and subsequently administered to the patient). For in
vivo applications, an N/P ratio of at least 0.1 (0.2, 0.3, 0.4,
0.5, 0.6), preferably of a range of about 0.1 (0.2, 0.3, 0.4, 0.5,
or 0.6) to 1.5 is preferred. Even more preferred is an N/P ratio
range of 0.1 or 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9.
[0225] The N/P ratio significantly influences the surface charge of
the resulting complex consisting of cationic or polycationic
compounds or of a polymeric carrier and a nucleic acid cargo e.g.
the at least one RNA coding for at least one antigen comprised in
the RNA vaccine or of an adjuvant nucleic acid. Thus, it is
preferable that the resulting polymeric carrier cargo complex is
positively charged for in vitro applications and negatively or
neutrally charged for in vivo applications. The surface charge of
the resulting polymeric carrier cargo complex can be indicated as
Zetapotential which may be measured by Doppler electrophoresis
method using a Zetasizer Nano (Malvern Instruments, Malvern,
UK).
[0226] The at least one RNA of the RNA vaccine encoding at least
one antigen in the inventive vaccine/inhibitor combination may also
be associated with a vehicle, transfection or complexation agent
for increasing the transfection efficiency and/or the
immunostimulatory properties of the at least one RNA.
[0227] In this context, it is particularly preferred that the at
least one RNA of the RNA vaccine encoding at least one antigen in
the inventive vaccine/inhibitor combination is complexed at least
partially with a cationic or polycationic compound and/or a
polymeric carrier, preferably cationic proteins or peptides.
Partially means that only a part of the at least one RNA is
complexed with a cationic compound and that the rest of the at
least one RNA of the RNA vaccine is comprised in the inventive
vaccine/inhibitor combination in uncomplexed form ("free" or
"naked"). Preferably, the ratio of complexed RNA to:uncomplexed RNA
in the RNA vaccine of the inventive vaccine/inhibitor combination
is selected from a range of about 5:1 (w/w) to about 1:10 (w/w),
more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w),
even more preferably from a range of about 3:1 (w/w) to about 1:5
(w/w) or 1:3 (w/w), and most preferably the ratio of complexed RNA
to free RNA in the RNA vaccine of the inventive vaccine/inhibitor
combination is selected from a ratio of about 1:1 (w/w).
[0228] The at least one complexed RNA in the RNA vaccine of the
inventive vaccine/inhibitor combination, is preferably prepared
according to a first step by complexing the at least one RNA with a
cationic or polycationic compound and/or with a polymeric carrier,
preferably as defined herein, in a specific ratio to form a stable
complex. In this context, it is highly preferable, that no free
cationic or polycationic compound or polymeric carrier or only a
negligibly small amount thereof remains in the component of the
complexed RNA after complexing the RNA. Accordingly, the ratio of
the RNA and the cationic or polycationic compound and/or the
polymeric carrier in the component of the complexed RNA is
typically selected in a range that the RNA is entirely complexed
and no free cationic or polycationic compound or polymeric carrier
or only a negligibly small amount thereof remains in the
composition.
[0229] Preferably, the ratio of the at least one RNA (e.g. mRNA)
comprising at least one open reading frame coding for at least one
antigen to the cationic or polycationic compound and/or the
polymeric carrier, preferably as defined herein, is selected from a
range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably
from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of
about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w) to about
1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about
2:1 (w/w). Alternatively, the ratio of the RNA to the cationic or
polycationic compound and/or the polymeric carrier, preferably as
defined herein, in the component of the complexed RNA, may also be
calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio)
of the entire complex. In the context of the present invention, an
N/P-ratio is preferably in the range of about 0.1-10, preferably in
a range of about 0.3-4 and most preferably in a range of about
0.5-2 or 0.7-2 regarding the ratio of cationic or polycationic
compound and/or polymeric carrier:RNA, preferably as defined
herein, in the complex, and most preferably in a range of about
0.7-1.5, 0.5-1 or 0.7-1, and even most preferably in a range of
about 0.3-0.9 or 0.5-0.9 preferably provided that the cationic or
polycationic compound in the complex is a cationic or polycationic
cationic or polycationic protein or peptide and/or the polymeric
carrier as defined above. In this specific embodiment, the
complexed RNA is also emcompassed in the term "adjuvant
component".
[0230] In another embodiment, the at least one antigen-providing
RNA in the RNA vaccine of the inventive vaccine/inhibitor
combination as defined above may be formulated together with an
adjuvant. Such an adjuvant may be preferably a further nucleic acid
that is not encoding a further antigen but is able to stimulate an
unspecific immune response, i.e. innate immune response, by
interacting with any part of the innate immune system. Such a
nucleic acid stimulating an unspecific immune response is termed
herein as "adjuvant nucleic acid".
[0231] In this context, an adjuvant nucleic acid preferably
comprises or consists of an oligo- or a polynucleotide; more
preferably an adjuvant nucleic acid comprising or consisting of an
RNA or a DNA; even more preferably such an adjuvant nucleic acid
comprising or consisting of an RNA or a DNA being complexed with a
cationic or polycationic compound and/or with a polymeric carrier
as defined herein; optionally in a weight ratio selected from a
range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably
from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of
about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to about
1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about
2:1 (w/w) of adjuvant nucleic acid to cationic or polycationic
compound and/or with a polymeric carrier; or optionally in a
nitrogen/phosphate ratio of the cationic or polycationic compound
and/or polymeric carrier to adjuvant nucleic acid in the range of
about 0.1-10, preferably in a range of about 0.3-4, most preferably
in a range of about 0.7-1 or 0.5-1, and even most preferably in a
range of about 0.3-0.9 or 0.5-0.9. Such a complexed adjuvant
nucleic acid is also encompassed in the term "adjuvant
component":
[0232] In the specific case that the induction of IFN-.alpha. is
intended, an N/P ratio of at least 0.1 (0.2, 0.3, 0.4, 0.5, or 0.6)
or an N/P ratio range of 0.1 to 1 is preferred or more preferred is
an N/P ratio range of 0.1 or 0.2 to 0.9 or an N/P ratio range of
0.5 to 0.9. Otherwise, if the induction of TNF.alpha. would be
intended, an N/P ratio of 1 to 20 is particularly preferred.
[0233] In other words, the RNA vaccine of the vaccine/inhibitor
combination according to the invention may comprise the at least
one RNA encoding at least one antigen, and a further nucleic acid
that is acting as an adjuvant which is called the adjuvant nucleic
acid. Of course the RNA vaccine of the inventive vaccine/inhibitor
combination is not limited to comprise only one adjuvant nucleic
acid but may comprise several different such nucleic acids. Both
kinds of nucleic acid, the antigen-encoding RNA and the adjuvant
nucleic acid, may be, independently from each other, complexed with
a carrier as defined herein. Therefore, a cationic or polycationic
compound and/or a polymeric carrier used to complex the at least
one RNA of the RNA vaccine encoding at least one antigen or the
adjuvant nucleic acid, may be selected from any cationic or
polycationic compound and/or polymeric carrier as defined
herein.
