U.S. patent application number 13/106548 was filed with the patent office on 2011-12-22 for application of mrna for use as a therapeutic against tumour diseases.
This patent application is currently assigned to CUREVAC GMBH. Invention is credited to Ingmar HOERR, Steve PASCOLO, Florian VON DER MULBE.
Application Number | 20110311472 13/106548 |
Document ID | / |
Family ID | 7709862 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110311472 |
Kind Code |
A1 |
HOERR; Ingmar ; et
al. |
December 22, 2011 |
APPLICATION OF MRNA FOR USE AS A THERAPEUTIC AGAINST TUMOUR
DISEASES
Abstract
The present invention relates to a pharmaceutical composition
comprising at least one mRNA comprising at least one coding region
for at least one antigen from a tumour, in combination with an
aqueous solvent and preferably a cytokine, e.g. GM-CSF, and a
process for the preparation of the pharmaceutical composition. The
pharmaceutical composition according to the invention is used in
particular for therapy and/or prophylaxis against cancer.
Inventors: |
HOERR; Ingmar; (Tubingen,
DE) ; VON DER MULBE; Florian; (Stuttgart, DE)
; PASCOLO; Steve; (Tubingen, DE) |
Assignee: |
CUREVAC GMBH
|
Family ID: |
7709862 |
Appl. No.: |
13/106548 |
Filed: |
May 12, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10870110 |
Jun 18, 2004 |
|
|
|
13106548 |
|
|
|
|
PCT/EP02/14577 |
Dec 19, 2002 |
|
|
|
10870110 |
|
|
|
|
Current U.S.
Class: |
424/85.1 ;
424/277.1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61K 39/001129 20180801; A61P 37/04 20180101; A61K 2039/572
20130101; A61P 35/00 20180101; A61K 9/0021 20130101; A61K 38/193
20130101; A61K 2039/70 20130101; A61P 35/04 20180101; A61K
2039/55522 20130101; A61K 39/0011 20130101; A61K 2039/53 20130101;
A61K 48/00 20130101; A61K 38/193 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/85.1 ;
424/277.1 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61P 35/00 20060101 A61P035/00; A61P 37/04 20060101
A61P037/04; A61K 31/7088 20060101 A61K031/7088 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2001 |
DE |
101 62 480.8 |
Claims
1. A pharmaceutical composition comprising at least one mRNA
comprising at least one coding region for at least one antigen from
a tumour, in combination with an aqueous solvent.
2. The pharmaceutical composition according to claim 1, wherein the
coding region for the antigen(s) from a tumour and/or the 5' and/or
the 3' untranslated region of the mRNA is modified compared with
the wild-type mRNA such that it has no destabilizing sequence
element.
3. The pharmaceutical composition according to claim 1, wherein the
mRNA has a 5' cap structure and/or a poly(A.sup.+) tail of at least
about 25 nucleotides and/or at least one internal ribosomal entry
site (IRES) and/or at least one 5'-stabilizing sequence and/or at
least one 3'-stabilizing sequence.
4. The pharmaceutical composition according to claim 3, wherein the
5'- and/or the 3'-stabilizing sequence(s) is/are chosen from the
group consisting of untranslated sequences (UTR) of the
.beta.-globin gene and a stabilizing sequence of the general
formula (C/U)CCAN.sub.xCCC(U/A)Py.sub.xUC(C/U)CC.
5. The pharmaceutical composition according to claim 1, wherein the
mRNA contains at least one analogue of naturally occurring
nucleotides.
6. The pharmaceutical composition according to claim 5, wherein the
analogue is chosen from the group consisting of phosphorothioates,
phosphoroamidates, peptide nucleotides, methylphosphonates,
7-deazaguanosine, 5-methylcytosine and inosine.
7. The pharmaceutical composition according to claim 1, wherein the
antigen(s) from a tumour is/are a polyepitope of antigens from a
tumour.
8. The pharmaceutical composition according to claim 7, wherein the
polyepitope is modified by deletion, addition and/or substitution
of one or more amino acid radicals.
9. The pharmaceutical composition according to claim 1, wherein the
mRNA additionally codes for at least one cytokine.
10. The pharmaceutical composition according to one claim 1, which
also comprises one or more adjuvants.
11. The pharmaceutical composition according to claim 10, wherein
the adjuvant is chosen from the group consisting of
lipopolysaccharide, TNF-.alpha., CD40 ligand, GP96,
oligonucleotides with the CpG motif, aluminium hydroxide, Freund's
adjuvant, lipopeptides and cytokines.
12. The pharmaceutical composition according to claim 11, wherein
the cytokine is GM-CSF.
13. The pharmaceutical composition according to claim 1, wherein
the mRNA is present in a form complexed or fused with at least one
cationic or polycationic agent.
14. The pharmaceutical composition according to claim 13, wherein
the cationic or polycationic agent is chosen from the group
consisting of protamine, poly-L-lysine, poly-L-arginine and
histones.
15. The pharmaceutical composition according to claim 1, which also
comprises at least one RNase inhibitor.
16. The pharmaceutical composition according to claim 15, wherein
the RNase inhibitor is RNasin.
17. The pharmaceutical composition according to one claim 1, which
comprises a majority of mRNA molecules which represent a cDNA
library, or a part thereof, of a tumour tissue.
18. The pharmaceutical composition according to claim 17, wherein
the part of the cDNA library codes for the tumour-specific
antigens.
19. Pharmaceutical composition according to claim 1, wherein the
antigen(s) from a tumour is/are chosen from the group consisting of
707-AP, AFP, ART-4, BAGE, .beta.-catenin/m, Bcr-abl, CAMEL, CAP-1,
CASP-8, CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1,
G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I,
HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE,
LDLR/FUT, MAGE, MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, -2,
-3, NA88-A, NY-ESO-1, p190 minor bcr-abl, Pml/RAR.alpha., PRAME,
PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, TEL/AML1,
TPI/m, TRP-1, TRP-2, TRP-2/INT2 and WT1.
20. The pharmaceutical composition according to claim 1, wherein
the mRNA contains a sequence region which serves to increase the
translation rate.
21. The pharmaceutical composition according to claim 1, comprising
at least one further pharmaceutically acceptable carrier and/or at
least one further pharmaceutically acceptable vehicle.
22. The pharmaceutical composition according to claim 1, for
therapy and/or prophylaxis of cancer.
23. A process for the preparation of a pharmaceutical composition
according to claim 1, comprising the steps: (a) preparation of a
cDNA library, or a part thereof, from tumour tissue of a patient,
(b) preparation of a matrix for in vitro transcription of RNA with
the aid of the cDNA library or a part thereof and (c) in vitro
transcribing of the matrix.
24. The process according to claim 23, wherein the part of the cDNA
library of the tumour tissue codes for the tumour-specific
antigens.
25. The process according to claim 24, in which the sequences of
the tumour-specific antigens are ascertained before step (a).
26. The process according to claim 25, wherein the ascertaining of
the sequences of the tumour-specific antigens comprises an
alignment with a cDNA library from healthy tissue.
27. The process according to claim 25, wherein the ascertaining of
the sequences of the tumour-specific antigens comprises a diagnosis
by a microarray.
Description
[0001] The present invention relates to a pharmaceutical
composition comprising at least one mRNA comprising at least one
coding region for at least one antigen from a tumour, in
combination with an aqueous solvent and preferably a cytokine, e.g.
GM-CSF, and a process for the preparation of the pharmaceutical
composition. The pharmaceutical composition according to the
invention is used in particular for therapy and/or prophylaxis
against cancer.
[0002] Gene therapy and genetic vaccination are molecular medicine
methods which, when used in the therapy and prevention of diseases,
will have considerable effects on medical practice. Both methods
are based on the introduction of nucleic acids into cells or into
tissues of the patient and on subsequent processing of the
information coded by the nucleic acids introduced, i.e. expression
of the desired polypeptides.
[0003] The conventional procedure of methods of gene therapy and of
genetic vaccination to date is the use of DNA to insert the
required genetic information into the cell. Various methods for
introducing DNA into cells have been developed in this connection,
such as e.g. calcium phosphate transfection, polyprene
transfection, protoplast fusion, electroporation, microinjection
and lipofection, whereas lipofection in particular having emerged
as a suitable method.
[0004] A further method which has been proposed in particular in
the case of genetic vaccination methods is the use of DNA viruses
as DNA vehicles. Such viruses have the advantage that because of
their infectious properties a very high transfection rate can be
achieved. The viruses used are genetically modified, so that no
functional infectious particles are formed in the transfected cell.
In spite of this safety precaution, however, a certain risk of
uncontrolled propagation of the genes having a gene therapy action
and the viral genes introduced cannot be ruled out because of
possible recombination events.
[0005] The DNA introduced into the cell is conventionally
integrated into the genome of the transfected cell to a certain
extent. On the one hand this phenomenon can exert a desired effect,
since a long-lasting action of the DNA introduced can thereby be
achieved. On the other hand, the integration into the genome
results in a substantial risk of gene therapy. Thus e.g., the DNA
introduced may be inserted into an intact gene, which represents a
mutation which interferes or even completely switches off the
function of the endogenous gene. On the one hand enzyme systems
which are essential for the cell may be switched off by such
integration events, and on the other hand there is also the danger
of a transformation of the cell modified in this way into a
degenerated state if a gene which is decisive for regulation of
cell growth is modified by the integration of the foreign DNA. A
risk of the development of cancer therefore cannot be ruled out
when using DNA viruses as gene therapeutics and vaccines. In this
connection it is also to be noted that for effective expression of
the genes introduced into the cell, the corresponding DNA vehicles
contain a strong promoter, e.g. the viral CMV promoter. Integration
of such promoters into the genome of the treated cell can lead to
undesirable changes in the regulation of gene expression in the
cell.
[0006] A further disadvantage of the use of DNA as gene
therapeutics and vaccines is the induction of pathogenic anti-DNA
antibodies in the patient, causing a possibly fatal immune
response.
[0007] In contrast to DNA, the use of RNA as a gene therapeutic or
vaccine is to be classified as substantially safer. In particular,
RNA does not involve the risk of being integrated into the genome
of the transfected cell in a stable manner. Furthermore, no viral
sequences, such as promoters, are necessary for effective
transcription. Moreover, RNA is degraded considerably more easily
in vivo. Apparently because of the relatively short half-life of
RNA in the blood circulation compared with DNA, no anti-RNA
antibodies have been detected to date. RNA can therefore be
regarded as the molecule of choice for molecular medicine therapy
methods.
[0008] Nevertheless, medical methods based on RNA expression
systems still require a solution to some fundamental problems
before they are used more widely. One of the problems of using RNA
is reliable cell- or tissue-specific efficient transfer of the
nucleic acid. Since RNA usually proves to be very unstable in
solution, it has not hitherto been possible, or has been possible
only in a very inefficient manner, to use RNA as a therapeutic or
vaccine by the conventional methods which are used with DNA.
[0009] RNA-degrading enzymes, so-called RNAases (ribonucleases),
are responsible for the instability. Even the smallest impurities
of ribonucleases are sufficient to degrade RNA in solution
completely. The natural degradation of mRNA in the cytoplasm of
cells is very finely regulated. Several mechanisms are known in
this respect. Thus, the terminal structure is of decisive
importance for a functional mRNA. At the 5'-end is the so-called
"cap structure" (a modified guanosine nucleotide), and at the
3'-end a sequence of up to 200 adenosine nucleotides (the so-called
poly-A tail). The RNA is recognized as mRNA and the degradation is
regulated via these structures. Moreover, there are further
processes which stabilize or destabilize RNA. Many of these
processes are still unknown, but an interaction between the RNA and
proteins often appears to be decisive for this. For example, an
"mRNA surveillance system" has recently been described (Hellerin
and Parker, Annu. Rev. Genet. 1999, 33: 229 to 260), in which
incomplete or nonsense mRNA is recognized by certain feedback
protein interactions in the cytosol and is rendered accessible to
degradation, the majority of these processes being performed by
exonucleases.
[0010] Some measures for increasing the stability of RNA and
thereby rendering possible its use as a gene therapeutic or RNA
vaccine have been proposed in the prior art.
[0011] To solve the abovementioned problems of the instability of
RNA ex vivo, EP-A-1083232 proposes a process for introduction of
RNA, in particular mRNA, into cells and organisms, in which the RNA
is in the form of a complex with a cationic peptide or protein.
[0012] WO 99/14346 describes further processes for stabilizing
mRNA. In particular, modifications of the mRNA which stabilize the
mRNA species against the degradation by RNases are proposed. Such
modifications concern on the one hand stabilization by sequence
modifications, in particular reduction of the C and/or U content by
base elimination or base substitution. On the other hand, chemical
modifications, in particular the use of nucleotide analogues, and
5'- and 3'-blocking groups, an increased length of the poly-A tail
and complexing of the mRNA with stabilizing agents and combinations
of the measures mentioned, are proposed.
[0013] The U.S. Pat. No. 5,580,859 and U.S. Pat. No. 6,214,804
disclose, inter alia, mRNA vaccines and therapeutics in the context
of "transient gene therapy" (TGT). Various measures for increasing
the translation efficiency and the mRNA stability based above all
on untranslated sequence regions are described.
[0014] Bieler and Wagner (in: Schleef (ed.), Plasmids for Therapy
and Vaccination, chapter 9, pages 147 to 168, Wiley-VCH, Weinheim,
2001) report on the use of synthetic genes in connection with gene
therapy methods using DNA vaccines and lentiviral vectors. The
construction of a synthetic gag gene derived from HIV-1, in which
the codons were modified (alternative codon usage) compared with
the wild-type sequence such that they corresponded to the use of
codons which are to be found in highly expressed mammalian genes,
is described. By this means, the A/T content in particular was
reduced compared with the wild-type sequence. The authors find in
particular an increased expression rate of the synthetic gag gene
in transfected cells. Furthermore, in mice an increased formation
of antibodies against the gag protein was observed in mice
immunized with the synthetic DNA construct, and also an increased
cytokine release in vitro in transfected spleen cells of mice.
