U.S. patent application number 14/375215 was filed with the patent office on 2015-04-30 for pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen.
This patent application is currently assigned to CureVac GMBH. The applicant listed for this patent is CureVac GmbH. Invention is credited to Patrick Baumhof, Mariola Fotin-Mleczek, Karl-Josef Kallen, Thomas Kramps, Sohnke Voss.
Application Number | 20150118264 14/375215 |
Document ID | / |
Family ID | 47631406 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150118264 |
Kind Code |
A1 |
Baumhof; Patrick ; et
al. |
April 30, 2015 |
PHARMACEUTICAL COMPOSITION COMPRISING A POLYMERIC CARRIER CARGO
COMPLEX AND AT LEAST ONE PROTEIN OR PEPTIDE ANTIGEN
Abstract
The present invention is directed to a pharmaceutical
composition including (e.g. for use as an adjuvant) a polymeric
carrier cargo complex, comprising as a carrier a polymeric carrier
formed by disulfide-crosslinked cationic components; and as a cargo
at least one nucleic acid molecule, and at least one antigen that
is selected from an antigen from a pathogen associated with
infectious disease; an antigen associated with allergy or allergic
disease; an antigen associated with autoimmune disease; or an
antigen associated with a cancer or tumour disease, or in each case
a fragment, variant and/or derivative of said antigen. The
pharmaceutical composition allows for efficient induction of an
adaptive immune response directed against said antigen. The present
invention furthermore provides kits, as well as the use of the
pharmaceutical composition or the kit as a vaccine, particularly in
the treatment of infectious diseases, allergies, autoimmune
diseases and tumour or cancer diseases.
Inventors: |
Baumhof; Patrick;
(Dusslingen, DE) ; Kramps; Thomas; (Tubingen,
DE) ; Voss; Sohnke; (Neckargemund, DE) ;
Kallen; Karl-Josef; (Konigsdorf, DE) ; Fotin-Mleczek;
Mariola; (Sindelfingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac GmbH |
Tubingen |
|
DE |
|
|
Assignee: |
CureVac GMBH
Tubingen
DE
|
Family ID: |
47631406 |
Appl. No.: |
14/375215 |
Filed: |
January 31, 2013 |
PCT Filed: |
January 31, 2013 |
PCT NO: |
PCT/EP2013/000291 |
371 Date: |
July 29, 2014 |
Current U.S.
Class: |
424/209.1 ;
424/204.1; 424/224.1; 424/227.1; 424/275.1; 424/277.1; 530/300;
530/350 |
Current CPC
Class: |
A61K 39/001109 20180801;
A61K 39/001153 20180801; C12N 2760/16134 20130101; A61K 39/001106
20180801; A61K 39/39 20130101; A61K 39/001164 20180801; C12N
2760/16171 20130101; A61P 31/12 20180101; A61K 39/001188 20180801;
A61K 39/001104 20180801; C12N 2760/20171 20130101; A61K 2039/55511
20130101; A61K 39/001191 20180801; A61P 31/14 20180101; A61P 31/16
20180101; Y02A 50/416 20180101; C12N 2730/10171 20130101; A61K
39/001182 20180801; A61K 39/145 20130101; A61K 39/001168 20180801;
A61K 47/646 20170801; A61K 48/0041 20130101; A61K 2039/575
20130101; A61K 39/001156 20180801; A61P 31/22 20180101; A61K
39/001193 20180801; A61P 31/20 20180101; A61P 37/00 20180101; A61K
39/001195 20180801; A61K 39/001135 20180801; A61P 35/00 20180101;
A61P 37/08 20180101; A61K 2039/572 20130101; C12N 2760/20134
20130101; A61K 39/29 20130101; A61K 2039/53 20130101; A61K
39/001194 20180801; A61K 39/001192 20180801; A61K 2039/6093
20130101; A61P 31/00 20180101; Y02A 50/30 20180101; A61K 39/001186
20180801; A61K 39/205 20130101; A61K 39/0011 20130101; A61K 39/12
20130101; A61K 2039/55561 20130101; C12N 2710/20071 20130101; A61P
31/04 20180101; A61K 39/35 20130101; A61P 37/02 20180101; A61K
47/645 20170801; A61K 2039/5252 20130101; C12N 2710/20034 20130101;
C12N 2730/10134 20130101; A61K 39/00117 20180801; A61K 2039/55516
20130101; A61P 31/06 20180101; C12N 7/00 20130101; A61K 39/001157
20180801; A61K 39/001151 20180801 |
Class at
Publication: |
424/209.1 ;
424/275.1; 424/224.1; 424/227.1; 424/204.1; 424/277.1; 530/300;
530/350 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 48/00 20060101 A61K048/00; A61K 39/35 20060101
A61K039/35; A61K 39/00 20060101 A61K039/00; C12N 7/00 20060101
C12N007/00; A61K 39/145 20060101 A61K039/145; A61K 39/29 20060101
A61K039/29; A61K 39/12 20060101 A61K039/12; A61K 47/48 20060101
A61K047/48; A61K 39/205 20060101 A61K039/205 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 2012 |
EP |
PCT/EP2012/000420 |
Claims
1. A pharmaceutical composition comprising: (A) a polymeric carrier
cargo complex, comprising: a) a polymeric carrier comprising
disulfide-crosslinked cationic components, preferably formed by
disulfide-crosslinked cationic components, as a carrier; and b) at
least one nucleic acid molecule as a cargo, and (B) at least one
protein or peptide antigen that is selected from the group
consisting of: (i) an antigen from a pathogen associated with
infectious disease; (ii) an antigen associated with allergy or
allergic disease; (iii) an antigen associated with autoimmune
disease; and (iv) an antigen associated with a cancer or tumour
disease or a fragment, variant and/or derivative of said protein or
peptide antigen.
2. The pharmaceutical composition of claim 1, wherein component (B)
is not covalently linked to component (A).
3. The pharmaceutical composition of claim 1, wherein said protein
or peptide antigen is from a pathogen selected from the list
consisting of Rabies virus, Hepatitis B virus, human Papilloma
virus (hPV), Bacillus anthracis, Respiratory syncytial virus (RSV),
Herpes simplex virus (HSV), Influenza virus and Mycobacterium
tuberculosis.
4. The pharmaceutical composition of claim 1, wherein said protein
or peptide antigen is selected from the list consisting of: The
Hemagglutinin (HA), the Neuraminidase (NA), the Nucleoprotein (NP),
the M1 protein, the M2 protein, the NS1 protein, the NS2 protein
(the NEP protein: nuclear export protein), the PA protein, the PB1
protein (polymerase basic 1 protein), the PB1-F2 protein and the
PB2 protein of Influenza virus; The nucleoprotein (N), the
phosphoprotein (P), the matrix protein (M), the glycoprotein (G),
and the viral RNA polymerase (L), in each case of Rabies virus; the
Hepatitis B surface antigen (HBsAg), the Hepatitis B core antigen
(HbcAg), the Hepatitis B virus DNA polymerase, the HBx protein, the
preS2 middle surface protein, the large S protein, the virus
protein VP1, the virus protein VP2, the virus protein VP3, and the
virus protein VP4, in each case of Hepatitis B virus; the E1
protein, the E2 protein, the E3 protein, the E4 protein, the E5
protein, the E6 protein, the E7 protein, the E8 protein, the L1
protein, and the L2 protein, in each case of human Papilloma virus
(hPV); the protective antigen (PA), the edema factor (EF), the
lethal factor (LF), and the S-layer homology proteins (SLH), in
each case of Bacillus anthracis; the Fusion (F) protein, the
nucleocapsid (N) protein, the phosphoprotein (P), the matrix (M)
protein, the glycoprotein (G), the large protein (L; RNA
polymerase), the non-structural protein 1 (NS1), the non-structural
protein 2 (N52), the small hydrophobic (SH) protein, the elongation
factor M2-1, and the transcription regulation protein M2-2, in each
case of respiratory syncytial virus (RSV); the Glycoprotein L
(UL1), the Uracil-DNA glycosylase UL2, the UL3 protein, the UL4
protein, the DNA replication protein UL5, the Portal protein UL6,
the Virion maturation protein UL7, the DNA helicase UL8, the
Replication origin-binding protein UL9, the Glycoprotein M (UL10),
the UL11 protein, the Alkaline exonuclease UL12, the
Serine-threonine protein kinase UL13, the Tegument protein UL14,
the Terminase (UL15), the Tegument protein UL16, the UL17 protein,
the Capsid protein VP23 (UL18), the Major capsid protein VP5
(UL19), the Membrane protein UL20, the Tegument protein UL21, the
Glycoprotein H (UL22), the Thymidine Kinase UL23, the UL24 protein,
the UL25 protein, the Capsid protein P40 (UL26, VP24, VP22A), the
Glycoprotein B (UL27), the ICP 18.5 protein (UL28), the Major
DNA-binding protein ICP8 (UL29), the DNA polymerase UL30, the
Nuclear matrix protein UL31, the Envelope glycoprotein UL32, the
UL33 protein, the Inner nuclear membrane protein UL34, the Capsid
protein VP26 (UL35), the Large tegument protein UL36, the Capsid
assembly protein UL37, the VP 19C protein (UL38), the
Ribonucleotide reductase (Large subunit) UL39, the Ribonucleotide
reductase (Small subunit) UL40, the Tegument protein/Virion host
shutoff VHS protein (UL41), the DNA polymerase processivity factor
UL42, the Membrane protein UL43, the Glycoprotein C (UL44), the
Membrane protein UL45, the Tegument proteins VP11/12 (UL46), the
Tegument protein VP13/14 (UL47), the Virion maturation protein VP16
(UL48, Alpha-TIF), the Envelope protein UL49, the dUTP
diphosphatase UL50, the Tegument protein UL51, the DNA
helicase/primase complex protein UL52, the Glycoprotein K (UL53),
the Transcriptional regulation protein 1E63 (ICP27, UL54), the UL55
protein, the UL56 protein, the Viral replication protein ICP22
(1E68, US1), the US2 protein, the Serine/threonine-protein kinase
US3, the Glycoprotein G (US4), the Glycoprotein J (US5), the
Glycoprotein D (US6), the Glycoprotein I (US7), the Glycoprotein E
(US8), the Tegument protein US9, the Capsid/Tegument protein US10,
the Vmw21 protein (US11), the ICP47 protein (IE12, US12), the Major
transcriptional activator ICP4 (1E175, RS1), the E3 ubiquitin
ligase ICP0 (IE110), the Latency-related protein 1 (LRP1), the
Latency-related protein 2 (LRP2), the Neurovirulence factor RL1
(ICP34.5), and the Latency-associated transcript (LAT), in each
case of Herpes simplex virus (HSV); or the ESAT-6 protein, the
ESX-1 protein, the CFP10 protein, the TB 10.4 protein, the MPT63
protein, the MPT64 protein, the MPT83 protein, the MTB12 protein,
the MTB8 protein, the AG85A protein, the AG85B protein, the
Rpf-like proteins, the KATG protein, the PPE18 protein, the MTB32
protein, the MTB39 protein, the Crystallin, the HSP65 protein, the
PST-S protein, and the HBHA protein, the 10 kDa filtrate antigen
EsxB, the serine protease PepA, the fibronectin-binding protein D
FbpD, the secreted protein MPT51, the periplasmic phosphate-binding
lipoprotein PSTS1 (PBP-1), the periplasmic phosphate-binding
lipoprotein PSTS3 (PBP-3, Phos-1), the PPE family protein PPE14,
the PPE family protein PPE68, the protein MTB72F, the molecular
chaperone DnaK, the cell surface lipoprotein MPT83, the lipoprotein
P23, the Phosphate transport system permease protein PstA, the 14
kDa antigen, the fibronectin-binding protein C FbpC1, the Alanine
dehydrogenase TB43, and the Glutamine synthetase 1, in each case of
Mycobacterium tuberculosis.
5. The pharmaceutical composition of claim 1, wherein said protein
or peptide antigen is associated with allergy or allergic disease
and is derived from a source selected from the list consisting of
grass pollen, tree pollen, flower pollen, herb pollen, dust mite,
mold, animals, food, and insect venom.
6. The pharmaceutical composition of claim 1, wherein said protein
or peptide antigen is associated with autoimmune disease and is
selected from the list consisting of: myelin basic protein (MBP),
proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein
(MOG), in each case associated with multiple sclerosis (MS); CD44,
preproinsulin, proinsulin, insulin, glutamic acid decaroxylase
(GAD65), tyrosine phosphatase-like insulinoma antigen 2 (IA2), zinc
transporter ((ZnT8), and heat shock protein 60 (HSP6O), in each
case associated with diabetes Typ I; interphotoreceptor
retinoid-binding protein (IRBP) associated with autoimmune uveitis;
acetylcholine receptor AchR, and insulin-like growth factor-1
receptor (IGF-1R), in each case associated with Myasthenia gravis;
M-protein from beta-hemolytic streptocci (pseudo-autoantigen)
associated with Rheumatic Fever; Macrophage migration inhibitory
factor associated with Arthritis; Ro/La RNP complex, alpha- and
beta-fodrin, islet cell autoantigen, poly(ADP)ribose polymerase
(PARP), NuMA, NOR-90, Ro60 autoantigen, and p27 antigen, in each
case associated with Sjogren's syndrome; Ro60 autoantigen,
low-density lipoproteins, Sm antigens of the U-1 small nuclear
ribonucleoprotein complex (B/B', D1, D2, D3, E, F, G), and RNP
ribonucleoproteins, in each case associated with lupus
erythematosus; oxLDL, beta(2)GPI, HSP6O/65, and oxLDL/beta(2)GPI,
in each case associated with Atherosclerosis; cardiac
beta(1)-adrenergic receptor associated with idiopathic dilated
cardiomyopathy (DCM); histidyl-tRNA synthetase (HisRS) associated
with myositis; topoisomerase I associated with scleroderma; IL-17;
or heat shock proteins.
7. The pharmaceutical composition of claim 1, wherein said protein
or peptide antigen is associated with a cancer or tumour disease
and is selected from the list consisting of p53, CA125, EGFR,
Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3, Mesothelin, MUC-1,
NY-ESO-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA VEGF, VEGFR1,
VEGFR2, Ras, CEA and WT1.
8. The pharmaceutical composition of claim 1, wherein said
polymeric carrier cargo complex is for use as an adjuvant.
9. The pharmaceutical composition of claim 1, wherein said nucleic
acid molecule cargo is an immunostimulatory nucleic acid.
10. The pharmaceutical composition of claim 1, wherein said nucleic
acid molecule cargo is RNA; preferably wherein said nucleic acid
molecule cargo is an immunostimulatory RNA (isRNA).
11. The pharmaceutical composition of claim 1, wherein the cationic
components of the polymeric carrier and the nucleic acid molecule
cargo comprised in said polymeric carrier cargo complex are
provided in an N/P ratio in the range of 0.1-20, or in the range of
0.1-5, or in the range of 0.1-1, or in the range of 0.5-0.9.
12. The pharmaceutical composition of claim 1, wherein the cationic
components of the polymeric carrier comprised in said polymeric
carrier cargo complex are cationic peptides.
13. The pharmaceutical composition of claim 12, wherein said
cationic peptides are selected from peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x,
wherein l+m+n+o+x=3-100, and l, m, n or o=independently of each
other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60,
61-70, 71-80, 81-90 and 91-100, provided that the overall content
of Arg, Lys, His and Orn represents at least 10% of all amino acids
of the cationic peptide; and Xaa is any amino acid selected from
native (=naturally occurring) or non-native amino acids except of
Arg, Lys, His or Orn; and x=any number selected from 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30,
31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that the
overall content of Xaa does not exceed 90% of all amino acids of
the cationic peptide, or said cationic peptides are selected from
peptides according to subformula (IIIa)
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa').sub.x
(Cys).sub.y} or said cationic peptides from peptides according to
subformula (Ib)
Cys.sub.1{(Arg).sub.1;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys-
.sub.2 wherein (Arg).sub.1; (Lys).sub.m; (His).sub.n; (Orn).sub.o;
and x are as defined above; Xaa' is any amino acid selected from
native (=naturally occurring) or non-native amino acids except of
Arg, Lys, His, Orn; or Cys and y is any number selected from 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, provided that
the overall content of Arg (Arginine), Lys (Lysine), His
(Histidine) and Orn (Ornithine) represents at least 10% of all
amino acids of the oligopeptide and wherein Cys.sub.1 and Cys.sub.2
are Cysteines proximal to, or terminal to
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x.
14. The pharmaceutical composition of claim 1 wherein said
polymeric carrier cargo complex does not include said protein or
peptide antigen.
15. A kit or kit of parts comprising: a) a polymeric carrier cargo
complex as defined according to claim 1; and b) at least one
protein or peptide antigen or fragment, variant and/or derivative
thereof as defined according claim 1.
16-19. (canceled)
20. A pharmaceutical package, including: (A) a polymeric carrier
cargo complex as defined according to claim 1; and (B) instructions
describing the use of said polymeric carrier cargo complex in
therapy in combination with at least one protein or peptide antigen
or fragment, variant and/or derivative thereof as defined according
to claim 1.
21. A pharmaceutical package, including: (A) at least one protein
or peptide antigen or fragment, variant and/or derivative thereof
as defined according to claim 1; and (B) instructions describing
the use of said protein or peptide antigen or fragment, variant
and/or derivative thereof in therapy in combination with a
polymeric carrier cargo complex as defined according to claim
1.
22. The pharmaceutical package of claim 20, wherein said
instructions further describe the use in the therapy of (i)
infectious disease; (ii) allergy or allergic disease; (iii)
autoimmune disease; or (iv) cancer or tumour disease.
Description
[0001] The present invention is directed to a pharmaceutical
composition comprising a polymeric carrier cargo complex and at
least one antigen. The polymeric carrier cargo complex preferably
comprises a carrier and a cargo, wherein the carrier is a
disulfide-crosslinked cationic component and the cargo at least one
nucleic acid molecule. The at least one antigen is preferably
selected from an antigen from a pathogen associated with infectious
disease, an antigen associated with allergy or allergic disease, an
antigen associated with autoimmune disease, or an antigen
associated with a cancer or tumour disease, or in each case a
fragment, variant and/or derivative of said antigen. Such an
inventive pharmaceutical composition may be, e.g., a vaccine
wherein the polymeric carrier cargo complex may serve as an
adjuvant to support an immune response to the antigen. Accordingly,
such a pharmaceutical composition allows for efficient induction of
an adaptive immune response directed against the at least one
antigen comprised therein, particularly of a Th1-shifted immune
response.
[0002] The present invention furthermore provides kits or kits of
parts comprising the components of the inventive pharmaceutical
composition, as well as the use of the inventive pharmaceutical
composition or the inventive kit or kit of parts as a vaccine,
particularly in the treatment of infectious diseases, allergies,
autoimmune diseases and tumour or cancer diseases. Furthermore the
invention provides: (a) a polymeric carrier cargo complex for use
in therapy in combination with at least one antigen or a fragment,
variant and/or derivative thereof; and (b) at least one antigen or
a fragment, variant and/or derivative thereof for use in therapy in
combination with a polymeric carrier cargo complex, in each case
(a) and (b), particularly for use in therapy of infectious
diseases, allergies, autoimmune diseases and tumour or cancer
diseases.
[0003] Many diseases today require administration of adjuvants to
provide an innate immune response to support an adaptive immune
response, particularly in the context of vaccinations. Some but not
necessarily all of these diseases additionally or alternatively
require administration of peptide-, protein-, and nucleic
acid-based drugs, e.g. the transfection of nucleic acids into cells
or tissues. These requirements usually represent different aspects
in the treatment of such diseases and are typically difficult to
address in one approach. As a consequence, the prior art usually
handles such aspects via separate approaches.
[0004] In the above context, vaccination is generally believed to
be one of the most effective and cost-efficient ways to prevent or
treat diseases. Nevertheless, several problems in vaccine
development have proved difficult to solve: Vaccines are often
inefficient for the very young and the very old; many vaccines need
to be given several times, and the protection they confer wanes
over time, requiring booster administrations, and, for some
diseases such as HIV, development of efficient vaccines is urgently
needed. As generally accepted, many of these vaccines would be
enabled or improved if they could elicit a stronger and more
durable immune response.
[0005] Accordingly, the development of new efficient and safe
pharmaceutical compositions that include adjuvants for vaccination
purposes which support induction and maintenance of an adaptive
immune response by initiating or boosting a parallel innate immune
response represents a main challenging problem.
[0006] Adjuvants are usually defined as compounds that can increase
and/or modulate the intrinsic immunogenicity of an antigen. To
reduce negative side effects, new vaccines have a more defined
composition that often leads to lower immunogenicity compared with
previous whole-cell or virus-based vaccines. Adjuvants are
therefore required to assist new vaccines to induce potent and
persistent immune responses, with the additional benefit that less
antigen and fewer injections are needed. Now it is clear that the
adaptive immune response mainly depends on the level and
specificity of the initial danger signals perceived by innate
immune cells following infection or vaccination (Guy, B. (2007),
Nat Rev Microbiol 5(7): 505-17.). In particular for new generation
vaccine candidates, which will increasingly comprise highly
purified recombinant proteins and, although very safe, are poorly
immunogenic, efficient adjuvants will become increasingly
necessary.
[0007] Unfortunately, only a few licensed adjuvants are available
so far. Most prominent is Alum, which is known to be safe, but also
represents a very weak adjuvant. Many further adjuvants have been
developed, e.g. including the administration of pathogens,
CpG-nucleotides, etc. Most of these new or "established" adjuvants,
however, still do not satisfy the above requirements, since many
new and emerging problems have to be considered and solved. These
problems inter alia include new and re-emerging infectious
diseases, repeated administrations, threat of pandemic flu,
etc.
[0008] Furthermore, the new vaccine targets are usually more
difficult to develop and--due to their specifically tailored immune
responses--require more potent adjuvants to enable success.
Moreover, there are still a significant number of important
pathogens for which we do not even have effective vaccines at
present. This represents a very challenging future target. To
enable vaccine development against such targets, more potent
pharmaceutical compositions that include adjuvants and such targets
will be necessary. Therefore, the new adjuvants in such
compositions will need to offer advantages, including more
heterologous antibody responses, covering pathogen diversity,
induction of potent functional antibody responses, ensuring
pathogen killing or neutralization and induction of more effective
T cell responses, for direct and indirect pathogen killing,
particularly the induction of cytotoxic T cells which are part of a
Th1 immune response. In addition, adjuvants may be necessary to
achieve more pragmatic effects, including antigen dose reduction
and overcoming antigen competition in combination vaccines.
Moreover, against the background of an aging population, which is
increasingly susceptible to infectious diseases, new adjuvants will
be necessary to overcome the natural deterioration of the immune
response with age (O'Hagan, D. T. and E. De Gregorio (2009), Drug
Discov Today 14(11-12): 541-51.).
[0009] The review of O'Hagan (2009; supra) summarizes some reasons
for the urgent need of new effective adjuvants e.g. the requirement
of a lower antigen dose in vaccines, the necessity to increase the
breadth of an immune response and the heterologous activity, to
enable complex combination vaccines, and to overcome antigenic
competition, to overcome limited immune response in some groups of
the population, such as the elderly, the young children, and
infants, patients with chronic diseases and the immunocompromised,
to increase effector T cell response and antibody titers, to induce
protective responses more rapidly and also to extend the duration
of response by enhancing memory B and T cell responses.
[0010] Summarizing the above, new efficient and safe pharmaceutical
compositions that include immunostimulating agents or adjuvants are
required, which are preferably efficient in inducing an innate
immune response, particularly in inducing the anti-viral cytokine
IFN-alpha; and which are also efficient in supporting an adaptive
immune response; safe, i.e. not associated with any long-term
effects; which are well tolerated; which are available via a simple
synthetic pathway; which exhibit low cost storage conditions
(particularly feasible lyophilisation); which require simple and
inexpensive components; which are biodegradable; which are
compatible with many different kinds of vaccine antigens; which are
capable of codelivery of antigen and immune potentiator, etc.
[0011] As already explained above adjuvants or immunostimulating
agents usually act via their capability to induce an innate immune
response. The innate immune system forms the dominant system of
host defense in most organisms and comprises barriers such as
humoral and chemical barriers including, e.g., inflammation, the
complement system and cellular barriers. The innate immune system
is typically based on a small number of receptors, called pattern
recognition receptors. They recognize conserved molecular patterns
that distinguish foreign organisms, like viruses, bacteria, fungi
and parasites, from cells of the host. Such pathogen-associated
molecular patterns (PAMP) include viral nucleic acids, components
of bacterial and fungal walls, flagellar proteins, and more. The
first family of pattern recognition receptors (PAMP receptors)
studied in detail was the Toll-like receptor (TLR) family. TLRs are
transmembrane proteins which recognize ligands of the extracellular
milieu or of the lumen of endosomes. Following ligand-binding they
transduce the signal via cytoplasmic adaptor proteins which leads
to triggering of a host-defence response and entailing production
of antimicrobial peptides, proinflammatory chemokines and
cytokines, antiviral cytokines, etc. (see e.g. Meylan, E.,).
Tschopp, et at (2006), Nature 442(7098): 39-44). Further relevant
components of the immune system include e.g. the endosomal TLRs,
cytoplasmic receptors, Type I interferons and cytoplasmic
receptors. Therefore, the immunostimulating agents or adjuvants are
defined herein preferably as inducers of an innate immune response,
which activate pattern recognition receptors (PAMP receptors).
Hereby, a cascade of signals is elicited, which e.g. may result in
the release of cytokines (e.g. IFN-alpha) supporting the innate
immune response. Accordingly, it is preferably a feature of an
immunostimulating agent or adjuvant to bind to such receptors and
activate such PAMP receptors. Ideally, such as an agent or adjuvant
additionally supports the adaptive immune response by e.g. shifting
the immune response such that the preferred class of Th cells is
activated. Depending on the disease or disorder to be treated a
shift to a Th1-based immune response may be preferred or, in other
cases, a shift to a Th2 immune response may be preferred.
[0012] In the prior art there are some promising adjuvant
candidates which fulfil at least some, but not all, of the above
defined required characteristics.
[0013] As an example, among the above developed new adjuvants, some
nucleic acids like CpG DNA oligonucleotides or isRNA
(immunostimulating RNA) turned out to be promising candidates for
new immunostimulating agents or adjuvants as they allow the
therapeutic or prophylactic induction of an innate immune response.
Comprehensibly, such nucleic acid based adjuvants usually have to
be delivered effectively to the site of action to allow induction
of an effective innate immune response without unnecessary loss of
adjuvant activity and, in some cases, without the necessity to
increase the administered volume above systemically tolerated
levels.
[0014] One approach to solve this issue may be the transfection of
cells which are part of the innate immune system (e.g. dendritic
cells, plasmacytoid dendritic cells (pDCs)) with immunostimulatory
nucleic acids, which are ligands of PAMP receptors, (e.g. Toll-like
receptors (TLRs)), and thus may lead to immunostimulation by the
nucleic acid ligand. Further approaches may be the direct
transfection of nucleic acid based adjuvants. All of these
approaches, however, are typically impaired by inefficient delivery
of the nucleic acid and consequently diminished adjuvant activity,
in particular when administered locally.
[0015] However, one main disadvantage of such nucleic acid based
adjuvant approaches until today is their limited ability to cross
the plasma membrane of mammalian cells, resulting in poor cellular
access and inadequate therapeutic efficacy. Until today this hurdle
represents a major challenge for nucleic acid transfection based
applications, e.g. biomedical developments and accordingly the
commercial success of many biopharmaceuticals (see e.g. Foerg, C.
& Merkle, H. P., J Pharm Sci 97, 144-62 (2008).
[0016] Transfection of nucleic acids or genes into cells or tissues
has been investigated up to date in the context of in vitro
transfection purposes and in the context of gene therapeutic
approaches. However, no adjuvants are available so far which are
based on such gene delivery techniques which are efficient and
safe, in particular no licensed adjuvants. This is presumably due
to the complex requirements of adjuvants in general in combination
with stability issues to be solved in the case of nucleic acid
based adjuvants.
[0017] Nevertheless, transfection of nucleic acids or genes into
cells or tissues for eliciting an (innate and/or adaptive) immune
response appears to provide a promising approach to provide new
adjuvants.
[0018] However, many of these approaches utilize transfection of
nucleic acids or genes into cells or tissues without the purpose to
induce an innate immune response. There are even some gene
therapeutic therapies, which have to strictly avoid induction of an
innate immune response. Even in the rare cases, where vaccination
is carried out to induce an adaptive antigen-specific immune
response using administration of nucleic acids, e.g. in tumour
vaccinations using DNA or mRNA encoded antigens, induction of an
adaptive immune response is typically carried out as an active
immunization against the encoded antigen but not as an accompanying
adjuvant therapy and thus may require additional administration of
a separate adjuvant to induce an innate immune response.
[0019] Even if a lot of transfection methods are known in the art,
transfer or insertion of nucleic acids or genes into an
individual's cells still represents a major challenge today and is
not yet solved satisfactorily. To address this complex issue a
variety of methods were developed in the last decade. These include
transfection by calcium phosphate, cationic lipids, cationic
polymers, and liposomes. Further methods for transfection are
electroporation and viral transduction.
[0020] However, as known to a skilled person, systems for transfer
or insertion of nucleic acids or genes have to fulfil several
requirements for in vivo applications which include efficient
nucleic acid delivery into an individual's cells with high
functionality, protection of the nucleic acid against ubiquitously
occurring nucleases, release of the nucleic acid in the cell, no
safety concerns, feasible manufacturing in a commercially
acceptable form amenable to scale-up and storage stability under
low cost conditions (e.g feasible lyophilisation). These
requirements are to be added to the complex requirements of an
adjuvant particularly if it is in the form of a nucleic acid as
outlined above.
[0021] Some successful strategies for the transfer or insertion of
nucleic acids or genes available today rely on the use of viral
vectors, such as adenoviruses, adeno-associated viruses,
retroviruses, and herpes viruses. Viral vectors are able to mediate
gene transfer with high efficiency and the possibility of long-term
gene expression. However, the acute immune response ("cytokine
storm"), immunogenicity, and insertion mutagenesis uncovered in
gene therapy clinical trials have raised serious safety concerns
about some commonly used viral vectors.
[0022] Another solution to the problem of transfer or insertion of
nucleic acids or genes may be found in the use of non-viral
vectors. Although non-viral vectors are not as efficient as viral
vectors, many non-viral vectors have been developed to provide a
safer alternative. Methods of non-viral nucleic acid delivery have
been explored using physical (carrier-free nucleic acid delivery)
and chemical approaches (synthetic vector-based nucleic acid
delivery). Physical approaches usually include needle injection,
electroporation, gene gun, ultrasound, and hydrodynamic delivery,
employ a physical force that permeates the cell membrane and
facilitates intracellular gene transfer. The chemical approaches
typically use synthetic or naturally occurring compounds (e.g.
cationic lipids, cationic polymers, lipid-polymer hybrid systems)
as carriers to deliver the nucleic acid into the cells. Although
significant progress has been made in the basic science and
applications of various nonviral nucleic acid delivery systems, the
majority of non-viral approaches are still much less efficient than
viral vectors, especially for in vivo gene delivery (see e.g. Gao,
X., Kim, K. & Liu, D., AAPSJ9, E92-104 (2007)).
[0023] Such transfection agents as defined above typically have
been used successfully solely in in vitro reactions. For
application of nucleic acids in vivo, however, further requirements
have to be fulfilled. For example, complexes between nucleic acids
and transfection agents have to be stable in physiological salt
solutions with respect to agglomerisation. Furthermore, such
complexes typically must not interact with parts of the complement
system of the host and thus must not be immunogenic itself as the
carrier itself shall not induce an adaptive immune response in the
individual. Additionally, the complex shall protect the nucleic
acid from early extracellular degradation by ubiquitously occurring
nucleases.
[0024] In the art many transfection reagents are available,
especially cationic lipids, which show excellent transfection
activity in cell culture. However, most of these transfection
reagents do not perform well in the presence of serum, and only a
few are active in vivo. A dramatic change in size, surface charge,
and lipid composition occurs when lipoplexes are exposed to the
overwhelming amount of negatively charged and often amphipathic
proteins and polysaccharides that are present in blood, mucus,
epithelial lining fluid, or tissue matrix. Once administered in
vivo, lipoplexes tend to interact with negatively charged blood
components and form large aggregates that could be absorbed onto
the surface of circulating red blood cells, trapped in a thick
mucus layer, or embolized in microvasculatures, preventing them
from reaching the intended target cells in the distal location.
Some even undergo dissolution after they are introduced to the
blood circulation (see e.g. Gao, X., Kim, K. & Liu, D., AAPS
P9, E92-104 (2007)).
[0025] One more promising approach utilizes cationic polymers.
Cationic polymers turned out to be efficient in transfection of
nucleic acids, as they can tightly complex and condense a
negatively charged nucleic acid. Thus, a number of cationic
polymers have been explored as carriers for in vitro and in vivo
gene delivery. These include polyethylenimine (PEI), polyamidoamine
and polypropylamine dendrimers, polyallylamine, cationic dextran,
chitosan, cationic proteins and cationic peptides. Although most
cationic polymers share the function of condensing DNA into small
particles and facilitate cellular uptake via endocytosis through
charge-charge interaction with anionic sites on cell surfaces,
their transfection activity and toxicity differs dramatically.
[0026] Only in one approach in the art, the immunostimulatory
effect of RNA complexed to short cationic peptides was demonstrated
by Fotin-Mleczek et al. (WO 2009/030481). These formulations appear
to efficiently induce the cytokine production in immunocompetent
cells. Unfortunately Fotin-Mleczek et al. did not assess the
induction of the preferable anti-viral cytokine IFN-.alpha. by
these complexes. Additionally, these complexes turned out to be
unstable during lyophilisation.
[0027] In the above context, cationic polymers exhibit better
transfection efficiency with rising molecular weight. However, a
rising molecular weight also leads to a rising toxicity of the
cationic polymer. In this above context, (high molecular weight)
PEI is perhaps the most active and most studied polymer for
transfection of nucleic acids, in particular for gene delivery
purposes. Unfortunately, it exhibits the same drawback due to its
non-biodegradable nature and toxicity. Furthermore, even though
polyplexes formed by high molecular weight polymers exhibit
improved stability under physiological conditions, data have
indicated that such polymers can hinder vector unpacking. To
overcome this negative impact, Read et al. (see Read, M. L. et al.,
J Gene Med. 5, 232-245 (2003); and Read, M. L. et al., Nucleic
Acids Res 33, e86 (2005)) developed a new type of synthetic vector
based on a linear reducible polycation (RPC) prepared by oxidative
polycondensation of the peptide Cys-Lys.sub.10-Cys. This peptide
Cys-Lys.sub.10-Cys can be cleaved by the intracellular environment
to facilitate release of nucleic acids. In this context, Read et
al. (2003, supra) could show that polyplexes formed by these RPCs
are destabilised by reducing conditions enabling efficient release
of DNA and mRNA. However, examining the transfection efficiency in
vitro Read et al. (2003, supra) also observed that N/P (nitrogen to
phosphor atoms) ratios of 2 were unsatisfying and higher N/P ratios
were necessary to improve transfection efficiency. Additonally,
Read et al. (2003, supra) observed that chloroquine or the cationic
lipid DOTAP was additionally necessary to enhance transfection
efficiency to adequate levels. As a consequence, Read et al. (2005,
supra) included histidine residues into the RPCs which have a known
endosomal buffering capacity and showed that such histidine-rich
RPCs can be cleaved by the intracellular reducing environment. This
approach enabled efficient cytoplasmic delivery of a broad range of
nucleic acids, including plasmid DNA, mRNA and siRNA molecules
without the requirement for the endosomolytic agent
chloroquine.
[0028] Unfortunately, neither Read et al (2003, supra) nor Read et
al (2005, supra) did assess as to whether RPCs can be directly used
for in vivo applications. In their study in 2005, transfections
were performed in the absence of serum to avoid masking the ability
of histidine residues to enhance gene transfer that may have arisen
from binding of serum proteins to polyplexes restricting cellular
uptake. Preliminary experiments, however, indicated that the
transfection properties of histidine-rich RPC polyplexes can be
affected by the presence of serum proteins with a 50% decrease in
GFP-positive cells observed in 10% FCS. For in vivo application
Read et al. (2005, supra) proposed modifications with the
hydrophilic polymer poly-[N-(2hydroxy-propyl)methacrylamide].
Unfortunately, they could not prevent aggregation of polyplexes and
binding of polycationic complexes to serum proteins. Furthermore,
strong cationic charged complexes are formed (positive zeta
potential) when complexing the nucleic acid due to the large excess
of cationic polymer, which is characterized by the high N/P ratio.
Accordingly, such complexes are only of limited use in vivo due to
their strong tendency of salt induced agglomeration and
interactions with serum contents (opsonization). Additionally,
these (positively charged) complexes may excite complement
activation, when used for purposes of gene therapy. It has also
turned out that these positively charged RPC based complexes showed
poor translation of the nucleic acid cargo subsequent to local
administration into the dermis.
[0029] In an approach similar to Read et al McKenzie et al.
(McKenzie, D. L., K. Y. Kwok, et al. (2000), J Biol Chem 275(14):
9970-7 and McKenzie, D. L., E. Smiley, et al. (2000), Bioconjug
Chem 11(6): 901-9) developed cross-linking peptides as gene
delivery agents by inserting multiple cysteines into short
synthetic peptides. In their studies they examined the optimal
complex formation with DNA and as a result they could show that an
N/P ratio of at least 2 is necessary for fully formed peptide DNA
condensates. Therefore only positively charged complexes appeared
to show optimal DNA condensation. In contrast to these data they
proposed the development of negatively charged complexes for in
vivo gene delivery, since it was shown in previous studies that
intravenous application of electropositive DNA condensates leads to
rapid opsonisation and nonspecific biodistribution to lung and
liver (Collard, W. T., Evers, D. L., McKenzie, D. L., and Rice, K.
G. (2000), Carbohydr. Res. 323, 176-184). Therefore, McKenzie et
al. (2000; supra) proposed the derivatization of the carriers with
polyethylene glycol and targeting ligands. To be noted, the
approach of McKenzie et al. (2000, supra) is additionally subject
of a patent (U.S. Pat. No. 6,770,740B1), which particularly
discloses the transfection of coding nucleic acids, antisense
nucleic acids and ribozymes.
[0030] Thus, in vivo application of nucleic acids appears to be
still one of the most challenging problems because plasma proteins
with anionic charges may non-specifically bind to positively
charged complexes and rapidly remove them e.g. via the
reticulo-endothelial system. Opsonization and activation of the
complement system by cationic complexes are additional
physiological phenomena that can participate in lowering the
efficacy of in vivo administered cationic complexes. This
particularly applies to administration of nucleic acid-based drugs,
e.g. the transfection of nucleic acids into cells or tissues,
particularly if the expression of an encoded protein or peptide or
transcription of an RNA of the transfected nucleic acid is
intended.
[0031] Summarizing the above, the prior art does not provide
feasible means or methods, which allow to establish efficient and
safe pharmaceutical compositions that include adjuvants for
vaccination purposes, particularly if a Th1-shifted immune response
is desired.
[0032] Accordingly, it is the object of the present invention to
provide such means or methods, which address one or more of these
problems.
[0033] The object underlying the present invention is solved by the
subject matter of the present invention, preferably by the subject
matter of the attached claims.
[0034] For the sake of clarity and readability the following
definitions are provided. Any technical features disclosed thereby
can be part of each and every embodiment of the invention.
Additional definitions and explanations can be provided in the
context of this disclosure.
[0035] Nucleic acid: The term nucleic acid means typically any DNA-
or RNA-molecule and is used synonymous with polynucleotide.
Furthermore, modifications or derivatives of the nucleic acid as
defined herein are explicitly included in the general term "nucleic
acid". For example, PNA is also included in the term "nucleic
acid".
[0036] Monocistronic RNA: A monocistronic RNA may typically be a
RNA, preferably a mRNA, that encodes only one open reading frame.
An open reading frame in this context is a sequence of several
nucleotide triplets (codons) that can be translated into a peptide
or protein.
[0037] Bi-/multicistronic RNA: RNA, preferably a mRNA, that
typically may have two (bicistronic) or more (multicistronic) open
reading frames (ORF). An open reading frame in this context is a
sequence of several nucleotide triplets (codons) that can be
translated into a peptide or protein.
[0038] 5'-Cap structure: A 5' Cap is typically a modified
nucleotide, particularly a guanine nucleotide, added to the 5'-end
of a RNA-molecule. Preferably, the 5'-Cap is added using a
5'-5'-triphosphate linkage.
[0039] Poly(C) sequence: A poly(C) sequence is typically a long
sequence of cytosine nucleotides, typically about 10 to about 200
cytidine nucleotides, preferably about 10 to about 100 cytidine
nucleotides, more preferably about 10 to about 70 cytidine
nucleotides or even more preferably about 20 to about 50 or even
about 20 to about 30 cytidine nucleotides. A poly(C) sequence may
preferably be located 3' of the coding region comprised by a
nucleic acid.
[0040] Poly(A) tail: A poly(A) tail also called "3'-poly(A) tail"
is typically a long sequence of adenine nucleotides of up to about
400 adenosine nucleotides, e.g. from about 25 to about 400,
preferably from about 50 to about 400, more preferably from about
50 to about 300, even more preferably from about 50 to about 250,
most preferably from about 60 to about 250 adenosine nucleotides,
added to the 3'-end of a RNA.
[0041] Stabilized nucleic acid: A stabilized nucleic acid,
typically, may be essentially resistant to in vivo degradation
(e.g. degradation by an exo- or endo-nuclease) and/or ex vivo
degradation (e.g. by the manufacturing process prior to vaccine
administration, e.g. in the course of the preparation of the
vaccine solution to be administered). Stabilization of mRNA can,
e.g., be achieved by providing a 5'-Cap structure, a Poly(A) tail,
a poly (C) tail, or any other UTR modification. It can also be
achieved by backbone modification or modification of the
G/C-content of the nucleic acid. Various other methods are
conceivable in the context of the invention.
[0042] Modification of a nucleic acid (modified nucleic acid):
Modification of a nucleic acid molecule typically may contain
backbone modifications, sugar modifications or base modifications.
A backbone modification in connection with the present invention
is, typically, a modification in which phosphates of the backbone
of the nucleotides contained in the nucleic acid molecule may be
chemically modified. A sugar modification in connection with the
present invention, typically, may be a chemical modification of the
sugar of the nucleotides of the nucleic acid. Furthermore, a base
modification in connection with the present invention, typically,
may be a chemical modification of the base moiety of the
nucleotides of the nucleic acid molecule. Therefore a modified
nucleic acid may also be defined herein as a nucleic acid molecule
which may include nucleotide analogues. Furthermore a modification
of a nucleic acid molecule may contain a lipid modification. Such a
lipid-modified nucleic acid typically may comprise a nucleic acid
as defined herein. Such a lipid-modified nucleic acid molecule
typically may further comprise at least one linker covalently
linked with that nucleic acid molecule, and at least one lipid
covalently linked with the respective linker. Alternatively, the
lipid-modified nucleic acid molecule may comprise at least one
nucleic acid molecule as defined herein and at least one
(bifunctional) lipid covalently linked (without a linker) with that
nucleic acid molecule. According to a third alternative, the
lipid-modified nucleic acid molecule may comprise a nucleic acid
molecule as defined herein, at least one linker covalently linked
with that nucleic acid molecule, and at least one lipid covalently
linked with the respective linker, and also at least one
(bifunctional) lipid covalently linked (without a linker) with that
nucleic acid molecule.
[0043] A modification of a nucleic acid may also comprise the
modification of the G/C content of the coding region of a nucleic
acid molecule, especially if the nucleic acid molecule is in the
form of an mRNA. In this context it is particularly preferred that
the G/C content of the coding region of the nucleic acid molecule
is increased, compared to the G/C content of the coding region of
its particular wild type coding sequence, i.e. the unmodified mRNA.
The encoded amino acid sequence of the nucleic acid sequence is
preferably not modified compared to the coded amino acid sequence
of the particular wild type mRNA. The modification of the
G/C-content of the nucleic acid molecule, especially if the nucleic
acid molecule is in the form of an mRNA or codes for an mRNA, is
based on the fact that the sequence of any mRNA region to be
translated is important for efficient translation of that mRNA.
Thus, the composition and the sequence of various nucleotides are
important. In particular, sequences having an increased G
(guanosine)/C (cytosine) content are more stable than sequences
having an increased A (adenosine)/U (uracil) content. Therefore,
the codons of the coding sequence or mRNA are therefore varied
compared to its wild type coding sequence or mRNA, while retaining
the translated amino acid sequence, such that they include an
increased amount of G/C nucleotides. In respect to the fact that
several codons code for one and the same amino acid (so-called
degeneration of the genetic code), the most favourable codons for
the stability can be determined (so-called alternative codon
usage). Preferably, the G/C content of the coding region of the
nucleic acid molecule, especially if the nucleic acid is in the
form of an mRNA or codes for an mRNA, is increased by at least 7%,
more preferably by at least 15%, particularly preferably by at
least 20%, compared to the G/C content of the coded region of the
wild type mRNA. According to a specific embodiment, at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even
more preferably at least 80% and most preferably at least 90%, 95%
or even 100% of the substitutable codons in the region coding for a
protein or peptide as defined herein or its fragment, variant
and/or derivative thereof or the whole sequence of the wild type
mRNA sequence or coding sequence are substituted, thereby
increasing the G/C content of said sequence. In this context, it is
particularly preferable to increase the G/C content of the nucleic
acid molecule, especially if the nucleic acid is in the form of an
mRNA or codes for an mRNA, to the maximum (i.e. 100% of the
substitutable codons), in particular in the region coding for a
protein, compared to the wild type sequence. Furthermore, a
modification of the nucleic acid, especially if the nucleic acid is
in the form of an mRNA or codes for an mRNA, is based on the
finding that the translation efficiency is also determined by a
different frequency in the occurrence of tRNAs in cells. The
frequency in the occurrence of tRNAs in a cell, and thus the codon
usage in said cell, is dependent on the species the cell is derived
from. Accordingly, a yeast cell generally exhibits a different
codon usage than a mammalian cell, such as a human cell. Thus, if
so-called "rare codons" are present in the nucleic acid molecule
(with respect to the respective expression system), especially if
the nucleic acid is in the form of an mRNA or codes for an mRNA, to
an increased extent, the corresponding modified nucleic acid
molecule is translated to a significantly poorer degree than in the
case where codons coding for relatively "frequent" tRNAs are
present. Therefore, especially if the modified nucleic acid
molecule is in the form of an mRNA or codes for an mRNA, the coding
region of the modified nucleic acid is preferably modified compared
to the corresponding region of the wild type mRNA or coding
sequence such that at least one codon of the wild type sequence
which codes for a tRNA which is relatively rare in the cell is
exchanged for a codon which codes for a tRNA which is relatively
frequent in the cell and carries the same amino acid as the
relatively rare tRNA. By this modification, the sequences of the
nucleic acid molecule, especially if the nucleic acid is in the
form of an mRNA or codes for an mRNA, is modified such that codons
for which frequently occurring tRNAs are available are inserted. In
other words, by this modification all codons of the wild type
sequence which code for a tRNA which is relatively rare in the cell
can in each case be exchanged for a codon which codes for a tRNA
which is relatively frequent in the cell and which, in each case,
carries the same amino acid as the relatively rare tRNA. Which
tRNAs occur relatively frequently in the cell and which, in
contrast, occur relatively rarely is known to a person skilled in
the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6):
660-666. It is particularly preferred that a nucleic acid sequence
coding for a protein used in the present invention is codon
optimized for the human codon usage. The codons which use for the
particular amino acid the tRNA which occurs the most frequently,
e.g. the Gly codon, which uses the tRNA which occurs the most
frequently in the (human) cell, are particularly preferred. In this
context, it is particularly preferable to link the sequential G/C
content which is increased, in particular maximized, in the
modified nucleic acid molecule, especially if the nucleic acid is
in the form of an mRNA or codes for an mRNA, with the "frequent"
codons without modifying the amino acid sequence of the protein
encoded by the coding region of the nucleic acid molecule. This
preferred embodiment allows provision of a particularly efficiently
translated and stabilized (modified) nucleic acid, especially if
the nucleic acid is in the form of an mRNA or codes for an
mRNA.
[0044] Derivative of a nucleic acid molecule: A derivative of a
nucleic acid molecule may typically be understood herein as a
modified nucleic acid, as defined above.
[0045] Nucleotide analogues: Nucleotide analogues, typically, are
nucleotides structurally similar (analogue) to naturally occurring
nucleotides which include phosphate backbone modifications, sugar
modifications, or modifications of the nucleobase.
[0046] UTR modification: A UTR modification is, typically, a
modification of the 5' and/or 3' region of a nucleic acid molecule,
particularly a coding nucleic acid molecule. Therein, "UTR"
typically means "untranslated region". An UTR may, e.g., contain,
comprise or consist of a stabilizing sequence (UTR modification).
These stabilizing sequences in the 5' and/or 3' untranslated
regions may have the effect of increasing the half-life of the
nucleic acid in the cytosol. These stabilizing sequences may have
100% sequence identity to naturally occurring sequences which occur
in viruses, bacteria and eukaryotes, but may also be partly or
completely synthetic. The untranslated sequences (UTR) of the
(alpha-) globin gene, e.g. from Homo sapiens or Xenopus laevis may
be mentioned as an example of stabilizing sequences which may be
used for a stabilized nucleic acid. 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 RNA which codes for (alpha-)globin,
type(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf.
Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
Such stabilizing sequences can of course be used individually or in
combination with one another and also in combination with other
stabilizing sequences known to a person skilled in the art. In the
context of the present invention, a UTR modification preferably
means a modification of a coding nucleic acid, such as a gene or
mRNA, by adding or exchanging a 5'- and/or 3'-UTR, preferably by
adding or exchanging for a stabilizing 5'- and/or 3'-UTR, e.g., as
specified above.
[0047] Nucleic acid synthesis: Nucleic acid molecules used
according to the invention as defined herein may be prepared using
any method known in the art, including synthetic methods such as
e.g. solid phase synthesis, as well as in vitro methods, such as in
vitro transcription reactions.
[0048] For preparation of a nucleic acid molecule, especially if
the nucleic acid is in the form of an mRNA, a corresponding DNA
molecule may, e.g., be transcribed in vitro. This DNA matrix
preferably comprises a suitable promoter, e.g. a T7 or SP6
promoter, for in vitro transcription, which is followed by the
desired nucleotide sequence coding for the nucleic acid molecule,
e.g. mRNA, to be prepared, and a termination signal for in vitro
transcription. The DNA molecule, which forms the matrix of the at
least one RNA of interest, may be prepared by fermentative
proliferation and subsequent isolation as part of a plasmid which
can be replicated in bacteria. Plasmids which may be mentioned as
suitable for the present invention are e.g. the plasmids pT7 Ts
(GenBank accession number U26404; Lai et al, Development 1995, 121:
2349 to 2360), pGEM.RTM. series, e.g. pGEM.RTM.-1 (GenBank
accession number X65300; from Promega) and pSP64 (GenBank accession
number X65327); cf. also Mezei and Storts, Purification of PCR
Products, in: Griffin and Griffin (ed.), PCR Technology: Current
Innovation, CRC Press, Boca Raton, Fla., 2001.
[0049] Protein: A protein typically consists of one or more
polypeptides folded into 3-dimensional form, facilitating a
biological function.
[0050] Peptide: A peptide is typically a short polymer of amino
acid monomers, linked by peptide bonds. It typically contains less
than 50 monomer units. Nevertheless, the term peptide is not a
disclaimer for molecules having more than 50 monomer units. Long
peptides are also called polypeptides, typically having between 50
and 600 monomeric units, more specifically between 50 and 300
monomeric units. Furthermore a "peptide" is defined herein also to
include any peptidyl molecule, including peptide analogues.
[0051] Peptide analogues: A peptide analogue may, typically,
comprise naturally or non-naturally occurring amino acids which may
be used for the purpose of the invention. For example they can
comprise amino acids selected from an isostere or a chiral analog
(D-amino acid or L-amino acid) of an amino acid. Additionally, the
analog may comprise one or more amino acids, preferably selected
from hydroxyproline, .beta.-alanine, 2,3-diaminopropionic acid,
.alpha.-aminoisobutyric acid, N-methylglycine (sarcosine),
ornithine, citrulline, t-butylalanine, t-butylglycine, N-methyl
isoleucine, phenylglycine, cyclohexylalanine, norleucine,
naphthylalanine, pyridylananine 3-benzothienyl alanine
4-chlorophenylalanine, 2-fluorophenylalanine,
3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,
1,2,3,4-tetrahydro-tic isoquinoline-3-carboxylic acid
[beta]-2-thienylalanine, methionine sulfoxide, homoarginine,
N-acetyl lysine, 2,4-diamino butyric acid, p-aminophenylalanine,
N-methylvaline, homocysteine, homoserine, .epsilon.-amino hexanoic
acid, .delta.-amino valeric acid, 2,3-diaminobutyric acid. A
peptide analogue as defined herein may further contain modified
peptides. The term specifically includes peptide back-bone
modifications (i.e., amide bond mimetics) known to those skilled in
the art. Such modifications include modifications of the amide
nitrogen, the .alpha.-carbon, amide carbonyl, complete replacement
of the amide bond, extensions, deletions or backbone crosslinks.
Several peptide backbone modifications are known, including
.PSI.[CH2S], .PSI.CH2NH], .PSI.[CSNH2], .PSI.[NHCO], .PSI.[COCH2],
and .PSI.[(E) or (Z) CH.dbd.CH]. In the nomenclature used above,
indicates the absence of an amide bond. The structure that replaces
the amide group is specified within the brackets. Other
modifications include, for example, an N-alkyl (or aryl)
substitution (.PSI.[CONR]), or backbone crosslinking to construct
lactams and other cyclic structures, C-terminal hydroxymethyl
modifications, O-modified modifications (e.g., C-terminal
hydroxymethyl benzyl ether), N-terminal modifications including
substituted amides such as alkylaniides and hydrazides.
[0052] Peptide synthesis: A peptide, a peptide analogue, or a
derivative thereof is preferably synthesized using a chemical
method known to the skilled artisan. For example, synthetic
peptides are prepared using known techniques of solid phase, liquid
phase, or peptide condensation, or any combination thereof, and can
include natural and/or unnatural amino acids. Generally, chemical
synthesis methods comprise the sequential addition of one or more
amino acids to a growing peptide chain. Normally, either the amino
or carboxyl group of the first amino acid is protected by a
suitable protecting group. The protected or derivatized amino acid
can then be either attached to an inert solid support or utilized
in solution by adding the next amino acid in the sequence having
the complementary (amino or carboxyl) group suitably protected,
under conditions that allow for the formation of an amide linkage.
The protecting group is then removed from the newly added amino
acid residue and the next amino acid (suitably protected) is then
added, and so forth. After the desired amino acids have been linked
in the proper sequence, any remaining protecting groups (and any
solid support, if solid phase synthesis techniques are used) are
removed sequentially or concurrently, to render the final
polypeptide. These methods are suitable for synthesis of a peptide
used for the purpose of the present invention (such as a peptide
analogue) or derivative thereof. Typical protecting groups include
t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc)
benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl;
benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl,
t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl,
cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
Typical solid supports are cross-linked polymeric supports. These
can include divinylbenzene cross-linked-styrene-based polymers, for
example, divinylbenzene-hydroxymethylstyrene copolymers,
divinylbenzene-chloromethylstyrene copolymers and
divinylbenzene-benzhydrylaminopolystyrene copolymers.
