U.S. patent application number 12/682187 was filed with the patent office on 2010-12-02 for composition for treating prostate cancer (pca).
This patent application is currently assigned to Cure Vac GmbH. Invention is credited to Ingmar Hoerr, Thomas Lander, Jochen Probst.
Application Number | 20100305196 12/682187 |
Document ID | / |
Family ID | 39472544 |
Filed Date | 2010-12-02 |
United States Patent
Application |
20100305196 |
Kind Code |
A1 |
Probst; Jochen ; et
al. |
December 2, 2010 |
COMPOSITION FOR TREATING PROSTATE CANCER (PCa)
Abstract
The present invention relates to an active (immunostimulatory)
composition comprising at least one RNA, preferably an mRNA,
encoding at least two (preferably different) antigens capable of
eliciting an (adaptive) immune response in a mammal wherein the
antigens are selected from the group consisting of PSA
(Prostate-Specific Antigen), PSMA (Prostate-Specific Membrane
Antigen), PSCA (Prostate Stem Cell Antigen), and STEAP (Six
Transmembrane Epithelial Antigen of the Prostate). The invention
furthermore relates to a vaccine comprising said active
(immunostimulatory) composition, and to the use of said active
(immunostimulatory) composition (for the preparation of a vaccine)
and/or of the vaccine for eliciting an (adaptive) immune response
for the treatment of prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto. Finally, the invention
relates to kits, particularly to kits of parts, containing the
active (immunostimulatory) composition and/or the vaccine.
Inventors: |
Probst; Jochen;
(Wolfschlugen, DE) ; Hoerr; Ingmar; (Tubingen,
DE) ; Lander; Thomas; (Konigstein 1. Taunus,
DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
Cure Vac GmbH
Tubingen
DE
|
Family ID: |
39472544 |
Appl. No.: |
12/682187 |
Filed: |
October 8, 2008 |
PCT Filed: |
October 8, 2008 |
PCT NO: |
PCT/EP2008/008504 |
371 Date: |
August 10, 2010 |
Current U.S.
Class: |
514/44R ;
536/23.5 |
Current CPC
Class: |
A61K 39/001195 20180801;
A61K 2039/53 20130101; A61P 37/04 20180101; A61K 2039/575 20130101;
A61K 2039/70 20130101; A61K 39/00 20130101; A61K 39/0011 20130101;
A61K 2039/572 20130101; A61K 39/39 20130101; A61P 13/08 20180101;
A61P 35/00 20180101; A61K 39/001194 20180801; A61K 39/001193
20180801; A61K 2039/55516 20130101 |
Class at
Publication: |
514/44.R ;
536/23.5 |
International
Class: |
A61K 31/711 20060101
A61K031/711; C07H 21/02 20060101 C07H021/02; A61P 35/00 20060101
A61P035/00; A61P 37/04 20060101 A61P037/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 2007 |
EP |
PCT EP2007 008771 |
Claims
1. Active (immunostimulatory) composition comprising at least one
RNA, encoding at least two, three or four different antigens, a)
wherein at least one antigen is selected from: STEAP (Six
Transmembrane Epithelial Antigen of the Prostate); and b) wherein
the further antigen(s) is (are) selected from at least one antigen
of any of the following antigens or specific combinations thereof:
PSA (Prostate-Specific Antigen), or PSMA (Prostate-Specific
Membrane Antigen), or PSCA (Prostate Stem Cell Antigen); or PSA and
PSMA, or PSA and PSCA, or PSMA and PSCA; or PSA, PSMA and PSCA.
2. Active (immunostimulatory) composition according to claim 1
comprising at least one RNA, encoding four different antigens
selected from PSA, PSMA, PSCA and STEAP.
3. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA comprises a length of 250 to 20000
nucleotides.
4. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA is an mRNA.
5. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA is a monocistronic, bicistronic or
even multicistronic RNA.
6. Active (immunostimulatory) composition according to claim 5,
wherein the at least two antigens are each encoded by a
monocistronic RNA.
7. Active (immunostimulatory) composition according to claim 5,
wherein the at least two antigens are encoded by a bi- or
multicistronic RNA.
8. Active (immunostimulatory) composition according to claim 5,
wherein the at least two antigens are encoded by a mixture of
monocistronic, bicistronic and/or even multicistronic RNAs.
9. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA comprises a RNA sequence encoding a
fragment, a variant or an epitope of an antigen as defined in claim
1.
10. Active (immunostimulatory) composition according to claim 1,
wherein at least one RNA comprises an RNA selected from RNAs being
identical or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%
identical to the RNA sequences of FIG. 2, 5, 8, or 11 (SEQ ID NOs:
2, 5, 8, or 11).
11. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA is a modified RNA, in particular a
stabilized mRNA.
12. Active (immunostimulatory) composition according to claim 11,
wherein the G/C content of the coding region of the at least one
RNA is increased compared to the G/C content of the coding region
of the wild-type RNA, the coded amino acid sequence of the at least
one RNA preferably not being modified compared to the coded amino
acid sequence of the wild-type RNA.
13. Active (immunostimulatory) composition according to claim 11,
wherein the A/U content in the environment of the ribosome binding
site of the at least one RNA is increased compared with the A/U
content in the environment of the ribosome binding site of the
wild-type RNA.
14. Active (immunostimulatory) composition according to claim 11,
wherein the coding region and/or the 5' and/or 3' untranslated
region of the modified mRNA is modified compared to the wild-type
RNA such that it contains no destabilizing sequence elements, the
coded amino acid sequence of the modified mRNA preferably not being
modified compared to the wild-type RNA.
15. Active (immunostimulatory) composition according to claim 11,
wherein the modified mRNA has a 5' cap structure and/or a poly(A)
tail, preferably of 10 to 200 adenosine nucleotides, and/or a
poly(C) tail, preferably of 10 to 200 cytosine nucleotides, and/or
at least one IRES and/or at least one 5' and/or 3' stabilizing
sequence.
16. Active (immunostimulatory) composition according to claim 1,
wherein at least one RNA comprises an RNA selected from RNAs being
identical or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%
identical to the RNA sequences of FIG. 1, 3, 4, 6, 7, 9, 10 or 12
(SEQ ID NOs: 1, 3, 4, 6, 7, 9, or 12).
17. Active (immunostimulatory) composition according to claim 1,
wherein the at least one RNA is complexed with one or more
polycations, preferably with protamine or oligofectamine, most
preferably with protamine.
18. Active (immunostimulatory) composition according to claim 1,
wherein the active composition additionally comprises at least one
adjuvant.
19. Active (immunostimulatory) composition according to claim 18,
wherein the at least one adjuvant is selected from the group
consisting of: cationic or polycationic compounds, comprising
cationic or polycationic peptides or proteins, including protamine,
nucleoline, spermin or spermidine, poly-L-lysine (PLL),
poly-arginine, basic polypeptides, cell penetrating peptides
(CPPs), including HIV-binding peptides, Tat, 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, pIsI, FGF, Lactoferrin,
Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived
peptides, SAP, protamine, spermine, spermidine, or histones,
cationic polysaccharides, including chitosan, polybrene, cationic
polymers, including polyethyleneimine (PEI), cationic lipids,
including DOTMA:
[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,
DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, 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:
rae-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,
CLIP9:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylamm-
onium, oligofectamine, or cationic or polycationic polymers,
including modified polyaminoacids, including
.beta.-aminoacid-polymers or reversed polyamides, modified
polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridinium
bromide)), modified acrylates, including pDMAEMA
(poly(dimethylaminoethyl methylacrylate)), modified Amidoamines
including pAMAM (poly(amidoamine)), modified polybetaminoester
(PBAE), including diamine end modified 1,4 butanediol
diacrylate-co-5-amino-1-pentanol polymers, dendrimers, including
polypropylamine dendrimers or pAMAM based dendrimers, polyimine(s),
including PEI: poly(ethyleneimine), poly(propyleneimine),
polyallylamine, sugar backbone based polymers, including
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 selected of a cationic polymer as mentioned before,
and of one or more hydrophilic- or hydrophobic blocks (e.g
polyethyleneglycole); or cationic or polycationic proteins or
peptides, selected from following proteins or peptides having the
following total formula (I): (Arg).sub.1; (Lys).sub.m; (His).sub.n;
(Orn).sub.o; (Xaa).sub.x, 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;
or nucleic acids having the formula (II): G.sub.lX.sub.mG.sub.n,
wherein: G is guanosine, uracil or an analogue of guanosine or
uracil; X is guanosine, uracil, adenosine, thymidine, cytosine or
an analogue of the above-mentioned nucleotides; l is an integer
from 1 to 40, wherein when l=1 G is guanosine or an analogue
thereof, when l>1 at least 50% of the nucleotides are guanosine
or an analogue thereof; m is an integer and is at least 3; wherein
when m=3.times. is uracil or an analogue thereof, when m>3 at
least 3 successive uracils or analogues of uracil occur; n is an
integer from 1 to 40, wherein when n=1 G is guanosine or an
analogue thereof, when n>1 at least 50% of the nucleotides are
guanosine or an analogue thereof; or nucleic acids having the
formula (III): C.sub.lX.sub.mC.sub.n, wherein: C is cytosine,
uracil or an analogue of cytosine or uracil; X is guanosine,
uracil, adenosine, thymidine, cytosine or an analogue of the
above-mentioned nucleotides; l is an integer from 1 to 40, wherein
when l=1 C is cytosine or an analogue thereof, when l>1 at least
50% of the nucleotides are cytosine or an analogue thereof; m is an
integer and is at least 3; wherein when m=3.times. is uracil or an
analogue thereof, when m>3 at least 3 successive uracils or
analogues of uracil occur; n is an integer from 1 to 40, wherein
when n=1 C is cytosine or an analogue thereof, when n>1 at least
50% of the nucleotides are cytosine or an analogue thereof.
20. Vaccine, comprising an active (immunostimulatory) composition
according to claim 1.
21. Vaccine according to claim 20, wherein the active
(immunostimulatory) composition elicits an adaptive immune
response.
22. Vaccine according to claim 20, wherein the vaccine further
comprises a pharmaceutically acceptable carrier.
23. Vaccine according to claim 21, wherein the vaccine further
comprises a pharmaceutically acceptable carrier.
24. Use of an active (immunostimulatory) composition according to
claim 1 for preparing a vaccine for the treatment of prostate
cancer (PCa), preferably of neoadjuvant and/or hormone-refractory
prostate cancers, and diseases or disorders related thereto.
25. Kit, preferably kit of parts, comprising the active
(immunostimulatory) composition according to claim 1, and
optionally technical instructions with information on the
administration and dosage of the active composition and/or the
vaccine.
Description
[0001] The present invention relates to an active
(immunostimulatory) composition comprising at least one RNA,
preferably an mRNA, encoding at least two (preferably different)
antigens capable of eliciting an (adaptive) immune response in a
mammal, wherein the antigens are selected from the group consisting
of PSA (Prostate-Specific Antigen), PSMA (Prostate-Specific
Membrane Antigen), PSCA (Prostate Stem Cell Antigen), and STEAP
(Six Transmembrane Epithelial Antigen of the Prostate). The
invention furthermore relates to a vaccine comprising said active
(immunostimulatory) composition, and to the use of said active
(immunostimulatory) composition (for the preparation of a vaccine)
and/or of the vaccine for eliciting an (adaptive) immune response
for the treatment of prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto. Finally, the invention
relates to kits, particularly to kits of parts, containing the
active (immunostimulatory) composition and/or the vaccine.
[0002] At present, prostate cancer is the second most commonly
diagnosed cancer and the fourth leading cause of cancer-related
death in men in the developed countries worldwide. Effective
curative treatment modalities are debilitating, and are only
currently available for localised disease. In hormone-refractory
prostate cancer, no agent has been shown to prolong survival beyond
approximately 1 year (see e.g. Pavlenko, M., A. K. Roos, et al
(2004). "A phase I trial of DNA vaccination with a plasmid
expressing prostate-specific antigen in patients with
hormone-refractory prostate cancer." Br J Cancer 91(4): 688-94.).
In some highly developed western countries such as the United
States of America, prostate cancer is at present even the most
commonly diagnosed malignancy and the third leading cause of cancer
related death among men in the United States (see e.g. Jemal, A.,
R. Siegel, et al. (2006). "Cancer statistics, 2006." CA Cancer J
Clin 56(2): 106-30.) and in Europe, respectively (see e.g.
Thomas-Kaskel, A. K., C. F. Waller, et al. (2007). "Immunotherapy
with dendritic cells for prostate cancer." Int J Cancer 121(3):
467-73). Most of the diagnosed tumors are adeno-carcinomas which
initially proliferate in a hormone-dependent manner. Prostate
cancer is a disease in which cancer develops in the prostate, a
gland in the male reproductive system. It occurs when cells of the
prostate mutate and begin to multiply out of control. Typical
antigens which have been shown to be overexpressed by prostate
cancer cells as compared to normal counterparts are inter alia
antigens like PSA, PSMA, PAP, PSCA, HER-2 and Ep-CAM. These
prostate cancer cells may spread (metastasize) from the prostate to
other parts of the body, especially the bones and lymph nodes.
Prostate cancer may cause pain, difficulty in urinating, erectile
dysfunction and other symptoms. Typically, prostate cancer develops
most frequently in men over fifty, which represent the most common
group of patients. However, prostate cancer remains most often
undiscovered, even if determination would be possible.
Determination of prostate cancer typically occurs by physical
examination or by screening blood tests, such as the PSA (prostate
specific antigen) test. When suspected to prostate cancer the
cancer is typically confirmed by removing a piece of the prostate
(biopsy) and examining it under a microscope. Further tests, such
as X-rays and bone scans, may be performed to determine whether
prostate cancer has spread.
[0003] Treatment of prostate cancer still remains an unsolved
challenge. Conventional therapy methods may be applied for
treatment of prostate cancer such as surgery, radiation therapy,
hormonal therapy, occasionally chemotherapy, proton therapy, or
some combination of these. However, the age and underlying health
of the man as well as the extent of spread, appearance under the
microscope, and response of the cancer to initial treatment are
important in determining the outcome of the disease. Since prostate
cancer is a disease, typically diagnosed with respect to older men,
many will die of other causes before a slowly advancing prostate
cancer can spread or cause symptoms. This makes treatment selection
difficult. The decision whether or not to treat localized prostate
cancer (a tumor that is contained within the prostate) with
curative intent is a patients trade-off between the expected
beneficial and harmful effects in terms of patient survival and
quality of life.
[0004] However, the above therapy methods, such as surgery,
radiation therapy, hormonal therapy, and chemotherapy, etc., all
suffer from severe limitations. By way of example, surgical removal
of the prostate, or prostatectomy, is a common treatment either for
early stage prostate cancer or for cancer which has failed to
respond to radiation therapy. It may cause nerve damage that
significantly alters the quality of life. The most common serious
complications are loss of urinary control and impotence. However,
even if the prostate cancer could be removed successfully, spread
of prostate cancer throughout the organism remains an unsolved
problem.
[0005] Radiation therapy is commonly used in prostate cancer
treatment. It may be used instead of surgery for early cancers, and
it may also be used in advanced stages of prostate cancer to treat
painful bone metastases. Radiation treatments also can be combined
with hormonal therapy for intermediate risk disease, when radiation
therapy alone is less likely to cure the cancer. However, radiation
therapy also bears high risks and often leads to a complete loss of
immune defence due to destruction of the patients immune system.
Furthermore, radiation therapy is typically applied locally at the
site of cancer growth and thus may not avoid the above problem of
spread of prostate cancer throughout the organism. If applied
systemically, radiation therapy may lead to severe damages to cells
and immune system.
[0006] Chemotherapy was a long time considered as a less effective
sort of treatment for prostate cancers since only very few patients
even respond to this sort of therapy. However, some patients
(responders), having a metastasizing prostate carcinoma, may
benefit from chemotherapy. The response rate is at about 20% and
chemotherapy will thus play a role during treatment of the tumor
relapse and failing of hormonal therapy. However, chemotherapy will
typically be only palliative and does not lead to a total
elimination of the prostate cancer in the patient. Typical
chemotherapeutic agents include cyclophosphamid, doxorubicin,
5-fluoruracil, adriamycin, suramin and other agents, however, none
of these allowed a significant longer survival of the patients. In
a recent study, published 2004 in the New England Journal of
Medicine, longer survival of median 2.5 months could be
demonstrated for patients which received a dosis of the agent
docetaxel every three weeks.
[0007] Hormonal therapy typically uses medications or a combination
of hormonal therapy with surgery to block prostate cancer cells
from getting dihydrotestosterone (DHT), a hormone produced in the
prostate and required for the growth and spread of most prostate
cancer cells. Blocking DHT often causes prostate cancer to stop
growing and even shrink. However, hormonal therapy rarely cures
prostate cancer because cancers which initially respond to hormonal
therapy typically become resistant after one to two years. E.g.
palliative androgen deprivation therapy can induce remissions in up
to 80% of the patients, but after 15-20 months, tumor cells become
hormone-insensitive and androgen-independent prostate cancer
develops. In this situation treatment options are rare, as
chemotherapy has been of limited efficacy (see above). Hormonal
therapy is therefore usually used when cancer has spread from the
prostate. It may also be given to certain men undergoing radiation
therapy or surgery to help prevent return of their cancer.
[0008] So for this the need for alternative treatment strategies
for patients with normal and also with relapsed or advanced
prostate cancer is high. As one approach, the above discussed
standard therapies used for organ-confined prostate cancer,
including radical prostatectomy or radiation therapy such as
external (beam) irradiation and brachytherapy may under some
circumstances incorporate also neoadjuvant or adjuvant hormonal
therapy (see e.g. Totterman, T. H., A. Loskog, et al. (2005). "The
immunotherapy of prostate and bladder cancer." BJU Int 96(5):
728-35.). While these therapies are relatively effective in the
short term, a significant proportion (30-40%) of patients having
initially localized disease will ultimately relapse. For metastatic
prostate cancer the main therapy is androgen ablation. While this
usually provides cytoreduction and palliation, progression to
hormone-refractory disease typically occurs within 14-20 months.
Many clinical studies have been reported in the field of
chemotherapy for advanced androgen-independent prostate cancer.
Only recently two trials have revealed that chemotherapy marginally
improves the overall survival of patients with hormone-refractory
disease.
[0009] Summarizing the above, standard techniques such as the above
mentioned surgery, radiation therapy, hormonal therapy,
occasionally chemotherapy, proton therapy, etc., if applied alone,
do not appear to be suitable for an efficient treatment of prostate
cancer (PCa). One improved way of treatment may therefore include
such treatments or supplementary treatments, which can complement
these standard techniques. Thus, it is suggested here to use the
adaptive immune system in an approach for the treatment or
supplementary treatment of prostate cancer (PCa).
[0010] As known in the art, the immune system plays an important
role in the treatment and prevention of numerous diseases.
According to the present stage of knowledge, various mechanisms are
provided by mammalians to protect the organism by identifying and
killing, e.g., tumor cells. For the purposes of the present
invention, these tumor cells have to be detected and distinguished
from the organism's normal (healthy) cells and tissues.
[0011] The immune systems of vertebrates such as humans consist of
many types of proteins, cells, organs, and tissues, which interact
in an elaborate and dynamic network. As part of this complex immune
response, the vertebrate system adapts over time to recognize
particular pathogens or tumor cells more efficiently. The
adaptation process creates immunological memories and allows even
more effective protection during future encounters. This process of
adaptive or acquired immunity forms the basis for vaccination
strategies.
[0012] The adaptive immune system is antigen-specific and requires
the recognition of specific "self" or "non-self" antigens during a
process called antigen presentation. Antigen specificity allows for
the generation of responses that are tailored to specific pathogens
or pathogen-infected cells or tumor cells. The ability to mount
these tailored responses is maintained in the body by so called
"memory cells". Should a pathogen infect the body more than once,
these specific memory cells are used to quickly eliminate it. The
adaptive immune system thus allows for a stronger immune response
as well as for an immunological memory, where each pathogen or
tumor cell is "remembered" by one or more signature antigens.