Finally, an induction of a cytotoxic immune response was to be
found in mice immunized with the gag expression plasmid. The
authors of this article attribute the improved properties of their
DNA vaccine substantially to a change, caused by the optimized
codon usage, to the nucleo-cytoplasmic transportation of the mRNA
expressed by the DNA vaccine. In contrast, the authors consider the
effect of the modified codon usage on the translation efficiency to
be low.
[0015] The present invention is therefore based on the object of
providing a new system for gene therapy and genetic vaccination for
tumours which overcomes the disadvantages associated with the
properties of DNA therapeutics and vaccines.
[0016] This object is solved by the embodiments of the present
invention characterized in the claims.
[0017] In particular, a pharmaceutical composition comprising at
least one mRNA comprising at least one coding region for at least
one antigen from a tumour, in combination with an aqueous solvent,
is provided.
[0018] According to the invention, the expression "antigen from a
tumour" means that the corresponding antigen is expressed in cells
associated with a tumour. According to the invention, antigens from
tumours are therefore in particular those which are produced in the
degenerated cells themselves. These are preferably antigens located
on the surface of the cells. Furthermore, however, antigens from
tumours are also those which are expressed in cells which are
(were) not themselves (or originally themselves) degenerated but
are associated with the tumour in question. These also include e.g.
antigens which are connected with tumour-supplying vessels or
(re)formation thereof, in particular those antigens which are
associated with neovascularization or angiogenesis, e.g. growth
factors, such as VEGF, bFGF etc. Such antigens connected with a
tumour furthermore also include those from cells of the tissue
embedding the tumour. Corresponding antigens of connective tissue
cells, e.g. antigens of the extracellular matrix, are to be
mentioned here.
[0019] According to the invention, in the pharmaceutical
composition one (or more) mRNAs is used for therapy or inoculation,
i.e. vaccination, for treatment or prevention (prophylaxis) of
cancer diseases. The vaccination is based on the introduction of an
antigen (or several antigens) of a tumour, in the present case the
genetic information for the antigen in the form of the mRNA which
codes for the antigen(s), into the organism, in particular into the
cell. The mRNA contained in the pharmaceutical composition is
translated into the (tumour) antigen, i.e. the polypeptide or
antigenic peptide coded by the modified mRNA is expressed, as a
result of which an immune response directed against this
polypeptide or antigenic polypeptide is stimulated. In the present
case of the use as genetic vaccines for treatment of cancer, the
immune response is therefore achieved by introduction of the
genetic information for antigens from a tumour, in particular
proteins which are expressed exclusively on cancer cells, in that a
pharmaceutical composition according to the invention which
comprises an mRNA which codes for such a cancer antigen is
administered. By this means, the cancer antigen(s) is (are)
expressed in the organism, as a result of which an immune response
which is directed effectively against the cancer cells is
provoked.
[0020] In its use as a vaccine, the pharmaceutical composition
according to the invention is to be considered in particular for
treatment of cancer diseases (the mRNA preferably coding for a
tumour-specific surface antigen (TSSA), e.g. for treatment of
malignant melanoma, colon carcinoma, lymphomas, sarcomas,
small-cell pulmonary carcinoma, blastomas etc. Specific examples of
tumour antigens are, inter alia, 707-AP, AFP, ART-4, BAGE,
.beta.-catenine/m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m,
CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AMLI1, G250, GAGE, GnT-V, Gp100,
HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT
(or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A,
MC1R, myosine/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1, p190 minor
bcr-abl, Pml/RAR.alpha., PRAME, PSA, PSM, RAGE, RU1 or RU2, SAGE,
SART-1 or SART-3, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and
WT1.
[0021] According to a further preferred embodiment, the antigen(s)
from a tumour is or are a polyepitope of the antigen (s) from a
tumour. A "polyepitope" of an antigen or several antigens is an
amino acid sequence in which several or many regions of the
antigen(s) which interact with the antigen-binding part of an
antibody or with a T cell receptor are represented. In this
context, the polyepitope can be complete and non-modified. However,
according to the present invention it can also be modified, in
particular to optimize the antibody/antigen and T cell
receptor/antigen interaction, respectively. A modification compared
with the wild-type polyepitope can include e.g. a deletion,
addition and/or substitution of one or more amino acid residues.
Accordingly, in the mRNA of the present invention which codes for
the modified polyepitope, one or more nucleotides is/are removed,
added and/or replaced, compared with the mRNA which codes for the
wild-type polyepitope.
[0022] In order to increase the stability of the (m)RNA contained
in the pharmaceutical composition of the present invention, each
(m)RNA contained in the pharmaceutical composition preferably has
one or more modifications, in particular chemical modifications,
which contribute towards increasing the half-life of the (m)RNA
(one or more) in the organism or improve the transfer of the (m)RNA
(one or more) into the cell.
[0023] For example, in the sequences of eukaryotic mRNAs, there are
destabilizing sequence elements (DSE) to which signal proteins bind
and regulate the enzymatic degradation of the mRNA in vivo. For
further stabilization of the modified mRNA preferably contained in
the pharmaceutical composition according to the invention, where
appropriate in the region which codes for at least one antigen from
a tumour one or more modifications compared with the corresponding
region of the wild-type mRNA are carried out, so that no
destabilizing sequence elements are present. According to the
invention, it is of course also preferable, where appropriate, to
eliminate from the mRNA DSEs present in the untranslated regions
(3'- and/or 5'-UTR).
[0024] Such destabilizing sequences are e.g. AU-rich sequences
("AURES"), which occur in 3'-UTR sections of numerous unstable
mRNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to
1674). The RNA molecules contained in the pharmaceutical
composition according to the invention are therefore preferably
modified compared with the wild-type mRNA such that they contain 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 eliminated in the modified mRNA of the pharmaceutical
composition according to the invention.
[0025] A skilled person in the art is familiar with various
processes which are suitable for substitution of codons in the
modified mRNA according to the invention. In the case of relatively
short coding regions (which code for biologically active or
antigenic peptides) e.g. the total mRNA can be synthesized
chemically using standard techniques.
[0026] Nevertheless, base substitutions are preferably introduced,
using a DNA matrix for the preparation of the modified mRNA with
the aid of techniques of the usual targeted mutagenesis; Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y., 2001.
[0027] In this process, for the preparation of the mRNA, a
corresponding DNA molecule is therefore transcribed in vitro. This
DNA matrix has a suitable promoter, e.g. a T7 or SP6 promoter, for
the in vitro transcription, which is followed by the desired
nucleotide sequence for the mRNA to be prepared and a termination
signal for the in vitro transcription. According to the invention,
the DNA molecule which forms the matrix of the RNA construct to be
prepared is 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 Access Number
U26404; Lai et al., Development 1995, 121: 2349 to 2360; cf. also
FIG. 8), pGEM.RTM. serie, e.g. pGEM.RTM.-1 (GenBank Access Number
X65300; from Promega) and pSP64 (Genbank Access 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.
[0028] Using short synthetic DNA oligonucleotides which contain
short single-stranded transitions at the cleavage sites formed or
genes prepared by chemical synthesis the desired nucleotide
sequence can thus be cloned into a suitable plasmid by molecular
biology methods with which a skilled person in the art is familiar
(cf. Maniatis et al., see above). The DNA molecule is then excised
the plasmid, in which it can be present in one or multiple copy, by
digestion with restriction endonucleases.
[0029] The modified mRNA contained in the pharmaceutical
composition according to the invention can moreover have a 5'-cap
structure (a modified guanosine nucleotide). Examples of cap
structures which may be mentioned are m7G(5')ppp
(5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
[0030] According to a further preferred embodiment of the present
invention, the modified mRNA contains a poly(A.sup.+) tail of at
least about 25, in particular at least about 30, preferably at
least about 50 nucleotides, more preferably at least about 70
nucleotides, particularly preferably at least about 100
nucleotides. However, the poly(A.sup.+) tail can also comprise 200
and more nucleotides.
[0031] For efficient translation of the mRNA, effective binding of
the ribosomes to the ribosome binding site (Kozak sequence:
GCCGCCAACCAUGG, AUG forms the start codon) is necessary. In this
respect, it has been found that an increased A/U content around
this site renders possible a more efficient ribosome binding to the
mRNA.
[0032] It is furthermore possible to insert one or more so-called
IRES ("internal ribosomal entry site) into the mRNA. An IRES can
thus function as the single ribosome binding site, but it can also
serve to provide an mRNA which codes several peptides or
polypeptides which are to be translated by the ribosomes
independently of one another ("muulticistronic" or "polycistronic"
mRNA). Examples of IRES sequences which can be used according to
the invention are those from picornaviruses (e.g. FMDV),
pestviruses (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).
[0033] According to a further preferred embodiment of the present
invention, the mRNA has, in the 5'- and/or 3'-untranslated regions,
stabilizing sequences which are capable of increasing the half-life
of the mRNA in the cytosol.
[0034] These stabilizing sequences can have a 100% sequence
homology to naturally occurring sequences which occur in viruses,
bacteria and eukaryotes, but can also be partly or completely of
synthetic nature. Examples of stabilizing sequences which can be
used in the present invention and which may be mentioned are the
untranslated sequences (UTR) of the .beta.-globin gene, e.g. from
Homo sapiens or Xenopus laevis. Another example of a stabilizing
sequence has the general formula
(C/U)CCAN.sub.xCCC(U/A)Py.sub.xUC(C/U)CC, which is contained in the
3'-UTR of the very stable mRNA which codes for .alpha.-globin,
.alpha.-(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 skilled
person in the art.
[0035] For further stabilization of the mRNA, it is moreover
preferred to contain at least one analogue of naturally occurring
nucleotides. This is based on the fact that the RNA-degrading
enzymes occurring in the cells preferentially recognize naturally
occurring nucleotides as a substrate. The degradation of RNA can
therefore be made difficult by insertion of nucleotide analogues,
whereby the effect on the translation efficiency on insertion of
these analogues, in particular in the coding region of the mRNA,
can have a positive or negative effect on the translation
efficiency.
[0036] In a list which is in no way conclusive, examples which may
be mentioned of nucleotide analogues which can be used according to
the invention are phosphoroamidates, phosphorothioates, peptide
nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine
and inosine. The preparation of such analogues is known to a
skilled person in the art e.g. from the 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. According to the invention, such analogues can occur in
untranslated and translated regions of the modified mRNA.
[0037] Furthermore, effective transfer of the preferably modified
mRNA into the cells to be treated or the organism to be treated can
be improved if the mRNA is associated with a cationic or
polycationic agent, in particular a corresponding peptide or
protein, or bound thereto. The mRNA is therefore present in the
pharmaceutical composition according to the invention preferably in
a form complexed or condensed with such an agent. In particular,
the use of protamine as a polycationic, nucleic acid-binding
protein is particularly effective in this context. The use of other
cationic peptides or proteins, such as poly-L-lysine,
poly-L-arginine or histones, is furthermore also possible. This
procedure for stabilizing the modified mRNA is described in
EP-A-1083232, the disclosure content of which in this respect is
included in its full scope in the present invention.
[0038] The mRNA modified according to the invention can moreover
also contain, in addition to the peptide or polypeptide which is
antigenic or active in gene therapy, at least one further
functional section which e.g. codes for a cytokine which promotes
the immune response, (monokine, lymphokine, interleukin or
chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12, IFN-.alpha., IFN-.gamma., GM-CFS, LT-.alpha. or
growth factors, such as hGH).
[0039] The pharmaceutical composition according to the invention
can further comprise one or more adjuvants to increase the
immunogenicity. "Adjuvant" here is to be understood as meaning any
chemical or biological compound which promotes a specific immune
response. Various mechanisms are possible in this respect,
depending on the various types of adjuvants used. For example,
compounds which promote endocytosis of the modified mRNA contained
in the pharmaceutical composition by dendritic cells (DC) form a
first class of adjuvants which can be used. Other compounds which
allow the maturation of the DC, e.g. lipopolysaccharides,
TNF-.alpha. or CD40 ligand, are a further class of suitable
adjuvants. Generally, any agent which influences the immune system
of the nature of a "warning signal" (LPS, GP96, oligonucleotides
with the CpG motif) or cytokines, in particular GM-CSF, can be used
as an adjuvant which allow an immune response against an antigen
which is coded by the modified mRNA to be increased and/or
influenced in a targeted manner. In particular, the abovementioned
cytokines are preferred in this context. Further known adjuvants
are aluminium hydroxide, Freund's adjuvant and the abovementioned
stabilizing cationic peptides or polypeptides, such as protamine.
Lipopeptides, such as Pam3Cys, are also particularly suitable for
use as adjuvants in the pharmaceutical composition of the present
invention; c.f. Deres et al., Nature 1989, 342: 561-564.
[0040] Further particularly suitable adjuvants are moreover (other)
RNA or also mRNA species, which can be added to the pharmaceutical
composition of the present invention to increase the
immunogenicity. Such adjuvant RNA is advantageously chemically
modified for stabilization ("cis modification" or "cis
stabilization"), for example by the abovementioned nucleotide
analogues, in particular phosphorothioate-modified nucleotides, or
by the above further measures for stabilization of RNA. A further
advantageous possibility of stabilization is complexing or
association ("trans association" or "trans modification" and "trans
stabilization", respectively) with the abovementioned cationic or
polycationic agents, e.g. with protamine.
[0041] According to a further advantageous embodiment, the
stability of the RNA molecules contained in the pharmaceutical
composition (mRNA, coding for a tumour antigen, and optionally
adjuvant (m)RNA) is increased by one or more RNase inhibitors.
Preferred RNase inhibitors are peptides or proteins, in particular
those from the placenta (e.g. from the human placenta) or pancreas.