[0053] Recombinant peptide or protein production: A peptide or
protein or derivative thereof may be produced using recombinant
protein or peptide production. To facilitate the production of a
recombinant peptide or protein, at least one nucleic acid encoding
the same is preferably isolated or synthesized. Typically, the
nucleic acid encoding the recombinant protein or peptide is
isolated using a known method, such as, for example, amplification
(e.g., using PCR) or isolated from nucleic acid from an organism
using one or more restriction enzymes or isolated from a library of
nucleic acids. For expressing a protein or peptide by recombinant
means, a protein/peptide-encoding nucleic acid is placed in
operable connection with a promoter or other regulatory sequence
capable of regulating expression in a cell-free system or cellular
system. For example, nucleic acid comprising a sequence that
encodes a peptide or protein is placed in operable connection with
a suitable promoter and maintained in a suitable cell for a time
and under conditions sufficient for expression to occur. Typical
expression vectors for in vitro expression, cell-free expression or
cell-based expression have been described and are well known for
the skilled person. In this context cell-free expression systems
may include E. coli S30 fraction, rabbit reticulocyte lysate and
wheat germ extract and a cellular system may be selected from
bacterial (e.g. E. coli), insect, plant, or mammalian cells (e.g.,
293, COS, CHO, 1OT cells, 293T cells).
[0054] Secretory signal peptide: Such signal peptides are
sequences, which typically exhibit a length of about 15 to 30 amino
acids and are preferably located at the N-terminus of the encoded
peptide, without being limited thereto. Signal peptides as defined
herein preferably allow the transport of the protein or peptide
into a defined cellular compartment, preferably the cell surface,
the endoplasmic reticulum (ER) or the endosomal-lysosomal
compartment.
[0055] Carrier/polymeric carrier: A carrier in the context of the
invention may typically be a compound that facilitates transport
and/or complexation of another compound. A polymeric carrier is
typically a carrier that is formed of a polymer. A carrier, in the
context of the present invention, is preferably suitable as carrier
for nucleic acid molecules, e.g. for mediating dissolution in
physiological acceptable liquids, transport and cellular uptake of
the nucleic acid molecules or a vector. Accordingly, a carrier, in
the context of the present invention, may be a component which may
be suitable for depot and delivery of a nucleic acid molecule or
vector. Such carriers may be, for example, cationic or polycationic
carriers or compounds which may serve as transfection or
complexation agent. Particularly preferred carriers or polymeric
carriers in this context are cationic or polycationic compounds,
including protamine, nucleoline, spermine or spermidine, or other
cationic peptides or proteins, such as poly-L-lysine (PLL),
poly-arginine, basic polypeptides, cell penetrating peptides
(CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),
Tat-derived peptides, Penetratin, VP22 derived or analog peptides,
HSV VP22 (Herpes simplex), MAP, KALA or protein transduction
domains (PTDs), PpT620, prolin-rich peptides, arginine-rich
peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,
Calcitonin peptide(s), Antennapedia-derived peptides (particularly
from Drosophila antennapedia), pAntp, pIsl, FGF, Lactoferrin,
Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, ha-derived
peptides, SAP, or histones. In the context of the present
invention, such cationic or polycationic carriers are preferably
cationic or polycationic peptides or proteins, which preferably
comprise or are additionally modified to comprise at least one
moiety, which is capable of forming a disulfide bond, preferably an
--SH moiety.
[0056] Cationic component: The term "cationic component" typically
refers to a charged molecule, which is positively charged (cation)
at a pH value of about typically 1 to 9, preferably of a pH value
of or below 9 (e.g. 5 to 9), of or below 8 (e.g. 5 to 8), of or
below 7 (e.g. 5 to 7), most preferably at physiological pH values,
e.g. about 7.3 to 7.4. Accordingly, a cationic peptide, protein or
polymer according to the present invention is positively charged
under physiological conditions, particularly under physiological
salt conditions of the cell in vivo. A cationic peptide or protein
contains a larger number of cationic amino acids, e.g. a larger
number of Arg, His, Lys or Orn, than negatively charged or neutral
amino acids. In a preferred embodiment, a cationic peptide or
protein in the context of the present invention contains a larger
number of cationic amino acids, e.g. a larger number of Arg, His,
Lys or Orn, than other residues. The definition "cationic" may also
refer to "polycationic" components.
[0057] The charge of a compound, complex or component, such as the
cationic component or the polymeric carrier cargo complex (A) as
defined herein is preferably determined or assessed under
physiological conditions, e.g. at a pH of between about 5.5 and
7.5, preferably at a pH of between about 6.0 and 7.4, such as about
7.0, at a temperature of between about 25.degree. C. and 40.degree.
C., preferably at a temperature of about 35 and 38.degree. C., such
as about 37.degree. C., at a physiological salt concentration of,
e.g. between about 130 and 160 mM, preferably between about 137 mM
and 150 mM, such as at about 137 mM. Particularly preferred
conditions for determining or assessing the charge of a compound,
complex or component as defined herein are the conditions found in
a 100% Ringer lactate solution at 25.degree. C.
[0058] Zetapotential: The "zetapotential" is a widely used
parameter for the electrical surface charge of a particle. It is
typically determined by moving the charged particle through an
electrical field. In the context of the present invention, the
zetapotential is the preferred parameter for characterizing the
charge of a particle, e.g. of complex (A) of the pharmaceutical
compositions according to the present invention. Thus, in the
context of the present invention, the charge of a particle is
preferably determined by determining the zetapotential by the laser
Doppler electrophoresis method using a Zetasizer Nano instrument
(Malvern Instruments, Malvern, UK) at 25.degree. C. and a
scattering angle of 173.degree.. The surface charge of a given
particle also depends on the ionic strength of the utilized matrix
(e.g. salt containing buffer) and the pH of the solution.
Therefore, the actual zetapotential of a given complex (A) at a
charge ratio (N/P) may differ slightly between different buffers
used for injection. For the measurement, the particles, such as
complex (A) of the pharmaceutical compositions according to the
present invention are preferably suspended in Ringer Lactate
solution. The present invention claims therefore the use of a
negativley charged complex (A) under the conditions of a given
injection buffer, preferably under the conditions of a Ringer
lactate solution, assessed by its Zetapotential. A Ringer lactate
solution according to the present invention preferably contains 130
mmol/L sodium ions, 109 mmol/L chloride ions, 28 mmol/L lactate, 4
mmol/L potassium ions and 1.5 mmol/L cacium ion. The sodium,
chloride, potassium and lactate typically come from NaCl (sodium
chloride), NaC.sub.3H.sub.5O.sub.3 (sodium lactate), CaCl.sub.2
(calcium chloride), and KCl (potassium chloride). The osmolarity of
the Ringer lactate solution is 273 mOsm/L and the pH is adjusted to
6.5.
[0059] Pharmaceutically effective amount: A pharmaceutically
effective amount in the context of the invention is typically
understood to be an amount that is sufficient to induce an immune
response.
[0060] Immune system: The immune system may protect organisms, for
example, from infection. If a pathogen breaks through a physical
barrier of an organism and enters this organism, the innate immune
system provides an immediate, but non-specific response. If
pathogens evade this innate response, vertebrates possess a second
layer of protection, the adaptive immune system. Here, the immune
system adapts its response during an infection to improve its
recognition of the pathogen. This improved response is then
retained after the pathogen has been eliminated, in the form of an
immunological memory, and allows the adaptive immune system to
mount faster and stronger attacks each time this pathogen is
encountered. According to this, the immune system comprises the
innate and the adaptive immune system. Each of these two parts
contains so called humoral and cellular components.
[0061] Immune response: An immune response may typically either be
a specific reaction of the adaptive immune system to a particular
antigen (so called specific or adaptive immune response) or an
unspecific reaction of the innate immune system (so called
unspecific or innate immune response). In essence, the invention is
associated with specific reactions (adaptive immune responses) of
the adaptive immune system. However, this specific response can be
supported by an additional unspecific reaction (innate immune
response). Therefore, the invention also relates to a compound or
composition for simultaneous stimulation of the innate and the
adaptive immune system to evoke an efficient adaptive immune
response.
[0062] Adaptive immune response: The adaptive immune response is
typically understood to be antigen-specific. Antigen specificity
allows for the generation of responses that are tailored to
specific antigens, antigen-expressing cells, pathogens or
pathogen-infected cells. The ability to mount these tailored
responses is maintained in the body by "memory cells". Should a
pathogen infect the body more than once, these specific memory
cells are used to quickly eliminate it. In this context, the first
step of an adaptive immune response is the activation of naive
antigen-specific T cells or different immune cells able to induce
an antigen-specific immune response by antigen-presenting cells.
This occurs in the lymphoid tissues and organs through which naive
T cells are constantly passing. Cell types that can serve as
antigen-presenting cells are inter alia dendritic cells,
macrophages, and B cells. Each of these cells has a distinct
function in eliciting immune responses. Dendritic cells take up
antigens by phagocytosis and macropinocytosis and are stimulated by
contact with e.g. a foreign antigen to migrate to the local
lymphoid tissue, where they differentiate into mature dendritic
cells. Macrophages ingest particulate antigens such as bacteria and
are induced by infectious agents or other appropriate stimuli to
express MHC molecules. The unique ability of B cells to bind and
internalize soluble protein antigens via their receptors may also
be important to induce T cells. Presenting the antigen on MHC
molecules leads to activation of T cells which induces their
proliferation and differentiation into armed effector T cells. The
most important function of effector T cells is the killing of
infected cells by CD8+ cytotoxic T cells and the activation of
macrophages by Th1 cells which together make up cell-mediated
immunity, and the activation of B cells by both Th2 and Th1 cells
to produce different classes of antibody, thus driving the humoral
immune response. T cells recognize an antigen by their T cell
receptors which do not recognize and bind antigen directly, but
instead recognize short peptide fragments e.g. of pathogen-derived
protein antigens, which are bound to MHC molecules on the surfaces
of other cells.
[0063] Adaptive immune system: The adaptive immune system is,
typically, composed of highly specialized, systemic cells and
processes that eliminate or prevent pathogenic growth. The adaptive
immune response provides the vertebrate immune system with the
ability to recognize and remember specific pathogens (to generate
immunity), and to mount stronger attacks each time the pathogen is
encountered. The system is highly adaptable because of somatic
hypermutation (a process of accelerated somatic mutations), and
V(D)) recombination (an irreversible genetic recombination of
antigen receptor gene segments). This mechanism allows a small
number of genes to generate a vast number of different antigen
receptors, which are then uniquely expressed on each individual
lymphocyte. Because the gene rearrangement leads to an irreversible
change in the DNA of each cell, all of the progeny (offspring) of
that cell will then inherit genes encoding the same receptor
specificity, including the Memory B cells and Memory T cells that
are the keys to long-lived specific immunity. Immune network theory
is a theory of how the adaptive immune system works, that is based
on interactions between the variable regions of the receptors of T
cells, B cells and of molecules made by T cells and B cells that
have variable regions.
[0064] Innate immune system: Typically, the innate immune system,
also known as non-specific immune system, is understood to comprise
the cells and mechanisms that defend the host from infection by
other organisms in a non-specific manner. This means that the cells
of the innate system recognize and respond to pathogens in a
generic way, but unlike the adaptive immune system, it does not
confer long-lasting or protective immunity to the host. The innate
immune system may be e.g. activated by ligands of
pathogen-associated molecular patterns (PAMP) receptors, e.g.
Toll-like receptors (TLRs) or other auxiliary substances such as
lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines,
monokines, lymphokines, interleukins or chemokines, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,
IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,
IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF,
LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human
Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2,
TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or
TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I like
receptor, an immunostimulatory nucleic acid, an immunostimulatory
RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral
agent. Typically a response of the innate immune system includes
recruiting immune cells to sites of infection, through the
production of chemical factors, including specialized chemical
mediators, called cytokines; activation of the complement cascade;
identification and removal of foreign substances present in organs,
tissues, the blood and lymph, by specialized white blood cells;
activation of the adaptive immune system through a process known as
antigen presentation; and/or acting as a physical and chemical
barrier to infectious agents.
[0065] Cellular immunity/cellular immune response: Cellular
immunity relates typically to the activation of macrophages,
natural killer cells (NK), antigen-specific cytotoxic
T-lymphocytes, and the release of various cytokines in response to
an antigen. In a more general way, cellular immunity is not related
to antibodies but to the activation of cells of the immune system.
A cellular immune response is characterized e.g. by activating
antigen-specific cytotoxic T-lymphocytes that are able to induce
apoptosis in body cells displaying epitopes of an antigen on their
surface, such as virus-infected cells, cells with intracellular
bacteria, and cancer cells displaying tumor antigens; activating
macrophages and natural killer cells, enabling them to destroy
pathogens; and stimulating cells to secrete a variety of cytokines
that influence the function of other cells involved in adaptive
immune responses and innate immune responses.
[0066] Humoral immunity/humoral immune response: Humoral immunity
refers typically to antibody production and the accessory processes
that may accompany it. A humoral immune response may be typically
characterized, e.g., by Th2 activation and cytokine production,
germinal center formation and isotype switching, affinity
maturation and memory cell generation. Humoral immunity also
typically may refer to the effector functions of antibodies, which
include pathogen and toxin neutralization, classical complement
activation, and opsonin promotion of phagocytosis and pathogen
elimination.
[0067] Antigen: According to the present invention, the term
"antigen" refers typically to a substance which may be recognized
by the immune system and may be capable of triggering an
antigen-specific immune response, e.g. by formation of antibodies
or antigen-specific T-cells as part of an adaptive immune response.
Typically, an antigen is a protein or peptide, but may also be a
sugar, lipid, nucleic acid etc. structure. In this context, the
first step of an adaptive immune response is the activation of
naive antigen-specific T cells by antigen-presenting cells. This
occurs in the lymphoid tissues and organs through which naive T
cells are constantly passing. The three cell types that can serve
as antigen-presenting cells are dendritic cells, macrophages, and B
cells. Each of these cells has a distinct function in eliciting
immune responses. Tissue dendritic cells take up antigens by
phagocytosis and macropinocytosis and are stimulated by infection
to migrate to the local lymphoid tissue, where they differentiate
into mature dendritic cells. Macrophages ingest particulate
antigens such as bacteria and are induced by infectious agents to
express MHC class II molecules. The unique ability of B cells to
bind and internalize soluble protein antigens via their receptors
may be important to induce T cells. By presenting the antigen on
MHC molecules leads to activation of T cells which induces their
proliferation and differentiation into armed effector T cells. The
most important function of effector T cells is the killing of
infected cells by CD8.sup.+ cytotoxic T cells and the activation of
macrophages by TH1 cells which together make up cell-mediated
immunity, and the activation of B cells by both TH2 and TH1 cells
to produce different classes of antibody, thus driving the humoral
immune response. T cells recognize an antigen by their T cell
receptors which does not recognize and bind antigen directly, but
instead recognize short peptide fragments e.g. of pathogens'
protein antigens, which are bound to MHC molecules on the surfaces
of other cells.
[0068] T cells fall into two major classes that have different
effector functions. The two classes are distinguished by the
expression of the cell-surface proteins CD4 and CD8. These two
types of T cells differ in the class of MHC molecule that they
recognize. There are two classes of MHC molecules--MHC class I and
MHC class II molecules--which differ in their structure and
expression pattern on tissues of the body. CD4+T cells bind to a
MHC class II molecule and CD8.sup.+ T cells to a MHC class I
molecule. MHC class I and MHC class II molecules have distinct
distributions among cells that reflect the different effector
functions of the T cells that recognize them. MHC class I molecules
present peptides from pathogens, commonly viruses to CD8.sup.+ T
cells, which differentiate into cytotoxic T cells that are
specialized to kill any cell that they specifically recognize.
Almost all cells express MHC class I molecules, although the level
of constitutive expression varies from one cell type to the next.
But not only pathogenic peptides from viruses are presented by MHC
class I molecules, also self-antigens like tumour antigens are
presented by them. MHC class I molecules bind peptides from
proteins degraded in the cytosol and transported in the endoplasmic
reticulum. Thereby MHC class I molecules on the surface of cells
infected with viruses or other cytosolic pathogens display peptides
from these pathogen. The CD8+T cells that recognize MHC class
I:peptide complexes are specialized to kill any cells displaying
foreign peptides and so rid the body of cells infected with viruses
and other cytosolic pathogens. The main function of CD4.sup.+ T
cells (CD4.sup.+ helper T cells) that recognize MHC class II
molecules is to activate other effector cells of the immune system.
Thus MHC class II molecules are normally found on B lymphocytes,
dendritic cells, and macrophages, cells that participate in immune
responses, but not on other tissue cells. Macrophages, for example,
are activated to kill the intravesicular pathogens they harbour,
and B cells to secrete immunoglobulins against foreign molecules.
MHC class II molecules are prevented from binding to peptides in
the endoplasmic reticulum and thus MHC class II molecules bind
peptides from proteins which are degraded in endosomes. They can
capture peptides from pathogens that have entered the vesicular
system of macrophages, or from antigens internalized by immature
dendritic cells or the immunoglobulin receptors of B cells.
Pathogens that accumulate in large numbers inside macrophage and
dendritic cell vesicles tend to stimulate the differentiation of
TH1 cells, whereas extracellular antigens tend to stimulate the
production of TH2 cells. TH1 cells activate the microbicidal
properties of macrophages and induce B cells to make IgG antibodies
that are very effective of opsonising extracellular pathogens for
ingestion by phagocytic cells, whereas TH2 cells initiate the
humoral response by activating naive B cells to secrete IgM, and
induce the production of weakly opsonising antibodes such as IgG1
and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgA and IgE
(mouse and human).
[0069] Vaccine: A vaccine is typically understood to be a
prophylactic or therapeutic material providing at least one antigen
or antigenic function. The antigen or antigenic function may
stimulate the body's adaptive immune system to provide an adaptive
immune response.
[0070] Immunostimulating agent: The term "immunostimulating agent"
is typically understood not to include agents as e.g. antigens (of
whatever chemical structure), which elicit an adaptive/cytotoxic
immune response, e.g. a "humoral" or "cellular" immune response, in
other words elicit immune responses (and confer immunity by
themselves) which are characterized by a specific response to
structural properties of an antigen recognized to be foreign by
immune competent cells. Rather, by "immunostimulating agent", it is
typically understood to mean agents/compounds/complexes which do
not trigger any adaptive/cytotoxic immune response by themselves,
but which may exclusively enhance such an adaptive/cytotoxic immune
reponse in an unspecific way, by e.g. activating "PAMP" receptors
and thereby triggering the release of cytokines which support the
actual adaptive/cytotoxic immune response. Accordingly, any
immunostimulation by agents (e.g. antigens) which evoke an adaptive
and/or cytotoxic immune response by themselves (conferring immunity
by themselves directly or indirectly) is typically disclaimed by
the phrase "immunostimulating agent".
[0071] Adjuvant: The term "adjuvant" is typically understood not to
comprise agents which confer immunity by themselves. Accordingly,
adjuvants may, typically, not confer immunity by themselves, but
assist the immune system in various ways to enhance the
antigen-specific immune response by e.g. promoting presentation of
an antigen to the immune system. Hereby, an adjuvant may preferably
e.g. modulate the antigen-specific immune response by e.g. shifting
the dominating Th1-based antigen specific response to a more
Th2-based antigen specific response or vice versa. Accordingly, the
terms "immunostimulating agent" and "adjuvant" in the context of
the present invention are typically understood to mean agents,
compounds or complexes which do not confer immunity by themselves,
but exclusively support the immune reponse in an unspecific way (in
contrast to an antigen-specific immune response) by effects, which
modulate the antigen-specific (adaptive cellular and/or humoral
immune response) by unspecific measures, e.g. cytokine
expression/secretion, improved antigen presentation, shifting the
nature of the arms of the immune response etc. Accordingly, any
agents evoking by themselves immunity are typically disclaimed by
the terms "adjuvant" or "immunostimulating agent".
[0072] Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in
the context of the invention may typically be a RNA that is able to
induce an innate immune response itself. It usually does not have
an open reading frame and thus does not provide a peptide-antigen
but elicits an innate immune response e.g. by binding to a specific
kind of pathogen-associated molecular patterns (PAMP) receptors
(e.g. Toll-like-receptor (MR) or other suitable receptors).
However, of course also mRNAs having an open reading frame and
coding for a peptide/protein (e.g. an antigenic function) may
induce an innate immune response.
[0073] Fragment of a sequence: a fragment of a sequence is
typically a shorter portion of a full-length sequence of e.g. a
nucleic acid sequence or an amino acid sequence. Accordingly, a
fragment of a sequence, typically, consists of a sequence that is
identical to the corresponding stretch or corresponding stretches
within the full-length sequence. A preferred fragment of a sequence
in the context of the present invention, consists of a continuous
stretch of entities, such as nucleotides or amino acids,
corresponding to a continuous stretch of entities in the molecule
the fragment is derived from, which represents at least 5%,
preferably at least 20%, preferably at least 30%, more preferably
at least 40%, more preferably at least 50%, even more preferably at
least 60%, even more preferably at least 70%, and most preferably
at least 80% of the total (i.e. full-length) molecule from which
the fragment is derived. Thus, for example, a fragment of a protein
or peptide antigen preferably corresponds to a continuous stretch
of entities in the protein or peptide antigen the fragment is
derived from, which represents at least 5%, preferably at least
20%, preferably at least 30%, more preferably at least 40%, more
preferably at least 50%, even more preferably at least 60%, even
more preferably at least 70%, and most preferably at least 80% of
the total (i.e. full-length) protein or peptide antigen. It is
particularly preferred that the fragment of a sequence is a
functional fragment, i.e. that the fragment fulfills one or more of
the functions fulfilled by the sequence the fragment is derived
from. For example, a fragment of a protein or peptide antigen
preferably exhibits at least one antigenic function (e.g. is
capable of eliciting a specific immune reaction against at least
one antigen determinant in said protein or peptide antigen) of the
protein or peptide antigen the fragment is derived from.
[0074] Fragments of proteins: "Fragments" of proteins or peptides,
i.e., fragments of amino acid sequences, in the context of the
present invention may, typically, comprise a sequence of a protein
or peptide as defined herein, which is, with regard to its amino
acid sequence (or its encoding nucleic acid molecule),
N-terminally, C-terminally and/or intrasequentially truncated
compared to the amino acid sequence of the original (native)
protein (or its encoded nucleic acid molecule). Such truncation may
thus occur either on the amino acid level or correspondingly on the
nucleic acid level. A sequence identity with respect to such a
fragment as defined herein may therefore preferably refer to the
entire protein or peptide as defined herein or to the entire
(coding) nucleic acid molecule of such a protein or peptide.
[0075] Likewise, "fragments" of nucleic acid sequences in the
context of the present invention may comprise a sequence of a
nucleic acid as defined herein, which is, with regard to its
nucleic acid molecule 5'-, 3'- and/or intrasequentially truncated
compared to the nucleic acid molecule of the original (native)
nucleic acid molecule. A sequence identity with respect to such a
fragment as defined herein may therefore preferably refer to the
entire nucleic acid as defined herein.
[0076] Preferred fragments of proteins or peptides in the context
of the present invention may furthermore comprise a sequence of a
protein or peptide as defined herein, which has a length of about 6
to about 20 or even more amino acids, e.g. fragments as processed
and presented by MHC class I molecules, preferably having a length
of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6,
7, 11, or 12 amino acids), or fragments as processed and presented
by MHC class II molecules, preferably having a length of about 13
or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even
more amino acids, wherein these fragments may be selected from any
part of the amino acid sequence. These preferred fragments are
typically recognized by T-cells in form of a complex consisting of
the peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form. Fragments of
proteins or peptides may comprise at least one epitope of those
proteins or peptides. Furthermore, also domains of a protein, like
the extracellular domain, the intracellular domain or the
transmembrane domain and shortened or truncated versions of a
protein may be understood to comprise a fragment of a protein.
[0077] Epitope: (also called "antigen determinant"): T cell
epitopes or parts of the proteins in the context of the present
invention may comprise fragments preferably having a length of
about 6 to about 20 or even more amino acids, e.g. fragments as
processed and presented by MHC class I molecules, preferably having
a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or
even 11, or 12 amino acids), or fragments as processed and
presented by MHC class II molecules, preferably having a length of
about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20
or even more amino acids, wherein these fragments may be selected
from any part of the amino acid sequence. These fragments are
typically recognized by T cells in form of a complex consisting of
the peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form.
[0078] B cell epitopes are typically fragments located on the outer
surface of (native) protein or peptide antigens as defined herein,
preferably having 5 to 15 amino acids, more preferably having 5 to
12 amino acids, even more preferably having 6 to 9 amino acids,
which may be recognized by antibodies, i.e. in their native
form.
[0079] Such epitopes of proteins or peptides may furthermore be
selected from any of the herein mentioned variants of such proteins
or peptides. In this context antigenic determinants can be
conformational or discontinuous epitopes which are composed of
segments of the proteins or peptides as defined herein that are
discontinuous in the amino acid sequence of the proteins or
peptides as defined herein but are brought together in the
three-dimensional structure or continuous or linear epitopes which
are composed of a single polypeptide chain.
[0080] Variant: A variant of an entity, such as a variant of a
sequence, e.g. of a nucleotide or amino acid sequence, refers to a
modified entity, such as a modified sequence, e.g. a modified
nucleotide or amino acid sequence. For example, a variant of a
sequence may exhibit one or more nucleotide or amino acid
deletions, insertions, additions and/or substitutions compared to
the sequence the variant is derived from. Preferably, a variant of
a sequence in the context of the present invention is at least 40%,
preferably at least 50%, more preferably at least 60%, more
preferably at least 70%, even more preferably at least 80%, even
more preferably at least 90%, most preferably at least 95%
identical to the sequence the variant is derived from. Accordingly,
a variant of a peptide or protein antigen in the context of the
present invention is preferably at least 40%, preferably at least
50%, more preferably at least 60%, more preferably at least 70%,
even more preferably at least 80%, even more preferably at least
90%, most preferably at least 95% identical to the sequence of the
protein or peptide antigen the variant is derived from. Preferably,
the variant is a functional variant, i.e. that the variant fulfills
one or more of the functions fulfilled by the sequence the variant
is derived from. For example, a variant of a protein or peptide
antigen preferably exhibits at least one antigenic function (e.g.
is capable of eliciting a specific immune reaction against at least
one antigen determinant in said protein or peptide antigen) of the
protein or peptide antigen the variant is derived from.
[0081] "Variants" of proteins or peptides as defined in the context
of the present invention may be generated, having an amino acid
sequence which differs from the original sequence in one or more
mutation(s), such as one or more substituted, inserted and/or
deleted amino acid(s). Preferably, these fragments and/or variants
have the same biological function or specific activity compared to
the full-length native protein, e.g. its specific antigenic
property. "Variants" of proteins or peptides as defined in the
context of the present invention may, e.g., comprise conservative
amino acid substitution(s) compared to their native, i.e.
non-mutated physiological, sequence. Those amino acid sequences as
well as their encoding nucleotide sequences in particular fall
under the term variants as defined herein. Substitutions in which
amino acids, which originate from the same class, are exchanged for
one another are called conservative substitutions. In particular,
these are amino acids having aliphatic side chains, positively or
negatively charged side chains, aromatic groups in the side chains
or amino acids, the side chains of which can enter into hydrogen
bridges, e.g. side chains which have a hydroxyl function. This
means that e.g. an amino acid having a polar side chain is replaced
by another amino acid having a likewise polar side chain, or, for
example, an amino acid characterized by a hydrophobic side chain is
substituted by another amino acid having a likewise hydrophobic
side chain (e.g. serine (threonine) by threonine (serine) or
leucine (isoleucine) by isoleucine (leucine)). Insertions and
substitutions are possible, in particular, at those sequence
positions which cause no modification to the three-dimensional
structure or do not affect the binding region. Modifications to a
three-dimensional structure by insertion(s) or deletion(s) can
easily be determined e.g. using CD spectra (circular dichroism
spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of
Polypeptides, in: Modern Physical Methods in Biochemistry,
Neuberger et al (ed.), Elsevier, Amsterdam).
[0082] Additionally variants of proteins or peptides may comprise
peptide analogues as defined herein. Furthermore, variants of
proteins or peptides as defined herein, which may be encoded by a
nucleic acid molecule, may also comprise those sequences, wherein
nucleotides of the nucleic acid are exchanged according to the
degeneration of the genetic code, without leading to an alteration
of the respective amino acid sequence of the protein or peptide,
i.e. the amino acid sequence or at least part thereof may not
differ from the original sequence in one or more mutation(s) within
the above meaning.
[0083] Sequence identity: In order to determine the percentage to
which two sequences are identical, e.g. nucleic acid sequences or
amino acid sequences as defined herein, such as the amino acid
sequences encoded by a nucleic acid sequence of the polymeric
carrier cargo complex or a nucleic acid sequence or amino acid
sequence of an antigen as defined herein, the cargo nucleic acid
sequence or the amino acid sequences themselves, the sequences can
be aligned in order to be subsequently compared to one another.
Therefore, e.g. a position of a first sequence may be compared with
the corresponding position of the second sequence. If a position in
the first sequence is occupied by the same component as is the case
at a position in the second sequence, the two sequences are
identical at this position. If this is not the case, the sequences
differ at this position. If insertions occur in the second sequence
in comparison to the first sequence, gaps can be inserted into the
first sequence to allow a further alignment. If deletions occur in
the second sequence in comparison to the first sequence, gaps can
be inserted into the second sequence to allow a further alignment.
The percentage to which two sequences are identical is then a
function of the number of identical positions divided by the total
number of positions including those positions which are only
occupied in one sequence. The percentage to which two sequences are
identical can be determined using a mathematical algorithm. A
preferred, but not limiting, example of a mathematical algorithm
which can be used is the algorithm of Karlin et al. (1993), PNAS
USA, 90:5873-5877 or Altschul et al (1997), Nucleic Acids Res.,
25:3389-3402. Such an algorithm is integrated in the BLAST program.
Sequences which are identical to the sequences of the present
invention to a certain extent can be identified by this program. A
"variant" of a protein or peptide may have, e.g., at least 70%,
75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a
stretch of 10, 20, 30, 50, 75 or 100 amino acids, preferably over
the full length sequence, of such protein or peptide. Analogously,
a "variant" of a nucleic acid sequence may have, e.g., at least
70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a
stretch of 10, 20, 30, 50, 75 or 100 nucleotides, preferably over
the full length sequence, of such nucleic acid sequence.
[0084] Derivative of a protein or peptide: A derivative of a
peptide or protein is typically understood to be a molecule that is
derived from another molecule, such as said peptide or protein. A
"derivative" of a peptide or protein also encompasses fusions
comprising a peptide or protein used in the present invention. For
example, the fusion comprises a label, such as, for example, an
epitope, e.g., a FLAG epitope or a V5 epitope or an HA epitope. For
example, the epitope is a FLAG epitope. Such a tag is useful for,
for example, purifying the fusion protein. The term "derivative" of
a peptide or protein also encompasses a derivatised peptide or
protein, such as, for example, a peptide or protein modified to
contain one or more-chemical moieties other than an amino acid. The
chemical moiety may be linked covalently to the peptide or protein
e.g., via an amino terminal amino acid residue, a carboxyl terminal
amino acid residue, or at an internal amino acid residue. Such
modifications include the addition of a protective or capping group
on a reactive moiety in the peptide or protein, addition of a
detectable label, and other changes that do not adversely destroy
the activity of the peptide or protein compound. For example, a
derivative may comprise a PEG moiety, radionuclide, coloured latex,
etc. A derivative generally possesses or exhibits an improved
characteristic relative to a e.g., enhanced protease resistance
and/or longer half-life and/or enhanced transportability between
cells or tissues of the human or animal body and/or reduced adverse
effect(s) and/or enhanced affinity or immunogenicity. WO
2010/003193 describes various methodologies to provide peptide or
protein derivatives which may be employed separately or in
combination using standard procedures known to the person of
ordinary skill, including derivatisation of a protein or peptide by
e.g. PEGylation, HESylation, or glycosylation.
[0085] According to a first aspect, one or more objects underlying
the present invention are solved by a pharmaceutical composition
including: [0086] (A) a polymeric carrier cargo complex,
comprising: [0087] a) a polymeric carrier comprising
disulfide-crosslinked cationic components, preferably formed by
disulfide-crosslinked cationic components; and [0088] b) at least
one nucleic acid molecule, and [0089] (B) at least one antigen that
is selected from the group consisting of: [0090] (i) an antigen
from a pathogen associated with infectious disease; [0091] (ii) an
antigen associated with allergy or allergic disease; [0092] (iii)
an antigen associated with autoimmune disease; and [0093] (iv) an
antigen associated with a cancer or tumour disease, or a fragment,
variant and/or derivative of said antigen.
[0094] Preferably, component (B) is not covalently linked, in
particular not by a disulfide bond, with component (A). Thus,
component (B) is preferably not covalently linked, such as by a
disulfide bond, to the polymeric carrier and/or the at least one
nucleic acid molecule. Preferably, the at least one antigen is not
covalently linked to the polymeric carrier cargo complex, in
particular not to the polymeric carrier of the polymeric carrier
cargo complex. For example, preferably, the at least one antigen,
such as a protein or peptide antigen, is not covalently linked to
the polymeric carrier cargo complex, such as to the polymeric
carrier, by a disulfide bond. However, in an embodiment, wherein
component (A) and component (B) are linked via disulfide bonds,
such linkage is preferably not realized via a crosslinker, such as
via a 3,6-Dioxa-1,8-octanedithiol (DODT) crosslinker. Furthermore,
in an embodiment, wherein component (A) and component (B) are
linked via disulfide bonds, component (B) is preferably not
ovalbumine or a fragment of ovalbumine.
[0095] The advantage of component (B), e.g. a protein antigen, not
being covalently linked to the polymeric carrier is that the
structure of the antigen, e.g. the protein antigen, will not be
disturbed and its immunogenicity will be preserved. If the antigen,
e.g. the protein antigen, is covalently linked to the polymeric
carrier, e.g. by disulfide bonds, the tertiary structure of the
antigen may be changed, or even denatured, which may destroy the
structure of conformational epitopes and may render the protein
less immunogenic or non-immunogenic. Epitopes that are recognized
by the immune system may be linear epitopes consisting of a
continuous stretch of amino acids, or conformational epitopes that
have a specific three-dimensional shape consisting of amino acids
from distinct parts of the protein.
[0096] Furthermore, preferably, the polymeric carrier, in
particular the cationic components of the polymeric carrier, and
the at least one nucleic acid molecule of the polymeric carrier
cargo complex are not covalently linked, but are preferably
associated via other bonds than covalent bonds, such as by ionic
bonds and/or van der Waals bonds. Thus, it is preferred that, in
the polymeric carrier cargo complex, only the cationic components
are covalently linked with each other, but that the nucleic acid
molecules are associated non-covalently with the polymeric
carrier.
[0097] Moreover, in a preferred embodiment, components (A) and (B)
do not form a micelle structure together, in particular, the
polmeric carrier preferably does not form a micelle structure.
[0098] In certain embodiments of all aspects of the invention, the
polymeric carrier cargo complex is for use as an adjuvant. For
example, it is used as an adjuvant, and/or has adjuvant properties,
as may be readily determined by the person of ordinary skill using
routine methodologies, and including methodologies as described
herein.
[0099] As a first ingredient the inventive pharmaceutical
composition includes (e.g. as an adjuvant) at least one polymeric
carrier cargo complex, comprising [0100] a) (as a carrier) a
polymeric carrier comprising disulfide-crosslinked cationic
components, preferably formed by disulfide-crosslinked cationic
components, and [0101] b) (as a cargo) at least one nucleic acid
molecule.
[0102] The polymeric carrier cargo complex comprised in the
inventive pharmaceutical composition allows provision of a more
efficient and/or safer adjuvant for vaccination purposes.
Advantageously, the polymeric carrier cargo complex is suited for
in vivo delivery of nucleic acids, in particular for compacting and
stabilizing a nucleic acid for the purposes of nucleic acid
transfection, such as exhibiting one or more reduced negative side
effects of high-molecular weight polymers as discussed above, such
as poor biodegradability or high toxicity, agglomeration, low
transfection activity in vivo, etc. The polymeric carrier cargo
complex also provides for improved nucleic acid transfer in vivo
particularly via intradermal or intramuscular routes, including
serum stability, salt stability, efficiency of uptake, reduced
complement activation, nucleic acid release, etc. Such a polymeric
carrier cargo complex, furthermore may support induction and
maintenance of an adaptive immune response by initiating or
boosting a parallel innate immune response. Additionally, the
polymeric carrier cargo complex may exhibit improved storage
stability, particularly during lyophilisation.
[0103] The polymeric carrier cargo complex as defined above
comprises as one component a polymeric carrier formed by
disulfide-crosslinked cationic components. The term "cationic
component" typically refers to a charged molecule, which is
positively charged (cation) at a pH value of about 1 to 9,
preferably of a pH value of or below 9, of or below 8, of or below
7, most preferably at physiological pH values, e.g. about 7.3 to
7.4. Accordingly, a cationic peptide, protein or polymer according
to the present invention is positively charged under physiological
conditions, particularly under physiological salt conditions of the
cell in vivo. The definition "cationic" may also refer to
"polycationic" components.
[0104] In this context, the cationic components, which form basis
for the polymeric carrier of the polymeric carrier cargo complex by
disulfide-crosslinkage, are typically selected from any suitable
cationic or polycationic peptide, protein or polymer suitable for
this purpose, particular any cationic or polycationic peptide,
protein or polymer capable to complex a nucleic acid as defined
according to the present invention, and thereby preferably
condensing the nucleic acid. The cationic or polycationic peptide,
protein or polymer is preferably a linear molecule, however,
branched cationic or polycationic peptides, proteins or polymers
may also be used.
[0105] Each cationic or polycationic protein, peptide or polymer of
the polymeric carrier contains at least one --SH moiety, most
preferably at least one cysteine residue or any further chemical
group exhibiting an --SH moiety, capable to form a disulfide
linkage upon condensation with at least one further cationic or
polycationic protein, peptide or polymer as cationic component of
the polymeric carrier as mentioned herein.
[0106] Each cationic or polycationic protein, peptide or polymer or
any further component of the polymeric carrier is preferably linked
to its neighbouring component(s) (cationic proteins, peptides,
polymers or other components) via disulfide-crosslinking.
Preferably, the disulfide-crosslinking is a (reversible) disulfide
bond (--S--S--) between at least one cationic or polycationic
protein, peptide or polymer and at least one further cationic or
polycationic protein, peptide or polymer or other component of the
polymeric carrier. The disulfide-crosslinking is typically formed
by condensation of --SH-moieties of the components of the polymeric
carrier particularly of the cationic components. Such an
--SH-moiety may be part of the structure of the cationic or
polycationic protein, peptide or polymer or any further component
of the polymeric carrier prior to disulfide-crosslinking or may be
added prior to disulfide-crosslinking by a modification as defined
below. In this context, the sulphurs adjacent to one component of
the polymeric carrier, necessary for providing a disulfide bond,
may be provided by the component itself, e.g. by a --SH moiety as
defined herein or may be provided by modifying the component
accordingly to exhibit a --SH moiety. These --SH-moieties are
typically provided by each of the component, e.g. via a cysteine or
any further (modified) amino acid or compound of the component,
which carries a --SH moiety. In the case that the cationic
component or any further component of the polymeric carrier is a
peptide or protein it is preferred that the --SH moiety is provided
by at least one cysteine residue. Alternatively, the component of
the polymeric carrier may be modified accordingly with a --SH
moiety, preferably via a chemical reaction with a compound carrying
a --SH moiety, such that each of the components of the polymeric
carrier carries at least one such --SH moiety. Such a compound
carrying a --SH moiety may be e.g. an (additional) cysteine or any
further (modified) amino acid or compound of the component of the
polymeric carrier, which carries a --SH moiety. Such a compound may
also be any non-amino compound or moiety, such as a non amino acid
compound or moiety, which contains or allows to introduce a --SH
moiety into the component as defined herein. Such non-amino
compounds, e.g. non amino acid compounds, may be attached to the
component of the polymeric carrier according to the present
invention via chemical reactions or binding of compounds, e.g. by
binding of a 3-thio propionic acid or 2-iminothiolane (Traut's
reagent), by amide formation (e.g. carboxylic acids, sulphonic
acids, amines, etc.), by Michael addition (e.g maleinimide
moieties, .alpha.,.beta. unsatured carbonyls, etc.), by click
chemistry (e.g. azides or alkines), by alkene/alkine methatesis
(e.g. alkenes or alkines), imine or hydrozone formation (aldehydes
or ketons, hydrazins, hydroxylamins, amines), complexation
reactions (avidin, biotin, protein G) or components which allow
S.sub.n-type substitution reactions (e.g halogenalkans, thiols,
alcohols, amines, hydrazines, hydrazides, sulphonic acid esters,
oxyphosphonium salts) or other chemical moieties which can be
utilized in the attachment of further components. In some cases,
the --SH moiety may be masked by protecting groups during chemical
attachment to the component. Such protecting groups are known in
the art and may be removed after chemical coupling. In each case,
the --SH moiety, e.g. of a cysteine or of any further (modified)
amino acid or compound, may be present at the terminal ends or
internally at any position of the component of the polymeric
carrier. As defined herein, each of the components of the polymeric
carrier, preferably each of the cationic components of the
polymeric carrier, typically exhibits at least one --SH-moiety, but
may also contain two, three, four, five, or even more
--SH-moieties.
[0107] In a preferred embodiment, the polymeric carrier, the cargo
nucleic acid molecule and/or the antigen, such as the protein or
peptide antigen, are not modified by introducing new coupling sites
for forming disulfide bonds, such as by introducing new
--SH-moieties, in particular the polymeric carrier, the cargo
nucleic acid molecule and/or the antigen, such as the protein or
peptide antigen, are preferably not modified by dithiopyridine.
Thus, in a particular preferred embodiment, the polymeric carrier,
the cargo nucleic acid molecule and/or the antigen do not comprise
dithiopyridine.
[0108] In a further preferred embodiment, the polymeric carrier
does not comprise a polyethylene glycol (PEG) moiety, in particular
preferably the cationic components of the polymeric carrier
preferably do not comprise a PEG moiety. However, if the polymeric
carrier comprises a PEG moiety, the cationic component is
preferably not poly-L-lysine.
[0109] Additionally to binding of cationic components a --SH moiety
may be used to attach further components to the polymeric carrier
as defined herein, particularly an amino acid component, e.g.
antigen epitopes, antigens, antibodies, cell penetrating peptides
(e.g. TAT), ligands, etc. If the polymeric carrier comprises
further components in addition to the cationic components, it is
preferred that the additional component is not ovalbumin or a
fragment of ovalbumin, in particular, if the additional component
is an amino acid component.
[0110] As defined above, the polymeric carrier of the polymeric
carrier cargo complex is formed by disulfide-crosslinked cationic
(or polycationic) components.
[0111] According to one first alternative, at least one cationic
(or polycationic) component of the polymeric carrier may be
selected from cationic or polycationic peptides or proteins. Such
cationic or polycationic peptides or proteins preferably exhibit a
length of about 3 to 100 amino acids, preferably a length of about
3 to 50 amino acids, more preferably a length of about 3 to 25
amino acids, e.g. a length of about 3 to 10; 5 to 20; 5 to 15; 8 to
15, 16 or 17; 10 to 15, 16, 17, 18, 19, or 20; or 15 to 25 amino
acids. Alternatively or additionally, such cationic or polycationic
peptides or proteins may exhibit a molecular weight of about 0.01
kDa to about 100 kDa, including a molecular weight of about 0.5 kDa
to about 100 kDa, preferably of about 10 kDa to about 50 kDa, even
more preferably of about 10 kDa to about 30 kDa. In this context
also analogues and derivatives of proteins or peptides as defined
herein are explicitly encompassed.
[0112] In the specific case that the cationic component of the
polymeric carrier comprises or consists of a cationic or
polycationic peptide or protein, the cationic properties of the
cationic or polycationic peptide or protein or of the entire
polymeric carrier, if the polymeric carrier is composed of cationic
or polycationic peptides or proteins, may be determined based on
its content of cationic amino acids, in particular based on its
content of cationic amino acids in excess over anionic or neutral
amino acids, and thus, based on its net positive charge.
Preferably, the content of cationic amino acids in the cationic or
polycationic peptide or protein and/or the polymeric carrier is at
least 10%, 20%, or 30%, preferably at least 40%, more preferably at
least 50%, 60% or 70%, but also preferably at least 80%, 90%, or
even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or
may be in the range of about 10% to 90%, more preferably in the
range of about 15% to 75%, even more preferably in the range of
about 20% to 50%, e.g. 20%, 30%, 40% or 50%, or in a range formed
by any two of the afore mentioned values, provided, that the
content of all amino acids, e.g. cationic, lipophilic, hydrophilic,
aromatic and further amino acids, in the cationic or polycationic
peptide or protein, or in the entire polymeric carrier, if the
polymeric carrier is entirely composed of cationic or polycationic
peptides or proteins, is 100%.
[0113] In this context, cationic amino acids are preferably the
naturally occurring amino acids Arg (Arginine), Lys (Lysine), His
(Histidine), and Orn (Ornithin). However, in a broader sense any
(non-natural) amino acid carrying a cationic charge on its side
chain may also be envisaged to carry out the invention. However,
those cationic amino acids are preferred which comprise side chains
which are positively charged under physiological pH conditions. In
a more preferred embodiment, these amino acids are Arg, Lys, and
Orn.
[0114] Preferably, such cationic or polycationic peptides or
proteins of the polymeric carrier, which comprise or are
additionally modified to comprise at least one --SH moeity, are
selected from, without being restricted thereto, cationic peptides
or proteins such as protamine, nucleoline, spermine or spermidine,
oligo- or poly-L-lysine (PLL), basic polypeptides, oligo or
poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs,
such as Transportan, or MPG peptides, HIV-binding peptides, Tat,
HIV-1 Tat (HIV), Tat-derived peptides, members of the 0 family,
e.g. Penetratin, Antennapedia-derived peptides (particularly from
Drosophila antennapedia), pAntp, pIsI, etc., antimicrobial-derived
CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived
peptides, SAP, MAP, KALA, PpTG20, Loligomere, FGF, Lactoferrin,
histones, VP22 derived or analog peptides, HSV, VP22 (Herpes
simplex), MAP, KALA or protein transduction domains (PTDs, PpT620,
prolin-rich peptides, arginine-rich peptides, lysine-rich peptides,
Pep-1, L-oligomers, Calcitonin peptide(s), etc.
[0115] Alternatively or additionally, such cationic or polycationic
peptides or proteins of the polymeric carrier, which comprise or
are additionally modified to comprise at least one --SH moeity, are
selected from, without being restricted thereto, following cationic
peptides having the following sum formula (I):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x};
wherein l+m+n+o+x=3-100, and l, m, n or o independently of each
other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60,
61-70, 71-80, 81-90 and 91-100 provided that the overall content of
Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine)
represents at least 10% of all amino acids of the oligopeptide; and
Xaa is any amino acid selected from native (=naturally occurring)
or non-native amino acids except of Arg, Lys, His or Orn; and x is
any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,
71-80, 81-90, provided, that the overall content of Xaa does not
exceed 90% of all amino acids of the oligopeptide. Any of amino
acids Arg, Lys, His, Orn and Xaa may be positioned at any place of
the peptide. In this context cationic peptides or proteins in the
range of 7-30 amino acids are particular preferred. Even more
preferred peptides of this formula are oligoarginines such as e.g.
Arg.sub.7, Arg.sub.8, Arg.sub.9, Arg.sub.12, His.sub.3Arg.sub.9,
Arg.sub.9His.sub.3, His.sub.3Arg.sub.9His.sub.3,
His.sub.6Arg.sub.9His.sub.6 His.sub.3Arg.sub.4His.sub.3,
His.sub.6Arg.sub.4His.sub.6, TyrSer.sub.2Arg.sub.9Ser.sub.2Tyr,
(ArgLysHis).sub.4, Tyr(ArgLysHis).sub.2Arg, etc.
[0116] According to a particular preferred embodiment, such
cationic or polycationic peptides or proteins of the polymeric
carrier having the empirical sum formula (I) as shown above, may,
without being restricted thereto, comprise at least one of the
following subgroup of formulae:
Arg.sub.7, Arg.sub.8, Arg.sub.9, Arg.sub.10, Arg.sub.11,
Arg.sub.12, Arg.sub.13, Arg.sub.14, Arg.sub.15-30; Lys.sub.7,
Lys.sub.8, Lys.sub.9, Lys.sub.10, Lys.sub.11, Lys.sub.12,
Lys.sub.13, Lys.sub.14, Lys.sub.15-30; His.sub.7, His.sub.8,
His.sub.9, His.sub.10, His.sub.11, His.sub.12, His.sub.13,
His.sub.14, His.sub.15-30; Orn.sub.7, Orn.sub.8, Orn.sub.9,
Orn.sub.10, Orn.sub.11, Orn.sub.12, Orn.sub.13, Orn.sub.14,
Orn.sub.15-30.
[0117] According to a further particularly preferred embodiment,
cationic or polycationic peptides or proteins of the polymeric
carrier, having the empirical sum formula (I) as shown above and
which comprise or are additionally modified to comprise at least
one --SH moeity, may be preferably selected from, without being
restricted thereto, at least one of the following subgroup of
formulae. The following formulae (as with empirical formula (I)) do
not specify any amino acid order, but are intended to reflect
empirical formulae by exclusively specifying the (number of) amino
acids as components of the respective peptide. Accordingly, as an
example, empirical formula Arg.sub.(7-29)Lys.sub.1 is intended to
mean that peptides falling under this formula contain 7 to 19 Arg
residues and 1 Lys residue of whatsoever order. If the peptides
contain 7 Arg residues and 1 Lys residue, all variants having 7 Arg
residues and 1 Lys residue are encompassed. The Lys residue may
therefore be positioned anywhere in the e.g. 8 amino acid long
sequence composed of 7 Arg and 1 Lys residues. The subgroup
preferably comprises:
Arg.sub.(4-29)Lys.sub.1, Arg.sub.(4-29)His.sub.1,
Arg.sub.(4-29Orn.sub.1, Lys.sub.(4-29)His.sub.1,
Lys.sub.(4-29)Orn.sub.1, His.sub.(4-29)Orn.sub.1,
Arg.sub.(3-28)Lys.sub.2, Arg.sub.(3-28)His.sub.2,
Arg.sub.(3-28)Orn.sub.2, Lys.sub.(3-28)His.sub.2,
Lys.sub.(3-28)Orn.sub.2, His.sub.(3-28),Orn.sub.2,
Arg.sub.(2-27),Lys.sub.3, Arg.sub.(2-27)His.sub.3,
Arg.sub.(2-27)Orn.sub.3, Lys.sub.(2-27)His.sub.3,
Lys.sub.(2-27)Orn.sub.3, His.sub.(2-27)Orn.sub.3,
Arg.sub.(1-26)Lys.sub.4, Arg.sub.(1-26)His.sub.4,
Arg.sub.(1-26)Orn.sub.4, Lys.sub.(1-26)His.sub.4,
Lys.sub.(1-26)Orn.sub.4, His.sub.(1-26)Orn.sub.4,
Arg.sub.(3-28)Lys.sub.1His.sub.1, Arg.sub.(3-28)Lys.sub.1Orn.sub.1,
Arg.sub.(3-28)His.sub.1Orn.sub.1,
Arg.sub.(1)Lys.sub.(3-28)His.sub.1,
Arg.sub.(1)Lys.sub.(3-28)Orn.sub.1,
Lys.sub.(3-28)His.sub.1Orn.sub.1, Arg.sub.1Lys.sub.1His.sub.(3-28),
Arg.sub.1His.sub.(3-28)Orn.sub.1, Lys.sub.1His.sub.(3-28)Orn.sub.1;
Arg.sub.(2-27)Lys.sub.2His.sub.1, Arg.sub.(2-27)Lys.sub.1His.sub.2,
Arg.sub.(2-27)Lys.sub.2Orn.sub.1, Arg.sub.(2-27)Lys.sub.1Orn.sub.2,
Arg.sub.(2-27)His.sub.2Orn.sub.1, Arg.sub.(2-27)His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.(2-27)His.sub.1, Arg.sub.lLys.sub.(2-27)His.sub.2,
Arg.sub.2Lys.sub.(2-27)Orn.sub.1, Agr.sub.1Lys.sub.(2-27)Orn.sub.2,
Lys.sub.(2-27)His.sub.2Orn.sub.1, Lys.sub.(2-27)His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.1His.sub.(2-27), Arg.sub.1Lys.sub.2His.sub.(2-27),
Arg.sub.2His.sub.(2-27)Orn.sub.1, Arg.sub.1His.sub.(2-27)Orn.sub.2,
Lys.sub.2His.sub.(2-27)Orn.sub.1, Lys.sub.1His.sub.(2-27)Orn.sub.2;
Arg.sub.(1-26)Lys.sub.3His.sub.1, Arg.sub.(1-26)Lys.sub.2His.sub.2,
Arg.sub.(1-26)Lys.sub.1His.sub.3, Arg.sub.(1-26)Lys.sub.3Orn.sub.1,
Arg.sub.(1-26)Lys.sub.2Orn.sub.2, Arg.sub.(1-26)Lys.sub.1Orn.sub.3,
Arg.sub.(1-26)His.sub.3Orn.sub.1, Arg.sub.(1-26)His.sub.2Orn.sub.2,
Arg.sub.(1-26)His.sub.1Orn.sub.3, Arg.sub.3Lys.sub.(1-26)His.sub.1,
Arg.sub.2LYs.sub.(1-26)His.sub.2, Arg.sub.1Lys.sub.(1-26)His.sub.3,
Arg.sub.3Lys.sub.(1-26)Orn.sub.1, Arg.sub.2Lys.sub.(1-26)Orn.sub.2,
Arg.sub.1Lys.sub.(1-26)Orn.sub.3, Lys.sub.(1-26)His.sub.3Orn.sub.1,
Lys.sub.(1-26)His.sub.2Orn.sub.2, Lys.sub.(1-26)His.sub.1Orn.sub.3,
Arg.sub.3Lys.sub.1His.sub.(1-26), Arg.sub.2Lys.sub.2His.sub.(1-26),
Arg.sub.1Lys.sub.3His.sub.(1-26), Arg.sub.3His.sub.(1-26)Orn.sub.1,
Arg.sub.2His.sub.(1-26)Orn.sub.2, Arg.sub.1His.sub.(1-26)Orn.sub.3,
Lys.sub.3His.sub.(1-26)Orn.sub.1, Lys.sub.2His.sub.(1-26)Orn.sub.2,
Lys.sub.1His.sub.(1-26)Orn.sub.3;
Arg.sub.(2-27)Lys.sub.1His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(2-27)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(2-27)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.1Orn.sub.(2-27);
Arg.sub.(1-26)Lys.sub.2His.sub.1Orn.sub.1,
Arg.sub.(1-26)Lys.sub.1His.sub.2Orn.sub.1,
Arg.sub.(1-26)Lys.sub.1His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.(1-26)His.sub.1Orn.sub.1,
Arg.sub.1Lys.sub.(1-26)His.sub.2Orn.sub.1,
Arg.sub.1Lys.sub.(1-26)His.sub.1Orn.sub.2,
Arg.sub.2Lys.sub.1His.sub.(1-26)Orn.sub.1,
Arg.sub.1Lys.sub.2His.sub.(1-26)Orn.sub.1,
Arg.sub.1Lys.sub.1His.sub.(1-26)Orn.sub.2,
Arg.sub.2Lys.sub.1His.sub.1Orn.sub.(1-26),
Arg.sub.1Lys.sub.2His.sub.1Orn.sub.(1-26),
Arg.sub.1Lys.sub.1His.sub.2Orn.sub.(1-26);
[0118] According to a further particular preferred embodiment,
cationic or polycationic peptides or proteins of the polymeric
carrier, having the empirical sum formula (I) as shown above and
which comprise or are additionally modified to comprise at least
one --SH moeity, may be, without being restricted thereto, selected
from the subgroup consisting of generic formulas Arg.sub.7, (also
termed as R.sub.7), Arg.sub.9 (also termed R.sub.9), Arg.sub.12
(also termed as R.sub.12).