[0234] In case the RNA vaccine of the inventive vaccine/inhibitor
combination (or the inventive vaccine/inhibitor combination)
comprises an antigen-providing RNA and additionally an adjuvant
nucleic acid, the immune response that is evoked by administration
of such a vaccine comprises activation of both parts of the immune
system, the adaptive immune system as well as the innate immune
system.
[0235] A substantial factor for a suitable adaptive immune response
is the stimulation of different T cell sub-populations.
T-lymphocytes are typically divided into two sub-populations, the
T-helper 1 cells, in the following Th1-cells, and the T-helper 2
cells, in the following Th2-cells, with which the immune system is
capable of destroying intracellular and extracellular pathogens
(e.g. antigens). Thereby Th1-cells are responsible for
intracellular pathogen destruction by assisting the cellular immune
response by activation of macrophages and cytotoxic T cells.
Th2-cells, on the other hand, are mainly for extracellular
pathogen-elimination and promote the humoral immune response by
stimulation of B-cells for conversion into plasma cells and by
formation of antibodies (e.g. against antigens). The two T-helper
cell populations differ in the pattern of the effector proteins
(cytokines) produced by them.
[0236] The Th1-cell/Th2-cell ratio is of great importance in the
induction and maintenance of an adaptive immune response. In
connection with the present invention, the Th1-cell/Th2-cell ratio
of the (adaptive) immune response is preferably shifted in the
direction towards the cellular response (Th1 response) and a
cellular immune response is thereby induced. Stimulation of this
response of the adaptive immune system is mainly provoked by the
translation of the antigen-providing RNA and the resulting presence
of the peptide or protein antigens within the organism.
[0237] The innate immune system which may support such an adaptive
immune response and which may induce or support a shift towards a
Th1 response may be activated by ligands of Toll-like receptors
(TLRs). TLRs are a family of highly conserved pattern recognition
receptor (PRR) polypeptides that recognize pathogen-associated
molecular patterns (PAMPs) and play a critical role in innate
immunity in mammals. Currently at least thirteen family members,
designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4,
TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), have
been identified. Furthermore, a number of specific TLR ligands have
been identified. It was e.g. found that unmethylated bacterial DNA
and synthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi
H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl.
Acad. Sci. USA 98, 9237-42). Furthermore, it has been reported that
ligands for certain TLRs include certain nucleic acid molecules and
that certain types of RNA are immunostimulatory in a
sequence-independent or sequence-dependent manner, wherein these
various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or
TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.
[0238] In the context of the invention, the activation of the
innate immune system can be provided by an adjuvant nucleic acid,
preferably an immunostimulatory RNA (isRNA) as defined herein,
comprised in the inventive vaccine/inhibitor combination,
preferably comprised in the RNA vaccine.
[0239] According to the above, in a further preferred embodiment of
the invention, the RNA vaccine of the inventive vaccine/inhibitor
combination is formulated to comprise [0240] a) said at least one
RNA comprising at least one open reading frame coding for at least
one antigen; preferably in form of a mono-, bi- or multicistronic
RNA, optionally being stabilized, optionally being optimized for
translation and/or optionally being complexed with a cationic or
polycationic compound or a polymeric carrier; [0241] b) optionally
an adjuvant component, comprising or consisting of said at least
one RNA comprising at least one open reading frame coding for at
least one antigen and/or at least one adjuvant nucleic acid,
complexed with a cationic or polycationic compound and/or with a
polymeric carrier, and [0242] c) optionally a pharmaceutically
acceptable carrier as defined herein.
[0243] In this context, it is particularly preferred that the
optionally comprised adjuvant component comprises the same RNA as
comprised in the RNA vaccine of the inventive vaccine/inhibitor
combination as antigen-providing RNA e.g. mRNA coding for at least
one antigen.
[0244] Furthermore, the RNA vaccine of the inventive
vaccine/inhibitor combination may comprise further components for
facilitating administration and uptake of the components of the RNA
vaccine. Such further components may be an appropriate carrier or
vehicle, or e.g. additional adjuvants for supporting any immune
response as defined herein.
[0245] According to one further embodiment, the components of the
RNA vaccine e.g. the at least one RNA coding for at least one
antigen and an adjuvant component, of the inventive
vaccine/inhibitor combination, may be formulated together or
separately in the same or different compositions.
[0246] As a second component, the inventive vaccine/inhibitor
combination includes as inhibitor a composition comprising a PD-1
pathway inhibitor targeting any member of the PD-1 signaling
pathway, preferably targeting PD-1, PD-L1 or PD-L2.
[0247] Programmed Death-1 (PD-1, PDCD1) is a type I transmembrane
protein belonging to the extended CD28 family of T cell regulators.
PD-1 lacks the membrane-proximal cysteine residue required for
homodimerization of other members of the CD28 family. Structural
and biochemical analyses showed that PD-1 is monomeric in solution
as well as on the cell surface (Okazaki and Honjo, 2007. Int
Immunol. 19(7):813-24). PD-1 is expressed on activated T cells, B
cells and monocytes. The broader expression of PD-1 contrasts with
restricted expression of other CD28 family members to T cells,
suggesting that PD-1 regulates a wider spectrum of immune responses
compared with other CD28 family members.
[0248] PD-1 negatively regulates antigen receptor signaling by
recruiting the protein tyrosine phosphatase SHP-2 upon interacting
with either of two ligands, PD-L1 or PD-L2.
[0249] PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are type I
transmembrane glycoproteins composed of IgC- and IgV-type
extracellular domains. PD-L1 and PD-L2 share 40% amino acid
identity while human and mouse orthologs of PD-L1 and PD-L2 share
70% amino acid identity. Both PD-L1 and PD-L2 have short
cytoplasmic tails with no known motif for signal transduction,
suggesting that these ligands do not transduce any signal upon
interaction with PD-1.
[0250] The interaction of PD-L1 and PD-1 provides a crucial
negative co-stimulatory signal to T cells and functions as cell
death inducer. Interaction between low concentration of PD-1 and
PD-L1 leads to the transmission of an inhibitory signal that
inhibits the proliferation of antigen-specific CD8+ cells. At
higher concentrations this interaction does not inhibit T cell
proliferation but reduces the production of multiple cytokines.
Thus, binding to PD-L1 can antagonize the B7-CD28 signal when
antigenic stimulation is weak and plays a key role in
downregulating T cell responses.
[0251] The role of PD-1 and PD-1 ligands in inhibiting T cell
activation and proliferation suggested that these proteins may
serve as therapeutic targets for treatments of inflammation, cancer
or infectious diseases. Depending on the desired therapeutic
outcome, an up- or down-modulation of the PD-1 pathway is required.