[0013] The major components of the adaptive immune system in
vertebrates predominantly include lymphocytes on the cellular level
and antibodies on the molecular level. Lymphocytes as cellular
components of the adaptive immune system include B cells and T
cells which are derived from hematopoietic stem cells in the bone
marrow. B cells are involved in the humoral response, whereas T
cells are involved in cell mediated immune response. Both B cells
and T cells carry receptor molecules that recognize specific
targets. T cells recognize a "non-self" target, such as a
pathogenic target structure, only after antigens (e.g. small
fragments of a pathogen) have been processed and presented in
combination with a "self" receptor called a major
histocompatibility complex (MHC) molecule. In contrast, the B cell
antigen-specific receptor is an antibody molecule on the B cell
surface, and recognizes pathogens as such when antibodies on its
surface bind to a specific foreign antigen. This antigen/antibody
complex is taken up by the B cell and processed by proteolysis into
peptides. The B cell then displays these antigenic peptides on its
surface MHC class II molecules. This combination of MHC and antigen
attracts a matching helper T cell, which releases lymphokines and
activates the B cell. As the activated B cell then begins to
divide, its offspring secretes millions of copies of the antibody
that recognizes this antigen. These antibodies circulate in blood
plasma and lymph, bind to pathogens or tumor cells expressing the
antigen and mark them for destruction by complement activation or
for uptake and destruction by phagocytes. As a cellular component
of the adaptive immune system cytotoxic T cells (CD8.sup.+) may
also form a CTL-response. Cytotoxic T cells (CD8.sup.+) can
recognize peptides from endogenous pathogens and self-antigens
bound by MHC type I molecules. CD8.sup.+-T cells carry out their
killing function by releasing cytotoxic proteins in the cell.
[0014] Mechanisms of the immune system may thus form targets for
curative treatments of various diseases. Appropriate methods are
typically based on the administration of adjuvants to elicit an
innate immune response or on the administration of antigens or
immunogens in order to evoke an adaptive immune response. As
antigens are typically based on specific components of pathogens
(e.g. surface proteins) or fragments thereof, administration of
nucleic acids to the patient which is followed by the expression of
desired polypeptides, proteins or antigens is envisaged as
well.
[0015] As an example, vaccination studies based on known prostate
related antigens have been carried out in Noguchi et al. (2003) and
(2004) (see e.g. Noguchi, M., K. Itoh, et al. (2004). "Phase I
trial of patient-oriented vaccination in HLA-A2-positive patients
with metastatic hormone-refractory prostate cancer." Cancer Sci
95(1): 77-84; and Noguchi, M., K. Kobayashi, et al. (2003).
"Induction of cellular and humoral immune responses to tumor cells
and peptides in HLA-A24 positive hormone-refractory prostate cancer
patients by peptide vaccination." Prostate 57(1): 80-92. Noguchi et
al. 2003 and Noguchi et al. 2004). Noguchi et al. (2003) and (2004)
carried out two phase I studies with a multipeptide trial of
vaccination in metastatic hormone-resistant prostate cancer
patients showing increased cellular as well as humoral immune
responses to the selected targets. The vaccination strategy was
safe, well tolerated with no major toxic effects. However,
stabilization or reduction of prostate specific antigen (PSA)
levels was also observed and only one patient showed disappearance
of a bone metastasis. The main limitation of this approach that
makes it difficult for clinical applications relies on the need of
a priori knowledge of the patient's HLA haplotype as well as of
peptide expression by prostate cancer cells.
[0016] Some other recent approaches utilize cell based vaccination
strategies, e.g. the use of different antigens in vaccination
strategies or the use of dendritic cells loaded with different
antigens or fragments thereof. According to one example,
vaccination of prostate cancer patients has been tested in clinical
trials with autologous dendritic cells pulsed with recombinant
human PSA (see e.g. Barrou, B., G. Benoit, et al. (2004).
"Vaccination of prostatectomized prostate cancer patients in
biochemical relapse, with autologous dendritic cells pulsed with
recombinant human PSA." Cancer Immunol Immunother 53(5): 453-60).
During vaccination of advanced prostate cancer patients with PSCA
and PSA peptide-loaded dendritic cells 5 from 10 patients showed an
immune response against at least one antigen (see e.g.
Thomas-Kaskel, A. K., R. Zeiser, et al. (2006). "Vaccination of
advanced prostate cancer patients with PSCA and PSA peptide-loaded
dendritic cells induces DTH responses that correlate with superior
overall survival." Int J Cancer 119(10): 2428-34.).
[0017] In another example, Murphy et al. (1996) carried out
vaccination of prostate cancer patients in a corresponding phase I
trial with two HLA-A*0201 PSMA epitopes to compare vaccination with
peptide alone or with pulsed DCs. The results showed that more
patients responded to the vaccination, if the patients were
vaccinated with pulsed DCs. This study showed that vaccination with
DCs loaded with peptides or proteins leads at least partially to
detectable immune responses as well as a temporary PSC declines or
stabilization (see e.g. Murphy, G., B. Tjoa, et al. (1996). "Phase
I clinical trial: T-cell therapy for prostate cancer using
autologous dendritic cells pulsed with HLA-A0201-specific peptides
from prostate-specific membrane antigen." Prostate 29(6):
371-80).
[0018] Vaccination of prostate cancer patients may also be carried
out with combinations of peptides loaded on dendritic cells, e.g.
with peptide cocktail-loaded dendritic cells (see e.g. Fuessel, S.,
A. Meye, et al. (2006). "Vaccination of hormone-refractory prostate
cancer patients with peptide cocktail-loaded dendritic cells:
results of a phase I clinical trial." Prostate 66(8): 811-21). The
cocktail contained peptides from PSA, PSMA, Survivin, Prostein and
Trp-p8 (transient receptor potential p8). Clinical trials were also
carried out with an dendritic cell-based multi-epitope
immunotherapy of hormone-refractory prostate carcinoma (see e.g.
Waeckerle-Men, Y., E. Uetz-von Allmen, et al. (2006). "Dendritic
cell-based multi-epitope immunotherapy of hormone-refractory
prostate carcinoma." Cancer Immunol Immunother 55(12): 1524-33).
Waeckerle-Men, Y., E. Uetz-von Allmen, et al. (2006) tested
vaccination of hormone-refractory prostate carcinoma with peptides
from PSCA, PAP (prostatic acid phosphatase), PSMA and PSA.
[0019] While vaccination with antigenic proteins or peptides, e.g.
when loaded on dendritic cells, is a common method for eliciting an
immune response, immunization or vaccination may also be based on
the use of nucleic acids in order to incorporate the required
genetic information into the cell. In general, various methods have
been developed for introducing nucleic acids into cells, such as
calcium phosphate transfection, polyprene transfection, protoplast
fusion, electroporation, microinjection and lipofection,
lipofection having in particular proven to be a suitable
method.
[0020] According to one example vaccination treatment of prostate
cancer may be based on the transfection of total mRNA derived from
the autologous tumor into DCs (see Heiser et al. (2002) (see e.g.
Heiser, A., D. Coleman, et al. (2002). "Autologous dendritic cells
transfected with prostate-specific antigen RNA stimulate CTL
responses against metastatic prostate tumors." J Clin Invest
109(3): 409-17.). This strategy has the advantage of targeting
multiple HLA class I and class II patient specific tumor associated
antigens (TAAs) without prior HLA typing. Moreover, even stromal
antigens could be targeted by this strategy since mRNA was obtained
from surgical samples and not from tumor cell lines. As an example,
Heiser et al. developed a DC-based immunotherapy protocol in which
DCs were transfected with mRNA encoding PSA. The vaccination was
well tolerated and induced an increased T cell response to PSA.
However, such DC-based anti prostate cancer vaccines appear to
generate a strong T cell response, which may be accompanied by
clinical response though the frequency of the latter still remains
unsatisfactory
[0021] DNA may also be utilized as a nucleic acid in vaccination
strategies in order to incorporate the required genetic information
into the cell. According to a specific example, DNA viruses may be
used as a DNA vehicle. Because of their infectious properties, such
viruses achieve a very high transfection rate. The viruses used are
genetically modified in such a manner that no functional infectious
particles are formed in the transfected cell. E.g., phase I trials
were carried out in a study of Eder et al. (2000) using recombinant
vaccinia viruses expressing PSA. The authors demonstrated T cell
immune responses to PSA and also serum PSA stabilizations in
selected patients. (see e.g. Eder, J. P., P. W. Kantoff, et al.
(2000). "A phase I trial of a recombinant vaccinia virus expressing
prostate-specific antigen in advanced prostate cancer." Clin Cancer
Res 6(5): 1632-8.). The inflammatory response triggered by the
highly immunogenic peptides from the recombinant virus may enhance
the immunogenicity of the foreign protein but it was shown that the
immune system reduces the replication of the recombinant virus and
thereby limits the clinical outcome. Even though recombinant
vaccines have shown immunogenicity and evidence for a tumor
response was shown in several trials, these results, however, need
to be substantiated.
[0022] According to a further approach, vaccination of
hormone-refractory prostate cancer patients was carried out with
DNA plasmids expressing PSA (see e.g. Pavlenko, M., A. K. Roos, et
al. (2004). "A phase I trial of DNA vaccination with a plasmid
expressing prostate-specific antigen in patients with
hormone-refractory prostate cancer." Br J Cancer 91(4): 688-94).
Garcia-Hernandez et al. (2007) showed that therapeutic and
prophylactic vaccination with a plasmid or a virus-like replicon
coding for STEAP (Six Transmembrane Epithelial Antigen of the
Prostate) prolonged the survival in tumor-challenged mice (see e.g.
Garcia-Hernandez Mde, L., A. Gray, et al. (2007). "In vivo effects
of vaccination with six-transmembrane epithelial antigen of the
prostate: a candidate antigen for treating prostate cancer." Cancer
Res 67(3): 1344-51). Recently STEAP was identified as indicator
protein for advanced human prostate cancer, which is highly
overexpressed in human prostate cancer. Its function is currently
unknown.
[0023] While using DNA as a carrier of genetic information, it is,
however, not possible to rule out the risk of uncontrolled
propagation of the introduced gene or of viral genes, for example
due to potential recombination events. This also entails the risk
of the DNA being inserted into an intact gene of the host cell's
genome by e.g. recombination, with the consequence that this gene
may be mutated and thus completely or partially inactivated or the
gene may give rise to misinformation. In other words, synthesis of
a gene product which is vital to the cell may be completely
suppressed or alternatively a modified or incorrect gene product is
expressed. One particular risk occurs if the DNA is integrated into
a gene which is involved in the regulation of cell growth. In this
case, the host cell may become degenerate and lead to cancer or
tumor formation. Furthermore, if the DNA introduced into the cell
is to be expressed, it is necessary for the corresponding DNA
vehicle to contain a strong promoter, such as the viral CMV
promoter. The integration of such promoters into the genome of the
treated cell may result in unwanted alterations of the regulation
of gene expression in the cell. Another risk of using DNA as an
agent to induce an immune response (e.g. as a vaccine) is the
induction of pathogenic anti-DNA antibodies in the patient into
whom the foreign DNA has been introduced, so bringing about a
(possibly fatal) immune response.
[0024] Summarizing the results of the above approaches it is
doubtless that some improvement is achieved for the treatment of
prostate cancer (PCa), even though there is still--given the high
mortality rates--a strong need for further, alternative or improved
ways of treatment.
[0025] Thus overall, there is room and a need for an efficient
system, which may be used to effectively stimulate the immune
system to allow treatment of prostate cancer (PCa), while avoiding
the problems of uncontrolled propagation of an introduced gene due
to DNA based compositions.
[0026] It is thus an object of the present invention to provide a
composition, which a) allows treatment of prostate cancer (PCa) by
stimulating the immune system, while b) avoiding the above
mentioned disadvantages.
[0027] This object is solved by the subject matter of the present
invention, particularly by an active (immunostimulatory)
composition comprising at least one RNA, encoding at least two,
three or even four (preferably different) antigens selected from
the group comprising the following antigens: [0028] PSA
(Prostate-Specific Antigen)=KLK3 (Kallikrein-3), [0029] PSMA
(Prostate-Specific Membrane Antigen), [0030] PSCA (Prostate Stem
Cell Antigen), [0031] STEAP (Six Transmembrane Epithelial Antigen
of the Prostate).
[0032] Surprisingly, it has been found that a specific combination
of at least two antigens, antigenic proteins or antigenic peptides
of the afore mentioned group, preferably of two, three or four of
these antigens, antigenic proteins or antigenic peptides, as
contained in an active (immunostimulatory) composition according to
the present invention, is capable to effectively stimulate the
(adaptive) immune system to allow treatment of prostate cancer
(PCa), preferably of neoadjuvant and/or hormone-refractory prostate
cancers, and diseases or disorders related thereto. Herein, the
terms antigens, antigenic proteins or antigenic peptides may be
used synomously. In the context of the present invention, an
inventive active (immunostimulatory) composition shall be further
understood as a composition, which is able to elicit an immune
response, preferably an adaptive immune response as defined herein,
due to one of the component(s) contained or encoded by the
components of the active (immunostimulatory) composition,
preferably by the at least one RNA, preferably (m)RNA, encoding the
at least two (preferably different) antigens.
[0033] Antigens like PSA, PSMA and PSCA have been shown to be
overexpressed by prostate cancer cells as compared to normal
counterparts. These antigens therefore represent possible targets
of immunotherapy (see e.g. Marrari, A., M. Iero, et al. (2007).
"Vaccination therapy in prostate cancer." Cancer Immunol Immunother
56(4): 429-45.)
[0034] The at least one RNA of the active (immunostimulatory)
composition may be PSA. In the context of this invention "PSA" is
"Prostate-specific antigen" and may be synomously named KLK3
(Kallikrein-3) in the literature. Prostate-specific antigen (PSA)
is a 33 kDa protein and an androgen-regulated kallikrein-like,
serine protease that is produced exclusively by the epithelial
cells of all types of prostatic tissue, benign and malignant.
Particularly, PSA is highly expressed by normal prostatic
epithelial cells and represents one of the most characterized tumor
associated antigens in prostate cancer. Physiologically, it is
present in the seminal fluid at high concentration and functions to
cleave the high molecular weight protein responsible for the
seminal coagulum into smaller polypeptides. This action results in
liquefaction of the coagulum. PSA is also present in the serum and
can be measured reliably by either a monoclonal immunoradiometric
assay or a polyclonal radioimmunoassay. PSA is the most widely used
tumor marker for screening, diagnosing, and monitoring prostate
cancer today. In particular, several immunoassays for the detection
of serum PSA are in widespread clinical use. Recently, a reverse
transcriptase-polymerase chain reaction (RT-PCR) assay for PSA mRNA
in serum has been developed. However, PSA is not recognized as a
disease-specific marker, as elevated levels of PSA are detectable
in a large percentage of patients with BPH and prostatitis (25-86%)
(Gao et al, 1997, Prostate 31 : 264-281), as well as in other
nonmalignant disorders and in some normal men, a factor which
significantly limits the diagnostic specificity of this marker. In
the context of this invention the preferred sequence of the at
least one RNA, preferably of the mRNA, encoding PSA (prostate
specific antigen)--if being used in the active (immunostimulatory)
composition according to the invention--contains or comprises a
sequence as deposited under accession number NM.sub.--001648, more
preferably, it contains or comprises a sequence as shown in FIG. 1
(SEQ ID NO: 1), and--even more preferably--the at least one RNA, if
encoding PSA (prostate specific antigen), contains or comprises a
coding sequence as shown in any of FIG. 2 or 3 (SEQ ID NOs: 2 or
3). According to a further preferred embodiment, the at least one
RNA of the active (immunostimulatory) composition may alternatively
or additionally encode a PSA antigen sequence selected from a
fragment, a variant or an epitope of a PSA sequence as deposited
under accession number NM.sub.--001648 or as shown in any of FIG.
1, 2 or 3 (SEQ ID NOs: 1, 2 or 3).
[0035] The at least one RNA of the active (immunostimulatory)
composition may be PSMA. In the context of this invention "PSMA" is
"Prostate-specific membrane antigen" and may be synomously named
FOLH1 (Folate hydrolase 1) or "PSM". PSMA is a 100 kDa type II
transmembrane glycoprotein, wherein PSMA expression is largely
restricted to prostate tissues, but detectable levels of PSMA mRNA
have been observed in brain, salivary gland, small intestine, and
renal cell carcinoma (Israeli et al., 1993, Cancer Res 53 :
227-230). PSMA is highly expressed in most primary and metastatic
prostate cancers, but is also expressed in most normal
intraepithelial neoplasia specimens (Gao et al. (1997), supra).
Particularly, PSMA is highly expressed in prostate cancer cells and
nonprostatic solid tumor neovasculature and is a target for
anticancer imaging and therapeutic agents. PSMA acts as a glutamate
carboxypeptidase (GCPII) on small molecule substrates, including
folate, the anticancer drug methotrexate, and the neuropeptide
N-acetyl-L-aspartyl-L-glutamate. In prostate cancer, PSMA
expression has been shown to correlate with disease progression,
with highest levels expressed in hormone-refractory and metastatic
disease. The cellular localization of PSMA is cytoplasmic and/or
membranous. PSMA is considered a biomarker for prostate cancer
(PCa) and is under intense investigation for use as an imaging and
therapeutic target. In the context of this invention the preferred
sequence of the at least one RNA, preferably of the mRNA, encoding
PSMA (prostate specific membrane antigen)--if being used in the
active (immunostimulatory) composition according to the
invention--contains or comprises a sequence as deposited under
accession number NM.sub.--004476, more preferably, it contains or
comprises a sequence as shown in FIG. 4 (SEQ ID NO: 4), and--even
more preferably--the at least one RNA, if encoding PSMA (prostate
specific antigen), contains or comprises a coding sequence as shown
in any of FIG. 5 or 6 (SEQ ID NO: 5 or 6). According to a further
preferred embodiment, the at least one RNA of the active
(immunostimulatory) composition may alternatively or additionally
encode a PSMA antigen sequence selected from a fragment, a variant
or an epitope of a PSMA sequence as deposited under accession
number NM.sub.--004476 or as shown in any of FIG. 5 or 6 (SEQ ID
NOs: 5 or 6).
[0036] The at least one RNA of the active (immunostimulatory)
composition may be PSCA. In the context of this invention "PSCA" is
"prostate stem cell antigen". PSCA is widely over-expressed across
all stages of prostate cancer, including high grade prostatic
intraepithelial neoplasia (PIN), androgen-dependent and
androgen-independent prostate tumors. The PSCA gene shows 30%
homology to stem cell antigen-2, a member of the Thy-I/Ly-6 family
of glycosylphosphatidylinositol (GPI)-anchored cell surface
antigens, and encodes a 123 amino acid protein with an
amino-terminal signal sequence, a carboxy-terminal GPI-anchoring
sequence, and multiple N-glycosylation sites. PSCA mRNA expression
is highly upregulated in both androgen dependent and androgen
independent prostate cancer xenografts. In situ mRNA analysis
localizes PSCA expression to the basal cell epithelium, the
putative stem cell compartment of the prostate. Flow cytometric
analysis demonstrates that PSCA is expressed predominantly on the
cell surface and is anchored by a GPI linkage. Fluorescent in situ
hybridization analysis localizes the PSCA gene to chromosome 8q24.
2, a region of allelic gain in more than 80% of prostate cancers.
PSCA may be used as a prostate cancer marker to discriminate
between malignant prostate cancers, normal prostate glands and
non-malignant neoplasias. For example, PSCA is expressed at very
high levels in prostate cancer in relation to benign prostatic
hyperplasia (BPH). In the context of this invention the preferred
sequence of the at least one RNA, preferably of the mRNA, encoding
PSCA (prostate stem cell antigen)--if being used in the active
(immunostimulatory) composition according to the
invention--contains or comprises a sequence as deposited under
accession number NM.sub.--005672, more preferably, it contains or
comprises a sequence as shown in FIG. 7 (SEQ ID NO: 7), and--even
more preferably--the at least one RNA, if encoding PSCA (prostate
stem cell antigen), contains or comprises a coding sequence as
shown in any of FIG. 8 or 9 (SEQ ID NOs: 8 or 9). According to a
further preferred embodiment, the at least one RNA of the active
(immunostimulatory) composition may alternatively or additionally
encode a PSCA antigen sequence selected from a fragment, a variant
or an epitope of a PSCA sequence as deposited under accession
number NM.sub.--005672 or as shown in any of FIG. 8 or 9 (SEQ ID
NOs: 8 or 9).