[0119] According to a one further particular preferred embodiment,
the cationic or polycationic peptide or protein of the polymeric
carrier, when defined according to formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (I)) as shown above and which comprise or are additionally
modified to comprise at least one --SH moeity, may be, without
being restricted thereto, selected from subformula (Ia):
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa').sub.x(Cys).sub.y-
} formula (Ia)
wherein (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o; and x are
as defined herein, Xaa' is any amino acid selected from native
(=naturally occurring) or non-native amino acids except of Arg,
Lys, His, Orn or Cys and y is any number selected from 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and 81-90, provided that
the overall content of Arg (Arginine), Lys (Lysine), His
(Histidine) and Orn (Ornithine) represents at least 10% of all
amino acids of the oligopeptide.
[0120] This embodiment may apply to situations, wherein the
cationic or polycationic peptide or protein of the polymeric
carrier, e.g. when defined according to empirical formula
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula (I)) as shown above, comprises or has been modified with
at least one cysteine as --SH moiety in the above meaning such that
the cationic or polycationic peptide as cationic component carries
at least one cysteine, which is capable to form a disulfide bond
with other components of the polymeric carrier.
[0121] According to another particular preferred embodiment, the
cationic or polycationic peptide or protein of the polymeric
carrier, when defined according to formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (I)) as shown above, may be, without being restricted
thereto, selected from subformula (Ib):
Cys.sub.1{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cy-
s.sub.2 (formula (Ib))
wherein empirical formula
{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}
(formula (I)) is as defined herein and forms a core of an amino
acid sequence according to (semiempirical) formula (I) and wherein
Cys.sub.1 and Cys.sub.2 are Cysteines proximal to, or terminal to
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x.
Exemplary examples may comprise any of the above sequences flanked
by two Cys and following sequences:
TABLE-US-00001 Cys(Arg.sub.7)Cys, Cys(Arg.sub.8)Cys,
Cys(Arg.sub.9)Cys, Cys(Arg.sub.10)Cys, Cys(Arg.sub.11)Cys,
Cys(Arg.sub.12)Cys, Cys(Arg.sub.13)Cys, Cys(Arg.sub.14)Cys,
Cys(Arg.sub.15)Cys, Cys(Arg.sub.16)Cys, Cys(Arg.sub.17)Cys,
Cys(Arg.sub.18)Cys, Cys(Arg.sub.19)Cys, Cys(Arg.sub.20)Cys (SEQ ID
NOs: 1-14): CysArg.sub.7Cys (SEQ ID NO. 1)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.8Cys (SEQ ID NO. 2)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.9Cys: (SEQ ID
NO. 3) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.10Cys
(SEQ ID NO. 4) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys
CysArg.sub.11Cys (SEQ ID NO. 5)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Cys
CysArg.sub.12Cys: (SEQ ID NO. 6)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Cys
CysArg.sub.13Cys: (SEQ ID NO. 7)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Cys
CysArg.sub.14Cys: (SEQ ID NO. 8)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Cys
CysArg.sub.15Cys: (SEQ ID NO. 9)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Cys CysArg.sub.16Cys: (SEQ ID NO. 10)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.17Cys: (SEQ ID NO. 11)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.18Cys: (SEQ ID NO. 12)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.19Cys: (SEQ ID NO. 13)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg.sub.20Cys: (SEQ ID NO. 14)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Cys
[0122] This embodiment may apply to situations, wherein the
cationic or polycationic peptide or protein of the polymeric
carrier, e.g. when defined according to empirical formula
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x
(formula (I)) as shown above, has been modified with at least two
cysteines as --SH moieties in the above meaning such that the
cationic or polycationic peptide of the polymeric carrier cargo
complex as cationic component carries at least two (terminal)
cysteines, which are capable to form a disulfide bond with other
components of the polymeric carrier.
[0123] According to a second alternative, at least one cationic (or
polycationic) component of the polymeric carrier may be selected
from e.g. any (non-peptidic) cationic or polycationic polymer
suitable in this context, provided that this (non-peptidic)
cationic or polycationic polymer exhibits or is modified to exhibit
at least one --SH-moiety, which provide for a disulfide bond
linking the cationic or polycationic polymer with another component
of the polymeric carrier as defined herein. Thus, likewise as
defined herein, the polymeric carrier may comprise the same or
different cationic or polycationic polymers.
[0124] In the specific case that the cationic component of the
polymeric carrier comprises a (non-peptidic) cationic or
polycationic polymer the cationic properties of the (non-peptidic)
cationic or polycationic polymer may be determined upon its content
of cationic charges when compared to the overall charges of the
components of the cationic polymer. Preferably, the content of
cationic charges, preferably the net cationic charges (i.e. upon
subtraction of anionic and neutral charges), in the cationic
polymer at a (physiological) pH as defined herein is at least 10%,
20%, or 30%, preferably at least 40%, more preferably at least 50%,
60% or 70%, but also preferably at least 80%, 90%, or even 95%,
96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in
the range of about 10% to 90%, more preferably in the range of
about 30% to 100%, even preferably in the range of about 50% to
100%, e.g. 50, 60, 70, 80%, 90% or 100%, or in a range formed by
any two of the afore mentioned values, provided, that the content
of all charges, e.g. positive and negative charges at a
(physiological) pH as defined herein, in the entire cationic
polymer is 100%.
[0125] Preferably, the (non-peptidic) cationic component of the
polymeric carrier represents a cationic or polycationic polymer,
typically exhibiting a molecular weight of about 0.1 or 0.5 kDa to
about 100 kDa, preferably of about 1 kDa to about 75 kDa, more
preferably of about 5 kDa to about 50 kDa, even more preferably of
about 5 kDa to about 30 kDa, or a molecular weight of about 10 kDa
to about 50 kDa, even more preferably of about 10 kDa to about 30
kDa. Additionally, the (non-peptidic) cationic or polycationic
polymer typically exhibits at least one -SH-moiety, which is
capable to form a disulfide linkage upon condensation with either
other cationic components or other components of the polymeric
carrier as defined herein.
[0126] Said cationic or polycationic peptides or proteins may be
prepared by all methods known to a person of ordinary skill or by
recombinant peptide or protein production or by peptide synthesis
as described herein.
[0127] In the above context, the (non-peptidic) cationic component
of the polymeric carrier may be selected from acrylates, modified
acrylates, such as pDMAEMA (poly(dimethylaminoethyl
methylacrylate)), chitosanes, aziridines or 2-ethyl-2-oxazoline
(forming oligo ethylenimines or modifed oligoethylenimines),
polymers obtained by reaction of bisacrylates with amines forming
oligo beta aminoesters or poly amido amines, or other polymers like
polyesters, polycarbonates, etc. Each molecule of these
(non-peptidic) cationic or polycationic polymers typically exhibits
at least one --SH-moiety, wherein these at least one --SH-moiety
may be introduced into the (non-peptidic) cationic or polycationic
polymer by chemical modifications, e.g. using imonothiolan, 3-thio
propionic acid or introduction of --SH-moieties containing amino
acids, such as cysteine or any further (modified) amino acid. Such
--SH-moieties are preferably as already defined above.
[0128] In the context of the polymeric carrier, the cationic
components, which form basis for the polymeric carrier by
disulfide-crosslinkage, may be the same or different from each
other. It is also particularly preferred that the polymeric carrier
of the present invention comprises mixtures of cationic peptides,
proteins or polymers and optionally further components as defined
herein, which are crosslinked by disulfide bonds as described
herein. Particularly preferred cationic components of the polymeric
carrier in the context of the present invention are cationic
peptides or proteins.
[0129] In this context, the polymeric carrier cargo complex due to
its variable polymeric carrier advantageously allows to combine
desired properties of different (short) cationic or polycationic
peptides, proteins or polymers or other components. The polymeric
carrier, e.g., allows to efficiently compact nucleic acids for the
purpose of efficient transfection of nucleic acids, for adjuvant
therapy, for the purposes of gene therapy, for gene knock-down or
others strategies without loss of activity, particularly exhibiting
an efficient transfection of a nucleic acid into different cell
lines in vitro but particularly transfection in vivo. The polymeric
carrier and thus the polymeric carrier cargo complex is furthermore
not toxic to cells, provides for efficient release of its nucleic
acid cargo, is stable during lyophilization and is applicable as
immunostimulating agent or adjuvant. Preferably, the polymer
carrier cargo complex may induce the anti-viral cytokine
IFN-alpha.
[0130] In particular, the polymeric carrier formed by
disulfide-linked cationic components allows considerably to vary
its peptide or polymeric content and thus to modulate its
biophysical/biochemical properties, particularly the cationic
properties of the polymeric carrier, quite easily and fast, e.g. by
incorporating as cationic components the same or different cationic
peptide(s) or polymer(s) and optionally adding other components
into the polymeric carrier. Even though consisting of quite small
non-toxic monomer units the polymeric carrier forms a long cationic
binding sequence providing a strong condensation of the nucleic
acid cargo and complex stability. Under the reducing conditions of
the cytosole (e.g. cytosolic GSH), the complex is rapidly degraded
into its (cationic) components, which are further degraded (e.g.
oligopeptides). This supports deliberation of the nucleic acid
cargo in the cytosol. Due to degradation into small oligopeptides
or polymers in the cytosol, no toxicity is observed as known for
high-molecular oligopeptides or polymers, e.g. from high-molecular
polyarginine.
[0131] Accordingly, the polymeric carrier of the polymeric carrier
cargo complex may comprise different (short) cationic or
polycationic peptides, proteins or polymers selected from cationic
or polycationic peptides, proteins or (non-peptidic) polymers as
defined above, optionally together with further components as
defined herein.
[0132] Additionally, the polymeric carrier of the polymeric carrier
cargo complex as defined above, more preferably at least one of the
different (short) cationic or polycationic peptides or
(non-peptidic) polymers forming basis for the polymeric carrier via
disulfide-crosslinking, may be, preferably prior to the
disulfide-crosslinking, modified with at least one further
component. Alternatively, the polymeric carrier as such may be
modified with at least one further component. It may also
optionally comprise at least one further component, which typically
forms the polymeric carrier disulfide together with the other
(short) cationic or polycationic peptides as defined above via
disulfide crosslinking.
[0133] To allow modification of a cationic or polycationic peptide
or a (non-peptidic) polymer as defined above, each of the
components of the polymeric carrier may (preferably already prior
to disulfide-crosslinking) also contain at least one further
functional moiety, which allows attaching such further components
as defined herein. Such functional moieties may be selected from
functionalities which allow the attachment of further components,
e.g. functionalities as defined herein, e.g. by amide formation
(e.g. carboxylic acids, sulphonic acids, amines, etc.), by Michael
addition (e.g maleinimide moieties, .alpha.,.beta. unsatured
carbonyls, etc.), by click chemistry (e.g. azides or alkines), by
alkene/alkine methatesis (e.g. alkenes or alkines), imine or
hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins,
amines), complexation reactions (avidin, biotin, protein G) or
components which allow S.sub.n-type substitution reactions (e.g
halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,
sulphonic acid esters, oxyphosphonium salts) or other chemical
moieties which can be utilized in the attachment of further
components.
[0134] According to a particularly preferred embodiment, the
further component, which may be contained in the polymeric carrier
or which may be used to modify the different (short) cationic or
polycationic peptides or (non-peptidic) polymers forming basis for
the polymeric carrier of the polymeric carrier cargo complex is an
amino acid component (AA), which may e.g. modify the
biophysical/biochemical properties of the polymeric carrier as
defined herein. According to the present invention, the amino acid
component (AA) comprises a number of amino acids preferably in a
range of about 1 to 100, preferably in a range of about 1 to 50,
more preferably selected from a number comprising 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14 or 15-20, or may be selected from a
range formed by any two of the afore mentioned values. In this
context the amino acids of amino acid component (AA) can be chosen
independently from each other. For example if in the polymeric
carrier two or more (AA) components are present they can be the
same or can be different from each other.
[0135] The amino acid component (AA) may contain or may be flanked
(e.g. terminally) by a --SH containing moiety, which allows
introducing this component (AA) via a disulfide bond into the
polymeric carrier as defined herein. In the specific case that the
--SH containing moiety represents a cysteine, the amino acid
component (AA) may also be read as -Cys-(AA)-Cys-wherein Cys
represents Cysteine and provides for the necessary --SH-moiety for
a disulfide bond. The --SH containing moiety may be also introduced
into amino acid component (AA) using any of modifications or
reactions as shown above for the cationic component or any of its
components.
[0136] Furthermore, the amino acid component (AA) may be provided
with two --SH-moieties (or even more), e.g. in a form represented
by formula HS-(AA)-SH to allow binding to two functionalities via
disulfide bonds, e.g. if the amino acid component (AA) is used as a
linker between two further components (e.g. as a linker between two
cationic polymers). In this case, one --SH moiety is preferably
protected in a first step using a protecting group as known in the
art, leading to an amino acid component (AA) of formula
HS-(AA)-S-protecting group. Then, the amino acid component (AA) may
be bound to a further component of the polymeric carrier, to form a
first disulfide bond via the non-protected --SH moiety. The
protected --SH-moiety is then typically deprotected and bound to a
further free --SH-moiety of a further component of the polymeric
carrier to form a second disulfide bond.
[0137] Alternatively, the amino acid component (AA) may be provided
with other functionalities as already described above for the other
components of the polymeric carrier, which allow binding of the
amino acid component (AA) to any of components of the polymeric
carrier.
[0138] In embodiments, wherein the amino acid component (AA) is
linked via disulfide bonds to the polymeric carrier, it is
preferred that the disulfide linkage is not realized via a
crosslinker, such as a 3,6-Dioxa-1,8-octanedithiol (DODT)
crosslinker.
[0139] Thus, the amino acid component (AA) may be bound to further
components of the polymeric carrier with or without using a
disulfide linkage. Binding without using a disulfide linkage may be
accomplished by any of the reactions described above, preferably by
binding the amino acid component (AA) to the other component of the
polymeric carrier using an amid-chemistry as defined herein. If
desired or necessary, the other terminus of the amino acid
component (AA), e.g. the N- or C-terminus, may be used to couple
another component, e.g. a ligand L. For this purpose, the other
terminus of the amino acid component (AA) preferably comprises or
is modified to comprise a further functionality, e.g. an
alkyn-species (see above), which may be used to add the other
component via e.g. click-chemistry. If the ligand is bound via an
acid-labile bond, the bond is preferably cleaved off in the
endosome and the polymeric carrier presents amino acid component
(AA) at its surface.
[0140] The amino acid component (AA) may occur as a further
component of the polymeric carrier as defined above, e.g. as a
linker between cationic components e.g. as a linker between one
cationic peptide and a further cationic peptide, as a linker
between one cationic polymer and a further cationic polymer, as a
linker between one cationic peptide and a cationic polymer, all
preferably as defined herein, or as an additional component of the
polymeric carrier, e.g. by binding the amino acid component (AA) to
the polymeric carrier or a component thereof, e.g. via side chains,
SH-moieties or via further moieties as defined herein, wherein the
amino acid component (AA) is preferably accordingly modified.
[0141] According to a further and particularly preferred
alternative, the amino acid component (AA), may be used to modify
the polymeric carrier, particularly the content of cationic
components in the polymeric carrier as defined above.
[0142] In this context it is preferable, that the content of
cationic components in the polymeric carrier is at least 10%, 20%,
or 30%, preferably at least 40%, more preferably at least 50%, 60%
or 70%, but also preferably at least 80%, 90%, or even 95%, 96%,
97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the
range of about 30% to 100%, more preferably in the range of about
50% to 100%, even preferably in the range of about 70% to 100%,
e.g. 70, 80, 90 or 100%, or in a range formed by any two of the
afore mentioned values, provided, that the content of all
components in the polymeric carrier is 100%.
[0143] In the context of the present invention, the amino acid
component (AA) may be selected from the following alternatives.
[0144] According to a first alternative, the amino acid component
(AA) may be an aromatic amino acid component (AA). The
incorporation of aromatic amino acids or sequences as amino
aromatic acid component (AA) into the polymeric carrier of the
present invention enables a different (second) binding of the
polymeric carrier to the nucleic acid due to interactions of the
aromatic amino acids with the bases of the nucleic acid cargo in
contrast to the binding thereof by cationic charged sequences of
the polymeric carrier molecule to the phosphate backbone. This
interaction may occur e.g. by intercalations or by minor or major
groove binding. This kind of interaction is not prone to
decompaction by anionic complexing partners (e.g. Heparin,
Hyaluronic acids) which are found mainly in the extracellular
matrix in vivo and is also less susceptible to salt effects.
[0145] For this purpose, the amino acids in the aromatic amino acid
component (AA) may be selected from either the same or different
aromatic amino acids e.g. selected from Tip, Tyr, or Phe.
[0146] Additionally, the aromatic amino acid component (AA) may
contain or may be flanked by a --SH containing moiety, which allows
introducing this component via a disulfide bond as a further part
of the polymeric carrier as defined above, e.g. as a linker. Such a
--SH containing moiety may be any moiety as defined herein suitable
to couple one component as defined herein to a further component as
defined herein. As an example, such a --SH containing moiety may be
a cysteine.
[0147] Additionally, the aromatic amino acid component (AA) may
contain or represent at least one proline, which may serve as a
structure breaker of longer sequences of Trp, Tyr and Phe in the
aromatic amino acid component (AA), preferably two, three or more
prolines.
[0148] According to a second alternative, the amino acid component
(AA) may be a hydrophilic (and preferably non charged polar) amino
acid component (AA). The incorporation of hydrophilic (and
preferably non charged polar) amino acids or sequences as amino
hydrophilic (and preferably non charged polar) acid component (AA)
into the polymeric carrier of the present invention enables a more
flexible binding to the nucleic acid cargo. This leads to a more
effective compaction of the nucleic acid cargo and hence to a
better protection against nucleases and unwanted decompaction. It
also allows provision of a (long) polymeric carrier which exhibits
a reduced cationic charge over the entire carrier and in this
context to better adjusted binding properties, if desired or
necessary.
[0149] For this purpose, the amino acids in the hydrophilic (and
preferably non charged polar) amino acid component (AA) may be
selected from either the same or different hydrophilic (and
preferably non charged polar) amino acids e.g. selected from Thr,
Ser, Asn or Gln.
[0150] Additionally, the hydrophilic (and preferably non-charged
polar) amino acid component (AA) may contain or may be flanked by a
--SH containing moiety, which allows introducing this component via
a disulfide bond as a further part of generic formula (I) above,
e.g. as a linker. Such a --SH containing moiety may be any moiety
as defined herein suitable to couple one component as defined
herein to a further component as defined herein. As an example,
such a --SH containing moiety may be a cysteine.
[0151] Additionally, the hydrophilic (and preferably non-charged
polar) amino acid component (AA) may contain at least one proline,
which may serve as a structure breaker of longer sequences of Ser,
Thr and Asn in the hydrophilic (and preferably non charged polar)
amino acid component (AA), preferably two, three or more
prolines.
[0152] According to a third alternative, the amino acid component
(AA) may be a lipohilic amino acid component (AA). The
incorporation of lipohilic amino acids or sequences as amino
lipohilic acid component (AA) into the polymeric carrier of the
present invention enables a stronger compaction of the nucleic acid
cargo and/or the polymeric carrier and its nucleic acid cargo when
forming a complex. This is particularly due to interactions of one
or more polymer strands of the polymeric carrier, particularly of
lipophilic sections of lipohilic amino acid component (AA) and the
nucleic acid cargo. This interaction will preferably add an
additional stability to the complex between the polymeric carrier
and its nucleic acid cargo. This stabilization may somehow be
compared to a sort of non covalent crosslinking between different
polymer strands. Especially in aqueous environment this interaction
is typically strong and provides a significant effect.
[0153] For this purpose, the amino acids in the lipophilic amino
acid component (AA) may be selected from either the same or
different lipophilic amino acids e.g. selected from Leu, Val, Ile,
Ala, Met.
[0154] Additionally, the lipophilic amino acid component (AA) may
contain or may be flanked by a --SH containing moiety, which allows
introducing this component via a disulfide bond as a further part
of the polymeric carrier above, e.g. as a linker. Such a --SH
containing moiety may be any moiety as defined herein suitable to
couple one component as defined herein to a further component as
defined herein. As an example, such a --SH containing moiety may be
a cysteine.
[0155] Additionally, the lipophilic amino acid component (AA) may
contain at least one proline, which may serve as a structure
breaker of longer sequences of Leu, Val, Ile, Ala and Met in the
lipophilic amino acid component (AA), preferably two, three or more
prolines.
[0156] Finally, according to a fourth alternative, the amino acid
component (AA) may be a weak basic amino acid component (AA). The
incorporation of weak basic amino acids or sequences as weak basic
amino acid component (AA) into the polymeric carrier of the present
invention may serve as a proton sponge and facilitates endosomal
escape (also called endosomal release) (proton sponge effect).
Incorporation of such a weak basic amino acid component (AA)
preferably enhances transfection efficiency.
[0157] For this purpose, the amino acids in the weak basic amino
acid component (AA) may be selected from either the same or
different weak amino acids e.g. selected from histidine or
aspartate (aspartic acid).
[0158] Additionally, the weak basic amino acid component (AA) may
contain or may be flanked by a --SH containing moiety, which allows
introducing this component via a disulfide bond as a further part
of generic formula (I) above, e.g. as a linker. Such a --SH
containing moiety may be any moiety as defined herein suitable to
couple one component as defined herein to a further component as
defined herein.
[0159] Additionally, the weak basic amino acid component (AA) may
contain at least one proline, which may serve as a structure
breaker of longer sequences of histidine or aspartate (aspartic
acid) in the weak basic amino acid component (AA), preferably two,
three or more prolines.
[0160] According to a fifth alternative, the amino acid component
(AA) may be a signal peptide or signal sequence, a localization
signal or sequence, a nuclear localization signal or sequence
(NLS), an antibody, a cell penetrating peptide, (e.g. TAT), etc.
Preferably such an amino acid component (AA) is bound to the
polymeric carrier or to another component of the polymeric carrier
via a (reversible) disulfide bond. In this context, the signal
peptide or signal sequence, a localization signal or sequence, a
nuclear localization signal or sequence (NLS), an antibody, a cell
penetrating peptide, (e.g. TAT), etc. additionally comprises at
least one --SH-moiety. In this context, a signal peptide, a
localization signal or sequence or a nuclear localization signal or
sequence (NLS), may be used to direct the inventive polymeric
carrier cargo complex to specific target cells (e.g. hepatocytes or
antigen-presenting cells) and preferably allows a translocalization
of the polymeric carrier to a specific target, e.g. into the cell,
into the nucleus, into the endosomal compartment, sequences for the
mitochondrial matrix, localisation sequences for the plasma
membrane, localisation sequences for the Golgi apparatus, the
nucleus, the cytoplasm and the cytosceleton, etc. Such signal
peptide, a localization signal or sequence or a nuclear
localization signal may be used for the transport of any of the
herein defined nucleic acids, preferably an RNA or a DNA, more
preferably an shRNA or a pDNA, e.g. into the nucleus. Without being
limited thereto, such a signal peptide, a localization signal or
sequence or a nuclear localization signal may comprise, e.g.,
localisation sequences for the endoplasmic reticulum. Examples of
secretory signal peptides as defined herein include, without being
limited thereto, signal peptides of classical or non-classical
MHC-molecules (e.g. signal sequences of MHC I and II molecules,
e.g. of the MHC class I molecule HLA-A*0201), signal peptides of
cytokines or immunoglobulins as defined herein, signal peptides of
the invariant chain of immunoglobulins or antibodies as defined
herein, signal peptides of Lamp1, Tapasin, Erp57, Calreticulin,
Calnexin, and further membrane associated proteins or of proteins
associated with the endoplasmic reticulum (ER) or the
endosomal-lysosomal compartment. Particularly preferably, signal
peptides of MHC class I molecule HLA-A*0201 may be used according
to the present invention. Such an additional component may be bound
e.g. to a cationic polymer or to any other component of the
polymeric carrier as defined herein. Preferably this signal
peptide, localization signal or sequence or nuclear localization
signal or sequence (NLS), is bound to the polymeric carrier or to
another component of the polymeric carrier via a (reversible)
disulfide bond. For this purpose the (AA) component additionally
comprises at least one --SH moiety as defined herein. The binding
to any of components of the polymeric carrier may also be
accomplished using an acid-labile bond, preferably via a side chain
of any of components of the polymeric carrier, which allows to
detach or release the additional component at lower pH-values, e.g.
at physiological pH-values as defined herein.
[0161] Additionally, according to another alternative, the amino
acid component (AA) may be a functional peptide or protein, which
may modulate the functionality of the polymeric carrier
accordingly. Such functional peptides or proteins as the amino acid
component (AA) preferably comprise any peptides or proteins as
defined herein, e.g. as defined below as therapeutically active
proteins. According to one alternative, such further functional
peptides or proteins may comprise so called cell penetrating
peptides (CPPs) or cationic peptides for transportation.
Particularly preferred are CPPs, which induce a pH-mediated
conformational change in the endosome and lead to an improved
release of the polymeric carrier (in complex with a nucleic acid)
from the endosome by insertion into the lipid layer of the
liposome. These cell penetrating peptides (CPPs) or cationic
peptides for transportation, may include, without being limited
thereto protamine, nucleoline, spermine or spermidine, oligo- or
poly-L-lysine (PLL), basic polypeptides, oligo or poly-arginine,
cell penetrating peptides (CPPs), chimeric CPPs, such as
Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat
(HIV), Tat-derived peptides, members of the penetratin family, e.g.
Penetratin, Antennapedia-derived peptides (particularly from
Drosophila antennapedia), pAntp, plsl, etc., antimicrobial-derived
CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, ha-derived
peptides, SAP, MAP, KALA, PpTG20, Loligomere, FGF, Lactoferrin,
histones, VP22 derived or analog peptides, HSV, VP22 (Herpes
simplex), MAP, KALA or protein transduction domains (PTDs, PpT620,
prolin-rich peptides, arginine-rich peptides, lysine-rich peptides,
Pep-1, L-oligomers, Calcitonin peptide(s), etc. Such an amino acid
component (AA) may also be bound to any component of the polymeric
carrier as defined herein. Preferably it is bound to the polymeric
carrier or to another component of the polymeric carrier via a
(reversible) disulfide bond. For the above purpose, the amino acid
component (AA) preferably comprises at least one --SH moiety as
defined herein. The binding to any of the components of the
polymeric carrier may also be accomplished using an SH-moiety or an
acid-labile bond, preferably via a side chain of any of components
of the polymeric carrier which allows to detach or release the
additional component at lower pH-values, e.g. at physiological
pH-values as defined herein.
[0162] According to a last alternative, the amino acid component
(AA) may consist of any peptide or protein which can execute any
favourable function in the cell. Particularly preferred are
peptides or proteins selected from therapeutically active proteins
or peptides, from antigens, e.g. tumour antigens, pathogenic
antigens (animal antigens, viral antigens, protozoan antigens,
bacterial antigens, allergic antigens), autoimmune antigens, or
further antigens, from allergens, from antibodies, from
immunostimulatory proteins or peptides, from antigen-specific
T-cell receptors, or from any other protein or peptide suitable for
a specific (therapeutic) application as defined below for coding
nucleic acids. Particularly preferred are peptide epitopes from the
at least one antigen (an antigen from a pathogen associated with
infectious disease; an antigen associated with allergy or allergic
disease; an antigen associated with autoimmune disease; or an
antigen associated with a cancer or tumour disease) as defined
herein.
[0163] In the event, the amino acid component (AA) is covalently
attached to the polymeric carrier cargo complex, in particular to
the polymeric carrier, the amino acid component (AA) is preferably
not ovalbumin or a fragment of ovalbumin. Preferably, the amino
acid component is not ovalbumin or a fragment of ovalbumin.
[0164] Due to the peptidic nature of the amino acid component also
the definition of peptide, protein, or fragment, variant and
derivative thereof applies accordingly and are explicitly
encompassed.
[0165] Furthermore, said (AA) components may be prepared by all
methods known to a person of ordinary skill or by recombinant
peptide or protein production or by peptide synthesis as described
herein.
[0166] The polymeric carrier may comprise at least one of the above
mentioned cationic or polycationic peptides, proteins or polymers
or further components, e.g. (AA), wherein any of the above
alternatives may be combined with each other, and may be formed by
polymerizing same in a polymerization condensation reaction via
their --SH-moieties.
[0167] According to another embodiment the polymeric carrier of the
polymeric carrier cargo complex or single components thereof, e.g.
of the above mentioned cationic or polycationic peptides, proteins
or polymers or further components, e.g. (AA), may be further
modified with a ligand, preferably a carbohydrate, more preferably
a sugar, even more preferably mannose. Preferably this ligand is
bound to the polymeric carrier or to a component of the polymeric
carrier via a (reversible) disulfide bond or via Michael addition.
In the case that the ligand is bound by a disulfide bond the ligand
additionally comprises at least one --SH-moiety. These ligands may
be used to direct the polymeric carrier cargo complex to specific
target cells (e.g. hepatocytes or antigen-presenting cells). In
this context mannose is particular preferred as ligand in the case
that dendritic cells are the target especially for vaccination or
adjuvant purposes.
[0168] According to a further embodiment of the invention, the
polymeric carrier cargo complex may comprise (AA) components as
defined above which do not comprise --SH moieties. These (AA)
components can be added before or during the complexation reaction
of the at least one nucleic acid molecule. Thereby, the (AA)
component(s) is/are (non-covalently) incorporated into the
polymeric carrier cargo complex without inclusion of the (AA)
component(s) in the polymeric carrier itself by (covalent)
polymerization.
[0169] According to one specific embodiment, the entire polymeric
carrier cargo complex may be formed by a polymerization
condensation (of at least one) of the above mentioned cationic or
polycationic peptides, proteins or polymers or further components,
e.g. (AA), via their--SH-moieties in a first step and complexing
the nucleic acid to such a polymeric carrier in a second step. The
polymeric carrier may thus contain a number of at least one or even
more of the same or different of the above defined cationic or
polycationic peptides, proteins or polymers or further components,
e.g. (AA), the number preferably determined by the above range.
[0170] According to one alternative specific embodiment, the
polymeric carrier cargo complex is formed by carrying out the
polymerization condensation of at least one of the above mentioned
cationic or polycationic peptides, proteins or polymers or further
components, e.g. (AA), via their --SH-moieties simultaneously to
complexing the nucleic acid cargo to the (in situ prepared)
polymeric carrier. Likewise, the polymeric carrier may thus also
here contain a number of at least one or even more of the same or
different of the above defined cationic or polycationic peptides,
proteins or polymers or further components, e.g. (AA), the number
preferably determined by the above range.
[0171] The polymeric carrier cargo complex additionally comprises
as a cargo at least one nucleic acid (molecule). In the context of
the present invention, such a nucleic acid molecule may be any
suitable nucleic acid, selected e.g. from any (single-stranded or
double-stranded) DNA, preferably, without being limited thereto,
e.g. genomic DNA, single-stranded DNA molecules, double-stranded
DNA molecules, coding DNA, DNA primers, DNA probes,
immunostimulatory DNA, a (short) DNA oligonucleotide ((short)
oligodesoxyribonucleotides), or may be selected e.g. from any PNA
(peptide nucleic acid) or may be selected e.g. from any
(single-stranded or double-stranded) RNA, preferably, without being
limited thereto, a (short) RNA oligonucleotide ((short)
oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), an
immunostimulatory RNA, a small interfering RNA (siRNA), an
antisense RNA, a micro RNA, a small nuclear RNA (snRNA), a
small-hairpin (sh) RNA or riboswitches, ribozymes or aptamers; etc.
The nucleic acid molecule of the polymeric carrier cargo complex
may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a
messenger RNA (mRNA), or a viral RNA (vRNA). Preferably, the
nucleic acid molecule of the polymeric carrier cargo complex is an
RNA. More preferably, the nucleic acid molecule of the polymeric
carrier cargo complex is a (linear) single-stranded RNA, even more
preferably an mRNA or an immunostimulatory RNA. In the context of
the present invention, an mRNA is typically an RNA, which is
composed of several structural elements, e.g. an optional 5'-CAP
structure, an optional 5'-UTR region, an upstream positioned
ribosomal binding site followed by a coding region, an optional
3'-UTR region, which may be followed by a poly-A tail (and/or a
poly-C-tail). An mRNA may occur as a mono-, di-, or even
multicistronic RNA, i.e. a RNA which carries the coding sequences
of one, two or more proteins or peptides. Such coding sequences in
di-, or even multicistronic mRNA may be separated by at least one
IRES sequence, e.g. as defined herein.
[0172] Furthermore, the nucleic acid of the polymeric carrier cargo
complex may be a single- or a double-stranded nucleic acid
(molecule) (which may also be regarded as a nucleic acid (molecule)
due to non-covalent association of two single-stranded nucleic
acid(s) (molecules)) or a partially double-stranded or partially
single stranded nucleic acid, which are at least partially self
complementary (both of these partially double-stranded or partially
single stranded nucleic acid molecules are typically formed by a
longer and a shorter single-stranded nucleic acid molecule or by
two single stranded nucleic acid molecules, which are about equal
in length, wherein one single-stranded nucleic acid molecule is in
part complementary to the other single-stranded nucleic acid
molecule and both thus form a double-stranded nucleic acid molecule
in this region, i.e. a partially double-stranded or partially
single stranded nucleic acid (molecule). Preferably, the nucleic
acid (molecule) may be a single-stranded nucleic acid molecule.
Furthermore, the nucleic acid (molecule) may be a circular or
linear nucleic acid molecule, preferably a linear nucleic acid
molecule.
[0173] According to one alternative, the nucleic acid molecule of
the polymeric carrier cargo complex may be a coding nucleic acid,
e.g. a DNA or RNA. Such a coding DNA or RNA may be any DNA or RNA
as defined herein. Preferably, such a coding DNA or RNA may be a
single- or a double-stranded DNA or RNA, more preferably a
single-stranded DNA or RNA, and/or a circular or linear DNA or RNA,
more preferably a linear DNA or RNA. Even more preferably, the
coding DNA or RNA may be a (linear) single-stranded DNA or RNA.
Most preferably, the nucleic acid molecule according to the present
invention may be a ((linear) single-stranded) messenger RNA (mRNA).
Such an mRNA may occur as a mono-, di-, or even multicistronic RNA,
i.e. an RNA which carries the coding sequences of one, two or more
proteins or peptides. Such coding sequences in di-, or even
multicistronic mRNA may be separated by at least one IRES sequence,
e.g. as defined herein.
[0174] Coding Nucleic Acids:
[0175] The nucleic acid molecule of the polymeric carrier cargo
complex may encode a protein or a peptide, which may be selected,
without being restricted thereto, e.g. from therapeutically active
proteins or peptides, including adjuvant proteins, from antigens,
e.g. tumour antigens, pathogenic antigens (e.g. selected, from
animal antigens, from viral antigens, from protozoal antigens, from
bacterial antigens), allergenic antigens, autoimmune antigens, or
further antigens, from allergens, from antibodies, from
immunostimulatory proteins or peptides, from antigen-specific
T-cell receptors, or from any other protein or peptide suitable for
a specific (therapeutic) application, wherein the coding nucleic
acid may be transported into a cell, a tissue or an organism and
the protein may be expressed subsequently in this cell, tissue or
organism. In this context, the coding nucleic acid may additionally
code for a signal peptide as defined herein.
a) Therapeutically Active Proteins
[0176] In the context of the present invention, therapeutically
active proteins or peptides may be encoded by the nucleic acid
molecule of the herein defined polymeric carrier cargo complex.
Therapeutically active proteins are defined herein as proteins
which have an effect on healing, prevent prophylactically or treat
therapeutically a disease, preferably as defined herein, or are
proteins of which an individual is in need of. These may be
selected from any naturally or synthetically designed occurring
recombinant or isolated protein known to a skilled person from the
prior art. Without being restricted thereto therapeutically active
proteins may comprise proteins, capable of stimulating or
inhibiting the signal transduction in the cell, e.g. cytokines,
lymphokines, monokines, growth factors, receptors, signal
transduction molecules, transcription factors, etc; anticoagulants;
antithrombins; antiallergic proteins; apoptotic factors or
apoptosis related proteins, therapeutic active enzymes and any
protein or peptide connected with any acquired disease or any
hereditary disease or favourable for the treatment of any acquired
disease or any hereditary disease.
[0177] A therapeutically active protein, which may be encoded by
the nucleic acid molecule of the herein defined polymeric carrier
cargo complex, may also be an adjuvant protein. In this context, an
adjuvant protein is preferably to be understood as any protein,
which is capable to elicit an innate immune response as defined
herein. Preferably, such an innate immune response comprises
activation of a pattern recognition receptor, such as e.g. a
receptor selected from the Toll-like receptor (TLR) family,
including e.g. a Toll like receptor selected from human TLR1 to
TLR10 or from murine Toll like receptors TLR1 to TLR13. More
preferably, the adjuvant protein is selected from human adjuvant
proteins or from pathogenic adjuvant proteins, selected from the
group consisting of, without being limited thereto, bacterial
proteins, protozoan proteins, viral proteins, or fungal proteins,
animal proteins, in particular from bacterial adjuvant proteins. In
addition, nucleic acids encoding human proteins involved in
adjuvant effects (e.g. ligands of pattern recognition receptors,
pattern recognition receptors, proteins of the signal transduction
pathways, transcription factors or cytokines) may be used as
well.
b) Antigens
[0178] The nucleic acid molecule of the herein defined polymeric
carrier cargo complex may alternatively encode an antigen. In the
context of the present invention, antigens as encoded by the
nucleic acid molecule of the herein defined polymeric carrier cargo
complex typically comprise any antigen, antigenic epitope or
antigenic peptide, falling under the above definition, more
preferably protein and peptide antigens, e.g. tumour antigens,
allergenic antigens, auto-immune self-antigens, pathogenic
antigens, etc. In particular antigens as encoded by the nucleic
acid molecule of the herein defined polymeric carrier cargo complex
may be antigens generated outside the cell, more typically antigens
not derived from the host organism (e.g. a human) itself (i.e.
non-self antigens) but rather derived from host cells outside the
host organism, e.g. viral antigens, bacterial antigens, fungal
antigens, protozoological antigens, animal antigens, allergenic
antigens, etc. Allergenic antigens (allergy antigens) are typically
antigens, which cause an allergy in a human and may be derived from
either a human or other sources. Additionally, antigens as encoded
by the nucleic acid molecule of the herein defined polymeric
carrier cargo complex may be furthermore antigens generated inside
the cell, the tissue or the body. Such antigens include antigens
derived from the host organism (e.g. a human) itself, e.g. tumour
antigens, self-antigens or auto-antigens, such as auto-immune
self-antigens, etc., but also (non-self) antigens as defined
herein, which have been originally been derived from host cells
outside the host organism, but which are fragmented or degraded
inside the body, tissue or cell, e.g. by (protease) degradation,
metabolism, etc. In this context, an antigen as encoded by the
nucleic acid cargo comprised in the polymeric carrier cargo complex
is defined as described below for the at least one antigen, the
second ingredient of the inventive pharmaceutical composition.
[0179] Particularly preferred in this context is, that the antigen
or a fragment, variant and/or derivative thereof encoded by the
nucleic acid cargo is the same antigen as the at least one antigen
as defined herein as comprised in the inventive pharmaceutical
composition as second ingredient. In alternative embodiments
however, the antigen or a fragment, variant and/or derivative
thereof encoded by the nucleic acid cargo is a different antigen as
the at least one antigen as defined herein as comprised in the
inventive pharmaceutical composition as second ingredient. In the
specific case that an antigen is encoded by the nucleic acid cargo,
the nucleic acid molecule together with the polymeric carrier
serves as adjuvant or imunostimulating agent to induce an
unspecific innate immune response, whereas the encoded protein or
peptide antigen which is expressed by the nucleic acid cargo serves
as antigen to induce an antigen-specific adaptive immune
response.
c) Antibodies
[0180] According to a further alternative, the nucleic acid
molecule of the herein defined polymeric carrier cargo complex may
encode an antibody or an antibody fragment. According to the
present invention, such an antibody may be selected from any
antibody, e.g. any recombinantly produced or naturally occurring
antibodies, known in the art, in particular antibodies suitable for
therapeutic, diagnostic or scientific purposes, or antibodies which
have been identified in relation to specific cancer diseases.
Herein, the term "antibody" is used in its broadest sense and
specifically covers monoclonal and polyclonal antibodies (including
agonist, antagonist, and blocking or neutralizing antibodies) and
antibody species with polyepitopic specificity. According to the
invention, the term "antibody" typically comprises any antibody
known in the art (e.g. IgM, IgD, IgG, IgA and IgE antibodies), such
as naturally occurring antibodies, antibodies generated by
immunization in a host organism, antibodies which were isolated and
identified from naturally occurring antibodies or antibodies
generated by immunization in a host organism and recombinantly
produced by biomolecular methods known in the art, as well as
chimeric antibodies, human antibodies, humanized antibodies,
bispecific antibodies, intrabodies, i.e. antibodies expressed in
cells and optionally localized in specific cell compartments, and
fragments and variants of the aforementioned antibodies. In
general, an antibody consists of a light chain and a heavy chain
both having variable and constant domains. The light chain consists
of an N-terminal variable domain, V.sub.L, and a C-terminal
constant domain, C.sub.L. In contrast, the heavy chain of the IgG
antibody, for example, is comprised of an N-terminal variable
domain, V.sub.H, and three constant domains, C.sub.H1, C.sub.H2 and
C.sub.H3.
[0181] In the context of the present invention, antibodies as
encoded by the nucleic acid molecule of the herein defined
polymeric carrier cargo complex may preferably comprise full-length
antibodies, i.e. antibodies composed of the full heavy and full
light chains, as described above. However, derivatives of
antibodies such as antibody fragments, variants or adducts may also
be encoded by the nucleic acid molecule of the herein defined
polymeric carrier cargo complex. Antibody fragments are preferably
selected from Fab, Fab', F(ab').sub.2, Fc, Facb, pFc', Fd and Fv
fragments of the aforementioned (full-length) antibodies. In
general, antibody fragments are known in the art. For example, a
Fab ("fragment, antigen binding") fragment is composed of one
constant and one variable domain of each of the heavy and the light
chain. The two variable domains bind the epitope on specific
antigens. The two chains are connected via a disulfide linkage. A
scFv ("single chain variable fragment") fragment, for example,
typically consists of the variable domains of the light and heavy
chains. The domains are linked by an artificial linkage, in general
a polypeptide linkage such as a peptide composed of 15-25 glycine,
proline and/or serine residues.
[0182] In the present context it is preferable that the different
chains of the antibody or antibody fragment are encoded by a
multicistronic nucleic acid molecule. Alternatively, the different
strains of the antibody or antibody fragment are encoded by several
monocistronic nucleic acid(s) (sequences).
siRNA:
[0183] According to a further alternative, the nucleic acid
molecule of the herein defined polymeric carrier cargo complex may
be in the form of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is
of interest particularly in connection with the phenomenon of RNA
interference. The in vitro technique of RNA interference (RNAi) is
based on double-stranded RNA molecules (dsRNA), which trigger the
sequence-specific suppression of gene expression (Zamore (2001)
Nat. Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490:
Hannon (2002) Nature 41: 244-251). In the transfection of mammalian
cells with long dsRNA, the activation of protein kinase R and
RnaseL brings about unspecific effects, such as, for example, an
interferon response (Stark et al. (1998) Annu. Rev. Biochem. 67:
227-264; He and Katze (2002) Viral Immunol. 15: 95-119). These
unspecific effects are avoided when shorter, for example 21- to
23-mer, so-called siRNA (small interfering RNA), is used, because
unspecific effects are not triggered by siRNA that is shorter than
30 bp (Elbashir et al. (2001) Nature 411: 494-498).
[0184] The nucleic acid molecule of the herein defined polymeric
carrier cargo complex may thus be a double-stranded RNA (dsRNA)
having a length of from 17 to 29, preferably from 19 to 25, and
preferably is at least 90%, more preferably 95% and especially 100%
(of the nucleotides of a dsRNA) complementary to a section of the
nucleic acid molecule of a (therapeutically relevant) protein or
antigen described (as active ingredient) hereinbefore or of any
further protein as described herein, either a coding or a
non-coding section, preferably a coding section. Such a (section of
the) nucleic acid molecule may be termed herein a "target sequence"
and may be any nucleic acid molecule as defined herein, preferably
a genomic DNA, a cDNA, a RNA, e.g. an mRNA, etc. 90% complementary
means that with a length of a dsRNA described herein of, for
example, 20 nucleotides, the dsRNA contains not more than 2
nucleotides showing no complementarity with the corresponding
section of the target sequence. The sequence of the double-stranded
RNA used according to the invention is, however, preferably wholly
complementary in its general structure with a section of the target
sequence. In this context the nucleic acid molecule of the
polymeric carrier cargo complex may be a dsRNA having the general
structure 5-(N.sub.17-29)-3', preferably having the general
structure 5'-(N.sub.19-25)-3', more preferably having the general
structure 5'-(N.sub.19-24)-3', or yet more preferably having the
general structure 5'-(N.sub.21-23)-3 wherein for each general
structure each N is a (preferably different) nucleotide of a
section of the target sequence, preferably being selected from a
continuous number of 17 to 29 nucleotides of a section of the
target sequence, and being present in the general structure
5'-(N.sub.17-29)-3' in their natural order. In principle, all the
sections having a length of from 17 to 29, preferably from 19 to
25, base pairs that occur in the target sequence can serve for
preparation of a dsRNA as defined herein. Equally, dsRNAs used as
nucleic acid molecule of the polymeric carrier cargo complex can
also be directed against nucleotide sequences of a (therapeutically
relevant) protein or antigen described (as active ingredient)
hereinbefore that do not lie in the coding region, in particular in
the 5' non-coding region of the target sequence, for example,
therefore, against non-coding regions of the target sequence having
a regulatory function. The target sequence of the dsRNA used as
nucleic acid molecule of the polymeric carrier cargo complex can
therefore lie in the translated and untranslated region of the
target sequence and/or in the region of the control elements of a
protein or antigen described hereinbefore. The target sequence for
a dsRNA used as the nucleic acid molecule of the polymeric carrier
cargo complex can also lie in the overlapping region of
untranslated and translated sequence; in particular, the target
sequence can comprise at least one nucleotide upstream of the start
triplet of the coding region, e.g. of a genomic DNA, a cDNA, a RNA,
or an mRNA, etc.
Immunostimulatory Nucleic Acids:
a) Immunostimulatory CpG Nucleic Acids:
[0185] According to another alternative, the nucleic acid molecule
of the herein defined polymeric carrier cargo complex may be in the
form of a(n) (immunostimulatory) CpG nucleic acid, in particular
CpG-RNA or CpG-DNA, which preferably induces an innate immune
response. A CpG-RNA or CpG-DNA used according to the invention can
be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded
CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a
double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid used
according to the invention is preferably in the form of CpG-RNA,
more preferably in the form of single-stranded CpG-RNA (ss
CpG-RNA). Also preferably, such CpG nucleic acids have a length as
described above. Preferably the CpG motifs are unmethylated. In a
preferred embodiment, the CpG nucleic acid is not a CpG-DNA
consisting of the sequence 5'TCCATGACGTTCCTGACGTT-3' (SEQ ID NO:
123), in particular if the protein or peptide antigen is ovalbumin
or a fragment of ovalbumin. In a further preferred embodiment, the
CpG nucleic acid is not a sequence comprising SEQ ID NO: 123.
Preferably, the CpG nucleic acid is not a CpG-DNA. In some
embodiments of the present invention, the polymeric carrier cargo
complex does not comprise a CpG-DNA, preferably does not comprise a
CpG nucleic acid. In some embodiments of the present invention, the
pharmaceutical composition does not comprise a CpG-DNA, preferably
does not comprise a CpG nucleic acid.
b) Immunostimulatory RNA (isRNA):
[0186] Likewise, according to a further alternative, the
(immunostimulatory) nucleic acid molecule of the polymeric carrier
cargo complex may be in the form of an immunostimulatory RNA
(isRNA), which preferably elicits an innate immune response. Such
an immunostimulatory RNA may be any (double-stranded or
single-stranded) RNA, e.g. a coding RNA, as defined herein.
Preferably, the immunostimulatory RNA may be a single-stranded, a
double-stranded or a partially double-stranded RNA, more preferably
a single-stranded RNA, and/or a circular or linear RNA, more
preferably a linear RNA. More preferably, the immunostimulatory RNA
may be a (linear) single-stranded RNA. Even more preferably, the
immunostimulatory RNA may be a (long) (linear) single-stranded)
non-coding RNA. In this context it is particular preferred that the
isRNA carries a triphosphate at its 5'-end which is the case for in
vitro transcribed RNA. An immunostimulatory RNA may also occur as a
short RNA oligonucleotide as defined herein. An immunostimulatory
RNA as used herein may furthermore be selected from any class of
RNA molecules, found in nature or being prepared synthetically, and
which can induce an innate immune response and may support an
adaptive immune response induced by an antigen. In this context, an
immune response may occur in various ways. A substantial factor for
a suitable (adaptive) immune response is the stimulation of
different T-cell sub-populations. T-lymphocytes are typically
divided into two sub-populations, the T-helper 1 (Th1) cells and
the T-helper 2 (Th2) cells, with which the immune system is capable
of destroying intracellular (Th1) and extracellular (Th2) pathogens
(e.g. antigens). The two Th cell populations differ in the pattern
of the effector proteins (cytokines) produced by them. Thus, Th1
cells assist the cellular immune response by activation of
macrophages and cytotoxic T-cells. Th2 cells, on the other hand,
promote the humoral immune response by stimulation of 6-cells for
conversion into plasma cells and by formation of antibodies (e.g.
against antigens). The Th1/Th2 ratio is therefore of great
importance in the induction and maintenance of an adaptive immune
response. In connection with the present invention, the Th1/Th2
ratio of the (adaptive) immune response is preferably shifted in
the direction towards the cellular response (Th1 response) and a
cellular immune response is thereby induced. According to one
example, the innate immune system which may support an adaptive
immune response, may be activated by ligands of Toll-like receptors
(TLRs). TLRs are a family of highly conserved pattern recognition
receptor (PRR) polypeptides that recognize pathogen-associated
molecular patterns (PAMPs) and play a critical role in innate
immunity in mammals. Currently at least thirteen family members,
designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4,
TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), have
been identified. Furthermore, a number of specific TLR ligands have
been identified. It was e.g. found that unmethylated bacterial DNA
and synthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi
H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc
NatlAcadSci USA 98, 9237-42). Furthermore, it has been reported
that ligands for certain TLRs include certain nucleic acid
molecules and that certain types of RNA are immunostimulatory in a
sequence-independent or sequence-dependent manner, wherein these
various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or
TLR8, or intracellular receptors such as RIG-I, MDA-5, etc. E.g.
Lipford et al. determined certain G,U-containing
oligoribonucleotides as immunostimulatory by acting via TLR7 and
TLR8 (see WO 03/086280). The immunostimulatory G,U-containing
oligoribonucleotides described by Lipford et al. were believed to
be derivable from RNA sources including ribosomal RNA, transfer
RNA, messenger RNA, and viral RNA.
[0187] The immunostimulatory RNA (isRNA) used as the nucleic acid
molecule of the herein defined polymeric carrier cargo complex may
thus comprise any RNA sequence known to be immunostimulatory,
including, without being limited thereto, RNA sequences
representing and/or encoding ligands of TLRs, preferably selected
from human family members TLR1-TLR10 or murine family members
TLR1-TLR13, more preferably selected from (human) family members
TLR1-TLR10, even more preferably from TLR7 and TLR8, ligands for
intracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see
e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA
helicases: two parallel ways to trigger antiviral responses. Mol.
Cell 22, 561-569), or any other immunostimulatory RNA sequence.
Furthermore, (classes of) immunostimulatory RNA molecules, used as
the nucleic acid molecule of the polymeric carrier cargo complex
may include any other RNA capable of eliciting an innate immune
response. Without being limited thereto, such an immunostimulatory
RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA),
messenger RNA (mRNA), and viral RNA (vRNA), preferably the
immunostimulatory RNA is a non-coding RNA. Such an
immunostimulatory RNA may comprise a length of 1000 to 5000, of 500
to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to
100, of 5 to 50 or of 5 to 30 nucleotides.
[0188] According to a particularly preferred embodiment, such
immunostimulatory nucleic acid sequence is preferably RNA
preferably consisting of or comprising a nucleic acid sequence of
formula (II) or (III):
G.sub.lX.sub.mG.sub.n, (formula (II))
wherein: [0189] G is guanosine, uracil or an analogue of guanosine
or uracil; [0190] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0191]
l is an integer from 1 to 40,
[0192] wherein
[0193] when l=1 G is guanosine or an analogue thereof,
[0194] when l>1 at least 50% of the nucleotides are guanosine or
an analogue thereof; [0195] m is an integer and is at least 3;
[0196] wherein [0197] when m=3.times. is uracil or an analogue
thereof, [0198] when m>3 at least 3 successive uracils or
analogues of uracil occur; [0199] n is an integer from 1 to 40,
[0200] wherein [0201] when n=1 G is guanosine or an analogue
thereof, [0202] when n>1 at least 50% of the nucleotides are
guanosine or an analogue thereof.
[0202] C.sub.lX.sub.mC.sub.n, (formula (III))
wherein: [0203] C is cytosine, uracil or an analogue of cytosine or
uracil; [0204] X is guanosine, uracil, adenosine, thymidine,
cytosine or an analogue of the above-mentioned nucleotides; [0205]
l is an integer from 1 to 40, [0206] wherein [0207] when l=1 C is
cytosine or an analogue thereof, [0208] when l>1 at least 50% of
the nucleotides are cytosine or an analogue thereof; [0209] m is an
integer and is at least 3; [0210] wherein [0211] when m=3.times. is
uracil or an analogue thereof, [0212] when m>3 at least 3
successive uracils or analogues of uracil occur; [0213] n is an
integer from 1 to 40, [0214] wherein [0215] when n=1 C is cytosine
or an analogue thereof, [0216] when n>1 at least 50% of the
nucleotides are cytosine or an analogue thereof.