Such RNase inhibitors can also be in a recombinant form. A specific
example of an RNase inhibitor is RNasin.RTM., which is commercially
obtainable, e.g. from Promega. Such RNase inhibitors can be used
generally for stabilizing RNA. A pharmaceutical composition
comprising at least one RNA, in particular mRNA, which codes for at
least one antigen, and at least one RNase inhibitor as defined
above, optionally in combination with a pharmaceutically acceptable
solvent, carrier and/or vehicle, is therefore also provided
generally according to the invention. Corresponding antigens in a
general form and solvents, carriers and vehicles are defined below.
In respect of preferred tumour antigens, reference is made to the
statements in this respect concerning the preferred pharmaceutical
composition comprising at least one mRNA which codes for at least
one antigen from a tumour.
[0042] The pharmaceutical composition according to the invention
preferably comprises, in addition to the aqueous solvent and the
mRNA, one or more further pharmaceutically acceptable carrier(s)
and/or one or more further pharmaceutically acceptable vehicle(s).
Corresponding routes for suitable formulation and preparation of
the pharmaceutical composition according to the invention are
disclosed in "Remington's Pharmaceutical Sciences" (Mack Pub. Co.,
Easton, Pa., 1980), which is a constituent in its full content of
the disclosure of the present invention. Possible carrier
substances for parenteral administration are e.g., in addition to
sterile water or sterile saline solutions as aqueous solvents, also
polyalkylene glycols, hydrogenated naphthalene and, in particular,
biocompatible lactide polymers, lactide/glycolide copolymers or
polyoxyethylene/polyoxypropylene copolymers. Compositions according
to the invention can comprise filler substances or substances such
as lactose, mannitol, substances for covalent linking of polymers,
such as e.g. polyethylene glycol, to inhibitors according to the
invention, complexing with metal ions or inclusion of materials in
or on particular preparations of a polymer compound, such as e.g.
polylactate, polyglycolic acid or hydrogel, or on liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte fragments or spheroplasts. The particular embodiments
of the compositions are chosen according to the physical
properties, for example in respect of solubility, stability,
bioavailability or degradability. Controlled or constant release of
the active compound component according to the invention in the
composition includes formulations based on lipophilic depots (e.g.
fatty acids, waxes or oils). Coatings of substances according to
the invention or compositions comprising such substances, that is
to say coatings with polymers (e.g. polyoxamers or polyoxamines)
are also disclosed in the context of the present invention.
Substances or compositions according to the invention can
furthermore have protective coatings, e.g. protease inhibitors or
permeability-increasing agents. Preferred aqueous carrier materials
are e.g. water for injection (WFI) or water buffered with
phosphate, citrate or acetate etc., whereby the pH typically being
adjusted to 5.0 to 800, preferably 6.0 to 7.0. The aqueous solvent
or the further carrier(s) or the further vehicle(s) will
additionally preferably comprise salt constituents, e.g. sodium
chloride, potassium chloride or other components which render the
solution e.g. isotonic. Aqueous solvents or the further carrier(s)
or the further vehicle(s) can furthermore comprise, in addition to
the above-mentioned constituents, additional components, such as
human serum albumin (HSA), Polysorbate 80, sugars or amino
acids.
[0043] The method and mode of administration and the dosage of the
pharmaceutical composition according to the invention depend on the
disease to be treated and the stage of advancement thereof, and
also the body weight, the age and the sex of the patient.
[0044] The concentration of the modified mRNA in such formulations
can therefore vary within a wide range from 1 .mu.g to 100 mg/ml.
The pharmaceutical composition according to the invention is
preferably administered to the patient parenterally, e.g.
intravenously, intraarterially, subcutaneously or intramuscularly.
It is also possible to administer the pharmaceutical composition
topically or orally. The pharmaceutical composition according to
the invention is preferably administered intradermally. A
transdermal administration with the aid of electric currents or by
osmotic forces is furthermore possible. The pharmaceutical
composition of the present invention can moreover be injected
locally into a tumour.
[0045] Thus, a method for treatment or a vaccination method for
prevention of cancer diseases or the above-mentioned diseases which
comprises administration of the pharmaceutical composition
according to the invention to a patient, in particular a human, is
thus also provided according to the invention.
[0046] According to a preferred embodiment of the treatment or
vaccination method or in the use, defined above, of the mRNA
according to the invention which codes for at least one antigen
from a tumour for the preparation of a pharmaceutical composition
for treatment and/or prevention of cancer diseases one or more
cytokine(s) is administered to the patient, in addition to the
pharmaceutical composition according to the invention.
[0047] A treatment or vaccination method comprising administration
of at least one RNA, preferably mRNA, which code(s) for at least
one antigen from a tumour (in accordance with the above definition)
and is (are) optionally stabilized in accordance with the above
statements, and at least one cytokine, e.g. one or more of the
abovementioned cytokines, in particular GM-CSF, to a patient, in
particular a human, is therefore also provided generally according
to the invention. The method is used in particular for treatment
and/or prevention of corresponding cancer diseases (e.g. the above
cancer diseases). The present invention is accordingly also
directed generally to a pharmaceutical composition comprising at
least one RNA, preferably mRNA, which code(s) for at least one
antigen from a tumour (according to the above definition) and is
(are) optionally stabilized in accordance with the above
statements, and at least one cytokine, e.g. one or more of the
abovementioned cytokines, such as GM-CSF, preferably in combination
with a pharmaceutically acceptable carrier and/or vehicle, e.g. an
aqueous solvent, or one or more of the carriers, solvents or
vehicles defined above. The use of cytokines, e.g. one or more of
the abovementioned cytokines, in particular GM-CSF, in combination
with one or more RNA molecule (s as defined above, for treatment
and/or prevention of cancer diseases (e.g. cancer diseases listed
above) is thus also disclosed according to the invention.
[0048] According to a further preferred embodiment of the present
invention, the cytokine, e.g. GM-CSF, is administered
simultaneously with or, which is more preferable, before or after
the pharmaceutical composition comprising the mRNA which codes for
at least one antigen from a tumour (or is used for the preparation
of a corresponding medicament for simultaneous administration with
or for administration before or after the abovementioned (m)RNA).
The administration of the cytokine, in particular GM-CSF, is very
particularly preferably carried out shortly before (e.g. about 15
min or less, e.g. about 10 or about 5 min) or a relatively short
time (e.g. about 5, 10, 15, 30, 45 or 60 min) after or a longer
time (e.g. about 2, 6, 12, 24 or 36 h) after the administration of
the pharmaceutical composition defined above or generally after the
(m)RNA of at least one which codes for at least one antigen from a
tumour.
[0049] The application of the cytokine, e.g. GM-CSF, can be carried
out in this context by the same route as the pharmaceutical
composition according to the invention or the at least one (m)RNA
which codes for at least one antigen from a tumour or in a manner
separate from this. Suitable administration routes and also the
suitable formulation possibilities in respect of the cytokine(s)
can be found from the above statements in respect of the
pharmaceutical compositions according to the invention. In the case
of a human patient, a GM-CSF dose of 100 micrograms/m.sup.2 in
particular is advisable. The administration of the cytokine, e.g.
GM-CSF, is particularly preferably carried out by an s.c.
injection.
[0050] The pharmaceutical compositions of the present invention or
the RNA which codes for an antigen from a tumour and where
appropriate, in association therewith, the cytokine(s) are
preferably administered in the form of interval doses. For example,
a dose of a pharmaceutical composition according to the invention
can be administered in relatively short intervals, e.g., daily,
every second day, every third day etc., or, which is more
preferable, in longer intervals, e.g. once weekly, once in two
weeks, once in three weeks, once a month etc. The intervals can
also be changeable in this context, whereby it being necessary in
particular to take into account the immunological parameters of the
patient. For example, the administration of a pharmaceutical
composition according to the invention (and where appropriate, in
association therewith, also the administration of the cytokine(s))
can follow a treatment plan in which the interval is shorter, e.g.
once in two weeks, at the start of the treatment and then,
depending on the course of treatment or the appropriately
determined immunological parameters of the patient, the interval is
lengthened to e.g. once a month. A therapy plan tailor-made to the
particular individual can thus be applied according to the patient,
in particular his condition and his immunological parameters.
[0051] The present invention also provides a process for the
preparation of the pharmaceutical composition defined above,
comprising the steps: [0052] (a) preparation of a cDNA library, or
a part thereof, from tumour tissue of a patient, [0053] (b)
preparation of a matrix for in vitro transcription of RNA with the
aid of the cDNA library or a part thereof and [0054] (c) in vitro
transcribing of the matrix.
[0055] The tumour tissue of the patient can be obtained e.g. by a
simple biopsy. However, it can also be provided by surgical removal
of tumour-invaded tissue. The preparation of the cDNA library or a
part thereof according to step (a) of the preparation process of
the present invention can moreover be carried out after the
corresponding tissue has been deep-frozen for storage, preferably
at temperatures below -70.degree. C. For preparation of the cDNA
library or a part thereof, isolation of the total RNA, e.g. from a
tumour tissue biopsy, is first carried out. Processes for this are
described, e.g. in Maniatis et al., supra. Corresponding kits are
furthermore commercially obtainable for this, e.g. from Roche AG
(e.g. the product "High Pure RNA Isolation Kit"). The corresponding
poly(A.sup.+) RNA is isolated from the total RNA in accordance with
processes known to a person skilled in the art (cf. e.g. Maniatis
et al., supra). Appropriate kits are also commercially obtainable
for this. An example is the "High Pure RNA Tissue Kit" from Roche
AG, Starting from the poly(A.sup.+) RNA obtained in this way, the
cDNA library is then prepared (in this context cf. also e.g.
Maniatis et al., supra). For this step in the preparation of the
cDNA library also, commercially obtainable kits are available to a
person skilled in the art, e.g. the "SMART PCR cDNA Synthesis Kit"
from Clontech Inc. The individual sub-steps from the poly(A.sup.+)
RNA to the double-stranded cDNA is shown schematically in FIG. 11
by the example of the process in accordance with the "SMART PCR
cDNA Synthesis Kit" from Clontech Inc.
[0056] According to step (b) of the above preparation process,
starting from the cDNA library (or a part thereof), a matrix is
synthesized for the in vitro transcription. According to the
invention, this is effected in particular by cloning the cDNA
fragments obtained into a suitable RNA production vector. The
suitable DNA matrix and the plasmids which are preferred according
to the invention are already mentioned above in connection with the
preparation of the mRNA for the pharmaceutical composition
according to the invention.
[0057] For in vitro transcription of the matrix prepared in step
(b) according to the invention, these are first linearized with a
corresponding restriction enzyme, if they are present as circular
plasmid (c)DNA. Preferably, the construct cleaved in this way is
purified once more, e.g. by appropriate phenol/chloroform and/or
chloroform/phenol/isoamyl alcohol mixtures, before the actual in
vitro transcription. By this means it is ensured in particular that
the DNA matrix is in a protein-free form. The enzymatic synthesis
of the RNA is then carried out starting from the purified matrix.
This sub-step takes place in an appropriate reaction mixture
comprising the linearized, protein-free DNA matrix in a suitable
buffer, to which a ribonuclease inhibitor is preferably added,
using a mixture of the required ribonucleotide triphosphates (rATP,
rCTP, rUTP and rGTP) and a sufficient amount of a RNA polymerase,
e.g. T7 polymerase. The reaction mixture is present here in
RNase-free water. Preferably, a CAP analogue is also added during
the actual enzymatic synthesis of the RNA. After an incubation of
an appropriately long period, e.g. 2 h, at 37.degree. C., the DNA
matrix is degraded by addition of RNase-free DNase, incubation
preferably being carried out again at 37.degree. C.
[0058] Preferably, the RNA prepared in this way is precipitated by
means of am ammonium acetate/ethanol and, where appropriate, washed
once or several, times with RNase-free ethanol. Finally, the RNA
purified in this way is dried and, according to a preferred
embodiment, is taken up in RNase-free water. The RNA prepared in
this way can moreover be subjected to several extractions with
phenol/chloroform or phenol/chloroform/isoamyl alcohol.
[0059] According to a further preferred embodiment of the
preparation process defined above, only a part of a total cDNA
library is obtained and converted into corresponding mRNA
molecules. According to the invention, a so-called subtraction
library can therefore also be used as part of the total cDNA
library in order to provide the mRNA molecules according to the
invention. A preferred part of the cDNA library of the tumour
tissue codes for the tumour-specific antigens. For certain tumours,
the corresponding antigens are known. According to a further
preferred embodiment, the part of the cDNA library which codes for
the tumour-specific antigens can first be defined (i.e. before step
(a) of the process defined above). This is preferably effected by
determining the sequences of the tumour-specific antigens by an
alignment with a corresponding cDNA library from healthy
tissue.
[0060] The alignment according to the invention comprises in
particular a comparison of the expression pattern of the healthy
tissue with that of the tumour tissue in question, corresponding
expression patterns can be determined at the nucleic acid level
e.g. with the aid of suitable hybridization experiments. For this
e.g. the corresponding (m)RNA or cDNA libraries of the tissue can
in each case be separated in suitable agarose or polyacrylamide
gels, transferred to membranes and hybridized with corresponding
nucleic acid probes, preferably oligonucleotide probes, which
represent the particular genes (northern and southern blots,
respectively). A comparison of the corresponding hybridizations
thus provides those genes which are expressed either exclusively by
the tumour tissue or to a greater extent therein.