[0037] The at least one RNA of the active (immunostimulatory)
composition may be STEAP. In the context of this invention "STEAP"
is six transmembrane epithelial antigen of the prostate and may be
synomously named STEAP1. STEAP or STEAP-1 is a novel cell surface
protein and is expressed predominantly in human prostate tissue and
in other common malignancies including prostate, bladder, colon,
and ovarian carcinomas, and in Ewing's sarcoma, suggesting that it
could function as an almost universal tumor antigen. Particularly,
STEAP is highly expressed in primary prostate cancer, with
restricted expression in normal tissues. STEAP positivity in marrow
samples was highly correlated with survival with new metastasis in
Kaplan Meier analysis (p=0.001). In the context of this invention
the preferred sequence of the at least one RNA, preferably of the
mRNA, encoding STEAP (six transmembrane epithelial antigen of the
prostate) (or STEAP1)--if being used in the active
(immunostimulatory) composition according to the
invention--contains or comprises a sequence as deposited under
accession number NM.sub.--012449, more preferably, it contains or
comprises a sequence as shown in FIG. 10 (SEQ ID NO: 10), and--even
more preferably--the at least one RNA, if encoding STEAP (six
transmembrane epithelial antigen of the prostate), contains or
comprises a coding sequence as shown in any of FIG. 11 or 12 (SEQ
ID NO: 11 or 12). According to a further preferred embodiment, the
at least one RNA of the active (immunostimulatory) composition may
alternatively or additionally encode a STEAP antigen sequence
selected from a fragment, a variant or an epitope of a STEAP
sequence as deposited under accession number NM.sub.--012449 or as
shown in any of FIG. 11 or 12 (SEQ ID NOs: 11 or 12).
[0038] Antigens, antigenic proteins or antigenic peptides as
defined above which may be encoded by the at least one RNA of the
active (immunostimulatory) composition according to the present
invention, may comprise fragments or variants of those sequences.
Such fragments or variants may typically comprise a sequence having
a sequence homology with one of the above mentioned antigens,
antigenic proteins or antigenic peptides or sequences or their
encoding nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, preferably at least 70%, more preferably at least 80%,
equally more preferably at least 85%, even more preferably at least
90% and most preferably at least 95% or even 97%, to the entire
wild-type sequence, either on nucleic acid level or on amino acid
level.
[0039] "Fragments" of antigens, antigenic proteins or antigenic
peptides in the context of the present invention may comprise a
sequence of an antigen, antigenic protein or antigenic peptide as
defined above, which is, with regard to its amino acid sequence (or
its encoded nucleic acid sequence), N-terminally, C-terminally
and/or intrasequentially truncated compared to the amino acid
sequence of the original (native) protein (or its encoded nucleic
acid sequence). Such truncation may thus occur either on the amino
acid level or correspondingly on the nucleic acid level. A sequence
homology with respect to such a fragment as defined above may
therefore preferably refer to the entire antigen, antigenic protein
or antigenic peptide as defined above or to the entire (coding)
nucleic acid sequence of such an antigen, antigenic protein or
antigenic peptide.
[0040] Fragments of antigens, antigenic proteins or antigenic
peptides in the context of the present invention may furthermore
comprise a sequence of an antigen, antigenic protein or antigenic
peptide as defined above, which has 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 6, 7, 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.
[0041] Fragments of antigens, antigenic proteins or antigenic
peptides as defined herein may also comprise epitopes of those
antigens, antigenic proteins or antigenic peptides. Epitopes (also
called "antigen determinants") in the context of the present
invention are typically fragments located on the outer surface of
(native) antigens, antigenic proteins or antigenic peptides 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 or B-cell
receptors, i.e. in their native form. Such epitopes of antigens,
antigenic proteins or antigenic peptides may furthermore be
selected from any of the herein mentioned variants of such
antigens, antigenic proteins or antigenic peptides. In this context
antigenic determinants can be conformational or discontinous
epitopes which are composed of segments of the antigens, antigenic
proteins or antigenic peptides as defined herein that are
discontinuous in the amino acid sequence of the antigens, antigenic
proteins or antigenic 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.
[0042] "Variants" of antigens, antigenic proteins or antigenic
peptides as defined above may be encoded by the at least one RNA of
the active (immunostimulatory) composition according to the present
invention, wherein nucleic acids of the at least one (m)RNA,
encoding the antigen, antigenic protein or antigenic peptide as
defined above, are exchanged. Thereby, an antigen, antigenic
protein or antigenic peptide 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 fragments and/or variants
have the same biological function or specific activity compared to
the full-length native antigen or antigenic protein, e.g. its
specific antigenic property.
[0043] The at least one RNA of the active (immunostimulatory)
composition according to the present invention may also encode an
antigen or an antigenic protein as defined above, wherein the
encoded amino acid sequence comprises conservative amino acid
substitution(s) compared to its physiological sequence. Those
encoded amino acid sequences as well as their encoding nucleotide
sequences in particular fall under the term variants as defined
above. 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).
[0044] Furthermore, variants of antigens, antigenic proteins or
antigenic peptides as defined above, which may be encoded by the at
least one RNA of the active (immunostimulatory) composition
according to the present invention, may also comprise those
sequences, wherein nucleic acids of the at least one (m)RNA are
exchanged according to the degeneration of the genetic code,
without leading to an alteration of respective amino acid sequence
of the antigen, antigenic protein or antigenic 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.
[0045] In order to determine the percentage to which two sequences
(nucleic acid sequences, e.g. RNA or mRNA sequences as defined
herein, or amino acid sequences, preferably their encoded amino
acid sequences, e.g. the amino acid sequences of the antigens,
antigenic proteins or antigenic peptides as defined above) are
identical, the sequences can be aligned in order to be subsequently
compared to one another. Therefore, e.g. gaps can be inserted into
the sequence of the first sequence and the component at the
corresponding position of the second sequence can be compared. If a
position in the first sequence is occupied by the same component as
is the case at a position in the second sequence, the two sequences
are identical at this position. The percentage to which two
sequences are identical is a function of the number of identical
positions divided by the total number of positions. 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.
[0046] The active (immunostimulatory) composition according to the
present invention comprises, as defined above, at least one RNA,
encoding least two (preferably different) antigens selected from
any of the antigens of the above group, since according to the
invention a specific combination of at least two (preferably
different) antigens of the afore mentioned group is capable to
effectively stimulate the (adaptive) immune system to allow
treatment of prostate cancer (PCa). However, the present invention
may also provide such active (immunostimulatory) compositions,
comprising at least one RNA, encoding three or four (preferably
different) antigens selected from any of the antigens of the above
group, wherein any combination of these antigens is possible and
encompassed by the present invention.
[0047] More preferably, the present invention may also provide an
active (immunostimulatory) composition, comprising at least one
RNA, encoding at least three or four (preferably different)
antigens selected from any of the antigens of the above group,
wherein any combination of these antigens is possible.
[0048] Accordingly, due to another particularly preferred
embodiment, the at least one RNA of the active (immunostimulatory)
composition of the present invention, may encode at least two
(preferably different) antigens selected from any of the antigens
of the above mentioned group comprising (at least) any one of the
following combinations of antigens: [0049] PSA and PSMA, or [0050]
PSA and PSCA, or [0051] PSA and STEAP, or [0052] PSMA and PSCA, or
[0053] PSMA and STEAP, or [0054] PSCA and STEAP, [0055] or [0056]
PSA, PSMA and PSCA, or [0057] PSA, PSMA and STEAP, or [0058] PSMA,
PSCA and STEAP, or [0059] PSA, PSCA and STEAP, or [0060] or [0061]
PSA, PSMA, PSCA and STEAP.
[0062] According to a further preferred embodiment, the present
invention provides an active (immunostimulatory) composition
comprising at least one RNA, encoding at least two, three or four
(preferably different) antigens, [0063] a) wherein at least one
antigen is selected from: [0064] STEAP (Six Transmembrane
Epithelial Antigen of the Prostate); and [0065] b) wherein the
further antigen(s) is (are) selected from at least one antigen of
any of the following specific antigens or combinations thereof:
[0066] PSA (Prostate-Specific Antigen), or [0067] PSMA
(Prostate-Specific Membrane Antigen), or [0068] PSCA (Prostate Stem
Cell Antigen);
[0069] or [0070] PSA and PSMA, or [0071] PSA and PSCA, or [0072]
PSMA and PSCA;
[0073] or [0074] PSA, PSMA and PSCA.
[0075] According to an even more preferred embodiment the present
invention provides an active (immunostimulatory) composition
comprising at least one RNA, encoding four (preferably different)
antigens selected from PSA, PSMA, PSCA and STEAP.
[0076] The at least one RNA of the active (immunostimulatory)
composition according to the present invention is typically any
RNA, preferably, without being limited thereto, a coding RNA, a
circular or linear RNA, a single- or a double-stranded RNA (which
may also be regarded as a RNA due to non-covalent association of
two single-stranded RNA) 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 in this region, i.e. a partially double-stranded or partially
single stranded RNA with respect to the entire RNA sequence). More
preferably, the at least one RNA of the active (immunostimulatory)
composition according to the present invention is a single-stranded
RNA, even more preferably a linear RNA. Most preferably, the at
least RNA of the active (immunostimulatory) composition according
to the present invention is a messenger RNA (mRNA). In this
context, a messenger RNA (mRNA) is typically a RNA, which is
composed of (at least) several structural elements, e.g. an
optional 5'-UTR region, an upstream positioned ribosomal binding
site followed by a coding region, an optional 3'-UTR region, which
may be followed by a poly-A tail (and/or a poly-C-tail).
[0077] Due to one particularly preferred embodiment, each of the at
least two (preferably different) antigens of the active
(immunostimulatory) composition of the present invention, may be
encoded by one (monocistronic) RNA, preferably one (monocistronic)
mRNA. In other words, the active (immunostimulatory) composition of
the present invention may contain at least two (monocistronic)
RNAs, preferably mRNAs, wherein each of these two (monocistronic)
RNAs, preferably mRNAs, may encode just one (preferably different)
antigen, selected from one of the above mentioned groups or
subgroups, preferably in one of the above mentioned
combinations.
[0078] According to another particularly preferred embodiment, the
active (immunostimulatory) composition of the present invention,
may comprise (at least) one bi- or even multicistronic RNA,
preferably mRNA, i.e. (at least) one RNA which carries two or even
more of the coding sequences of at the least two (preferably
different) antigens, selected from one of the above mentioned
groups or subgroups, preferably in one of the above mentioned
combinations. Such coding sequences of the at least two (preferably
different) antigens of the (at least) one bi- or even
multicistronic RNA may be separated by at least one IRES (internal
ribosomal entry site) sequence, as defined below. Thus, the term
"encoding at least two (preferably different) antigens" may mean,
without being limited thereto, that the (at least) one (bi- or even
multicistronic) RNA, preferably a mRNA, may encode e.g. at least
two, three or four (preferably different) antigens of the above
mentioned group(s) of antigens or their fragments or variants
within the above definitions. More preferably, without being
limited thereto, the (at least) one (bi- or even multicistronic)
RNA, preferably mRNA, may encode e.g. at least two, three or four
(preferably different) antigens of the above mentioned subgroup(s)
of antigens or their fragments or variants within the above
definitions. In this context, a so-called IRES (internal ribosomal
entry site) sequence as defined above can function as a sole
ribosome binding site, but it can also serve to provide a bi- or
even multicistronic RNA as defined above which codes for several
proteins, 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).
[0079] According to a further particularly preferred embodiment,
the active (immunostimulatory) composition of the present
invention, may comprise a mixture of at least one monocistronic
RNA, preferably mRNA, as defined above, and at least one bi- or
even multicistronic RNA, preferably mRNA, as defined above. The at
least one monocistronic RNA and/or the at least one bi- or even
multicistronic RNA preferably encode different antigens or their
fragments or variants within the above definitions, the antigens
preferably being selected from one of the above mentioned groups or
subgroups of antigens, more preferably in one of the above
mentioned combinations. However, the at least one monocistronic RNA
and the at least one bi- or even multicistronic RNA may preferably
also encode (in part) identical antigens selected from one of the
above mentioned groups or subgroups of antigens, preferably in one
of the above mentioned combinations, provided that the active
(immunostimulatory) composition of the present invention as a whole
provides at least two (preferably different) antigens as defined
above. Such an embodiment may be advantageous e.g. for a staggered,
e.g. time dependent, administration of the active
(immunostimulatory) composition of the present invention to a
patient in need thereof. The components of such an active
(immunostimulatory) composition of the present invention,
particularly the different RNAs encoding the at least two
(preferably different) antigens, may be e.g. contained in
(different parts of) a kit of parts composition or may be e.g.
administered separately as components of different active
(immunostimulatory) compositions according to the present
invention.
[0080] Preferably, the at least one RNA of the active
(immunostimulatory) composition, encoding at least two (preferably
different) antigens selected from the above defined group of
antigens, more preferably in the above combinations, typically
comprises a length of about 50 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.
[0081] According to one embodiment, the at least one RNA of the
active (immunostimulatory) composition, encoding at least two
(preferably different) antigens selected from the above defined
group(s) or subgroup(s) of antigens, more preferably in the above
combinations, may be in the form of a modified RNA, wherein any
modification, as defined herein, may be introduced into the at
least one RNA of the active (immunostimulatory) composition.
Modifications as defined herein preferably lead to a stabilized at
least one RNA of the active (immunostimulatory) composition of the
present invention.
[0082] According to a first embodiment, the at least one RNA of the
active (immunostimulatory) composition of the present invention may
thus be provided as a "stabilized RNA", preferably a stabilized
mRNA, that is to say as an (m)RNA that is essentially resistant to
in vivo degradation (e.g. by an exo- or endo-nuclease). Such
stabilization can be effected, for example, by a modified phosphate
backbone of the at least one (m)RNA of the active
(immunostimulatory) composition of the present invention. A
backbone modification in connection with the present invention is a
modification in which phosphates of the backbone of the nucleotides
contained in the RNA are chemically modified. Nucleotides that may
be preferably used in this connection contain e.g. a
phosphorothioate-modified phosphate backbone, preferably at least
one of the phosphate oxygens contained in the phosphate backbone
being replaced by a sulfur atom. Stabilized (m)RNAs may further
include, for example: non-ionic phosphate analogues, such as, for
example, alkyl and aryl phosphonates, in which the charged
phosphonate oxygen is replaced by an alkyl or aryl group, or
phosphodiesters and alkylphosphotriesters, in which the charged
oxygen residue is present in alkylated form. Such backbone
modifications typically include, without implying any limitation,
modifications from the group consisting of methylphosphonates,
phosphoramidates and phosphorothioates (e.g.
cytidine-5'-O-(1-thiophosphate)).
[0083] The at least one RNA of the active (immunostimulatory)
composition of the present invention may additionally or
alternatively also contain sugar modifications. A sugar
modification in connection with the present invention is a chemical
modification of the sugar of the nucleotides of the at least one
RNA and typically includes, without implying any limitation, sugar
modifications selected from the group consisting of
2'-deoxy-2'-fluoro-oligoribonucleotide
(2'-fluoro-2'-deoxycytidine-5'-tri phosphate,
2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamine
oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate,
2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O-alkyl
oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide
(2'-O-methylcytidine-5'-triphosphate,
2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotide,
and isomers thereof (2'-aracytidine-5'-triphosphate,
2'-arauridine-5'-triphosphate), or azidotriphosphate
(2'-azido-2'-deoxycytidine-5'-triphosphate,
2'-azido-2'-deoxyuridine-5'-triphosphate).
[0084] The at least one RNA of the active (immunostimulatory)
composition of the present invention may additionally or
alternatively also contain at least one base modification, which is
preferably suitable for increasing the expression of the protein
coded for by the at least one RNA sequence significantly as
compared with the unaltered, i.e. natural (=native), RNA sequence.
Significant in this case means an increase in the expression of the
protein compared with the expression of the native RNA sequence by
at least 20%, preferably at least 30%, 40%, 50% or 60%, more
preferably by at least 70%, 80%, 90% or even 100% and most
preferably by at least 150%, 200% or even 300% or more. In
connection with the present invention, a nucleotide having such a
base modification is preferably selected from the group of the
base-modified nucleotides consisting of
2-amino-6-chloropurineriboside-5'-triphosphate,
2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate,
2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate,
5-aminoallylcytidine-5'-triphosphate,
5-aminoallyluridine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-tri phosphate,
5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate,
6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate,
6-chloropurineriboside-5'-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
8-azaadenosine-5'-tri phosphate, 8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N1-methyladenosine-5'-triphosphate,
N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-tri
phosphate, O6-methylguanosine-5'-triphosphate,
pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate,
xanthosine-5'-triphosphate. Particular preference is given to
nucleotides for base modifications selected from the group of
base-modified nucleotides consisting of
5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, and pseudouridine-5'-tri
phosphate.
[0085] According to another embodiment, the at least one RNA of the
active (immunostimulatory) composition of the present invention can
likewise be modified (and preferably stabilized) by introducing
further modified nucleotides containing modifications of their
ribose or base moieties. Generally, the at least one (m)RNA of the
active (immunostimulatory) composition of the present invention may
contain any native (=naturally occurring) nucleotide, e.g.
guanosine, uracil, adenosine, and/or cytosine or an analogue
thereof. In this connection, nucleotide analogues are defined as
non-natively occurring variants of naturally occurring nucleotides.
Accordingly, analogues are chemically derivatized nucleotides with
non-natively occurring functional groups, which are preferably
added to or deleted from the naturally occurring nucleotide or
which substitute the naturally occurring functional groups of a
nucleotide. Accordingly, each component of the naturally occurring
nucleotide may be modified, namely the base component, the sugar
(ribose) component and/or the phosphate component forming the
backbone (see above) of the RNA sequence. Analogues of guanosine,
uracil, adenosine, and cytosine include, without implying any
limitation, any naturally occurring or non-naturally occurring
guanosine, uracil, adenosine, thymidine or cytosine that has been
altered chemically, for example by acetylation, methylation,
hydroxylation, etc., including 1-methyl-adenosine,
1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine,
2,6-diaminopurine, 2'-Amino-2'-deoxyadenosine,
2'-Amino-2'-deoxycytidine, 2'-Amino-2'-deoxyguanosine,
2'-Amino-2'-deoxyuridine, 2-Amino-6-chloropurineriboside,
2-Aminopurine-riboside, 2'-Araadenosine, 2'-Aracytidine,
2'-Arauridine, 2'-Azido-2'-deoxyadenosine,
2'-Azido-2'-deoxycytidine, 2'-Azido-2'-deoxyguanosine,
2'-Azido-2'-deoxyuridine, 2-Chloroadenosine,
2'-Fluoro-2'-deoxyadenosine, 2'-Fluoro-2'-deoxycytidine,
2'-Fluoro-2-deoxyguanosine, 2'-Fluoro-2'-deoxyuridine,
2'-Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,
2-methyl-thio-N-6-isopenenyl-adenosine,
2'-O-Methyl-2-aminoadenosine, 2'-O-Methyl-2'-deoxyadenosine,
2'-O-Methyl-2'-deoxycytidine, 2'-O-Methyl-2'-deoxyguanosine,
2'-O-Methyl-2'-deoxyuridine, 2'-O-Methyl-5-methyluridine,
2'-O-Methyl inosine, 2'-O-Methylpseudouridine, 2-Thiocytidine,
2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine,
4-Thiouridine, 5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine,
5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine,
5-carboxymethylaminomethyl-2-thio-uracil,
5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine,
5-Fluoro-uridine, 5-Iodouridine, 5-methoxycarbonylmethyl-uridine,
5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine,
6-Azauridine, 6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside,
6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside,
7-Deaza-2'-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine,
8-Azaadenosine, 8-Bromoadenosine, 8-Bromo-guanosine,
8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazoleriboside,
Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine,
N1-Methyladenosine, N6-([6-Aminohexyl]carbamoylmethyl)-adenosine,
N6-isopentenyl-adenosine, N6-methyl-adenosine,
N7-Methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester,
Puromycin, Queosine, Uracil-5-oxyacetic acid, Uracil-5-oxyacetic
acid methyl ester, Wybutoxosine, Xanthosine, and Xylo-adenosine.
The preparation of such analogues is known to a person skilled in
the art, for example from U.S. Pat. No. 4,373,071, U.S. Pat. No.
4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.
Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No.
4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S.
Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642. In the
case of an analogue as described above, particular preference may
be given according to the invention to those analogues that
increase the immunogenity of the RNA of the inventive active
(immunostimulatory) composition and/or do not interfere with a
further modification of the RNA that has been introduced.
[0086] According to a particular embodiment, the at least one RNA
of the active (immunostimulatory) composition of the present
invention can contain a lipid modification. Such a lipid-modified
RNA typically comprises an RNA as defined herein, encoding at least
two antigens selected from the group of antigens as defined above,
preferably in the above combinations. Such a lipid-modified RNA
typically further comprises at least one linker covalently linked
with that RNA, and at least one lipid covalently linked with the
respective linker. Alternatively, the lipid-modified RNA comprises
an (at least one) RNA as defined herein and at least one
(bifunctional) lipid covalently linked (without a linker) with that
RNA. According to a third alternative, the lipid-modified RNA
comprises an RNA as defined herein, at least one linker covalently
linked with that RNA, 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 RNA.