[0217] The nucleic acids of formula (II) or (III), which may be
used the nucleic acid cargo of the polymeric carrier cargo complex
may be relatively short nucleic acid molecules with a typical
length of approximately from 5 to 100 (but may also be longer than
100 nucleotides for specific embodiments, e.g. up to 200
nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably
a length of approximately from 5 to 70, more preferably a length of
approximately from 8 to 60 and, more preferably a length of
approximately from 15 to 60 nucleotides, more preferably from 20 to
60, most preferably from 30 to 60 nucleotides. If the nucleic acid
of the nucleic acid cargo complex has a maximum length of e.g. 100
nucleotides, m will typically be <=98. The number of nucleotides
G in the nucleic acid of formula (II) is determined by l or n. l
and n, independently of one another, are each an integer from 1 to
40, wherein when l or n=1 G is guanosine or an analogue thereof,
and when l or n>1 at least 50% of the nucleotides are guanosine
or an analogue thereof. For example, without implying any
limitation, when l or n=4 G.sub.l or G.sub.n can be, for example, a
GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.;
when l or n=5 G.sub.l or G.sub.n can be, for example, a GGGUU,
GGUGU, GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG,
GGUGG, GUGGG, UGGGG, or GGGGG, etc.; etc. A nucleotide adjacent to
X.sub.m in the nucleic acid of formula (II) according to the
invention is preferably not a uracil. Similarly, the number of
nucleotides C in the nucleic acid of formula (III) according to the
invention is determined by l or n. l and n, independently of one
another, are each an integer from 1 to 40, wherein when l or n=1 C
is cytosine or an analogue thereof, and when I or n>1 at least
50% of the nucleotides are cytosine or an analogue thereof. For
example, without implying any limitation, when l or n=4, C.sub.l or
C.sub.n can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU,
CCUC, CUCC, UCCC or CCCC, etc.; when l or n=5 C.sub.l or C.sub.n
can be, for example, a CCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC,
UUCCC, CUCUC, CCCCU, CCCUC, CCUCC, CUCCC, UCCCC, or CCCCC, etc.;
etc. A nucleotide adjacent to X.sub.m in the nucleic acid of
formula (V) according to the invention is preferably not a uracil.
Preferably, for formula (II), when l or n>1, at least 60%, 70%,
80%, 90% or even 100% of the nucleotides are guanosine or an
analogue thereof, as defined above. The remaining nucleotides to
100% (when guanosine constitutes less than 100% of the nucleotides)
in the flanking sequences G.sub.l and/or G.sub.n are uracil or an
analogue thereof, as defined hereinbefore. Also preferably, l and
n, independently of one another, are each an integer from 2 to 30,
more preferably an integer from 2 to 20 and yet more preferably an
integer from 2 to 15. The lower limit of l or n can be varied if
necessary and is at least 1, preferably at least 2, more preferably
at least 3, 4, 5, 6, 7, 8, 9 or 10. This definition applies
correspondingly to formula (III).
[0218] According to a particularly preferred embodiment, a nucleic
acid according to any of formulas (II) or (III) above, which may be
used as nucleic acid of the polymeric carrier cargo complex, may be
selected from a sequence consisting of or comprising any of the
following sequences:
TABLE-US-00002 (SEQ ID NO: 15) GGUUUUUUUUUUUUUUUGGG; (SEQ ID NO:
16) GGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 17)
GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 18)
GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQ ID NO: 19)
GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQ ID NO: 20)
GGGGGGGGGUUUGGGGGGGG; (SEQ ID NO: 21) GGGGGGGGUUUUGGGGGGGG; (SEQ ID
NO: 22) GGGGGGGUUUUUUGGGGGGG; (SEQ ID NO: 23) GGGGGGGUUUUUUUGGGGGG;
(SEQ ID NO: 24) GGGGGGUUUUUUUUGGGGGG; (SEQ ID NO: 25)
GGGGGGUUUUUUUUUGGGGG; (SEQ ID NO: 26) GGGGGGUUUUUUUUUUGGGG; (SEQ ID
NO: 27) GGGGGUUUUUUUUUUUGGGG; (SEQ ID NO: 28) GGGGGUUUUUUUUUUUUGGG;
(SEQ ID NO: 29) GGGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 30)
GGGGUUUUUUUUUUUUUUGG; (SEQ ID NO: 31) GGUUUUUUUUUUUUUUUUGG; (SEQ ID
NO: 32) GUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 33)
GGGGGGGGGGUUUGGGGGGGGG; (SEQ ID NO: 34) GGGGGGGGGUUUUGGGGGGGGG;
(SEQ ID NO: 35) GGGGGGGGUUUUUUGGGGGGGG; (SEQ ID NO: 36)
GGGGGGGGUUUUUUUGGGGGGG; (SEQ ID NO: 37) GGGGGGGUUUUUUUUGGGGGGG;
(SEQ ID NO: 38) GGGGGGGUUUUUUUUUGGGGGG; (SEQ ID NO: 39)
GGGGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 40) GGGGGGUUUUUUUUUUUGGGGG;
(SEQ ID NO: 41) GGGGGGUUUUUUUUUUUUGGGG; (SEQ ID NO: 42)
GGGGGUUUUUUUUUUUUUGGGG; (SEQ ID NO: 43) GGGGGUUUUUUUUUUUUUUGGG;
(SEQ ID NO: 44) GGGUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 45)
GGUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 46) GGGGGGGGGGGUUUGGGGGGGGGG;
(SEQ ID NO: 47) GGGGGGGGGGUUUUGGGGGGGGGG; (SEQ ID NO: 48)
GGGGGGGGGUUUUUUGGGGGGGGG; (SEQ ID NO: 49) GGGGGGGGGUUUUUUUGGGGGGGG;
(SEQ ID NO: 50) GGGGGGGGUUUUUUUUGGGGGGGG; (SEQ ID NO: 51)
GGGGGGGGUUUUUUUUUGGGGGGG; (SEQ ID NO: 52) GGGGGGGGUUUUUUUUUUGGGGGG;
(SEQ ID NO: 53) GGGGGGGUUUUUUUUUUUGGGGGG; (SEQ ID NO: 54)
GGGGGGGUUUUUUUUUUUUGGGGG; (SEQ ID NO: 55) GGGGGGUUUUUUUUUUUUUGGGGG;
(SEQ ID NO: 56) GGGGGGUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 57)
GGGGUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 58) GGGUUUUUUUUUUUUUUUUUUGGG;
(SEQ ID NO: 59) GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 60)
GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 61)
GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 62)
GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 63)
GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 64)
GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 65)
GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQ ID NO: 66)
GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQ ID NO: 67)
GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQ ID NO: 68)
GGUUUGG; (SEQ ID NO: 69) GGUUUUGG; (SEQ ID NO: 70) GGUUUUUGG; (SEQ
ID NO: 71) GGUUUUUUGG; (SEQ ID NO: 72) GGUUUUUUUGG; (SEQ ID NO: 73)
GGUUUUUUUUGG; (SEQ ID NO: 74) GGUUUUUUUUUGG; (SEQ ID NO: 75)
GGUUUUUUUUUUGG; (SEQ ID NO: 76) GGUUUUUUUUUUUGG; (SEQ ID NO: 77)
GGUUUUUUUUUUUUGG; (SEQ ID NO: 78) GGUUUUUUUUUUUUUGG; (SEQ ID NO:
79) GGUUUUUUUUUUUUUUGG; (SEQ ID NO: 80) GGUUUUUUUUUUUUUUUGG; (SEQ
ID NO: 81) GGGUUUGGG; (SEQ ID NO: 82) GGGUUUUGGG; (SEQ ID NO: 83)
GGGUUUUUGGG; (SEQ ID NO: 84) GGGUUUUUUGGG; (SEQ ID NO: 85)
GGGUUUUUUUGGG; (SEQ ID NO: 86) GGGUUUUUUUUGGG; (SEQ ID NO: 87)
GGGUUUUUUUUUGGG; (SEQ ID NO: 88) GGGUUUUUUUUUUGGG; (SEQ ID NO: 89)
GGGUUUUUUUUUUUGGG; (SEQ ID NO: 90) GGGUUUUUUUUUUUUGGG; (SEQ ID NO:
91) GGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 92)
GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUU UUUUUUGGG; (SEQ ID
NO: 93) GGGUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 94)
GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGU UUGGG; (short
GU-rich, SEQ ID NO: 95) GGUUUUUUUUUUUUUUUGGG
or
TABLE-US-00003 (SEQ ID NO: 96)
CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCCUUUUUUUUU UUUUUUCCC (SEQ ID
NO: 97) CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCC C (SEQ
ID NO: 98) CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCC
or from a sequence having at least 60%, 70%, 80%, 90%, or even 95%
sequence identity with any of these sequences.
[0219] According to a further particularly preferred embodiment,
such immunostimulatory nucleic acid sequences, particularly isRNA,
consist of or comprise a nucleic acid of formula (IV) or (V):
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a, (formula (IV))
wherein: [0220] G is guanosine (guanine), uridine (uracil) or an
analogue of guanosine (guanine) or uridine (uracil), preferably
guanosine (guanine) or an analogue thereof; [0221] X is guanosine
(guanine), uridine (uracil), adenosine (adenine), thymidine
(thymine), cytidine (cytosine), or an analogue of these nucleotides
(nucleosides), preferably uridine (uracil) or an analogue thereof;
[0222] N is a nucleic acid sequence having a length of about 4 to
50, preferably of about 4 to 40, more preferably of about 4 to 30
or 4 to 20 nucleic acids, each N independently being selected from
guanosine (guanine), uridine (uracil), adenosine (adenine),
thymidine (thymine), cytidine (cytosine) or an analogue of these
nucleotides (nucleosides); [0223] a is an integer from 1 to 20,
preferably from 1 to 15, most preferably from 1 to 10; [0224] l is
an integer from 1 to 40, [0225] wherein when l=1, G is guanosine
(guanine) or an analogue thereof, [0226] when l>1, at least 50%
of these nucleotides (nucleosides) are guanosine (guanine) or an
analogue thereof; [0227] m is an integer and is at least 3; [0228]
wherein when m=3, X is uridine (uracil) or an analogue thereof, and
[0229] when m>3, at least 3 successive uridines (uracils) or
analogues of uridine (uracil) occur; [0230] n is an integer from 1
to 40, [0231] wherein when n=1, G is guanosine (guanine) or an
analogue thereof, [0232] when n>1, at least 50% of these
nucleotides (nucleosides) are guanosine (guanine) or an analogue
thereof; [0233] u,v may be independently from each other an integer
from 0 to 50, [0234] preferably wherein when u=0, v.gtoreq.1, or
[0235] when v=0, u.gtoreq.1; wherein the nucleic acid molecule of
formula (IV) has a length of at least 50 nucleotides, preferably of
at least 100 nucleotides, more preferably of at least 150
nucleotides, even more preferably of at least 200 nucleotides and
most preferably of at least 250 nucleotides.
[0235] (N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a, (formula
(V))
wherein: [0236] C is cytidine (cytosine), uridine (uracil) or an
analogue of cytidine (cytosine) or uridine (uracil), preferably
cytidine (cytosine) or an analogue thereof; [0237] X is guanosine
(guanine), uridine (uracil), adenosine (adenine), thymidine
(thymine), cytidine (cytosine) or an analogue of the
above-mentioned nucleotides (nucleosides), preferably uridine
(uracil) or an analogue thereof; [0238] N is each a nucleic acid
sequence having independent from each other a length of about 4 to
50, preferably of about 4 to 40, more preferably of about 4 to 30
or 4 to 20 nucleic acids, each N independently being selected from
guanosine (guanine), uridine (uracil), adenosine (adenine),
thymidine (thymine), cytidine (cytosine) or an analogue of these
nucleotides (nucleosides); [0239] a is an integer from 1 to 20,
preferably from 1 to 15, most preferably from 1 to 10; [0240] l is
an integer from 1 to 40, [0241] wherein when l=1, C is cytidine
(cytosine) or an analogue thereof, [0242] when l>1, at least 50%
of these nucleotides (nucleosides) are cytidine (cytosine) or an
analogue [0243] thereof; [0244] m is an integer and is at least 3;
[0245] wherein when m=3, X is uridine (uracil) or an analogue
thereof, [0246] when m>3, at least 3 successive uridines
(uracils) or analogues of uridine (uracil) occur; [0247] n is an
integer from 1 to 40, [0248] wherein when n=1, C is cytidine
(cytosine) or an analogue thereof, [0249] when n>1, at least 50%
of these nucleotides (nucleosides) are cytidine (cytosine) or an
analogue thereof. [0250] u, v may be independently from each other
an integer from 0 to 50, [0251] preferably wherein when u=0,
v.gtoreq.1, or [0252] when v=0, u.gtoreq.1; wherein the nucleic
acid molecule of formula (V) according to the invention has a
length of at least 50 nucleotides, preferably of at least 100
nucleotides, more preferably of at least 150 nucleotides, even more
preferably of at least 200 nucleotides and most preferably of at
least 250 nucleotides.
[0253] For formula (V), any of the definitions given above for
elements N (i.e. N.sub.u and N.sub.v) and X (X.sub.m), particularly
the core structure as defined above, as well as for integers a, I,
m, n, u and v, similarly apply to elements of formula (IV)
correspondingly, wherein in formula (V) the core structure is
defined by C.sub.lX.sub.mC.sub.n. The definition of bordering
elements N.sub.u and N.sub.v is identical to the definitions given
above for N.sub.u and N.sub.v.
[0254] According to a very particularly preferred embodiment, the
nucleic acid molecule according to formula (IV) comprises,
preferably consists of, e.g. any of the following sequences:
TABLE-US-00004 (SEQ ID NO: 99)
UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAA GUACACG (SEQ
ID NO: 100) UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAA
GUACACGAUCGCUUCGAGAACCUGGAUCCAAAAAAAAAAAAAAACCCACGCAAGGA
UCUUCAUGUGC (SEQ ID NO: 101)
GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAU
AUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAU
UCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUC
AGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACU CUAUUAGAUC
(SEQ ID NO: 102)
GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAU
AUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAU
UCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUC
AGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACU
CUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAA
GUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUA
GACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUAC
CCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCAC
UCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUAC
UUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUA G (SEQ ID
NO: 103) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAU
AUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAU
UCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUC
AGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACU
CUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAA
GUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUA
GACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAU UAC
CCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCAC
UCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUAC
UUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUA
GAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCU
UAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUAC
GGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCG
GGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGC
GCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCG
UUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACA
GCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGG
UGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGAC
ACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCC
AAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUAAAAGCUUGAAUU (SEQ ID NO: 104)
GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGU
AUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCU
AUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCC
CCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGA
UGCUGGCCCAGAUC (R722A or isRNA722A; SEQ ID NO: 105)
GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGU
AUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCU
AUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCC
CCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGA
UGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCU
UUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUAC
AGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGU
UUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGU
CACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUU
UUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAG GUCUGCUCUA.
(R722B or isRNA722B; SEQ ID NO: 122)
GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGU
AUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCU
AUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCC
CCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGA
UGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCU
UUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUAC
AGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGU
UUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGU
CACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUU
UUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAG GUCUGCUCUAG
(SEQ ID NO: 106)
GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGU
AUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCU
AUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCC
CCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGA
UGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCU
UUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUAC
AGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGU
UUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGU
CACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUU
UUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAG
GUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUU
UUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGU
UGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUU
UUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGC
GCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUU
UUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACG
UCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUU
UUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAU
AGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUU
UUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGU
UAAGCUUGAAUU
or a nucleic acid sequence having at least 60%, preferably at least
70%, preferably at least 80%, more preferably at least 90%, and
most preferably at least 95% identity to any of the above defined
sequences.
[0255] According to another very particularly preferred embodiment,
the nucleic acid molecule according to formula (V) comprises,
preferably consists of, e.g. any of the following sequences:
TABLE-US-00005 (SEQ ID NO: 107)
UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAG AAGUACACG
or
TABLE-US-00006 (SEQ ID NO: 108)
UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUUCCCUGCGUUCCUA
GAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAAAAAAAAAGGG
ACGCAAGGAUCUUCAUGUGC
or a nucleic acid sequence having at least 60%, preferably at least
70%, preferably at least 80%, more preferably at least 90%, and
most preferably at least 95% identity to any of the above defined
sequences.
[0256] In a further preferred embodiment, the nucleic acid molecule
of the herein defined polymeric carrier cargo complex may also
occur in the form of a modified nucleic acid.
[0257] According to a first embodiment, the nucleic acid molecule
of the herein defined polymeric carrier cargo complex may be
provided as a "stabilized nucleic acid", preferably as a stabilized
RNA or DNA, more preferably as a RNA that is essentially resistant
to in vivo degradation (e.g. by an exo- or endo-nuclease) as
defined above.
[0258] According to another embodiment, the nucleic acid cargo of
the herein defined polymeric carrier cargo complex may be modified
as defined herein, and/or stabilized, especially if the nucleic
acid molecule is in the form of a coding nucleic acid e.g. an mRNA,
by modifying the G/C content of the nucleic acid molecule,
particularly an mRNA, preferably of the coding region thereof as
defined herein.
[0259] Nucleic acid molecules used herein as cargo comprised in the
polymeric carrier cargo complex as defined herein may be prepared
using any method known in the art, including the methods for
nucleic acid synthesis as defined herein.
[0260] Furthermore, the present invention explicitly encloses
variants and fragments of nucleic acid molecules as defined herein
comprised as nucleic acid cargo in the polymeric carrier cargo
complex.
[0261] Particularly preferred nucleic acid cargo molecules in the
context of the present invention are nucleic acid molecules
comprising, preferably consisting of, a nucleic acid sequence
according to SEQ ID NO. 105 or 122 or a sequence which is at least
60%, preferably at least 70%, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95% identical
to SEQ ID NO. 105 or 122.
[0262] In the polymeric carrier cargo complex, the cationic
component of the polymeric carrier as defined herein and the
nucleic acid cargo are typically provided in a molar ratio of about
1 to 10000, preferably in a molar ratio of about 5 to 5000, more
preferably in a molar ratio of about 10 to 2500, even more
preferably in a molar ratio of about 25 to 2000, and most
preferably in a molar ratio of about 25 to 1000 of polymeric
carrier to nucleic acid.
[0263] Furthermore, in the polymeric carrier cargo complex, the
cationic component of the polymeric carrier as defined herein and
the nucleic acid cargo are preferably provided in an N/P-ratio of
at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5 or 2. Preferably,
the N/P-ratio lies within a range of about 0.1, 0.3, 0.4, 0.5,
0.75, 1.0, 1.5 or 2 to 20, preferably in a range of about 0.2 (0.5
or 0.75 or 1.0) to 12, more preferably in an N/P-ratio of about 0.4
(0.75 or 1.0) to 10, and even more preferably in an N/P ratio of
about 0.4 (0.75 or 1.0) to 5. Most preferably the N/P ratio lies in
a ratio between 0.1 and 0.9. In this context, the N/P ratio is a
measure of the ionic charge of the cationic (side chain) component
of the polymeric carrier or of the polymeric carrier as such. In
particular, if the cationic properties of the cationic component
are generated by nitrogens (e.g. of the amino acid side chains),
the N/P ratio expresses the ratio of basic nitrogen atoms to
phosphate residues in the nucleotide backbone, considering that
(side chain) nitrogen atoms in the cationic component of the
polymeric carrier contribute to positive charges and phosphate of
the phosphate backbone of the nucleic acid contribute to the
negative charge. Generally, one phosphate provides one negative
charge, e.g. one nucleotide in the cargo nucleic acid molecule
provides one negative charge. A formula is given in the Examples.
The N/P-ratio is defined as the nitrogen/phosphate ratio
(N/P-ratio) of the entire inventive polymeric carrier cargo
complex. This is typically illustrative for the content/amount of
cationic components, in the polymeric carrier and characteristic
for the content/amount of nucleic acids bound or complexed in the
inventive polymeric carrier cargo complex. It may be calculated on
the basis that, for example, 1 .mu.g RNA typically contains about 3
nmol phosphate residues, provided that RNA exhibits a statistical
distribution of bases. Additionally, 1 nmol peptide typically
contains about x nmol nitrogen residues, dependent on the molecular
weight and the number of its (cationic) amino acids.
[0264] In this context, it is preferable that in the polymeric
carrier cargo complex, the cationic component of the polymeric
carrier as defined herein and the nucleic acid cargo are provided
in an N/P-ratio of at least about 1 or, preferably, of a range of
about 1 to 20 for in vitro applications (e.g. in the case cells
extracted from the patient would be treated in vitro with the
inventive pharmaceutical composition and subsequently administered
to the patient).
[0265] For in vivo applications of the inventive pharmaceutical
composition, an N/P ratio of at least 0.1 (0.2, 0.3, 0.4, 0.5,
0.6), preferably of a range of about 0.1 (0.2, 0.3, 0.4., 0.5, or
0.6) to 1.5 is preferred. Even more preferred is an N/P ratio range
of 0.1 or 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9.
[0266] In the specific case that the induction of IFN-.alpha. is
intended, an N/P ratio of at least 0.1 (0.2, 0.3, 0.4, 0.5, or 0.6)
or an N/P ratio range of 0.1 to 1 is preferred or more preferred is
an N/P ratio range of 0.1 or 0.2 to 0.9 or an N/P ratio range of
0.5 to 0.9. Otherwise, if the induction of TNF.alpha. would be
intended, an N/P ratio of 1 to 20 is particularly preferred.
[0267] The N/P ratio significantly influences the surface charge of
the resulting polymeric carrier cargo complex. Thus, it is
preferable that the resulting polymeric carrier cargo complex is
positively charged for in vitro applications and negatively or
neutrally charged for in vivo applications. The surface charge of
the resulting polymeric carrier cargo complex can be indicated as
Zetapotential which may be measured by Doppler electrophoresis
method using a Zetasizer Nano (Malvern Instruments, Malvern, UK).
Generally, an N/P ratio of below 1 results in a negative
Zetapotential, and an N/P ratio of above 1 results in a positive
Zetapotential (within the scope of typical measurement errors).
[0268] In some embodiments, the surface charge of the polymeric
carrier cargo complex, preferably the Zetapotential, is positive,
i.e. above 0 mV, such as above 1 mV, above 2 mV, above 4 mV, above
5 mV, or above 10 mV. In alternative embodiments, the surface
charge of the polymeric carrier cargo complex, preferably the
Zetapotential, is negative, i.e. below 0 mV, such as below -1 mV,
below -2 mV, below -4 mV, below -5 mV, or below -10 mV, such as
between about -1 mV and -50 mV, between about -2 mV and -40 mV, or
between about -5 mV and -30 mV.
[0269] The polymeric carrier cargo complex as used in the present
invention, such as for use as an adjuvant, is preferably capable of
triggering a non-antigen-specific, (innate) immune reaction (as
provided by the innate immune system), preferably in an
immunostimulating manner. An immune reaction can generally be
brought about in various ways. An important factor for a suitable
immune response is the stimulation of different T-cell
sub-populations. T-lymphocytes typically differentiate into two
sub-populations, the T-helper 1 (Th1) cells and the T-helper 2
(Th2) cells, with which the immune system is capable of destroying
intracellular (Th1) and extracellular (Th2) pathogens (e.g.
antigens). The two Th cell populations differ in the pattern of
effector proteins (cytokines) produced by them. Thus, Th1 cells
assist the cellular immune response by activation of macrophages
and cytotoxic T-cells. Th2 cells, on the other hand, promote the
humoral immune response by stimulation of B-cells for conversion
into plasma cells and by formation of antibodies (e.g. against
antigens). The Th1/Th2 ratio is therefore of great importance in
the immune response. In connection with the present invention, the
Th1/Th2 ratio of the immune response is preferably displaced by the
immune-stimulating agent, in particular the polymeric carrier cargo
complex, in the direction towards the cellular response, that is to
say the Th1 response, and a predominantly cellular immune response
is thereby induced. As described above, the polymeric carrier cargo
complex can induce an unspecific innate immune response, which may
allow the support of a specific adaptive immune response elicited
by the antigen.
[0270] Determination of the (innate) immunostimulatory or adjuvant
capacity of a component in the inventive pharmaceutical
composition:
[0271] For the determination of the immunostimulatory capacity of
an immunostimulating agent or adjuvant (in particular of a
polymeric carrier cargo complex as used in the present invention)
several methods are known in the art and may be used. E.g., in
vitro methods are advantageous to utilise for compounds as to their
capacity to induce cytokines, which are (exclusively or at least
typically) part of the innate immune system and thereby (as an
additional arm of the immune system) typically improve the
induction of an antigen-specific immune response caused by an
antigen. For this purpose, e.g. PBMCs may be isolated from blood
samples and stimulated with the particular immunostimulating agent
or adjuvant. After incubation, secretion of the desired cytokines
(e.g. as a reaction of an activation of the PAMP receptors) being
typically part of the innate immune system (and not of the
antigen-specific immune system) is determined by ELISA. These
selected cytokines may be used in the art as determinants of the
induction of an innate immune response in the body. In this
context, the secretion of TNF-alpha and IFN-alpha is preferably
measured to determine the unspecific (innate immune response)
evoked by a compound or complex. Especially, IFN-alpha plays an
important role in the induction of an unspecific immune response
after viral infection and can be used as an indicators of induction
of a Th1-shifted adaptive immune response, which is particularly
preferred in the context of the treatment of cancer or tumour
diseases. Accordingly, it is particularly preferred that the
immunostimulatory compound or complex tested in the screening
assay, induces the secretion of e.g. IFN-alpha. Such a compound or
complex may then be applied e.g. for the use as an immunotimualting
agent (triggering the unspecific (innate) immune response) in
vaccination therapies.
[0272] IFN-alpha is part of the family of type I interferons. Type
I interferons (IFN) are pleiotropic cytokines that are essential
for supporting anti-viral immune responses. They induce apoptosis
of virus-infected cells and cellular resistance to viral infection,
in addition to activating natural killer (NK) and T cells. Type I
interferons have effects on a large set of cytokines and chemokines
that i.a. influence immunocyte maturation, homing, effector
functions and apoptosis. Typically, a major role of IFN-alpha is
the induction of a priming state affecting the production and
regulation of other mediators, including cytokines. For example,
IFN-alpha.beta. signaling upregulates IFN-alpha.gamma. production
by dendritic cells (DCs) and T cells and thereby favours the
induction and maintenance of Th1 cells. Shifting of an immune
response in direction of a Th1 immune response may become
particularly important, once protein or peptide vaccines are used,
because these vaccines usually induce a Th2-based immune response
which consequently prevents or decreases the induction of cytotoxic
T cells.
[0273] Therefore, it is preferred that a compound or complex to be
used as an adjuvant in the context of the present invention may
preferably have the property of shifting an antigen-specific immune
response caused by a antigen to a Th1-based immune response. The
direction of an immune response induced by an antigen is usually
measured by determination of the induction of several subtypes of
antigen-specific antibodies and the induction of antigen-specific
cytotoxic CD8.sup.+ T cells. In this context, the subtype antibody
IgG1 represents the induction of a Th2-based immune response and
the induction of the subtype antibody IgG2a and the induction of
cytotoxic T cells represent the induction of a Th1-based immune
response. The induction of antigen-specific antibodies is typically
determined by measurement of the antibody titer in the blood of the
vaccine by ELISA. The induction of antigen-specific cytotoxic T
cells is typically determined by measurement of IFN-gamma secretion
in splenocytes after stimulation with antigen-specific peptides by
ELISPOT. In this context, the induction of IFN-gamma secretion
provides evidence that antigen-specific cytotoxic T cells are
present in the spleen and which can specifically attack cells that
present epitopes of the antigen on MHC I molecules on their
surface.
[0274] For the determination of beneficial properties of an
adjuvant, in vivo vaccinations are typically performed. Therewith,
it is possible to investigate if the adjuvant or immunostimulatory
compound or complex improves an antigen-specific immune response
caused by the vaccine and, furthermore, if it can shift an
antigen-specific immune response in the desired direction to
display adjuvant properties. Particularly, in the induction of an
anti-tumoral immune response the induction of a Th1-shifted immune
response, especially the induction of cytotoxic T cells is believed
to play a major role, because the induction of antigen-specific
cytotoxic T cells are believed to represent an indispensable
prerequisite for the successful combat of a tumour.
[0275] Accordingly, the methods to screen for, test and/or
investigate compound or complexes which exhibit properties as
adjuvants are well known in the art and may readily be applied e.g.
by ELISA tests measuring the immune response elicited by the tested
compounds/complexes.
[0276] In another aspect, the present invention relates to a method
of preparing a pharmaceutical composition of the invention, said
method comprising the steps of: (i) providing at least one
polymeric carrier cargo complex as defined anywhere herein; (ii)
providing an antigen as defined anywhere herein; and (iii)
combining said polymeric carrier cargo complex and said antigen.
The combining step of (iii) may occur briefly before administration
to a patient (such as about 1, 5, 15, 30 or 60 minutes prior to, up
to 72 hours before, said administration), or may occur during
manufacture of said pharmaceutical composition. The respective
person of ordinary skill (e.g. a doctor or health professional, or
a manufacturer) will be aware of the routine methodologies suitable
for such combining step.
[0277] In the context of the present invention, a method of
preparing the polymeric carrier cargo complex as defined herein may
comprise the following steps: [0278] a) providing at least one
cationic protein or peptide as defined herein and/or at least one
cationic or polycationic polymer and optionally at least one amino
acid component (AA) as defined herein, each comprising at least one
--SH moiety, [0279] b) providing at least one nucleic acid molecule
as defined herein, preferably in the above mentioned ratios, [0280]
c) mixing the components provided in steps a) and b), preferably in
a basic or neutral milieu as defined herein, preferably in the
presence of oxygen or a further starter as defined herein,
preferably at a pH, at a temperature and at time as defined herein,
and thereby condensing and thus polymerizing the cationic
components provided in step a) with each other via disulfide bonds
(in a polymerization condensation or polycondensation) to obtain
the polymeric carrier and complexing the nucleic acid molecule
provided in step b) with the cationic components provided in step
a), [0281] d) optionally purifying the polymeric carrier cargo
complex obtained according to step c), preferably using a method as
defined herein, [0282] e) optionally lyophilization of the
polymeric carrier cargo complex obtained according to step c) or
d).
[0283] The method of preparing the polymeric carrier cargo complex
as described herein may comprise a multi-step condensation
polymerization or polycondensation reaction via --SH moieties of
the educts e.g. cationic peptides or polymers as defined herein and
optionally further amino acid components (AA) in step c). The
condensation polymerization or polycondensation reaction which
occurs simultaneously to the complexation or electrostratic binding
of the nucleic acid molecule preferably leads to the polymeric
carrier cargo complex wherein the polymeric carrier is a
condensation polymer, wherein the single components are linked by
disulfide bonds.
[0284] As described herein in a step a) of the method of preparing
the polymeric carrier cargo complex, at least one cationic or
polycationic protein or peptide as defined herein and/or at least
one cationic or polycationic polymer as defined herein are
provided, preferably in the ratios indicated above. These
components are mixed in step c) with the nucleic acid molecule
provided in step b), preferably in a basic or neutral milieu as
defined herein, preferably in the presence of oxygen or a further
starter as defined herein, preferably at a pH, and at a temperature
and at a time as defined herein, and thereby condensing and thus
polymerizing these components with each other via disulfide bonds
(in a polymerization condensation or polycondensation) to obtain a
polymeric carrier complexed to the nucleic acid molecule as defined
herein.
[0285] According to an alternative, in step a) of the method of
preparing the polymeric carrier cargo complex at least one cationic
or polycationic protein or peptide and/or at least one cationic or
polycationic polymer are provided as defined herein, and optionally
at least one amino acid component (AA), are provided in step a) as
defined herein, and are used for a polymerization condensation or
polycondensation and complexation reaction prior to adding the
nucleic acid of step b) but using the same polymerization
conditions outlined for step c). The polymerized polymeric carrier
and the nucleic acid of step b) are then mixed in step c).
Preferably, the components are all provided in the ratios indicated
above and mixed, preferably in a basic or neutral milieu as defined
herein, preferably in the presence of oxygen or a further starter
as defined herein, preferably at a pH, at a temperature and at time
as defined herein. Upon mixing and starting the reaction, the
components are condensed and thus polymerized with each other via
disulfide bonds (in a polymerization condensation or
polycondensation) to obtain a polymeric carrier complexed to the
nucleic acid molecule as defined herein.
[0286] In both of the above alternatives, different polymeric
carriers, particularly different peptides and/or different
polymers, may be selected in the condensation polymerization as
indicated above. In this context, the selection of different
component(s) of the polymeric carrier is typically dependent upon
the desired properties of the final polymeric carrier and the
desired cationic strength of the final polymeric carrier.
Accordingly, the content of cationic components, may furthermore be
"diluted" or modified in the above alternative of step a) e.g. by
introducing an amino acid component (AA) as defined herein,
preferably in the above defined ratios. Thereby, a modified
polymeric carrier may be obtained, wherein the cationic character
of the unmodified polymeric carrier typically remains in the
limitations as defined herein. The properties of the final
polymeric carrier may thus be adjusted as desired with properties
of components (AA) by inserting amino acid component (AA) as
defined herein in steps a).
[0287] In step c), the at least one cationic or polycationic
protein or peptide as defined herein and/or at least one cationic
or polycationic polymer as defined herein, and optionally at least
one amino acid component (AA) and the at least one nucleic acid as
defined herein, are preferably contained in a basic or neutral
milieu in the step a) of the inventive method of preparing the
inventive polymeric carrier cargo complex. Such a basic or neutral
milieu typically exhibits a pH range of about 5 to about 10,
preferably a pH range of about 6 to about 9, more preferably a pH
range of about 7 to about 8, e.g. about 6.5, 7, 7.5, 8, 8.5, or 9
or any range selected from any two of these or the aforementioned
values.
[0288] Furthermore, the temperature of the solution in step c) is
preferably in a range of about 5.degree. C. to about 60.degree. C.,
more preferably in a range of about 15.degree. C. to about
40.degree. C., even more preferably in a range of about 20.degree.
C. to about 30.degree. C., and most preferably in a range of about
20.degree. C. to about 25.degree. C., e.g. about 25.degree. C.
[0289] In step c) of the method of preparing the polymeric carrier
cargo complex as described herein, buffers may be used as suitable.
Preferred buffers may comprise, but are not limited to, preferably
are selected from carbonate buffers, borate buffers, Bicine buffer,
CHES buffer, CAPS buffer, Ethanolamine containing buffers, HEPES,
MOPS buffer, Phosphate buffer, PIPES buffer, Tris buffer, Tricine
buffer, TAPS buffer, and/or TES buffer as buffering agents.
Particularly preferred is a carbonate buffer.
[0290] Upon mixing the components, preferably in the presence of
oxygen, preferably in the presence of a basic or neutral mileu as
defined herein, the condensation polymerization or polycondensation
reaction and the complexation of the at least one nucleic acid
molecule is started. For this purpose, the mixture in step c) is
preferably exposed to oxygen or may be started using a further
starter, e.g. a catalytic amount of an oxidizing agent, e.g. DMSO,
etc. Upon start of the condensation polymerization or
polycondensation reaction of the at least one cationic or
polycationic protein or peptide and/or at least one cationic or
polycationic polymer and optionally at least one amino acid
component (AA) as defined herein, are condensed and thus
polymerized with each other via disulfide bonds (polymerization
condensation or polycondensation). In this reaction step a)
preferably linear polymers are created using monomers with at least
one reactive --SH moiety, i.e. at least one cationic or
polycationic protein or peptide and/or at least one cationic or
polycationic polymer and optionally at least one amino acid
component (AA) as defined herein, each component exhibiting at
least one free --SH-moieties as defined herein, e.g. at their
terminal ends. However, components with more than one, preferably
two free --SH-moieties may be used, which may lead to branched
polymers. Simultaneously to the polymerization reaction the
cationic polymers bind to the at least one nucleic acid molecule
and thereby complexing it.
[0291] According to one alternative, the polymeric carrier cargo
complex additionally may be modified with a component (AA) as
defined herein.
[0292] According to a first example, a component (AA) (e.g. a
ligand) is attached to the cationic component prior to providing
the cationic component in step a) via any functionality as defined
herein, e.g. a --SH moiety. This component (AA) or (e.g. a ligand)
is preferably attached to the cationic component at one terminus of
these components. If the attachment is carried out via --SH bonds,
the cationic components are preferably provided with two (or even
more) --SH-moieties. The component (AA) or (e.g. a ligand)
preferably carries only one --SH moiety. In this case, one --SH
moiety of the cationic component is preferably protected in a first
step using a protecting group as known in the art. Then, the
cationic component may be bound to a component L to form a first
disulfide bond via the non-protected --SH moiety. The protected
--SH-moiety of the cationic component is then typically deprotected
for further reactions.
[0293] Alternatively, the above mentioned component (AA) or (e.g. a
ligand) may be used in step c) to be coupled with the cationic
components provided in step a) above, e.g. via disulfide bonds
without blocking the free --SH moieties. But in this context all
methods known to a skilled person or defined herein may be used to
attach the component (AA) to the cationic component or to the
polymeric carrier.
[0294] Alternatively, a component (AA) or (e.g. a ligand) can be
bound to the polymeric carrier cargo complex after step c) via any
functionality as defined herein, e.g. a --SH moiety. In this
context it is preferable that the component (AA) (e.g. a ligand) is
bound via free --SH moieties of the polymeric carrier
components.
[0295] According to step c) of the method of preparing the
polymeric carrier cargo complex as described herein, at least one
nucleic acid molecule as defined herein is mixed with the cationic
components provided in step b), preferably in the above mentioned
ratios. Typically, in the polymeric carrier cargo complex, the
cationic components as defined herein, and the at least one nucleic
acid molecule are provided in a molar ratio of about 5 to 10000,
preferably in a molar ratio of about 5 to 5000, more preferably in
a molar ratio of about 10 to 2500, even more preferably in a molar
ratio of about 10 to 1000 cationic polymer to nucleic acid. The N/P
ratios are preferably as indicated above. In this context, it is
particularly preferred that the N/P ratios are selected thereby
avoiding agglomeration and toxicity in vivo.
[0296] In a specific embodiment, (AA) components as defined above
which do not comprise --SH moieties can be added in step c) which
are thereby incorporated into the polymeric carrier cargo complex
without polymerization by (terminal) --SH moieties. Thereby these
(AA) components are typically not covalently linked and included
non-covalently in the complex as a further component. Particularly
preferred in this context is the incorporation of the at least one
antigen or a fragment, variant and/or derivative thereof, provided
as protein or peptide in the polymeric carrier cargo complex as
(AA) component. This embodiment is particularly preferred if AA is
ovalbumin or a fragment of ovalbumin. Thus, in a particularly
preferred embodiment, if AA is ovalbumin or a fragment thereof, AA
is not covalently linked to the polymeric carrier cargo complex,
for example, AA is not covalently linked to the polymeric carrier
cargo complex by disulfide bonds.
[0297] According to a further step d) of the method of preparing
the polymeric carrier cargo complex as described herein, the
polymeric carrier cargo complex obtained according to step c) is
optionally purified. Purification may occur by using
chromatographic methods, such as HPLC, FPLC, GPS, dialysis,
etc.
[0298] According to a further step e) of the method of preparing
the polymeric carrier cargo complex as described herein, the
polymeric carrier cargo complex obtained according to step c) or d)
is optionally lyophilized. For this purpose any suitable
cryoprotectant or lyoprotectant may be added to the polymeric
carrier cargo complex obtained in step c) or d).
[0299] The method of preparing the polymeric carrier cargo complex
as defined herein is particularly suitable to adapt the chemical
properties of the desired polymeric carrier cargo complex due to
specific selection of its components of the polymeric carrier
thereby avoiding agglomeration and toxicity in vivo.
[0300] As a second ingredient the inventive pharmaceutical'
composition comprises at least one antigen selected from an antigen
from a pathogen associated with infectious disease; an antigen
associated with allergy or allergic disease; an antigen associated
with autoimmune disease; or an antigen associated with a cancer or
tumour disease, or in each case a fragment, variant and/or
derivative of said antigen.
[0301] This at least one antigen can be provided as protein or
peptide, as nucleic acid coding for the at least one antigen, or as
antigenic cells, antigenic cellular fragments, cellular fractions;
cell wall components modified, attenuated or inactivated (e.g.
chemically or by irradiation) pathogens (virus, bacteria etc.)
comprising the at least one antigen.
[0302] In certain embodiments, the antigen included as a second
ingredient in the pharmaceutical composition is a peptide or
protein antigen, or a fragment, variant and/or derivative of said
peptide or protein antigen.
a) Antigens from a Pathogen Associated with Infectious Disease:
[0303] Antigens from a pathogen associated with infectious disease
are derived from a pathogen which is associated with the induction
of an infectious disease. In certain embodiments, said antigen is a
peptide or protein antigen, or a fragment, variant and/or
derivative of said peptide or protein antigen, and/or is comprised
in, provided as and/or derived from (e.g. a preparation of)
inactivated or attenuated said pathogen, (e.g. a virus such as any
one described herein). In this context, the (e.g. peptide or
protein) antigen may be comprised in provided as and/or derived
from (e.g. a preparation of) an attenuated or inactivated pathogen
(e.g. a virus such as any one described herein) associated with
infectious disease.
[0304] In alternative embodiments of all aspects of the invention,
an antigen (e.g. a peptide or protein antigen) used in the present
invention is not one comprised in (e.g. a preparation of)
inactivated or attenuated virus (such as any one described herein,
or any pathogen described herein); and/or is one that is not
provided as (e.g. a preparation of) inactivated or attenuated said
virus or pathogen; and/or is one that is not derived from (e.g. a
preparation of) inactivated or attenuated said virus or pathogen.
For example, the antigen used in any aspect of the present
invention may be, or may be provided as, an isolated and/or
purified protein or peptide antigen. As will be understood by the
person of ordinary skill, an isolated (and/or purified) antigen
includes such antigens that are present (or provided) in a
(starting) composition that has less than about 40%, 30%, 20%, 10%,
5%, 2% or 1% non-desired or specified other components such as
other proteins/peptides or impurities.
[0305] In particular embodiments, the (e.g. protein or peptide)
antigen used in the present invention is a recombinant antigen, for
example one that is prepared using recombinant production, such as
using those methodologies described herein. In alternative
embodiments, the (e.g. protein or peptide) antigen used in the
present invention is a synthetic antigen, for example one that is
prepared using peptide synthesis, such as using those methodologies
described herein.
[0306] Antigens from a pathogen associated with infectious disease
are selected from antigens from the pathogens Acinetobacter
baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma
braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum,
Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia
genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK
virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella
pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp,
Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia
cepacia and other Burkholderia species, Burkholderia mallei,
Burkholderia pseudomallei, Caliciviridae family, Campylobacter
genus, Candida albicans, Candida spp, Chlamydia trachomatis,
Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion,
Clonorchis sinensis, Clostridium botulinum, Clostridium difficile,
Clostridium perfringens, Clostridium perfringens, Clostridium spp,
Clostridium tetani, Coccidioides spp, coronaviruses,
Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo
hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium
genus, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and
DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus
genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus,
Entamoeba histolytica, Enterococcus genus, Enterovirus genus,
Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71),
Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli
0157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola
gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,
Francisella tularensis, Fusobacterium genus, Geotrichum candidum,
Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,
Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,
Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus,
Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis
E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2),
Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea
werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and
Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human
papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese
encephalitis virus, JC virus, Junin virus, Kingella kingae,
Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella
pneumophila, Leishmania genus, Leptospira genus, Listeria
monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo
virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus
yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),
Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,
Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma
pneumoniae, Naegleria fowleri, Necator americanus, Neisseria
gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia
spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae
family, Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus
westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus,
Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory
syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari,
Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii,
Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella
virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS
coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus,
Hantavirus, Sporothrix schenckii, Staphylococcus genus,
Staphylococcus genus, Streptococcus agalactiae, Streptococcus
pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis,
Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV),
Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema
pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton
spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi,
Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella
zoster virus (VZV), Variola major or Variola minor, vCJD prion,
Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile
virus, Western equine encephalitis virus, Wuchereria bancrofti,
Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and
Yersinia pseudotuberculosis.
[0307] In this context particularly preferred are antigens from the
pathogens selected from Rabies virus, Hepatitis B virus, human
Papilloma virus (hPV), Bacillus anthracis, respiratory syncytial
virus (RSV), herpes simplex virus (HSV), Influenza virus and
Mycobacterium tuberculosis.