[0061] According to a further preferred embodiment, the
hybridization experiments mentioned are carried out with the aid of
a diagnosis by microarrays (one or more microarrays). A
corresponding DNA microarray comprises a defined arrangement, in
particular in a small or very small space, of nucleic acid, in
particular oligonucleotide, probes, each probe representing e.g. in
each case a gene, the presence or absence of which is to be
investigated in the corresponding (m)RNA or cDNA library. In an
appropriate microarrangement, hundreds, thousands and even tens to
hundreds of thousands of genes can be represented in this way. For
analysis of the expression pattern of the particular tissue, either
the poly(A.sup.+) RNA or, which is preferable, the corresponding
cDNA is then marked with a suitable marker, in particular
fluorescence markers are used for this purpose, and brought into
contact with the microarray under suitable hybridization
conditions. If a cDNA species binds to a probe molecule present on
the microarray, in particular an oligonucleotide probe molecule, a
more or less pronounced fluorescence signal, which can be measured
with a suitable detection apparatus, e.g. an appropriately designed
fluorescence spectrometer, is accordingly observed. The more the
cDNA (or RNA) species is represented in the library, the greater
will be the signal, e.g. the fluorescence signal. The corresponding
microarray hybridization experiment (or several or many of these)
is fare) carried out separately for the tumour tissue and the
healthy tissue. The genes expressed exclusively or to an increased
extent by the tumour tissue can therefore be concluded from the
difference between the signals read from the microarray
experiments. Such DNA microarray analyses are described e.g. in
Schena (2002), Microarray Analysis, ISBN 0-471-41443-3, John Wiley
& Sons, Inc., New York, the disclosure content in this respect
of this document being included in its full scope in the present
invention.
[0062] However, the establishing of tumour tissue-specific
expression patterns is in no way limited to analyses at the nucleic
acid level. Methods known from the prior art which serve for
expression analysis at the protein level are of course also
familiar to a person skilled in the art. There may be mentioned
herein particular techniques of 2D gel electrophoresis and mass
spectrometry, whereby these techniques advantageously also can be
combined with protein biochips (i.e., microarrays at the protein
level, in which e.g. a protein extract from healthy or tumour
tissue is brought into contact with antibodies and/or peptides
applied to the microarray substrate). With regard to the mass
spectroscopy methods, MALDI-TOF ("matrix assisted laser
desorption/ionization-time of flight") methods are to be mentioned
in this respect. The techniques mentioned for protein chemistry
analysis to obtain the expression pattern of tumour tissue in
comparison with healthy tissue are described e.g. in Rehm (2000)
Der Experimentator: Proteinbiochemie/Proteomics [The Experimenter:
Protein Biochemistry/Proteomics], Spektrum Akademischer Verlag,
Heidelberg, 3rd ed., to the disclosure content of which in this
respect reference is expressly made expressis verbis in the present
invention. With regard to protein microarrays, reference is
moreover again made to the statements in this respect in Schena
(2002), supra.
[0063] The figures show:
[0064] FIG. 1 shows a graphical view of the results of a tumour
vaccination, with RNA, of mice (rat Her-2/neu transgenic animals)
which develop mammary carcinomas spontaneously. The tumour
multiplicity is plotted on the y-axis against the age of the mice
on the x-axis. Untreated mice (n=4), which served as a control, all
had tumours at an age of 6 months. Three mice were injected with
DNA which codes for Her-2/neu, one mouse being tumour-free after 10
months. As a further negative control, 4 mice received an antisense
mRNA complementary to the mRNA for Her-2/neu. These mice also all
had tumours after 6 months (not shown). In contrast, one of 4 mice
which were injected with mRNA which codes for Her-2/neu (i.e., the
sense strand) was tumour-free after 9 months.
[0065] FIG. 2 shows a graphical view of the results of experiments
relating to beta-galactosidase (beta-Gal)-specific CTL (cytotoxic T
lymphocyte) activity by immunization with an mRNA which codes for
beta-Gal, under the influence of GM-CSF. BALB/c mice were immunized
with 25 .mu.g of mRNA which codes for beta-Gal by injection into
the inner auricula. The splenocytes were stimulated with beta-Gal
protein in vitro and the CTL activity was determined 6 days after
the in vitro stimulation using a standard .sup.51Cr release test.
The target cells were P815 (H.sub.2.sup.d) cells which were charged
(.box-solid.) with the synthetic peptide TPHPARIGL, which
corresponds to the H.sub.2.sup.d epitope of beta-Gal, or were not
charged (.tangle-solidup.). In each case three or two animals were
treated per group. Animals which were injected i.d. in both
auriculae with only injection buffer served as a negative control.
Animals which were injected i.d. in both auriculae with 10 .mu.g of
a plasmid which codes for beta-Gal in PBS served as a positive
control ("DNA"). The test groups received RNA which codes for
beta-Gal by itself or in combination with GM-CSF, which was
injected 24 h ("GM-CSF t-1"), 2 h before the RNA injection ("GM-CSF
t0") or 24 h after the RNA injection ("GM-CSF t+1") into the same
site (into the auriculae) or at another site (s.c. on the back). In
each case three different effector/target cell ratios (200, 44, 10)
were tested.
[0066] FIG. 3 shows further graphical views of the results of ELISA
standard tests specific for IFN-gamma (A) and IL-4 (B), which
document the corresponding cytokine production of splenocytes which
were restimulated with beta-Gal protein in vitro. BALB/c mice were
immunized as already described above for FIG. 2. The splenocytes
were stimulated with beta-Gal protein in vitro, the corresponding
culture supernatants were obtained and the IFN-gamma or IL-4
concentration was determined using an ELISA standard test.
[0067] FIG. 4 shows further graphical views which demonstrate the
antibody response of mice immunized according to the invention.
BALB/c mice were immunized as described for FIG. 2. Two weeks after
the boost, blood was taken and the blood serum was obtained
therefrom. Beta-Gal-specific IgG1 (A) and IgG2a antibodies (B) were
determined with the aid of an ELISA test. In each case the
extinction (OD) at 405 nm which results from the conversion of the
substrate ABTS in the ELISA test is shown on the y-axis. The
extinctions shown are the values from which the corresponding
values of mice treated with injection buffer are subtracted.
[0068] FIG. 5 shows microscope sections, stained with X-Gal, of the
auricula of mice which have been injected i.d. into the auricula
with mRNA which codes for beta-galactosidase. 12 hours after the
injection of 25 .mu.g RNA in HEPES-NaCl injection buffer, the ears
were removed and sections stained with X-Gal were prepared. Blue
cells indicate a beta-galactosidase activity. As can be seen from
the two sections, only few blue cells are present.
[0069] FIG. 6 shows a section, corresponding to FIG. 5, through an
auricula of a mouse which was injected into the auricula with mRNA
which codes for beta-galactosidase and was stabilized with
protamine. The microscope section stained with X-Gal show a few
cells stained blue.
[0070] FIG. 7 shows two further sections through the auricula of
mice, two images being produced per section in order to represent a
larger area. In this case, mRNA which codes for beta-galactosidase,
in a buffer, to which 10 U RNasin, an enzymatic RNase inhibitor
from the pancreas (obtainable from Roche or Promega) was added
directly before the injection, was injected into the auricula.
Compared with the sections of FIG. 5 and FIG. 6, significantly more
blue-stained regions of cells with beta-galactosidase activity are
to be recognized.
[0071] FIG. 8 shows a schematical view of the plasmid pT7TS, which
was used for the in vitro transcription. Constructs according to
the invention were cloned into the BglII and SpeI sites, the
relative position of which to one another is shown. The region
shaded in black contains the 5' untranslated region of the
beta-globin gene from Xenopus laevis, while the region shaded in
grey represents a corresponding 3' untranslated region of the
beta-globin gene from X. laevis. The relative position of the T7
promoter, the PstI site used for sequencing, the poly(A.sup.+) tail
(A.sub.30C.sub.30) and, with an arrow, the transcription direction
are furthermore indicated.
[0072] FIG. 9 shows in a flow chart, by way of example, the course
of an RNA vaccination therapy according to the invention with
assisting administration of GM-CSF. The mRNA molecules which code
for one or more tumour antigens (MUC1, Her-2/neu, tilomerase,
MAGE-1) or a mRNA which codes for a control antigen (influenza
matrix protein (IMP), a viral antigen) are administered i.d. to the
patient on days 0, 14, 28 and 42. In addition, one day after the
RNA inoculation the patient is injected s.c. with GM-CFS
(Leucomax.RTM. (100 .mu.g/m.sup.2) from Novartis/Essex Pharma).
When the course is stable or there is an objective tumour response
(complete remission (CR) or partial remission (PR)), the patients
receive the vaccinations s.c. once a month. After the fourth
injection (day 49), the response of the tumour is evaluated
radiologically, by laboratory chemistry or sonographically, and the
immunological phenomena induced by the therapy are evaluated. From
day 70, the immunization therapy is continued at intervals of 4
weeks. On day 0, 14, 28, 42 and 49, blood samples are taken for
determination of appropriate laboratory parameters, the
differential blood count (Diff-BB), FACS analysis and cytokines.
Restaging of the patient takes place from day 49 and where
appropriate every further 4 to 8 weeks.
[0073] FIG. 10 shows a flow chart of the construction of
autologous, stabilized RNA according to the preparation process of
the present invention. Tumour tissue is first obtained, e.g. by
biopsy. The total RNA is extracted from this. A cDNA library is
constructed with the aid of the poly(A.sup.+) RNA obtained from the
RNA extraction. Starting from this, after preparation of a
corresponding DNA matrix, the autologous, stabilized RNA is
obtained by means of in vitro transcription.
[0074] FIG. 11 shows a reaction scheme of the steps for preparation
of a cDNA library, starting from poly(A.sup.+) RNA, for the SMART
PCR cDNA Synthesis Kit from Clontech Inc. by way of example.
[0075] FIG. 12 shows a photograph of an agarose gel which shows the
typical size fractionation of a cDNA library compiled from human
placenta tissue. A length marker with fragments of the length shown
on the left is plotted in track M. The "DS cDNA" track contains the
cDNA library. Those fragments which correspond to the expected size
fraction (about 200 bp to 4,000 bp) are used for the in vitro
transcription.
[0076] FIG. 13 shows by way of example a treatment plan for the
tumour therapy according to the invention by injection of a tumour
mRNA library, here in combination with GM-CSF, for patients with
malignant melanoma. Autologous, stabilized RNA prepared from the
patient's own tumour tissue is used for this. This amplified
autologous tumour RNA is administered to the patient i.d. on days
0, 14, 28 and 42. In addition, one day after the RNA injection the
patient is injected s.c. with GM-CSF (Leucomax.RTM. 100
.mu.g/m.sup.2 Novartis/Essex Pharma). Two weeks after the fourth
injection (day 56), the response of the tumour is evaluated by a
staging analysis (inter alia sonography, thorax X-ray, CT etc.) and
by assessment of the immunological parameters induced by the
therapy. When the course of the disease is stable or there is an
objective tumour response (CR or PR), the patient receives in each
case a further vaccination every four weeks. Further restaging
analyses are carried out on day 126 and then at intervals of 12
weeks.
[0077] FIG. 14 shows once more schematically of the general course
of a therapy with the pharmaceutical composition according to the
invention with autologous, amplified tumour RNA, i.e., the RNA
contained in the pharmaceutical composition represents a cDNA
library of the tumour tissue. A sample of the tumour tissue is
first obtained, e.g. via a biopsy. The total and then the
poly(A.sup.+) RNA are prepared from the tissue by appropriate
extractions. Starting from the poly(A.sup.+) RNA, a cDNA library is
constructed and is cloned into a vector suitable for subsequent in
vitro transcription. An RNA vaccine is then obtained by in vitro
transcription, and is injected into the patient from whom the
tumour tissue has been taken to combat the tumour.
[0078] The following embodiment examples explain the present
invention in more detail, without limiting it.
EXAMPLES
Example 1
Tumour Vaccination with RNA in an Animal Model
Materials and Methods
[0079] Capped mRNA which codes for a shortened version of the
Her-2/neu protein of the rat ("ECD-TM-neu-rat", containing the
extracellular domain and the transmembrane region, but not the
cytoplasmic region) was prepared, using the "SP6 mMessagemMachine"
(Ambion) with the aid of a plasmid which substantially corresponded
to the structure shown in FIG. 8, but contained an SP6 promoter
instead of the TV promoter and in which the ECD-TM-neu-rat
construct was inserted after the SP6 RNA polymerase promoter. The
mRNA prepared was dissolved in injection buffer (150 mM NaCl, 10 mM
HEPES) at a concentration of 0.8 mg/l and the solution was mixed
with protamine sulfate (Sigma) (1 mg protamine per 1 mg RNA). 50
.mu.l of this solution were injected into the auriculae (in each
case 25 .mu.l per ear) of mice. Eight injections were performed, in
each case one at the age of 6, 8, 13, 15, 20, 22, 27 and 29 weeks.
Mice to which corresponding injections with injection buffer, with
plasmid DNA which codes for ECD-TM-neu rat or with an antisense
mRNA corresponding to the mRNA according to the invention were
administered served as controls.
Results
[0080] Female BalB-neu T mice (BalB/c mice which express the
oncogene Her-2/neu of the rat; cf. Rovero et al. (2000) J. Immunol.
165(9):5133-5142) which develop mammary carcinomas spontaneously
were immunized with RNA which codes for a shortened version of the
Her-2/neu protein ("ECD-TM-neu-rat", containing the extracellular
domains and the transmembrane region, but not the cytoplasmic
region). Four mice treated with injection buffer served as a
negative control. A further group of three mice was injected with
DNA which codes for the shortened Her-2/neu. Four mice received the
mRNA which codes, according to the invention, for the tumour
antigen Her-2/neu (shortened version of ECD-TM, see above). Four
mice which were injected with the corresponding antisense RNA
served as a further control group. As shown in FIG. 1, in the
animals of the untreated control group a tumour multiplicity of on
average 10 was observed after 26 weeks, whereby all animals having
palpable breast tumours at the age of about 20 weeks. In contrast,
in the case of immunization with the mRNA which codes for
ECD-TM-neu-rat, a significant slowing down of the formation of
carcinomas is to be observed, in particular a tumour multiplicity
of 10 is achieved only at the age of 30 weeks. Furthermore, the
size of the tumours is also reduced (not shown). Of the 4 mice
treated with the mRNA according to the invention, one was still
tumour-free after 9 months. That group of mice which had been
injected with the antisense mRNA all showed tumours at the age of 6
months. The comparison group of mice injected with plasmid DNA
which codes for the shortened version of Her-2/neu also showed a
carcinoma formation which was slowed down compared with the
untreated control group (cf. also in respect of corresponding
plasmid. DNA experiments on intramuscular injection: Di Carlo et
al. (2001) Clin. Cancer Res. 7 (3rd supplement): 830s-837s), but
the formation of carcinomas up to the 27th week was not slowed down
to the same extent as in the case of immunization with mRNA
according to the invention which codes for the shortened version of
Her-2/neu. Furthermore, in the case of immunization with DNA, the
abovementioned disadvantages, in particular the risk of integration
of the DNA into the genome, the formation of anti-DNA antibodies
etc., are to be taken into account.