[0087] The lipid contained in the at least one RNA of the inventive
active (immunostimulatory) composition (complexed or covalently
bound thereto) is typically a lipid or a lipophilic residue that
preferably is itself biologically active. Such lipids preferably
include natural substances or compounds such as, for example,
vitamins, e.g. alpha-tocopherol (vitamin E), including
RRR-alpha-tocopherol (formerly D-alpha-tocopherol),
L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E
succinate (VES), or vitamin A and its derivatives, e.g. retinoic
acid, retinol, vitamin D and its derivatives, e.g. vitamin D and
also the ergosterol precursors thereof, vitamin E and its
derivatives, vitamin K and its derivatives, e.g. vitamin K and
related quinone or phytol compounds, or steroids, such as bile
acids, for example cholic acid, deoxycholic acid, dehydrocholic
acid, cortisone, digoxygenin, testosterone, cholesterol or
thiocholesterol. Further lipids or lipophilic residues within the
scope of the present invention include, without implying any
limitation, polyalkylene glycols (Oberhauser et al., Nucl. Acids
Res., 1992, 20, 533), aliphatic groups such as, for example,
C.sub.1-C.sub.20-alkanes, C.sub.1-C.sub.20-alkenes or
C.sub.1-C.sub.20-alkanol compounds, etc., such as, for example,
dodecanediol, hexadecanol or undecyl residues (Saison-Behmoaras et
al., EMBO J, 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,
327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids
such as, for example, phosphatidylglycerol,
diacylphosphatidylglycerol, phosphatidylcholine,
dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine,
di-hexadecyl-rac-glycerol, sphingolipids, cerebrosides,
gangliosides, or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res.,
1990, 18, 3777), polyamines or polyalkylene glycols, such as, for
example, polyethylene glycol (PEG) (Manoharan et al., Nucleosides
& Nucleotides, 1995, 14, 969), hexaethylene glycol (HEG),
palmitin or palmityl residues (Mishra et al., Biochim. Biophys.
Acta, 1995, 1264, 229), octadecylamines or
hexylaminocarbonyl-oxycholesterol residues (Crooke et at, J.
Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes, terpenes,
alicyclic hydrocarbons, saturated and mono- or poly-unsaturated
fatty acid residues, etc.
[0088] The at least one RNA of the active (immunostimulatory)
composition of the present invention may likewise be stabilized in
order to prevent degradation of the RNA in vivo by various
approaches. It is known in the art that instability and (fast)
degradation of mRNA or of RNA in vivo in general may represent a
serious problem in the application of RNA based compositions. 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 mRNA in the cytoplasm of
cells is very finely regulated and RNase contaminations may be
generally removed by special treatment prior to use of said
sompositions, in particular with diethyl pyrocarbonate (DEPC). A
number of mechanisms of natural degradation are known in this
connection in the prior art, which may be utilized as well. E.g.,
the terminal structure is typically of critical importance for a
mRNA in vivo. As an example, at the 5' end of naturally occurring
mRNAs there is usually a so-called "cap structure" (a modified
guanosine nucleotide), and at the 3' end is typically a sequence of
up to 200 adenosine nucleotides (the so-called poly-A tail).
[0089] The at least one RNA of the active (immunostimulatory)
composition of the present invention, particularly if provided as
an mRNA, can therefore be stabilized against degradation by RNases
by the addition of a so-called "5' cap" structure. Particular
preference is given in this connection to an m7G(5')ppp
(5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure.
However, such a modification is introduced only if a modification,
for example a lipid modification, has not already been introduced
at the 5' end of the (m)RNA of the inventive immunostimulatory
composition or if the modification does not interfere with the
immunogenic properties of the (unmodified or chemically modified)
(m)RNA.
[0090] According to a further preferred embodiment, the at least
one RNA of the active (immunostimulatory) composition of the
present invention may contain, especially if the RNA is in the form
of an mRNA, a poly-A tail on the 3' terminus of typically about 10
to 200 adenosine nucleotides, preferably about 10 to 100 adenosine
nucleotides, more preferably about 20 to 100 adenosine nucleotides
or even more preferably about 40 to 80 adenosine nucleotides.
[0091] According to a further preferred embodiment, the at least
one RNA of the active (immunostimulatory) composition of the
present invention may contain, especially if the RNA is in the form
of an mRNA, a poly-C tail on the 3' terminus of typically about 10
to 200 cytosine nucleotides, preferably about 10 to 100 cytosine
nucleotides, more preferably about 20 to 70 cytosine nucleotides or
even more preferably about 20 to 60 or even 10 to 40 cytosine
nucleotides.
[0092] According to another embodiment, the at least one RNA of the
active (immunostimulatory) composition of the present invention may
be modified, and thus stabilized, especially if the RNA is in the
form of an mRNA, by modifying the G/C content of the RNA,
preferably of the coding region of the at least one RNA.
[0093] In a particularly preferred embodiment of the present
invention, the G/C content of the coding region of the at least one
(m)RNA of the active (immunostimulatory) composition of the present
invention is modified, particularly increased, compared to the G/C
content of the coding region of its particular wild-type (m)RNA,
i.e. the unmodified (m)RNA. The encoded amino acid sequence of the
at least one (m)RNA is preferably not modified compared to the
coded amino acid sequence of the particular wild-type (m)RNA.
[0094] This modification of the at least one (m)RNA of the active
(immunostimulatory) composition of the present invention is based
on the fact that the sequence of any (m)RNA region to be translated
is important for efficient translation of that (m)RNA. Thus, the
composition and the sequence of various nucleotides is 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. According to the
invention, the codons of the (m)RNA are therefore varied compared
to its wild-type (m)RNA, 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 favorable codons for the stability can be
determined (so-called alternative codon usage).
[0095] Depending on the amino acid to be encoded by the at least
one (m)RNA, there are various possibilities for modification of the
(m)RNA sequence, compared to its wild-type sequence. In the case of
amino acids which are encoded by codons which contain exclusively G
or C nucleotides, no modification of the codon is necessary. Thus,
the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG)
and Gly (GGC or GGG) require no modification, since no A or U is
present.
[0096] In contrast, codons which contain A and/or U nucleotides can
be modified by substitution of other codons which code for the same
amino acids but contain no A and/or U. Examples of these are:
[0097] the codons for Pro can be modified from CCU or CCA to CCC or
CCG;
[0098] the codons for Arg can be modified from CGU or CGA or AGA or
AGG to CGC or CGG;
[0099] the codons for Ala can be modified from GCU or GCA to GCC or
GCG;
[0100] the codons for Gly can be modified from GGU or GGA to GGC or
GGG.
[0101] In other cases, although A or U nucleotides cannot be
eliminated from the codons, it is however possible to decrease the
A and U content by using codons which contain a lower content of A
and/or U nucleotides. Examples of these are:
[0102] the codons for Phe can be modified from UUU to UUC;
[0103] the codons for Leu can be modified from UUA, UUG, CUU or CUA
to CUC or CUG;
[0104] the codons for Ser can be modified from UCU or UCA or AGU to
UCC, UCG or AGC;
[0105] the codon for Tyr can be modified from UAU to UAC;
[0106] the codon for Cys can be modified from UGU to UGC;
[0107] the codon for His can be modified from CAU to CAC;
[0108] the codon for Gln can be modified from CAA to CAG;
[0109] the codons for Ile can be modified from AUU or AUA to
AUC;
[0110] the codons for Thr can be modified from ACU or ACA to ACC or
ACG;
[0111] the codon for Asn can be modified from AAU to AAC;
[0112] the codon for Lys can be modified from AAA to AAG;
[0113] the codons for Val can be modified from GUU or GUA to GUC or
GUG;
[0114] the codon for Asp can be modified from GAU to GAC;
[0115] the codon for Glu can be modified from GAA to GAG;
[0116] the stop codon UAA can be modified to UAG or UGA.
[0117] In the case of the codons for Met (AUG) and Trp (UGG), on
the other hand, there is no possibility of sequence
modification.
[0118] The substitutions listed above can be used either
individually or in all possible combinations to increase the G/C
content of the at least one (m)RNA of the active
(immunostimulatory) composition of the present invention compared
to its particular wild-type (m)RNA (i.e. the original sequence).
Thus, for example, all codons for Thr occurring in the wild-type
sequence can be modified to ACC (or ACG). Preferably, however, for
example, combinations of the above substitution possibilities are
used:
[0119] substitution of all codons coding for Thr in the original
sequence (wild-type (m)RNA) to ACC (or ACG) and
[0120] substitution of all codons originally coding for Ser to UCC
(or UCG or AGC);
[0121] substitution of all codons coding for Ile in the original
sequence to AUC and
[0122] substitution of all codons originally coding for Lys to AAG
and
[0123] substitution of all codons originally coding for Tyr to
UAC;
[0124] substitution of all codons coding for Val in the original
sequence to GUC (or GUG) and
[0125] substitution of all codons originally coding for Glu to GAG
and
[0126] substitution of all codons originally coding for Ala to GCC
(or GCG) and
[0127] substitution of all codons originally coding for Arg to CGC
(or CGG);
[0128] substitution of all codons coding for Val in the original
sequence to GUC (or GUG) and
[0129] substitution of all codons originally coding for Glu to GAG
and
[0130] substitution of all codons originally coding for Ala to GCC
(or GCG) and
[0131] substitution of all codons originally coding for Gly to GGC
(or GGG) and
[0132] substitution of all codons originally coding for Asn to
AAC;
[0133] substitution of all codons coding for Val in the original
sequence to GUC (or GUG) and
[0134] substitution of all codons originally coding for Phe to UUC
and
[0135] substitution of all codons originally coding for Cys to UGC
and
[0136] substitution of all codons originally coding for Leu to CUG
(or CUC) and
[0137] substitution of all codons originally coding for Gln to CAG
and
[0138] substitution of all codons originally coding for Pro to CCC
(or CCG); etc.
[0139] Preferably, the G/C content of the coding region of the at
least one (m)RNA of the active (immunostimulatory) composition of
the present invention 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 (m)RNA
which codes for an antigen, antigenic protein or antigenic peptide
as deinined herein or its fragment or variant thereof. 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 an antigen, antigenic protein or
antigenic peptide as deinined herein or its fragment or variant
thereof or the whole sequence of the wild type (m)RNA sequence are
substituted, thereby increasing the GC/content of said
sequence.
[0140] In this context, it is particularly preferable to increase
the G/C content of the at least one (m)RNA of the active
(immunostimulatory) composition of the present invention 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.
[0141] According to the invention, a further preferred modification
of the at least one (m)RNA of the active (immunostimulatory)
composition of the present invention is based on the finding that
the translation efficiency is also determined by a different
frequency in the occurrence of tRNAs in cells. Thus, if so-called
"rare codons" are present in the at least one (m)RNA of the active
(immunostimulatory) composition of the present invention to an
increased extent, the corresponding modified at least one (m)RNA
sequence is translated to a significantly poorer degree than in the
case where codons coding for relatively "frequent" tRNAs are
present.
[0142] According to the invention, in the modified at least one
(m)RNA of the active (immunostimulatory) composition of the present
invention, the region which codes for the adjuvant protein is
modified compared to the corresponding region of the wild-type
(m)RNA 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 at least one
(m)RNA of the active (immunostimulatory) composition of the present
invention is modified such that codons for which frequently
occurring tRNAs are available are inserted. In other words,
according to the invention, 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.
[0143] 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. 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.
[0144] According to the invention, it is particularly preferable to
link the sequential G/C content which is increased, in particular
maximized, in the modified at least one (m)RNA of the active
(immunostimulatory) composition of the present invention, with the
"frequent" codons without modifying the amino acid sequence of the
protein encoded by the coding region of the (m)RNA. This preferred
embodiment allows provision of a particularly efficiently
translated and stabilized (modified) at least one (m)RNA of the
active (immunostimulatory) composition of the present
invention.
[0145] The determination of a modified at least one (m)RNA of the
active (immunostimulatory) composition of the present invention as
described above (increased G/C content; exchange of tRNAs) can be
carried out using the computer program explained in WO
02/098443--the disclosure content of which is included in its full
scope in the present invention. Using this computer program, the
nucleotide sequence of any desired (m)RNA can be modified with the
aid of the genetic code or the degenerative nature thereof such
that a maximum G/C content results, in combination with the use of
codons which code for tRNAs occurring as frequently as possible in
the cell, the amino acid sequence coded by the modified at least
one (m)RNA preferably not being modified compared to the
non-modified sequence. Alternatively, it is also possible to modify
only the G/C content or only the codon usage compared to the
original sequence. The source code in Visual Basic 6.0 (development
environment used: Microsoft Visual Studio Enterprise 6.0 with
Servicepack 3) is also described in WO 02/098443.
[0146] In a further preferred embodiment of the present invention,
the A/U content in the environment of the ribosome binding site of
the at least one (m)RNA of the active (immunostimulatory)
composition of the present invention is increased compared to the
A/U content in the environment of the ribosome binding site of its
particular wild-type (m)RNA. This modification (an increased A/U
content around the ribosome binding site) increases the efficiency
of ribosome binding to the at least one (m)RNA. An effective
binding of the ribosomes to the ribosome binding site (Kozak
sequence: GCCGCCACCAUGG (SEQ ID NO: 27), the AUG forms the start
codon) in turn has the effect of an efficient translation of the at
least one (m)RNA.
[0147] According to a further embodiment of the present invention
the at least one (m)RNA of the active (immunostimulatory)
composition of the present invention may be modified with respect
to potentially destabilizing sequence elements. Particularly, the
coding region and/or the 5' and/or 3' untranslated region of this
at least one (m)RNA may be modified compared to the particular
wild-type (m)RNA such that is contains no destabilizing sequence
elements, the coded amino acid sequence of the modified at least
one (m)RNA preferably not being modified compared to its particular
wild-type (m)RNA. It is known that, for example, in sequences of
eukaryotic RNAs destabilizing sequence elements (DSE) occur, to
which signal proteins bind and regulate enzymatic degradation of
RNA in vivo. For further stabilization of the modified at least one
(m)RNA, optionally in the region which encodes for an antigen,
antigenic protein or antigenic peptide as defined herein, one or
more such modifications compared to the corresponding region of the
wild-type (m)RNA can therefore be carried out, so that no or
substantially no destabilizing sequence elements are contained
there. According to the invention, DSE present in the untranslated
regions (3'- and/or 5'-UTR) can also be eliminated from the at
least one (m)RNA of the active (immunostimulatory) composition of
the present invention by such modifications.
[0148] Such destabilizing sequences are e.g. AU-rich sequences
(AURES), which occur in 3'-UTR sections of numerous unstable RNAs
(Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674).
The at least one (m)RNA of the active (immunostimulatory)
composition of the present invention is therefore preferably
modified compared to the wild-type (m)RNA such that the at least
one (m)RNA contains no such destabilizing sequences. This also
applies to those sequence motifs which are recognized by possible
endonucleases, e.g. the sequence GAACAAG, which is contained in the
3'-UTR segment of the gene which codes for the transferrin receptor
(Binder et al., EMBO J. 1994, 13: 1969 to 1980). These sequence
motifs are also preferably removed in the at least one (m)RNA of
the active (immunostimulatory) composition of the present
invention.
[0149] Also preferably according to the invention, the at least one
(m)RNA of the active (immunostimulatory) composition of the present
invention has, in a modified form, at least one IRES as defined
above and/or at least one 5' and/or 3' stabilizing sequence, in a
modified form, e.g. to enhance ribosome binding or to allow
expression of different encoded antigens located on an at least one
(bi- or even multicistronic) RNA of the active (immunostimulatory)
composition of the present invention.
[0150] According to the invention, the at least one (m)RNA of the
active (immunostimulatory) composition of the present invention
furthermore preferably has at least one 5' and/or 3' stabilizing
sequence. These stabilizing sequences in the 5' and/or 3'
untranslated regions have the effect of increasing the half-life of
the at least one (m)RNA in the cytosol. These stabilizing sequences
can have 100% sequence homology 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 globin gene, e.g. from Homo sapiens or Xenopus laevis may be
mentioned as an example of stabilizing sequences which can be used
in the present invention for a stabilized RNA. Another example of a
stabilizing sequence has the general formula
(C/U)CCAN.sub.xCCC(U/A)Py.sub.xUC(C/U)CC (SEQ ID NO: 28), which is
contained in the 3'UTR of the very stable RNA which codes for
globin, (I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase
(cf. Holcik et al, Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to
2414). Such stabilizing sequences can of course be used
individually or in combination with one another and also in
combination with other stabilizing sequences known to a person
skilled in the art. The at least one (m)RNA of the active
(immunostimulatory) composition of the present invention is
therefore preferably present as globin UTR (untranslated
regions)-stabilized RNA, in particular as globin UTR-stabilized
RNA.
[0151] Nevertheless, substitutions, additions or eliminations of
bases are preferably carried out with the at least one RNA of the
active (immunostimulatory) composition of the present invention,
using a DNA matrix for preparation of the at least one RNA of the
active (immunostimulatory) composition of the present invention by
techniques of the well known site directed mutagenesis or with an
oligonucleotide ligation strategy (see e.g. Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y., 2001). In such
a process, for preparation of the at least one (m)RNA, a
corresponding DNA molecule may 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 for the at least one RNA to be prepared
and a termination signal for in vitro transcription. The DNA
molecule, which forms the matrix of an 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.
[0152] The stabilization of the at least one RNA of the active
(immunostimulatory) composition of the present invention can
likewise by carried out by associating or complexing the at least
one RNA with, or binding it to, a cationic compound, in particular
a polycationic compound, for example a (poly)cationic peptide or
protein. In particular the use of protamine, nucleoline, spermin or
spermidine as the polycationic, nucleic-acid-binding protein to the
RNA is particularly effective. Furthermore, the use of other
cationic peptides or proteins, such as poly-L-lysine or histones,
is likewise possible. This procedure for stabilizing RNA is
described in EP-A-1083232, the disclosure of which is incorporated
by reference into the present invention in its entirety. Further
preferred cationic substances which can be used for stabilizing the
RNA of the active (immunostimulatory) composition of the present
invention include cationic polysaccharides, for example chitosan,
polybrene, polyethyleneimine (PEI) or poly-L-lysine (PLL), etc.
Association or complexing of the at least one RNA of the inventive
active (immunostimulatory) composition with cationic compounds,
e.g. cationic proteins or cationic lipids, e.g. oligofectamine as a
lipid based complexation reagent) preferably increases the transfer
of the at least one RNA present as a pharmaceutically active
component into the cells to be treated or into the organism to be
treated. It is also referred to the disclosure herein with regard
to the stabilizing effect for the at least one RNA of the active
(immunostimulatory) composition of the present invention by
complexation, which holds for the stabilization of RNA as well.
[0153] According to another particularly preferred embodiment, the
at least RNA of the active (immunostimulatory) composition may
additionally or alternatively encode a secretory signal peptide.
Such signal peptides are sequences, which typically exhibit a
length of about 15 to 30 amino acids and are preferably located at
the N-terminus of the encoded peptide, without being limited
thereto. Signal peptides as defined herein preferably allow the
transport of the antigen, antigenic protein or antigenic peptide as
encoded by the at least one RNA of the active (immunostimulatory)
composition into a defined cellular compartiment, preferably the
cell surface, the endoplasmic reticulum (ER) or the
endosomal-lysosomal compartiment. Examples of secretory signal
peptide sequences as defined herein include, without being limited
thereto, signal sequences of classical or non-classical
MHC-molecules (e.g. signal sequences of MHC I and II molecules,
e.g. of the MHC class I molecule HLA-A*0201), signal sequences of
cytokines or immunoglobulines as defined herein, signal sequences
of the invariant chain of immunoglobulines or antibodies as defined
herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin,
Calnexin, and further membrane associated proteins or of proteins
associated with the endoplasmic reticulum (ER) or the
endosomal-lysosomal compartiment. Particularly preferably, signal
sequences of MHC class I molecule HLA-A*0201 may be used according
to the present invention.
[0154] Any of the above modifications may be applied to the at
least one RNA of the active (immunostimulatory) composition of the
present invention, and further to any (m)RNA as used in the context
of the present invention and may be, if suitable or necessary, be
combined with each other in any combination, provided, these
combinations of modifications do not interfere with each other in
the respective at least one RNA. A person skilled in the art will
be able to take his choice accordingly.