Up-modulation of the immune system is particularly required in the
treatment of cancers and chronic infections. This can be achieved
for example by PD-1 blockade or inhibiting the PD-1 pathway.
Inhibition of the PD-1 pathway can be achieved, for example, by an
antibody directed at PD-1 or a PD-1 ligand. In this context the
PD-1 pathway inhibitor may remove T cell dysfunction resulting from
PD-1 signaling and thereby restore or enhance T cell function (e.g.
proliferation, cytokine production, target cell killing). In
addition, anergic T cells which are unresponsive to antigen
stimulation may be reactivated.
[0252] In the context of the present invention, the PD-1 pathway
inhibitor may be in specific embodiments an antibody, particularly
an antagonistic antibody or a nucleic acid-encoded antibody
(intrabody), an siRNA, an antisense RNA, a protein comprising (or a
nucleic acid coding for) an amino acid sequence capable of binding
to PD-1 but preventing PD-1 signaling (e.g. a fusion protein of a
fragment of PD-L1 or PD-L2 and the Fc part of an immunoglobulin), a
soluble protein (or a nucleic acid coding for a soluble protein)
competing with membrane-bound PD-1 for binding of its ligands PD-L1
and PD-L2; or a small molecule inhibitor capable of inhibiting PD-1
pathway signaling.
[0253] Therefore, in a preferred embodiment of the present
invention, the PD-1 pathway inhibitor is an antibody (or a nucleic
acid coding for an antibody) directed against PD-1, preferably an
antibody specifically binding to the extracellular domain of PD-1
and thereby inhibiting PD-1 signaling. Preferably, such an
antagonistic antibody binds close to the PD-L1 binding site on
PD-1, thus inhibiting the binding of PD-L1 to PD-1.
[0254] Particularly preferred are the anti-PD1 antibodies Nivolumab
(MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin
Oncol. 28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011),
(Berger et al., 2008. Clin Cancer Res. 14(10):3044-51; PMID:
18483370); and MK-3475 (SCH 900475).
[0255] In a further preferred embodiment, the PD-1 pathway
inhibitor is an antibody (or a nucleic acid coding for an antibody)
directed against a PD-1 ligand, preferably an antibody specifically
binding to the extracellular domain of the PD-1 or PD-2 ligand.
Preferably, such antibody binds proximal to and disruptive of the
PD-1 or PD-2 binding site on the ligand.
[0256] Particularly preferred are the anti-PD-L1 antibodies
MDX-1105/BMS-936559 (Brahmer et al. 2012. N Engl J Med.
366(26):2455-65; PMID: 22658128); MPDL3280A/RG7446, or
MEDI4736.
[0257] In a further preferred embodiment, the PD-1 pathway
inhibitor is a protein comprising (or a nucleic acid coding for) an
amino acid sequence capable of binding to PD-1 but preventing PD-1
pathway signaling, particularly preferred in this context is a
fusion protein of a fragment of PD-L1 or PD-L2 ligand.
[0258] In this context, a particularly preferred embodiment is a
fusion protein comprising the extracellular domain of PD-L1 or
PD-L2 or a fragment thereof capable of binding to PD-1 and an Fc
portion of an immunoglobulin. An example of such a fusion protein
is represented by AMP-224 (extracellular domain of murine
PD-L2/B7-DC fused to the unmodified Fc portion of murine IgG2a
protein; Mkrtichyan et al., 2012. J Immunol. 189(5):2338-47; PMID:
22837483).
[0259] In the context of the present invention, the administration
of the vaccine and the inhibitor may occur either simultaneously or
timely staggered, either at the same site of administration or at
different sites of administration, as further outlined below.
[0260] To ensure that the separate mechanisms elicited by the RNA
vaccine and the PD-1 pathway inhibitor are not negatively
influenced by each other, the PD-1 pathway inhibitor and the RNA
vaccine are preferably administered separated in time (in a
time-staggered manner), i.e. sequentially, and/or are administered
at different administration sites. This means that the RNA vaccine
may be administrated e.g. prior, concurrent or subsequent to the
PD-1 pathway inhibitor, or vice versa. Alternatively or
additionally, the RNA vaccine and the PD-1 pathway inhibitor may be
administered at different administration sites, or at the same
administration site, preferably, when administered in a time
staggered manner. According to a particularly preferred embodiment,
the RNA vaccine is to be administered first and the PD-1 pathway
inhibitor is to be administered subsequent to the RNA vaccine. This
procedure ensures that the immune cells such as antigen-presenting
cells and T cells have already encountered the antigen before the
immune system is stimulated by inhibition of the PD-1 pathway, even
though a concurrent administration or an administration, wherein
the PD-1 pathway inhibitor is to be administered prior to the RNA
vaccine, may lead to the same or at least comparable results.
[0261] Accordingly, in a further embodiment, the inventive
vaccine/inhibitor combination furthermore comprises a
pharmaceutically acceptable carrier and/or vehicle.
[0262] Such a pharmaceutically acceptable carrier typically
includes the liquid or non-liquid basis of a composition comprising
the components of the inventive vaccine/inhibitor combination. If
the composition is provided in liquid form, the carrier will
preferably be pyrogen-free water; isotonic saline or buffered
(aqueous) solutions, e.g. phosphate, citrate etc. buffered
solutions. The injection buffer may be hypertonic, isotonic or
hypotonic with reference to the specific reference medium, i.e. the
buffer may have a higher, identical or lower salt content with
reference to the specific reference medium, wherein preferably such
concentrations of the afore mentioned salts may be used, which do
not lead to damage of cells due to osmosis or other concentration
effects. Reference media are e.g. liquids occurring in "in vivo"
methods, such as blood, lymph, cytosolic liquids, or other body
liquids, or e.g. liquids, which may be used as reference media in
"in vitro" methods, such as common buffers or liquids. Such common
buffers or liquids are known to a skilled person. Ringer-Lactate
solution is particularly preferred as a liquid basis.
[0263] However, one or more compatible solid or liquid fillers or
diluents or encapsulating compounds, which are suitable for
administration to a patient to be treated, may be used as well for
the vaccine/inhibitor combination according to the invention. The
term "compatible" as used here means that these constituents of the
vaccine/inhibitor combination are capable of being mixed with the
RNA vaccine and/or the PD-1 pathway inhibitor in such a manner that
no interaction occurs which would substantially reduce the
pharmaceutical effectiveness of the vaccine/inhibitor combination
under typical use conditions.