Example 2
Influence of GM-CSF on RNA Vaccination
Materials and Methods
Mice
[0081] BALB-c AnNCr1BR (H-2.sup.d) mice (female) 6-10 weeks old
were obtained from Charles River (Sulzfeld, Germany).
Plasmids and Preparation of RNA
[0082] The ORF (LacZ) which codes for beta-galactosidase, flanked
by 5'- and 3'-untranslated sequences from the beta-globin gene of
X. Laevis, was into the plasmid pT7TS (P.A. Creek, Austin, Tex.,
USA), in order to prepare the plasmid pT7TS-kozak-5' beta
gl-lacZ-3' beta gl-A30C30 (cf. Hoerr et al. (2000) Eur. J. Immunol.
30: 1-7). A schematical view of the general structure of the
plasmid pT7TS with the flanking 5' and 3' untranslated sequences
from the beta-globin gene of X. laevis is shown in FIG. 8.
[0083] The plasmid prepared in this way was linearized with PstI
and transcribed in vitro using the m-MessagemMachineT7 Kit (Ambion,
Austin, Tex. USA). The RNA prepared in this way was purified by
means of LiCl precipitation, phenol/chloroform extraction and
ammonium acetate precipitation. Finally, the purified RNA was
resuspended in injection buffer (150 mM NaCl, 10 mM HEPES) in a
concentration of 025 mg/ml.
Media and Cell Culture
[0084] P815 and P13.1 cells were cultured in RPMI 1640
(Bio-Whittaker, Verviers, Belgium), supplemented with 10%
heat-inactivated foetal calf serum (FCS) (PAN systems, Germany), 2
mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.
[0085] CTL cultures were kept in RPMI 1640 medium, supplemented
with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin, 0.05 .mu.M beta-mercaptoethanol, 50 mg/ml gentamycin,
MEM non-essential amino acids (100.times.) and 1 mM sodium
pyruvate. The CTL were restimulated for one week with 1 mg/ml
beta-galactosidase protein (Sigma, Taufkirchen, Germany). On day 4,
4 ml of culture supernatant were carefully pipetted off and
replaced by fresh medium containing 10 U/ml riL-2 (final
concentration).
Immunization
[0086] 3 BALB/c mice per group were anesthetized with 20 mg
pentobarbital i.p. per mouse. The mice were then injected i.d. in
both auriculae with 25 mg of mRNA which codes for
beta-galactosidase (beta-Gal) in injection buffer (150 mM NaCl, 10
mM HEPES). In some cases, granulocyte macrophage colony-stimulating
factor (GM-CSF) was additionally injected into the same site or
into an injection site away from this (into the auricula or s.c.
into the back) 24 h or 2 h before or 24 h after the RNA injection.
As a positive control, animals were injected i.d. in both auriculae
with in each case 10 mg of a DNA plasmid which codes for beta-gal
in PBS. A group of animals to which only injection buffer was
administered i.d. into both auriculae served as a negative control.
Two weeks after the first injection, a boost injection was
performed in each case in the same manner as the first injection.
Two weeks after the boost injection, blood was taken, the mice were
sacrificed and the spleen was removed.
.sup.51Cr Release Test
[0087] Splenocytes obtained from the spleen were stimulated with
beta-gal protein in vitro and the CTL activity was determined after
6 days using a 6-hours .sup.51Cr standard test as described in
Rammensee et al. (1989) Immunogenetics 30: 296-302. Summarized
briefly, target cells were marked with .sup.51Cr and charged with
the peptide TPHPARIGL for 20 min at room temperature. After
co-incubation of effector and target cells (at in each case three
different ratios of effector:target cells: 200, 44 and 10) in
circular plates with 96 wells for 6 h, 50 ml of 200 ml of culture
supernatant were pipetted into a Luma scintillation plate (Packard)
with 96 wells and, after drying, the radioactivity was measured
with a scintillation counter (1405 Microbeta Plus). The percentage
specific release was determined from the amount of .sup.51Cr
released into the medium (A) minus the spontaneous release (B)
divided by the total release (C) (using Triton X-100) minus the
spontaneous release (B): Percent specific lysis=100
(A-B)/(C-B).
Cytokine ELISA
[0088] After 4 days of restimulation with beta-gal protein, the
supernatant of the splenocyte culture was pipetted off and stored
at -50.degree. C. until used. 100 ml anti-mouse-anti-IFN-gamma or
-IL-4 scavenger antibodies (Becton Dickinson, Heidelberg, Germany)
were pipetted out overnight at 4.degree. C. on MaxiSorb plates
(Nalge Nunc International, Nalge, Denmark) at a, concentration of 1
mg/ml, in coating buffer (0.02% NaN.sub.3, 15 mM Na.sub.2CO.sub.3,
15 mM NaHCO.sub.3, pH 9.6). After washing three times with washing
buffer (0.05% Tween 20 in PBS), the plates were saturated with 200
ml of blocking buffer (0.05% Tween 20, 1% BSA in PBS) for 2 h at
37.degree. C. After washing three times with washing buffer, 100 ml
of the cell culture supernatants were incubated for 5 h at
37.degree. C. The plates were then washed four times with washing
buffer, 100 ml of biotinylated anti-mouse-anti-IFN-gamma or -IL-4
detection antibodies (Becton Dickinson, Heidelberg, Germany) per
well at a concentration of 0.5 mg/ml in blocking buffer were
pipetted and incubation was carried out for 1 h at room
temperature. After washing three times with washing buffer, 100 ml
of a 1/1,000 dilution of streptavidin-HRP (BD Biosciences,
Heidelberg, Germany) were added into each well. After 30 min at
room temperature, the plates were washed three times with washing
buffer and twice with bidistilled water. Thereafter, 100 ml of the
ABTS substrate were added into each well. After 15-30 min at room
temperature, the extinction at 405 on was measured with a Sunrise
ELISA reader (Tecan, Crailsheim, Germany)
Antibody ELISA
[0089] Two weeks after the boost injection, blood was taken from
the mice via the orbital vein and blood serum was prepared. 100 ml
of beta-gal protein at a concentration of 100 mg/ml in coating
buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl.sub.2, pH 7.5) were
pipetted out for 2 h at 37.degree. C. on to MaxiSorb plates (Nalge
Nunc International, Nalge, Denmark). The plates were then washed
three times with 200 ml of washing buffer (0.05 M Tris-HCl, 0.15 M
NaCl, 0.01 M EDTA, 0.1% Tween 20, 1% BSA, pH 7.4) and saturated
with protein with 200 ml of washing buffer overnight at 4.degree.
C. The plates were washed three times with washing buffer and blood
sera were added in a dilution of 1/10, 1/30 or 1/90 in washing
buffer. After 1 h at 37.degree. C., the plates were washed three
times with washing buffer and 100 ml of 1/1,000 dilutions of goat
anti-mouse IgG1 or IgG2a antibodies (Caltag, Burlington, Calif.,
USA) were added. After 1 h at room temperature, the wells were
washed three times with washing buffer and 100 ml of ABTS substrate
per well were added. After 15-30 min at room temperature the
extinction at 405 nm was measured with a Sunrise ELISA reader
(Tecan, Crailsheim, Germany).
Results and Discussion
[0090] It was confirmed that direct injection of RNA which codes
for beta-galactosidase into the auricula of mice induces an
anti-beta-galactosidase immune response, substantially of the Th2
type. Production of anti-beta-galactosidase immunoglobulins of the
IG1 type (FIG. 3A) and secretion of IL-4 (FIG. 3B) was found in
splenocytes, stimulated with beta-galactosidase, from mice which
had been injected with the RNA which codes for beta-galactosidase.
To increase the efficiency of the RNA vaccine, the cytokine GM-CSF
was additionally administered. This cytokine increases the
efficiency of some DNA vaccines. It was furthermore found that the
time of the GM-CSF injection influences the type of the immune
response, compared with DNA injection (Kusakabe (2000) J. Immunol.
164: 3102-3111). It was found according to the invention that
GM-CSF can enhance the immune response brought about by an RNA
vaccination. The injection of GM-CSF one day before the injection
of RNA shows scarcely any influence on the strength or the type of
the immune response. In contrast, injection of GM-CSF 2 hours
before injection of the RNA enhances the immune response (cf. the
IL-4 release in FIG. 38 in the 2 mice injected with GM-CSF at time
T=0), but does not influence the Th2 polarity. On the other hand,
if GM-CSF is injected one day after the RNA vaccine into the same
site or into a site away from this (not shown), not only is the
immune response enhanced overall (cf. the antibody response
according to FIG. 3), the immune response is polarized to the Th1
type (cf. the IFN-gamma production by splenocytes stimulated with
beta-gal protein according to FIG. 3A, the production of IgG2a
antibodies against beta-Gal according to FIG. 3B and the production
of activated CTL according to FIG. 1). The injection of GM-CSF some
minutes or some hours after the RNA injection should result in the
same effect (enhancement and polarization) on the immune
response.
Example 3
Effect of an RNase Inhibitor on mRNA Expression In Vivo
[0091] Naked or protamine-associated or -complexed mRNA which codes
for beta-galactosidase (prepared as described in example 2) was
injected into the auricula of mice in an amount of 25 mg of RNA in
injection buffer (150 MI NaCl, 10 mM HEPES). Further mice were
injected with the mRNA which codes for beta-galactosidase, together
with 10 U of the RNase inhibitor RNasin (an enzymatic RNase
inhibitor extracted from the pancrease, obtainable from Roche or
Promega). The RNase inhibitor was mixed with the RNA solution
directly before the injection. After 12 hours, the ears were in
each case removed from the mice. Thin microscope sections of the
auriculae were prepared and were stained with X-gal. Injection of
naked or protamine-associated mRNA leads to a detectable
beta-galactosidase activity in a few cells in the corresponding
thin sections (blue cells in FIGS. 5 and 6). Some cells have thus
taken up the exogenous RNA here and translated it into the protein.
When the mRNA which codes for beta-galactosidase was in the form
protected with the RNase inhibitor RNasin, very many more blue
cells were observed than in the case of the naked or
protamine-associated RNA (FIG. 7). Since RNasin inhibits RNases,
the half-life of the injected mRNA molecules in vivo is prolonged,
where the environment (interstitial tissue) is contaminated with
RNases. Such a stabilization of the RNA leads to an increased
uptake by the surrounding cells and therefore to an increased
expression of the protein coded by the exogenous RNA. This
phenomenon can therefore also be utilized for an enhanced immune
response to an antigen coded by the mRNA injected.
Example 4
RNA Vaccination of Patients with Malignant Diseases
Introduction
[0092] Cytotoxic T lymphocytes (CTL) recognize antigens as short
peptides (8-9 amino acids) which are expressed bound to MHC class 1
glycoproteins on the cell surface (1). These peptides are fragments
of intracellular protein molecules. However, there are indications
that antigens taken up exogenously by macropinocytosis or
phagocytosis can lead to the CD8.sup.+ T cell-mediated immune
response. The proteins are cleaved into proteosomes and the
peptides formed by this means are transported out of the cytosol
into the lumen of the endoplasmic reticulum and bound to MHC class
I molecules.
[0093] The proteins processed in this way are transported as
peptide/MHC class I complex to the cell surface and presented to
the CTL. This process takes place in every cell and in this way
makes it possible for the immune system to monitor accurately each
individual cell for the presence of proteins which are foreign to
the body or modified or embryonic, regardless of whether they
originate from intracellular pathogenic germs, oncogenes or
dysregulated genes. By this means, cytotoxic lymphocytes are
capable of recognizing and lysing infected and neoplastic cells,
respectively (2, 3).
[0094] In recent years various tumour-associated antigens (TAA) and
peptides which are recognized by CTL and therefore lead to lysis of
tumour cells have been successfully isolated (21-27). These TAA are
capable of stimulating T cells and inducing antigen-specific CTL,
if they are expressed as a complex of HLA molecule and peptide on
antigen-presenting cells (APC).
[0095] In numerous studies carried out mainly on patients with
malignant melanoma, it has been possible to demonstrate that
malignant cells lose the expression of TAA as the tumour disease
proceeds. Similar circumstances are also observed with vaccinations
with individual tumour antigens. Under vaccination therapies,
selection of tumour cells may also occur, which renders possible an
escape from the immune system and a progression of the disease in
spite of therapy. The use of several different tumour antigens as
envisaged in the treatment plan according to the invention of the
present example should prevent selection of tumour cells and escape
of the malignant cells from the immune system due to loss of
antigens.
[0096] A method with which DC can be transfected with RNA from a
plasmid which codes for a tumour antigen has recently been
developed (Nair et al., 1998, Nair et al., 2000). Transfection of
DC with RNA for CEA or telomerase led to induction of
antigen-specific CTL. This process renders it possible to induce
CTL and T helper cells against several epitopes on various HLA
molecules from a tumour antigen. A further advantage of this
strategy is the fact that neither the characterization of the
tumour antigens or epitopes used nor definition of the HLA
haplotype of the patient is a prerequisite. By a polyvalent vaccine
of this type, the probability of the occurrence of so-called clonal
"tumour escape" phenomena could be reduced significantly.
Furthermore, T cell-mediated immune responses against antigens
processed and presented by the natural route and with possibly a
higher immune dominance could be induced by this approach. By
additional participation of MHC class II-restricted epitopes, the
induced tumour-specific immune response could be enhanced and
maintained for longer.