[0155] According to another embodiment, the active
(immunostimulatory) composition according to the invention may
comprise an adjuvant. In this context, an adjuvant may be
understood as any compound, which is suitable to support
administration and delivery of the active (immunostimulatory)
composition according to the invention. Furthermore, such an
adjuvant may, without being bound thereto, initiate or increase an
immune response of the innate immune system, i.e. a non-specific
immune response. With other words, when administered, the active
(immunostimulatory) composition according to the invention
typically initiates an adaptive immune response due to the at least
two antigens encoded by the at least one RNA contained in the
inventive active (immunostimulatory) composition. Additionally, the
active (immunostimulatory) composition according to the invention
may generate an (supportive) innate immune response due to addition
of an adjuvant as defined herein to the active (immunostimulatory)
composition according to the invention. Such an adjuvant may be
selected from any adjuvant known to a skilled person and suitable
for the present case, i.e. supporting the induction of an immune
response in a mammal. Preferably, the adjuvant may be selected from
the group consisting of, without being limited thereto, 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-leucylamino-b-D-glucopyranosyl)-N-octadecyl-do-
decanoyl-amide 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-D-glutamine
(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)
zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP
(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-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); interferon-gamma;
interleukin-1beta; interleukin-2; interleukin-7; interleukin-12;
ISCOMS'; ISCOPREP 7.0.3..TM.; liposomes; LOXORIBINE.TM.
(7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile
enterotoxin-protoxin); microspheres and microparticles of any
composition; MF59.TM.; (squalene-water 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-OCH.sub.3); MURAPALMITINE.TM.
and D-MURAPALMITINE.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.
(.beta.-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic
acid and glycolic acid; microspheres/nanospheres); PLURONIC
L121.TM.; PMMA (polymethyl methacrylate); 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-Ala-dipalmitoxypro-
pylamide); Theronyl-MDP (Termurtide.TM. or [thr 1]-MDP;
N-acetylmuramyl-L-threonyl-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, 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.
[0156] Suitable adjuvants may also be selected from cationic or
polycationic compounds wherein the adjuvant is preferably prepared
upon complexing the at least one RNA of the inventive active
(immunostimulatory composition) with the cationic or polycationic
compound. Association or complexing the RNA of the active
(immunostimulatory) composition with cationic or polycationic
compounds as defined herein preferably provides adjuvant properties
and confers a stabilizing effect to the at least one RNA of the
active (immunostimulatory) composition. Particularly such
preferred, such cationic or polycationic compounds are selected
from cationic or polycationic peptides or proteins, including
protamine, nucleoline, spermin 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, Tat, 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, protamine, spermine, spermidine, or histones. Further
preferred cationic or polycationic compounds 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,
CLI P9:
rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammoni-
um, oligofectamine, 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 polybetaminoester (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 of a
cationic polymer as mentioned above) and of one or more
hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole);
etc.
[0157] Additionally, preferred cationic or polycationic proteins or
peptides, which can be used as an adjuvant by complexing the at
least one RNA of the active (immunostimulatory) composition, may be
selected from following proteins or peptides having the following
total formula (I): (Arg).sub.l; (Lys).sub.n; (His).sub.n,
(Orn).sub.o; (Xaa).sub.x, wherein l+m+n+o+x=8-15, and l, m, n or
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 oligoarginines in this context are e.g.
Arg.sub.7, Arg.sub.8, Arg.sub.9, Arg.sub.7, 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.
[0158] Suitable adjuvants may furthermore be selected from nucleic
acids having the formula (II): G.sub.lX.sub.mG.sub.n, wherein: G is
guanosine, uracil or an analogue of guanosine or uracil; X is
guanosine, uracil, adenosine, thymidine, cytosine or an analogue of
the above-mentioned nucleotides; l is an integer from 1 to 40,
wherein when l=1 G is guanosine or an analogue thereof, when l>1
at least 50% of the nucleotides are guanosine or an analogue
thereof; m is an integer and is at least 3; wherein when m=3.times.
is uracil or an analogue thereof, when m>3 at least 3 successive
uracils or analogues of uracil occur; n is an integer from 1 to 40,
wherein when n=1 G is guanosine or an analogue thereof, when n>1
at least 50% of the nucleotides are guanosine or an analogue
thereof.
[0159] Other suitable adjuvants may furthermore be selected from
nucleic acids having the formula (III): C.sub.lX.sub.mC.sub.n,
wherein: C is cytosine, uracil or an analogue of cytosine or
uracil; X is guanosine, uracil, adenosine, thymidine, cytosine or
an analogue of the above-mentioned nucleotides; l is an integer
from 1 to 40, wherein when l=1 C is cytosine or an analogue
thereof, when l>1 at least 50% of the nucleotides are cytosine
or an analogue thereof; m is an integer and is at least 3; wherein
when m=3.times. is uracil or an analogue thereof, when m>3 at
least 3 successive uracils or analogues of uracil occur; n is an
integer from 1 to 40, wherein when n=1 C is cytosine or an analogue
thereof, when n>1 at least 50% of the nucleotides are cytosine
or an analogue thereof.
[0160] According to one preferred embodiment, the present invention
may furthermore provide a vaccine containing the active
(immunostimulatory) composition according to the invention. The
inventive vaccine may additionally contain a pharmaceutically
acceptable carrier and/or further auxiliary substances and
additives and/or adjuvants. According to a particularly preferred
embodiment, the antigens encoded by the at least one RNA of the
active (immunostimulatory) composition, contained in the inventive
vaccine, are selected from the above mentioned group.
[0161] The inventive vaccine typically comprises a safe and
effective amount of the at least one RNA of the active
(immunostimulatory) composition as defined above encoding at least
two antigens as defined above, more preferably encoding at least
two antigens selected from any of the above groupmost preferably in
any of the indicated combinations. As used herein, "safe and
effective amount" means an amount of the at least one RNA of the
active (immunostimulatory) composition in the vaccine as defined
above, that is sufficient to significantly induce a positive
modification of prostate cancer (PCa), preferably of neoadjuvant
and/or hormone-refractory prostate cancers, and diseases or
disorders related thereto. At the same time, however, a "safe and
effective amount" is small enough to avoid serious side-effects,
that is to say to permit a sensible relationship between advantage
and risk. The determination of these limits typically lies within
the scope of sensible medical judgment. In relation to the
inventive vaccine, the expression "safe and effective amount"
preferably means an amount of the RNA (and thus of the encoded at
least two antigens) that is suitable for stimulating the adaptive
immune system in such a manner that no excessive or damaging immune
reactions are achieved but, preferably, also no such immune
reactions below a measurable level. Such a "safe and effective
amount" of the at least one RNA of the active (immunostimulatory)
composition in the vaccine as defined above may furthermore be
selected in dependence of the type of RNA, e.g. monocistronic, bi-
or even multicistronic RNA, since a bi- or even multicistronic RNA
may lead to a significantly higher expression of the encoded
antigen(s) than use of an equal amount of a monocistronic RNA. A
"safe and effective amount" of the at least one RNA of the active
(immunostimulatory) composition as defined above, which is
contained in the inventive vaccine, will furthermore vary in
connection with the particular condition to be treated and also
with the age and physical condition of the patient to be treated,
the severity of the condition, the duration of the treatment, the
nature of the accompanying therapy, of the particular
pharmaceutically acceptable carrier used, and similar factors,
within the knowledge and experience of the accompanying doctor. The
vaccine according to the invention can be used according to the
invention for human and also for veterinary medical purposes, as a
pharmaceutical composition or as a vaccine.
[0162] The vaccine according to the invention typically contains a
pharmaceutically acceptable carrier. The expression
"pharmaceutically acceptable carrier" as used herein preferably
includes the liquid or non-liquid basis of the inventive vaccine.
If the inventive vaccine is provided in liquid form, the carrier
will typically be pyrogen-free water; isotonic saline or buffered
(aqueous) solutions, e.g phosphate, citrate etc. buffered
solutions. Particularly for injection of the inventive vaccine,
water or preferably a buffer, more preferably an aqueous buffer,
may be used, containing a sodium salt, preferably at least 50 mM of
a sodium salt, a calcium salt, preferably at least 0.01 mM of a
calcium salt, and optionally a potassium salt, preferably at least
3 mM of a potassium salt. According to a preferred embodiment, the
sodium, calcium and, optionally, potassium salts may occur in the
form of their halogenides, e.g. chlorides, iodides, or bromides, in
the form of their hydroxides, carbonates, hydrogen carbonates, or
sulfates, etc. Without being limited thereto, examples of sodium
salts include e.g. NaCl, NaI, NaBr, Na.sub.2CO.sub.3, NaHCO.sub.3,
Na.sub.2SO.sub.4, examples of the optional potassium salts include
e.g. KCl, KI, KBr, K.sub.2CO.sub.3, KHCO.sub.3, K.sub.2SO.sub.4,
and examples of calcium salts include e.g. CaCl.sub.2, CaI.sub.2,
CaBr.sub.2, CaCO.sub.3, CaSO.sub.4, Ca(OH).sub.2. Furthermore,
organic anions of the aforementioned cations may be contained in
the buffer. According to a more preferred embodiment, the buffer
suitable for injection purposes as defined above, may contain salts
selected from sodium chloride (NaCl), calcium chloride (CaCl.sub.2)
and optionally potassium chloride (KCl), wherein further anions may
be present additional to the chlorides. CaCl.sub.2 can also be
replaced by another salt like KCl. Typically, the salts in the
injection buffer are present in a concentration of at least 50 mM
sodium chloride (NaCl), at least 3 mM potassium chloride (KCl) and
at least 0.01 mM calcium chloride (CaCl.sub.2). 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. in "in vivo" methods occurring liquids 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.
[0163] However, one or more compatible solid or liquid fillers or
diluents or encapsulating compounds may be used as well, which are
suitable for administration to a person. The term "compatible" as
used herein means that the constituents of the inventive vaccine
are capable of being mixed with the at least one RNA of the active
(immunostimulatory) composition, encoding at least two antigens as
defined above, in such a manner that no interaction occurs which
would substantially reduce the pharmaceutical effectiveness of the
inventive vaccine under typical use conditions. Pharmaceutically
acceptable carriers, fillers and diluents must, of course, have
sufficiently high purity and sufficiently low toxicity to make them
suitable for administration to a person to be treated. Some
examples of compounds which can be used as pharmaceutically
acceptable carriers, fillers or constituents thereof are sugars,
such as, for example, lactose, glucose and sucrose; starches, such
as, for example, corn starch or potato starch; cellulose and its
derivatives, such as, for example, sodium carboxymethylcellulose,
ethylcellulose, cellulose acetate; powdered tragacanth; malt;
gelatin; tallow; solid glidants, such as, for example, stearic
acid, magnesium stearate; calcium sulfate; vegetable oils, such as,
for example, groundnut oil, cottonseed oil, sesame oil, olive oil,
corn oil and oil from theobroma; polyols, such as, for example,
polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene
glycol; alginic acid.
[0164] The choice of a pharmaceutically acceptable carrier is
determined in principle by the manner in which the inventive
vaccine is administered. The inventive vaccine can be administered,
for example, systemically or locally. Routes for systemic
administration in general include, for example, transdermal, oral,
parenteral routes, including subcutaneous, intravenous,
intramuscular, intraarterial, intradermal and intraperitoneal
injections and/or intranasal administration routes. Routes for
local administration in general include, for example, topical
administration routes but also intradermal, transdermal,
subcutaneous, or intramuscular injections or intralesional,
intracranial, intrapulmonal, intracardial, and sublingual
injections. More preferably, vaccines may be administered by an
intradermal, subcutaneous, or intramuscular route.
Compositions/vaccines are therefore preferably formulated in liquid
or solid form. The suitable amount of the inventive vaccine to be
administered can be determined by routine experiments with animal
models. Such models include, without implying any limitation,
rabbit, sheep, mouse, rat, dog and non-human primate models.
Preferred unit dose forms for injection include sterile solutions
of water, physiological saline or mixtures thereof. The pH of such
solutions should be adjusted to about 7.4. Suitable carriers for
injection include hydrogels, devices for controlled or delayed
release, polylactic acid and collagen matrices. Suitable
pharmaceutically acceptable carriers for topical application
include those which are suitable for use in lotions, creams, gels
and the like. If the inventive vaccine is to be administered
perorally, tablets, capsules and the like are the preferred unit
dose form. The pharmaceutically acceptable carriers for the
preparation of unit dose forms which can be used for oral
administration are well known in the prior art. The choice thereof
will depend on secondary considerations such as taste, costs and
storability, which are not critical for the purposes of the present
invention, and can be made without difficulty by a person skilled
in the art.
[0165] The inventive vaccine can additionally contain one or more
auxiliary substances in order to further increase the
immunogenicity. A synergistic action of the at least one RNA of the
active (immunostimulatory) composition as defined above and of an
auxiliary substance, which may be optionally also contained in the
inventive vaccine as described above, is preferably achieved
thereby. Depending on the various types of auxiliary substances,
various mechanisms can come into consideration in this respect. For
example, compounds that permit the maturation of dendritic cells
(DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand,
form a first class of suitable auxiliary substances. In general, it
is possible to use as auxiliary substance any agent that influences
the immune system in the manner of a "danger signal" (LPS, GP96,
etc.) or cytokines, such as GM-CFS, which allow an immune response
produced by the immune-stimulating adjuvant according to the
invention to be enhanced and/or influenced in a targeted manner.
Particularly preferred auxiliary substances are cytokines, such as
monokines, lymphokines, interleukins or chemokines,
that--additional to induction of the adaptive immune response by
the encoded at least two antigens--promote the innate immune
response, such as 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, INF-alpha, IFN-beta,
INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth
factors, such as hGH.
[0166] Further additives which may be included in the inventive
vaccine 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.
[0167] The inventive vaccine can also additionally contain any
further compound, which is known to be immune-stimulating due to
its binding affinity (as ligands) to human Toll-like receptors
TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due
to its binding affinity (as ligands) to murine Toll-like receptors
TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,
TLR12 or TLR13.
[0168] Another class of compounds, which may be added to an
inventive vaccine in this context, may be CpG nucleic acids, in
particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA 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 is
preferably in the form of CpG-RNA, more preferably in the form of
single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid
preferably contains at least one or more (mitogenic)
cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According
to a first preferred alternative, at least one CpG motif contained
in these sequences, that is to say the C (cytosine) and the G
(guanine) of the CpG motif, is unmethylated. All further cytosines
or guanines optionally contained in these sequences can be either
methylated or unmethylated. According to a further preferred
alternative, however, the C (cytosine) and the G (guanine) of the
CpG motif can also be present in methylated form.
[0169] According to a further preferred object of the present
invention, the inventive active (immunostimulatory) composition may
be used (for the preparation of a vaccine according to the present
invention) for the treatment of prostate cancer (PCa), preferably
of neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto.
[0170] According to a further preferred object of the present
invention, the inventive vaccine or the at least one RNA encoding
at least two (preferably) different antigens as defined herein may
be used for the treatment of prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto.
[0171] In this context also included in the present invention are
methods of treating prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto, by administering to a
patient in need thereof a pharmaceutically effective amount of an
inventive vaccine, or a pharmaceutically effective amount of an
inventive active (immunostimulatory) composition. Such a method
typically comprises an optional first step of preparing the
inventive active (immunostimulatory) composition, or the inventive
vaccine, and a second step, comprising administering (a
pharmaceutically effective amount of) said inventive active
(immunostimulatory) composition or said inventive vaccine to a
patient in need thereof. A patient in need thereof will be
typically selected from any mammal. In the context of the present
invention, a mammal is preferably a mammal, selected from the group
comprising, without being limited thereto, e.g. goat, cattle,
swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse,
hamster, rabbit and, particularly, human, wherein the mammal
typically suffers from prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto.
[0172] The invention relates also to the use of the inventive
active (immunostimulatory) composition or the at least one RNA
encoding at least two (preferably) different antigens as defined
herein (for the preparation of an inventive vaccine), preferably
for eliciting an immune response in a mammal, preferably for the
treatment of prostate cancer (PCa), more preferably of neoadjuvant
and/or hormone-refractory prostate cancers, and diseases or
disorders related thereto.
[0173] Similarly, the invention also relates also to the use of the
inventive vaccine per se or the at least one RNA encoding at least
two (preferably) different antigens as defined herein for eliciting
an adaptive immune response in a mammal, preferably for the
treatment of prostate cancer (PCa), preferably of neoadjuvant
and/or hormone-refractory prostate cancers, and diseases or
disorders related thereto.
[0174] Prevention or treatment of prostate cancer (PCa), preferably
of neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto, may be carried out by
administering the inventive active (immunostimulatory) composition
and/or the inventive vaccine at once or in a time staggered manner,
e.g. as a kit of parts, each part containing at least one
preferably different antigen. For administration, preferably any of
the administration routes may be used as defined above. E.g., one
may treat prostate cancer (PCa), preferably of neoadjuvant and/or
hormone-refractory prostate cancers, and diseases or disorders
related thereto, by inducing or enhancing an adaptive immune
response on the basis of at least two (specifically selected)
antigens encoded by the at least one RNA of the inventive active
(immunostimulatory) composition. Administering of the inventive
active (immunostimulatory) composition and/or the inventive vaccine
may then occur prior, concurrent and/or subsequent to administering
another inventive active (immunostimulatory) composition and/or
inventive vaccine as defined herein which may contain another
combination of RNAs encoding different antigens, wherein each
antigen encoded by the at least one RNA of the inventive active
(immunostimulatory) composition may preferably be suitable for the
therapy of prostate cancer (PCa), preferably of neoadjuvant and/or
hormone-refractory prostate cancers, and diseases or disorders
related thereto. In this context, a therapy as defined herein may
also comprise the modulation of a disease associated to prostate
cancer (PCa), preferably of neoadjuvant and/or hormone-refractory
prostate cancers, and of diseases or disorders related thereto,
[0175] According to one further embodiment, the present invention
furthermore comprises the use of the inventive active
(immunostimulatory) composition or the at least one RNA encoding at
least two (preferably) different antigens as defined herein (for
the preparation of an (inventive) vaccine) for modulating,
preferably to induce or enhance, an immune response in a mammal as
defined above, more preferably to treat and/or to support the
treatment of prostate cancer (PCa), preferably of a neoadjuvant
and/or hormone-refractory prostate cancer, or of diseases or
disorders related thereto. In this context, support of the
treatment of prostate cancer (PCa) may be any combination of a
conventional prostate cancer therapy method of such as surgery,
radiation therapy, hormonal therapy, occasionally chemotherapy,
proton therapy, or some combination of these, and a therapy using
the inventive active (immunostimulatory) composition as defined
herein. Support of the treatment of prostate cancer (PCa) may be
also envisaged in any of the other embodiments defined herein.
[0176] Administration of the inventive active (immunostimulatory)
composition or the at least one RNA encoding at least two
(preferably) different antigens as defined herein or the inventive
vaccine may be carried out in a time staggered treatment. A time
staggered treatment may be e.g. administration of the inventive
active (immunostimulatory) composition or the at least one RNA
encoding at least two (preferably) different antigens as defined
herein or the inventive vaccine prior, concurrent and/or subsequent
to a conventional therapy of prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto, e.g. by administration of
the inventive medicament or the active inventive
(immunostimulatory) composition or vaccine prior, concurrent and/or
subsequent to a therapy or an administration of a therapeutic
suitable for the treatment of prostate cancer (PCa), preferably of
neoadjuvant and/or hormone-refractory prostate cancers, and
diseases or disorders related thereto. Such time staggered
treatment may be carried out using e.g. a kit, preferably a kit of
parts as defined below.
[0177] Time staggered treatment may additionally or alternatively
also comprise an administration of the inventive active
(immunostimulatory) composition or vaccine, preferably of the at
least one RNA encoding at least two (preferably different) antigens
as defined above, in a form, wherein the at least one RNA encoding
at least two (preferably different) antigens as defined above,
preferably forming part of the inventive active (immunostimulatory)
composition or vaccine, is administered parallel, prior or
subsequent to another at least one RNA encoding at least two
(preferably different) antigens as defined above, preferably
forming part of the same inventive active (immunostimulatory)
composition or vaccine. Preferably, the administration (of all at
least one RNAs) occurs within an hour, more preferably within 30
minutes, even more preferably within 15, 10, 5, 4, 3, or 2 minutes
or even within 1 minute. Such time staggered treatment may be
carried out using e.g. a kit, preferably a kit of parts as defined
below.