[0264] Furthermore, the inventive vaccine/inhibitor combination may
comprise one or more additional adjuvants which are suitable to
initiate or increase an immune response of the innate immune
system, i.e. a non-specific immune response, particularly by
binding to pathogen-associated molecular patterns (PAMPs). With
other words, when administered, the RNA vaccine preferably elicits
an innate immune response due to the adjuvant, optionally contained
therein. Nevertheless, the adjuvant may also be part of another
component of the inventive vaccine/inhibitor combination than the
RNA vaccine. Preferably, such an adjuvant may be selected from an
adjuvant known to a skilled person and suitable for the present
case, i.e. supporting the induction of an innate immune response in
a mammal, e.g. an adjuvant nucleic acid or an adjuvant component as
defined above or an adjuvant as defined in the following.
[0265] Therefore, such an adjuvant may also be selected from any
adjuvant known to a skilled person and suitable for the present
case, i.e. supporting the induction of an innate immune response in
a mammal and/or suitable for depot and delivery of the components
of the inventive vaccine/inhibitor combination. Preferred as
adjuvants suitable for depot and delivery are cationic or
polycationic compounds as defined above. Likewise, the adjuvant may
be selected from the group consisting of, e.g., cationic or
polycationic compounds as defined above, from chitosan, TDM, MDP,
muramyl dipeptide, pluronics, alum solution, aluminium hydroxide,
ADJUMER.TM. (polyphosphazene); aluminium phosphate gel; glucans
from algae; algammulin; aluminium hydroxide gel (alum); highly
protein-adsorbing aluminium hydroxide gel; low viscosity aluminium
hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80
(0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);
AVRIDINE.TM. (propanediamine); BAY R1005.TM.
((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-am-
ide hydroacetate); CALCITRIOL.TM. (1-alpha,25-dihydroxy-vitamin
D3); calcium phosphate gel; CAP.TM. (calcium phosphate
nanoparticles); cholera holotoxin,
cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of
the cholera toxin; CRL 1005 (block copolymer P1205);
cytokine-containing liposomes; DDA (dimethyldioctadecylammonium
bromide); DHEA (dehydroepiandrosterone); DMPC
(dimyristoylphosphatidylcholine); DMPG
(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic
acid sodium salt); Freund's complete adjuvant; Freund's incomplete
adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i)
N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine
(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)
zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP
(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47
alanyl-D-isoglutamine); imiquimod
(1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher.TM.
(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol
dipalmitate); DRVs (immunoliposomes prepared from
dehydration-rehydration vesicles); interferongamma;
interleukin-1beta; interleukin-2; interleukin-7; interleukin-12;
ISCOMS.TM.; ISCOPREP 7.0.3..TM.; liposomes; LOXORIBINE.TM.
(7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E. coli labile
enterotoxin-protoxin); microspheres and microparticles of any
composition; MF59.TM.; (squalenewater emulsion); MONTANIDE ISA
51.TM. (purified incomplete Freund's adjuvant); MONTANIDE ISA
720.TM. (metabolisable oil adjuvant); MPL.TM.
(3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE
liposomes
((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glyce-
ro-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt);
MURAMETIDE.TM. (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE.TM. and
DMURAPALMITINE.TM. (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);
NAGO (neuraminidase-galactose oxidase); nanospheres or
nanoparticles of any composition; NISVs (non-ionic surfactant
vesicles); PLEURAN.TM. (.quadrature.-glucan); PLGA, PGA and PLA
(homo- and co-polymers of lactic acid and glycolic acid;
microspheres/nanospheres); PLURONIC L121.TM.; PMMA
(polymethylmethacrylate); PODDS.TM. (proteinoid microspheres);
polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic
acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein
cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.);
STIMULON.TM. (QS-21); Quil-A (Quil-A saponin); S-28463
(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethano-
l); SAF-1.TM. ("Syntex adjuvant formulation"); Sendai
proteoliposomes and Sendai containing lipid matrices; Span-85
(sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and
Tween 85); squalene or Robane.RTM.
(2,6,10,15,19,23-hexamethyltetracosan and
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);
stearyltyrosine (octadecyltyrosine hydrochloride); Theramid.RTM.
(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxyprop-
ylamide); Theronyl-MDP (Termurtide.TM. or [thr 1]-MDP;
N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs or
virus-like particles); Walter-Reed liposomes (liposomes containing
lipid A adsorbed on aluminium hydroxide), and lipopeptides,
including Pam3Cys, in particular aluminium salts, such as
Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF,
IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers,
including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes,
including Stealth, cochleates, including BIORAL; plant derived
adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants
suitable for costimulation including Tomatine, biopolymers,
including PLG, PMM, Inulin, microbe derived adjuvants, including
Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences,
CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13,
ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine,
IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin,
GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV
fusion protein, cdiGMP; and adjuvants suitable as antagonists
including CGRP neuropeptide.
[0266] An adjuvant is preferably selected from adjuvants, which
support induction of a Th1-immune response or maturation of naive
T-cells, such as GM-CSF, IL-12, IFNg, any adjuvant nucleic acid as
defined above, preferably an immunostimulatory RNA, CpG DNA,
etc.
[0267] In a further preferred embodiment, it is also possible that
the inventive vaccine/inhibitor combination contains besides the
antigen-providing RNA and the PD-1 pathway inhibitor further
components which are selected from the group comprising: further
antigens or further antigen-providing nucleic acids; a further
immunotherapeutic agent; one or more auxiliary substances; or any
further compound, which is known to be immunostimulating due to its
binding affinity (as ligands) to human Toll-like receptors; and/or
an adjuvant nucleic acid, preferably an immunostimulatory RNA
(isRNA).
[0268] Accordingly, in another preferred embodiment, the inventive
vaccine/inhibitor combination furthermore comprises at least one
adjuvant, an auxiliary substance selected from lipopolysaccharides,
TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines,
interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,
IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha,
IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha,
growth factors, and hGH, a ligand of human Toll-like receptor TLR1,
TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of
murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like
receptor, a ligand of a RIG-I like receptor, an adjuvant nucleic
acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial
agent, or an anti-viral agent.
[0269] The vaccine/inhibitor combination as defined according to
the present invention may furthermore comprise further additives or
additional compounds. Further additives which may be included in
the inventive vaccine/inhibitor combination, such as in the RNA
vaccine and/or the composition comprising a PD-1 pathway inhibitor,
are emulsifiers, such as, for example, Tween.RTM.; wetting agents,
such as, for example, sodium lauryl sulfate; colouring agents;
taste-imparting agents, pharmaceutical carriers; tablet-forming
agents; stabilizers; antioxidants; preservatives, RNase inhibitors
and/or an anti-bacterial agent or an anti-viral agent.
[0270] The inventive vaccine/inhibitor combination typically
comprises a "safe and effective amount" of the components of the
inventive vaccine/inhibitor combination as defined herein. As used
herein, a "safe and effective amount" preferably means an amount of
the components, preferably of the at least one RNA encoding at
least one antigen and the PD-1 pathway inhibitor, that is
sufficient to significantly induce a positive modification or
prevention of a disease or disorder as defined herein. At the same
time, however, a "safe and effective amount" is small enough to
avoid serious side-effects and to permit a sensible relationship
between advantage and risk. The determination of these limits
typically lies within the scope of sensible medical judgment.