[0097] A treatment scheme according to the invention for tumour
vaccination of patients with advanced malignant diseases (mammary,
ovarian, colorectal, pancreatic and renal cell carcinomas) is
provided by way of example. In this, RNA which has been prepared
from plasmids which code for MUC1, Her-2/neu, telomerase and MAGE-1
tumour antigens and influenza matrix protein (IMP) (positive
control) is administered intradermally to patients with the
abovementioned malignant diseases. A CTL induction in vivo is
thereby rendered possible, in order to prevent the progression of
the disease or to effect the regression thereof in this way. The
tumour antigens mentioned are expressed on the malignant cells of
mammary, ovarian, colorectal, pancreatic and renal cell
carcinomas.
[0098] According to the treatment plan (cf. the following
statements in this respect and FIG. 9), the RNA species prepared in
the laboratory which code for CEA, MUC1, Her-2/neu, telomerase,
Mage-1 and IMP are administered to the patient i.d., initially
4.times. on days 0, 14, 28 and 42, In addition, GM-CSF
(Leucomax.RTM., 100 .mu.g/m.sup.2, Novartis/Essex Pharma) is
administered s.c. to the patient in each case one day after the RNA
inoculation.
[0099] The treatment according to the invention is an immunisation
approach which requires only minimal, interventions on the patient
(injection). Therapy is conducted ambulant and is suitable for many
tumour patients, without the limitation to particular HLA types or
defined T cell epitopes. Furthermore, polyclonal CD4.sup.+-T
helpers and also CD8.sup.+-CTL can be induced by this therapy.
Treatment Plan
[0100] The RNAs for several tumour antigens (MUC1, Her-2/neu,
telomerase, MAGE-1) and for a control antigen, influenza matrix
protein (IMP, a viral antigen) are administered i.d. to the patient
on days 0, 14, 28 and 42. In addition, the patients receive GM-CSF
(Leucomax.RTM. (100 .mu.g/m.sup.2) Novartis/Essex Pharma) s.c. in
each case one day after the RNA inoculation. When the course of the
disease is stable or there is an objective tumour response
(complete remission (CR) or partial remission (PR)), where
appropriate the patients receive the vaccinations s.c. once a
month. After the fourth injection (day 49), the response of the
tumour is evaluated radiologically, by laboratory chemistry and/or
sonographically, and the immunological phenomena induced by the
therapy are evaluated.
[0101] From day 70, the immunization therapy is continued at
intervals of 4 weeks.
[0102] On days 0, 14, 28, 42 and 49, in each case blood samples are
taken for laboratory parameters, Diff-BB, FACS analysis and
cytokines (50 ml in total). Restaging of the patients takes place
from day 49 and where appropriate every further 4 to 8 weeks.
[0103] The treatment plan is shown schematically in FIG. 9. [0104]
Laboratory: clotting, electrolytes, LDH, .beta.2-d, CK, liver
enzymes, bilirubin, creatinine, uric acid, total protein, CRP,
tumour markers (Ca 12-5, Ca 15-3, CEA, Ca 19-9): 15 ml of blood.
[0105] Diff-BB: differential blood count with smear (5 ml of EDTA
blood). [0106] Cytokines: 10 ml of serum [0107] FACS: 10 ml of
heparin blood. [0108] ELIspot: 20 ml of heparin blood. [0109]
Multitest: analysis of the DTH reaction. [0110] DTH: ("delayed type
hypersensitivity", delayed T cell-mediated reaction) analysis of
the reaction to intradermally administered RNA. In addition a skin
biopsy should be performed in the event of a positive DTH reaction
(local anaesthesia is not necessary for this).
Preparation of RNA from Plasmids
[0111] For production of a vaccine based on mRNA, only precursors
which are chemically synthesized and purified from bacteria are
required. This is preferably effected in a specially equipped RNA
production unit. This is in a sealed-off room which is declared an
RNase-free zone, i.e. work with RNase (e.g. purification of
plasmids) must not be carried out. Contamination with naturally
occurring RNases is also constantly checked. This room is fitted
out with new apparatuses (4.degree. C. and -20.degree. C.
refrigerators, heating block, sterile bench, centrifuges, pipettes)
which have never been used for biological or clinical work. This
RNA production unit is used exclusively for enzymatic production
(in vitro transcription) of mRNA (without bacterial, viral or cell
culture work). The end product comprises a sterile RNA solution in
HEPES/NaCl buffer. Quality analyses are carried out on a
formaldehyde-agarose gel. In addition, the RNA concentration and
the content of proteins are determined photometrically
(OD.sub.320<0.1; ratio of OD.sub.260/OD.sub.280>1.8 in pure
RNA). Possible contamination by LPS is analysed in the LAL test.
All RNA samples are subjected to sterile filtration before
administration.
Plasmid Constructs
[0112] The chosen genes (CEA, mucin1, Her-2/neu, telomerase,
Mage-A1 and influenza matrix) are amplified via a PCR using a
heat-stable high-performance enzyme (pfu, Stratagene). The genes
originate from tumour cDNA (mucin1, Her-2/neu, telomerase), or they
have been cloned into bacterial vectors (influenza matrix and
MAGE-A1). The PCR fragments are cleaved with restriction enzymes
(mucin1: BglII-SpeI; Her-2/neu: HinDIIIblunt-SpeI; telomerase:
BglII-SpeI; MAGE-A1: BamHI-SpeI; influenza matrix protein:
BglII-SpeI) and cloned into the T7TS-Plasmid (cf. FIG. 8) via the
BglII and SpeI restriction sites. Plasmids of high purity are
obtained via the Endo-free Maxipreparation Kit (Qiagen, Hilden,
Germany). The sequence of the vector is controlled via a
double-strand sequencing from the T7 promoter up to the PstI site
and documented. Plasmids with a correct inserted gene sequence
without mutations are used for the in vitro transcription. (Control
via the published sequences: Accession Numbers: M11730 for
Her-2/neu, NM.sub.--002456 for MUC1, NM.sub.--003219 for telomerase
TERT, V01099 for influenza matrix and M77481 for MAGE-A1)
In Vitro Transcription
Production of Linear, Protein-Free DNA
[0113] 500 .mu.g of each plasmid are linearized in a volume of 0.8
ml via digestion with the restriction enzyme PstI in a 2 ml
Eppendorf reaction vessel. This cleaved construct is transferred
into the RNA production unit. 1 ml of a mixture of
phenol/chloroform/isoamyl alcohol is added to the linearized DNA.
The reaction vessel is vortexed for 2 minutes and centrifuged at
15,000 rpm for 3 minutes. The aqueous phase is removed and mixed
with 0.7 ml 2-propanol in a 2 ml reaction vessel. This vessel is
centrifuged at 15,000 rpm for 15 minutes, the supernatant is
discarded and 1 ml 75% ethanol is added. The reaction vessel is
centrifuged at 15,000 rpm for 10 minutes and the ethanol is
removed. The vessel is centrifuged for a further 2 minutes and the
residues of the ethanol are removed with a microlitre pipette tip.
The DNA pellet is then dissolved in 1 .mu.g/ml in RNase-free
water.
Enzymatic Synthesis of the RNA
[0114] The following reaction mixture is prepared in a 50 ml Falcon
tube: 100 .mu.g linearized protein-free DNA, 1 ml 5.times. buffer
(200 mM Tris-HCl (pH 7.9), 30 mM MgCl.sub.2, 10 mM spermidine, 50
mM NaCl, 50 mM DTT), 200 .mu.l ribonuclease (RNase) inhibitor
(recombinant, 50,000 U), 1 ml rNTP mix (in each case 10 mM ATP,
CTP, UTP; 2 mM GTP), 1 ml CAP analogue (8 mM), 150 .mu.l T7
polymerase (3,000 U) and 2.55 ml RNase-free water. The total volume
is 5 ml. The mixture is incubated at 37.degree. C. for 2 hours in a
heating block. Thereafter, 100 U of RNase-free DNase are added and
the mixture is incubated again at 37.degree. C. for 30 minutes. The
DNA matrix is enzymatically degraded by this procedure.
Description and Origin of the Individual Components
[0115] T7 polymerase: purified from an E. coli strain which
contains a plasmid with the gene for the polymerase. This RNA
polymerase uses as the substrate only promoter sequences of the T7
phage; Fermentas. NTPs: synthesized chemically and purified via
HPLC. Purity more than 96%; Fermentas. CAP analogue: synthesized
chemically and purified via HPLC. Purity more than 90%; Institute
of Organic Chemistry of the University of Tubingen. RNase
inhibitor: RNasin, for injection, prepared recombinantly (E. coli);
Promega. DNase: Pulmozym.RTM. ("dornase alfa"); Roche
Purification
[0116] The RNA treated with DNase is mixed with 20 ml of a solution
of 3.3 ml 5 M NH.sub.4OAc plus 16.65 ml of ethanol. The mixture is
incubated at -20.degree. C. for 1 hour and centrifuged at 4,000 rpm
for 1 hour. The supernatant is removed and the pellet is washed
with 5 ml of 75% RNase-free ethanol. The vessel is centrifuged
again at 4,000 rpm for 15 minutes and the supernatant is removed.
The vessel is centrifuged again under the previous conditions and
the ethanol which remains is removed with a microlitre pipette tip.
The reaction vessel is opened and the pellet is dried under a
sterile bench in the sterile environment.
[0117] 1 ml of RNase-free water is added to the dried RNA. The
pellet is incubated at 4.degree. C. for at least 4 hours. 2 .mu.l
of the aqueous solution are subjected to a quantitative analysis
(determination of the UV absorption at 260 nm). 2 ml of a
phenol/chloroform/isoamyl alcohol solution are added to 1 ml of
aqueous RNA solution. The mixture is vortexed for 2 minutes and
centrifuged at 4,000 rpm for 2 minutes. The aqueous phase is
removed with a microlitre pipette and transferred into a new
reaction vessel. 4 ml of a solution of 0.66 ml 0.5 M NH.sub.4OAc
plus 3.33 ml ethanol are added. The mixture is incubated at
-20.degree. C. for 1 hour and centrifuged at 4,000 rpm for 1 hour.
The supernatant is removed and the pellet is washed with 75%
RNase-free ethanol. The vessel is centrifuged again at 4,000 rpm
for minutes and the supernatant is removed. The vessel is
centrifuged again under the previous conditions and the ethanol
which remains is removed with a microlitre pipette tip. The
reaction vessel is opened and the pellet is dried under a sterile
bench in the sterile environment.
[0118] The RNA is dissolved in RNase-free water and adjusted to a
concentration of 10 mg/ml. It is incubated for 12 hours at
4.degree. C. A final concentration of 2 mg/ml is achieved by
addition of injection buffer (150 mM NaCl, 10 mN HEPES). The end
product is preferably subjected to sterile filtration under GMP
conditions before use.
Application of the RNA
[0119] Each patient receives at two different sites an intradermal
(i.d.) injection of in each case 150 .mu.l of the injection
solution in which in each case 100 .mu.g of antigen-coding mRNA
(CEA, Her-2/neu, MAGE-A1, mucin 1, telomerase, influenza matrix
protein) are present in solution.
[0120] After the primary immunization, a booster immunization is
carried out every 14 days, for the inoculations then to be repeated
at a monthly interval. In each case one day after the RNA
injection, GM-CSF (Leucomax.RTM., Sandoz/Essex Pharma) is
administered subcutaneously (s.c.) to the patient.
[0121] If a clinical response is present or the disease is
stabilized, this therapy is continued at monthly intervals.
Further Immunological Investigations In Vitro (Optional)
[0122] Flow cytometry analyses of PBMC for quantification of CTL
precursors; .sup.51Cr release tests; Soluble receptor and cytokine
levels in the serum; DTH reaction (skin reaction to intradermally
injected RNA, "delayed type hypersensitivity", T
lymphocyte-mediated reaction); and Skin biopsy samples from the
injection site for histological analysis for T cell infiltration
(pathology)
Parameters for Evaluation of the Efficacy
[0123] To be able to answer the question of the efficacy of this
immunotherapy, the induction of tumour-specific T cells and a
measurable tumour remission is used. Parameters are T cell
reactions measured in vitro and in vivo and changes in the size of
bidimensionally recordable tumour manifestation or laboratory
chemistry parameters of the course of the disease.
[0124] Objective remission is defined as the best response in the
form of a complete or partial remission, corresponding to the
criteria listed below. The remission rate is calculated from the
ratio of the number of patients with objective remission and the
total number of evaluable patients.
[0125] A change in the immune status, determined by immunotyping of
peripheral mononuclear cells, an increase in the antigen-specific
CTL precursor frequency in the peripheral blood and the induction
of a persistent tumour-specific T cell activity are assessed as the
immunological response to the therapy. For this purpose, in vitro
induction cultures are established for activation of
tumour-specific CTL.
Remission Criteria (Acc. to UICC)
[0126] Complete remission (CR): Complete regression of all
measurable tumour manifestation, documented by 2 control
investigations at least 4 weeks apart. [0127] Partial remission
(PR): Decrease in size of the total area dimensions (product of two
tumour diameters or linear measurement of one-dimensionally
measurable lesions of all tumour findings by 50% for at least 4
weeks). No new occurrence of tumour manifestations or progression
of a tumour finding. [0128] "No Change" (NC): Decrease of all the
measurable tumour manifestations by less than 50% or increase in a
tumour finding. [0129] Progression (PD): Increase in size of the
tumour parameters in at least one focus or new occurrence of a
tumour manifestation.
REFERENCES
[0129] [0130] 1. Rammensee H G, Falk K, Rotzschke O: Peptides
naturally presented by MHC class I molecules. Annu Rev Immunol 11:
213, 1993. [0131] 2. Bevan M. J: Antigen presentation to cytotoxic
T lymphocytes in vivo. J Exp Med 182: 639, 1995. [0132] 3. Rock K.