[0308] Furthermore, the antigen from a pathogen associated with
infectious disease may be selected from the following antigens:
Outer membrane protein A OmpA, biofilm associated protein Bap,
transport protein MucK (Acinetobacter baumannii, Acinetobacter
infections)); variable surface glycoprotein VSG,
microtubule-associated protein MAPP15, trans-sialidase TSA
(Trypanosoma brucei, African sleeping sickness (African
trypanosomiasis)); HIV p24 antigen, HIV Eenvelope proteins (Gp120,
Gp41, Gp160), polyprotein GAG, negative factor protein Nef,
trans-activator of transcription Tat (HIV (Human immunodeficiency
virus), AIDS (Acquired immunodeficiency syndrome));
galactose-inhibitable adherence protein GIAP, 29 kDa antigen Eh29,
GaVGaINAc lectin, protein CRT, 125 kDa immunodominant antigen,
protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica,
Amoebiasis); Major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3,
MSP4, MSP5), type IV secreotion system proteins (VirB2, VirB7,
VirB11, VirD4) (Anaplasma genus, Anaplasmosis); protective Antigen
PA, edema factor EF, lethal facotor LF, the S-layer homology
proteins SLH (Bacillus anthracis, Anthrax); acranolysin,
phospholipase D, collagen-binding protein CbpA (Arcanobacterium
haemolyticum, Arcanobacterium haemolyticum infection); nucleocapsid
protein NP, glycoprotein precursor GPC, glycoprotein GP1,
glycoprotein GP2 (Junin virus, Argentine hemorrhagic fever);
chitin-protein layer proteins, 14 kDa suarface antigen A14, major
sperm protein MSP, MSP polymerization-organizing protein MPOP, MSP
fiber protein 2 MFP2, MSP polymerization-activating kinase MPAK,
ABA-1-like protein ALB, protein ABA-1, cuticulin CUT-1 (Ascaris
lumbricoides, Ascariasis); 41 kDa allergen Asp v13, allergen Asp
f3, major conidial surface protein rodlet A, protease Pep1p,
GPI-anchored protein Gel1p, GPI-anchored protein Crf1p (Aspergillus
genus, Aspergillosis); family VP26 protein, VP29 protein
(Astroviridae, Astrovirus infection); Rhoptry-associated protein 1
RAP-1, merozoite surface antigens MSA-1, MSA-2 (a1, a2, b, c),
12D3, 1105, 21B4, P29, variant erythrocyte surface antigen VESA1,
Apical Membrane Antigen 1 AMA-1 (Babesia genus, Babesiosis);
hemolysin, enterotoxin C, PXO1-51, glycolate oxidase,
ABC-transporter, penicillin-bingdn protein, zinc transporter family
protein, pseudouridine synthase Rsu, plasmid replication protein
RepX, oligoendopeptidase F, prophage membrane protein, protein
HemK, flagellar antigen H, 28.5-kDa cell surface antigen (Bacillus
cereus, Bacillus cereus infection); large T antigen LT, small T
antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus
infection); 29 kDa-protein, caspase-3-like antigens; glycoproteins
(Blastocystis hominis, Blastocystis hominis infection); yeast
surface adhesin WI-1 (Blastomyces dermatitidis, Blastomycosis);
nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP
(Machupo virus, Bolivian hemorrhagic fever); outer surface protein
A OspA, outer surface protein OspB, outer surface protein OspC,
decorin binding protein A DbpA, decorin binding protein B DbpB,
flagellar filament 41 kDa core protein Fla, basic membrane protein
A precursor BmpA (Immunodominant antigen P39), outer surface 22 kDa
lipoprotein precursor (antigen IPLA7), variable surface lipoprotein
vIsE (Borrelia genus, Borrelia infection); Botulinum neurotoxins
BoNT/A1, BoNT/A2, BoNT/A3, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F,
BoNT/G, recombinant botulinum toxin F Hc domain FHc (Clostridium
botulinum, Botulism (and Infant botulism)); nucleocapsid,
glycoprotein precursor (Sabia virus, Brazilian hemorrhagic fever);
copper/Zinc superoxide dismutase SodC, bacterioferritin Bfr, 505
ribosomal protein RpIL, OmpA-like transmembrane domain-containing
protein Omp31, immunogenic 39-kDa protein M5 P39, zinc ABC
transporter periplasmic zinc-bnding protein znuA, periplasmic
immunogenic protein Bp26, 30S ribosomal protein S12 RpsL,
glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer-membrane
immunogenic protein precursor Omp25, invasion protein B lalB,
trigger factor Tig, molecular chaperone DnaK, putative
peptidyl-prolyl cis-trans isomerase SurA, lipoprotein Omp19, outer
membrane protein MotY Omp16, conserved outer membrane protein D15,
malate dehydrogenase Mdh, component of the Type-IV secretion system
(T4SS) VirJ, lipoprotein of unknown function BAB1.sub.--0187
(Brucella genus, Brucellosis); members of the ABC transporter
family (LoIC, OppA, and PotF), putative lipoprotein releasing
system transmembrane protein LoICJE, flagellin FliC, Burkholderia
intracellular motility A BimA, bacterial Elongation factor-Tu
EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding
protein (Burkholderia cepacia and other Burkholderia species,
Burkholderia infection); mycolyl-transferase Ag85A, heat-shock
protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3,
heat-shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer);
norovirus major and minor viral capsid proteins VP1 and VP2, genome
polyprotein, Sapoviurus capsid protein VP1, protein Vp3, geome
polyprotein (Caliciviridae family, Calicivirus infection (Norovirus
and Sapovirus)); major outer membrane protein PorA, flagellin FIaA,
surface antigen CjaA, fibronectin binding protein CadF,
aspartate/glutamate-binding ABC transporter protein Peb1A, protein
FspA1, protein FspA2 (Campylobacter genus, Campylobacteriosis);
glycolytic enzyme enolase, secreted aspartyl proteinases SAP1-10,
glycophosphatidylinositol (GPI)-linked cell wall protein, protein
Hyr1, complement receptor 3-related protein CR3-RP, adhesin Als3p,
heat shock protein 90 kDa hsp90, cell surface hydrophobicity
protein CSH (usually Candida albicans and other Candida species,
Candidiasis); 17-kDa antigen, protein P26, trimeric autotransporter
adhesins TAAs, Bartonella adhesin A BadA, variably expressed
outer-membrane proteins Vomps, protein Pap3, protein HbpA,
envelope-associated protease HtrA, protein OMP89, protein GroEL,
protein LaIB, protein OMP43, dihydrolipoamide succinyltransferase
SucB (Bartonella henselae, Cat-scratch disease); amastigote surface
protein-2, amastigote-specific surface protein SSP4, cruzipain,
trans-sialidase TS, trypomastigote surface glycoprotein TSA-1,
complement regulatory protein CRP-10, protein G4, protein G2,
paraxonemal rod protein PAR2, paraflagellar rod component Par1,
mucin-Associated Surface Proteins MPSP (Trypanosoma cruzi, Chagas
Disease (American trypanosomiasis)); envelope glycoproteins (gB,
gC, gE, gH, gI, gK, gL) (Varicella zoster virus (VZV), Chickenpox);
major outer membrane protein MOMP, probable outer membrane protein
PMPC, outer membrane complex protein B OmcB, heat shock proteins
Hsp60 HSP10, protein IncA, proteins from the type III secretion
system, ribonucleotide reductase small chain protein NrdB, plasmid
protein Pgp3, chlamydial outer protein N CopN, antigen CT521,
antigen CT425, antigen CT043, antigen TC0052, antigen TC0189,
antigen TCO582, antigen TC0660, antigen TC0726, antigen TC0816,
antigen TC0828 (Chlamydia trachomatis, Chlamydia); low calcium
response protein E LCrE, chlamydial outer protein N CopN,
serine/threonine-protein kinase PknD, acyl-carrier-protein
S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb,
major outer membrane protein MOMP, outer membrane protein 2 Omp2,
polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5,
Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15,
Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21) (Chlamydophila
pneumoniae, Chlamydophila pneumoniae infection); cholera toxin B
CTB, toxin coregulated pilin A TcpA, toxin coregulated pilin TcpF,
toxin co-regulated pilus biosynthesis ptrotein F TcpF, cholera
enterotoxin subunit A, cholera enterotoxin subunit B, Heat-stable
enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer
membrane protein U Porin ompU, Poring B protein, polymorphic
membrane protein-D (Vibrio cholerae, Cholera); propionyl-CoA
carboxylase PCC, 14-3-3 protein, prohibitin, cysteine proteases,
glutathione transferases, gelsolin, cathepsin L proteinase CatL,
Tegumental Protein 20.8 kDa TP20.8, tegumental protein 31.8 kDa
TP31.8, lysophosphatidic acid phosphatase LPAP, (Clonorchis
sinensis, Clonorchiasis); surface layer proteins SLPs, glutamate
dehydrogenase antigen GDH, toxin A, toxin B, cysteine protease
Cwp84, cysteine protease Cwp13, cysteine protease Cwp19, Cell Wall
Protein CwpV, flagellar protein FliC, flagellar protein FliD
(Clostridium difficile, Clostridium difficile infection);
rhinoviruses: capsid proteins VP1, VP2, VP3, VP4; coronaviruses:
sprike proteins S, envelope proteins E, membrane proteins M,
nucleocapsid proteins N (usually rhinoviruses and coronaviruses,
Common cold (Acute viral rhinopharyngitis; Acute coryza)); prion
protein Prp (CJD prion, Creutzfeldt-Jakob disease (CJD)); envelope
protein Gc, envelope protein Gn, nucleocapsid proteins
(Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic
fever (CCHF)); virulence-associated DEAD-box RNA helicase VAD1,
galactoxylomannan-protein GaIXM, glucuronoxylomannan GXM,
mannoprotein MP (Cryptococcus neoformans, Cryptococcosis); acidic
ribosomal protein P2 CpP2, mucin antigens Muc1, Muc2, Muc3 Muc4,
Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherence
protein CP23, surface protein CP12, surface protein CP21, surface
protein CP40, surface protein CP60, surface protein CP15,
surface-associated glycopeptides gp40, surface-associated
glycopeptides gp15, oocyst wall protein AB, profilin PRF, apyrase
(Cryptosporidium genus, Cryptosporidiosis); fatty acid and retinol
binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP
(TMP), cysteine proteinase ACEY-1, cysteine proteinase ACCP-1,
surface antigen Ac-16, secreted protein 2 ASP-2, metalloprotease 1
MTP-1, aspartyl protease inhibitor API-1, surface-associated
antigen SAA-1, adult-specific secreted factor Xa serine protease
inhibitor anticoagulant AP, cathepsin D-like aspartic protease
ARR-1 (usually Ancylostoma braziliense; multiple other parasites,
Cutaneous larva migrans (CLM)); cathepsin L-like proteases,
53/25-kDa antigen, 8 kDa family members, cysticercus protein with a
marginal trypsin-like activity TsAg5, oncosphere protein TSOL18,
oncosphere protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate
dehydrogenase B LDHB (Taenia solium, Cysticercosis); pp65 antigen,
membrane protein pp15, capsid-proximal tegument protein pp150,
protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM,
glycoprotein gN, glcoprotein H, glycoprotein B gB, protein UL83,
protein UL94, protein UL99 (Cytomegalovirus, Cytomegalovirus
infection); capsid protein C, premembrane protein prM, membrane
protein M, envelope protein E (domain I, domain II, domain II),
protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A,
protein 2K, protein NS4B, protein NS5 (Dengue viruses (DEN-1,
DEN-2, DEN-3 and DEN-4)-Flaviviruses, Dengue fever); 39 kDa protein
(Dientamoeba fragilis, Dientamoebiasis); diphtheria toxin precursor
Tox, diphteria toxin DT, pilin-specific sortase SrtA, shaft pilin
protein SpaA, tip pilin protein SpaC, minor pilin protein SpaB,
surface-associated protein DIP1281 (Corynebacterium diphtheriae,
Diphtheria); glycoprotein GP, nucleoprotein NP, minor matrix
protein VP24, major matrix protein VP40, transcription activator
VP30, polymerase cofactor VP35, RNA polymerase L (Ebolavirus
(EBOV), Ebola hemorrhagic fever); prion protein (vCJD prion,
Variant Creutzfeldt-Jakob disease (vCJD, nvCJD)); UvrABC system
protein B, protein Flp1, protein Flp2, protein Flp3, protein TadA,
hemoglobin receptor HgbA, outer membrane protein TdhA, protein
CpsRA, regulator CpxR, protein SapA, 18 kDa antigen, outer membrane
protein NcaA, protein LspA, protein LspA1, protein LspA2, protein
LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hp,
major outer membrane protein OMP, outer membrane protein OmpA2
(Haemophilus ducreyi, Chancroid); aspartyl protease 1 Pep1,
phospholipase B PLB, alpha-mannosidase 1 AMN1,
glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein
Pmp1, proline-rich antigen Pra, humal T-cell reative protein TcrP
(Coccidioides immitis and Coccidioides posadasii,
Coccidioidomycosis); allergen Tri r 2, heat shock protein 60 Hsp60,
fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri t1,
protein IV, glycerol-3-phosphate dehydrogenase Gpd1, osmosensor
HwSho1A, osmosensor HwSho1B, histidine kinase HwHhk7B, allergen
Mala s 1, allergen Mala s 11, thioredoxin Trx Mala s 13, allergen
Mala f, allergen Mala s (usually Trichophyton spp, Epidermophyton
spp., Malassezia spp., Hortaea werneckii, Dermatophytosis); protein
EG95, protein EG10, protein EG18, protein EgA31, protein EM18,
antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3,
8-kDa protein, myophilin, heat shock protein 20 HSP20, glycoprotein
GP-89, fatty acid binding protein FAPB (Echinococcus genus,
Echinococcosis); major surface protein 2 MSP2, major surface
protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane
protein OMP, outer membrande protein 19 OMP-19, major antigenic
protein MAP1, major antigenic protein MAP1-2, major antigenic
protein MAP1B, major antigenic protein MAP1-3, Erum2510 coding
protein, protein GroEL, protein GroES, 30-kDA major outer membrane
proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa
protein (Ehrlichia genus, Ehrlichiosis); secreted antigen SagA,
sagA-like proteins SaIA and SaIB, collagen adhesin Sun, surface
proteins Fms1 (EbpA(fm), Fms5 (EbpB(fm), Fms9 (EpbC(fm) and Fms10,
protein EbpC(fm), 96 kDa immunoprotective glycoprotein G1
(Enterococcus genus, Enterococcus infection); genome polyprotein,
polymerase 3D, viral capsid protein VP1, viral capsid protein VP2,
viral capsid protein VP3, viral capsid protein VP4, protease 2A,
protease 3C (Enterovirus genus, Enterovirus infection); outer
membrane proteins OM, 60 kDa outer membrane protein, cell surface
antigen OmpA, cell surface antigen OmpB (sca5), 134 kDa outer
membrane protein, 31 kDa outer membrane protein, 29.5 kDa outer
membrane protein, cell surface protein SCA4, cell surface protein
Adr1 (RP827), cell surface protein Adr2 (RP828), cell surface
protein SCA1, Invasion protein invA, cell division protein fts,
secretion proteins sec 0family, virulence proteins virB, tlyA,
tlyC, parvulin-like protein Plp, preprotein translocase SecA,
120-kDa surface protein antigen SPA, 138 kD complex antigen, major
100-kD protein (protein I), intracytoplasmic protein D, protective
surface protein antigen SPA (Rickettsia prowazekii, Epidemic
typhus); Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A,
EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP)), latent membrane
proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane
antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease
EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gp110,
glycoprotein gp42, glycoprotein gHgL, glycoprotein gB (Epstein-Barr
Virus (EBV), Epstein-Barr Virus Infectious Mononucleosis); cpasid
protein VP2, capsid protein VP1, major protein NS1 (Parvovirus B19,
Erythema infectiosum (Fifth disease)); pp65 antigen, glycoprotein
105, major capsid protein, envelope glycoprotein H, protein U51
(Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7),
Exanthem subitum); thioredoxin-glutathione reductase TGR,
cathepsins L1 and L2, Kunitz-type protein KTM, leucine
aminopeptidase LAP, cysteine proteinase Fast, saposin-like
protein-2 SAP-2, thioredoxin peroxidases TN, Prx-1, Prx-2,
cathepsin I cysteine proteinase CL3, protease cathepsin L CL1,
phosphoglycerate kinase PGK, 27-kDa secretory protein, 60 kDa
protein HSP35alpha, glutathione transferase GST, 28.5 kDa
tegumental antigen 28.5 kDa TA, cathepsin B3 protease CatB3, Type I
cystatin stefin-1, cathepsin L5, cathepsin L1g and cathepsin B,
fatty acid binding protein FABP, leucine aminopeptidases LAP (
Fasciola hepatica and Fasciola gigantica, Fasciolosis); prion
protein (FFI prion, Fatal familial insomnia (FFI)); venom allergen
homolog-like protein VAL-1, abundant larval transcript ALT-1,
abundant larval transcript ALT-2, thioredoxin peroxidase TPX,
vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2,
antigenic protein SXP (peptides N, N1, N2, and N3), activation
associated protein-1 ASP-1, Thioredoxin TRX, transglutaminase
BmTGA, glutathione-S-transferases GST, myosin, vespid allergen
homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate
dehydrogenase GAPDH, cuticular collagen Col-4, secreted larval
acidic proteins SLAPs, chitinase CHI-1, maltose binding protein
MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba,
tropomyosin TMY-1, nematode specific gene product OvB20,
onchocystatin CPI-2, Cox-2 (Filarioidea superfamily, Filariasis);
phospholipase C PLC, heat-labile enterotoxin B, Iota toxin
component Ib, protein CPE1281, pyruvate ferredoxin oxidoreductase,
elongation factor G EF-G, perfringolysin O Pfo,
glyceraldehyde-3-phosphate dehydrogenase GapC,
Fructose-bisphosphate aldolase Alf2, clostridium perfringens
enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid
ETd, protein HP, large cytotoxin TpeL,
endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm
(Clostridium perfringens, Food poisoning by Clostridium
perfringens); leukotoxin IktA, adhesion FadA, outer membrane
protein RadD, high-molecular weight arginine-binding protein
(Fusobacterium genus, Fusobacterium infection); phospholipase C
PLC, heat-labile enterotoxin B, Iota toxin component Ib, protein
CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G
EF-G, perfringolysin O Pfo, glyceraldehyde-3-phosphate
dehydrogenase GapC, fructose-bisphosphate aldolase Alf2,
clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha
toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL,
endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm
(usually Clostridium perfringens; other Clostridium species, Gas
gangrene (Clostridial myonecrosis)); lipase A, lipase B, peroxidase
Dec1 (Geotrichum candidum, Geotrichosis); priori protein (GSS
prion, Gerstmann-Straussler-Scheinker syndrome (GSS)); cyst wall
proteins CWP1, CWP2, CWP3, variant surface protein VSP, VSP1, VSP2,
VSP3, VSP4, VSP5, VSP6, 56 kDa antigen; pyruvate ferredoxin
oxidoreductase PFOR, alcohol dehydrogenase E ADHE, alpha-giardin,
alpha8-giardin, alpha1-guiardin, beta-giardin, cystein proteases,
glutathione-S-transferase GST, arginine deiminase ADI,
fructose-1,6-bisphosphat aldolase FBA, Giardia trophozoite antigens
GTA (GTA1, GTA2), ornithine carboxyl transferase OCT, striated
fiber-asseblin-like protein SALP, uridine phosphoryl-like protein
UPL, alpha-tubulin, beta-tubulin (Giardia intestinalis,
Giardiasis); members of the ABC transporter family (LolC, OppA, and
PotF), putative lipoprotein releasing system transmembrane protein
LolC/E, flagellin FIiC, Burkholderia intracellular motility A BimA,
bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein,
boaA coding protein (Burkholderia mallei, Glanders); cyclophilin
CyP, 24 kDa third-stage larvae protien GS24, excretion-secretion
products ESPs (40, 80, 120 and 208 kDa) (Gnathostoma spinigerum and
Gnathostoma hispidum, Gnathostomiasis); pilin proteins, minor
pilin-associated subunit pilC, major pilin subunit and variants
pilE, pilS, phase variation protein porA, Porin B PorB, protein
TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major
outer membrane protein PI, outer membrane proteins PIA and PIB, W
antigen, surface protein A NspA, transferrin binding protein TbpA,
transferrin binding protein TbpB, PBP2, mtrR coding protein, ponA
coding protein, membrane permease FbpBC, FbpABC protein system,
LbpAB proteins, outer membrane protein Opa, outer membrane
transporter FetA, iron-repressed regulator MpeR (Neisseria
gonorrhoeae, Gonorrhea); outer membrane protein A OmpA, outer
membrane protein C OmpC, outer membrane protein K17 OmpK17
(Klebsiella granulomatis, Granuloma inguinale (Donovanosis));
fibronectin-binding protein Sfb, fibronectin/fibrinogen-binding
protein FBP54, fibronectin-binding protein FbaA, M protein type 1
Emm1, M protein type 6 Emm6, immunoglobulin-binding protein 35
Sib35, Surface protein R28 Spr28, superoxide dismutase SOD, C5a
peptidase ScpA, antigen I/II AgI/II, adhesin AspA, G-related
alpha2-macroglobulin-binding protein GRAB, surface fibrillar
protein M5 (Streptococcus pyogenes, Group A streptococcal
infection); C protein .beta. antigen, arginine deiminase proteins,
adhesin BibA, 105 kDA protein BPS, surface antigens c, surface
antigens R, surface antigens X, trypsin-resistant protein R1,
trypsin-resistant protein R3, trypsin-resistant protein R4, surface
immunogenic protein Sip, surface protein Rib, Leucine-rich repeats
protein LrrG, serine-rich repeat protein Srr-2, C protein
alpha-antigen Bca, Beta antigen Bag, surface antigen Epsilon,
alpha-like protein ALP1, alpha-like protein ALP5 surface antigen
delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like
protein ALP4, Cbeta protein Bac (Streptococcus agalactiae, Group B
streptococcal infection); transferrin-binding protein 2 Tbp2,
phosphatase P4, outer membrane protein P6, peptidoglycan-associated
lipoprotein Pal, protein D, protein E, adherence and penetration
protein Hap, outer membrane protein 26 Omp26, outer membrane
protein P5 (Fimbrin), outer membrane protein D15, outer membrane
protein OmpP2, 5'-nucleotidase NucA, outer membrane protein P1,
outer membrane protein P2, outer membrane lipoprotein Pcp,
Lipoprotein E, outer membrane protein P4, fuculokinase fucK,
[Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein 0145,
alpha-galactosylceramide (Haemophilus influenzae, Haemophilus
influenzae infection); polymerase 3D, viral capsid protein VP1,
viral capsid protein VP2, viral capsid protein VP3, viral capsid
protein VP4, protease 2A, protease 3C (Enteroviruses, mainly
Coxsackie A virus and Enterovirus 71 (EV71), Hand, foot and mouth
disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn,
glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein G1,
nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus,
Hantavirus, Hantavirus Pulmonary Syndrome (HPS)); heat shock
protein HspA, heat shock protein HspB, citrate synthase GltA,
protein UreB, heat shock protein Hsp60, neutrophil-activating
protein NAP, catalase KatA, vacuolating cytotoxin VacA, urease
alpha UreA, urease beta Ureb, protein Cpn10, protein groES, heat
shock protein Hsp10, protein MopB, cytotoxicity-associated 10 kDa
protein CAG, 36 kDa antigen, beta-lactamase HcpA, Beta-lactamase
HcpB (Helicobacter pylori, Helicobacter pylori infection); integral
membrane proteins, aggregation-prone proteins, O-antigen,
toxin-antigens Stx2B, toxin-antigen Stx1B, adhesion-antigen
fragment Int28, protein EspA, protein EspB, Intimin, protein Tir,
protein IntC300, protein Eae (Escherichia coli O157:H7, O111 and
O104:H4, Hemolytic-uremic syndrome (HUS)); RNA polymerase L,
protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein
S, envelope glycoprotein G1, nucleoprotein NP, protein N,
polyprotein M (Bunyaviridae family, Hemorrhagic fever with renal
syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein
N, fusion protein F, polymerase L, protein W, proteinC,
phosphoprotein p, non-structural protein V (Henipavirus (Hendra
virus Nipah virus), Henipavirus infections); polyprotein,
glycoproten Gp2, hepatitis A surface antigen HBAg, protein 2A,
virus protein VP1, virus protein VP2, virus protein VP3, virus
protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D
(Hepatitis A Virus, Hepatitis A); hepatitis B surface antigen
HBsAg, Hepatitis B core antigen HbcAg, polymerase, protein Hbx,
preS2 middle surface protein, surface protein L, large S protein,
virus protein VP1, virus protein VP2, virus protein VP3, virus
protein VP4 (Hepatitis B Virus, Hepatitis B); envelope glycoprotein
E1 gp32 gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid
protein C, core protein Core, polyprotein, virus protein VP1, virus
protein VP2, virus protein VP3, virus protein VP4, antigen G,
protein NS3, protein NS5A, (Hepatitis C Virus, Hepatitis C); virus
protein VP1, virus protein VP2, virus protein VP3, virus protein
VP4, large hepaptitis delta antigen, small hepaptitis delta antigen
(Hepatitis D Virus, Hepatitis D); virus protein VP1, virus protein
VP2, virus protein VP3, virus protein VP4, capsid protein E2
(Hepatitis E Virus, Hepatitis E); glycoprotein L UL1, uracil-DNA
glycosylase UL2, protein UL3, protein UL4, DNA replication protein
UL5, portal protein UL6, virion maturation protein UL7, DNA
helicase ULB, replication origin-binding protein UL9, glycoprotein
M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine
protein kinase UL13, tegument protein UL14, terminase UL15,
tegument protein UL16, protein UL17, capsid protein VP23 UL18,
major capsid protein VP5 UL19, membrane protein UL20, tegument
protein UL21, Glycoprotein H (UL22), Thymidine Kinase UL23, protein
UL24, protein UL25, capsid protein P40 (UL26, VP24,
VP22A),gGlycoprotein B (UL27), ICP18.5 protein (UL28), major
DNA-binding protein ICP8 (UL29), DNA polymerase UL30, nuclear
matrix protein UL31, envelope glycoprotein UL32, protein UL33,
inner nuclear membrane protein UL34, capsid protein VP26 (UL35),
large tegument protein UL36, capsid assembly protein UL37, VP19C
protein (UL38), ribonucleotide reductase (Large subunit) UL39,
ribonucleotide reductase (Small subunit) UL40, tegument
protein/virion host shutoff VHS protein (UL41), DNA polymerase
processivity factor UL42, membrane protein UL43, glycoprotein C
(UL44), membrane protein UL45, tegument proteins VP11/12 (UL46),
tegument protein VP13/14 (UL47), virion maturation protein VP16
(UL48, Alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50,
tegument protein UL51, DNA helicase/primase complex protein UL52,
glycoprotein K (UL53), transcriptional regulation protein 1E63
(ICP27, UL54), protein UL55, protein UL56, viral replication
protein ICP22 (1E68, US1), protein US2, serine/threonine-protein
kinase US3, glycoprotein G (US4),gGlycoprotein (US5), glycoprotein
D (US6),glycoprotein I (US7), glycoprotein E (US8), tegument
protein US9, capsid/tegument protein US10, Vmw21 protein (US11),
ICP47 protein (1E12, US12), major transcriptional activator ICP4
(1E175, RS1), E3 ubiquitin ligase ICP0 (1E110), latency-related
protein 1 LRP1, latency-related protein 2 LRP2, neurovirulence
factor RL1 (ICP34.5), latency-associated transcript LAT (Herpes
simplex virus 1 and 2 (HSV-1 and HSV-2), Herpes simplex); heat
shock protein Hsp60, cell surface protein H1C, dipeptidyl peptidase
type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like
protein (Histoplasma capsulatum, Histoplasmosis); fatty acid and
retinol binding protein-1 FAR-1, tissue inhibitor of
metalloproteinase TIMP (TMP), cysteine proteinase ACEY-1, cysteine
proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2,
metalloprotease 1 MTP-1, aspartyl protease inhibitor API-1,
surface-associated antigen SAA-1, surface-associated antigen SAA-2,
adult-specific secreted factor Xa, serine protease inhibitor
anticoagulant AP, cathepsin D-like aspartic protease ARR-1,
glutathione S-transferase GST, aspartic protease APR-1,
acetylcholinesterase AChE (Ancylostoma duodenale and Necator
americanus, Hookworm infection); protein NS1, protein NP1, protein
VP1, protein VP2, protein VP3 (Human bocavirus (HBoV), Human
bocavirus infection); major surface protein 2 MSP2, major surface
protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane
protein OMP, outer membrande protein 19 OMP-19, major antigenic
protein MAP1, major antigenic protein MAP1-2, major antigenic
protein MAP1B, major antigenic protein MAP1-3, Erum2510 coding
protein, protein GroEL, protein GroES, 30-kDA major outer membrane
proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa
protein (Ehrlichia ewingii, Human ewingii ehrlichiosis); major
surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type
IV secreotion system proteins VirB2, VirB7, VirB11, VirD4
(Anaplasma phagocytophilum, Human granulocytic anaplasmosis (HGA));
protein NS1, small hydrophobic protein NS2, SH protein, fusion
protein F, glycoprotein G, matrix protein M, matrix protein M2-1,
matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase
L (Human metapneumovirus (hMPV), Human metapneumovirus infection);
major surface protein 2 MSP2, major surface protein 4 MSP4, MSP
variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer
membrande protein 19 OMP-19, major antigenic protein MAP1, major
antigenic protein MAP1-2, major antigenic protein MAP1B, major
antigenic protein MAP1-3, Erum2510 coding protein, protein GroEL,
protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa
protein, GE 130-kDa protein, GE 160-kDa protein (Ehrlichia
chaffeensis, Human monocytic ehrlichiosis); replication protein E1,
regulatory protein E2, protein E3, protein E4, protein E5, protein
E6, protein E7, protein E8, major capsid protein L1, minor capsid
protein L2 (Human papillomavirus (HPV), Human papillomavirus (HPV)
infection); fusion protein F, hemagglutinin-neuramidase HN,
glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein
N, polymerase L (Human parainfluenza viruses (HPIV), Human
parainfluenza virus infection); "hemagglutinin HA, neuraminidase
NA, nucleoprotein NP, matrix protein M1, matrix protein M2, protein
NS1, polymerase complex PA, PB1, PB2, nuclear export protein NEP;;"
(Orthomyxoviridae family, Influenza (flu)); genome polyprotein,
protein E, protein M, capsid protein C (Japanese encephalitis
virus, Japanese encephalitis); RTX toxin, type IV piIi, major pilus
subunit PiIA, regulatory transcription factors PiIS and PiIR,
protein sigma54, outer membrane proteins (Kingella kingae, Kingella
kingae infection); prion protein (Kuru prion, Kuru); nucleoprotein
N, polymerase L, matrix protein Z, glycoprotein GP (Lassa virus,
Lassa fever); peptidoglycan-associated lipoprotein PAL, 60 kDa
chaperonin Cpn60 (groEL, HspB), type IV pilin PilE, outer membrane
protein MIP, major outer membrane protein MompS, zinc
metalloproteinase MSP (Legionella pneumophila, Legionellosis
(Legionnaires' disease, Pontiac fever)); P4 nuclease, protein WD,
ribonucleotide reductase M2, surface membrane glycoprotein Pg46,
cysteine proteinase CP, glucose-regulated protein 78 GRP-78,
stage-specific S antigen-like protein A2, ATPase F1, beta-tubulin,
heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein
BT1, nucleoside hydrolase NH, cell surface protein B1, ribosomal
protein P1-like protein P1, sterol 24-c-methyltransferase SMT, LACK
protein, histone H1, SPB1 protein, thiol specific antioxidant TSA,
protein antigen STI1, signal peptidase SP, histone H2B, suface
antigen PSA-2, cystein proteinase b Cpb (
Leishmania genus, Leishmaniasis); major membrane protein I,
serine-rich antigen-45 kDa, 10 kDa caperonin GroES, HSP kDa
antigen, amino-oxononanoate synthase AONS, protein recombinase A
RecA, Acetyl-/propionyl-coenzyme A carboxylase alpha, alanine
racemase, 60 kDa chaperonin 2, ESAT-6-like protein EcxB (L-ESAT-6),
protein Lsr2, protein ML0276, Heparin-binding hemagglutinin HBHA,
heat-shock protein 65 Hsp65, mycP1 or ML0041 coding protein, htrA2
or ML0176 coding protein, htrA4 or ML2659 coding protein, gcp or
ML0379 coding protein, cIpC or ML0235 coding protein (Mycobacterium
leprae and Mycobacterium lepromatosis, Leprosy); outer membrane
protein LipL32, membrane protein LIC10258, membrane protein LP30,
membrane protein LIC12238, Ompa-like protein Lsa66, surface protein
LigA, surface protein LigB, major outer membrane protein OmpL1,
outer membrane protein LipL41, protein LigAni, surface protein
LcpA, adhesion protein LipL53, outer membrane protein UpL32,
surface protein Lsa63, flagellin FIaB1, membran lipoprotein LipL21,
membrane protein pL40, leptospiral surface adhesin Lsa27, outer
membrane protein OmpL36, outer membrane protein OmpL37, outer
membrane protein OmpL47, outer membrane protein OmpL54,
acyltransferase LpxA (Leptospira genus, Leptospirosis);
listeriolysin O precursor Hly (LLO), invasion-associated protein
lap (P60), Listeriolysin regulatory protein PrfA, Zinc
metalloproteinase MpI, Phosphatidylinositol-specific phospholipase
C PLC (PICA, PIcB), 0-acetyltransferase Oat, ABC-transporter
permease Im.G.sub.--1771, adhesion protein LAP, LAP receptor Hsp60,
adhesin LapB, haemolysin listeriolysin 0 LLO, protein ActA,
Internalin A InIA, protein InIB (Listeria monocytogenes,
Listeriosis); outer surface protein A OspA, outer surface protein
OspB, outer surface protein OspC, decorin binding protein A DbpA,
decorin binding protein B DbpB, flagellar filament 41 kDa core
protein Fla, basic membrane protein A BmpA (Immunodominant antigen
P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7),
variable surface lipoprotein vIsE (usually Borrelia burgdorferi and
other Borrelia species, Lyme disease (Lyme borreliosis)); venom
allergen homolog-like protein VAL-1, abundant larval transcript
ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase
TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2,
antigenic protein SXP (peptides N, N1, N2, and N3), activation
associated protein-1 ASP-1, thioredoxin TRX, transglutaminase
BmTGA, glutathione-S-transferases GST, myosin, vespid allergen
homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate
dehydrogenase GAPDH, cuticular collagen Col-4, Secreted Larval
Acidic Proteins SLAPs, chitinase CHI-1, maltose binding protein
MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba,
tropomyosin TMY-1, nematode specific gene product OvB20,
onchocystatin CPI-2, protein Cox-2 (Wuchereria bancrofti and Brugia
malayi, Lymphatic filariasis (Elephantiasis)); glycoprotein GP,
matrix protein Z, polymerase L, nucleoprotein N (Lymphocytic
choriomeningitis virus (LCMV), Lymphocytic choriomeningitis);
thrombospondin-related anonymous protein TRAP, SSP2 Sporozoite
surface protein 2, apical membrane antigen 1 AMA1, rhoptry membrane
antigen RMA1, acidic basic repeat antigen ABRA, cell-traversal
protein PF, protein Pvs25, merozoite surface protein 1 MSP-1,
merozoite surface protein 2 MSP-2, ring-infected erythrocyte
surface antigen RESALiver stage antigen 3 LSA-3, protein Eba-175,
serine repeat antigen 5 SERA-5, circumsporozoite protein CS,
merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP8,
enolase PF10, hepatocyte erythrocyte protein 17 kDa HEP17,
erythrocyte membrane protein 1 EMP1, protein Kbetamerozoite surface
protein 4/5 MSP 4/Sheat shock protein Hsp90, glutamate-rich protein
GLURP, merozoite surface protein 4 MSP-4, protein STARP,
circumsporozoite protein-related antigen precursor CRA (Plasmodium
genus, Malaria); nucleoprotein N, membrane-associated protein VP24,
minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L,
matrix protein VP40, envelope glycoprotein GP (Marburg virus,
Marburg hemorrhagic fever (MHF)); protein C, matrix protein M,
phosphoprotein P, non-structural protein V, hemagglutinin
glycoprotein H, polymerase L, nucleoprotein N, fusion protein F
(Measles virus, Measles); members of the ABC transporter family
(LoIC, OppA, and PotF), putative lipoprotein releasing system
transmembrane protein LoIC/E, flagellin FliC, Burkholderia
intracellular motility A BimA, bacterial Elongation factor-Tu
EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding
protein (Burkholderia pseudomallei, Melioidosis (Whitmore's
disease)); pilin proteins, minor pilin-associated subunit pilC,
major pilin subunit and variants pilE, pilS, phase variation
protein porA, Porin B PorB, protein TraD, Neisserial outer membrane
antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer
membrane proteins PIA and PIB, W antigen, surface protein A NspA,
transferrin binding protein TbpA, transferrin binding protein TbpB,
PBP2, mtrR coding protein, ponA coding protein, membrane permease
FbpBC, FbpABC protein system, LbpAB proteins, outer membrane
protein Opa, outer membrane transporter FetA, iron-repressed
regulator MpeR, factor H-binding protein fHbp, adhesin NadA,
protein NhbA, repressor FarR (Neisseria meningitidis, Meningococcal
disease); 66 kDa protein, 22 kDa protein (usually Metagonimus
yokagawai, Metagonimiasis); polar tube proteins (34, 75, and 170
kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon),
kinesin-related protein, RNA polymerase II largest subunit, similar
of integral membrane protein YIPA, anti-silencing protein 1, heat
shock transcription factor HSF, protein kinase, thymidine kinase,
NOP-2 like nucleolar protein (Microsporidia phylum,
Microsporidiosis); CASP8 and FADD-like apoptosis regulator,
Glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, Poly(A)
polymerase catalytic subunit PAPL, Major envelope protein P43K,
early transcription factor 70 kDa subunit VETFS, early
transcription factor 82 kDa subunit VETFL, metalloendopeptidase
G1-type, nucleoside triphosphatase I NPH1, replication protein
A28-like MC134L, RNA polymease 7 kDa subunit RPO7 (Molluscum
contagiosum virus (MCV), Molluscum contagiosum (MC)); matrix
protein M, phosphoprotein P/V, small hydrophobic protein SH,
nucleoprotein N, protein V, fusion glycoprotein F,
hemagglutinin-neuraminidase HN, RNA polymerase L (Mumps virus,
Mumps); Outer membrane proteins OM, cell surface antigen OmpA, cell
surface antigen OmpB (sca5), cell surface protein SCA4, cell
surface protein SCA1, intracytoplasmic protein D, crystalline
surface layer protein SLP, protective surface protein antigen SPA
(Rickettsia typhi,Murine typhus (Endemic typhus)); adhesin P1,
adhesion P30, protein p116, protein P40, cytoskeletal protein HMW1,
cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152 coding
protein, MPN426 coding protein, MPN456 coding protein,
MPN-500coding protein (Mycoplasma pneumoniae, Mycoplasma
pneumonia); NocA, Iron dependent regulatory protein, VapA, VapD,
VapF, VapG, caseinolytic protease, filament tip-associated 43-kDa
protein, protein P24, protein P61, 15-kDa protein, 56-kDa protein
(usually Nocardia asteroides and other Nocardia species,
Nocardiosis); venom allergen homolog-like protein VAL-1, abundant
larval transcript ALT-1, abundant larval transcript ALT-2,
thioredoxin peroxidase TPX, vespid allergen homologue VAH,
thiordoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N,
N1, N2, and N3), activation associated protein-1 ASP-1, Thioredoxin
TRX, transglutaminase BmTGA, glutathione-S-transferases GST,
myosin, vespid allergen homologue VAH, 175 kDa collagenase,
glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen
Col-4, Secreted Larval Acidic Proteins SLAPs, chitinase CHI-1,
maltose binding protein MBP, glycolytic enzyme
fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode
specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Onchocerca
volvulus, Onchocerciasis (River blindness)); 43 kDa secreted
glycoprotein, glycoprotein gp0, glycoprotein gp75, antigen Pb27,
antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70,
heat shock protein Hsp90, protein P10, triosephosphate isomerase
TPI, N-acetyl-glucosamine-binding lectin Paracoccin, 28 kDa protein
Pb28 (Paracoccidioides brasiliensis, Paracoccidioidomycosis (South
American blastomycosis)); 28-kDa cruzipain-like cystein protease
Pw28CCP (usually Paragonimus westermani and other Paragonimus
species, Paragonimiasis); outer membrane protein OmpH, outer
membrane protein Omp28, protein PM1539, protein PM0355, protein
PM1417, repair protein MutL, protein BcbC, prtein PM0305, formate
dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase,
lipoprotein PIpE, adhesive protein Cp39, heme aquisition system
receptor HasR, 39 kDa capsular protein, iron-regulated OMP IROMP,
outer membrane protein OmpA87, fimbrial protein Ptf, fimbrial
subunit protein PtfA, transferrin binding protein TbpI, esterase
enzyme MesA, Pasteurella multocida toxin PMT, adhesive protein Cp39
(Pasteurella genus, Pasteurellosis); "filamentous hemagglutinin
FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor
PtxD, pertactin precursor Pm, toxin subunit 1 PtxA, protein Cpn60,
protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis
toxin subunit 3 precursor PNC, pertussis toxin subunit 5 precursor
PtxE, pertactin Pm, protein Fim2, protein Fim3;" (Bordetella
pertussis, Pertussis (Whooping cough)); "F1 capsule antigen,
virulence-associated V antigen, secreted effector protein LcrV, V
antigen, outer membrane protease Pla, secreted effector protein
YopD, putative secreted protein-tyrosine phosphatase YopH, needle
complex major subunit YscF, protein kinase YopO, putative
autotransporter protein YapF, inner membrane ABC-transporter YbtQ
(Irp7), putative sugar binding protein YPO0612, heat shock protein
90 HtpG, putative sulfatase protein YdeN, outer-membrane
lipoprotein carrier protein LoIA, secretion chaperone YerA,
putative lipoprotein YP00420, hemolysin activator protein HpmB,
pesticin/yersiniabactin outer membrane receptor Psn, secreted
effector protein YopE, secreted effector protein YopF, secreted
effector protein YopK, outer membrane protein YopN, outer membrane
protein YopM, Coagulase/fibrinolysin precursor Pla;" (Yersinia
pestis, Plague); protein PhpA, surface adhesin PsaA, pneumolysin
Ply, ATP-dependent protease Clp, lipoate-protein ligase LplA, cell
wall surface anchored protein psrP, sortase SrtA, glutamyl-tRNA
synthetase GltX, choline binding protein A CbpA, pneumococcal
surface protein A PspA, pneumococcal surface protein C PspC,
6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA,
Murein hydrolase LytB, proteon LytC, protease A1 (Streptococcus
pneumoniae, Pneumococcal infection); major surface protein B,
kexin-like protease KEX1, protein A12, 55 kDa antigen P55, major
surface glycoprotein Msg (Pneumocystis jirovecii, Pneumocystis
pneumonia (PCP)); genome polyprotein, polymerase 3D, viral capsid
protein VP1, viral capsid protein VP2, viral capsid protein VP3,
viral capsid protein VP4, protease 2A, protease 3C (Poliovirus,
Poliomyelitis); protein Nfa1, exendin-3, secretory lipase,
cathepsin B-like protease, cysteine protease, cathepsin,
peroxiredoxin, protein Cry1Ac (usually Naegleria fowleri, Primary
amoebic meningoencephalitis (PAM)); agnoprotein, large T antigen,
small T antigen, major capsid protein VP1, minor capsid protein Vp2
(JC virus, Progressive multifocal leukoencephalopathy); low calcium
response protein E LCrE, chlamydial outer protein N CopN,
serine/threonine-protein kinase PknD, acyl-carrier-protein
S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb,
major outer membrane protein MOMP, outer membrane protein 2 Omp2,
polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5,
Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15,
Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21) (Chlamydophila psittaci,
Psittacosis); outer membrane protein P1, heat shock protein B HspB,
peptide ABC transporter, GTP-binding protein, protein IcmB,
ribonuclease R, phosphatas SixA, protein DsbD, outer membrane
protein TolC, DNA-binding protein PhoB, ATPase DotB, heat shock
protein B HspB, membrane protein Com1, 28 kDa protein,
DNA-3-methyladenine glycosidase I, pouter membrane protein OmpH,
outer membrane protein AdaA, glycine cleavage system T-protein
(Coxiella burnetii, Q fever); nucleoprotein N, large structural
protein L, phophoprotein P, matrix protein M, glycoprotein G
(Rabies virus, Rabies); fusionprotein F, nucleoprotein N, matrix
protein M, matrix protein M2-1, matrix protein M2-2, phophoprotein
P, small hydrophobic protein SH, major surface glycoprotein G,
polymerase L, non-structural protein 1 NS1, non-structural protein
2 NS2 (Respiratory syncytial virus (RSV), Respiratory syncytial
virus infection); genome polyprotein, polymerase 3D, viral capsid
protein VP1, viral capsid protein VP2, viral capsid protein VP3,
viral capsid protein VP4, protease 2A, protease 3C (Rhinovirus,
Rhinovirus infection); outer membrane proteins OM, cell surface
antigen OmpA, cell surface antigen OmpB (sca5), cell surface
protein SCA4, cell surface protein SCA1, protein PS120,
intracytoplasmic protein D, protective surface protein antigen SPA
(Rickettsia genus, Rickettsial infection); outer membrane proteins
OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5),
cell surface protein SCA4, cell surface protein SCA1,
intracytoplasmic protein D (Rickettsia akari, Rickettsialpox);
envelope glycoprotein GP, polymerase L, nucleoprotein N,
non-structural protein NSS (Rift Valley fever virus, Rift Valley
fever (RVF)); outer membrane proteins OM, cell surface antigen
OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4,
cell surface protein SCA1, intracytoplasmic protein D (Rickettsia
rickettsii, Rocky mountain spotted fever (RMSF)); "non-structural
protein 6 NS6, non-structural protein 2 NS2, intermediate capsid
protein VP6, inner capsid protein VP2, non-structural protein 3
NS3, RNA-directed RNA polymerase L, protein VP3, non-structural
protein 1 NS1, non-structural protein 5 NS5, outer capsid
glycoprotein VP7, non-structural glycoprotein 4 NS4, outer capsid
protein VP4;;" (Rotavirus, Rotavirus infection); polyprotein P200,
glycoprotein E1, glycoprotein E2, protein NS2, capsid protein C
(Rubella virus, Rubella); chaperonin GroEL (MopA), inositol
phosphate phosphatase SopB, heat shock protein HslU, chaperone
protein DnaJ, protein TviB, protein IroN, flagellin FliC, invasion
protein SipC, glycoprotein gp43, outer membrane protein LamB, outer
membrane protein PagC, outer membrane protein TolC, outer membrane
protein NmpC, outer membrane protein FadL, transport protein SadA,
transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF
(Salmonella
genus, Salmonellosis); "protein 14, non-structural protein NS7b,
non-structural protein NS8a, protein 9b, protein 3a, nucleoprotein
N, non-structural protein NS3b, non-structural protein NS6, protein
7a, non-structural protein NS8b, membrane protein M, envelope small
membrane protein EsM, replicase polyprotein 1a, spike glycoprotein
S, replicase polyprotein lab;" (SARS coronavirus, SARS (Severe
Acute Respiratory Syndrome)); serin protease, Atypical Sarcoptes
Antigen 1 ASA1, glutathione S-transferases GST, cystein protease,
serine protease, apolipoprotein (Sarcoptes scabiei, Scabies);
glutathione 5-transferases GST, paramyosin, hemoglbinase SM32,
major egg antigen, 14 kDa fatty acid-binding protein Sm14, major
larval surface antigen P37, 22,6 kDa tegumental antigen, calpain
CANP, triphospate isomerase Tim, surface protein 9B, outer capsid
protein VP2, 23 kDa integral membrane protein Sm23,
Cu/Zn-superoxide dismutase, glycoprotein Gp, myosin (Schistosoma
genus, Schistosomiasis (Bilharziosis)); 60 kDa chaperonin, 56 kDa
type-specific antigen, pyruvate phosphate dikinase,
4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi,
Scrub typhus); dehydrogenase GuaB, invasion protein Spa32, invasin
IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH,
invasin Ipa) (Shigella genus, Shigellosis (Bacillary dysentery));
protein P53, virion protein US10 homolog, transcriptional regulator
1E63, transcriptional transactivator 1E62, protease P33, alpha
trans-inducing factor 74 kDa protein, deoxyuridine 5'-triphosphate
nucleotidohydrolase, transcriptional transactivator 1E4, membrane
protein UL43 homolog, nuclear phosphoprotein UL3 homolog, nuclear
protein UL4 homolog, replication origin-binding protein, membrane
protein 2, phosphoprotein 32, protein 57,DNA polymerase
processivity factor, portal protein 54, DNA primase, tegument
protein UL14 homolog, tegument protein UL21 homolog, tegument
protein UL55 homolog, tripartite terminase subunit UL33 homolog,
tripartite terminase subunit UL15 homolog, capsid-binding protein
44, virion-packaging protein 43 (Varicella zoster virus (VZV),
Shingles (Herpes zoster)); truncated 3-beta hydroxy-5-ene steroid
dehydrogenase homolog, virion membrane protein A13, protein A19,
protein A31, truncated protein A35 homolog, protein A37.5 homolog,
protein A47, protein A49, protein A51, semaphorin-like protein A43,
serine proteinase inhibitor 1, serine proteinase inhibitor 2,
serine proteinase inhibitor 3, protein A6, protein B15, protein C1,
protein C5, protein C6, protein F7, protein F8, protein F9, protein
F11, protein F14, protein F15, protein F16 (Variola major or
Variola minor, Smallpox (Variola)); adhesin/glycoprotein gp70,
proteases (Sporothrix schenckii, Sporotrichosis); heme-iron binding
protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein
MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA,
enterotoxin type B EntB, enterotoxin type C EntC1, enterotoxin type
C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, Toxic
shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding
protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus
genus, Staphylococcal food poisoning); heme-iron binding protein
IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA,
fibronectin-binding protein A FnbA, enterotoxin type A EntA,
enterotoxin type B EntB, enterotoxin type C EntC1, enterotoxin type
C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, Toxic
shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding
protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus
genus, Staphylococcal infection); antigen Ss-IR, antigen NIE,
strongylastacin, Na+-K+ATPase Sseat-6, tropomysin SsTmy-1, protein
LEC-5, 41 kDa aantigen P5, 41-kDa larval protein, 31-kDa larval
protein, 28-kDa larval protein (Strongyloides stercoralis,
Strongyloidiasis); glycerophosphodiester phosphodiesterase GlpQ
(Gpd), outer membrane protein TmpB, protein Tp92, antigen TpF1,
repeat protein Tpr, repeat protein F TprF, repeat protein G TprG,
repeat protein I Tpr1, repeat protein J TprJ, repeat protein K
TprK, treponemal membrane protein A TmpA, lipoprotein, 15 kDa
Tpp15, 47 kDa membrane antigen, miniferritin TpF1, adhesin Tp0751,
lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane
protein TP0136, outer membrane protein TP0155, outer membrane
protein TP0326, outer membrane protein TP0483, outer membrane
protein TP0956 (Treponema pallidum, Syphilis); Cathepsin L-like
proteases, 53/25-kDa antigen, 8 kDa family members, cysticercus
protein with a marginal trypsin-like activity TsAg5, oncosphere
protein TSOL18, oncosphere protein TSOL45-1 .ANG., lactate
dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia genus,
Taeniasis); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S
layer protein, flavoprotein beta-subunit CT3, phospholipase
(lecithinase), phosphocarrier protein HPr (Clostridium tetani,
Tetanus (Lockjaw)); genome polyprotein, protein E, protein M,
capsid protein C (Tick-borne encephalitis virus (TBEV), Tick-borne
encephalitis); 58-kDa antigen, 68-kDa antigens, Toxocara larvae
excretory-secretory antigen TES, 32-kDa glycoprotein, glycoprotein
TES-70, glycoprotein GP31, excretory-secretory antigen TcES-57,
perienteric fluid antigen Pe, soluble extract antigens Ex,
excretory/secretory larval antigens ES, antigen TES-120,
polyprotein allergen TBA-1, cathepsin L-like cysteine protease
c-cpl-1, 26-kDa protein (Toxocara canis or Toxocara cati,
Toxocariasis (Ocular Larva Migrans (OLM) and Visceral Larva Migrans
(VLM))); microneme proteins (MIC1, MIC2, MIC3, MIC4, MIC5, MIC6,
MIC7, MIC8), rhoptry protein Rop2, rhoptry proteins (Rop1, Rop2,
Rop3, Rop4, Rop5, Rop6, Rop7, Rop16, Rjop17), protein SR1,surface
antigen P22, major antigen p24, major surface antigen p30, dense
granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8,
GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2 related
antigen, nucleoside-triphosphatase 1, nucleoside-triphosphatase 2,
protein Stt3, HesB-like domain-containing protein, rhomboid-like
protease 5, toxomepsin 1 (Toxoplasma gondii, Toxoplasmosis); 43 kDa
secreted glycoprotein, 53 kDa secreted glycoprotein, paramyosin,
antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigens,
caveolin-1 CAV-1, 49 kDa newborn larva antigen, prosaposin
homologue, serine protease, serine proteinase inhibitor, 45-kDa
glycoprotein Gp45 (Trichinella spiralis, Trichinellosis); Myb-like
transcriptional factors (Myb1, Myb2, Myb3), adhesion protein AP23,
adhesion protein AP33, adhesin protein AP33-3, adhesins AP51,
adhesin AP65, adhesion protein AP65-1, alpha-actinin,
kinesin-associated protein, teneurin, 62 kDa proteinase,
subtilisin-like serine protease SUB1, cysteine proteinase gene 3
CP3, alpha-enolase Enol, cysteine proteinase CP30, heat shock
proteins (Hsp70, Hsp60), immunogenic protein P270, (Trichomonas
vaginalis, Trichomoniasis); beta-tubulin, 47-kDa protein, secretory
leucocyte-like proteinase-1 SLP-1, 50-kDa protein TT50, 17 kDa
antigen, 43/47 kDa protein (Trichuris trichiura, Trichuriasis
(Whipworm infection)); protein ESAT-6 (EsxA), 10 kDa filtrate
antigen EsxB, secreted antigen 85-B FBPB, fibronectin-binding
protein A FbpA (Ag85A), serine protease PepA, PPE family protein
PPE18, fibronectin-binding protein D FbpD, immunogenic protein
MPT64, secreted protein MPT51, catalase-peroxidase-peroxynitritase
T KATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3,
Phos-1), iron-regulated heparin binding hemagglutinin Hbha, PPE
family protein PPE14, PPE family protein PPE68, protein Mtb72F,
protein Apa, immunogenic protein MPT63, periplasmic
phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone
DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate
transport system permease protein pstA, 14 kDa antigen,
fibronectin-binding protein C FbpC1, Alanine dehydrogenase TB43,
Glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4
protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like
proteins, protein MTB32, protein MTB39, crystallin, heat-shock
protein HSP65, protein PST-S(usually Mycobacterium tuberculosis,
Tuberculosis); outer membrane protein FobA, outer membrane protein
FobB, intracellular growth locus IgIC1, intracellular growth locus
IgIC2, aminotransferase Wbtl, chaperonin GroEL, 17 kDa major
membrane protein TUL4, lipoprotein LpnA, chitinase family 18
protein, isocitrate dehydrogenase, Nif3 family protein, type IV
pili glycosylation protein, outer membrane protein tolC, FAD
binding family protein, type IV pilin multimeric outer membrane
protein, two component sensor protein KdpD, chaperone protein DnaK,
protein TolQ (Francisella tularensis, Tularemia); "MB antigen,
urease, protein GyrA, protein GyrB, protein ParC, protein ParE,
lipid associated membrane proteins LAMP, thymidine kinase TK,
phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C,
surface-expressed 96-kDa antigen;" (Ureaplasma urealyticum,
Ureaplasma urealyticum infection); non-structural polyprotein,
structural polyprotein, capsid protein CP, protein E1, protein E2,
protein E3, protease P1, protease P2, protease P3 (Venezuelan
equine encephalitis virus, Venezuelan equine encephalitis);
glycoprotein GP, matrix protein Z, polymerase L, nucleoprotein N
(Guanarito virus, Venezuelan hemorrhagic fever); polyprotein,
protein E, protein M, capsid protein C, protease NS3, protein NS1,
protein NS2A, protein AS2B, brotein NS4A, protein NS4B, protein NS5
(West Nile virus, West Nile Fever); cpasid protein CP, protein E1,
protein E2, protein E3, protease P2 (Western equine encephalitis
virus, Western equine encephalitis); genome polyprotein, protein E,
protein M, capsid protein C, protease NS3, protein NS1, protein
NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (Yellow
fever virus, Yellow fever); putative Yop targeting protein YobB,
effector protein YopD, effector protein YopE, protein YopH,
effector protein YopJ, protein translocation protein YopK, effector
protein YopT, protein YpkA, flagellar biosyntheses protein FlhA,
peptidase M48, potassium efflux system KefA, transcriptional
regulatoer RovA, adhesin Ifp, translocator portein LcrV, protein
PcrV, invasin Inv, outer membrane protein OmpF-like porin, adhesin
YadA, protein kinase C, phospholipase C1, protein PsaA,
mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa
(Yersinia pseudotuberculosis, Yersinia pseudotuberculosis
infection); effector protein YopB, 60 kDa chaperonin, protein WbcP,
tyrosin-protein phosphatase YopH, protein YopQ, enterotoxin,
Galactoside permease, reductaase NrdE, protein YasN, Invasin Inv,
adhesin YadA, outer membrane porin F OmpF, protein UspA1, protein
EibA, protein Hia, cell surface protein Ail, chaperone SycD,
protein LcrD, protein LcrG, protein LcrV, protein SycE, protein
YopE, regulator protein TyeA, protein YopM, protein YopN, protein
YopO, protein YopT, protein YopD, protease CIpP, protein MyfA,
protein FilA, and protein PsaA (Yersinia enterocolitica,
Yersiniosis).
(in brackets are the particular pathogen of which the antigen(s)
is/are derived and the infectious disease with which the antigen is
associated)
[0309] In specific embodiments according to the present invention,
following antigens of pathogens associated with infectious disease
are particularly preferred: [0310] The nucleoprotein (N), the
phosphoprotein (P), the matrix protein (M), the glycoprotein (G),
and the viral RNA polymerase (L), in each case of Rabies virus;
[0311] the Hepatitis B surface antigen (HBsAg), the Hepatitis B
core antigen (HbcAg), the Hepatitis B virus DNA polymerase, the HBx
protein, the preS2 middle surface protein, the large S protein, the
virus protein VP1, the virus protein VP2, the virus protein VP3,
and the virus protein VP4, in each case of Hepatitis B virus;
[0312] the E1 protein, the E2 protein, the E3 protein, the E4
protein, the E5 protein, the E6 protein, the E7 protein, the E8
protein, the L1 protein, and the L2 protein, in each case of human
Papilloma virus (hPV); [0313] the protective antigen (PA), the
edema factor (EF), the lethal factor (LF), and the S-layer homology
proteins (SLH), in each case of Bacillus anthracis; [0314] the
Fusion (F) protein, the nucleocapsid (N) protein, the
phosphoprotein (P), the matrix (M) protein, the glycoprotein (G),
the large protein (L; RNA polymerase), the non-structural protein 1
(NS1), the non-structural protein 2 (NS2), the small hydrophobic
(SH) protein, the elongation factor M2-1, and the transcription
regulation protein M2-2, in each case of respiratory syncytial
virus (RSV); [0315] the Glycoprotein L (UL1), the Uracil-DNA
glycosylase UL2, the UL3 protein, the UL4 protein, the DNA
replication protein UL5, the Portal protein UL6, the Virion
maturation protein UL7, the DNA helicase UL8, the Replication
origin-binding protein UL9, the Glycoprotein M (UL10), the UL11
protein, the Alkaline exonuclease UL12, the Serine-threonine
protein kinase UL13, the Tegument protein UL14, the Terminase
(UL15), the Tegument protein UL16, the UL17 protein, the Capsid
protein VP23 (UL18), the Major capsid protein VP5 (UL19), the
Membrane protein UL20, the Tegument protein UL21, the Glycoprotein
H (UL22), the Thymidine Kinase UL23, the UL24 protein, the UL25
protein, the Capsid protein P40 (UL26, VP24, VP22A), the
Glycoprotein B (UL27), the ICP18.5 protein (UL28), the Major
DNA-binding protein ICP8 (UL29), the DNA polymerase UL30, the
Nuclear matrix protein UL31, the Envelope glycoprotein UL32, the
UL33 protein, the Inner nuclear membrane protein UL34, the Capsid
protein VP26 (UL35), the Large tegument protein UL36, the Capsid
assembly protein UL37, the VP19C protein (UL38), the Ribonucleotide
reductase (Large subunit) UL39, the Ribonucleotide reductase (Small
subunit) UL40, the Tegument protein/Virion host shutoff VHS protein
(UL41), the DNA polymerase processivity factor UL42, the Membrane
protein UL43, the Glycoprotein C (UL44), the Membrane protein UL45,
the Tegument proteins VP11/12 (UL46), the Tegument protein VP13/14
(UL47), the Virion maturation protein VP16 (UL48, Alpha-TIF), the
Envelope protein UL49, the dUTP diphosphatase UL50, the Tegument
protein UL51, the DNA helicase/primase complex protein UL52, the
Glycoprotein K (UL53), the Transcriptional regulation protein 1E63
(ICP27, UL54), the UL55 protein, the UL56 protein, the Viral
replication protein ICP22 (1E68, US1), the US2 protein, the
Serine/threonine-protein kinase US3, the Glycoprotein G (US4), the
Glycoprotein) (US5), the Glycoprotein D (US6), the Glycoprotein I
(US7), the Glycoprotein E (US8), the Tegument protein US9, the
CapsicVTegument protein US10, the Vmw21 protein (US11), the ICP47
protein (1E12, US12), the Major transcriptional activator ICP4
(1E175, RS1), the E3 ubiquitin ligase ICPO (1E110), the
Latency-related protein 1 (LRP1), the Latency-related protein 2
(LRP2), the Neurovirulence factor RL1 (ICP34.5), and the
Latency-associated transcript (LAT), in each case of Herpes simplex
virus (HSV); or [0316] the ESAT-6 protein, the ESX-1 protein, the
CFP10 protein, the TB10.4 protein, the MPT63 protein, the MPT64
protein, the MPT83 protein, the MTB12 protein, the MTB8 protein,
the AG85A protein, the AG85B protein, the Rpf-like proteins, the
KATG protein, the PPE18 protein, the MTB32 protein, the MTB39
protein, the Crystallin, the HSP65 protein, the PST-S protein, and
the HBHA protein, the 10 kDa filtrate antigen EsxB, the serine
protease PepA, the fibronectin-binding protein D FbpD, the secreted
protein MPT51, the periplasmic phosphate-binding lipoprotein PSTS1
(PBP-1), the periplasmic phosphate-binding lipoprotein PSTS3
(PBP-3, Phos-1), the PPE family protein PPE14, the PPE family
protein PPE68, the protein MTB72F, the molecular chaperone DnaK,
the cell surface lipoprotein MPT83, the lipoprotein P23, the
Phosphate transport system permease protein PstA, the 14 kDa
antigen, the fibronectin-binding protein C FbpC1, the Alanine
dehydrogenase TB43, and the Glutamine synthetase 1, in each case of
Mycobacterium tuberculosis. b) Antigens associated with allergy or
allergic disease (allergenic antigens or allergens):
[0317] According to another alternative, one further class of
antigens comprises allergenic antigens. Such allergenic antigens
may be selected from antigens derived from different sources, e.g.
from animals, plants, fungi, bacteria, etc. Sources of allergens in
this context include e.g. grasses, pollens, molds, drugs, or
numerous environmental triggers, etc. Allergenic antigens typically
belong to different classes of compounds, such as nucleic acids and
their fragments, proteins or peptides and their fragments,
carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc.
Of particular interest in the context of the present invention are
protein or peptide antigens and their fragments or epitopes, or
nucleic acids and their fragments, particularly nucleic acids and
their fragments, encoding such protein or peptide antigens and
their fragments or epitopes.
[0318] In alternative embodiments, said antigen is a peptide or
protein antigen, or a fragment, variant and/or derivative of said
peptide or protein antigen, such as a peptide or protein antigen
comprised in a preparation extracted from said source. In
alternative embodiments, a peptide or protein antigen used in the
present invention is not one comprised in a preparation extracted
from said source, and/or is one that is not obtained from a
preparation extracted from said source.