[0271] The inventive vaccine/inhibitor combination may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term parenteral as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-nodal, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional, intracranial, transdermal,
intradermal, intrapulmonal, intraperitoneal, intracardial,
intraarterial, and sublingual injection or infusion techniques.
Preferably the RNA vaccine is administered by intradermal or
intramuscular application and the PD-1 pathway inhibitor is
preferably administered by intramuscular or intraperitoneal
injection, more preferably by intravenous infusion, in case it is
in form of an antibody.
[0272] According to a further aspect, the object underlying the
present invention is solved by a pharmaceutical composition
comprising a vaccine and an inhibitor, in particular as vaccine an
RNA vaccine comprising at least one RNA comprising at least one
open reading frame coding for at least one antigen and as an
inhibitor a composition comprising a PD-1 pathway inhibitor, both
preferably as defined above. Likewise, the pharmaceutical
composition is preferably formulated and administered as defined
above for the components of the inventive vaccine/inhibitor
combination. Such a pharmaceutical composition may further comprise
any ingredient as defined above for the inventive vaccine/inhibitor
combination.
[0273] Accordingly, the combination of the RNA vaccine and the PD-1
pathway inhibitor as defined according to the present invention may
occur either as one composition, e.g. the pharmaceutical
composition according to the present invention, or may occur in
more than one compositions, e.g. as a kit of parts, wherein the
different components form different parts of such kit of parts.
These different components, such as the vaccine and the inhibitor,
may be formulated each as a pharmaceutical composition or as a
composition as defined above. Preferably, each of the different
parts of the kit comprises a different component, e.g. one part
comprises the RNA vaccine as defined herein, one further part
comprises the PD-1 pathway inhibitor as defined herein, etc.
[0274] Therefore, according to a further aspect, the present
invention also provides kits, particularly kits of parts. Such
kits, particularly kits of parts, typically comprise as components
alone or in combination with further components as defined herein
an RNA vaccine comprising at least one RNA comprising at least one
open reading frame coding for at least one antigen and, preferably
in a different part of the kit, a PD-1 pathway inhibitor as defined
herein. The inventive vaccine/inhibitor combination as defined
herein, optionally in combination with further components as
defined herein, such as an additional adjuvant, may occur in one or
different parts of the kit. As an example, e.g. at least one part
of the kit may comprise the RNA vaccine comprising at least one RNA
encoding at least one antigen as defined herein, at least one
further part of the kit may comprise the PD-1 pathway inhibitor as
defined herein, and optionally at least one further part of the kit
may comprise an additional adjuvant as described herein. The kit or
kit of parts may furthermore contain technical instructions with
information on the administration and dosage of the inventive
vaccine/inhibitor combination, the inventive pharmaceutical
composition or of any of its components or parts.
[0275] The inventive vaccine/inhibitor combination, the inventive
pharmaceutical composition or the inventive kit of parts comprising
an RNA vaccine and a PD-1 pathway inhibitor may be used for human
and also for veterinary medical purposes, preferably for human
medical purposes.
[0276] Therefore, according to a further aspect, the present
invention is directed to the first medical use of the inventive
vaccine/inhibitor combination, the inventive pharmaceutical
composition and the inventive kit of parts comprising an RNA
vaccine and a PD-1 pathway inhibitor as defined herein.
Accordingly, the inventive vaccine/inhibitor combination, the
inventive pharmaceutical composition and the inventive kit of parts
comprising an RNA vaccine and a PD-1 pathway inhibitor as defined
herein may be used as a medicament.
[0277] According to another aspect, the present invention is
directed to the second medical use of the inventive
vaccine/inhibitor combination, the inventive pharmaceutical
composition and the inventive kit of parts comprising an RNA
vaccine and a PD-1 pathway inhibitor as defined herein. Thus, the
inventive vaccine/inhibitor combination, the inventive
pharmaceutical composition and the inventive kit of parts
comprising an RNA vaccine and a PD-1 pathway inhibitor as defined
herein may be used for the treatment and/or amelioration of various
diseases, particularly of cancer and tumor diseases and infectious
diseases as defined herein.
[0278] Cancer or tumour diseases in this context preferably include
e.g. melanomas, malignant melanomas, colon carcinomas, lymphomas,
sarcomas, blastomas, renal carcinomas, gastrointestinal tumors,
gliomas, prostate tumors, bladder cancer, rectal tumors, stomach
cancer, oesophageal cancer, pancreatic cancer, liver cancer,
mammary carcinomas (=breast cancer), uterine cancer, cervical
cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia
(ALL), chronic myeloid leukaemia (CIVIL), chronic lymphocytic
leukaemia (CLL), hepatomas, various virus-induced tumors such as,
for example, papilloma virus-induced carcinomas (e.g. cervical
carcinoma=cervical cancer), adenocarcinomas, herpes virus-induced
tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma),
heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1- and
HTLV-2-induced lymphomas, acoustic neuroma, lung carcinomas (=lung
cancer=bronchial carcinoma), small-cell lung carcinomas, pharyngeal
cancer, anal carcinoma, glioblastoma, rectal carcinoma,
astrocytoma, brain tumors, retinoblastoma, basalioma, brain
metastases, medulloblastomas, vaginal cancer, pancreatic cancer,
testicular cancer, Hodgkin's syndrome, meningiomas, Schneeberger
disease, hypophysis tumor, Mycosis fungoides, carcinoids,
neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, renal
cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's
lymphomas, urethral cancer, CUP syndrome, head/neck tumors,
oligodendroglioma, vulval cancer, intestinal cancer, colon
carcinoma, oesophageal carcinoma (=oesophageal cancer), wart
involvement, tumors of the small intestine, craniopharyngeomas,
ovarian carcinoma, genital tumors, ovarian cancer (=ovarian
carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrial
carcinoma, liver metastases, penile cancer, tongue cancer, gall
bladder cancer, leukaemia, plasmocytoma, lid tumor, prostate cancer
(=prostate tumors), etc.
[0279] In specific embodiments the treatment of lung cancer (e.g.
non-small cell lung cancer or small cell lung cancer) or prostate
cancer is particularly preferred.