L: A new foreign policy: MHC class I molecules police the outside
world. Immunol Today 17:131, 1996. [0133] 4. Steinman, A. M: The
dendritic cell system and its role in immunogenicity. Annu. Rev
Immunol 9:271, 1991. [0134] 5. Steinman R M, Witmer-Pack M, Inaba
K: Dendritic cells: antigen presentation, accessory function and
clinical relevance. Adv Exp Med Biol 329:1, 1993. [0135] 6. Inaba
K, Metlay J P, Crowley M T, Steinman R M: Dendritic cells pulsed
with protein antigens in vitro can prime antigen-specific,
MHC-restricted T cells in situ. J Exp Med 172:631, 1990. [0136] 7.
Austyn M: New insight into the mobilisation and phagocytic activity
of dendritic cells. J Exp Med 183:1287, 1996. [0137] 8. Romani N,
Koide S, Crowley M, Witmer-Pack H, Livingstone A M, Fathman C G,
Steinman R M: Presentation of exogenous protein antigens by
dendritic cells to T cell clones. J Exp Med 169:1169, 1989. [0138]
9. Nair S, Zhou F, Reddy R, Huang L, Rouse B T: Soluble proteins
delivered to dendritic cells via pH-sensitive liposomes induce
primary cytotoxic T lymphocyte responses in vitro. J Exp Med
175:609, 1992. [0139] 10. Cohen P J, Cohen P A, Rosenberg S A, Katz
S I, Mule J J: Murine epidermal Langerhans cells and splenic
dendritic cells present tumor-associated antigens to primed T
cells. Eur J Immunol 24:315, 1994. [0140] 11. Porgador A, Gilboa E:
Bone-marrow-generated dendritic cells pulsed with a class
I-restricted peptide are potent inducers of cytotoxic T
lymphocytes. J Exp Med 182:255, 1995. [0141] 12. Celluzzi C M,
Mayordomo J I, Storkus W J, M. T. Lotze M T, and L. D. Falo L D:
Peptide-pulsed dendritic cells induce antigen-specific,
CTL-mediated protective tumor immunity. J Exp Med 183:283, 1996.
[0142] 13. Zitvogel L, Mayordomo J I, Tjandrawan T, DeLeo A B,
Clarke M R, Lotze M T, Storkus W J: Therapy of murine tumors with
tumor peptide-pulsed dendritic cells: dependence on T cells, B7
costimulation, and T helper cell 1-associated cytokines. J Exp Med
183:87, 1996. [0143] 14. Porgador A, Snyder D, Gilboa E: Induction
of antitumor immunity using bone marrow-generated dendritic cells.
J Immunol 156:2918, 1996. [0144] 15. Paglia P, Chiodoni C, Rodolfo
M, Colombo M P: Murine dendritic cells loaded in vitro with soluble
protein prime cytotoxic T lymphocytes against tumor antigen in
vivo. J Exp Med 183:317, 1996. [0145] 16. Brossart P, Goldrath A W,
Butz E A, Martin S, Bevan M J: Adenovirus mediated delivery of
antigenic epitopes into DC by a means of CTL induction. J Immunol
158: 3270, 1997. [0146] 17. Fisch P, Kohler C G, Garbe A, Herbst B,
Wider D, Kohler H, Schaefer H E, Mertelsmann R, Brugger W, Kanz L:
Generation of antigen-presenting cells for soluble protein antigens
ex vivo from peripheral blood CD34+hematopoetic progenitor cells in
cancer patients Eur J Immunol 26: 595, 1996. [0147] 18. Sallusto F,
Cella H, Danieli C, Lanzavecchia A: Dendritic cells use
macropinocytosis and the mannose receptor to concentrate
macromolecules in the Major Histocompatibility Complex class II
compartment: Down regulation by cytokines and bacterial products, J
Exp Med 182:389, 1995. [0148] 19. Bernhard H, Disis M L, Heimfeld
S, Hand S, Gralow J R, Cheever M A: Generation of immunostimulatory
dendritic cells from human CD34+ hematopoetic progenitor cells of
the bone marrow and peripheral blood. Cancer Res 55: 1099, 1995.
[0149] 20. Hsu F J, Benike C, Fagnoni F, Liles T M, Czerwinski D,
Taidi B, Engelman E G, Levy R: Vaccination of patients with B-cell
lymphoma using autologous antigen-pulsed dendritic cells. Nat Med
2: 52, 1996. [0150] 21. Robbins P F, Kawakami Y: Human tumor
antigens recognized by T cells. Curr Opin Immunol 8: 628, 1996.
[0151] 22. Linehan D C, Goedegebuure P S, Peoples G E, Rogers S O,
Eberlein T J: Tumor-specific and HLA-A2 restricted cytolysis by
tumor-associated lymphocytes in human metastatic breast cancer, J
Immunol 155: 4486, 1995. [0152] 23. Peoples G E, Goedegebure P S,
Smith R, Linehan D C, Yoshino I, Eberlein T J: Breast and ovarian
cancer specific cytotoxic T lymphocytes recognize the same
HER-2/-neu derived peptide. Proc Natl Acad Sci USA 92: 432, 1995.
[0153] 24. Fisk B, Blevins T L, Wharton J T, Ioannides C G:
Identification of an immunodominant peptide of HER-2/neu
protooncogene recognized by ovarian tumor-specific cytotoxic t
lymphocyte lines. J Exp Med 181: 2109, 1995. [0154] 25. Brossart P,
Stuhler G, Flad T, Stevanovic S, Rammensee H-G, Kanz L and Brugger
W. HER-2/neu derived peptides are tumor-associated antigens
expressed by human renal cell and colon carcinoma lines and are
recognized by in vitro induced specific cytotoxic T lymphocytes.
Cancer Res. 58: 732-736, 1998. [0155] 26. Apostolopoulos, V. and
McKenzie, I. F. C., Cellular mucins: targets for immunotherapy.
Crit. Rev. Immunol. 14: 293-302, 1995. [0156] 27. Brossart P,
Heinrich K S, Stevanovic S, Stuhler G, Behnke L, Reichardt V L,
Muhm A, Rammensee H-G, Kanz L, Brugger W. Identification of HLA-A2
restricted T cell epitopes derived from the MUC1 tumor antigen for
broadly applicable cancer vaccines. Blood 93: 4309-4317, 1999
[0157] 28. Brossart P, Wirths S, Stuhler G, Reichardt V L, Kanz L,
Brugger W. Induction of CTL responses in vivo after vaccinations
with peptide pulsed dendritic cells, Blood 96:3102-8, 2000 [0158]
29. Kugler A, Stuhler: G, Walden P, Zoller G, Zobywalski A,
Brossart P, Trefzer U, Ullrich S, Muller C A, Becker V, Gross A J,
Hemmerlein B, Kanz L, Muller G A, Ringert R H. Regression of human
metastatic renal cell carcinoma after vaccination with tumor
cell-dendritic cell hybrids. Nature Med 3: 332-336, 2000 (IF 25,58)
[0159] 30. Nestle F O, Alijagic S, Gilliet M, Sun Y, Grabbe S,
Dummer R, Burg G, Schadendorf D (1998) Vaccination of melanoma
patients with peptide- or tumor lysate-pulsed dendritic cells. Nat.
Med. 4:328 [0160] 31. Schuler-Thurner B, Dieckmann D, Keikavoussi
P, Bender A, Maczek C, Jonuleit H, Roder C, Haendle I, Leisgang W,
Dunbar R, Cerundolo V, von Den D P, Knop J, Brocker E B, Enk A,
Kampgen E, Schuler G (2000) Mage-3 and influenza-matrix
peptide-specific cytotoxic T cells are inducible in terminal stage
HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic
cells. J. Immunol. 165:3492 [0161] 32. Thurner B, Haendle I, Roder
C, Dieckmann D, Keikavoussi P, Jonuleit H, Bender A, Maczek C,
Schreiner D, von Den D P, Brocker E B, Steinman R, Enk A, Kampgen
E, Schuler G (1999) Vaccination with mage-3A1 peptide-pulsed
mature, monocyte-derived dendritic cells expands specific cytotoxic
T cells and induces regression of some metastases in advanced stage
IV melanoma. J. Exp. Med. 190:1669
Example 5
Vaccination with Autologous, Amplified Tumour RNA in Patients with
Malignant Melanoma
Introduction
[0162] The incidence of malignant melanoma has increased sharply
worldwide in recent years. If the melanoma disease is already in
the metastased stage at the time of diagnosis, there is currently
no therapy which has a positive influence on the further course of
the disease with sufficient certainty.
[0163] Vaccination therapies carried out to date using dendritic
cells are very labour-, cost- and time-intensive because of the
complicated culturing of the cells (GMP conditions). Furthermore,
the studies have hitherto concentrated predominantly on known
tumour-associated antigens (TAA), such as, for example, melan-A or
tyrosinase.
[0164] A number of various immunological phenomena, such as, inter
alia, the occurrence of spontaneous tumour regressions or
spontaneous involution of metastases, have made the melanoma the
prior candidate for testing immunotherapy investigations (Parkinson
et al., 1992). In addition to experiments on non-specific
stimulation of the immune system by means of interleukin-2,
mistletoe extracts, BCG and interferons, which have so far not led
to decisive breakthroughs in the therapy of advanced tumour
diseases, the strategy of induction of various highly specific
cytotoxic T lymphocytes (CTL) has been pursued in particular in
recent years. These CTL are capable of recognizing and killing
autologous melanoma cells (Boon et al., 1994; Houghton, 1994).
Studies of this process have shown that the CTL recognize defined
peptides in combination with MHC class I molecules. The
presentation of peptides by antigen-presenting cells (APC) is the
physiological route to generation of specific immune responses by
lymphocytes (Rammensee, 1993). Dendritic cells have proved to be
potent antigen-presenting cells which lead to an induction of the
immune response by two routes: The first is the direct presentation
of peptides towards CD8.sup.+-T lymphocytes and activation thereof
(Schuler & Steinmann, 1985; Inaba et al., 1987; Romani et al.,
1989), and the second is the generation of a protective immune
response, which is mediated by CD4.sup.+ helper lymphocytes, and
requires a presentation of peptides via MHC class II molecules
(Grabbe et al., 1991, 1992, 1995).
[0165] By means of peptide analysis, it was therefore possible to
identify in this way various tumour-associated antigens (TAA) which
are specific for the melanoma and, after presentation in
combination with the MHC molecule and recognition by the CTL, lead
to cytolysis of the tumour cells (Schadendorf et al., 1997, p.
21-27).
[0166] The use of autologous, dendritic cells was tested in the
context of a pilot study on melanoma patients in respect of its
potential to induce cytotoxic T lymphocytes effectively, rapidly
and reliably. In this study, 16 melanoma patients in stage IV who
had already been pretreated by chemotherapy were vaccinated with
peptide-charged dendritic cells. The response rates were above 30%
(5/16 patients) (Nestle et al., 199). In a further independent
study it was possible to demonstrate an even higher response rate
of more than 50% (6/11 patients) after immunization of melanoma
patients who had already been pretreated by chemotherapy with
MAGE-3A1-charged dendritic cells (Thurner et al., 1999). A
significant expansion of MAGE-A3-specific CD8.sup.+-T cells was
also observed in 8/11 patients. A regression of the metastases took
place in some cases after the DC vaccination. This was accompanied
by a CD8.sup.+-T cell infiltration. This showed that the T cells
induced were active in vivo. A disadvantage of this strategy is the
high outlay on costs and the laboratory (in particular GMP
conditions). Large amounts of blood from the patient are required
for the time-intensive generation of the DC. In the preparation of
the peptides, on the one hand only known tumour-associated antigens
can be used, and on the other hand various peptides are necessary,
depending on the HLA haplotype.
[0167] A further development of this approach is vaccination with
RNA-transfected DC (Nair et al., 19983, Nair et al, 2000). In the
meantime, numerous studies demonstrate that DC from mice and humans
which have been transfected with mRNA can induce an efficient CTL
response in vitro and in vivo and can lead to a significant
reduction in metastases (Boczkowski et al., 1996, 2000; Ashley et
al., 1997; Nair et al., 1998, 2000; Heiser et al., 2001; Mitchell
and Nair, 2000; Koido at al., 2000; Schmitt et al., 2001). A great
advantage in the use of RNA compared with peptides is that the most
diverse peptides can be processed and presented from one mRNA which
codes for a TAA. By a polyvalent vaccine of this type, the
probability of the occurrence of so-called clonal "tumour escape"
phenomena can be reduced significantly. Furthermore, T
cell-mediated immune responses against antigens processed and
presented by the natural route and with potentially a higher immune
dominance can be induced by this system. By additional
participation of MHC class II-restricted epitopes, the
tumour-specific immune response induced can be intensified and
maintained for longer. Nevertheless, this process also can be
carried out only with a high outlay on the laboratory (GMP
conditions) because of the necessary culturing of the autologous
DCs.
[0168] In the present strategy according to the invention,
vaccination is carried out with the RNA expression profile present
in the autologous tumour of the patient. The specific tumour
profile of the patient is thereby taken into account, unknown TAAs
also being included in the inoculation. Expensive culture of the
DCs is omitted, since RNAs (not transfected DCs) are used in the
vaccination.
[0169] A vaccination therapy using amplified autologous tumour RNA
on patients with metastased malignant melanoma, in particular stage
III/IV, is therefore provided according to the invention.
[0170] Tumour-specific cytotoxic T cells are induced in vive by the
vaccination, in order thus to achieve a clinico-therapeutic effect
(tumour response). This is an immunisation system which requires
only minimal interventions on the patient (injection). Therapy can
be conducted ambulant and is suitable for many tumour patients,
without the limitation to particular HLA types or defined T cell
epitopes. Furthermore, polyclonal CD4.sup.+-T helpers and also
CD8.sup.+-CTL can be induced by this therapy. From the point of
view of the strategy, it is decisive also that hitherto unknown
TAAs are taken into account in the vaccination protocol, and the
exclusive use of autologous material is particularly
advantageous.