[0178] According to a final embodiment, the present invention also
provides kits, particularly kits of parts, comprising the active
inventive (immunostimulatory) composition, and/or the inventive
vaccine, and optionally technical instructions with information on
the administration and dosage of the inventive active
(immunostimulatory) composition and/or the inventive vaccine. The
technical instructions may contain information about administration
and dosage of the inventive active (immunostimulatory) composition,
and/or the inventive vaccine. Such kits, preferably kits of parts,
may applied e.g. for any of the above mentioned applications or
uses, preferably for the use of at least one inventive active
(immunostimulatory) composition (for the preparation of an
inventive medicament, preferably a vaccine) for the treatment of
prostate cancer (PCa), preferably of neoadjuvant and/or
hormone-refractory prostate cancers, and diseases or disorders
related thereto. The kits may also be applied for the use of at
least one inventive active (immunostimulatory) composition (for the
preparation of an inventive vaccine) for the treatment of prostate
cancer (PCa), preferably of neoadjuvant and/or hormone-refractory
prostate cancers, and diseases or disorders related thereto,
wherein the inventive active (immunostimulatory) composition)
and/or the vaccine due to the encoded at least two antigens may be
capable to induce or enhance an immune response in a mammal as
defined above. Such kits may further be applied for the use of at
least one inventive active (immunostimulatory) composition, (for
the preparation of an inventive medicament, preferably a vaccine)
for modulating, preferably for eliciting, e.g. to induce or
enhance, an immune response in a mammal as defined above, and
preferably to support treatment of prostate cancer (PCa),
preferably of neoadjuvant and/or hormone-refractory prostate
cancers, and diseases or disorders related thereto. Kits of parts,
as a special form of kits, may contain one or more identical or
different active inventive (immunostimulatory) compositions and/or
one or more identical or different inventive vaccines in different
parts of the kit. Kits of parts may also contain an (e.g. one)
active inventive (immunostimulatory) composition, an (e.g. one)
inventive vaccine and/or the at least one RNA encoding at least one
antigen as defined above in different parts of the kit, e.g. each
part of the kit containing at least one RNA encoding a preferably
different antigen. Additionally, a combination of both types of
kits of parts is possible. Kits of parts may be used, e.g. when a
time staggered treatment is envisaged, e.g. when using different
formulations and/or increasing concentrations of the active
inventive (immunostimulatory) composition, the inventive vaccine
and/or the at least one RNA encoding at least one antigens as
defined above during the same treatment in vivo. Kits of parts may
also be used when a separated formulation or administration of the
different antigens of the inventive active (immunostimulatory)
composition (i.e. in parts) is envisaged or necessary (e.g. for
technical reasons), but e.g. a combined presence of the different
antigens in vivo is still to be achieved. Particularly kits of
parts as a special form of kits are envisaged, wherein each part of
the kit contains at least one preferably different antigen as
defined above, all parts of the kit of parts preferably forming the
active inventive (immunostimulatory) composition or the inventive
vaccine as defined herein. Such specific kits of parts may
particularly be suitable, e.g. if different antigens are formulated
separately as different parts of the kits, but are then
administered at once together or in a time staggered manner to the
mammal in need thereof. In the latter case administration of all of
the different parts of such a kit typically occurs within a short
time limit, such that all antigens are present in the mammal at
about the same time subsequent to administration of the last part
of the kit. Any of the above kits may be used in a treatment as
defined above.
ADVANTAGES OF THE PRESENT INVENTION
[0179] The present invention provides an active (immunostimulatory)
composition for the treatment of prostate cancer (PCa), wherein the
composition comprises at least one RNA, preferably an mRNA,
encoding at least two (preferably different) antigens capable of
eliciting an (adaptive) immune response in a mammal wherein the
antigens are selected from the group consisting of PSA
(Prostate-Specific Antigen), PSMA (Prostate-Specific Membrane
Antigen), PSCA (Prostate Stem Cell Antigen), and STEAP (Six
Transmembrane Epithelial Antigen of the Prostate). Such an active
(immunostimulatory) composition allows efficient treatment of
prostate cancer (PCa) or supplementary treatment when using
conventional therapies. It furthermore avoids the problem of
uncontrolled propagation of the introduced DNA sequences by the use
of RNA as an approach for curative methods. RNA as used in the
inventive active (immunostimulatory) composition has additional
considerable advantages over DNA expression systems e.g. in immune
response, immunization or vaccination. These advantages include,
inter alia, that RNA introduced into a cell is not integrated into
the genome. This avoids the risk of mutation of this gene, which
otherwise may be completely or partially inactivated or give rise
to misinformation. It further avoids other risks of using DNA as an
agent to induce an immune response (e.g. as a vaccine) such as the
induction of pathogenic anti-DNA antibodies in the patient into
whom the foreign DNA has been introduced, so bringing about a
(possibly fatal) immune response. In contrast, no anti-RNA
antibodies have yet been detected.
FIGURES
[0180] The following Figures are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
[0181] FIG. 1: depicts the plasmid construct RNActive
CAP-KLK3(GC)-muag-A70-C30 (SEQ ID NO: 1), encoding for PSA
(prostate specific antigen) (=KLK3). The construct contains
following sequence elements: [0182] a GC-optimized sequence for
stabilization and a better codon usage [0183]
.about.70.times.Adenosin at the 3'-terminal end (poly-A-tail),
[0184] .about.30.times.Cytosin at the 3'-terminal end
(poly-C-tail); This DNA construct corresponds to the coding mRNA
and served as a basis for preparation of the corresponding RNA
construct by in vitro transcription experiments.
[0185] FIG. 2: depicts the wildtype-coding sequence corresponding
to the RNA construct RNActive CAP-KLK3(GC)-muag-A70-C30 (SEQ ID NO:
2), encoding for PSA (prostate specific antigen) (=KLK3), i.e. the
coding sequence (CDS) encoding PSA (prostate specific antigen)
without GC-optimized sequence.
[0186] FIG. 3: depicts the GC-optimized coding sequence of the RNA
construct RNActive CAP-KLK3(GC)-muag-A70-C30 (SEQ ID NO: 3),
encoding for PSA (prostate specific antigen) (=KLK3), i.e. the
coding sequence (CDS) encoding PSA (prostate specific antigen) with
GC-optimized sequence.
[0187] FIG. 4: depicts the plasmid construct RNActive
CAP-FOLH1(GC)-muag-A70-C30 (SEQ ID NO: 4), encoding for PSMA
(prostate specific membrane antigen) (=FOLH1). The construct
contains following sequence elements: [0188] a GC-optimized
sequence for stabilization and a better codon usage [0189]
.about.70.times.Adenosin at the 3'-terminal end (poly-A-tail),
[0190] 30.times.Cytosin at the 3'-terminal end (poly-C-tail); This
DNA construct corresponds to the coding mRNA and served as a basis
for generation of the corresponding RNA construct by in vitro
transcription experiments.
[0191] FIG. 5: depicts the wt-coding sequence corresponding to the
RNA construct RNActive CAP-FOLH1(GC)-muag-A70-C30 (SEQ ID NO: 5),
encoding for PSMA (prostate specific membrane antigen)
(.dbd.FOLH1), i.e. the coding sequence (CDS) encoding PSMA
(prostate specific membrane antigen) (=FOLH1) without GC-optimized
sequence.
[0192] FIG. 6: depicts the GC-optimized coding sequence of the RNA
construct RNActive CAP-FOLH1(GC)-muag-A70-C30 (SEQ ID NO: 6),
encoding for PSMA (prostate specific membrane antigen)
(.dbd.FOLH1), i.e. the coding sequence (CDS) encoding PSMA
(prostate specific membrane antigen) (.dbd.FOLH1) with GC-optimized
sequence.
[0193] FIG. 7: depicts the plasmid construct RNActive
CAP-PSCA(GC)-muag-A70-C30 (SEQ ID NO: 7), encoding for PSCA
(prostate stem cell antigen). The construct contains following
sequence elements: [0194] a GC-optimized sequence for stabilization
and a better codon usage [0195] .about.70.times.Adenosin at the
3'-terminal end (poly-A-tail), [0196] 30.times.Cytosin at the
3'-terminal end (poly-C-tail); This DNA construct corresponds to
the coding mRNA and served as a basis for generation of the
corresponding RNA construct by in vitro transcription
experiments.
[0197] FIG. 8: depicts the wt-coding sequence corresponding to the
RNA construct RNActive CAP-PSCA(GC)-muag-A70-C30 (SEQ ID NO: 8),
encoding for PSCA (prostate stem cell antigen), i.e. the coding
sequence (CDS) encoding PSCA (prostate stem cell antigen) without
GC-optimized sequence.
[0198] FIG. 9: depicts the GC-optimized coding sequence of the RNA
construct RNActive CAP-PSCA(GC)-muag-A70-C30 (SEQ ID NO: 9),
encoding for PSCA (prostate stem cell antigen), i.e. the coding
sequence (CDS) encoding PSCA (prostate stem cell antigen) with
GC-optimized sequence.
[0199] FIG. 10: depicts the plasmid construct RNActive
CAP-STEAP(GC)-muag-A70-C30 (SEQ ID NO: 10), encoding for STEAP (Six
Transmembrane Epithelial Antigen of the Prostate). The construct
contains following sequence elements: [0200] a GC-optimized
sequence for stabilization and a better codon usage [0201]
.about.70.times.Adenosin at the 3'-terminal end (poly-A-tail),
[0202] 30.times.Cytosin at the 3'-terminal end (poly-C-tail); This
DNA construct corresponds to the coding mRNA and served as a basis
for generation of the corresponding RNA construct by in vitro
transcription experiments.
[0203] FIG. 11: depicts the wt-coding sequence corresponding to the
RNA construct RNActive CAP-STEAP(GC)-muag-A70-C30 (SEQ ID NO: 11),
encoding for STEAP (Six Transmembrane Epithelial Antigen of the
Prostate), i.e. the coding sequence (CDS) encoding STEAP (Six
Transmembrane Epithelial Antigen of the Prostate) without
GC-optimized sequence.
[0204] FIG. 12: depicts the GC-optimized coding sequence of the RNA
construct RNActive CAP-STEAP(GC)-muag-A70-C30 (SEQ ID NO: 12),
encoding for STEAP (Six Transmembrane Epithelial Antigen of the
Prostate), i.e. the coding sequence (CDS) encoding STEAP (Six
Transmembrane Epithelial Antigen of the Prostate) with GC-optimized
sequence.
[0205] FIG. 13: depicts the detection of an antigen-specific immune
response (B-cell immune response) by detecting antigen-specific
antibodies. As can be seen in FIG. 13, administration of an
RNA-Mix, i.e. of an PCa-RNA cocktail comprising mRNA coding for
PSA, PSMA, PSCA or STEAP, respectively, showed a significant
induction of an antigen-specific immune response (B-cell immune
response) due to significant formation of IgG2a antibodies against
PSA in comparison to samples containing a buffer or the
control.
[0206] FIG. 14: shows detection of an antigen-specific cellular
immune response by ELISPOT. As can be seen in FIG. 15, vaccination
of mice with an RNA-Mix, i.e. with an PCa-RNA cocktail comprising
mRNA coding for PSA, PSMA, PSCA or STEAP, respectively, or with
mRNA coding for PSA, PSMA, PSCA or STEAP, respectively, leads to a
significant induction of an antigen-specific immune response (CTL)
due to significant formation of INFgamma in comparison to native
mice and mice vaccinated with buffer.
[0207] FIG. 15: depicts immunization and tumor challenge by using
the inventive PCa-RNA cocktail comprising 5 .mu.g from mRNA coding
for PSA, PSMA, PSCA and STEAP respectively. As could be seen in
FIG. 15, the tumor volume is significantly reduced upon
immunization with the PCa-RNA cocktail according to a) 2.times.i.m.
(intramuscularly) comprising mRNA coding for PSA, PSMA, PSCA and
STEAP, respectively. The tumor volume is even more reduced, when
the PCa-RNA cocktail according to a) is administered 4.times.i.m.
(intramuscularly).
[0208] FIG. 16: depicts the induction of PSA-specific IgG1
antibodies. FIG. 16 particularly shows the presence of IgG1
antibodies specific for the tumor antigen PSA in mice which were
vaccinated with the mRNA vaccine consisting of 4 components,
containing GC-optimized mRNAs coding for the human prostate
differentiation antigens PSMA, STEAP, PSA and PSCA. Each was
formulated with the cationic peptide protamine. Control mice were
treated either with buffer (Ringer-lactate) or with irrelevant RNA
(Pp Luc) formulated with protamine analog to the mRNA vaccine. For
the analysis sera from 5 mice in each group were pooled and
titrated. Error bars represent mean deviations of two replicates
from the mean.
[0209] FIG. 17: shows the induction of PSA-specific IgG2a
antibodies. FIG. 17 particularly shows the presence of IgG2a
antibodies specific for the tumor antigen PSA in mice which were
vaccinated with the mRNA vaccine consisting of 4 components, each
containing GC-optimized mRNAs coding for the human prostate
differentiation antigens PSMA, STEAP, PSA and PSCA. Each was
formulated with the cationic peptide protamine. Control mice were
treated either with buffer (Ringer-lactate) or with irrelevant RNA
(Pp Luc) formulated with protamine analog to the mRNA vaccine. For
the analysis sera from 5 mice in each group were pooled and
titrated. Error bars represent mean deviations of two replicates
from the mean.
[0210] FIG. 18: describes the results of the induction of
PSCA-specific IgG1 antibodies. Particularly, FIG. 18 shows the
presence of IgG1 antibodies specific for the tumor antigen PSCA in
mice which were vaccinated with the mRNA vaccine consisting of 4
components, each containing GC-optimized mRNAs coding for the human
prostate differentiation antigens PSMA, STEAP, PSA and PSCA. Each
was formulated with the cationic peptide protamine. Control mice
were treated with buffer (Ringer-lactate). For the analysis sera
from 5 mice in each group were pooled and titrated. Error bars
represent mean deviations of two replicates from the mean.
[0211] FIG. 19: shows the induction of PSMA-specific cytotoxic T
cells. 5 mice per group were vaccinated either with the mRNA
vaccine consisting of 4 components, each containing GC-optimized
mRNAs coding for the human prostate differentiation antigens PSMA,
STEAP, PSA and PSCA or with irrelevant mRNA (Pp Luc) formulated
with protamine analog to the mRNA vaccine. Splenocytes from
vaccinated and control mice were isolated at day 6 after last
vaccination and stimulated either with PSMA-derived or with control
peptide library. The secretion of IFN-.gamma. was measured ex vivo
using ELISPOT technique. Lines represent median values and range of
5 mice per group analyzed separately. Statistical analysis was
performed with GraphPad Prism, p value was calculated with
one-sided Mann-Whitney test
[0212] FIG. 20: shows the induction of PSMA-specific cytotoxic T
cells in vivo. C57BL/6 mice were vaccinated intradermally in three
vaccination cycles with the mRNA vaccine consisting of 4
components, each containing GC-optimized mRNAs coding for the human
prostate differentiation antigens PSMA, STEAP, PSA and PSCA.
Control mice were vaccinated with control mRNA Pp Luc or treated
with buffer (Ringer-lactate). At day 6, after last injection,
30.times.10.sup.6 differentially labeled splenocytes (low and high
population, loaded with PSMA-derived or control peptide library,
mixed at the ratio of 1:1) were infused intravenously. 16 hours
later splenocytes from recipient mice were isolated and analyzed by
flow cytometry. The graph shows single data points for individual
mice. Lines represent the median and range of values. 5 mice per
group were analyzed. Statistical analysis was performed with
GraphPad Prism, p value was calculated with Mann Whitney test.
[0213] FIG. 21: shows the induction of PSA-specific memory
cytotoxic T cells ten weeks after the last vaccination. C57BL/6
mice were vaccinated in two vaccination cycles with the mRNA
vaccine consisting of 4 components, each containing GC-optimized
mRNAs coding for the human prostate differentiation antigens PSMA,
STEAP, PSA and PSCA. Control mice were treated with buffer
(Ringer-lactate). 10 weeks after last injection, 30.times.10.sup.6
differentially labeled splenocytes (low and high population, loaded
with PSA-derived or control peptide library, mixed at the ratio of
1:1) were transplanted intravenously. 16 hours later splenocytes
from recipient mice were isolated and analyzed by flow cytometry.
The graph shows single data points and the lines represent median
values from 6 mice per group analyzed separately. Statistical
analysis was performed with GraphPad Prism, p value was calculated
with Mann Whitney test.
[0214] FIG. 22: shows the induction of PSCA-specific memory
cytotoxic T cells ten weeks after the last vaccination. C57BL/6
mice were vaccinated in two vaccination cycles with the mRNA
vaccine consisting of 4 components, each containing GC-optimized
mRNAs coding for the human prostate differentiation antigens PSMA,
STEAP, PSA and PSCA. Control mice were treated with buffer
(Ringer-lactate). 10 weeks after last injection, 30.times.10.sup.6
differentially labeled splenocytes (low and high population, loaded
with PSCA-derived or control peptide library, mixed at the ratio of
1:1) were transplanted intravenously. 16 hours later splenocytes
from recipient mice were isolated and analyzed by flow cytometry.
The graph shows single data points and the lines represent median
values from 6 mice per group analyzed separately. Statistical
analysis was performed with GraphPad Prism, p value was calculated
with Mann Whitney test.
[0215] FIG. 23: shows the inhibition of tumor growth in mice
vaccinated with the mRNA vaccine consisting of 4 components, each
containing GC-optimized mRNAs coding for the human prostate
differentiation antigens PSMA, STEAP, PSA and PSCA. C57BL/6 mice
were vaccinated intradermally in two vaccination cycles with the
mRNA vaccine. Control mice were treated with buffer. 15 days post
last vaccination mice were challenged subcutaneously with
1.times.10.sup.6 syngeneic TRAMP-C1 tumor cells. The tumor growth
was monitored. Graph shows the logarithm of tumor volume measured
at day 52 after tumor challenge. Statistical analysis was performed
with GraphPad Prism, p value was calculated with Mann Whitney
test.
EXAMPLES
[0216] The following examples are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
1. Preparation of Encoding Plasmids:
[0217] In the following experiment DNA sequences, corresponding to
the respective mRNA sequences end encoding the antigens [0218] PSA
(Prostate-Specific Antigen), [0219] PSMA (Prostate-Specific
Membrane Antigen), [0220] PSCA (Prostate Stem Cell Antigen), and
[0221] STEAP (Six Transmembrane Epithelial Antigen of the
Prostate),
[0222] respectively, were prepared and used for in vitro
transcription and transfection experiments. Thereby, the DNA
sequence corresponding to the native antigen encoding mRNA
(sequences comprising the coding sequences according to FIGS. 2, 5,
8 and 11, i.e. SEQ ID NOs: 2, 5, 8 and 11) were GC-optimized for a
better codon-usage obtaining a sequence comprising the coding
sequences according to FIGS. 3, 6, 9 and 12, i.e. SEQ ID NOs: 3, 6,
9 and 12. Then, the coding sequences was transferred into an
GC-optimized construct (CureVac GmbH, Tubingen, Germany), which has
been modified with a poly-A-tag and a poly-C-tag (A70-C30).
[0223] The final constructs were termed: [0224] RNActive
CAP-KLK3(GC)-muag-A70-C30 (SEQ ID NO: 1), [0225] RNActive
CAP-FOLH1(GC)-muag-A70-C30 (SEQ ID NO: 4), [0226] RNActive
CAP-PSCA(GC)-muag-A70-C30 (SEQ ID NO: 7), and [0227] RNActive
CAP-STEAP(GC)-muag-A70-C30 (SEQ ID NO: 10), respectively.
[0228] The final constructs comprise a sequence according to
sequences as shown in FIGS. 1, 4, 7 and 10 (SEQ ID NOs: 1, 4, 7 and
10), respectively, which contain following sequence elements:
[0229] GC-optimized sequence for stabilization and a better codon
usage [0230] .about.70.times.Adenosin at the 3'-terminal end
(poly-A-tail), [0231] 30.times.Cytosin at the 3'-terminal end
(poly-C-tail);
2. In Vitro Transcription:
[0232] Based on the recombinant plasmid DNA obtained in Example 1
the RNA sequences were prepared by in vitro transcription.
Therefore, the recombinant plasmid DNA was linearized and
subsequently in vitro transcribed using the T7 RNA polymerase. The
DNA template was then degraded by DNase I digestion, and the RNA
was recovered by LiCl precipitation and further cleaned by HPLC
extraction (PUREMessenger.RTM., CureVac GmbH, Tubingen,
Germany).