[0319] Antigens associated with allergy or allergic disease
(allergens) are preferably derived from a source selected from the
list consisting of:
[0320] Acarus spp (Aca s 1, Aca s 10, Aca s 10.0101, Aca s 13, Aca
s 13.0101, Aca s 2, Aca s 3, Aca s 7, Aca s 8), Acanthocybium spp
(Aca so 1), Acanthocheilonema spp (Aca v 3, Aca v 3.0101), Acetes
spp (Ace ja 1), Actinidia spp (Act a 1, Act c 1, Act c 10, Act c
10.0101, Act c 2, Act c 4, Act c 5, Act c 5.0101, Act c 8, Act c
8.0101, Act c Chitinase, Act d 1, Act d 1.0101, Act d 10, Act d
10.0101, Act d 10.0201, Act d 11, Act d 11.0101, Act d 2, Act d
2.0101, Act d 3, Act d 3.0101, Act d 3.02, Act d 4, Act d 4.0101,
Act d 5, Act d 5.0101, Act d 6, Act d 6.0101, Act d 7, Act d
7.0101, Act d 8, Act d 8.0101, Act d 9, Act d 9.0101, Act d
Chitinase, Act e 1, Act e 5), Acyrthosiphon spp (Acy pi 7, Acy pi
7.0101, Acy pi 7.0102), Adenia spp (Ade v RIP), Aedes spp (Aed a 1,
Aed a 1.0101, Aed a 2, Aed a 2.0101, Aed a 3, Aed a 3.0101, Aed a
4, Aed a 7, Aed a 7.0101, Aed a 7.0102, Aed a 7.0103, Aed a 7.0104,
Aed a 7.0105, Aed a 7.0106, Aed a 7.0107, Aed a 7.0108, Aed a
7.0109, Aed a 7.0110, Aed a 7.0111, Aed al 1, Aed al 3, Aed al 37
kD, Aed v 37 kD, Aed v 63 kD), Aegilops spp (Aeg ta 28, Aeg ta
alpha_Gliadin, Aeg um 28, Aeg un 28), Aethaloperca spp (Aet ro 1),
Agropyron spp (Agr c 7), Agrostis spp (Agr ca 1, Agr ca 5, Agr g 1,
Agr g 4, Agr s 5), Agrobacterium spp (Agr sp CP4 EPSPS), Ailuropoda
spp (Ail me Phosvitin, Ail me TCTP), Aix spp (Aix ga 1, Aix sp 1),
Aleuroglyphus spp (Ale o 1, Ale o 10, Ale o 10.0101, Ale o 10.0102,
Ale o 13, Ale o 14, Ale o 2, Ale o 20, Ale o 3, Ale o 5, Ale o 7,
Ale o 8, Ale o 9), Allium spp (All a 3, All a Alliin lyase, All c
3, All c 30 kD, All c 4, All c Alliin lyase, All p Alliin lyase,
All s Alliin lyase), Alnus spp (Aln g 1, Aln g 1.0101, Aln g 1/Bet
v 1/Cor a 1 TPC7, Aln g 1/Bet v 1/Cor a 1 TPC9, Aln g 2, Aln g 4,
Aln g 4.0101), Alopochen spp (Alo ae 1), Alopecurus spp (Alo p 1,
Alo p 5), Alternaria spp (Alt a 1, Alt a 1.0101, Alt a 1.0102, Alt
a 10, Alt a 10.0101, Alt a 12, Alt a 12.0101, Alt a 13, Alt a
13.0101, Alt a 2, Alt a 3, Alt a 3.0101, Alt a 4, Alt a 4.0101, Alt
a 5, Alt a 5.0101, Alt a 6, Alt a 6.0101, Alt a 7, Alt a 7.0101,
Alt a 70 kD, Alt a 8, Alt a 8.0101, Alt a 9, Alt a MnSOD, Alt a
NTF2, Alt a TCTP, Alt ar 1, Alt arg 1, Alt b 1, Alt bl 1, Alt br 1,
Alt c 1, Alt ca 1, Alt ce 1, Alt ch 1, Alt ci 1, Alt co 1, Alt cr
1, Alt ct 1, Alt cu 1, Alt cy 1, Alt d 1, Alt du 1, Alt e 1, Alt et
1, Alt eu 1, Alt ga 1, Alt gr 1, Alt j 1, Alt 11, Alt lo 1, Alt m
1, Alt me 1, Alt mi 1, Alt mo 1, Alt o 1, Alt p 1, Alt ph 1, Alt po
1, Alt ps 1, Alt r 1, Alt s 1, Alt se 1, Alt sm 1, Alt so 1, Alt su
1, Alt t 1, Alt to 1, Alt to 1), Amaranthus spp (Ama r 2, Ama r
2.0101, Ama v 2, Ama v 2.0101, Ama v 2.0201), Ambrosia spp (Amb a
1, Amb a 1.0101, Amb a 1.0201, Amb a 1.0202, Amb a 1.0301, Amb a
1.0302, Amb a 1.0303, Amb a 1.0304, Amb a 1.0305, Amb a 1.0401, Amb
a 1.0402, Amb a 1.0501, Amb a 1.0502, Amb a 10, Amb a 10.0101, Amb
a 3, Amb a 3.0101, Amb a 4, Amb a 4.0101, Amb a 5, Amb a 5.0101,
Amb a 6, Amb a 6.0101, Amb a 7, Amb a 7.0101, Amb a 8, Amb a
8.0101, Amb a 8.0102, Amb a 9, Amb a 9.0101, Amb a 9.0102, Amb a
CPI, Amb p 1, Amb p 5, Amb p 5.0101, Amb p 5.0201, Amb t 5, Amb t
5.0101, Amb t 8), Ammothea spp (Amm h 7, Amm h 7.0101), Anadara spp
(Ana br 1), Ananas spp (Ana c 1, Ana c 1.0101, Ana c 2, Ana c
2.0101, Ana c 2.0101 (MUXF3)), Anas spp (Ana ca 1), Anarhichas spp
(Ana I 1), Anacardium spp (Ana o 1, Ana o 1.0101, Ana o 1.0102, Ana
o 2, Ana o 2.0101, Ana o 3, Ana o 3.0101), Anas spp (Ana p 1, Ana p
2, Ana p 3), Anguilla spp (Ang a 1, Ang j 1), Anisakis spp (Ani s
1, Ani s 1.0101, Ani s 10, Ani s 10.0101, Ani s 11, Ani s 11.0101,
Ani s 12, Ani s 12.0101, Ani s2, Ani s 2.0101, Ani s 24 kD, Ani s
3, Ani s 3.0101, Ani s 4, Ani s 4.0101, Ani s5, Ani s 5.0101, Ani s
6, Ani s 6.0101, Ani s 7, Ani s 7.0101, Ani s 8, Ani s 8.0101, Ani
s 9, Ani s 9.0101, Ani s CCOS3, Ani s Cytochrome B, Ani s FBPP, Ani
s NADHDS4L, Ani s NARaS, Ani s PEPB, Ani s Troponin), Annona spp
(Ann c Chitinase), Anopheles spp (Ano da 17, Ano da 17.0101, Ano da
27, Ano da 27.0101, Ano da 7, Ano da 7.0101, Ano g 7, Ano g
7.0101), Anser spp (Ans a 1, Ans a 2, Ans a 3, Ans in 1),
Anthoxanthum spp (Ant o 1, Ant o 1.0101, Ant o 12, Ant o 13, Ant o
2, Ant o 4, Ant o 5, Ant o 6, Ant o 7), Apis spp (Api c 1, Api c
1.0101, Api c 10, Api c 2, Api c 4, Api d 1, Api d 1.0101, Api d 4,
Api fl 4), Apium spp (Api g 1, Api g 1.0101, Api g 1.0201, Api g 2,
Api g 2.0101, Api g 3, Api g 3.0101, Api g 4, Api g 4.0101, Api g
5, Api g 5.0101, Api g 6, Api g 6.0101), Apis spp (Api m 1, Api m
1.0101, Api m 10, Api m 10.0101, Api m 11, Api m 11.0101, Api m
11.0201, Api m 13 kD, Api m 2, Api m 2.0101, Api m 3, Api m 3.0101,
Api m 4, Api m 4.0101, Api m 5, Api m 5.0101, Api m 6, Api m
6.0101, Api m 7, Api m 7.0101, Api m 8, Api m 8.0101, Api m 9, Api
m 9.0101, Api m A1-A2, Api m A1-A2-A3, Api m Apalbumin 1, Api m
Apalbumin 2, Api me 1, Api me 4), Arachis spp (Ara d 2, Ara d 6,
Ara f 3, Ara f 4, Ara h 1, Ara h 1.0101, Ara h 10, Ara h 10.0101,
Ara h 10.0102, Ara h 11, Ara h 11.0101, Ara h 2, Ara h 2.0101, Ara
h 2.0102, Ara h 2.0201, Ara h 2.0202, Ara h 3, Ara h 3.0101, Ara h
4, Ara h 4.0101, Ara h 5, Ara h 5.0101, Ara h 6, Ara h 6.0101, Ara
h 7, Ara h 7.0101, Ara h 7.0201, Ara h 7.0202, Ara h 8, Ara h
8.0101, Ara h 8.0201, Ara h 9, Ara h 9.0101, Ara h 9.0201, Ara h
Agglutinin, Ara h Oleosin 18 kD, Ara i 2, Ara i 6), Arabidopsis spp
(Ara t 3, Ara t 8, Ara t GLP), Archosargus spp (Arc pr 1),
Archaeopotamobius spp (Arc s 8, Arc s 8.0101), Aequipecten spp (Arg
i 1), Argas spp (Arg r 1, Arg r 1.0101), Ariopsis spp (Ari fe 1),
Armoracia spp (Arm r HRP), Arrhenatherum spp (Arr e 1, Arr e 5),
Artemisia spp (Art a 1, Art ap 1), Artemia spp (Art fr 1, Art fr
1.0101, Art fr 5, Art fr 5.0101), Arthrobacter spp (Art gl CO),
Achorion spp (Art gy 7), Artocarpus spp (Art h 17 kD, Art h 4),
Arthrospira spp (Art pl beta_Phycocyanin), Artemisia spp (Art v 1,
Art v 1.0101, Art v 1.0102, Art v 1.0103, Art v 1.0104, Art v
1.0105, Art v 1.0106, Art v 1.0107, Art v 2, Art v 2.0101, Art v 3,
Art v 3.0101, Art v 3.0201, Art v 3.0202, Art v 3.0301, Art v 4,
Art v 4.0101, Art v 4.0201, Art v 47 kD, Art v 5, Art v 5.0101, Art
v 6, Art v 6.0101, Art v 60 kD), Arthroderma spp (Art va 4),
Ascaris spp (Asc I 3, Asc I 3.0101, Asc I 3.0102, Asc I 34 kD, Asc
s 1, Asc s 1.0101, Asc s 3, Asc s 3.0101, Asc s GST), Aspergillus
spp (Asp aw Glucoamylase, Asp c 22, Asp f 1, Asp f 1.0101, Asp f
10, Asp f 10.0101, Asp f 11, Asp f 11.0101, Asp f 12, Asp f
12.0101, Asp f 13, Asp f 13.0101, Asp f 15, Asp f 15.0101, Asp f
16, Asp f 16.0101, Asp f 17, Asp f 17.0101, Asp f 18, Asp f
18.0101, Asp f 2, Asp f 2.0101, Asp f 22, Asp f 22.0101, Asp f 23,
Asp f 23.0101, Asp f 27, Asp f 27.0101, Asp f 28, Asp f 28.0101,
Asp f 29, Asp f 29.0101, Asp f 3, Asp f 3.0101, Asp f 34, Asp f
34.0101, Asp f 4, Asp f 4.0101, Asp f 5, Asp f 5.0101, Asp f 56 kD,
Asp f 6, Asp f 6.0101, Asp f 7, Asp f 7.0101, Asp f 8, Asp f
8.0101, Asp f 9, Asp f 9.0101, Asp f AfCalAp, Asp f AT_V, Asp f
Catalase, Asp f Chitosanase, Asp f CP, Asp f DPPV, Asp f FDH, Asp f
gamma_Actin, Asp f Glucosidase, Asp f GPI, Asp f GST, Asp f GT, Asp
f IAO, Asp f IPMI, Asp f LPL1, Asp f LPL3, Asp f Mannosidase, Asp f
MDH, Asp f PL, Asp f PUP, Asp f RPS3, Asp f SXR, Asp fl 13, Asp fl
13.0101, Asp fl 18, Asp fl 2, Asp fl 21, Asp fl 3, Asp fl 4, Asp fl
7, Asp fl 8, Asp fl 9, Asp me Seaprose, Asp n 14, Asp n 14.0101,
Asp n 18, Asp n 18.0101, Asp n 25, Asp n 25.0101, Asp n 30, Asp n
Glucoamylase, Asp n Hemicellulase, Asp n Pectinase, Asp o 13, Asp o
13.0101, Asp o 21, Asp o 21.0101, Asp o 3, Asp o 4, Asp o 7, Asp o
8, Asp o Lactase, Asp o Lipase, Asp oc 13, Asp r 1, Asp sa AP, Asp
sp Glucoamylase, Asp sp Glucoseoxidase, Asp sp PL, Asp sp PME, Asp
sy 13, Asp v 13, Asp v 13.0101, Asp v Catalase A, Asp v Enolase,
Asp v GAPDH, Asp v MDH, Asp v SXR), Asparagus spp (Aspa o 1, Aspa o
1.01, Aspa o 1.02, Aspa o 17 kD, Aspa o 4), Aspergillus spp (Aspe
ni 2, Aspe ni 3, Aspe ni 4, Aspe ni 7, Aspe ni 8, Aspe ni 9), Avena
spp (Ave s 1, Ave s 12, Ave s 13, Ave s 2, Ave s 4, Ave s 5, Ave s
7), Babylonia spp (Bab ja 1), Bacillus spp (Bac al Subtilisin, Bac
cl Subtilisin, Bac l Subtilisin, Bac li aA, Bac li Subtilisin),
Bactrocera spp (Bac ol 27, Bac ol 27.0101), Bacillus spp (Bac sp
aAl, Bac sp aA3, Bac sp Decarboxylase, Bac st amyM, Bac su
Subtilisin, Bac t Cry1Ab, Bac t Cry1Fa, Bac t Cry3Bb1, Bac t
Cry9c), Bagre spp (Bag ma 1), Balistes spp (Bal ca 1), Balanus spp
(Bal r 1, Bal r 1.0101), Beauveria spp (Bea b Ald, Bea b Enol, Bea
b f2, Bea b Hex), Bertholletia spp (Ber e 1, Ber e 1.0101, Ber e 2,
Ber e 2.0101), Beryx spp (Ber sp 1), Betula spp (Bet ab 1, Bet al
1, Bet ch 1, Bet co 1, Bet da 1, Bet gr 1, Bet hu 1, Bet le 1, Bet
me 1, Bet n 1, Bet p 1, Bet pa 1, Bet po 1, Bet pu 1, Bet pu 2, Bet
pu 4, Bet pu 6, Bet pu 7, Bet sc 1, Bet ut 1, Bet v 1, Bet v
1B1-B1-B1, Bet v 1 fv Mal 4x, Bet v 1.0101, Bet v 1.0102, Bet v
1.0103, Bet v 1.0201, Bet v 1.0301, Bet v 1.0401, Bet v 1.0402, Bet
v 1.0501, Bet v 1.0601, Bet v 1.0602, Bet v 1.0701, Bet v 1.0801,
Bet v 1.0901, Bet v 1.1001, Bet v 1.1101, Bet v 1.1201, Bet v
1.1301, Bet v 1.1401, Bet v 1.1402, Bet v 1.1501, Bet v 1.1502, Bet
v 1.1601, Bet v 1.1701, Bet v 1.1801, Bet v 1.1901, Bet v 1.2001,
Bet v 1.2101, Bet v 1.2201, Bet v 1.2301, Bet v 1.2401, Bet v
1.2501, Bet v 1.2601, Bet v 1.2701, Bet v 1.2801, Bet v 1.2901, Bet
v 1.3001, Bet v 1.3101, Bet v 2, Bet v 2.0101, Bet v 3, Bet v
3.0101, Bet v 4, Bet v 4.0101, Bet v 6, Bet v 6.0101, Bet v 6.0102,
Bet v 7, Bet v 7.0101, Bet v 8, Bet v Glucanase), Beta spp (Beta v
1, Beta v 1.0101, Beta v 2, Beta v 2.0101), Blattella spp (Bla g 1,
Bla g 1.0101, Bla g 1.0102, Bla g 1.0103, Bla g 1.0201, Bla g
1.0202, Bla g 2, Bla g 2.0101, Bla g 2.0201, Bla g 36 kD, Bla g 4,
Bla g 4.0101, Bla g 4.0201, Bla g 5, Bla g 5.0101, Bla g 5.0201,
Bla g 6, Bla g 6.0101, Bla g 6.0201, Bla g 6.0301, Bla g 7, Bla g
7.0101, Bla g 8, Bla g 8.0101, Bla g 9, Bla g Enolase, Bla g GSTD1,
Bla g RACK1, Bla g TPI, Bla g Trypsin, Bla g Vitellogenin), Blatta
spp (Bla o 1, Bla o 7), Blomia spp (Blo t 1, Blo t 1.0101, Blo t
1.0201, Blo t 10, Blo t 10.0101, Blo t 10.0102, Blo t 11, Blo t
11.0101, Blo t 12, Blo t 12.0101, Blo t 12.0102, Blo t 13, Blo t
13.0101, Blo t 14, Blo t 15, Blo t 18, Blo t 19, Blo t 19.0101, Blo
t 2, Blo t 2.0101, Blo t 2.0102, Blo t 2.0103, Blo t 20, Blo t 21,
Blo t 21.0101, Blo t 3, Blo t 3.0101, Blo t 4, Blo t 4.0101, Blo t
5, Blo t 5.0101, Blo t 6, Blo t 6.0101, Blo t 7, Blo t 8, Blo t 9,
Blo t HSP70), Bombus spp (Born ar 4, Born by 4, Born p 1, Born p
1.0101, Boni p 2, Born p 3, Born p 4, Born p 4.0101, Bom t 1, Bom t
1.0101, Bom t 4, Bom t 4.0101), Bombyx spp (Bomb m 1, Bomb m
1.0101, Bomb m 7, Bomb m 7.0101, Bomb m 7.0102, Bomb m 7.0103, Bomb
m 7.0104, Bomb m 7.0105, Bomb m 7.0106), Boophilus spp (Boo m 1,
Boo m 7, Boo m 7.0101), Bos spp (Bos d 2, Bos d 2.0101, Bos d
2.0102, Bos d 2.0103, Bos d 3, Bos d 3.0101, Bos d 4, Bos d 4.0101,
Bos d 5, Bos d 5.0101, Bos d 5.0102, Bos d 6, Bos d 6 (MDA), Bos d
6.0101, Bos d 7, Bos d 7.0101, Bos d 8, Bos d 8 alphaSl, Bos d 8
alphaS2, Bos d 8 beta, Bos d 8 kappa, Bos d alphaSl, Bos d
alpha21.0101, Bos d Chymosin, Bos d Fibrin, Bos d Gelatin, Bos d
HG, Bos d Insulin, Bos d Lactoferrin, Bos d Lactoperoxidase, Bos d
Myoglobin, Bos d OBP, Bos d OSCP, Bos d Phosvitin, Bos d PLA2, Bos
d PRVB, Bos d Thrombin, Bos d TI, Bos gr ALA, Bos gr Myoglobin),
Bothrops spp (Bot as 1, Bot at 1), Bouteloua spp (Bou g 1), Biting
spp (Boy ov 1), Brama spp (Bra du 1), Brassica spp (Bra j 1, Bra j
1.0101, Bra n 1, Bra n 1.0101, Bra n 4, Bra n 7, Bra n 8, Bra n PG,
Bra ni 8, Bra o 3, Bra o 3.0101, Bra r 1, Bra r 1.0101, Bra r 2,
Bra r 2.0101, Bra r 3, Bra r 4, Bra r 7), Bromus spp (Bro a 1, Bro
a 4), Brosme spp (Bro br 1), Bromus spp (Broil, Bro i 5, Bro i7),
Brugia spp (Bru m 3, Bru m 3.0101, Bru m Bm33), Bubalus spp (Bub b
ALA, Bub b BLG, Bub b Casein, Bub b Casein alphaSl, Bub b Casein
alphaS2, Bub b Casein beta, Bub b Casein kappa), Caenorhabditis spp
(Cae b 3, Cae b 3.0101, Cae br 3, Cae br 3.0101, Cae e 3, Cae e
3.0101, Cae e 3.0102, Cae re 13, Cae re 13.0101), Cajanus spp (Caj
c 1), Caligus spp (Cal cl 1, Cal cl 1.0101, Cal cl 1.0102), Calamus
spp (Cal le 1), Callinectes spp (Cal s 2), Camelus spp (Cam d ALA,
Cam d Casein, Cam d Casein alphaS1, Cam d Casein alphaS2, Cam d
Casein beta, Cam d Casein kappa), Camponotus spp (Cam fl 7, Cam fl
7.0101), Canis spp (Can f 1, Can f 1.0101, Can f 2, Can f 2.0101,
Can f 3, Can f 3.0101, Can f 4, Can f 4.0101, Can f 5, Can f
5.0101, Can f 6, Can f 6.0101, Can f Feld1-like, Can f Homs2-like,
Can f Phosvitin, Can f TCTP), Canthidermis spp (Can ma 1), Cancer
spp (Can mg 2, Can p 1), Cannabis spp (Can s 3), Candida spp (Cand
a 1, Cand a 1.0101, Cand a 3, Cand a 3.0101, Cand a CAAP, Cand a
CyP, Cand a Enolase, Cand a FPA, Cand a MnSOD, Cand a PGK, Cand b
2, Cand b 2.0101, Cand b FDH, Cand r Lipase), Capsicum spp (Cap a
1, Cap a 1.0101, Cap a 17 kD, Cap a 2, Cap a 2.0101, Cap a 30 kD,
Cap a Glucanase, Cap ch 17 kD), Caprella spp (Cap e 1), Capra spp
(Cap h ALA, Cap h BLG, Cap h Casein, Cap h Casein alphaS1, Cap h
Casein alphaS2, Cap h Casein beta, Cap h Casein kappa, Cap h GSA),
Capitulum spp (Cap m 1), Carassius spp (Car au 1), Carpinus spp
(Car b 1, Car b 1.0101, Car b 1.0102, Car b 1.0103, Car b 1.0104,
Car b 1.0105, Car b 1.0106, Car b 1.0107, Car b 1.0108, Car b
1.0109, Car b 1.0110, Car b 1.0111, Car b 1.0112, Car b 1.0113, Car
b 1.0201, Car b 1.0301, Car b 1.0302, Car b 2, Car b 4), Caranx spp
(Car cr 1), Carya spp (Car i 1, Car i 1.0101, Car i 2, Car i 4, Car
i 4.0101), Carcinus spp (Car ma 2), Caryota spp (Car mi 2), Carica
spp (Car p 1, Car p Chitinase, Car p Chymopapain, Car p
Endoproteinase), Castanea spp (Cas c 24 kD, Cas s 1, Cas s 1.0101,
Cas s 1.0102, Cas s 1.0103, Cas s 2, Cas s 5, Cas s 5.0101, Cas s
8, Cas s 8.0101, Cas s 9, Cas s 9.0101), Catharanthus spp (Cat r 1,
Cat r 1.0101, Cat r 17 kD, Cat r 2), Caulolatilus spp (Cau ch 1),
Cavia spp (Cav p 1, Cav p 1.0101, Cav p 2, Cav p 2.0101, Cav p 3,
Cav p 3.0101, Cav p Gelatin, Cav p GSA), Centropristis spp (Cen s
1), Cephalopholis spp (Cep so 1), Charybdis spp (Cha f 1, Cha f
1.0101), Chaetodipterus spp (Cha fa 1), Chamaecyparis spp (Cha o 1,
Cha o 1.0101, Cha o 2, Cha o 2.0101), Chenopodium spp (Che a 1, Che
a 1.0101, Che a 2, Che a 2.0101, Che a 3, Che a 3.0101), Chironomus
spp (Chi k 1, Chi k 10, Chi k 10.0101), Chinchilla spp (Chi I 21
kD_a, Chi I 21 kD_b), Chionoecetes spp (Chi o 1, Chi o 1.0101, Chi
o 2, Chi o 4, Chi o 6, Chi o alpha_Actin, Chi o SERCA), Chironomus
spp (Chi t 1, Chi t 1.0101, Chi t 1.0201, Chi t 2, Chi t 2.0101,
Chi t 2.0102, Chi t 3, Chi t 3.0101, Chi t 4, Chi t 4.0101, Chi t
5, Chi t 5.0101, Chi t 6, Chi t 6.0101, Chi t 6.0201, Chi t 7, Chi
t 7.0101, Chi t 8, Chi t 8.0101, Chi t 9, Chi t 9.0101), Chlamys
spp (Chl n 1), Chloephaga spp (Chl pi 1), Chortoglyphus spp (Cho a
10), Chrysomela spp (Chr tr 7, Chr tr 7.0101), Cicer spp (Cic a 2S
Albumin, Cic a Albumin),
Cichorium spp (Cic i 1), Cimex spp (Cim I Nitrophorin), Citrus spp
(Cit I 1, Cit I 3, Cit I 3.0101), Citrullus spp (Cit Ia 2, Cit Ia
MDH, Cit Ia TPI), Citrus spp (Cit r 3, Cit r 3.0101, Cit s 1, Cit s
1.0101, Cit s 2, Cit s 2.0101, Cit s 3, Cit s 3.0101, Cit s 3.0102,
Cit s IFR), Cladosporium spp (Cla c 14, Cla c 14.0101, Cla c 9, Cla
c 9.0101, Cla h 1, Cla h 10, Cla h 10.0101, Cla h 12, Cla h
12.0101, Cla h 2, Cla h 2.0101, Cla h 42 kD, Cla h 5, Cla h 5.0101,
Cla h 6, Cla h 6.0101, Cla h 7, Cla h 7.0101, Cla h 8, Cla h 8 CSP,
Cla h 8.0101, Cla h 9, Cla h 9.0101, Cla h abH, Cla h GST, Cla h
HCh1, Cla h HSP70, Cla h NTF2, Cla h TCTP), Clostridium spp (Clo hi
Collagenase, Clo t Toxoid), Clupea spp (Clu h 1, Clu h 1.0101, Clu
h 1.0201, Clu h 1.0301), Cocos spp (Coc n 2, Coc n 4, Coc n 5),
Coccidioides spp (Coc po 8), Coffea spp (Cof a 1, Cof a 1.0101),
Columba spp (Col I PSA), Coprinus spp (Cop c 1, Cop c 1.0101, Cop c
2, Cop c 2.0101, Cop c 3, Cop c 3.0101, Cop c 4, Cop c 5, Cop c
5.0101, Cop c 6, Cop c 7, Cop c 7.0101), Corylus spp (Cor a 1, Cor
a 1.0101, Cor a 1.0102, Cor a 1.0103, Cor a 1.0104, Cor a 1.0201,
Cor a 1.0301, Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, Cor a
1.0404, Cor a 10, Cor a 10.0101, Cor a 11, Cor a 11.0101, Cor a 12,
Cor a 12.0101, Cor a 13, Cor a 13.0101, Cor a 14, Cor a 14.0101,
Cor a 2, Cor a 2.0101, Cor a 2.0102, Cor a 8, Cor a 8.0101, Cor a
9, Cor a 9.0101), Corynebacterium spp (Cor d Toxoid), Corylus spp
(Cor he 1), Coryphaena spp (Cor hi 1), Coriandrum spp (Cor s 1, Cor
s 11 kD, Cor s 2), Cotoneaster spp (Cot I 3), Crangon spp (Cra c 1,
Cra c 1.0101, Cra c 2, Cra c 2.0101, Cra c 4, Cra c 4.0101, Cra c
5, Cra c 5.0101, Cra c 6, Cra c 6.0101, Cra c 8, Cra c 8.0101),
Crassostrea spp (Cra g 1), Cricetus spp (Cri c HSA), Crivellia spp
(Cri pa 1), Crocus spp (Cro s 1, Cro s 1.0101, Cro s 2, Cro s
2.0101, Cro s 3, Cro s 3.01, Cro s 3.02), Cryptomeria spp (Cry j 1,
Cry j 1.0101, Cry j 1.0102, Cry j 1.0103, Cry j 2, Cry j 2.0101,
Cry j 2.0102, Cry j 3, Cry j 3.1, Cry j 3.2, Cry j 3.3, Cry j 3.4,
Cry j 3.5, Cry j 3.6, Cry j 3.7, Cry j 3.8, Cry j 4, Cry j AP, Cry
j Chitinase, Cry j CPA9, Cry j IFR, Cry j LTP, Cry j P1-P2),
Cryphonectria spp (Cry p AP), Ctenocephalides spp (Cte f 1, Cte f
1.0101, Cte f 2, Cte f 2.0101, Cte f 3, Cte f 3.0101),
Ctenopharyngodon spp (Cte id 1), Cucumis spp (Cuc m 1, Cuc m
1.0101, Cuc m 2, Cuc m 2.0101, Cuc m 3, Cuc m 3.0101, Cuc m Lec17,
Cuc m MDH), Cucurbita spp (Cuc ma 18 kD, Cuc ma 2, Cuc p 2, Cuc p
AscO), Cucumis spp (Cuc s 2), Culicoides spp (Cul n 1, Cul n 10,
Cul n 11, Cul n 2, Cul n 3, CuI n 4, Cul n 5, Cul n 6, Cul n 7, Cul
n 8, Cul n 9, Cul n HSP70), Culex spp (Cul q 28 kD, Cul q 35 kD,
Cul q 7, CuI q 7.0101, Cul q 7.0102), Culicoides spp (Cul so 1),
Cuminum spp (Cum c 1, Cum c 2), Cupressus spp (Cup a 1, Cup a
1.0101, Cup a 1.02, Cup a 2, Cup a 3, Cup a 4, Cup a 4.0101, Cup s
1, Cup s 1.0101, Cup s 1.0102, Cup s 1.0103, Cup s 1.0104, Cup s
1.0105, Cup s 3, Cup s 3.0101, Cup s 3.0102, Cup s 3.0103, Cup s
8), Cochliobolus spp (Cur 11, Cur I 1.0101, Cur I 2, Cur I 2.0101,
Cur 13, Cur I 3.0101, Cur I 4, Cur I 4.0101, Cur I ADH, Cur I GST,
Cur I MnSOD, Cur I Oryzin, Cur I Trx, Cur I ZPS1), Cyanochen spp
(Cya cy 1), Cynoscion spp (Cyn ar 1), Cynosurus spp (Cyn cr 1, Cyn
cr 5), Cynodon spp (Cyn d 1, Cyn d 1.0101, Cyn d 1.0102, Cyn d
1.0103, Cyn d 1.0104, Cyn d 1.0105, Cyn d 1.0106, Cyn d 1.0107, Cyn
d 1.0201, Cyn d 1.0202, Cyn d 1.0203, Cyn d 1.0204, Cyn d 10, Cyn d
11, Cyn d 12, Cyn d 12.0101, Cyn d 13, Cyn d 15, Cyn d 15.0101, Cyn
d 2, Cyn d 22, Cyn d 22.0101, Cyn d 23, Cyn d 23.0101, Cyn d 24,
Cyn d 24.0101, Cyn d 4, Cyn d 5, Cyn d 6, Cyn d 7, Cyn d 7.0101),
Cynoscion spp (Cyn ne 1), Cynomys spp (Cyn sp Lipocalin), Cyprinus
spp (Cyp c 1, Cyp c 1.01, Cyp c 1.02), Daboia spp (Dab ru 1),
Dactylis spp (Dac g 1, Dac g 1.01, Dac g 1.0101, Dac g 1.02, Dac g
12, Dac g 13, Dac g 2, Dac g 2.0101, Dac g 3, Dac g 3.0101, Dac g
4, Dac g 4.0101, Dac g 5, Dac g 5.0101, Dac g 7), Dama spp (Dam d
CSA), Danio spp (Dan re 1, Dan re 2, Dan re alpha2l, Dan re CK),
Dasyatis spp (Das ak 1, Das am 1, Das sa 1), Daucus spp (Dau c 1,
Dau c 1.0101, Dau c 1.0102, Dau c 1.0103, Dau c 1.0104, Dau c
1.0105, Dau c 1.0201, Dau c 1.0301, Dau c 3, Dau c 4, Dau c 4.0101,
Dau c CyP), Decapterus spp (Dec ru 1), Dendronephthya spp (Den n 1,
Den n 1.0101), Dermatophagoides spp (Der 11, Der 11.0101, Der
11.0102, Der 11.0103, Der f 1.0104, Der f 1.0105, Der f 1.0106, Der
f 1.0107, Der f 1.0108, Der f 1.0109, Der f 1.0110, Der f 10, Der f
10.0101, Der f 10.0102, Der f 11, Der f 11.0101, Der f 13, Der f
13.0101, Der f 14, Der f 14.0101, Der f 15, Der f 15.0101, Der f
16, Der f 16.0101, Der f 17, Der f 17.0101, Der f 18, Der f
18.0101, Der f 2, Der f 2.0101, Der f 2.0102, Der f 2.0103, Der f
2.0104, Der f 2.0105, Der f 2.0106, Der f 2.0107, Der f 2.0108, Der
f 2.0109, Der f 2.0110, Der f 2.0111, Der f 2.0112, Der f 2.0113,
Der f 2.0114, Der f 2.0115, Der f 2.0116, Der f 2.0117, Der f 20,
Der f 21, Der f 22, Der f 22.0101, Der f 3, Der f 3.0101, Der f 4,
Der f 5, Der f 6, Der f 6.0101, Der f 7, Der f 7.0101, Der f 8, Der
f 9, Der f HSP70), Dermanyssus spp (Der g 10, Der g 10.0101),
Dermatophagoides spp (Der m 1, Der m 1.0101, Der p 1, Der p 1.0101,
Der p 1.0102, Der p 1.0103, Der p 1.0104, Der p 1.0105, Der p
1.0106, Der p 1.0107, Der p 1.0108, Der p 1.0109, Der p 1.0110, Der
p 1.0111, Der p 1.0112, Der p 1.0113, Der p 1.0114, Der p 1.0115,
Der p 1.0116, Der p 1.0117, Der p 1.0118, Der p 1.0119, Der p
1.0120, Der p 1.0121, Der p 1.0122, Der p 1.0123, Der p 1.0124, Der
p 10, Der p 10.0101, Der p 10.0102, Der p 10.0103, Der p 11, Der p
11.0101, Der p 13, Der p 14, Der p 14.0101, Der p 15, Der p 18, Der
p 2, Der p 2.0101, Der p 2.0102, Der p 2.0103, Der p 2.0104, Der p
2.0105, Der p 2.0106, Der p 2.0107, Der p 2.0108, Der p 2.0109, Der
p 2.0110, Der p 2.0111, Der p 2.0112, Der p 2.0113, Der p 2.0114,
Der p 2.0115, Der p 20, Der p 20.0101, Der p 21, Der p 21.0101, Der
p 23, Der p 23.0101, Der p 3, Der p 3.0101, Der p 4, Der p 4.0101,
Der p 5, Der p 5.0101, Der p 5.0102, Der p 6, Der p 6.0101, Der p
7, Der p 7.0101, Der p 8, Der p 8.0101, Der p 9, Der p 9.0101, Der
p 9.0102, Der p P1-P2, Der p P2-P1, Der s 1, Der s 2, Der s 3),
Dianthus spp (Dia c RIP), Dicranopteris spp (Dic I 2S Albumin),
Diospyros spp (Dio k 17 kD, Dio k 4, Dio k IFR), Dioscorea spp (Dio
p TSP), Diplodus spp (Dip ho 1), Distichlis spp (Dis s 1, Dis s 7),
Ditrema spp (Dit to 1), Dolichovespula spp (Dol a 1, Dol a 2, Dol a
5, Dol a 5.0101), Dolichos spp (Dol b Agglutinin), Dolichovespula
spp (Dol m 1, Dol m 1.0101, Dol m 1.02, Dol m 2, Dol m 2.0101, Dol
m 5, Dol m 5.0101, Dol m 5.02), Drosophila spp (Dro an 7, Dro an
7.0101, Dro er 7, Dro er 7.0101, Dro er 7.0102, Dro gr 7, Dro gr
7.0101, Dro gr 7.0102, Dro m 7, Dro m 7.0101, Dro m 7.0102, Dro m
7.0103, Dro m 7.0104, Dro m 7.0105, Dro m 7.0106, Dro m 7.0107, Dro
m 7.0108, Dro m 7.0109, Dro m 7.0110, Dro m 7.0111, Dro m 7.0112,
Dro m 7.0113, Dro m 9, Dro m MnSOD, Dro mo 7, Dro mo 7.0101, Dro pp
7, Dro pp 7.0101, Dro se 7, Dro se 7.0101, Dro si 7, Dro si 7.0101,
Dro si 7.0102, Dro vi 7, Dro vi 7.0101, Dro wi 7, Dro wi 7.0101,
Dro y 7, Dro y 7.0101, Dro y 7.0102, Dro y 7.0103), Echium spp (Ech
p Cytochrome C), Elaeis spp (Ela g 2, Ela g Bd31 kD), Elops spp
(Elo sa 1), Embellisia spp (Emb a 1, Emb i 1, Emb nz 1, Emb t 1),
Engraulis spp (Eng e 1), Enteroctopus spp (Ent d 1), Epinephelus
spp (Epi bI 1, Epi co 1, Epi fl 1, Epi mc 1, Epi mo 1), Epicoccum
spp (Epi p 1, Epi p 1.0101, Epi p 12 kD, Epi p GST), Epinephelus
spp (Epi po 1, Epi un 1), Equisetum spp (Equ a 17 kD), Equus spp
(Equ as 4, Equ as DSA, Equ bu 4, Equ c 1, Equ c 1.0101, Equ c 2,
Equ c 2.0101, Equ c 2.0102, Equ c 3, Equ c 3.0101, Equ c 4, Equ c
4.0101, Equ c 5, Equ c 5.0101, Equ c ALA, Equ c BLG, Equ c Casein,
Equ c Casein beta, Equ c Casein kappa, Equ c PRVB, Equ he 4, Equ z
ZSA), Erimacrus spp (Eri i 1, Eri i 1.0101, Eri i 1.0102),
Eriocheir spp (Eri s 1, Eri s 1.0101, Eri s 2), Erwinia spp (Erw ch
Asparaginase), Escherichia spp (Esc c Asparaginase, Esc c beta
GAL), Esox spp (Eso I 1), Euphausia spp (Eup p 1, Eup p 1.0101),
Euphasia spp (Eup s 1, Eup s 1.0101), Euroglyphus spp (Eur m 1, Eur
m 1.0101, Eur m 1.0102, Eur m 1.0103, Eur m 10, Eur m 14, Eur m
14.0101, Eur m 2, Eur m 2.0101, Eur m 2.0102, Eur m 3, Eur m
3.0101, Eur m 4, Eur m 4.0101), Evynnis spp (Evy j 1), Fagopyrum
spp (Fag e 1, Fag e 1.0101, Fag e 10 kD, Fag e 19 kD, Fag e 2, Fag
e 2.0101, Fag e TI), Fagus spp (Fag s 1, Fag s 1.0101, Fag s 2, Fag
s 4), Fagopyrum spp (Fag t 1, Fag t 10 kD, Fag t 2, Fag t 2.0101),
Felis spp (Fel d 1, Fel d 1.0101, Fel d 2, Fel d 2.0101, Fel d 3,
Fel d 3.0101, Fel d 4, Fel d 4.0101, Fel d 5, Fel d 5.0101, Fel d
6, Fel d 6.0101, Fel d 7, Fel d 7.0101, Fel d 8, Fel d 8.0101, Fel
d IgG), Fenneropenaeus spp (Fen c 1, Fen c 2, Fen me 1, Fen me
1.0101), Festuca spp (Fes e 1, Fes e 13, Fes e 4, Fes e 5, Fes e 7,
Fes p 1, Fes p 13, Fes p 4, Fes p 4.0101, Fes p 5, Fes r 1, Fes r
5), Ficus spp (Fic c 17 kD, Fic c 4, Fic c Ficin), Foeniculum spp
(Foe v 1, Foe v 2), Forsythia spp (For s 1), Forcipomyia spp (For t
1, For t 1.0101, For t 2, For t 2.0101, For t 7, For t FPA, For t
Myosin, For t TPI), Fragaria spp (Fra a 1, Fra a 1.0101, Fra a 3,
Fra a 3.0101, Fra a 3.0102, Fra a 3.0201, Fra a 3.0202, Fra a
3.0203, Fra a 3.0204, Fra a 3.0301, Fra a 4, Fra a 4.0101, Fra c
1), Fraxinus spp (Fra e 1, Fra e 1.0101, Fra e 1.0102, Fra e
1.0201, Fra e 12, Fra e 2, Fra e 3, Fra e 9), Fragaria spp (Fra v
1), Fusarium spp (Fus c 1, Fus c 1.0101, Fus c 2, Fus c 2.0101, Fus
c 3, Fus s 1, Fus s 45 kD, Fus sp Lipase), Gadus spp (Gad c 1, Gad
c 1.0101, Gad c APDH, Gad m 1, Gad m 1.0101, Gad m 1.0102, Gad m
1.0201, Gad m 1.0202, Gad m 45 kD, Gad m Gelatin, Gad ma 1), Gallus
spp (Gal d 1, Gal d 1.0101, Gal d 2, Gal d 2.0101, Gal d 3, Gal d
3.0101, Gal d 4, Gal d 4.0101, Gal d 5, Gal d 5.0101, Gal d 6, Gal
d 6.0101, Gal d Apo I, Gal d Apo VI, Gal d GPI, Gal d HG, Gal d
IgY, Gal d L-PGDS, Gal d Ovomucin, Gal d Phosvitin, Gal d PRVB, Gal
la 4), Galleria spp (Gal m 18 kD, Gal m 24 kD), Gallus spp (Gal so
4), Gammarus spp (Gam s TM), Gelonium spp (Gel m RIP), Geothelphusa
spp (Geo de 1), Glossina spp (Glo m 5, Glo m 5.0101, Glo m 7, Glo m
7.0101, Glo m 7.0102, Glo m 7.0103), Glycine spp (Gly a Bd3OK, Gly
ar Bd3OK, Gly ca Bd3OK, Gly cl Bd3OK, Gly cu Bd3OK, Gly cy Bd3OK),
Glycyphagus spp (Gly d 10, Gly d 10.0101, Gly d 13, Gly d 2, Gly d
2.0101, Gly d 2.0201, Gly d 2.03, Gly d 2/Lep d 2 L1, Gly d 2/Lep d
2 L2, Gly d 2/Lep d 2 L3, Gly d 2/Lep d 2 L4, Gly d 2/Lep d 2 R1,
Gly d 2/Lep d 2 R2, Gly d 2/Lep d 2 R3, Gly d 2/Lep d 2 R4, Gly d
2/Lep d 2 R5, Gly d 20, Gly d 3, Gly d 5, Gly d 5.01, Gly d 5.02,
Gly d 7, Gly d 8), Glycine spp (Gly f Bd3OK, Gly l Bd3OK, Gly m 1,
Gly m 1.0101, Gly m 1.0102, Gly m 2, Gly m 2.0101, Gly m 2S
Albumin, Gly m 3, Gly m 3.0101, Gly m 3.0102, Gly m 39 kD, Gly m 4,
Gly m 4.0101, Gly m 5, Gly m 5.0101, Gly m 5.0201, Gly m 5.0301,
Gly m 5.0302, Gly m 50 kD, Gly m 6, Gly m 6.0101, Gly m 6.0201, Gly
m 6.0301, Gly m 6.0401, Gly m 6.0501, Gly m 68 kD, Gly m
Agglutinin, Gly m Bd28K, Gly m Bd3OK, Gly m Bd6OK, Gly m CPI, Gly m
EAP, Gly m TI, Gly mi Bd3OK, Gly s Bd3OK, Gly t Bd30K, Gly to
Bd30K), Gossypium spp (Gos h Vicilin), Haemophilus spp (Hae in P6),
Haemaphysalis spp (Hae I 7, Hae I 7.0101, Hae q 7, Hae q 7.0101),
Haliotis spp (Hal a 1, Hal d 1, Hal di 1, Hal di PM, Hal m 1, Hal m
1.0101, Hal r 1, Hal r 49 kD, Hal ru 1), Harmonia spp (Har a 1, Har
a 1.0101, Har a 2, Har a 2.0101), Harpegnathos spp (Har sa 7, Har
sa 7.0101, Har sa 7.0102), Helianthus spp (Hel a 1, Hel a 1.0101,
Hel a 2, Hel a 2.0101, Hel a 2S Albumin, Hel a 3, Hel a 3.0101, Hel
a 4), Helix spp (Hel ap 1, Hel as 1, Hel as 1.0101),
Heligmosomoides spp (Hel p 3, Hel p 3.0101), Helianthus spp (Hel to
1), Hemanthias spp (Hem le 1), Hemifusus spp (Hem t 1), Heterodera
spp (Het g 3, Het g 3.0101), Hevea spp (Hevb 1, Hevb 1.0101, Hevb
10, Hevb 10.0101, Hevb 10.0102, Hey b 10.0103, Hey b 11, Hey b
11.0101, Hey b 11.0102, Hey b 12, Hey b 12.0101, Hey b 13, Hey b
13.0101, Hey b 14, Hey b 14.0101, Hey b 2, Hey b 2.0101, Hey b 3,
Hey b 3.0101, Hey b 4, Hey b 4.0101, Hey b 5, Hey b 5.0101, Hey b
6, Hey b 6.01, Hey b 6.02, Hey b 6.0202, Hey b 6.03, Hey b 7, Hey b
7.01, Hey b 7.02, Hey b 7.D2, Hey b 7.S2, Hey b 8, Hey b 8.0101,
Hey b 8.0102, Hey b 8.0201, Hey b 8.0202, Hey b 8.0203, Hey b
8.0204, Hey b 9, Hey b 9.0101, Hey b Citrate binding Protein, Hey b
GAPDH, Hey b HSP80, Hey b IFR, Hey b Proteasome subunit, Hey b
Rotamase, Hey b SPI, Hey b Trx, Hey b UDPGP), Hexagrammos spp (Hex
of 1), Hippoglossus spp (Hip h 1), Hippoglossoides spp (Hip pl 1),
Hippoglossus spp (Hip st 1), Hirudo spp (Hir me Hirudin), Holcus
spp (Hol I 1, Hol I 1.0101, Hol I 1.0102, Hol I 2, Hol I 4, Hol I
5, Hol I 5.0101, Hol I 5.0201), Holocnemus spp (Hol pl 9, Hol pl
Hemocyanin), Homarus spp (Horn a 1, Horn a 1.0101, Horn a 1.0102,
Horn a 1.0103, Horn a 3, Horn a 3.0101, Horn a 4, Hom a 6, Hom a
6.0101, Hom g 1, Hom g 2), Homo spp (Homs 1, Homs 1.0101, Horn s 2,
Horn s 2.0101, Horn s 3, Horn s 3.0101, Horn s 4, Horn s 4.0101,
Horn s 5, Horn s 5.0101, Horn s AAT, Horn s ACTH, Horn s
Adalimumab, Horn s ALA, Horn s alpha_Actin, Horn s
alpha-Galactosidase, Horn s APDH, Horn s Arylsulfatase B, Horn s
Casein, Horn s CyP A, Horn s CyP B, Horn s CyP C, Horn s DSF70,
Horn s DSG3, Horn s elF6, Horn s Etanercept, Horn s Factor IX, Horn
s Factor VII, Horn s Factor VIII, Horn s G-CSF, Horn s
Glucocerebrosidase, Horn s Glucosidase, Horn s HLA-DR-alpha, Horn s
HSA, Horn s Iduronidase, Horn s Idursulfase, Horn s IgA, Horn s
Insulin, Horn s Lactoferrin, Horn s Laminin gamma.sub.--2, Horn s
MnSOD, Horn s Oxytocin, Horn s P2, Horn s Phosvitin, Horn s
Profilin, Horn s PSA, Horn s RP1, Horn s TCTP, Horn s TL, Horn s
TPA, Horn s TPO, Horn s Transaldolase, Horn s Trx, Horn s
Tubulin-alpha, Horn s/Mus m Basiliximab, Horn s/Mus m Cetuximab,
Horn s/Mus m Cetuximab (Gal-Gal), Horn s/Mus m Infliximab, Horn
s/Mus m Natalizumab, Horn s/Mus m Omalizumab, Horn s/Mus m
Palivizumab, Horn s/Mus m Rituximab, Horn s/Mus m Tocilizumab, Horn
s/Mus m Trastuzumab), Hoplostethus spp (Hop a 1), Hordeum spp (Hor
v 1, Hor v 12, Hor v 12.0101, Hor v 13, Hor v 14, Hor v 15, Hor v
15.0101, Hor v 16, Hor v 16.0101, Hor v 17, Hor v 17.0101, Hor v 18
kD, Hor v 2, Hor v 21, Hor v 21.0101, Hor v 28, Hor v 33, Hor v 4,
Hor v 5, Hor v 5.0101, Hor v BDAI, Hor v BTI), Humicola spp (Hum in
Cellulase), Humulus spp (Hum j 1, Hum j 1.0101, Hum j 10 kD, Hum j
2), Huso spp (Hus h 1), Hylocereus spp (Hyl un LTP), Hymenocephalus
spp (Hym st 1), Hyperoglyphe spp (Hyp by 1), Hypophthalmichthys spp
(Hyp mo 1), Hypophthalmichthy spp (Hyp no 1), Ictalurus spp act fu
1, Id p 1), Imperata spp (Imp c 4, Imp c 5, Imp c VIIIe1), Ixodes
spp (Ixo r 2, Ixo sc 7, Ixo sc 7.0101), Jasus spp (Jas Ia 1, Jas Ia
1.0101, Jas Ia 1.0102), Juglans spp (Jug ca 1, Jug ca 2, Jug ci 1,
Jug ci 2, Jug n 1, Jug n 1.0101, Jug n 2, Jug n 2.0101, Jug r 1,
Jug r 1.0101, Jug r 2, Jug r 2.0101, Jug r 3, Jug r 3.0101, Jug r
4, Jug r 4.0101, Jug r 5),
Juniperus spp (Jun a 1, Jun a 1.0101, Jun a 1.0102, Jun a 2, Jun a
2.0101, Jun a 3, Jun a 3.0101, Jun c 1, Jun o 1, Jun o 4, Jun o
4.0101, Jun r 3, Jun r 3.1, Jun r 3.2, Jun v 1, Jun v 1.0101, Jun v
1.0102, Jun v 3, Jun v 3.0101, Jun v 3.0102, Jun v 4), Katsuwonus
spp (Kat p 1), Kyphosus spp (Kyp se 1), Lachnolaimus spp (Lac ma
1), Lachesis spp (Lac mu 1), Lactuca spp (Lac s 1, Lac s 1.0101),
Lagocephalus spp (Lag Ia 1), Larus spp (Lar a 1, Lar a 2, Lar a 3),
Larimichthys spp (Lar po 1), Lates spp (Lat c 1), Lateolabrax spp
(Lat ja 1), Lathyrus spp (Lat oc Agglutinin), Leiostomus spp (Lei
xa 1), Lens spp (Len c 1, Len c 1.0101, Len c 1.0102, Len c 1.0103,
Len c 2, Len c 2.0101, Len c 3, Len c 3.0101, Len c Agglutinin),
Leopardus spp (Leo p 1), Lepidoglyphus spp (Lep d 10, Lep d
10.0101, Lep d 12, Lep d 13, Lep d 13.0101, Lep d 2, Lep d 2.0101,
Lep d 2.0102, Lep d 2.0201, Lep d 2.0202, Lep d 3, Lep d 39 kD, Lep
d 5, Lep d 5.0101, Lep d 5.0102, Lep d 5.0103, Lep d 7, Lep d
7.0101, Lep d 8, Lep d alpha Tubulin), Lepomis spp (Lep gi 1),
Leptomelanosoma spp (Lep i 1), Lepomis spp (Lep ma 1), Lepisma spp
(Lep s 1, Lep s 1.0101, Lep s 1.0102), Lepeophtheirus spp (Lep sa
1, Lep sa 1.0101, Lep sa 1.0102, Lep sa 1.0103), Leptailurus spp
(Lep se 1), Lepidorhombus spp (Lep w 1, Lep w 1.0101), Lethocerus
spp (Let in 7, Let in 7.0101, Let in 7.0102), Leuciscus spp (Leu ce
1), Lewia spp (Lew in 1), Ligustrum spp (Lig v 1, Lig v 1.0101, Lig
v 1.0102, Lig v 2), Lilium spp (Lil I 2, Lil I PG), Limanda spp
(Lim fe 1), Limnonectes spp (Lim m 1), Limulus spp (Lim p 1, Lim p
1.0101, Lim p 2, Lim p LPA), Liposcelis spp (Lip b 1, Lip b
1.0101), Litchi spp (Lit c 1, Lit c 1.0101, Lit c IFR, Lit c TPI),
Lithobates spp (Lit ca 1), Litopenaeus spp (Lit se 1, Lit v 1, Lit
v 1.0101, Lit v 2, Lit v 2.0101, Lit v 3, Lit v 3.0101, Lit v 4,
Lit v 4.0101), Filiaria spp (Loa lo 3, Loa lo 3.0101), Lobotes spp
(Lob su 1), Locusta spp (Loc m 7, Loc m 7.0101), Loligo spp (Lol b
1, Lol e 1), Lolium spp (Lol m 2, Lol m 5, Lol p 1, Lol p 1.0101,
Lol p 1.0102, Lol p 1.0103, Lol p 10, Lol p 11, Lol p 11.0101, Lol
p 12, Lol p 13, Lol p 2, Lol p 2.0101, Lol p 3, Lol p 3.0101, Lol p
4, Lol p 4.0101, Lol p 5, Lol p 5.0101, Lol p 5.0102, Lol p 7, Lol
p CyP, Lol p Fr, Lol p Legumin), Lonomia spp (Lon o 7, Lon o
7.0101), Lophodytes spp (Lop cu 1), Lophonetta spp (Lop sp 1),
Lupinus spp (Lup a 1, Lup a alpha_Conglutin, Lup a delta_Conglutin,
Lup a gamma_Conglutin, Lup an 1, Lup an 1.0101, Lup an
alpha_Conglutin, Lup an delta_Conglutin, Lup an gamma_Conglutin,
Lup I 17 kD), Lutjanus spp (Lut a 1, Lut c 1, Lut cy 1, Lut gr 1,
Lut gu 1, Lut jo 1), Lutraria spp (Lut p 1), Lutjanus spp (Lut pu
1, Lut sy 1), Lycopersicon spp (Lyc e 1, Lyc e 1.0101, Lyc e 11S
Globulin, Lyc e 2, Lyc e 2.0101, Lyc e 2.0102, Lyc e 3, Lyc e
3.0101, Lyc e 4, Lyc e 4.0101, Lyc e ARP605, Lyc e Chitinase, Lyc e
Glucanase, Lyc e Peroxidase, Lyc e PG, Lyc e PME, Lyc e PR23, Lyc e
Vicilin), Maconellicoccus spp (Mac h 7, Mac h 7.0101), Macruronus
spp (Mac ma 1, Mac n 1), Maclura spp (Mac po 17 kD), Macrobrachium
spp (Mac ro 1, Mac ro 1.0101, Mac ro Hemocyanin), Macropus spp
(Macr s Gelatin), Malus spp (Mal d 1, Mal d 1.0101, Mal d 1.0102,
Mal d 1.0103, Mal d 1.0104, Mal d 1.0105, Mal d 1.0106, Mal d
1.0107, Mal d 1.0108, Mal d 1.0109, Mal d 1.0201, Mal d 1.0202, Mal
d 1.0203, Mal d 1.0204, Mal d 1.0205, Mal d 1.0206, Mal d 1.0207,
Mal d 1.0208, Mal d 1.0301, Mal d 1.0302, Mal d 1.0303, Mal d
1.0304, Mal d 1.0401, Mal d 1.0402, Mal d 1.0403, Mal d 2, Mal d
2.0101, Mal d 3, Mal d 3.0101, Mal d 3.0102, Mal d 3.0201, Mal d
3.0202, Mal d 3.0203, Mal d 4, Mal d 4.0101, Mal d 4.0102, Mal d
4.0201, Mal d 4.0202, Mal d 4.0301, Mal d 4.0302), Malpighia spp
(Mal g 4, Mal g Hevein), Malus spp (Mal p 1), Malassezia spp (Mala
f 2, Mala f 2.0101, Mala f 3, Mala f 3.0101, Mala f 4, Mala f
4.0101, Mala g 10, Mala s 1, Mala s 1.0101, Mala s 10, Mala s
10.0101, Mala s 11, Mala s 11.0101, Mala s 12, Mala s 12.0101, Mala
s 13, Mala s 13.0101, Mala s 5, Mala s 5.0101, Mala s 6, Mala s
6.0101, Mala s 7, Mala s 7.0101, Mala s 8, Mala s 8.0101, Mala s 9,
Mala s 9.0101), Manihot spp (Man e 5, Man e 5.0101, Man e FPA, Man
e GAPDH), Mangifera spp (Man i 1, Man i 14 kD, Man i 2, Man i 3,
Man i 3.01, Man i 3.02, Man i Chitinase), Marsupenaeus spp (Mar j
1, Mar j 1.0101, Mar j 2, Mar j 4), Matricaria spp (Mat c 17 kD),
Mecopoda spp (Mec e 7), Megalobrama spp (Meg am 2, Meg am CK),
Megathura spp (Meg c Hemocyanin), Megalops spp (Meg sp 1),
Melanogrammus spp (Mel a 1), Meleagris spp (Mel g 1, Mel g 2, Mel g
3, Mel g PRVB, Mel g TSA), Melicertus spp (Mel I 1), Menticirrhus
spp (Men am 1), Mercurialis spp (Mer a 1, Mer a 1.0101), Merluccius
spp (Mer ap 1, Mer au 1, Mer bi 1, Mer ca 1, Mer ga 1, Mer hu 1),
Merlangius spp (Mer me 1), Merluccius spp (Mer mr 1, Mer pa 1, Mer
po 1, Mer pr 1, Mer se 1), Meriones spp (Mer un 23 kD), Metarhizium
spp (Met a 30), Metapenaeopsis spp (Met ba 1), Metapenaeus spp (Met
e 1, Met e 1.0101, Met e 2), Metasequoia spp (Met gI 2),
Metapenaeus spp (Met j 1, Met j 2), Metanephrops spp (Met ja 1),
Metapenaeopsis spp (Met la 1), Metanephrops spp (Met t 2),
Micromesistius spp (Mic po 1), Micropogonias spp (Mic un 1),
Mimachlamys spp (Mim n 1), Momordica spp (Mom c RIP), Morus spp
(Mor a 17 kD, Mor a 4), Morone spp (Mor am 1), Morus spp (Mor n 3,
Mor n 3.0101), Morone spp (Mor sa 1, Mor sc 1), Mugil spp (Mug c
1), Muraenolepis spp (Mur mi 1), Musa spp (Mus a 1, Mus a 1.0101,
Mus a 2, Mus a 2.0101, Mus a 3, Mus a 3.0101, Mus a 4, Mus a
4.0101, Mus a 5, Mus a 5.0101, Mus a 5.0102), Mus spp (Mus m 1, Mus
m 1.0101, Mus m 1.0102, Mus m 2, Mus m Gelatin, Mus m IgG, Mus m
MSA, Mus m Muromonab, Mus m Phosvitin), Mustela spp (Mus p 17 kD),
Musa spp (Mus xp 1, Mus xp 2, Mus xp 5), Mycteroperca spp (Myc bo
1, Myc mi 1, Myc ph 1), Myceliophthora spp (Myc sp Laccase),
Myrmecia spp (Myr p 1, Myr p 1.0101, Myr p 2, Myr p 2.0101, Myr p
2.0102, Myr p 3, Myr p 3.0101), Mytilus spp (Myt e 1, Myt g 1, Myt
g PM), Myzus spp (Myz p 7, Myz p 7.0101), Nemorhedus spp (Nae go
Hya), Necator spp (Nec a Calreticulin), Nemipterus spp (Nem vi 1),
Neosartorya spp (Neo fi 1, Neo fi 22), Neochen spp (Neo ju 1),
Neoscona spp (Neo n 7, Neo n 7.0101), Nephelium spp (Nep I GAPDH),
Nephrops spp (Nep n 1, Nep n DF9), Neptunea spp (Nep po 1, Nep po
1.0101), Nicotiana spp (Nic t 8, Nic t Osmotin, Nic t Villin),
Nimbya spp (Nim c 1, Nim s 1), Nippostrongylus spp (Nip b Ag1),
Nycticebus spp (Nyc c 1), Octopus spp (Oct f 1, Oct 11, Oct v 1,
Oct v 1.0101, Oct v PM), Ocyurus spp (Ocy ch 1), Olea spp (Ole e 1,
Ole e 1.0101, Ole e 1.0102, Ole e 1.0103, Ole e 1.0104, Ole e
1.0105, Ole e 1.0106, Ole e 1.0107, Ole e 10, Ole e 10.0101, Ole e
11, Ole e 11.0101, Ole e 11.0102, Ole e 12, Ole e 13, Ole e 2, Ole
e 2.0101, Ole e 3, Ole e 3.0101, Ole e 36 kD, Ole e 4, Ole e
4.0101, Ole e 5, Ole e 5.0101, Ole e 6, Ole e 6.0101, Ole e 7, Ole
e 7.0101, Ole e 8, Ole e 8.0101, Ole e 9, Ole e 9.0101),
Ommastrephes spp (Omm b 1, Omm b 1.0101), Oncorhynchus spp (Onc ke
1, Onc ke 18 kD, Onc ke alpha2l, Onc ke Vitellogenin, Onc m 1, Onc
m 1.0101, Onc m 1.0201, Onc m alpha2l, Onc m Protamine, Onc m
Vitellogenin, Onc ma 1, Onc ma FPA, Onc ma FSA, Onc ma TPI, Onc n
1), Onchocerca spp (Onc o 3, Onc o 3.0101), Oncorhynchus spp (Onc
is 1), Onchocerca spp (Onc v 3, Onc v 3.0101), Oratosquilla spp
(Ora o 1, Ora 01.0101), Oreochromis spp (Ore a 1, Ore mo 1, Ore mo
2, Ore mo FPA, Ore mo SCAF7145, Ore nil, Ore ni 18 kD, Ore ni 45
kD), Ornithonyssus spp (Orn sy 10, Orn sy 10.0101, Orn sy 10.0102),
Oryctolagus spp (Ory c 1, Ory c 1.0101, Ory c 2, Ory c Casein, Ory
c Phosvitin, Ory c RSA), Oryza spp (Ory s 1, Ory s 1.0101, Ory s
11, Ory s 12, Ory s 12.0101, Ory s 13, Ory s 14, Ory s 17 kD, Ory s
19 kD, Ory s 2, Ory s 23, Ory s 3, Ory s 7, Ory s aA_TI, Ory s
GLP52, Ory s GLP63, Ory s Glyoxalase I, Ory s NRA), Ostrya spp (Ost
c 1, Ost c 1.0101), Ovis spp (Ovi a ALA, Ovi a BLG, Ovi a Casein,
Ovi a Casein alphaS1, Ovi a Casein alphaS2, Ovi a Casein beta, Ovi
a Casein kappa, Ovi a Phosvitin, Ovi a SSA), Pachycondyla spp (Pac
c 3), Pagrus spp (Pag m 1, Pag pa 1), Pampus spp (Pam ar 1, Pam c
1), Pandalus spp (Pan b 1, Pan b 1.0101), Pangasius spp (Pan bo 1),
Pandalus spp (Pan e 1, Pan e 1.0101, Pan e 4), Panulirus spp (Pan h
1, Pan by 1), Pangasius spp (Pan by 18 kD, Pan by 45 kD), Panulirus
spp (Pan j 1), Panthera spp (Pan I 1, Pan o 1, Pan p 1), Panulirus
spp (Pan s 1, Pan s 1.0101), Panthera spp (Pan t 1), Pan spp (Pan
tr TCTP), Papaver spp (Pap s 17 kD, Pap s 2, Pap s 34 kD), Papilio
spp (Pap xu 7, Pap xu 7.0101, Pap xu 7.0102), Paralichthys spp (Par
a 1), Parasilurus spp (Par as 1, Par c 1), Paralithodes spp (Par c
1.0101, Par c 1.0102, Par f 1), Parthenium spp (Par h 1),
Parietaria spp (Par j 1, Par j 1.0101, Par j 1.0102, Par j 1.0103,
Par j 1.0201, Par j 2, Par j 2.0101, Par j 2.0102, Par j 3, Par j
3.0101, Par j 3.0102, Par j 4, Par j 4.0101, Par 01-J2),
Paralichthys spp (Par le 1), Parietaria spp (Par m 1, Par o 1, Par
o 1.0101), Paralichthys spp (Par of 1, Par of alpha2l), Parahucho
spp (Par pe Vitellogenin), Passiflora spp (Pas e Chitinase, Pas e
Hevein), Paspalum spp (Pas n 1, Pas n 1.0101, Pas n 13),
Patinopecten spp (Pat y 1), Pediculus spp (Ped h 7, Ped h 7.0101),
Penaeus spp (Pen a 1, Pen a 1.0101, Pen a 1.0102, Pen a 1.0102
(103-117), Pen a 1.0102 (109-123), Pen a 1.0102 (1-15), Pen a