[0280] Infectious diseases in this context, preferably includes
viral, bacterial or protozoological infectious diseases. Such
infectious diseases, preferably (viral, bacterial or
protozoological) infectious diseases, are typically selected from
the list consisting of Acinetobacter infections, African sleeping
sickness (African trypanosomiasis), AIDS (Acquired immunodeficiency
syndrome), Amoebiasis, Anaplasmosis, Anthrax, Appendicitis,
Arcanobacterium haemolyticum infections, Argentine hemorrhagic
fever, Ascariasis, Aspergillosis, Astrovirus infections, Athlete's
foot, Babesiosis, Bacillus cereus infections, Bacterial meningitis,
Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides
infections, Balantidiasis, Baylisascaris infections, Bilharziosis,
BK virus infections, Black piedra, Blastocystis hominis infections,
Blastomycosis, Bolivian hemorrhagic fever, Borrelia infections
(Borreliosis), Botulism (and Infant botulism), Bovine tapeworm,
Brazilian hemorrhagic fever, Brucellosis, Burkholderia infections,
Buruli ulcer, Calicivirus infections (Norovirus and Sapovirus),
Campylobacteriosis, Candidiasis (Candidosis), Canine tapeworm
infections, Cat-scratch disease, Chagas Disease (American
trypanosomiasis), Chancroid, Chickenpox, Chlamydia infections,
Chlamydia trachomatis infections, Chlamydophila pneumoniae
infections, Cholera, Chromoblastomycosis, Climatic bubo,
Clonorchiasis, Clostridium difficile infections,
Coccidioidomycosis, Cold, Colorado tick fever (CTF), Common cold
(Acute viral rhinopharyngitis; Acute coryza), Condyloma acuminata,
Conjunctivitis, Creutzfeldt-Jakob disease (CJD), Crimean-Congo
hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis,
Cutaneous larva migrans (CLM), Cutaneous Leishmaniosis,
Cyclosporiasis, Cysticercosis, Cytomegalovirus infections, Dengue
fever, Dermatophytosis, Dientamoebiasis, Diphtheria,
Diphyllobothriasis, Donavanosis, Dracunculiasis, Early summer
meningoencephalitis (FSME), Ebola hemorrhagic fever,
Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infections),
Enterococcus infections, Enterovirus infections, Epidemic typhus,
Epiglottitis, Epstein-Barr Virus Infectious Mononucleosis, Erythema
infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,
Fasciolosis, Fatal familial insomnia (FFI), Fifth disease,
Filariasis, Fish poisoning (Ciguatera), Fish tapeworm, Flu, Food
poisoning by Clostridium perfringens, Fox tapeworm, Free-living
amebic infections, Fusobacterium infections, Gas gangrene,
Geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS),
Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma
inguinale (Donovanosis), Group A streptococcal infections, Group B
streptococcal infections, Haemophilus influenzae infections, Hand
foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome (HPS),
Helicobacter pylori infections, Hemolytic-uremic syndrome (HUS),
Hemorrhagic fever with renal syndrome (HFRS), Henipavirus
infections, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D,
Hepatitis E, Herpes simplex, Herpes simplex type I, Herpes simplex
type II, Herpes zoster, Histoplasmosis, Hollow warts, Hookworm
infections, Human bocavirus infections, Human ewingii ehrlichiosis,
Human granulocytic anaplasmosis (HGA), Human metapneumovirus
infections, Human monocytic ehrlichiosis, Human papillomavirus
(HPV) infections, Human parainfluenza virus infections,
Hymenolepiasis, Influenza, Isosporiasis, Japanese encephalitis,
Kawasaki disease, Keratitis, Kingella kingae infections, Kuru,
Lambliasis (Giardiasis), Lassa fever, Legionellosis (Legionnaires'
disease, Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,
Lice, Listeriosis, Lyme borreliosis, Lyme disease, Lymphatic
filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria,
Marburg hemorrhagic fever (MHF), Marburg virus, Measles,
Melioidosis (Whitmore's disease), Meningitis, Meningococcal
disease, Metagonimiasis, Microsporidiosis, Miniature tapeworm,
Miscarriage (prostate inflammation), Molluscum contagiosum (MC),
Mononucleosis, Mumps, Murine typhus (Endemic typhus), Mycetoma,
Mycoplasma hominis, Mycoplasma pneumonia, Myiasis, Nappy/diaper
dermatitis, Neonatal conjunctivitis (Ophthalmia neonatorum),
Neonatal sepsis (Chorioamnionitis), Nocardiosis, Noma, Norwalk
virus infections, Onchocerciasis (River blindness), Osteomyelitis,
Otitis media, Paracoccidioidomycosis (South American
blastomycosis), Paragonimiasis, Paratyphus, Pasteurellosis,
Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),
Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory
disease (PID), Pertussis (Whooping cough), Pfeiffer's glandular
fever, Plague, Pneumococcal infections, Pneumocystis pneumonia
(PCP), Pneumonia, Polio (childhood lameness), Poliomyelitis,
Porcine tapeworm, Prevotella infections, Primary amoebic
meningoencephalitis (PAM), Progressive multifocal
leukoencephalopathy, Pseudo-croup, Psittacosis, Q fever, Rabbit
fever, Rabies, Rat-bite fever, Reiter's syndrome, Respiratory
syncytial virus infections (RSV), Rhinosporidiosis, Rhinovirus
infections, Rickettsial infections, Rickettsialpox, Rift Valley
fever (RVF), Rocky mountain spotted fever (RMSF), Rotavirus
infections, Rubella, Salmonella paratyphus, Salmonella typhus,
Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies,
Scarlet fever, Schistosomiasis (Bilharziosis), Scrub typhus,
Sepsis, Shigellosis (Bacillary dysentery), Shingles, Smallpox
(Variola), Soft chancre, Sporotrichosis, Staphylococcal food
poisoning, Staphylococcal infections, Strongyloidiasis, Syphilis,
Taeniasis, Tetanus, Three-day fever, Tick-borne encephalitis, Tinea
barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp),
Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch),
Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis
(Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor
(Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans (OLM)
and Visceral Larva Migrans (VLM)), Toxoplasmosis, Trichinellosis,
Trichomoniasis, Trichuriasis (Whipworm infections), Tripper,
Trypanosomiasis (sleeping sickness), Tsutsugamushi disease,
Tuberculosis, Tularemia, Typhus, Typhus fever, Ureaplasma
urealyticum infections, Vaginitis (Colpitis), Variant
Creutzfeldt-Jakob disease (vCJD, nvCJD), Venezuelan equine
encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia,
Visceral Leishmaniosis, Warts, West Nile Fever, Western equine
encephalitis, White piedra (Tinea blanca), Whooping cough, Yeast
fungus spots, Yellow fever, Yersinia pseudotuberculosis infections,
Yersiniosis, and Zygomycosis.
[0281] Particularly preferred are diseases, particularly cancer or
tumor diseases, which are associated with an overexpression of at
least one member of the PD-1 pathway, preferably PD-1 receptor
and/or its ligands PD-L1 and PD-L2.
[0282] Particularly preferred in this context is the treatment of
melanoma, glioblastoma, and carcinomas of the pancreas, lung,
breast, colon, ovary, and renal cells, urothelial cancers, squamous
cell carcinomas of the head and neck and hepatocellular carcinoma
which are associated with an overexpression of PD-L1. Most
particularly preferred is the treatment of non-small cell lung
cancer (NSCLC) or small cell lung cancer associated with an
overexpression of any member of the PD-1 pathway, particularly of
PD-1 or PD-L1.