3. Complexation with Protamine
[0233] For transfection of the RNA into cells and organisms the RNA
obtained by in vitro transcription was preferably complexed, more
preferably with protamine upon mixing the RNA with protamine.
4. Immunization Experiments
[0234] For immunization the RNA obtained by the in vitro
transcription experiment as shown above (see Experiment 2) was
transfected into mice (Mice: C57 BL/6), preferably when complexed
with protamine. Vaccination occurred in different groups, wherein
one group (control group) mice was injected with buffer as control.
4 mice per group were immunized intradermally four times with 20
.mu.g mRNA (5 .mu.g per gene) complexed with protamine, wherein the
RNA codes for PSA, PSMA, PSCA and STEAP.
5. Detection of an Antigen-Specific Immune Response (B-Cell Immune
Response):
[0235] Detection of an antigen-specific immune response (B-cell
immune response) was carried out by detecting antigen-specific
antibodies. Therefore, blood samples were taken from the vaccinated
mice one week after the last vaccination and sera were prepared.
MaxiSorb plates (Nalgene Nunc International) were coated with human
PSA protein (0.5 .mu.g/well). After blocking with 1.times.PBS
containing 0.05% Tween-20 and 1% BSA the plates were incubated with
diluted mouse serum (1:30, 1.90, 1:270, 1:810). Subsequently a
biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was
added. After washing, the plate was incubated with Horseradish
peroxidase-streptavidin and subsequently the conversion of the ABTS
substrate (2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid)
was measured.
[0236] As can be seen in FIG. 13, administration of an RNA-Mix,
i.e. of an PCa-RNA cocktail comprising mRNA coding for PSA, PSMA,
PSCA or STEAP, respectively, (sequences as shown in FIGS. 1, 4, 7
and 10 (SEQ ID NOs: 1, 4, 7 and 10), respectively) showed a
significant induction of an antigen-specific immune response
(B-cell immune response) due to significant formation of IgG2a
antibodies against PSA in comparison to samples from control
mice.
6. Detection of an Antigen-Specific Cellular Immune Response by
ELISPOT:
[0237] 2 months after the last vaccination mice were sacrificed,
the spleens were removed and the splenocytes were isolated. The
splenocytes were incubated for 7 days in presence of IL-4 to select
dendritic cells. To determine an antigen-specific cellular immune
response INFgamma secretion was measured after re-stimulation. As
target cells splenocytes from a native mouse were used which were
electroporated with the PCa-mRNA-cocktail (Mix) or with mRNA coding
for PSA, PSMA, PSCA or STEAP (sequences as shown in FIGS. 1, 4, 7
and 10 (SEQ ID NOs: 1, 4, 7 and 10), respectively).
[0238] For detection of INFgamma a coat multiscreen plate
(Millipore) was incubated overnight with coating buffer 0.1 M
Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4
g/l NaHCO.sub.3) comprising antibody against INF.gamma. (BD
Pharmingen, Heidelberg, Germany). Target cells and effector cells
were incubated together in the plate in the ratio of 1:20 for 24 h.
The plate was washed with 1.times.PBS and incubated with a
biotin-coupled secondary antibody. After washing with
1.times.PBS/0.05% Tween-20 the substrate (5-Bromo-4-Cloro-3-Indolyl
Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma
Aldrich, Taufkirchen, Germany) was added to the plate and the
conversion of the substrate could be detected visually.
[0239] As can be seen in FIG. 14, vaccination of mice with the
PCa-mRNA-cocktail coding for PSA, PSMA, PSCA or STEAP, (sequences
as shown in FIGS. 1, 4, 7 and 10 (SEQ ID NOs: 1, 4, 7 and 10),
respectively) leads to a significant induction of an
antigen-specific immune response (CTL) against all four antigens
due to significant formation of INFgamma in comparison to native
mice and mice vaccinated with buffer.
7. Tumor Challenge:
[0240] a) Immunization: [0241] 20 .mu.g mRNA (an PCa-RNA cocktail
comprising 5 .mu.g from mRNA coding for PSA, PSMA, PSCA and STEAP
respectively, e.g. constructs RNActive CAP-KLK3(GC)-muag-A70-C30,
RNActive CAP-FOLH1(GC)-muag-A70-C30, RNActive
CAP-PSCA(GC)-muag-A70-C30, and RNActive CAP-STEAP(GC)-muag-A70-C30,
respectively (sequences as shown in FIGS. 1, 4, 7 and 10, i.e. SEQ
ID NOs: 1, 4, 7 and 10)) were injected intramuscularly in the mice.
The immunization was repeated 1 or 3 times within 7 weeks. 40 days
after the last immunization 1 Mio Tramp-C1 tumor cells were
injected subcutaneously in the mice. Within 50 days tumor volume
was determined.
[0242] b) Results [0243] As could be seen in FIG. 15, the tumor
volume is significantly reduced upon immunization with the PCa-RNA
cocktail according to a) comprising mRNA coding for PSA, PSMA, PSCA
and STEAP, respectively, 2.times.i.m. (intramuscularly). The tumor
volume is even more reduced, when the PCa-RNA cocktail according to
a) is administered 4.times.i.m. (intramuscularly). 8. Preparation
of an mRNA Vaccine and Induction of Antigen-Specific Cytotoxic
Antibodies and Antigen-Specific Cytotoxic T-Cells: 8.1 Preparation
of an mRNA Vaccine:
[0244] The mRNA vaccine consists of GC-optimized mRNAs coding for
the human prostate differentiation antigens PSMA, STEAP, PSA and
PSCA (according to SEQ ID NOs: 3, 6, 9 and 12), each antigen
formulated with the cationic peptide protamine at a mass ratio of
4:1 (RNA:protamine) dissolved in 80% (v/v) Ringer-lactate
solution.
8.2 Vaccination
[0245] C57BL/6 mice were vaccinated intradermally with 64 .mu.g (16
.mu.g per antigen) of the mRNA vaccine as described under 8.1
above. Control mice were treated either with buffer
(Ringer-lactate) or with irrelevant RNA (GC-enriched mRNA coding
for Photinus pyralis luciferase) formulated with protamine analog
to the mRNA vaccine. Vaccination comprised two or three
immunization cycles in week 1, 3, (and 7). Each cycle consisted of
4 injections on day 1, 2, 3 and 4 of the week.
8.3 Tumor Challenge
[0246] 15 days after completion of vaccination, 1.times.10.sup.6
TRAMP-C1 cells (transgenic adenocarcinoma of the mouse prostate
cell line 1, expressing mouse homologues to human PSMA, PSCA and
STEAP) per mouse were transplanted subcutaneously. Tumors were
palpable 4 weeks after tumor cell inoculation. The tumor growth was
monitored by measuring the tumor size with calipers.
8.4 Detection of Antigen-Specific Antibodies
[0247] 14 days after the last vaccination, blood samples (200
.mu.l) were taken retro-orbitally and serum was analyzed for the
presence of antigen specific antibody subtypes IgG1 and IgG2a using
the following ELISA protocol. 96-well ELISA plates were coated with
recombinant protein PSCA or human purified PSA (both: 10 .mu.g/ml
in coating buffer) and (after blocking and washing) incubated with
serum for 4 hours at 37.degree. C. After incubation with
biotin-labeled antibody against mouse IgG1 or IgG2a followed by
incubation with HRP-Streptavidin, the TMB-substrate was added. The
colorimetric reaction was measured at 450 nm using a Tecan ELISA
reader.
8.5 Elispot--Detection of CTL (Cytotoxic T Cell) Responses
[0248] For the detection of CTL (cytotoxic T cell) responses the
analysis of IFN-.gamma. secretion in response to a specific
stimulus can be visualized at a single cell level using the ELISPOT
technique.
[0249] Splenocytes from mice vaccinated with the mRNA vaccine as
described under 8.1 above and control mice were isolated 6 days
after the last vaccination in the third vaccination cycle and then
transferred into 96-well ELISPOT plates coated with an
.alpha.-IFN-.gamma. capture antibody. The cells were then
stimulated for 24 hours at 37.degree. C. either with a PSMA-derived
peptide library or with a HIV-derived library as a control. Both
libraries were used at a concentration of 1 .mu.g/peptide/ml. After
the incubation period the cells were washed out of the plate and
the IFN-.gamma. secreted by the cells was detected using a
biotinylated secondary antibody against murine IFN-.gamma.,
followed by streptavidin-AKP. Spots were visualized using BCIP/NBT
substrate and counted using an automated ELISPOT reader (Immunospot
Analyzer, CTL Analyzers LLC).
8.6 In Vivo Cytotoxicity
[0250] To detect the activity of cytotoxic T cells in vivo lysis of
specific target cells in the vaccinated and control mice was
analyzed. The assay was performed one week as well as 10 weeks
after last immunization to detect induction of memory cytotoxic T
lymphocytes.
[0251] Splenocytes from naive donor mice were isolated and labeled
with two different concentrations of the fluorescent dye CFSE.
Thereby two populations of high and low fluorescence intensity were
created. Cells with low fluorescence were loaded for 3 hours at
37.degree. C. with PSMA-, PSA- or PSCA-derived restricted peptide
libraries (1 .mu.g/ml/peptide). Control cells with high
fluorescence were loaded with HIV Pol-derived peptide library (1
.mu.g/ml/peptide) accordingly. Both populations were mixed in
cell:cell ratio of 1:1 and transplanted intravenously into naive or
vaccinated recipient mice. 16 hours later splenocytes from
recipient mice were isolated and analyzed for the presence of
fluorescent cells by flow cytometry. The shift of the ratio between
low and high fluorescent cells in vaccinated mice represented the
specific killing of target cells in vivo. An exact ratio between
the number of observed specific target cells and observed control
cells was estimated as a mean value in all control mice. This ratio
allows a prediction of expected specific targets in vaccinated mice
on the base of observed control cells. A ratio between observed and
predicted number of specific target represent the percentage of
remaining (non killed) specific targets.
[0252] The following rules were applied:
Ratio=specific targets (observed) in control mice/control targets
(observed) in control mice
Ratio.times.number of observed control targets in vaccinated
mice=predicted number of specific targets in vaccinated mice
% Killing=(1-(specific targets observed/specific targets
predicted)
8.7 Statistical Analysis
[0253] Statistical analysis was performed using GraphPad Prism
Software, Version 5.01. Due to the lack of normal distribution of
the sample populations and the small sample size, non-parametric
Mann Whitney tests were used to analyze the differences between the
test groups with a significance level of 5%.
8.9 Results and Discussion
[0254] Mice were vaccinated with the mRNA vaccine consisting of
GC-optimized mRNAs coding for the human prostate differentiation
antigens PSMA, STEAP, PSA and PSCA each formulated separately with
the cationic peptide protamine at a mass ratio of 4:1
(RNA:protamine). Control mice were treated either with buffer
(Ringer-lactate) or with irrelevant RNA (Pp Luc) formulated with
protamine analog to the mRNA vaccine.
[0255] a) Induction of Antigen-Specific Antibodies in Response to
Vaccination with the mRNA Vaccine:
[0256] Restricted by the availability of recombinant protein
required for the detection of antibodies the induction of specific
antibodies for two of the four antigens was tested. For both
analyzed proteins PSA and PSCA antigen specific antibodies in serum
of mice vaccinated with the mRNA vaccine was detected demonstrating
that both mRNAs are functional and immunogenic in vivo (see FIGS.
16 to 18).
[0257] b) Induction of Antigen-Specific Cytotoxic T Cells in
Response to Vaccination with the mRNA Vaccine:
[0258] Furthermore; the activation of cytotoxic T-cells in response
to the administration of the mRNA vaccine was analyzed applying two
independent functional assays: secretion of IFN-.gamma. and in vivo
cytotoxicity assay. IFN-.gamma. is the main mediator of Th1
response and secreted by activated CTLs. Therefore, the presence of
antigen-specific cytotoxic T-cells in splenocytes from vaccinated
mice was investigated using ELISPOT technique. As an antigenic
stimulus for splenocytes a restricted PSMA-derived peptide library
was used. The stimulation with this library led to high IFN-.gamma.
secretion but only in splenocytes from mice vaccinated with the
mRNA vaccine and not in control mice, vaccinated with mRNA coding
for irrelevant protein luciferase (Pp Luc). None of the splenocytes
reacted to the HIV-derived control peptide library (FIG. 19).
[0259] Antigen specific cellular immunity towards PSMA could be
confirmed by an in vivo cytotoxicity assay. Specific killing of
target cells loaded with a restricted PSMA-derived peptide library
was observed in all five mice vaccinated with the mRNA vaccine,
however the cytotoxic effect ranged between 12 and 90%. Mice
vaccinated with control RNA (Pp Luc) or injection solution
(Ringer-lactate buffer) were not able to eliminate infused target
cells (see FIG. 20).
[0260] In another experiment it was investigated whether
vaccination with the mRNA vaccine induces sustained memory effects
in vivo. To address this point the presence of antigen-specific
memory cytotoxic T cells was determined ten weeks after vaccination
was completed by an in vivo cytotoxicity test. Specific killing of
target cells was observed after vaccination for PSA as well as PSCA
(FIG. 21-22). Taken together the mRNA vaccine induces sustained
long lasting (at least 10 weeks) cellular immunity in mice.
[0261] Finally the ability of the mRNA vaccine to induce anti-tumor
responses in mice was tested. To address this point we used the
TRAMP-C1 tumor cell line. TRAMP-C1 expresses the mouse homologues
to the human antigens PSMA, PSCA and STEAP included in the mRNA
vaccine as antigen encoding mRNAs. Homology between mouse and man
varies between 50-80% for the different antigens. Due to the fact
that mice were vaccinated with mRNAs coding for human proteins, a
protection against TRAMP-C1 tumor cells can only be mediated by
cross-reactivity. As presented in FIG. 23 vaccination of mice with
the mRNA vaccine mediated the protection against transplanted
TRAMP-C1 tumor growth. This observation indicates the ability of
the mRNA vaccine to induce cross-reactivity in mice.
Sequence CWU 1
1
141990DNAArtificial SequenceDescription of sequence construct
RNActiveII KLK3(GC) = PSA (see Figure 1) 1gggagaaagc ttaccatgtg
ggtgccggtc gtgttcctga ccctcagcgt gacgtggatc 60ggcgccgcgc ccctgatcct
gtcgcggatc gtggggggct gggagtgcga gaagcacagc 120cagccctggc
aggtgctggt ggccagccgc ggccgggccg tgtgcggcgg cgtgctggtg
180cacccccagt gggtgctgac cgccgcccac tgcatccgga acaagagcgt
catcctgctg 240ggccggcaca gcctgttcca ccccgaggac accggccagg
tgttccaggt gagccacagc 300ttcccccacc ccctgtacga catgagcctc
ctgaagaacc ggttcctgcg gcccggcgac 360gacagcagcc acgacctgat
gctgctgcgg ctgagcgagc ccgccgagct gaccgacgcc 420gtgaaggtga
tggacctgcc gacccaggag cccgccctgg gcaccacctg ctacgccagc
480ggctggggga gcatcgagcc cgaggagttc ctcaccccca agaagctgca
gtgcgtggac 540ctgcacgtga tcagcaacga cgtgtgcgcc caggtgcacc
cccagaaggt gaccaagttc 600atgctgtgcg ccggccggtg gaccggcggc
aagagcacct gcagcggcga cagcggcggc 660cccctggtct gcaacggcgt
gctgcagggc atcaccagct ggggcagcga gccctgcgcc 720ctgcccgagc
gccccagcct gtacaccaag gtggtgcact accggaagtg gatcaaggac
780accatcgtgg ccaacccgtg accactagtt ataagactga ctagcccgat
gggcctccca 840acgggccctc ctcccctcct tgcaccgaga ttaataaaaa
aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaat attccccccc cccccccccc 960cccccccccc ccctctagac
aattggaatt 9902786DNAArtificial SequenceDescription of sequence
construct CDS KLK3(wt) = PSA (see Figure 2) 2atgtgggtcc cggttgtctt
cctcaccctg tccgtgacgt ggattggtgc tgcacccctc 60atcctgtctc ggattgtggg
aggctgggag tgcgagaagc attcccaacc ctggcaggtg 120cttgtggcct
ctcgtggcag ggcagtctgc ggcggtgttc tggtgcaccc ccagtgggtc
180ctcacagctg cccactgcat caggaacaaa agcgtgatct tgctgggtcg
gcacagcctg 240tttcatcctg aagacacagg ccaggtattt caggtcagcc
acagcttccc acacccgctc 300tacgatatga gcctcctgaa gaatcgattc
ctcaggccag gtgatgactc cagccacgac 360ctcatgctgc tccgcctgtc
agagcctgcc gagctcacgg atgctgtgaa ggtcatggac 420ctgcccaccc
aggagccagc actggggacc acctgctacg cctcaggctg gggcagcatt
480gaaccagagg agttcttgac cccaaagaaa cttcagtgtg tggacctcca