1.0102 (115-129), Pen a 1.0102 (121-135), Pen a 1.0102 (127-141),
Pen a 1.0102 (13-27), Pen a 1.0102 (133-147), Pen a 1.0102
(139-153), Pen a 1.0102 (145-159)), Farfantepenaeus spp (Pen a
1.0102 (151-165)), Penaeus spp (Pen a 1.0102 (157-171), Pen a
1.0102 (163-177), Pen a 1.0102 (169-183), Pen a 1.0102 (175-189),
Pen a 1.0102 (181-195), Pen a 1.0102 (187-201), Pen a 1.0102
(193-207), Pen a 1.0102 (19-33), Pen a 1.0102 (199-213), Pen a
1.0102 (205-219), Pen a 1.0102 (211-225), Pen a 1.0102 (217-231),
Pen a 1.0102 (223-237), Pen a 1.0102 (229-243)), Farfantepenaeus
spp (Pen a 1.0102 (235-249)), Penaeus spp (Pen a 1.0102 (241-255),
Pen a 1.0102 (247-261), Pen a 1.0102 (253-267), Pen a 1.0102
(25-39), Pen a 1.0102 (259-273), Pen a 1.0102 (265-279), Pen a
1.0102 (270-284), Pen a 1.0102 (31-45), Pen a 1.0102 (37-51), Pen a
1.0102 (43-57), Pen a 1.0102 (49-63)), Farfantepenaeus spp (Pen a
1.0102 (55-69)), Penaeus spp (Pen a 1.0102 (61-75), Pen a 1.0102
(67-81), Pen a 1.0102 (7-21), Pen a 1.0102 (73-87), Pen a 1.0102
(79-93), Pen a 1.0102 (85-99), Pen a 1.0102 (91-105), Pen a 1.0102
(97-111), Pen a 1.0103), Penicillium spp (Pen b 13, Pen b 13.0101,
Pen b 26, Pen b 26.0101, Pen c 1, Pen c 13, Pen c 13.0101, Pen c
18, Pen c 19, Pen c 19.0101, Pen c 2, Pen c 22, Pen c 22.0101, Pen
c 24, Pen c 24.0101, Pen c 3, Pen c 3.0101, Pen c 30, Pen c
30.0101, Pen c 32, Pen c 32.0101, Pen c MnSOD, Pen ch 13, Pen ch
13.0101, Pen ch 18, Pen ch 18.0101, Pen ch 20, Pen ch 20.0101, Pen
ch 31, Pen ch 31.0101, Pen ch 33, Pen ch 33.0101, Pen ch 35, Pen ch
35.0101, Pen ch MnSOD), Penaeus spp (Pen i 1, Pen i 1.0101, Pen m
1, Pen m 1.0101, Pen m 1.0102, Pen m 2, Pen m 2.0101, Pen m 3, Pen
m 3.0101, Pen m 4, Pen m 4.0101, Pen m 6, Pen m 6.0101),
Penicillium spp (Pen o 18, Pen o 18.0101), Penaeus spp (Pena o 1,
Pena o 1.0101), Periplaneta spp (Per a 1, Per a 1.0101, Per a
1.0102, Per a 1.0103, Per a 1.0104, Per a 1.0105, Per a 1.0201, Per
a 10, Per a 10.0101, Per a 2, Per a 3, Per a 3.0101, Per a 3.0201,
Per a 3.0202, Per a 3.0203, Per a 4, Per a 5, Per a 6, Per a
6.0101, Per a 7, Per a 7.0101, Per a 7.0102, Per a 7.0103, Per a 9,
Per a 9.0101, Per a Cathepsin, Per a FABP, Per a Trypsin, Per f 1,
Per f 7, Per f 7.0101), Perna spp (Per v 1), Persea spp (Pers a 1,
Pers a 1.0101, Pers a 4), Petroselinum spp (Pet c 1, Pet c 2, Pet c
3), Phalaris spp (Pha a 1, Pha a 1.0101, Pha a 5, Pha a 5.0101, Pha
a 5.02, Pha a 5.03, Pha a 5.04), Phaseolus spp (Pha v 3, Pha v
3.0101, Pha v 3.0201, Pha v aAl, Pha v aAI.0101, Pha v Chitinase,
Pha v PHA, Pha v Phaseolin), Phleum spp (Phl p 1, Phl p 1.0101, Phl
p 1.0102, Phl p 11, Phl p 11.0101, Phl p 12, Phl p 12.0101, Phl p
12.0102, Phl p 12.0103, Phl p 13, Phl p 13.0101, Phl p 2, Phl p
2.0101, Phl p 3, Phl p 3.0101, Phl p 3.0102, Phl p4, Phl p4.0101,
Phl p4.0102, Phl p4.0201, Phl p4.0202, Phl p4.0203, Phl p4.0204,
Phl p 5, Phl p 5.0101, Phl p 5.0102, Phl p 5.0103, Phl p 5.0104,
Phl p 5.0105, Phl p 5.0106, Phl p 5.0107, Phl p 5.0108, Phl p
5.0109, Phl p 5.0201, Phl p 5.0202, Phl p 5.0203, Phl p 5.0204, Phl
p 5.0205, Phl p 5.0206, Phl p 5.0207, Phl p 6, Phl p 6.0101, Phl p
6.0102, Phl p 7, Phl p 7.0101, Phl p P1-P2-P5-P6, Phl p P2-P6, Phl
p P5-P1, Phl p P6-P2), Phoenix spp (Pho d 2, Pho d 2.0101, Pho d 40
kD, Pho d 90 kD), Phodopus spp (Pho s 21 kD), Phoma spp (Pho t 1),
Phragmites spp (Phr a 1, Phr a 12, Phr a 13, Phr a 4, Phr a 5),
Phytolacca spp (Phy a RIP), Pimpinella spp (Pim a 1, Pim a 2),
Pinna spp (Pin a 1), Piper spp (Pip n 14 kD, Pip n 28 kD), Pisum
spp (Pis s 1, Pis s 1.0101, Pis s 1.0102, Pis s 2, Pis s 2.0101,
Pis s 5, Pis s Agglutinin, Pis s Albumin), Pistacia spp (Pis v 1,
Pis v 1.0101, Pis v 2, Pis v 2.0101, Pis v 2.0201, Pis v 3, Pis v
3.0101, Pis v 4, Pis v 4.0101, Pis v 5, Pis v 5.0101), Platanus spp
(Pla a 1, Pla a 1.0101, Pla a 2, Pla a 2.0101, Pla a 3, Pla a
3.0101, Pla a 8), Platichthys spp (Pla f 1), Plantago spp (Pla I 1,
Pla I 1.0101, Pla I 1.0102, Pla I 1.0103, Pla I Cytochrome C),
Platanus spp (Pla oc 1, Pla or 1, Pla or 1.0101, Pla or 2, Pla or
2.0101, Pla or 3, Pla or 3.0101, Pla or 4, Pla or CyP, Pla r 1),
Plectropomus spp (Ple ar 1), Pleospora spp (Ple h 1), Plectropomus
spp (Ple le 1), Plodia spp (Plo i 1, Plo i 1.0101, Plo i 2, Plo i
2.0101), Poa spp (Poa p 1, Poa p 1.0101, Poa p 10, Poa p 12, Poa p
13, Poa p 2, Poa p 4, Poa p 5, Poa p 5.0101, Poa p 6, Poa p 7),
Polistes spp (Pol a 1, Pol a 1.0101, Pol a 2, Pol a 2.0101, Pol a
5, Pol a 5.0101, Pol d 1, Pol d 1.0101, Pol d 1.0102, Pol d 1.0103,
Pol d 1.0104, Pol d 4, Pol d 4.0101, Pol d 5, Pol d 5.0101, Pol e
1, Pol e 1.0101, Pol e 2, Pol e 4, Pol e 4.0101, Pol e 5, Pol e
5.0101, Pol f 5, Pol f 5.0101, Pol g 1, Pol g 1.0101, Pol g 2, Pol
g 4, Pol g 5, Pol g 5.0101, Pol he MLT, Pol m 5, Pol m 5.0101),
Polypedilum spp (Pol n 1), Pollicipes spp (Pol po 1), Pollachius
spp (Pol vi 1), Polybia spp (Poly p 1, Poly p 1.0101, Poly p 2,
Poly p 5, Poly s 5, Poly s 5.0101), Pomatomus spp (Porn sa 1),
Pongo spp (Pon ab HSA), Pontastacus spp (Pon I 4, Pon I 4.0101, Pon
I 7, Pon I 7.0101), Portunus spp (Por s 1, Por s 1.0101, Por s
1.0102, Por tr 1, Por tr 1.0101), Protortonia spp (Pro ca 38 kD),
Procumbarus spp (Pro cl 1, Pro cl 1.0101, Pro cl 21 kD), Prosopis
spp (Pro j 20 kD), Prunus spp (Pru ar 1, Pru ar 1.0101, Pru ar 3,
Pru ar 3.0101, Pru av 1, Pru av 1.0101, Pru av 1.0201, Pru av
1.0202, Pru av 1.0203, Pru av 2, Pru av 2.0101, Pru av 3, Pru av
3.0101, Pru av 4, Pru av 4.0101, Pru c 1, Pru d 1, Pru d 2, Pru d
3, Pru d 3.0101, Pru d 4, Pru du 1, Pru du 2, Pru du 2S Albumin,
Pru du 3, Pru du 3.0101, Pru du 4, Pru du 4.0101, Pru du 4.0102,
Pru du 5, Pru du 5.0101, Pru du 6, Pru du 6.0101, Pru du 6.0201,
Pru du Conglutin, Pru p 1, Pru p 1.0101, Pru p 2, Pru p 2.0101, Pru
p 2.0201, Pru p 2.0301, Pru p 3, Pru p 3.0101, Pru p 3.0102, Pru p
4, Pru p 4.0101, Pru p 4.0201, Pru sa 3),
Psilocybe spp (Psi c 1, Psi c 1.0101, Psi c 2, Psi c 2.0101),
Psoroptes spp (Pso o 1, Pso o 10, Pso o 10.0101, Pso o 11, Pso o
13, Pso o 14, Pso a 2, Pso o 21, Pso o 3, Pso o 5, Pso o 7), Puma
spp (Pum c 1), Punica spp (Pun g 3), Pyrus spp (Pyr c 1, Pyr c
1.0101, Pyr c 3, Pyr c 3.0101, Pyr c 4, Pyr c 4.0101, Pyr c 5, Pyr
c 5.0101, Pyr py 2), Quercus spp (Que a 1, Que a 1.0101, Que a
1.0201, Que a 1.0301, Que a 1.0401, Que a 2, Que a 4), Rachycentron
spp (Rac ca 1), Rana spp (Ran e 1, Ran e 1.0101, Ran e 2, Ran e
2.0101), Ranina spp (Ran ra 1), Rangifer spp (Ran t BLG), Rattus
spp (Rat n 1, Rat n 1.0101, Rat n Casein, Rat n Gelatin, Rat n IgG,
Rat n Phosvitin, Rat n RSA, Rat n Transferrin), Rhizomucor spp (Rhi
m AP), Rhizopus spp (Rhi nv Lipase, Rhi o Lipase), Rhomboplites spp
(Rho au 1), Rhodotorula spp (Rho m 1, Rho m 1.0101, Rho m 2, Rho m
2.0101), Ricinus spp (Ric c 1, Ric c 1.0101, Ric c 2, Ric c 3, Ric
c 8, Ric c RIP), Rivulus spp (Riv ma 1), Robinia spp (Rob p 2, Rob
p 4, Rob p Glucanase), Rosa spp (Ros r 3), Roystonea spp (Roy e 2),
Rubus spp (Rub i 1, Rub i 1.0101, Rub i 3, Rub i 3.0101, Rub i
Chitinase, Rub i CyP), Saccharomyces spp (Sac c Carboxypeptidase Y,
Sac c CyP, Sac c Enolase, Sac c Glucosidase, Sac c Invertase, Sac c
MnSOD, Sac c P2, Sac c Profilin), Salvelinus spp (Sal f 1), Salsola
spp (Sal k 1, Sal k 1.0101, Sal k 1.0201, Sal k 1.0301, Sal k
1.0302, Sal k 2, Sal k 2.0101, Sal k 3, Sal k 3.0101, Sal k 4, Sal
k 4.0101, Sal k 4.0201, Sal k 5, Sal k 5.0101), Salvelinus spp (Sal
le Vitellogenin), Salmo spp (Sal s 1, Sal s 1.0101, Sal s 1.0201,
Sal s 2, Sal s 2.0101, Sal s Gelatin), Sambucus spp (Sam n 1),
Sander spp (San lu 1), Saponaria spp (Sap o RIP), Sardinops spp
(Sar m 1), Sarkidiornis spp (Sar ml 1), Sardina spp (Sar p 1),
Sarcoptes spp (Sar s 1, Sar s 14, Sar s 3, Sar s GST, Sar s PM),
Sardinops spp (Sar sa 1, Sar sa 1.0101), Schistosoma spp (Sch j
GST, Sch j PM, Sch j Sj22, Sch j Sj67, Sch ma Sm20, Sch ma Sm21,
Sch ma Sm22, Sch ma Sm31), Sciaenops spp (Sci oc 1), Scomber spp
(Sco a 1), Scombermorus spp (Sco ca 1), Scomberomorus spp (Sco g
1), Scomber spp (Sco j 1, Sco ma 1, Sco s 1), Scolopendra spp (Sco
y 7, Sco y 7.0101), Scylla spp (Scy o 1, Scy o 1.0101, Scy o 2, Scy
pa 1, Scy pa 2, Scy s 1, Scy s 1.0101, Scy s 2), Sebastes spp (Seb
fa 1, Seb in 1, Seb m 1, Seb m 1.0101, Seb m 1.0201), Secale spp
(Sec c 1, Sec c 12, Sec c 13, Sec c 2, Sec c 20, Sec c 20.0101, Sec
c 20.0201, Sec c 28, Sec c 3, Sec c 4, Sec c 4.0101, Sec c 4.0201,
Sec c 5, Sec c 5.0101, Sec c aA_TI, Sec c aA_TI.0101), Senecio spp
(Sen j MDH, Sen j PL), Sepia spp (Sep e 1, Sep e 1.0101),
Sepioteuthis spp (Sep I 1, Sep I 1.0101), Sepia spp (Sep m 1),
Seriola spp (Ser d 1, Ser la 1), Sergestes spp (Ser lu 1), Seriola
spp (Ser q 1, Ser ri 1), Sesamum spp (Ses i 1, Ses i 1.0101, Ses i
2, Ses i 2.0101, Ses i 3, Ses i 3.0101, Ses i 4, Ses i 4.0101, Ses
i5, Ses i 5.0101, Ses i 6, Ses i 6.0101, Ses i 7, Ses i 7.0101, Ses
i 8), Shigella spp (Shi bo GST, Shi dy GST), Simulia spp (Sim vi 1,
Sim vi 2, Sim vi 3, Sim vi 4, Sim vi 70 kD), Sinapis spp (Sin a 1,
Sin a 1.0101, Sin a 1.0104, Sin a 1.0105, Sin a 1.0106, Sin a
1.0107, Sin a 1.0108, Sin a 2, Sin a 2.0101, Sin a 3, Sin a 3.0101,
Sin a 4, Sin a 4.0101), Sinonovacula spp (Sin c 1, Sin c 1.0101),
Solenopsis spp (Sol g 2, Sol g 2.0101, Sol g 3, Sol g 3.0101, Sol g
4, Sol g 4.0101, Sol g 4.0201, Sol i 1, Sol i 1.0101, Sol i 2, Sol
i 2.0101, Sol i 3, Sol i 3.0101, Sol i 4, Sol i 4.0101), Solenocera
spp (Sol me 1), Solenopsis spp (Sol r 1, Sol r 2, Sol r 2.0101, Sol
r 3, Sol r 3.0101, Sol s 2, Sol s 2.0101, Sol s 3, Sol s 3.0101,
Sol s 4), Solea spp (Sol so 1, Sol so TPI), Solanum spp (Sola t 1,
Sola t 1.0101, Sola t 2, Sola t 2.0101, Sola t 3, Sola t 3.0101,
Sola t 3.0102, Sola t 4, Sola t 4.0101, Sola t 8, Sola t
Glucanase), Sorghum spp (Sor b 1, Sor h 1, Sor h 1.0101, Sor h 12,
Sor h 7), Sparus spp (Spa a 1), Sphyrna spp (Sph ti 1), Spirulina
spp (Spi mx beta_Phycocyanin), Spinacia spp (Spi o 2, Spi o
RuBisCO), Squilla spp (Squ ac 1, Squ ac 1.0101, Squ o 1, Squ o
1.0101), Staphylococcus spp (Sta a FBP, Sta a SEA, Sta a SEB, Sta a
SEC, Sta a SED, Sta a SEE, Sta a TSST), Stachybotrys spp (Sta c 3,
Sta c 3.0101, Sta c Cellulase, Sta c Hemolysin, Sta c SchS34, Sta c
Stachyrase A), Stemphylium spp (Ste b 1, Ste c 1, Ste v 1),
Stolephorus spp (Sto i 1), Struthio spp (Str c 1, Str c 2, Str c
3), Streptococcus spp (Str dy Streptokinase), Streptomyces spp (Str
g Pronase), Streptococcus spp (Str pn PspC), Strongylocentrotus spp
(Str pu 18 kD, Str pu Vitellogenin), Streptococcus spp (Str py
SPEA, Str py SPEC, Str py Streptokinase), Strongyloides spp (Str st
45 kD), Streptomyces spp (Str v PAT), Styela spp (Sty p 1),
Suidasia spp (Sui m 1, Sui m 13, Sui m 2, Sui m 3, Sui m 5, Sui m
5.01, Sui m 5.02, Sui m 5.03, Sui m 6, Sui m 7, Sui m 8, Sui m 9),
Sus spp (Sus s ACTH, Sus s ALA, Sus s Amylase, Sus s BLG, Sus s
Casein, Sus s Casein alphaS1, Sus s Casein alphaS2, Sus s Casein
beta, Sus s Casein kappa, Sus s Gelatin, Sus s HG, Sus s Insulin,
Sus s Lipase, Sus s Pepsin, Sus s Phosvitin, Sus s PRVB, Sus s PSA,
Sus s TCTP), Syntelopodeuma spp (Syn y 7, Syn y 7.0101), Syringa
spp (Syr v 1, Syr v 1.0101, Syr v 1.0102, Syr v 1.0103, Syr v 2,
Syr v 3, Syr v 3.0101), Tabanus spp (Tab y 1, Tab y 1.0101, Tab y
2, Tab y 2.0101, Tab y 5, Tab y 5.0101), Tadorna spp (Tad ra 1),
Talaromyces spp (Tal st 22, Tal st 3, Tal st 8), Taraxacum spp (Tar
o 18 kD), Taxodium spp (Tax d 2), Tegenaria spp (Teg d Hemocyanin),
Teladorsagia spp (Tel ci 3), Thaumetopoea spp (Tha p 1, Tha p
1.0101, Tha p 2, Tha p 2.0101), Theragra spp (The c 1), Thermomyces
spp (The I Lipase, The sp Lipase, The sp Xylanase), Thunnus spp
(Thu a 1, Thu a 1.0101, Thu a Collagen, Thu al 1, Thu at 1, Thu o
1, Thu o Collagen), Thuja spp (Thu oc 3, Thu p 1), Thunnus spp (Thu
t 1, Thu to 1), Thyrsites spp (Thy at 1), Thyrophygus spp (Thy y 7,
Thy y 7.0101), Todarodes spp (Tod p 1, Tod p 1.0101, Tod p 1.0102),
Toxoptera spp (Tox c 7, Tox c 7.0101), Toxocara spp (Tox ca TES120,
Tox ca TES26, Tox ca TES30), Toxoplasma spp (Tox g HSP70),
Trachypenaeus spp (Tra c 1), Trachinotus spp (Tra ca 1), Trachurus
spp (Tra j 1, Tra j Gelatin, Tra tr Gelatin), Triticum spp (Tri a
1, Tri a 10 kD, Tri a 12, Tri a 12.0101, Tri a 12.0102, Tri a
12.0103, Tri a 12.0104, Tri a 13, Tri a 14, Tri a 14.0101, Tri a
14.0201, Tri a 15, Tri a 15.0101, Tri a 18, Tri a 18.0101, Tri a
19, Tri a 19.0101, Tri a 2, Tri a 21, Tri a 21.0101, Tri a 23kd,
Tri a 25, Tri a 25.0101, Tri a 26, Tri a 26.0101, Tri a 27, Tri a
27.0101, Tri a 28, Tri a 28.0101, Tri a 29, Tri a 29.0101, Tri a
29.0201, Tri a 3, Tri a 30, Tri a 30.0101, Tri a 31, Tri a 31.0101,
Tri a 32, Tri a 32.0101, Tri a 33, Tri a 33.0101, Tri a 34, Tri a
34.0101, Tri a 35, Tri a 35.0101, Tri a 36, Tri a 36.0101, Tri a
37, Tri a 37.0101, Tri a 4, Tri a 4.0101, Tri a 4.0201, Tri a 5,
Tri a 7, Tri a aA_SI, Tri a alpha_Gliadin, Tri a bA, Tri a Bd36K,
Tri a beta_Gliadin, Tri a Chitinase, Tri a CM16, Tri a DH, Tri a
Endochitinase, Tri a gamma_Gliadin, Tri a Germin, Tri a Gliadin,
Tri a GST, Tri a LMW Glu, Tri a LMW-GS B16, Tri a LMW-GS P42, Tri a
LMW-GS P73, Tri a LTP2, Tri a omega2_Gliadin, Tri a Peroxidase, Tri
a Peroxidase 1, Tri a SPI, Tri a TLP, Tri a Tritin, Tri a XI),
Tritirachium spp (Tri al Proteinase K), Tribolium spp (Tri ca 17,
Tri ca 17.0101, Tri ca 7, Tri ca 7.0101), Trichostrongylus spp (Tri
co 3, Tri co 3.0101), Trichophyton spp (Tri eq 4), Trigonella spp
(Tri fg 1, Tri fg 2, Tri fg 3, Tri fg 4), Trichosanthes spp (Tri k
RIP), Trichiurus spp (Tri le 1), Triticum spp (Tri m Peroxidase),
Trichophyton spp (Tri me 2, Tri me 4), Trisetum spp (Tri p 1, Tri p
5), Trichinella spp (Tri ps 3, Tri ps 3.0101), Trichophyton spp
(Tri r 2, Tri r 2.0101, Tri r 4, Tri r 4.0101), Trichoderma spp
(Tri rs Cellulase), Triticum spp (Tri s 14), Trichophyton spp (Tri
sc 2, Tri sc 4, Tri so 2), Trichinella spp (Tri sp 3, Tri sp
3.0101, Tri sp 3.0102, Tri sp 3.0103, Tri sp 3.0104, Tri sp 3.0105,
Tri sp 3.0106), Trichophyton spp (Tri t 1, Tri t 1.0101, Tri t 4,
Tri t 4.0101), Triticum spp (Tri td 14, Tri td aA_TI), Trichoderma
spp (Tri v Cellulase), Trichophyton spp (Tri ve 4), Triatoma spp
(Tria p 1, Tria p 1.0101), Triplochiton spp (Trip s 1), Turbo spp
(Tur c 1, Tur c PM), Tyrophagus spp (Tyr p 1, Tyr p 10, Tyr p
10.0101, Tyr p 10.0102, Tyr p 13, Tyr p 13.0101, Tyr p 2, Tyr p
2.0101, Tyr p 24, Tyr p 24.0101, Tyr p 3, Tyr p 3.0101, Tyr p 4,
Tyr p 5, Tyr p 5.01, Tyr p 5.02, Tyr p 5.03, Tyr p 7, Tyr p alpha
Tubulin), Ulocladium spp (Ulo a 1, Ulo at 1, Ulo b 1, Ulo c 1, Ulo
co 1, Ulo cu 1, Ulo mu 1, Ulo ob 1, Ulo se 1, Ulo su 1, Ulo to 1),
Uncia spp (Unc u 1), Urophycis spp (Uro to 1), Vaccinium spp (Vac m
3), Varroa spp (Var j 13 kD), Venerupis spp (Ven ph 1, Ven ph
1.0101), Vespula spp (Ves f 1, Ves f 2, Ves f 5, Ves f 5.0101, Ves
g 1, Ves g 2, Ves g 5, Ves g 5.0101, Ves m 1, Ves m 1.0101, Ves m
2, Ves m 2.0101, Ves m 5, Ves m 5.0101, Ves m MLT, Ves p 1, Ves p
2, Ves p 5, Ves p 5.0101, Ves s 1, Ves s 1.0101, Ves s 2, Ves s 5,
Ves s 5.0101, Ves v 1, Ves v 1.0101, Ves v 2, Ves v 2.0101, Ves v
2.0201, Ves v 3, Ves v 3.0101, Ves v 5, Ves v 5.0101, Ves v 5-Pol a
5, Ves vi 5, Ves vi 5.0101), Vespa spp (Vesp c 1, Vesp c 1.0101,
Vesp c 2, Vesp c 5, Vesp c 5.0101, Vesp c 5.0102, Vesp m 1, Vesp m
1.0101, Vesp m 5, Vesp m 5.0101, Vesp ma 1, Vesp ma 2, Vesp ma 5,
Vesp ma MLT, Vesp v MLT), Vigna spp (Vig r 1, Vig r 1.0101, Vig r
17 kD, Vig r 5, Vig r 8S Globulin, Vig r Albumin, Vig r
beta-Conglycinin), Vitis spp (Vit v 1, Vit v 1.0101, Vit v 4, Vit v
5, Vit v Glucanase, Vit v TLP), Xiphias spp (Xip g 1, Xip g 1.0101,
Xip g 25 kD), Zea spp (Zea m 1, Zea m 1.0101, Zea m 11, Zea m 12,
Zea m 12.0101, Zea m 12.0102, Zea m 12.0103, Zea m 12.0104, Zea m
12.0105, Zea m 13, Zea m 14, Zea m 14.0101, Zea m 14.0102, Zea m 2,
Zea m 20S, Zea m 22, Zea m 25, Zea m 25.0101, Zea m 27 kD Zein, Zea
m 3, Zea m 4, Zea m 5, Zea m 50 kD Zein, Zea m 7, Zea m Chitinase,
Zea m G1, Zea m G2, Zea m PAO, Zea m Zm13), Zeus spp (Zeu fa 1),
Ziziphus spp (Ziz m 1, Ziz m 1.0101), Zoarces spp (Zoa a ISP III),
Zygophyllum spp (Zyg f 2)
[0321] In this context, the terms in brackets indicate the
particular preferred allergens from the particular source.
[0322] Most preferably the antigen associated with allergy or
allergic disease is preferably derived from a source selected from
the list consisting of grass pollen (e.g. pollen of rye), tree
pollen (e.g. pollen of hazel, birch, alder, ash), flower pollen,
herb pollen (e.g. pollen of mugwort), dust mite (e.g. Der f 1, Der
p 1, Eur m 1, Der m 1 Der f 2, Der p 2, Eur m 2, Tyr p 2, Lep d 2),
mold (e.g. allergens of Acremonium, Aspergillus, Cladosporium,
Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma,
or Alternaria), animals (e.g Fel d1, Fel d2, Fel d3, or Fel d4 of
cats), food (e.g. allergens of fish (e.g. bass, cod, flounder),
seafood (e.g. crab, lobster, shrimps), egg, wheat, nuts (e.g.
peanuts, almonds, cashews, walnuts), soya, milk, etc.) or insect
venom (e.g. allergens from the venom of wasps, bees, hornets, ants,
mosquitos, or ticks).
c) Antigens Associated with Autoimmune Disease:
[0323] Antigens associated with autoimmune disease are preferably
selected from autoantigens asscociated with autoimmune diseases
selected from Addison disease (autoimmune adrenalitis, Morbus
Addison), alopecia areata, Addison's anemia (Morbus Biermer),
autoimmune hemolytic anemia (AIHA), autoimmune hemolytic anemia
(AIHA) of the cold type (cold hemagglutinine disease, cold
autoimmune hemolytic anemia (AIHA) (cold agglutinin disease),
(CHAD)), autoimmune hemolytic anemia (AIHA) of the warm type (warm
AIHA, warm autoimmune haemolytic anemia (AIHA)), autoimmune
hemolytic Donath-Landsteiner anemia (paroxysmal cold
hemoglobinuria), antiphospholipid syndrome (APS), atherosclerosis,
autoimmune arthritis, arteriitis temporalis, Takayasu arteriitis
(Takayasu's disease, aortic arch disease), temporal
arteriitis/giant cell arteriitis, autoimmune chronic gastritis,
autoimmune infertility, autoimmune inner ear disease (AIED),
Basedow's disease (Morbus Basedow), Bechterew's disease (Morbus
Bechterew, ankylosing spondylitis, spondylitis ankylosans),
Behcet's syndrome (Morbus Behcet), bowel disease including
autoimmune inflammatory bowel disease (including colitis ulcerosa
(Morbus Crohn, Crohn's disease), cardiomyopathy, particularly
autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (DCM),
celiac sprue dermatitis (gluten mediated enteropathia), chronic
fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy (CIDP), chronic polyarthritis,
Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome,
CREST syndrome (syndrom with Calcinosis cutis, Raynaud phenomenon,
motility disorders of the esophagus, sklerodaktylia and
teleangiectasia), Crohn's disease (Morbus Crohn, colitis ulcerosa),
dermatitis herpetiformis during, dermatologic autoimmune diseases,
dermatomyositis, Diabetes, Diabetes mellitus Type 1 (type I
diabetes, insuline dependent Diabetes mellitus), Diabetes mellitus
Type 2 (type II diabetes), essential mixed cryoglobulinemia,
essential mixed cryoglobulinemia, fibromyalgia, fibromyositis,
Goodpasture syndrome (anti-GBM mediated glomerulonephritis), graft
versus host disease, Guillain-Barre syndrome (GBM,
Polyradikuloneuritis), haematologic autoimmune diseases, Hashimoto
thyroiditis, hemophilia, acquired hemophilia, hepatitis, autoimmune
hepatitis, particularly autoimmune forms of chronic hepatitis,
idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic
purpura, Immuno-thrombocytopenic purpura (Morbus Werlhof; ITP), IgA
nephropathy, infertility, autoimmune infertility, juvenile
rheumatoid arthritis (Morbus Still, Still syndrome), Lambert-Eaton
syndrome, lichen planus, lichen sclerosus, lupus erythematosus,
systemic lupus erythematosus (SLE), lupus erythematosus (discoid
form), Lyme arthritis (Lyme disease, borrelia arthritis), Meniere's
disease (Morbus Meniere); mixed connective tissue disease (MCTD),
multiple sclerosis (MS, encephalomyelitis disseminate, Charcot's
disease), Myasthenia gravis (myasthenia, MG), myosits,
polymyositis, neural autoimmune diseases, neurodermitis, pemphigus
vulgaris, bullous pemphigoid, scar forming pemphigoid;
polyarteriitis nodosa (periarteiitis nodosa), polychondritis
(panchondritis), polyglandular (autoimmune) syndrome (PGA syndrome,
Schmidt's syndrome), Polymyalgia rheumatica, primary
agammaglobulinemia, primary biliary cirrhosis PBC, primary
autoimmune cholangitis), progressive systemic sclerosis (PSS),
Psoriasis, Psoriasis vulgaris, Raynaud's phenomena, Reiter's
syndrome (Morbus Reiter, urethral conjunctive synovial syndrome)),
rheumatoid arthritis (RA, chronic polyarthritis, rheumatic disease
of the joints, rheumatic fever), sarcoidosis (Morbus Boeck,
Besnier-Boeck-Schaumann disease), stiff-man syndrome, Sclerodermia,
Scleroderma, Sjogren's syndrome, sympathetic ophtalmia; Transient
gluten intolerance, transplanted organ rejection, uveitis,
autoimmune uveiitis, Vasculitis, Vitiligo, (leucoderma, piebold
skin), and Wegner's disease (Morbus Wegner, Wegner's
granulomatosis)
Particularly preferred in this context are autoantigens selected
from: [0324] myelin basic protein (MBP), proteolipid protein (PLP),
and myelin oligodendrocyte glycoprotein (MOG), in each case
associated with multiple sclerosis (MS); [0325] CD44,
preproinsulin, proinsulin, insulin, glutamic acid decaroxylase
(GAD65), tyrosine phosphatase-like insulinoma antigen 2 (IA2), zinc
transporter ((ZnT8), and heat shock protein 60 (HSP60), in each
case associated with diabetes Typ I; [0326] interphotoreceptor
retinoid-binding protein (IRBP) associated with autoimmune uveitis;
[0327] acetylcholine receptor AchR, and insulin-like growth
factor-1 receptor (IGF-1R), in each case associated with Myasthenia
gravis; [0328] M-protein from beta-hemolytic streptocci
(pseudo-autoantigen) associated with Rheumatic Fever; [0329]
Macrophage migration inhibitory factor associated with Arthritis;
[0330] Ro/La RNP complex, alpha- and beta-fodrin, islet cell
autoantigen, poly(ADP)ribose polymerase (PARP), NuMA, NOR-90, Ro60
autoantigen, and p27 antigen, in each case associated with
Sjo{umlaut over (g)}ren's syndrome; [0331] Ro60 autoantigen,
low-density lipoproteins, Sm antigens of the U-1 small nuclear
ribonucleoprotein complex (B/B', D1, D2, D3, E, F, G), and RNP
ribonucleoproteins, in each case associated with lupus
erythematosus; [0332] oxLDL, beta(2)GPI, HSP60/65, and
oxLDL/beta(2)GPI, in each case associated with Atherosclerosis;
[0333] cardiac beta(1)-adrenergic receptor associated with
idiopathic dilated cardiomyopathy (DCM); [0334] histidyl-tRNA
synthetase (HisRS) associated with myositis; [0335] topoisomerase I
associated with scleroderma disease.
[0336] Furthermore, in other embodiments, said antigen is
associated with the respective autoimmune disease, like e.g. IL-17,
heat shock proteins, and/or any idiotype pathogenic T cell or
chemokine receptor which is expressed by immune cells involved in
the autoimmune response in said autoimmune disease (such as any
autoimmune diseases described herein).
d) Antigens Associated with a Cancer or Tumour Disease ("Tumour
Antigens"):
[0337] "Tumour antigens" in this context are antigens which are
preferably located on the surface of the (tumour) cell. Tumour
antigens may also be selected from proteins, which are
overexpressed in tumour cells compared to a normal cell.
Furthermore, tumour antigens also include antigens expressed in
cells which are (were) not themselves (or originally not
themselves) degenerated but are associated with the supposed
tumour. Antigens which are connected with tumour-supplying vessels
or (re)formation thereof, in particular those antigens which are
associated with neovascularization, e.g. growth factors, such as
VEGF, bFGF etc., are also included herein. Antigens connected with
a tumour furthermore include antigens from cells or tissues,
typically embedding the tumour. Further, some substances (usually
proteins or peptides) are expressed in patients suffering
(knowingly or not-knowingly) from a cancer disease and they occur
in increased concentrations in the body fluids of said patients.
These substances are also referred to as "tumour antigens", however
they are not antigens in the stringent meaning of an immune
response inducing substance. The class of tumour antigens can be
divided further into tumour-specific antigens (TSAs) and
tumour-associated-antigens (TAAs). TSAs can only be presented by
tumour cells and never by normal "healthy" cells. They typically
result from a tumour specific mutation. TAAs, which are more
common, are usually presented by both tumour and healthy cells.
These antigens are recognized and the antigen-presenting cell can
be destroyed by cytotoxic T cells. Additionally, tumour antigens
can also occur on the surface of the tumour in the form of, e.g., a
mutated receptor. In this case, they can be recognized by
antibodies. Particular preferred tumour antigens are selected from
the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,
alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,
alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4,
ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4,
BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,
calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19,
CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80,
CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m,
coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,
cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,
EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3,
ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3,
GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3,
GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,
HLA-A*0201-R171, HLA-All/m, HLA-A2/m, HNE, homeobox NKX3.1,
HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2,
hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin
receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,
KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3,
MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2,
MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17,
MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1,
MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A,
MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m,
mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1,
MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class Vm, NA88-A,
N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,
NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,
OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15,
p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1,
PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME,
PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA,
PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE,
SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1,
SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B,
SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta,
TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,
TRP-p8, tyrosinase, UPA, VEGF, VEGFR1, VEGFR-2/FLK-1, and WT1. Such
tumour antigens preferably may be selected from the group
consisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1,
MAGE-A3, Mesothelin, MUC-1, NY-ESO-1, GP100, MART-1, Tyrosinase,
PSA, PSCA, PSMA VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more
preferably from PAP, NY-ESO-1, MAGE-A3, WT1, and MUC-1.
[0338] In this context, and for certain embodiments of all aspects
of the present invention, the antigen associated with a cancer or
tumour disease, does not include (x) an idiotype immunoglobulin (an
idiotype antibody or an idiotype B cell receptor); or (y) an
idiotype T cell receptor, and optionally is not a fragment, variant
and/or derivative of such antigen.
[0339] Furthermore, the at least one antigen, if provided as
protein or peptide antigen is in certain embodiments not the model
antigen Ovalbumine or a fragment of Ovalbumine, such as the
Ovalbumine derived peptide SIINFEKL (SEQ ID NO: 116).
[0340] The at least one antigen in the inventive pharmaceutical
composition can be provided as protein or peptide or can be encoded
by a nucleic acid, e.g. a DNA (e.g. a plasmid DNA or viral DNA), or
an RNA (e.g. an mRNA or a viral RNA). Preferably, the at least one
antigen is provided as a protein or peptide, or a fragment, variant
and/or derivative of said protein or peptide antigen. In certain
embodiments, said protein or peptide antigen (or fragment, variant
and/or derivative of said protein or peptide antigen) is comprised
in, provided as or derived from a defined sample, for example a
sample having a known number and or composition of components. For
example, said protein or peptide antigen is not comprised in; or is
not provided as; or is not derived from, in each case a mixture of
(e.g. undefined) other components, such as a mixture being a
preparation of inactivated or attenuated virus or pathogen (such
as, in either case, any one describe herein). For example, the
antigen used in any aspect of the present invention may be, or may
be provided as, an isolated and/or purified protein or peptide
antigen. As will be understood by the person of ordinary skill, an
isolated (and/or purified) antigen includes such antigens that are
present (or provided) in a (starting) composition that has less
than about 40%, 30%, 20%, 10%, 5%, 2% or 1% non-desired or
specified other components such as other proteins/peptides or
impurities.
[0341] Protein or peptide antigens can, for example, be prepared as
follows.
[0342] Protein or peptide antigens as described above, can be
prepared using recombinant production methods, such as those
described herein, or e.g. with the aid of molecular biology methods
known to the person of ordinary skill. Such an antigen can be
described, as applicable, as a "recombinant protein antigen" and/or
a "recombinant peptide antigen".
[0343] Alternatively, a protein or peptide as described above (e.g
fragments, domains, epitopes or protein antigens and/or peptide
analogues) can be prepared using peptide synthesis methods such as
those described herein, or e.g. with other methodologies known to
the person of ordinary skill. Such an antigen can be described, as
applicable, as a "synthetic protein antigen" and/or a "synthetic
peptide antigen".
[0344] In case that the at least one antigen is provided as protein
or peptide antigen (or a fragment, variant and/or derivative
thereof), the peptide or protein antigen can be provided in a first
alternative in a separate component of the inventive pharmaceutical
composition. In this case the at least one protein or peptide
antigen is not part of the polymeric carrier cargo complex or in
other words: in this case the polymeric carrier cargo complex does
not include the at least one antigen. In a second alternative the
at least one protein or peptide antigen can be provided as
component of the polymeric carrier cargo complex. In this case the
peptide or protein antigen can be added to the polymeric carrier
cargo complex during the polymerization step c) of the method of
preparing of the polymeric carrier cargo complex as described
herein. Thus, the peptide or protein antigen is integrated in the
polymeric carrier cargo complex. Particularly preferred in this
context is that the peptide or protein antigen bears at least one
SH-moiety for polymerization with the other components of the
polymeric carrier in the polymeric carrier cargo complex.
Furthermore, in a further alternative a protein or peptide antigen
is provided as component of the polymeric carrier of the polymeric
carrier cargo complex and at least one additional protein or
peptide antigen (the same or a different) is provided in a separate
component of the inventive pharmaceutical composition which is not
part of the polymeric carrier cargo complex.
[0345] Additionally, the at least one antigen (or a fragment,
variant and/or derivative thereof) can be provided in the inventive
pharmaceutical composition in the form of nucleic acids coding for
the at least one antigen (or fragments, variants and/or derivatives
thereof).
[0346] In this context, the nucleic acids coding for the at least
one antigen (or fragments, variants and/or derivatives thereof) are
defined as disclosed above for the nucleic acid cargo comprised in
the polymeric carrier cargo complex used as an adjuvant in the
inventive pharmaceutical composition. Therefore, also fragments,
variants, derivatives and modifications of a nucleic acid as
defined herein are explicitly encompassed.
[0347] The at least one antigen (or a fragment, variant and/or
derivative thereof) if provided in the inventive pharmaceutical
composition in the form of nucleic acids coding for the at least
one antigen (or fragments, variants and/or derivatives thereof),
can be prepared with all methods for nucleic acid synthesis known
for a skilled person. Particularly preferred are methods for
nucleic acid synthesis as defined herein.
[0348] Also in this case two alternatives exist. The first
alternative provides the nucleic acid coding for the at least one
antigen as part of the polymeric carrier cargo complex (e.g. as
nucleic acid cargo molecule) and the second alternative provides
the nucleic acid coding for the at least one antigen as separate
component of the inventive pharmaceutical composition. Thus, in
this case the nucleic acid coding for the at least one antigen is
not part of the polymeric carrier cargo complex.
[0349] In a further embodiment of the present invention, the at
least one antigen (or a fragment, variant and/or derivative
thereof) coded by a nucleic acid can be provided as part of the
(adjuvant) polymeric carrier cargo complex (e.g. as nucleic acid
cargo coding for the at least one antigen) and additionally an
antigen coded by a nucleic acid can be provided in a separate
component which is not part of the polymeric carrier cargo
complex.
[0350] The invention further provides the alternative that at least
one antigen is provided as a nucleic acid (as part of the polymeric
carrier cargo complex or not) and that at least one additional
antigen is provided as protein or peptide antigen (as part of the
polymeric carrier cargo complex or not).
[0351] As a further embodiment the at least one antigen if provided
as protein or peptide or as a nucleic acid coding for the at least
one antigen may further comprise or code for a signal peptide as
defined herein.
[0352] As a further ingredient the pharmaceutical composition may
comprise at least one additional pharmaceutically active component.
A pharmaceutically active component in this connection is a
compound that has a therapeutic effect to heal, ameliorate or
prevent a particular indication, preferably tumour or cancer
diseases, autoimmune disease, allergies or infectious diseases.
Such compounds include, without implying any limitation, peptides
or proteins, preferably as defined herein, nucleic acids,
preferably as defined herein, (therapeutically active) low
molecular weight organic or inorganic compounds (molecular weight
less than 5000, preferably less than 1000), sugars, antigens or
antibodies, preferably as defined herein, therapeutic agents
already known in the prior art, antigenic cells, antigenic cellular
fragments, cellular fractions; cell wall components (e.g.
polysaccharides), modified, attenuated or de-activated (e.g.
chemically or by irradiation) pathogens (virus, bacteria etc.),
adjuvants, preferably as defined herein, etc.
[0353] Furthermore, the inventive pharmaceutical composition may
comprise a pharmaceutically acceptable carrier and/or vehicle. In
the context of the present invention, a pharmaceutically acceptable
carrier typically includes the liquid or non-liquid basis of the
pharmaceutical composition. If the pharmaceutical composition is
provided in liquid form, the carrier will typically be pyrogen-free
water; isotonic saline or buffered (aqueous) solutions, e.g
phosphate, citrate etc. buffered solutions. The injection buffer
may be hypertonic, isotonic or hypotonic with reference to the
specific reference medium, i.e. the buffer may have a higher,
identical or lower salt content with reference to the specific
reference medium, wherein preferably such concentrations of the
afore mentioned salts may be used, which do not lead to damage of
cells due to osmosis or other concentration effects. Reference
media are e.g. liquids occurring in "in vivd" methods, such as
blood, lymph, cytosolic liquids, or other body liquids, or e.g.
liquids, which may be used as reference media in "in vitro"
methods, such as common buffers or liquids. Such common buffers or
liquids are known to a skilled person.
[0354] However, one or more compatible solid or liquid fillers or
diluents or encapsulating compounds may be used as well for the
pharmaceutical composition, which are suitable for administration
to a patient to be treated. The term "compatible" as used here
means that these constituents of the pharmaceutical composition are
capable of being mixed with the polymeric carrier cargo complex as
defined herein in such a manner that no interaction occurs which
would substantially reduce the pharmaceutical effectiveness of the
pharmaceutical composition under typical use conditions.
Pharmaceutically acceptable carriers, fillers and diluents must, of
course, have sufficiently high purity and sufficiently low toxicity
to make them suitable for administration to a person to be treated.
Some examples of compounds which can be used as pharmaceutically
acceptable carriers, fillers or constituents thereof are sugars,
such as, for example, lactose, glucose and sucrose; starches, such
as, for example, corn starch or potato starch; cellulose and its
derivatives, such as, for example, sodium carboxymethylcellulose,
ethylcellulose, cellulose acetate; powdered tragacanth; malt;
gelatin; tallow; solid glidants, such as, for example, stearic
acid, magnesium stearate; calcium sulfate; vegetable oils, such as,
for example, groundnut oil, cottonseed oil, sesame oil, olive oil,
corn oil and oil from theobroma; polyols, such as, for example,
polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene
glycol; alginic acid.
[0355] According to a specific embodiment, the inventive
pharmaceutical composition may comprise an (additional) adjuvant.
In this context, an adjuvant may be understood as any compound,
which is suitable to initiate or increase an immune response of the
innate immune system, i.e. a non-specific immune response. With
other words, when administered, the pharmaceutical composition
typically elicits an innate immune response due to the adjuvant,
optionally contained therein. Such an adjuvant may be selected from
any adjuvant known to a skilled person and suitable for the present
case, i.e. supporting the induction of an innate immune response in
a mammal.
[0356] The inventive pharmaceutical composition may be administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term parenteral as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-nodal, intra-synovial,
infrasternal, intrathecal, intrahepatic, intralesional,
intracranial, transdermal, intradermal, intrapulmonal,
intraperitoneal, intracardial, intraarterial, and sublingual
injection or infusion techniques.
[0357] Preferably, the inventive pharmaceutical composition may be
administered by parenteral injection, more preferably by
subcutaneous, intravenous, intramuscular, intra-articular,
intra-nodal, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional, intracranial, transdermal,
intradermal, intrapulmonal, intraperitoneal, intracardial,
intraarterial, and sublingual injection or via infusion techniques.