Treatment Plan
[0171] The amplified autologous tumour RNA is administered to the
patient i.d. on days 0, 14, 28 and 42. In addition, the patients
receive GM-CSF (Leucomax.RTM. 100 .mu.g/m.sup.2, Novartis/Essex)
s.c. in each case one day after the RNA inoculation. Each patient
receives, at two different sites, an i.d. injection of in each case
150 .mu.l of the injection solution, in which in each case 100
.mu.g of autologous tumour RNA is dissolved.
[0172] 2 weeks after the fourth injection (day 56), where
appropriate the response of the tumour is evaluated by a staging
analysis (inter alia sonography thorax X-ray, CT etc.; in this
context see the statements below) and by assessment of the
immunological parameters induced by the therapy.
[0173] When the course of the disease is stable or there is an
objective tumour response (CR or PR), the patients receive the
vaccinations every four weeks. Further restaging analyses can be
envisaged e.g. on day 126 and then at an interval of 12 weeks.
[0174] A diagram of the treatment plan is shown in FIG. 13.
Preparation of Autologous Tumour RNA
[0175] The aim is the preparation of autologous poly(A.sup.+) RNA.
For this, poly(A.sup.+) RNA is isolated from the patient's own
tumour tissue. This RNA isolated is very unstable per se and its
amount is limited. The genetic information is therefore transcribed
into a considerably more stable cDNA library and thus conserved.
Starting from the patient's own cDNA library, stabilized autologous
RNA can be prepared for the entire treatment period. The procedure
according to the invention is shown schematically in FIG. 10.
Isolation of RNA
[0176] A process of Roche AG is used to isolate total RNA from a
tumour tissue biopsy. The High Pure RNA Isolation Kit (order number
1828665) is employed here in accordance with the manufacturer's
instructions. Poly(A.sup.+) RNA is isolated from the total RNA via
a further process of Roche AG with the High Pure RNA Tissue Kit
(order number 2033674).
Preparation of a cDNA Library
[0177] The cDNA library is constructed with the "SMART PCR cDNA
Synthesis Kit" (Clontech Inc., USA; order number PT3041-1) in
accordance with the manufacturer's instructions.
[0178] In this procedure, the single-stranded poly(A.sup.+) RNA is
subjected to reverse transcription via a specific primer. Via a
poly-C overhang at the 3'-end of the newly synthesized DNA, a
further primer can hybridize, via which the construct can be
amplified by a PCR. The double-stranded cDNA fragments are now
ready for cloning into a suitable RNA production vector (e.g.
pT7TS; cf. FIG. 8).
[0179] The process for the preparation of the cDNA library from the
poly(A.sup.+) RNA with the aid of the above kit is shown
schematically in FIG. 11.
Plasmid Constructs
[0180] The cDNA PCR fragments are cleaved with the restriction
enzymes NotI and SpeI and cloned into the corresponding restriction
sites of the pT7TS vector by a procedure analogous to that
described in example 4. Plasmids of high purity are obtained via
the Endo-free Maxipreparation Kit (Qiagen, Hilden, Germany).
Plasmids with a cloned-in gene sequence which corresponds to the
expected size fractionation (200 bp-4,000 bp) of the cDNA library
are used for the in vitro transcription. An example of a separation
of a representative cDNA library in an agarose gel is shown in FIG.
12.
In Vitro Transcription and RNA Administration
[0181] The in vitro transcription and the administration of the RNA
are carried out as described in the above example 4.
Investigations During the Treatment
[0182] Before each inoculation (on the day of the inoculation):
Physical examination (including RR, fever); Blood sample for
routine laboratory values [0183] 1. Blood count, differential blood
count: 3 ml [0184] 2. Electrolytes, LDH, CK, liver enzymes,
bilirubin, creatinine, uric acid, total protein, CRP: 5 ml [0185]
3. Blood sedimentation: 2 ml; and at repeat inoculations
additionally: Inspection of the injection sites.
[0186] On day 1 after each inoculation:
Physical examination (including RR, fever); and Inspection of the
injection sites.
[0187] In staging analyses on day 56 and 126 after the first
inoculation, then every 12 weeks, additionally:
Extended routine blood sample: 1. Tumour marker S100 (7 ml) 2.
Clotting values (3 ml); Blood sample for immune monitoring (30 ml);
General well-being (ECOG score);
[0188] Imaging methods (thorax X-ray, sonography, skeleton
scintigram, CT abdomen, pelvis, thorax, skull); and ECG
("EKG").
Further Immunological Investigations In Vitro
[0189] Where appropriate, the relative incidence of
antigen-specific CTL precursor cells in the peripheral blood of the
patient in the course of time of the vaccination therapy is
measured.
[0190] On the one hand CTL precursor cells which are directed
against antigens expressed to a particular degree by melanoma cells
(tyrosinase, MAGE-3, melan-A, GP100) are quantified here with FACS
analyses (tetramer staining). On the other hand ELIspot analyses
are carried out, these being designed such that CTL precursor cells
which are directed specifically against hitherto unknown antigens
are additionally recorded. For this, autologous dendritic cells
cultured from the peripheral blood of the patient are incubated
with the same RNA with which the inoculation has also been carried
out. These then serve as stimulator cells in the ELIspot analysis.
The measurement thus records the total vaccine spectrum. For these
analyses, blood samples of 30 ml in total (20 ml ELIspot, 1.0 ml
FACS analysis) can be envisaged for the immune monitoring in the
context of the staging analyses and additionally on days 0, 14, 28
and 42, as well as a single withdrawal of 100 ml on day 70 for
culture of the DC.
[0191] Furthermore, skin biopsy samples from the injection site can
be obtained for histological analysis in respect of a T cell
infiltration.
Parameters for Evaluation of the Efficacy
[0192] The efficacy of the therapy according to the invention is
evaluated with the aid of the parameters described above in example
4.
REFERENCES
[0193] Anichini, A, Mortarini, R., Maccalli, C., Squarcina, P.,
Fleishhauer, K., Mascheroni, L., Parmiani, G. (1996). Cytotoxic T
cells directed to tumor antigens not expressed on normal
melanocytes dominate HLA-A2-restricted immune repertoire to
melanoma. J. Immunol. 156, 208-217. [0194] Ashley, D M., Faiola,
B., Nair, S., Hale, L P., Bigner, D D, Gilboa, E. (1997) Bone
marrow-generated dendritic cells pulsed with tumor extracts or
tumor RNA induce anti-tumor immunity against central nervous system
tumors. J. Exp. Med. 186, 1177-1182 [0195] Boczkowski, D., Nair, S
K., Synder, D., Gilboa, E. (1996). Dendritic cells pulsed with RNA
are potent antigen-presenting cells in vitro and in vivo. J. Exp.
Med. 184, 465-472. [0196] Boczkowski, D., Nair, S K., Nam, J.,
Lyerly, K., Gilboa, E. (2000) Induction of tumor immunity and
cytotoxic T lymphocyte responses using dendritic cells transfected
with messenger RNA amplified from tumor cells. Cancer Res. 60,
1028-1034. [0197] Boon, T., Coulie, P., Marchand, M., Weynants, P.,
Wolfel, T., Brichard, V. (1994). Genes coding for tumor rejection
antigens: perspectives for specific immunotherapy. In Important
Advances in Oncology 1994. DeVita, V T, Hellman, S., Rosenberg, S
A, ed. (Philadelphia: Lippincott Co), pp. 53-69. [0198] Garbe, C,
Orfanos, C E (1989): Epidemiologie des malignen Melanoms in der
Bundesrepublik Deutschland im internationalen Vergleich
[Epidemiology of malignant melanoma in the Federal Republic of
Germany in an international comparison]. Onkologie 12, 253-262.
[0199] Grabbe, S., Bruvers, S., Gallo, R. L., Knisely, T. L.,
Nazareno, R., and Granstein, R. D. (1991). Tumor antigen
presentation by murine epidermal cells. J. Immunol. 146, 3656-3661.
[0200] Grabbe, S., Bruvers, S., Lindgren, A. M., Hosoi, J., Tan, K.
C., and Granstein, R. D. (1992). Tumor antigen presentation by
epidermal antigen-presenting cells in the mouse: modulation by
granulocyte-macrophage colony-stimulating factor, tumor necrosis
factor alpha, and ultraviolet radiation. J Leukoc. Biol. 52,
209-217. [0201] Grabbe, S., Beissert, S., Schwarz, T., and
Granstein, R. D. (1995). Dendritic cells as initiators of tumor
immune responses: a possible strategy for tumor immunotherapy?.
Immunol. Today 16, 117-121. [0202] Grunebach, F, Muller, MR,
Nenciona, A, Brugger, W, and Brossart, P (2002). Transfection of
dendritic cells with RNA induces cytotoxic T lymphocytes against
breast and renal cell carcinomas and reveals the immunodominance of
presented T cell epitopes. submitted. [0203] Heiser, A., Maurice, M
A., Yancey, D R., Coleman, D M., Dahm, P., Vieweg, J. (2001). Human
dendritic cells transfected with renal tumor RNA stimulate
polyclonal T cell responses against antigens expressed by primary
and metastatic tumors. Cancer Res. 61, 3388-3393. [0204] Heiser,
A., Maurice, M A., Yancey, D R., Wu, N Z., Dahm P., Pruitt, S K.,
Boczkowski, D., Nair, S K., Ballo, M S., Gilboa, E., Vieweg, J.
(2001). Induction of polyclonal prostate cancer-specific CTL using
dendritic cells transfected with amplified tumor RNA. J. Immunol.
166, 2953-2960. [0205] Hoerr, I, Obst, R, Rammensee, H G, Jung, G
(2000). In vivo application of RNA leads to induction of specific
cytotoxic T lymphocytes and antibodies. Eur J Immunol. 30, 1-7.
[0206] Houghton, A N (1994). Cancer antigens: immune recognition of
self and altered self. J. Exp. Med 180, 1-4 [0207] Inaba, K, Young,
J W and Steinman, R M (1987). Direct activation of CD8+ cytotoxic T
lymphocytes by dendritic cells. J. Exp. Med. 166, 182-194. [0208]
Koido, S., Kashiwaba, M., Chen, D., Gendler, S., Kufe, D., Gong, J.
(2000). Induction of antitumor immunity by vaccination of dendritic
cells transfected with MUC1 RNA. Immunol. 165, 5713-5719. [0209]
Mitchell, D A., Nair, S K. (2000), RNA-transfected dendritic cells
in cancer immunotherapy. J. Clin. Invest. 106, 1065-1069. [0210]
Nair, S., Boczkowski, S., Synder, D., Gilboa, E. (1998). Antigen
presenting cells pulsed with unfractionated tumor-derived peptides
are potent tumor vaccines. Eur. J. Immunol. 27, 589-597. [0211]
Nair, S., Heiser, A., Boczkowski, D., Majumdar, A., Naoe, M.,
Lebkowski, J S., Vieweg, J., Gilboa, E. (2000), Induction of
cytotoxic T cell responses and tumor immunity against unrelated
tumors using telomerase reverse transcriptase RNA transfected
dendritic cells. Nat. Med. 6, 1011-1017. [0212] Nestle, F. O.,
Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, F., Burg,
G., and Schadendorf, D. (1998). Vaccination of melanoma patients
with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med 4,
328-332. [0213] Parkinson, D R, Houghton, A N, Hersey, P, Borden, E
C (1992), Biologic therapy for melanoma. I Cutaneous melanoma,
Balch, C M, Houghton, A N, Milton G W, Soober, A J, Soong, S J, ed.
(Lippincott Co), pp. 522-541 [0214] Rammensee, H. G., Falk, K., and
Rotzschke, O. (1993). Peptides naturally presented by MHC class I
molecules. Annu. Rev. Immunol. 11, 213-244. [0215] Romani N, Koide
S, Crowley M, Witmer-Pack M, Livingstone A M, Fathman C G, Steinman
R M: Presentation of exogenous protein antigens by dendritic cells
to T cell clones. J Exp Med 169:1169, 1989. [0216] Schadendorf, D,
Grabbe, S, Nestle, F O (1997). Vaccination with Dendritic Cells--A
specific Immunomodulatory Approach. In Strategies for
Immunointervention in Dermatology. Burg, G, Dummer, R G, ed.
(Heidelberg, New York: Springer-Verlag), [0217] Schmitt, W E.,
Stassar, M J J G., Schmitt, W., Littlee, M., Cochlovius, B. (2001).
In vitro induction of a bladder cancer-specific T-cell response by
mRNA-transfected dendritic cells. J. Cancer Res. Clin. Oncol. 127,
203-206. [0218] Schmoll H J, Hoffken K, Possinger K (1997)
Kompendium Internistitische Onkologie [Compendium of Internal
Oncology], 2nd ed., Springer-Verlag Berlin, part 2, 1415. [0219]
Schuler G and Steinmann R M (1985). Murine epidermal Langerhans
cells mature into potent immunostimulatory dendritic cells in
vitro. J. Exp. Med. 161, 526-546. [0220] Thurner, B., Haendle, I.,
Roder, C. et al. (1999) Vaccination with Mage-3A1 peptide-pulsed
mature, monocyte-derived dendritic cells expands specific cytotoxic
T cells and induces regression of some metastases in advanced stage
IV melanoma. J. Exp. Med. 190, 1669-1678.
[0221] All publications, patents and patent documents are
incorporated by reference herein, as though individually
incorporated by reference.
Sequence CWU 1
1
4122RNAArtificial SequenceDescription of Artificial Sequence
Synthetic stabilizing sequence 1yccannnnnc ccwyyyyucy cc
22213RNAArtificial SequenceDescription of Artificial Sequence
Synthetic Kozak sequence 2gccgccacca ugg 13345DNAXenopus laevis
3gcttgttctt tttgcagaag ctcagaataa acgctcaact ttggc 454157DNAXenopus
laevis 4gactgactag gatctggtta ccactaaacc agcctcaaga acacccgaat
ggagtctcta 60agctacataa taccaactta cacttacaaa atgttgtccc ccaaaatgta
gccattcgta 120tctgctccta ataaaaagaa agtttcttca cattcta 157
* * * * *