[0283] In another embodiment the treatment of cancer or tumor
diseases associated with no or low expression of at least one
member of the PD-1 pathway, preferably PD-1 receptor and/or its
ligands PD-L1 and PD-L2 is particularly preferred. In this context
the RNA vaccine of the inventive vaccine/inhibitor combination is
able to induce the expression of at least one member of the PD-1
pathway, e.g. PD-L1 in the patient to be treated and therefore
enables the therapeutic activity of the PD-1 inhibitor.
[0284] According to another aspect, the present invention provides
a PD-1 pathway inhibitor as defined above, for use in therapy in
combination with an RNA vaccine comprising at least one RNA
comprising at least one open reading frame coding for at least one
antigen as defined above, for example, for use in a method of
treatment or prevention of tumor and/or cancer diseases or
infectious diseases as defined herein.
[0285] According to yet another aspect, the present invention
provides an RNA vaccine comprising at least one RNA comprising at
least one open reading frame coding for at least one antigen as
defined above, for use in therapy in combination with a PD-1
pathway inhibitor as defined above, for example, for use in a
method of treatment or prevention of tumor and/or cancer diseases
or infectious diseases as defined herein.
[0286] Furthermore, the present invention provides in a further
aspect a method for transfecting and/or treating a cell, a tissue
or an organism, thereby applying or administering the inventive
vaccine/inhibitor combination particularly for therapeutic
purposes. In this context, typically after preparing the inventive
vaccine/inhibitor combination, the inventive vaccine/inhibitor
combination is preferably administered to a cell, a tissue or an
organism, preferably using any of the administration modes as
described herein. The method for transfecting and/or treating a
cell may be carried out in vitro, in vivo or ex vivo.
[0287] Therefore, the invention also provides a method of treatment
comprising administering to a subject in need thereof a
therapeutically effective amount of an RNA vaccine comprising at
least one RNA comprising at least one open reading frame coding for
at least one antigen as defined above in combination with a
composition comprising a PD-1 pathway inhibitor as defined
above.
[0288] In a preferred embodiment, the method comprises the in vitro
transfection of isolated cells. The cells used therefore are
preferably human or animal cells, particularly cells of a primary
cell culture, which are then retransferred to a human or animal.
Prior to transfection, these cells are typically isolated from the
patient to be treated and cultivated.
[0289] In a further preferred embodiment, the method of treatment
does not comprise the in vitro transfection of isolated cells. In
this case, the RNA vaccine as defined above administered to the
subject in need thereof does not comprise isolated cells
transfected with the at least one RNA comprising at least one open
reading frame coding for at least one antigen comprised in the RNA
vaccine.
[0290] In the present invention, if not otherwise indicated,
different features of alternatives and embodiments may be combined
with each other. In the context of the present invention, term
"comprising" may be substituted with the term "consisting of",
where applicable.
BRIEF DESCRIPTION OF THE FIGURES
[0291] The figures shown in the following are merely illustrative
and shall describe the present invention in a further way. These
figures shall not be construed to limit the present invention
thereto.
[0292] FIG. 1: The RNA vaccine (OVA-RNActive R1710) acts
synergistically with anti-PD-1 antibody. C57 BL/6 mice were
challenged subcutaneously with 3.times.10.sup.5 syngenic E.G7-OVA
tumor cells on day 0 and then treated with either OVA RNActive
vaccine (32 .mu.g) alone or in combination with anti-PD-1 antibody
or control IgG (100 .mu.g i.p.) in accordance with the indicated
schedule.
[0293] FIG. 2: Survival proportions of mice bearing E.G7-OVA tumors
treated with different therapies. According to Example 2, mice were
treated with either OVA-RNActive vaccine or anti-PD-1 antibody
alone or in combination.
[0294] FIG. 3: G/C optimized mRNA sequence of R1710 coding for
Gallus gallus ovalbumin as comprised in the OVA-RNActive
vaccine.
EXAMPLES
[0295] The examples shown in the following are merely illustrative
and shall describe the present invention in a further way. These
examples shall not be construed to limit the present invention
thereto.
Example 1
Preparation of the mRNA Vaccine
1. Preparation of DNA and mRNA Constructs
[0296] For the present examples a DNA sequence, encoding Gallus
gallus ovalbumin mRNA (R1710) was prepared and used for subsequent
in vitro transcription reactions.
[0297] According to a first preparation, the DNA sequence coding
for the above mentioned mRNA was prepared. The construct was
prepared by modifying the wild type coding sequence by introducing
a GC-optimized sequence for stabilization, followed by a
stabilizing sequence derived from the alpha-globin-3'-UTR (muag
(mutated alpha-globin-3'-UTR)), a stretch of 64 adenosines
(poly-A-sequence), a stretch of 30 cytosines (poly-C-sequence), and
a histone stem loop. In SEQ ID NO: 2 (see FIG. 3) the sequence of
the corresponding mRNA is shown.
2. In Vitro Transcription
[0298] The respective DNA plasmid prepared according to Example 1
was transcribed in vitro using T7 polymerase. Subsequently the mRNA
was purified using PureMessenger.RTM. (CureVac, Tubingen,
Germany).
3. Reagents
[0299] Complexation Reagent: protamine
4. Preparation of the Vaccine
[0300] The mRNA R1710 was complexed with protamine by addition of
protamine to the mRNA in the ratio (1:2) (w/w) (adjuvant
component). After incubation for 10 min, the same amount of free
mRNA R1710 used as antigen-providing RNA was added.
[0301] OVA-RNActive vaccine (R1710): comprising an adjuvant
component consisting of mRNA coding for Gallus gallus ovalbumin
(R1710) according to SEQ ID NO. 2 complexed with protamine in a
ratio of 2:1 (w/w) and the antigen-providing free mRNA coding for
Gallus gallus ovalbumin (R1710) according to SEQ ID NO. (ratio 1:1;
complexed RNA:free RNA).
Example 2
Combination of an Anti-PD1 Antibody and an RNA Vaccine
[0302] On day zero, C57BL/6 mice were implanted subcutaneously
(right flank) with 3.times.10.sup.5 E.G7-OVA cells per mouse
(volume 100 .mu.l in PBS). E.G7-OVA is a mouse T cell lymphoma cell
line stably expressing Gallus gallus ovalbumin (OVA). Intradermal
vaccination with the RNA vaccine comprising OVA mRNA R1720 (32
.mu.g/mouse/vaccination day) (according to Example 1) or
Ringer-lactate (RiLa) as buffer control and treatment with the
anti-PD-1/CD279 monoclonal antibody (100 .mu.g i.p.) or an isotype
control according to Table 1 started on day 4 and was repeated on
days 7, 11, 14, 18 and 21. Animals received the antibody injection
in the morning and were vaccinated in the afternoon with a minimum
of four hours between the treatments.