tgttatttcc 540aatgacgtgt gtgcgcaagt tcaccctcag aaggtgacca
agttcatgct gtgtgctgga 600cgctggacag ggggcaaaag cacctgctcg
ggtgattctg ggggcccact tgtctgtaat 660ggtgtgcttc aaggtatcac
gtcatggggc agtgaaccat gtgccctgcc cgaaaggcct 720tccctgtaca
ccaaggtggt gcattaccgg aagtggatca aggacaccat cgtggccaac 780ccctga
7863786DNAArtificial SequenceDescription of sequence construct CDS
KLK3(GC) = PSA (see Figure 3) 3atgtgggtgc ccgtcgtgtt cctgaccctc
agcgtgacct ggatcggcgc cgccccgctg 60atcctgtccc ggatcgtcgg gggctgggag
tgcgagaagc acagccagcc ctggcaggtg 120ctcgtggcgt cccgcgggcg
ggccgtctgc ggcggggtgc tggtgcaccc ccagtgggtc 180ctgacggccg
cccactgcat ccgcaacaag agcgtgatcc tcctgggccg gcactccctg
240ttccaccccg aggacaccgg ccaggtgttc caggtcagcc actccttccc
gcaccccctc 300tacgacatga gcctgctgaa gaaccgcttc ctccggcccg
gggacgactc cagccacgac 360ctgatgctgc tccgcctgtc cgagcccgcc
gagctgaccg acgcggtgaa ggtgatggac 420ctcccgaccc aggagcccgc
cctgggcacg acctgctacg ccagcgggtg gggctccatc 480gagcccgagg
agttcctgac ccccaagaag ctccagtgcg tcgacctgca cgtgatcagc
540aacgacgtgt gcgcccaggt ccacccgcag aaggtgacca agttcatgct
gtgcgcgggg 600cggtggacgg gcggcaagtc cacctgcagc ggggactccg
gcgggcccct cgtgtgcaac 660ggcgtcctgc agggcatcac cagctggggg
tccgagccct gcgccctgcc cgagcgcccg 720agcctctaca ccaaggtggt
gcactaccgg aagtggatca aggacacgat cgtcgccaac 780ccctga
78642457DNAArtificial SequenceDescription of sequence construct
RNActiveII FOLH1(GC) = PSMA (see Figure 4) 4gggagaaagc ttaccatgtg
gaacctgctc cacgagaccg acagcgccgt ggcgacggcc 60cggcgcccgc ggtggctgtg
cgccggcgcc ctggtcctgg ccgggggctt cttcctgctg 120ggcttcctgt
tcggctggtt catcaagtcg agcaacgagg ccaccaacat cacccccaag
180cacaacatga aggccttcct cgacgagctg aaggccgaga acatcaagaa
gttcctgtac 240aacttcaccc agatccccca cctggccggg accgagcaga
acttccagct ggccaagcag 300atccagagcc agtggaagga gttcggcctg
gactcggtgg agctggcgca ctacgacgtg 360ctgctcagct accccaacaa
gacccacccc aactacatca gcatcatcaa cgaggacggc 420aacgagatct
tcaacaccag cctgttcgag cccccgcccc ccggctacga gaacgtgtcg
480gacatcgtgc cccccttcag cgccttcagc ccgcagggca tgcccgaggg
ggacctggtg 540tacgtgaact acgcccggac ggaggacttc ttcaagctgg
agcgcgacat gaagatcaac 600tgcagcggca agatcgtgat cgcccggtac
ggcaaggtgt tccggggcaa caaggtgaag 660aacgcccagc tggccggggc
caagggcgtg atcctgtact cggaccccgc cgactacttc 720gcccccggcg
tgaagagcta ccccgacggc tggaacctgc ccggcggggg cgtccagcgc
780ggcaacatcc tcaacctgaa cggcgccggc gacccgctga cccccgggta
ccccgcgaac 840gagtacgcct accggcgggg catcgccgag gccgtgggcc
tgcccagcat ccccgtgcac 900ccgatcggct actacgacgc ccagaagctg
ctggagaaga tgggcgggag cgccccgccc 960gactcgagct ggcggggcag
cctgaaggtg ccctacaacg tgggccccgg cttcaccggg 1020aacttctcga
cccagaaggt gaagatgcac atccacagca ccaacgaggt gacccgcatc
1080tacaacgtga tcggcaccct gcggggcgcc gtggagcccg accggtacgt
gatcctcggc 1140gggcaccgcg acagctgggt gttcggcggc atcgaccccc
agagcggcgc cgccgtggtc 1200cacgagatcg tgcggtcgtt cggcaccctg
aagaaggagg ggtggcggcc ccgccggacg 1260atcctgttcg ccagctggga
cgcggaggag ttcggcctgc tgggcagcac cgagtgggcc 1320gaggagaaca
gccggctgct gcaggagcgg ggcgtggcct acatcaacgc cgactcgagc
1380atcgagggca actacaccct ccgcgtggac tgcaccccgc tgatgtacag
cctggtgcac 1440aacctgacca aggagctgaa gagccccgac gaggggttcg
agggcaagtc gctgtacgag 1500agctggacca agaagagccc ctcgcccgag
ttcagcggca tgccccggat cagcaagctg 1560ggcagcggga acgacttcga
ggtgttcttc cagcggctgg gcatcgcctc gggccgcgcc 1620cggtacacca
agaactggga gacgaacaag ttcagcggct accccctcta ccacagcgtg
1680tacgagacct acgagctggt ggagaagttc tacgacccca tgttcaagta
ccacctgacc 1740gtggcccagg tgcggggcgg gatggtgttc gagctggcca
acagcatcgt gctgcccttc 1800gactgccgcg actacgccgt cgtgctgcgg
aagtacgccg acaagatcta ctcgatcagc 1860atgaagcacc cccaggagat
gaagacctac agcgtgagct tcgactcgct gttcagcgcg 1920gtgaagaact
tcaccgagat cgccagcaag ttctcggagc ggctccagga cttcgacaag
1980agcaacccga tcgtgctgcg catgatgaac gaccagctga tgttcctgga
gcgggccttc 2040atcgaccccc tgggcctgcc cgaccggccc ttctaccggc
acgtgatcta cgcccccagc 2100agccacaaca agtacgccgg cgagtcgttc
ccggggatct acgacgccct gttcgacatc 2160gagagcaagg tggaccccag
caaggcctgg ggcgaggtga agcgccagat ctacgtggcc 2220gccttcaccg
tgcaggccgc ggccgagacc ctgagcgagg tggcctgacc actagttata
2280agactgacta gcccgatggg cctcccaacg ggccctcctc ccctccttgc
accgagatta 2340ataaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2400aaaaaatatt cccccccccc cccccccccc
cccccccccc tctagacaat tggaatt 245752253DNAArtificial
SequenceDescription of sequence construct CDS FOLH1(wt) = PSMA (see
Figure 5) 5atgtggaatc tccttcacga aaccgactcg gctgtggcca ccgcgcgccg
cccgcgctgg 60ctgtgcgctg gggcgctggt gctggcgggt ggcttctttc tcctcggctt
cctcttcggg 120tggtttataa aatcctccaa tgaagctact aacattactc
caaagcataa tatgaaagca 180tttttggatg aattgaaagc tgagaacatc
aagaagttct tatataattt tacacagata 240ccacatttag caggaacaga
acaaaacttt cagcttgcaa agcaaattca atcccagtgg 300aaagaatttg
gcctggattc tgttgagcta gcacattatg atgtcctgtt gtcctaccca
360aataagactc atcccaacta catctcaata attaatgaag atggaaatga
gattttcaac 420acatcattat ttgaaccacc tcctccagga tatgaaaatg
tttcggatat tgtaccacct 480ttcagtgctt tctctcctca aggaatgcca
gagggcgatc tagtgtatgt taactatgca 540cgaactgaag acttctttaa
attggaacgg gacatgaaaa tcaattgctc tgggaaaatt 600gtaattgcca
gatatgggaa agttttcaga ggaaataagg ttaaaaatgc ccagctggca
660ggggccaaag gagtcattct ctactccgac cctgctgact actttgctcc
tggggtgaag 720tcctatccag atggttggaa tcttcctgga ggtggtgtcc
agcgtggaaa tatcctaaat 780ctgaatggtg caggagaccc tctcacacca
ggttacccag caaatgaata tgcttatagg 840cgtggaattg cagaggctgt
tggtcttcca agtattcctg ttcatccaat tggatactat 900gatgcacaga
agctcctaga aaaaatgggt ggctcagcac caccagatag cagctggaga
960ggaagtctca aagtgcccta caatgttgga cctggcttta ctggaaactt
ttctacacaa 1020aaagtcaaga tgcacatcca ctctaccaat gaagtgacaa
gaatttacaa tgtgataggt 1080actctcagag gagcagtgga accagacaga
tatgtcattc tgggaggtca ccgggactca 1140tgggtgtttg gtggtattga
ccctcagagt ggagcagctg ttgttcatga aattgtgagg 1200agctttggaa
cactgaaaaa ggaagggtgg agacctagaa gaacaatttt gtttgcaagc
1260tgggatgcag aagaatttgg tcttcttggt tctactgagt gggcagagga
gaattcaaga 1320ctccttcaag agcgtggcgt ggcttatatt aatgctgact
catctataga aggaaactac 1380actctgagag ttgattgtac accgctgatg
tacagcttgg tacacaacct aacaaaagag 1440ctgaaaagcc ctgatgaagg
ctttgaaggc aaatctcttt atgaaagttg gactaaaaaa 1500agtccttccc
cagagttcag tggcatgccc aggataagca aattgggatc tggaaatgat
1560tttgaggtgt tcttccaacg acttggaatt gcttcaggca gagcacggta
tactaaaaat 1620tgggaaacaa acaaattcag cggctatcca ctgtatcaca
gtgtctatga aacatatgag 1680ttggtggaaa agttttatga tccaatgttt
aaatatcacc tcactgtggc ccaggttcga 1740ggagggatgg tgtttgagct
agccaattcc atagtgctcc cttttgattg tcgagattat 1800gctgtagttt
taagaaagta tgctgacaaa atctacagta tttctatgaa acatccacag
1860gaaatgaaga catacagtgt atcatttgat tcactttttt ctgcagtaaa
gaattttaca 1920gaaattgctt ccaagttcag tgagagactc caggactttg
acaaaagcaa cccaatagta 1980ttaagaatga tgaatgatca actcatgttt
ctggaaagag catttattga tccattaggg 2040ttaccagaca ggccttttta
taggcatgtc atctatgctc caagcagcca caacaagtat 2100gcaggggagt
cattcccagg aatttatgat gctctgtttg atattgaaag caaagtggac
2160ccttccaagg cctggggaga agtgaagaga cagatttatg ttgcagcctt
cacagtgcag 2220gcagctgcag agactttgag tgaagtagcc taa
225362253DNAArtificial SequenceDescription of sequence construct
CDS FOLH1(GC) = PSMA (see Figure 6) 6atgtggaacc tgctccacga
gaccgacagc gccgtggcca ccgcgcggcg cccccggtgg 60ctgtgcgccg gcgccctggt
cctcgccggg ggcttcttcc tgctggggtt cctcttcggc 120tggttcatca
agtccagcaa cgaggccacg aacatcaccc cgaagcacaa catgaaggcg
180ttcctggacg agctgaaggc cgagaacatc aagaagttcc tctacaactt
cacccagatc 240ccccacctgg ccgggaccga gcagaacttc cagctggcca
agcagatcca gtcccagtgg 300aaggagttcg gcctcgacag cgtggagctg
gcgcactacg acgtgctgct ctcctacccc 360aacaagacgc accccaacta
catcagcatc atcaacgagg acggcaacga gatcttcaac 420acctccctgt
tcgagccgcc cccccccggg tacgagaacg tcagcgacat cgtgccgccc
480ttctccgcct tcagccccca gggcatgccc gagggggacc tggtgtacgt
caactacgcc 540cgcaccgagg acttcttcaa gctcgagcgg gacatgaaga
tcaactgctc cggcaagatc 600gtgatcgccc gctacgggaa ggtgttccgg
ggcaacaagg tcaagaacgc ccagctggcg 660ggcgccaagg gggtgatcct
gtacagcgac ccggccgact acttcgcccc cggcgtgaag 720tcctaccccg
acgggtggaa cctccccggc ggcggggtcc agcgcggcaa catcctgaac
780ctgaacgggg ccggcgaccc gctcaccccc gggtaccccg cgaacgagta
cgcctaccgg 840cgcggcatcg ccgaggccgt gggcctgccc agcatcccgg
tgcaccccat cgggtactac 900gacgcccaga agctgctcga gaagatgggc
gggtccgcgc cccccgacag ctcctggcgg 960ggcagcctga aggtcccgta
caacgtgggg cccggcttca cgggcaactt ctccacccag 1020aaggtgaaga
tgcacatcca cagcaccaac gaggtcaccc gcatctacaa cgtgatcggg
1080acgctgcggg gcgccgtgga gcccgaccgc tacgtcatcc tcgggggcca
ccgggacagc 1140tgggtgttcg ggggcatcga cccccagtcc ggcgccgccg
tggtccacga gatcgtgcgc 1200agcttcggga ccctgaagaa ggagggctgg
cggccgcgcc ggaccatcct gttcgcctcc 1260tgggacgcgg aggagttcgg
gctcctgggc agcaccgagt gggccgagga gaactcccgc 1320ctgctccagg
agcggggcgt cgcctacatc aacgccgaca gctccatcga ggggaactac
1380acgctgcgcg tggactgcac cccgctgatg tacagcctcg tgcacaacct
gaccaaggag 1440ctgaagtccc ccgacgaggg cttcgagggg aagagcctct
acgagtcctg gaccaagaag 1500agcccgtccc ccgagttcag cggcatgccc
cggatctcca agctggggag cggcaacgac 1560ttcgaggtct tcttccagcg
gctgggcatc gcgtccgggc gcgcccggta cacgaagaac 1620tgggagacca
acaagttcag cggctacccc ctctaccact ccgtgtacga gacctacgag
1680ctggtggaga agttctacga cccgatgttc aagtaccacc tgaccgtcgc
ccaggtgcgc 1740gggggcatgg tgttcgagct ggccaacagc atcgtcctcc
ccttcgactg ccgggactac 1800gccgtggtgc tgcgcaagta cgcggacaag
atctacagca tctccatgaa gcacccccag 1860gagatgaaga cgtacagcgt
ctccttcgac agcctgttct ccgccgtgaa gaacttcacc 1920gagatcgcca
gcaagttctc cgagcggctc caggacttcg acaagagcaa ccccatcgtg
1980ctgcgcatga tgaacgacca gctgatgttc ctcgagcggg ccttcatcga
cccgctgggg 2040ctgcccgacc gccccttcta ccggcacgtc atctacgccc
cctccagcca caacaagtac 2100gcgggcgagt ccttcccggg gatctacgac
gccctcttcg acatcgagag caaggtggac 2160ccctccaagg cctggggcga
ggtgaagcgc cagatctacg tcgccgcctt caccgtgcag 2220gcggccgccg
agaccctgag cgaggtggcc tga 22537576DNAArtificial SequenceDescription
of sequence construct RNActiveII PSCA(GC) (see Figure 7)
7gggagaaagc ttaccatgaa ggccgtgctg ctcgcgctgc tgatggccgg cctggccctg
60cagccgggga ccgccctgct gtgctacagc tgcaaggccc aggtctcgaa cgaggactgc
120ctgcaggtgg agaactgcac gcagctgggc gagcagtgct ggaccgcccg
gatccgcgcc 180gtgggcctgc tcaccgtgat cagcaagggc tgcagcctga
actgcgtgga cgacagccag 240gactactacg tgggcaagaa gaacatcacc
tgctgcgaca ccgacctgtg caacgccagc 300ggcgcccacg ccctgcagcc
cgcggccgcc atcctggccc tgctgcccgc cctgggcctg 360ctgctctggg
gccccggcca gctgtgacca ctagttataa gactgactag cccgatgggc
420ctcccaacgg gccctcctcc cctccttgca ccgagattaa taaaaaaaaa
aaaaaaaaaa 480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaatattc cccccccccc 540cccccccccc ccccccccct ctagacaatt ggaatt
5768372DNAArtificial SequenceDescription of sequence construct CDS
PSCA(wt) (see Figure 8) 8atgaaggctg tgctgcttgc cctgttgatg
gcaggcttgg ccctgcagcc aggcactgcc 60ctgctgtgct actcctgcaa agcccaggtg
agcaacgagg actgcctgca ggtggagaac 120tgcacccagc tgggggagca
gtgctggacc gcgcgcatcc gcgcagttgg cctcctgacc 180gtcatcagca
aaggctgcag cttgaactgc gtggatgact cacaggacta ctacgtgggc
240aagaagaaca tcacgtgctg tgacaccgac ttgtgcaacg ccagcggggc
ccatgccctg 300cagccggctg ctgccatcct tgcgctgctc cctgcactcg
gcctgctgct ctggggaccc 360ggccagctct ag 3729372DNAArtificial
SequenceDescription of sequence construct CDS PSCA(GC) (see Figure
9) 9atgaaggccg tgctgctcgc cctgctgatg gcgggcctcg ccctgcagcc
cgggaccgcc 60ctgctctgct acagctgcaa ggcccaggtc tccaacgagg actgcctgca
ggtggagaac 120tgcacccagc tgggcgagca gtgctggacg gcccggatcc
gcgcggtggg gctcctgacc 180gtcatcagca agggctgctc cctgaactgc
gtggacgaca gccaggacta ctacgtgggg 240aagaagaaca tcacctgctg
cgacaccgac ctctgcaacg cctccggcgc ccacgccctg 300cagccggcgg
ccgccatcct ggccctcctg cccgccctgg gcctcctgct gtgggggccc
360ggccagctct ga 372101224DNAArtificial SequenceDescription of
sequence construct RNActive II STEAP (GC) = STEAP1 (see Figure 10)
10gggagaaagc ttaccatgga gagccggaag gacatcacca accaggagga gctgtggaag
60atgaagccgc gccggaacct cgaggaggac gactacctgc acaaggacac gggcgagacc
120tcgatgctga agcggcccgt gctcctgcac ctgcaccaga ccgcccacgc
ggacgagttc 180gactgcccga gcgagctcca gcacacgcag gagctgttcc
cgcagtggca cctgcccatc 240aagatcgccg ccatcatcgc gagcctcacc
ttcctgtaca ccctgctccg cgaggtcatc 300cacccgctgg ccacgtcgca
ccagcagtac ttctacaaga tcccgatcct ggtgatcaac 360aaggtgctcc
ccatggtcag catcaccctg ctggccctcg tgtacctgcc gggggtgatc
420gcggccatcg tccagctgca caacggcacc aagtacaaga agttcccgca
ctggctcgac 480aagtggatgc tgacgcggaa gcagttcggc ctgctcagct
tcttcttcgc cgtgctgcac 540gcgatctact cgctgagcta ccccatgcgg
cgcagctacc ggtacaagct cctgaactgg 600gcctaccagc aggtgcagca
gaacaaggag gacgcctgga tcgagcacga cgtctggcgg 660atggagatct
acgtgtcgct ggggatcgtg ggcctcgcga tcctggccct gctcgccgtc
720accagcatcc cgagcgtgtc ggacagcctg acctggcgcg agttccacta
catccagagc 780aagctgggca tcgtgtcgct cctgctgggg acgatccacg
cgctcatctt cgcctggaac 840aagtggatcg acatcaagca gttcgtctgg
tacaccccgc ccaccttcat gatcgccgtg 900ttcctgccga tcgtggtcct
gatcttcaag agcatcctct tcctgccgtg cctgcggaag 960aagatcctca
agatccggca cggctgggag gacgtgacga agatcaacaa gaccgagatc
1020tgcagccagc tgtgaccact agttataaga ctgactagcc cgatgggcct
cccaacgggc 1080cctcctcccc tccttgcacc gagattaata aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaatattccc cccccccccc cccccccccc 1200ccccccctct agacaattgg aatt
1224111020DNAArtificial SequenceDescription of sequence construct
CDS STEAP(wt) = STEAP1 (see Figure 11) 11atggaaagca gaaaagacat
cacaaaccaa gaagaacttt ggaaaatgaa gcctaggaga 60aatttagaag aagacgatta
tttgcataag gacacgggag agaccagcat gctaaaaaga 120cctgtgcttt
tgcatttgca ccaaacagcc catgctgatg aatttgactg cccttcagaa
180cttcagcaca cacaggaact ctttccacag tggcacttgc caattaaaat
agctgctatt 240atagcatctc tgacttttct ttacactctt ctgagggaag
taattcaccc tttagcaact 300tcccatcaac aatattttta taaaattcca
atcctggtca tcaacaaagt cttgccaatg 360gtttccatca ctctcttggc
attggtttac ctgccaggtg tgatagcagc aattgtccaa 420cttcataatg
gaaccaagta taagaagttt ccacattggt tggataagtg gatgttaaca
480agaaagcagt ttgggcttct cagtttcttt tttgctgtac tgcatgcaat
ttatagtctg 540tcttacccaa tgaggcgatc ctacagatac aagttgctaa
actgggcata tcaacaggtc 600caacaaaata aagaagatgc ctggattgag
catgatgttt ggagaatgga gatttatgtg 660tctctgggaa ttgtgggatt
ggcaatactg gctctgttgg ctgtgacatc tattccatct 720gtgagtgact
ctttgacatg gagagaattt cactatattc agagcaagct aggaattgtt
780tcccttctac tgggcacaat acacgcattg atttttgcct ggaataagtg
gatagatata 840aaacaatttg tatggtatac acctccaact tttatgatag
ctgttttcct tccaattgtt 900gtcctgatat ttaaaagcat actattcctg
ccatgcttga ggaagaagat actgaagatt 960agacatggtt gggaagacgt
caccaaaatt aacaaaactg agatatgttc ccagttgtag 1020121020DNAArtificial
SequenceDescription of sequence construct CDS STEAP(GC) = STEAP1
(see Figure 12) 12atggagagcc ggaaggacat caccaaccag gaggagctgt
ggaagatgaa gccccgccgg 60aacctcgagg aggacgacta cctgcacaag gacaccggcg
agacgtccat gctgaagcgc 120ccggtgctcc tgcacctgca ccagaccgcc
cacgccgacg agttcgactg ccccagcgag 180ctccagcaca cccaggagct
gttcccccag tggcacctgc ccatcaagat cgcggccatc 240atcgcctccc
tcaccttcct gtacacgctg ctccgggagg tcatccaccc gctggccacc
300agccaccagc agtacttcta caagatcccc atcctggtga tcaacaaggt
gctccccatg 360gtctccatca ccctgctggc cctcgtgtac ctgcccgggg
tgatcgcggc catcgtccag
420ctgcacaacg gcaccaagta caagaagttc ccgcactggc tcgacaagtg
gatgctgacg 480cgcaagcagt tcgggctgct cagcttcttc ttcgccgtgc
tgcacgccat ctactccctg 540agctacccca tgcggcgctc ctaccggtac
aagctcctga actgggcgta ccagcaggtg 600cagcagaaca aggaggacgc
ctggatcgag cacgacgtct ggcgcatgga gatctacgtg 660agcctgggca
tcgtggggct cgccatcctg gccctgctcg ccgtcacctc catccccagc
720gtgtccgaca gcctgacctg gcgggagttc cactacatcc agtccaagct
gggcatcgtg 780agcctcctgc tgggcaccat ccacgcgctc atcttcgcct
ggaacaagtg gatcgacatc 840aagcagttcg tctggtacac gcccccgacc
ttcatgatcg ccgtgttcct gcccatcgtg 900gtcctgatct tcaagtccat
cctcttcctg ccctgcctgc gcaagaagat cctcaagatc 960cggcacgggt
gggaggacgt gaccaagatc aacaagaccg agatctgcag ccagctgtga
10201313RNAArtificial Sequencedescription of sequence Kozak
sequence (see description p. 28) 13gccgccacca ugg
131413RNAArtificial Sequencedescription of sequence generic
stabilizing sequence contained in the 3 prime UTR of the very
stable RNA which codes for alpha-globin, alpha(I)-collagen,
15-lipoxygenase or for tyrosine hydroxylase (see description p. 34)
14nccacccnuc ncc 13
* * * * *