TABLE-US-00001 TABLE 1 Animal groups Number Injected RNA per
Injected antibody per Group of mice vaccination day and mouse
treatment day and mouse A 10 100% Ringer Lactate -- (RiLa) buffer B
10 32 .mu.g -- C 6 -- 100 .mu.g anti-PD-1 D 6 32 .mu.g 100 .mu.g
anti-PD-1 E 6 32 .mu.g 100 .mu.g control-IgG2a
[0303] The anti-PD-1/CD279 antibody (clone RMP1-14, rat IgG2a) and
the isotype control antibody (clone 2A3, rat IgG2a) were purchased
from BioXCell (West Lebanon, N.H., USA).
[0304] Tumour growth was monitored by measuring the tumour size in
2 dimensions (length and width) using a calliper (starting on day
4). Tumour volume was calculated according to the following
formula:
volume ( mm 3 ) = length ( mm ) .times. .pi. .times. width 2 ( mm 2
) 6 ##EQU00001##
[0305] The results are shown in FIGS. 1 and 2.
[0306] As can be seen in FIG. 1, the OVA mRNA vaccine (OVA-RNActive
R1710) alone or in combination with control-IgG delayed tumor
growth by approximately 5 days compared to the buffer-treated
control group. Treatment with anti-PD-1 antibody alone was
comparable to vaccination alone, whereas simultaneous application
of the RNA vaccine/anti-PD-1 combination led to significant
inhibition of tumor growth, indicating a synergistic effect. Lines
represent the development of mean tumor volume and error bars the
SEM. Statistical analysis was based on the 2 way Anova with
Bonferroni posttest.
[0307] As can be seen in FIG. 2, the administration of the OVA mRNA
vaccine (OVA-RNActive R1710) or anti-PD-1 antibody alone had
already a significant effect on survival, whereas simultaneous
application of the RNA vaccine/anti-PD-1 combination led to 50%
survival (3 of 6 animals), indicating a synergistic effect.
Sequence CWU 1
1
18144DNAArtificial Sequencemuag (mutated (alpha-) globin 3' UTR)
1gcccgatggg cctcccaacg ggccctcctc ccctccttgc accg
4421378RNAArtificial SequencemRNA corresponding to artificial DNA
construct 2gggagaaagc uuaccauggg cagcaucggg gccgcgucga uggaguucug
cuucgacgug 60uucaaggagc ugaaggucca ccacgccaac gagaacaucu ucuacugccc
gaucgccauc 120augagcgcgc ucgccauggu guaccugggc gccaaggaca
gcacccggac gcagaucaac 180aagguggucc gcuucgacaa gcugcccggc
uucggggacu cgaucgaggc gcagugcggc 240accagcguga acgugcacag
cucgcuccgg gacauccuga accagaucac caagccgaac 300gacgucuaca
gcuucagccu ggccucgcgg cucuacgccg aggagcgcua cccgauccug
360cccgaguacc ugcagugcgu gaaggagcuc uaccggggcg ggcuggagcc
gaucaacuuc 420cagacggcgg ccgaccaggc ccgggagcug aucaacagcu
ggguggagag ccagaccaac 480ggcaucaucc gcaacguccu ccagccgucg
agcguggaca gccagaccgc gauggugcug 540gucaacgcca ucguguucaa
gggccugugg gagaagacgu ucaaggacga ggacacccag 600gccaugcccu
uccgggugac cgagcaggag ucgaagccgg uccagaugau guaccagauc
660gggcucuucc ggguggcgag cauggccagc gagaagauga agauccugga
gcugccguuc 720gccucgggca cgaugagcau gcucgugcug cugcccgacg
aggucagcgg ccucgagcag 780cuggagucga ucaucaacuu cgagaagcug
accgagugga ccagcagcaa cgugauggag 840gagcgcaaga ucaaggugua
ccucccgcgg augaagaugg aggagaagua caaccugacg 900ucgguccuga
uggcgauggg gaucaccgac guguucagca gcucggccaa ccucagcggc
960aucagcucgg ccgagagccu gaagaucagc caggcggugc acgccgccca
cgcggagauc 1020aacgaggccg gccgggaggu cguggggucg gccgaggcgg
gcguggacgc cgccagcguc 1080agcgaggagu uccgcgcgga ccacccguuc
cuguucugca ucaagcacau cgccaccaac 1140gccgugcucu ucuucggccg
gugcgugucg cccugaccac uaguuauaag acugacuagc 1200ccgaugggcc
ucccaacggg cccuccuccc cuccuugcac cgagauuaau aaaaaaaaaa
1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaugcauc 1320cccccccccc cccccccccc cccccccccc aaaggcucuu
uucagagcca ccagaauu 137837PRTArtificial Sequencecationic peptide
3Arg Arg Arg Arg Arg Arg Arg 1 5 48PRTArtificial Sequencecationic
peptide 4Arg Arg Arg Arg Arg Arg Arg Arg 1 5 59PRTArtificial
Sequencecationic peptide 5Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5
612PRTArtificial Sequencecationic peptide 6His His His Arg Arg Arg
Arg Arg Arg Arg Arg Arg 1 5 10 712PRTArtificial Sequencecationic
peptide 7Arg Arg Arg Arg Arg Arg Arg Arg Arg His His His 1 5 10
815PRTArtificial Sequencecationic peptide 8His His His Arg Arg Arg
Arg Arg Arg Arg Arg Arg His His His 1 5 10 15 915PRTArtificial
Sequencecationic peptide 9Tyr Ser Ser Arg Arg Arg Arg Arg Arg Arg
Arg Arg Ser Ser Tyr 1 5 10 15 1012PRTArtificial Sequencecationic
peptide 10Arg Lys His Arg Lys His Arg Lys His Arg Lys His 1 5 10
118PRTArtificial Sequencecationic peptide 11Tyr Arg Lys His Arg Lys
His Arg 1 5 1212PRTArtificial Sequencecationic peptide 12Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 1321PRTArtificial
Sequencecationic peptide 13His His His His His His Arg Arg Arg Arg
Arg Arg Arg Arg Arg His 1 5 10 15 His His His His His 20
1410PRTArtificial Sequencecationic peptide 14His His His Arg Arg
Arg Arg His His His 1 5 10 1516PRTArtificial Sequencecationic
peptide 15His His His His His His Arg Arg Arg Arg His His His His
His His 1 5 10 15 1615PRTArtificial Sequencecationic peptide 16Tyr
Ser Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Tyr 1 5 10 15
1712PRTArtificial Sequencecationic peptide 17Arg Lys His Arg Lys
His Arg Lys His Arg Lys His 1 5 10 188PRTArtificial
Sequencecationic peptide 18Tyr Arg Lys His Arg Lys His Arg 1 5
* * * * *