Particularly preferred is intradermal, subcutaneous and
intramuscular injection. Sterile injectable forms of the
pharmaceutical compositions may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as carboxymethyl
cellulose or similar dispersing agents that are commonly used in
the formulation of pharmaceutically acceptable dosage forms
including emulsions and suspensions. Other commonly used
surfactants, such as Tweens, Spans and other emulsifying agents or
bioavailability enhancers which are commonly used in the
manufacture of pharmaceutically acceptable solid, liquid, or other
dosage forms may also be used for the purposes of formulation of
the pharmaceutical composition.
[0358] The inventive pharmaceutical composition as defined herein
may also be administered orally in any orally acceptable dosage
form including, but not limited to, capsules, tablets, aqueous
suspensions or solutions. In the case of tablets for oral use,
carriers commonly used include lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. For
oral administration in a capsule form, useful diluents include
lactose and dried cornstarch. When aqueous suspensions are required
for oral use, the active ingredient, i.e. the polymeric carrier
cargo complex, is combined with emulsifying and suspending agents.
If desired, certain sweetening, flavoring or coloring agents may
also be added.
[0359] The inventive pharmaceutical composition may also be
administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
e.g. including diseases of the skin or of any other accessible
epithelial tissue. Suitable topical formulations are readily
prepared for each of these areas or organs. For topical
applications, the pharmaceutical composition may be formulated in a
suitable ointment, containing the polymeric carrier cargo complex
suspended or dissolved in one or more carriers. Carriers for
topical administration include, but are not limited to, mineral
oil, liquid petrolatum, white petrolatum, propylene glycol,
polyoxyethylene, polyoxypropylene compound, emulsifying wax and
water. Alternatively, the pharmaceutical composition can be
formulated in a suitable lotion or cream. In the context of the
present invention, suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0360] The inventive pharmaceutical composition typically comprises
a "safe and effective amount" of the components of the
pharmaceutical composition, particularly of the polymeric carrier
cargo complex as defined herein or the nucleic acid as such. As
used herein, a "safe and effective amount" means an amount of the
polymeric carrier cargo complex as such that is sufficient to
significantly induce a positive modification of a disease or
disorder as defined herein. At the same time, however, a "safe and
effective amount" is small enough to avoid serious side-effects and
to permit a sensible relationship between advantage and risk. The
determination of these limits typically lies within the scope of
sensible medical judgment. A "safe and effective amount" of the
components of the pharmaceutical composition, particularly of the
polymeric carrier cargo complex or of the at least one antigen as
defined herein, will furthermore vary in connection with the
particular condition to be treated and also with the age and
physical condition of the patient to be treated, the body weight,
general health, sex, diet, time of administration, rate of
excretion, drug combination, the activity of the polymeric carrier
cargo complex or of the antigen, the severity of the condition, the
duration of the treatment, the nature of the accompanying therapy,
of the particular pharmaceutically acceptable carrier used, and
similar factors, within the knowledge and experience of the
accompanying doctor. The pharmaceutical composition may be used for
human and also for veterinary medical purposes, preferably for
human medical purposes, as a pharmaceutical composition in general
or as a vaccine.
[0361] The inventive pharmaceutical composition can additionally
contain one or more auxiliary substances in order to increase its
immunogenicity or immunostimulatory capacity, if desired. A
synergistic action of the (adjuvant) polymeric carrier cargo
complex as defined herein and of an auxiliary substance, which may
be optionally contained in the inventive pharmaceutical composition
as defined herein, is preferably achieved thereby. Depending on the
various types of auxiliary substances, various mechanisms can come
into consideration in this respect. For example, compounds that
permit the maturation of dendritic cells (DCs), for example
lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class
of suitable auxiliary substances. In general, it is possible to use
as auxiliary substance any agent that influences the immune system
in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines,
such as GM-CFS, which allow an immune response to be enhanced
and/or influenced in a targeted manner. Particularly preferred
auxiliary substances are cytokines, such as monokines, lymphokines,
interleukins or chemokines, that further promote the innate immune
response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta,
INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth
factors, such as hGH.
[0362] Further additives which may be included in the inventive
pharmaceutical composition are emulsifiers, such as, for example,
Tween.RTM.; wetting agents, such as, for example, sodium lauryl
sulfate; colouring agents; taste-imparting agents, pharmaceutical
carriers; tablet-forming agents; stabilizers; antioxidants;
preservatives.
[0363] The inventive pharmaceutical composition can also
additionally contain any further compound, which is known to be
immunostimulating due to its binding affinity (as ligands) to human
Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, or due to its binding affinity (as ligands) to murine
Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12 or TLR13.
[0364] The inventive pharmaceutical composition can also
additionally or alternatively contain an immunostimulatory RNA,
i.e. an RNA derived from an immunostimulatory RNA, which triggers
or increases an (innate) immune response. Preferably, such an
immunostimulatory RNA may be in general be as defined
hereinbefore.
[0365] Another class of compounds, which may be added to the
inventive pharmaceutical composition in this context, may be CpG
nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or
CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a
double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss
CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic
acid is preferably in the form of CpG-RNA, more preferably in the
form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid
preferably contains at least one or more (mitogenic)
cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According
to a first preferred alternative, at least one CpG motif contained
in these sequences, that is to say the C (cytosine) and the G
(guanine) of the CpG motif, is unmethylated. All further cytosines
or guanines optionally contained in these sequences can be either
methylated or unmethylated. According to a further preferred
alternative, however, the C (cytosine) and the G (guanine) of the
CpG motif can also be present in methylated form.
[0366] In the context of the present invention, the nucleic acid
cargo in the polymeric carrier cargo complex comprised in the
inventive pharmaceutical composition is preferably as defined
above. More preferably, the nucleic acid of the polymeric carrier
cargo complex, preferably contained in the pharmaceutical
composition, is typically an immunostimulatory nucleic acid as
defined herein, e.g. a CpG-DNA or an immunostimulatory RNA (isRNA),
preferably an isRNA. Alternatively or additionally, the nucleic
acid of the polymeric carrier cargo complex, preferably contained
in the pharmaceutical composition, is a coding nucleic acid
sequence as defined herein, preferably a cDNA or an mRNA, more
preferably encoding an adjuvant protein preferably as defined
herein. In this context, the polymeric carrier cargo complex,
typically initiates an innate immune response in the patient to be
treated.
[0367] In a specific embodiment in this context, it is preferred
that an adjuvant protein is a component of the polymeric carrier
cargo complex and, preferably, of the polymeric carrier. According
to a further aspect, the present invention also provides kits,
particularly kits of parts, comprising as components alone or in
combination with optional further ingredients, and including (as a
first component): [0368] (A) a polymeric carrier cargo complex,
comprising: [0369] a) (as a carrier) a polymeric carrier comprising
disulfide-crosslinked cationic components, preferably formed by
disulfide-crosslinked cationic components; and [0370] b) (as a
cargo) at least one nucleic acid molecule, [0371] and (as a second
component): [0372] (B) at least one antigen that is selected from:
[0373] (i) an antigen from a pathogen associated with infectious
disease; [0374] (ii) an antigen associated with allergy or allergic
disease; [0375] (iii) an antigen associated with autoimmune
disease; or [0376] (iv) an antigen associated with a cancer or
tumour disease, wherein in some embodiments the antigen associated
with a cancer or tumour disease is another than an antigen
comprising: (x) an idiotype immunoglobulin (e.g. an idiotype
antibody or an idiotype B cell receptor); and/or (y) at least one
idiotype T cell receptor, [0377] or a fragment, variant and/or
derivative of said antigen; in each case as defined anywhere
herein, and optionally technical instructions with information on
the administration and dosage of the polymeric carrier cargo
complex and the at least one antigen. Such kits, preferably kits of
parts, may be applied, e.g., for any of the applications or uses as
defined herein. Such kits, when occurring as a kit of parts, may
further contain each component of inventive pharmaceutical
composition in a different part of the kit.
[0378] In certain embodiments of the kits of the present invention,
the antigen is comprised in a vaccine.
[0379] The present invention furthermore provides several
applications and uses of the inventive pharmaceutical composition
(e.g. the adjuvanted vaccine) or of kits or kits of parts
comprising same as defined anywhere herein.
[0380] In this context, the present invention also provides a
method for transfecting and/or treating a cell, a tissue or an
organism, thereby applying or administering the inventive
pharmaceutical composition particularly for therapeutic purposes.
In this context, typically after preparing the inventive
pharmaceutical composition, the inventive pharmaceutical
composition is preferably administered to a cell, a tissue or an
organism, preferably using any of the administration modes as
described herein. The method for transfecting and/or treating a
cell may be carried out in vitro, in vivo or ex vivo.
[0381] Furthermore, the present invention provides the use of a
pharmaceutical composition or of kits or kits of parts in each case
as defined anywhere herein, in therapy and/or as a medicament,
preferably as a vaccine such as an adjuvanted vaccine.
[0382] Also in certain embodiments of all aspects of the present
invention, the at least one antigen is not selected from: (x) an
idiotype immunoglobulin (an idiotype antibody or an idiotype B cell
receptor); or (y) at least one idiotype T cell receptor; and
optionally is not a fragment, variant and/or derivative of such
antigen.
[0383] In this aspect of the present invention, particularly
preferred is the use of the inventive pharmaceutical composition or
of the kits or kits of parts comprising same as defined herein in
the treatment of infectious diseases, allergies or allergic
diseases, autoimmune diseases and cancer or tumour diseases, in
each case as defined anywhere herein.
[0384] In this context, infectious diseases are preferably viral,
bacterial or protozoological infectious diseases. Such infectious
diseases, preferably (viral, bacterial or protozoological)
infectious diseases, are typically selected from the list
consisting of Acinetobacter infections, African sleeping sickness
(African trypanosomiasis), AIDS (Acquired immunodeficiency
syndrome), Amoebiasis, Anaplasmosis, Anthrax, Appendicitis,
Arcanobacterium haemolyticum infections, Argentine hemorrhagic
fever, Ascariasis, Aspergillosis, Astrovirus infections, Athlete's
foot, Babesiosis, Bacillus cereus infections, Bacterial meningitis,
Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides
infections, Balantidiasis, Baylisascaris infections, Bilharziosis,
BK virus infections, Black piedra, Blastocystis hominis infections,
Blastomycosis, Bolivian hemorrhagic fever, Borrelia infectionss
(Borreliosis), Botulism (and Infant botulism), Bovine tapeworm,
Brazilian hemorrhagic fever, Brucellosis, Burkholderia infections,
Buruli ulcer, Calicivirus infections (Norovirus and Sapovirus),
Campylobacteriosis, Candidiasis (Candidosis), Canine tapeworm
infections, Cat-scratch disease, Chagas Disease (American
trypanosomiasis), Chancroid, Chickenpox, Chlamydia infections,
Chlamydia trachomatis infections, Chlamydophila pneumoniae
infections, Cholera, Chromoblastomycosis, Climatic bubo,
Clonorchiasis, Clostridium difficile infections,
Coccidioidomycosis, Cold, Colorado tick fever (CTF), Common cold
(Acute viral rhinopharyngitis; Acute coryza), Condyloma acuminata,
Conjunctivitis, Creutzfeldt-Jakob disease (CJD), Crimean-Congo
hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis,
Cutaneous larva migrans (CLM), Cutaneous Leishmaniosis,
Cyclosporiasis, Cysticercosis, Cytomegalovirus infections, Dengue
fever, Dermatophytosis, Dientamoebiasis, Diphtheria,
Diphyllobothriasis, Donavanosis, Dracunculiasis, Early summer
meningoencephalitis (FSME), Ebola hemorrhagic fever,
Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infections),
Enterococcus infections, Enterovirus infections, Epidemic typhus,
Epiglottitis, Epstein-Barr Virus Infectious Mononucleosis, Erythema
infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,
Fasciolosis, Fatal familial insomnia (FFI), Fifth disease,
Filariasis, Fish poisoning (Ciguatera), Fish tapeworm, Flu, Food
poisoning by Clostridium perfringens, Fox tapeworm, Free-living
amebic infections, Fusobacterium infections, Gas gangrene,
Geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS),
Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma
inguinale (Donovanosis), Group A streptococcal infections, Group B
streptococcal infections, Haemophilus influenzae infections, Hand
foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome (HPS),
Helicobacter pylori infections, Hemolytic-uremic syndrome (HUS),
Hemorrhagic fever with renal syndrome (HFRS), Henipavirus
infections, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D,
Hepatitis E, Herpes simplex, Herpes simplex type I, Herpes simplex
type II, Herpes zoster, Histoplasmosis, Hollow warts, Hookworm
infections, Human bocavirus infections, Human ewingii ehrlichiosis,
Human granulocytic anaplasmosis (HGA), Human metapneumovirus
infections, Human monocytic ehrlichiosis, Human papillomavirus
(HPV) infections, Human parainfluenza virus infections,
Hymenolepiasis, Influenza, Isosporiasis, Japanese encephalitis,
Kawasaki disease, Keratitis, Kingella kingae infections, Kuru,
Lambliasis (Giardiasis), Lassa fever, Legionellosis (Legionnaires'
disease, Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,
Lice, Listeriosis, Lyme borreliosis, Lyme disease, Lymphatic
filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria,
Marburg hemorrhagic fever (MHF), Marburg virus, Measles,
Melioidosis (Whitmore's disease), Meningitis, Meningococcal
disease, Metagonimiasis, Microsporidiosis, Miniature tapeworm,
Miscarriage (prostate inflammation), Molluscum contagiosum (MC),
Mononucleosis, Mumps, Murine typhus (Endemic typhus), Mycetoma,
Mycoplasma hominis, Mycoplasma pneumonia, Myiasis, Nappy/diaper
dermatitis, Neonatal conjunctivitis (Ophthalmia neonatorum),
Neonatal sepsis (Chorioamnionitis), Nocardiosis, Noma, Norwalk
virus infections, Onchocerciasis (River blindness), Osteomyelitis,
Otitis media, Paracoccidioidomycosis (South American
blastomycosis), Paragonimiasis, Paratyphus, Pasteurellosis,
Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),
Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory
disease (PID), Pertussis (Whooping cough), Pfeiffer's glandular
fever, Plague, Pneumococcal infections, Pneumocystis pneumonia
(PCP), Pneumonia, Polio (childhood lameness), Poliomyelitis,
Porcine tapeworm, Prevotella infections, Primary amoebic
meningoencephalitis (PAM), Progressive multifocal
leukoencephalopathy, Pseudo-croup, Psittacosis, Q fever, Rabbit
fever, Rabies, Rat-bite fever, Reiter's syndrome, Respiratory
syncytial virus infections (RSV), Rhinosporidiosis, Rhinovirus
infections, Rickettsial infections, Rickettsialpox, Rift Valley
fever (RVF), Rocky mountain spotted fever (RMSF), Rotavirus
infections, Rubella, Salmonella paratyphus, Salmonella typhus,
Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies,
Scarlet fever, Schistosomiasis (Bilharziosis), Scrub typhus,
Sepsis, Shigellosis (Bacillary dysentery), Shingles, Smallpox
(Variola), Soft chancre, Sporotrichosis, Staphylococcal food
poisoning, Staphylococcal infections, Strongyloidiasis, Syphilis,
Taeniasis, Tetanus, Three-day fever, Tick-borne encephalitis, Tinea
barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp),
Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch),
Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis
(Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor
(Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans (OLM)
and Visceral Larva Migrans (VLM)), Toxoplasmosis, Trichinellosis,
Trichomoniasis, Trichuriasis (Whipworm infections), Tripper,
Trypanosomiasis (sleeping sickness), Tsutsugamushi disease,
Tuberculosis, Tularemia, Typhus, Typhus fever, Ureaplasma
urealyticum infections, Vaginitis (Colpitis), Variant
Creutzfeldt-Jakob disease (vCJD, nvCJD), Venezuelan equine
encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia,
Visceral Leishmaniosis, Warts, West Nile Fever, Western equine
encephalitis, White piedra (Tinea blanca), Whooping cough, Yeast
fungus spots, Yellow fever, Yersinia pseudotuberculosis infections,
Yersiniosis, and Zygomycosis.
[0385] Allergies or allergic diseases are preferably selected from
pollen allergy (allergy against grass pollen, tree pollen (e.g.
pollen of hazel, birch, alder, ash), flower pollen, herb pollen
(e.g. pollen of mugwort)), dust mite allergy, mold allergy (e.g.
allergy against Acremonium, Aspergillus, Cladosporium, Fusarium,
Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma, or
Alternaria), pet allergy (allergy against animals; e.g against
cats, dogs, horses), food allergy (e.g. allergy against fish (e.g.
bass, cod, flounder), seafood (e.g. crab, lobster, shrimps), egg,
wheat, nuts (e.g. peanuts, almonds, cashews, walnuts), soya, milk,
etc.) or insect bite allergy (allergy against insect venom, e.g.
venom of wasps, bees, hornets, ants, mosquitos, or ticks).
[0386] According to another specific embodiment, diseases as
defined herein comprise autoimmune diseases as defined in the
following. Autoimmune diseases are preferably selected from Addison
disease (autoimmune adrenalitis, Morbus Addison), alopecia areata,
Addison's anemia (Morbus Biermer), autoimmune hemolytic anemia
(AIHA), autoimmune hemolytic anemia (AIHA) of the cold type (cold
hemagglutinine disease, cold autoimmune hemolytic anemia (AIHA)
(cold agglutinin disease), (CHAD)), autoimmune hemolytic anemia
(AIHA) of the warm type (warm AIHA, warm autoimmune haemolytic
anemia (AIHA)), autoimmune hemolytic Donath-Landsteiner anemia
(paroxysmal cold hemoglobinuria), antiphospholipid syndrome (APS),
atherosclerosis, autoimmune arthritis, arteriitis temporalis,
Takayasu arteriitis (Takayasu's disease, aortic arch disease),
temporal arteriitis/giant cell arteriitis, autoimmune chronic
gastritis, autoimmune infertility, autoimmune inner ear disease
(AIED), Basedow's disease (Morbus Basedow), Bechterew's disease
(Morbus Bechterew, ankylosing spondylitis, spondylitis ankylosans),
Behcet's syndrome (Morbus Behcet), bowel disease including
autoimmune inflammatory bowel disease (including colitis ulcerosa
(Morbus Crohn, Crohn's disease), cardiomyopathy, particularly
autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (DCM),
celiac sprue dermatitis (gluten mediated enteropathia), chronic
fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy (CIDP), chronic polyarthritis,
Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome,
CREST syndrome (syndrom with Calcinosis cutis, Raynaud phenomenon,
motility disorders of the esophagus, sklerodaktylia and
teleangiectasia), Crohn's disease (Morbus Crohn, colitis ulcerosa),
dermatitis herpetiformis during, dermatologic autoimmune diseases,
dermatomyositis, Diabetes, Diabetes mellitus Type 1 (type I
diabetes, insuline dependent Diabetes mellitus), Diabetes mellitus
Type 2 (type II diabetes), essential mixed cryoglobulinemia,
essential mixed cryoglobulinemia, fibromyalgia, fibromyositis,
Goodpasture syndrome (anti-GBM mediated glomerulonephritis), graft
versus host disease, Guillain-Barre syndrome (GBM,
Polyradikuloneuritis), haematologic autoimmune diseases, Hashimoto
thyroiditis, hemophilia, acquired hemophilia, hepatitis, autoimmune
hepatitis, particularly autoimmune forms of chronic hepatitis,
idiopathic pulmonary fibrosis (IPF), idiopathic thrombocytopenic
purpura, Immuno-thrombocytopenic purpura (Morbus Werlhof; ITP), IgA
nephropathy, infertility, autoimmune infertility, juvenile
rheumatoid arthritis (Morbus Still, Still syndrome), Lambert-Eaton
syndrome, lichen planus, lichen sclerosus, lupus erythematosus,
systemic lupus erythematosus (SLE), lupus erythematosus (discoid
form), Lyme arthritis (Lyme disease, borrelia arthritis), Meniere's
disease (Morbus Meniere); mixed connective tissue disease (MCTD),
multiple sclerosis (MS, encephalomyelitis disseminate, Charcot's
disease), Myasthenia gravis (myasthenia, MG), myosits,
polymyositis, neural autoimmune diseases, neurodermitis, pemphigus
vulgaris, bullous pemphigoid, scar forming pemphigoid;
polyarteriitis nodosa (periarteiitis nodosa), polychondritis
(panchondritis), polyglandular (autoimmune) syndrome (PGA syndrome,
Schmidt's syndrome), Polymyalgia rheumatica, primary
agammaglobulinemia, primary biliary cirrhosis PBC, primary
autoimmune cholangitis), progressive systemic sclerosis (PSS),
Psoriasis, Psoriasis vulgaris, Raynaud's phenomena, Reiter's
syndrome (Morbus Reiter, urethral conjunctive synovial syndrome)),
rheumatoid arthritis (RA, chronic polyarthritis, rheumatic disease
of the joints, rheumatic fever), sarcoidosis (Morbus Boeck,
Besnier-Boeck-Schaumann disease), stiff-man syndrome, Sclerodermia,
Scleroderma, Sjogren's syndrome, sympathetic ophtalmia; Transient
gluten intolerance, transplanted organ rejection, uveitis,
autoimmune uveiitis, Vasculitis, Vitiligo, (leucoderma, piebold
skin), and Wegner's disease (Morbus Wegner, Wegner's
granulomatosis).
[0387] Furthermore, cancer or tumor diseases are preferably
selected from melanomas, malignant melanomas, colon carcinomas,
lymphomas, sarcomas, blastomas, renal carcinomas, gastrointestinal
tumors, gliomas, prostate tumors, bladder cancer, rectal tumors,
stomach cancer, oesophageal cancer, pancreatic cancer, liver
cancer, mammary carcinomas (=breast cancer), uterine cancer,
cervical cancer, acute myeloid leukaemia (AML), acute lymphoid
leukaemia (ALL), chronic myeloid leukaemia (CML), chronic
lymphocytic leukaemia (CLL), hepatomas, various virus-induced
tumors such as, for example, papilloma virus-induced carcinomas
(e.g. cervical carcinoma=cervical cancer), adenocarcinomas, herpes
virus-induced tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell
lymphoma), heptatitis B-induced tumors (hepatocell carcinomas),
HTLV-1- and HTLV-2-induced lymphomas, acoustic neuroma, lung
carcinomas (=lung cancer=bronchial carcinoma), small-cell lung
carcinomas, pharyngeal cancer, anal carcinoma, glioblastoma, rectal
carcinoma, astrocytoma, brain tumors, retinoblastoma, basalioma,
brain metastases, medulloblastomas, vaginal cancer, pancreatic
cancer, testicular cancer, Hodgkin's syndrome, meningiomas,
Schneeberger disease, hypophysis tumor, Mycosis fungoides,
carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal
cancer, renal cancer, thymoma, corpus carcinoma, bone cancer,
non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck
tumors, oligodendroglioma, vulval cancer, intestinal cancer, colon
carcinoma, oesophageal carcinoma (=oesophageal cancer), wart
involvement, tumors of the small intestine, craniopharyngeomas,
ovarian carcinoma, genital tumors, ovarian cancer (=ovarian
carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrial
carcinoma, liver metastases, penile cancer, tongue cancer, gall
bladder cancer, leukaemia, plasmocytoma, lid tumor, prostate cancer
(=prostate tumors), etc.
[0388] In a further aspect, the present invention provides a
polymeric carrier cargo complex as defined anywhere herein, such as
one comprising: [0389] a) (as a carrier) a polymeric carrier
comprising disulfide-crosslinked cationic components, preferably
formed by disulfide-crosslinked cationic components; and [0390] b)
(as a cargo) at least one nucleic acid molecule for use in therapy
in combination with at least one antigen, preferably a protein or
peptide antigen or a fragment, variant and/or derivative thereof,
in each case as defined anywhere herein, particularly in the
treatment of infectious diseases, allergies or allergic diseases,
autoimmune diseases and cancer or tumour diseases as defined
above.
[0391] Additionally, the present invention provides at least one
antigen, preferably a protein or peptide antigen or a fragment,
variant and/or derivative thereof, in each case as defined anywhere
herein, for use in therapy in combination with a polymeric carrier
cargo complex as defined anywhere herein, such as one comprising:
[0392] a) (as a carrier) a polymeric carrier comprising
disulfide-crosslinked cationic components, preferably formed by
disulfide-crosslinked cationic components, and [0393] b) (as a
cargo) at least one nucleic acid molecule, particularly in the
treatment of infectious diseases, allergies or allergic diseases,
autoimmune diseases and cancer or tumour diseases as defined
above.
[0394] In certain embodiments of such aspects of the present
invention, the antigen is comprised in a vaccine, such as a
commercially available vaccine.
[0395] In this context, "in combination" means that the different
components (the polymeric carrier cargo complex and the at least
one antigen, or a fragment, variant and/or derivative thereof) can
be provided together in the same composition, or can be formulated
separately in different compositions, i.e. one composition
comprising or representing the polymeric carrier cargo complex as
defined herein, and one further composition comprising the at least
one antigen, or a fragment, variant and/or derivative thereof as
defined herein. If provided in different compositions the polymeric
carrier cargo complex and the at least one antigen or a fragment,
variant and/or derivative thereof may be administered separated in
time (in a time-staggered manner) and/or may be administered at
different administration sites and/or via different administration
routes. This means that the polymeric carrier cargo complex may be
administered e.g. prior, concurrent or subsequent to the at least
one antigen, or fragment, variant and/or derivative thereof, or
vice versa. Subsequent administration includes that each component
used in the therapy is administered within about 48 hours, 24
hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30
mins, 15 mins or 5 mins of each other.
[0396] In a further aspect, the present invention provides a
pharmaceutical package, including: [0397] (A) a polymeric carrier
cargo complex, comprising: [0398] a) (as a carrier) a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components, and
[0399] b) (as a cargo) at least one nucleic acid molecule, [0400]
as defined anywhere herein; [0401] and [0402] (B) instructions
describing the use of said polymeric carrier cargo complex in
therapy in combination with at least one antigen or fragment,
variant and/or derivative thereof as defined anywhere herein.
[0403] The pharmaceutical package may further comprise at least one
antigen or fragment, variant and/or derivative thereof as defined
anywhere herein.
[0404] Furthermore, the present invention provides in an additional
embodiment a pharmaceutical package, including: [0405] (A) at least
one antigen or fragment, variant and/or derivative thereof, in each
case as defined anywhere herein; [0406] and [0407] (B) instructions
describing the use of said antigen or fragment, variant and/or
derivative thereof in therapy in combination with a polymeric
carrier cargo complex as defined anywhere herein.
[0408] The pharmaceutical package may further comprise a polymeric
carrier cargo complex, comprising: [0409] a) (as a carrier) a
polymeric carrier comprising disulfide-crosslinked cationic
components, preferably formed by disulfide-crosslinked cationic
components, and [0410] b) (as a cargo) at least one nucleic acid
molecule, as defined anywhere herein.
[0411] In this context, the invention furthermore provides the use
of the components included in the above defined pharmaceutical
packages in the treatment of the particular disease (indication)
selected from an infectious disease, an allergy or allergic
disease, an autoimmune disease or a cancer or tumour disease as
defined above. The respective disease may be one as described
anywhere herein.
[0412] In the present invention, if not otherwise indicated,
different features of alternatives and embodiments may be combined
with each other, where suitable.
[0413] Taken together, in a preferred embodiment, the invention
relates to
a pharmaceutical composition comprising [0414] a polymeric carrier
cargo complex and [0415] at least one protein or peptide antigen
[0416] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0417] wherein the polymeric carrier cargo complex
comprises [0418] a polymeric carrier, [0419] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0420] more preferably one or more peptides according to formula
(I) (Arg).sub.l; (Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most preferably according to subformula (IA) or (IB)
thereof as defined above, [0421] and at least one nucleic acid
molecule, [0422] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0423] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen.
[0424] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0425] a polymeric carrier
cargo complex and [0426] at least one protein or peptide antigen
[0427] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0428] wherein the polymeric carrier cargo complex
comprises [0429] a polymeric carrier, [0430] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0431] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0432] and at least one nucleic acid
molecule, [0433] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0434] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen, [0435] and wherein the
protein or peptide antigen [0436] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from:
[0437] an antigen from a pathogen associated with infectious
disease, [0438] an antigen associated with allergy or allergic
disease, [0439] an antigen associated with autoimmune disease,
[0440] an antigen associated with a cancer or tumour disease.
[0441] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0442] a polymeric carrier
cargo complex and [0443] at least one protein or peptide antigen
[0444] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0445] wherein the polymeric carrier cargo complex
comprises [0446] a polymeric carrier, [0447] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0448] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0449] and at least one nucleic acid
molecule, [0450] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0451] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0452] wherein the
protein or peptide antigen [0453] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from:
[0454] an antigen from a pathogen associated with infectious
disease, [0455] an antigen associated with allergy or allergic
disease, [0456] an antigen associated with autoimmune disease,
[0457] an antigen associated with a cancer or tumour disease;
[0458] and preferably wherein the protein or peptide antigen and/or
the fragment, variant and/or derivative of said protein or peptide
antigen is not included in the polymeric carrier cargo complex.
[0459] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0460] a polymeric carrier
cargo complex and [0461] at least one protein or peptide antigen
[0462] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0463] wherein the polymeric carrier cargo complex
comprises [0464] a polymeric carrier, [0465] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0466] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0467] and at least one nucleic acid
molecule, [0468] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.m--C.sub.nN.sub.v).sub.a as defined above,
such as a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to any one of SEQ ID NOs. 15-108
and 122 or a sequence which is at least 60%, preferably at least
70%, preferably at least 80%, more preferably at least 90%, and
most preferably at least 95% identical to any one of SEQ ID NOs.
15-108 and 122, e.g. a nucleic acid molecule comprising or
consisting of a nucleic acid sequence according to SEQ ID NO. 105
or 122 or a sequence which is at least 60%, preferably at least
70%, preferably at least 80%, more preferably at least 90%, and
most preferably at least 95% identical to SEQ ID NO. 105 or 122,
[0469] or a nucleic acid molecule which encodes an antigen, more
preferably a nucleic acid molecule which encodes an antigen which
is the same antigen as the at least one protein or peptide antigen;
[0470] wherein the protein or peptide antigen [0471] or the
fragment, variant and/or derivative of said protein or peptide
antigen is selected from an antigen from a pathogen associated with
infectious disease, [0472] an antigen from a pathogen associated
with infectious disease, [0473] an antigen associated with allergy
or allergic disease, [0474] an antigen associated with autoimmune
disease, [0475] an antigen associated with a cancer or tumour
disease, [0476] preferably an antigen from a pathogen associated
with infectious disease, [0477] more preferably an antigen from a
pathogen selected from Rabies virus, Hepatitis B virus, human
Papilloma virus (hPV), Bacillus anthracis, Respiratory syncytial
virus (RSV), Herpes simplex virus (HSV), Influenza virus and
Mycobacterium tuberculosis, [0478] more preferably an antigen from
a pathogen selected from Rabies virus, Hepatitis B virus, human
Papilloma virus (hPV); [0479] and preferably wherein the protein or
peptide antigen and/or the fragment, variant and/or derivative of
said protein or peptide antigen is not included in the polymeric
carrier cargo complex.
[0480] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0481] a polymeric carrier
cargo complex and [0482] at least one protein or peptide antigen
[0483] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0484] wherein the polymeric carrier cargo complex
comprises [0485] a polymeric carrier, [0486] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0487] more preferably one or more peptides according to formula
(I) (Arg).sub.i;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x, as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0488] and at least one nucleic acid
molecule, [0489] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0490] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0491] and wherein the
protein or peptide antigen [0492] or the fragment, variant and/or
derivative of said protein or peptide antigen is [0493] selected
from an antigen from a pathogen associated with infectious disease,
[0494] an antigen associated with allergy or allergic disease,
[0495] an antigen associated with autoimmune disease, [0496] an
antigen associated with a cancer or tumour disease, [0497]
preferably an antigen from a pathogen associated with infectious
disease, [0498] more preferably an antigen from a pathogen selected
from Rabies virus, Hepatitis B virus, human Papilloma virus (hPV),
Bacillus anthracis, Respiratory syncytial virus (RSV), Herpes
simplex virus (HSV), Influenza virus and Mycobacterium
tuberculosis, [0499] even more preferably an antigen from Rabies
virus, most preferably an antigen from Rabies virus which is
selected from the nucleoprotein (N), the phosphoprotein (P), the
matrix protein (M), the glycoprotein (G), and the viral RNA
polymerase (1); [0500] and preferably wherein the protein or
peptide antigen and/or the fragment, variant and/or derivative of
said protein or peptide antigen is not included in the polymeric
carrier cargo complex.
[0501] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0502] a polymeric carrier
cargo complex and [0503] at least one protein or peptide antigen
[0504] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0505] wherein the polymeric carrier cargo complex
comprises [0506] a polymeric carrier, [0507] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0508] more preferably one or more peptides according to formula
(I) (Arg).sub.l(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, such as a nucleic acid molecule
comprising or consisting of a nucleic acid sequence according to
any one of SEQ ID NOs. 15-108 and 122 or a sequence which is at
least 60%, preferably at least 70%, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95% identical
to any one of SEQ ID NOs. 15-108 and 122, e.g. a nucleic acid
molecule comprising or consisting of a nucleic acid sequence
according to SEQ ID NO. 105 or 122 or a sequence which is at least
60%, preferably at least 70%, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95% identical
to SEQ ID NO. 105 or 122, [0509] and at least one nucleic acid
molecule, [0510] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n formula (III)
C.sub.lX.sub.mC.sub.n formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above,
[0511] or a nucleic acid molecule which encodes an antigen, more
preferably a nucleic acid molecule which encodes an antigen which
is the same antigen as the at least one protein or peptide antigen;
[0512] and wherein the protein or peptide antigen [0513] or the
fragment, variant and/or derivative of said protein or peptide
antigen is selected from an antigen from a pathogen associated with
infectious disease, [0514] an antigen associated with allergy or
allergic disease, [0515] an antigen associated with autoimmune
disease, [0516] an antigen associated with a cancer or tumour
disease, [0517] preferably an antigen from a pathogen associated
with infectious disease, [0518] more preferably an antigen from a
pathogen selected from Rabies virus, Hepatitis B virus, human
Papilloma virus (hPV), Bacillus anthracis, Respiratory syncytial
virus (RSV), Herpes simplex virus (HSV), Influenza virus and
Mycobacterium tuberculosis, [0519] even more preferably an antigen
from Hepatitis B virus, most preferably an antigen from Hepatitis B
virus which is selected from the Hepatitis B surface antigen
(HBsAg), the Hepatitis B core antigen (HbcAg), the Hepatitis B
virus DNA polymerase, the HBx protein, the preS2 middle surface
protein, the large S protein, the virus protein VP1, the virus
protein VP2, the virus protein VP3, and the virus protein VP4;
[0520] and preferably wherein the protein or peptide antigen and/or
the fragment, variant and/or derivative of said protein or peptide
antigen is not included in the polymeric carrier cargo complex.
[0521] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0522] a polymeric carrier
cargo complex and [0523] at least one protein or peptide antigen
[0524] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0525] wherein the polymeric carrier cargo complex
comprises [0526] a polymeric carrier, [0527] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0528] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o,;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0529] and at least one nucleic acid
molecule, [0530] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0531] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0532] and wherein the
protein or peptide antigen [0533] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0534]
an antigen associated with allergy or allergic disease, [0535] an
antigen associated with autoimmune disease, [0536] an antigen
associated with a cancer or tumour disease, [0537] preferably an
antigen from a pathogen associated with infectious disease, [0538]
more preferably an antigen from a pathogen selected from Rabies
virus, Hepatitis B virus, human Papilloma virus (hPV), Bacillus
anthracis, Respiratory syncytial virus (RSV), Herpes simplex virus
(HSV), Influenza virus and Mycobacterium tuberculosis, [0539] even
more preferably an antigen from Influenza virus, most preferably an
antigen from Influenza virus which is selected from the
Hemagglutinin (HA), the Neuraminidase (NA), the Nucleoprotein (NP),
the M1 protein, the M2 protein, the NS1 protein, the NS2 protein
(the NEP protein: nuclear export protein), the PA protein, the PB1
protein (polymerase basic 1 protein), the PB1-F2 protein and the
PB2 protein of Influenza virus; [0540] and preferably wherein the
protein or peptide antigen and/or the fragment, variant and/or
derivative of said protein or peptide antigen is not included in
the polymeric carrier cargo complex.
[0541] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0542] a polymeric carrier
cargo complex and [0543] at least one protein or peptide antigen
[0544] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0545] wherein the polymeric carrier cargo complex
comprises [0546] a polymeric carrier, [0547] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0548] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0549] and at least one nucleic acid
molecule, [0550] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0551] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0552] and wherein the
protein or peptide antigen [0553] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0554]
an antigen associated with allergy or allergic disease, [0555] an
antigen associated with autoimmune disease, [0556] an antigen
associated with a cancer or tumour disease, [0557] preferably an
antigen from a pathogen associated with infectious disease,
preferably an antigen from a pathogen selected from Rabies virus,
Hepatitis B virus, human Papilloma virus (hPV), Bacillus anthracis,
Respiratory syncytial virus (RSV), Herpes simplex virus (HSV),
Influenza virus and Mycobacterium tuberculosis, [0558] more
preferably an antigen from human Papilloma virus (hPV), even more
preferably an antigen from human Papilloma virus (hPV) which is
selected from the E1 protein, the E2 protein, the E3 protein, the
E4 protein, the E5 protein, the E6 protein, the E7 protein, the E8
protein, the L1 protein, and the L2 protein; [0559] and preferably
wherein the protein or peptide antigen and/or the fragment, variant
and/or derivative of said protein or peptide antigen is not
included in the polymeric carrier cargo complex.
[0560] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0561] a polymeric carrier
cargo complex and [0562] at least one protein or peptide antigen
[0563] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0564] wherein the polymeric carrier cargo complex
comprises [0565] a polymeric carrier, [0566] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0567] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0568] and at least one nucleic acid molecule,
[0569] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a. as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0570] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0571] and wherein the
protein or peptide antigen [0572] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0573]
an antigen associated with allergy or allergic disease, [0574] an
antigen associated with autoimmune disease, [0575] an antigen
associated with a cancer or tumour disease, [0576] preferably an
antigen from a pathogen associated with infectious disease, [0577]
preferably an antigen from a pathogen selected from Rabies virus,
Hepatitis B virus, human Papilloma virus (hPV), Bacillus anthracis,
Respiratory syncytial virus (RSV), Herpes simplex virus (HSV),
Influenza virus and Mycobacterium tuberculosis, [0578] more
preferably an antigen from Bacillus anthracis, even more preferably
an antigen from Bacillus anthracis which is selected from the
protective antigen (PA), the edema factor (EF), the lethal factor
(LF), and the S-layer homology proteins (SLH); and preferably
wherein the protein or peptide antigen and/or the fragment, variant
and/or derivative of said protein or peptide antigen is not
included in the polymeric carrier cargo complex.
[0579] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0580] a polymeric carrier
cargo complex and [0581] at least one protein or peptide antigen
[0582] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0583] wherein the polymeric carrier cargo complex
comprises [0584] a polymeric carrier, [0585] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0586] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0587] and at least one nucleic acid molecule,
[0588] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0589] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0590] and wherein the
protein or peptide antigen [0591] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0592]
an antigen associated with allergy or allergic disease, [0593] an
antigen associated with autoimmune disease, [0594] an antigen
associated with a cancer or tumour disease, [0595] preferably an
antigen from a pathogen associated with infectious disease, [0596]
more preferably an antigen from a pathogen selected from Rabies
virus, Hepatitis B virus, human Papilloma virus (hPV), Bacillus
anthracis, Respiratory syncytial virus (RSV), Herpes simplex virus
(HSV), Influenza virus and Mycobacterium tuberculosis, even more
preferably an antigen from Respiratory syncytial virus (RSV),
[0597] most preferably an antigen from Respiratory syncytial virus
(RSV) which is selected from the Fusion (F) protein, the
nucleocapsid (N) protein, the phosphoprotein (P), the matrix (M)
protein, the glycoprotein (G), the large protein (L; RNA
polymerase), the non-structural protein 1 (NS1), the non-structural
protein 2 (NS2), the small hydrophobic (SH) protein, the elongation
factor M2-1, and the transcription regulation protein M2-2; [0598]
and preferably wherein the protein or peptide antigen and/or the
fragment, variant and/or derivative of said protein or peptide
antigen is not included in the polymeric carrier cargo complex.
[0599] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0600] a polymeric carrier
cargo complex and [0601] at least one protein or peptide antigen
[0602] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0603] wherein the polymeric carrier cargo complex
comprises [0604] a polymeric carrier, [0605] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0606] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0607] and at least one nucleic acid molecule,
[0608] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0609] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0610] and wherein the
protein or peptide antigen [0611] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0612]
an antigen associated with allergy or allergic disease, [0613] an
antigen associated with autoimmune disease, [0614] an antigen
associated with a cancer or tumour disease, [0615] preferably an
antigen from a pathogen associated with infectious disease, [0616]
more preferably an antigen from a pathogen selected from Rabies
virus, Hepatitis B virus, human Papilloma virus (hPV), Bacillus
anthracis, Respiratory syncytial virus (RSV), Herpes simplex virus
(HSV), Influenza virus and Mycobacterium tuberculosis, [0617] even
more preferably an antigen from Herpes simplex virus (HSV), [0618]
most preferably an antigen from Herpes simplex virus (HSV) which is
selected from the Glycoprotein L (UL1), the Uracil-DNA glycosylase
UL2, the UL3 protein, the UL4 protein, the DNA replication protein
UL5, the Portal protein UL6, the Virion maturation protein UL7, the
DNA helicase UL8, the Replication origin-binding protein UL9, the
Glycoprotein M (UL10), the UL11 protein, the Alkaline exonuclease
UL12, the Serine-threonine protein kinase UL13, the Tegument
protein UL14, the Terminase (UL15), the Tegument protein UL16, the
UL17 protein, the Capsid protein VP23 (UL18), the Major capsid
protein VP5 (UL19), the Membrane protein UL20, the Tegument protein
UL21, the Glycoprotein H (UL22), the Thymidine Kinase UL23, the
UL24 protein, the UL25 protein, the Capsid protein P40 (UL26, VP24,
VP22A), the Glycoprotein B (UL27), the ICP18.5 protein (UL28), the
Major DNA-binding protein ICP8 (UL29), the DNA polymerase UL30, the
Nuclear matrix protein UL31, the Envelope glycoprotein UL32, the
UL33 protein, the Inner nuclear membrane protein UL34, the Capsid
protein VP26 (UL35), the Large tegument protein UL36, the Capsid
assembly protein UL37, the VP19C protein (UL38), the Ribonucleotide
reductase (Large subunit) UL39, the Ribonucleotide reductase (Small
subunit) UL40, the Tegument protein/Virion host shutoff VHS protein
(UL41), the DNA polymerase processivity factor UL42, the Membrane
protein UL43, the Glycoprotein C (UL44), the Membrane protein UL45,
the Tegument proteins VP11/12 (UL46), the Tegument protein VP13/14
(UL47), the Virion maturation protein VP16 (UL48, Alpha-TIF), the
Envelope protein UL49, the dUTP diphosphatase UL50, the Tegument
protein UL51, the DNA helicase/primase complex protein UL52, the
Glycoprotein K (UL53), the Transcriptional regulation protein 1E63
(ICP27, UL54), the UL55 protein, the UL56 protein, the Viral
replication protein ICP22 (1E68, US1), the US2 protein, the
Serine/threonine-protein kinase US3, the Glycoprotein G (US4), the
Glycoprotein J (US5), the Glycoprotein D (US6), the Glycoprotein I
(US7), the Glycoprotein E (US8), the Tegument protein US9, the
Capsid/Tegument protein US10, the Vmw21 protein (US11), the ICP47
protein (1E12, US12), the Major transcriptional activator ICP4
(1E175, RS1), the E3 ubiquitin ligase ICP0 (IE110), the
Latency-related protein 1 (LRP1), the Latency-related protein 2
(LRP2), the Neurovirulence factor RL1 (ICP34.5), and the
Latency-associated transcript (LAT); [0619] and preferably wherein
the protein or peptide antigen and/or the fragment, variant and/or
derivative of said protein or peptide antigen is not included in
the polymeric carrier cargo complex.
[0620] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0621] a polymeric carrier
cargo complex and [0622] at least one protein or peptide antigen
[0623] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0624] wherein the polymeric carrier cargo complex
comprises [0625] a polymeric carrier, [0626] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0627] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above, most preferably according to subformula (IA) or (IB)
thereof as defined above, [0628] and at least one nucleic acid
molecule, [0629] preferably a nucleic acid molecule comprising,
preferably consisting of a nucleic acid sequence according to
formula (II) G.sub.lX.sub.mG.sub.n, formula (III)
C.sub.lX.sub.mC.sub.n, formula (IV)
(N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0630] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0631] and wherein the
protein or peptide antigen [0632] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0633]
an antigen associated with allergy or allergic disease, [0634] an
antigen associated with autoimmune disease, [0635] an antigen
associated with a cancer or tumour disease, [0636] preferably an
antigen from a pathogen associated with infectious disease, [0637]
more preferably an antigen from a pathogen selected from Rabies
virus, Hepatitis B virus, human Papilloma virus (hPV), Bacillus
anthracis, Respiratory syncytial virus (RSV), Herpes simplex virus
(HSV), and Mycobacterium tuberculosis, [0638] even more preferably
an antigen from Mycobacterium tuberculosis, [0639] most preferably
an antigen from Mycobacterium tuberculosis which is selected from
from the ESAT-6 protein, the ESX-1 protein, the CFP10 protein, the
TB10.4 protein, the MPT63 protein, the MPT64 protein, the MPT83
protein, the MTB12 protein, the MTB8 protein, the AG85A protein,
the AG85B protein, the Rpf-like proteins, the KATG protein, the
PPE18 protein, the MTB32 protein, the MTB39 protein, the
Crystallin, the HSP65 protein, the PST-S protein, and the HBHA
protein, the 10 kDa filtrate antigen EsxB, the serine protease
PepA, the fibronectin-binding protein D FbpD, the secreted protein
MPT51, the periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1),
the periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3,
Phos-1), the PPE family protein PPE14, the PPE family protein
PPE68, the protein MTB72F, the molecular chaperone DnaK, the cell
surface lipoprotein MPT83, the lipoprotein P23, the Phosphate
transport system permease protein PstA, the 14 kDa antigen, the
fibronectin-binding protein C FbpC1, the Alanine dehydrogenase
TB43, and the Glutamine synthetase 1; [0640] and preferably wherein
the protein or peptide antigen and/or the fragement, variant and/or
derivative of said protein or peptide antigen is not included in
the polymeric carrier cargo complex.
[0641] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0642] a polymeric carrier
cargo complex and [0643] at least one protein or peptide antigen
[0644] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0645] wherein the polymeric carrier cargo complex
comprises [0646] a polymeric carrier, [0647] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0648] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0649] and at least one nucleic acid molecule,
[0650] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0651] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0652] and wherein the
protein or peptide antigen [0653] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0654]
an antigen associated with allergy or allergic disease, [0655] an
antigen associated with autoimmune disease, [0656] an antigen
associated with a cancer or tumour disease, [0657] preferably a
protein or peptide antigen which is associated with allergy or
allergic disease and derived from a source selected from the list
consisting of: grass pollen, tree pollen, flower pollen, herb
pollen, dust mite, mold, animals, food, and insect venom,
preferably tree pollen, flower pollen, herb pollen dust mite, food,
and insect venom, most preferably an allergen as listed above,
[0658] and preferably wherein the protein or peptide antigen and/or
the fragement, variant and/or derivative of said protein or peptide
antigen is not included in the polymeric carrier cargo complex.
[0659] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0660] a polymeric carrier
cargo complex and [0661] at least one protein or peptide antigen
[0662] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0663] wherein the polymeric carrier cargo complex
comprises [0664] a polymeric carrier, [0665] preferably a polymeric
carrier formed by disulfide-crosslinked cationic components, [0666]
more preferably one or more peptides according to formula (I)
(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0667] and at least one nucleic acid molecule,
[0668] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0669] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen, [0670] and wherein the
protein or peptide antigen [0671] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0672]
an antigen associated with allergy or allergic disease, [0673] an
antigen associated with autoimmune disease, [0674] an antigen
associated with a cancer or tumour disease, [0675] preferably an
antigen associated with autoimmune disease, more preferably an
antigen associated with multiple sclerosis (MS); diabetes Typ I;
autoimmune uveitis; Myasthenia gravis; Rheumatic Fever; Arthritis;
Sjogren's syndrome; lupus erythematosus; Atherosclerosis;
idiopathic dilated cardiomyopathy (DCM); myositis or scleroderma,
[0676] even more preferably an antigen selected from the group
comprising myelin basic protein (MBP), proteolipid protein (PLP),
and myelin oligodendrocyte glycoprotein (MOG), CD44, preproinsulin,
proinsulin, insulin, glutamic acid decaroxylase (GAD65), tyrosine
phosphatase-like insulinoma antigen 2 (IA2), zinc transporter
((ZnT8), and heat shock protein 60 (HSP60), interphotoreceptor
retinoid-binding protein (IRBP), acetylcholine receptor AchR, and
insulin-like growth factor-1 receptor (IGF-1R), M-protein from
beta-hemolytic streptocci (pseudo-autoantigen), Macrophage
migration inhibitory factor, Ro/La RNP complex, alpha- and
beta-fodrin, islet cell autoantigen, poly(ADP)ribose polymerase
(PARP), NuMA, NOR-90, Ro60 autoantigen, and p27 antigen, Ro60
autoantigen, low-density lipoproteins, Sm antigens of the U-1 small
nuclear ribonucleoprotein complex (B/B', D1, D2, D3, E, F, G), and
RNP ribonucleoproteins, oxLDL, beta(2)GPI, HSP60/65, and
oxLDL/beta(2)GPI, cardiac beta(1)-adrenergic receptor,
histidyl-tRNA synthetase (HisRS), topoisomerase I, IL-17; or heat
shock proteins; [0677] and preferably wherein the protein or
peptide antigen and/or the fragment, variant and/or derivative of
said protein or peptide antigen is not included in the polymeric
carrier cargo complex.
[0678] Provided the antigen is associated with an autoimmune
disease, it may be further preferred if the antigen is an
immunoglobulin idiotype or a T cell receptor idiotype of a lymphoid
cell, preferably of a B-cell or a T-cell. Such lymphoid cells, e.g.
B-cells or T-cells may be responsible for destruction of
body-cells, such as e.g. pancreatic beta-cells, if they are
erroneously programmed to recognise and fight self-epitopes of the
body. In this context, an immunoglobulin idiotype may be understood
to be a peptide or protein having the particular molecular shape of
the variable region of an immunoglobulin expressed by a particular
type of B-cells. Such an idiotype may be used as antigen for
eliciting an immune response directed against this particular type
of B-cells, for example against malignant B-cells. A T cell
receptor idiotype may be understood to be a peptide or protein
having the particular molecular shape of the variable region of a T
cell receptor expressed by a particular type of T-cells. Such an
idiotype may be used as antigen for eliciting an immune response
directed against this particular type of T-cells, for example
against malignant T-cells. The inventive pharmaceutical composition
may be used, for example, for vaccination against such
mis-programmed lymphoid cells. E.g. treatment of a patient
suffering from an autoimmune disease, such as e.g. Diabetes,
Crohn's disease, Multiple sclerosis or the like, may occur by
destruction of malfunctional lymphoid cells which attact the own
body and subsequent vaccination with the inventive pharmaceutical
composition.
[0679] In a further preferred embodiment, the invention relates
to
a pharmaceutical composition comprising [0680] a polymeric carrier
cargo complex and [0681] at least one protein or peptide antigen
[0682] or a fragment, variant and/or derivative of said protein or
peptide antigen; [0683] wherein the polymeric carrier cargo complex
comprises [0684] a polymeric carrier, [0685] preferably a polymeric
carrier comprising disulfide-crosslinked cationic components,
preferably formed by disulfide-crosslinked cationic components,
[0686] more preferably one or more peptides according to formula
(I) (Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x as
defined above most according to subformula (IA) or (IB) thereof as
defined above, [0687] and at least one nucleic acid molecule,
[0688] preferably a nucleic acid molecule comprising, preferably
consisting of a nucleic acid sequence according to formula (II)
G.sub.lX.sub.mG.sub.n, formula (III) C.sub.lX.sub.mC.sub.n, formula
(IV) (N.sub.uG.sub.lX.sub.mG.sub.nN.sub.v).sub.a or formula (V)
(N.sub.uC.sub.lX.sub.mC.sub.nN.sub.v).sub.a as defined above, such
as a nucleic acid molecule comprising or consisting of a nucleic
acid sequence according to any one of SEQ ID NOs. 15-108 and 122 or
a sequence which is at least 60%, preferably at least 70%,
preferably at least 80%, more preferably at least 90%, and most
preferably at least 95% identical to any one of SEQ ID NOs. 15-108
and 122, e.g. a nucleic acid molecule comprising or consisting of a
nucleic acid sequence according to SEQ ID NO. 105 or 122 or a
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122, [0689] or a nucleic
acid molecule which encodes an antigen, more preferably a nucleic
acid molecule which encodes an antigen which is the same antigen as
the at least one protein or peptide antigen; [0690] and wherein the
protein or peptide antigen [0691] or the fragment, variant and/or
derivative of said protein or peptide antigen is selected from an
antigen from a pathogen associated with infectious disease, [0692]
an antigen associated with allergy or allergic disease, [0693] an
antigen associated with autoimmune disease, [0694] an antigen
associated with a cancer or tumour disease, [0695] preferably an
antigen associated with a cancer or tumour disease, more preferably
an antigen selected from p53, CA125, EGFR, Her2/neu, hTERT, PAP,
MAGE-A1, MAGE-A3, Mesothelin, MUC-1, NY-ESO-1, GP100, MART-1,
Tyrosinase, PSA, PSCA, PSMA VEGF, VEGFR1, VEGFR2, Ras, CEA and WT1;
[0696] and preferably wherein the protein or peptide antigen and/or
the fragment, variant and/or derivative of said protein or peptide
antigen is not included in the polymeric carrier cargo complex.
[0697] In each of these embodiments the polymeric carrier cargo
complex is preferably for use as an adjuvant, wherein, more
preferably, the at least one nucleic acid molecule is an
immunostimulatory nucleic acid as defined herein, even more
preferably the at least one nucleic acid molecule is RNA, most
preferably an immunostimulatory RNA (isRNA). Particular preferred
nucleic acid cargos in the context of the present invention are
nucleic acid molecules comprising or consisting of a nucleic acid
sequence according to SEQ ID NO. 105 or 122 or a nucleic acid
sequence which is at least 60%, preferably at least 70%, preferably
at least 80%, more preferably at least 90%, and most preferably at
least 95% identical to SEQ ID NO. 105 or 122.
[0698] Further, in each of these embodiments, the polymeric carrier
cargo complex may be a polymeric carrier cargo complex wherein the
cationic components of the polymeric carrier and the nucleic acid
molecule cargo comprised in said polymeric carrier cargo complex
are provided in a N/P ratio in the range of 0.1-20, or in the range
of 0.1-5, or in the range of 0.1-1, or in the range of 0.5-0.9.
[0699] In some embodiment, it may be preferred, provided the
polymeric carrier cargo complex comprises a polymeric carrier,
preferably a polymeric carrier formed by disulfide-crosslinked
cationic components, and at least one nucleic acid molecule, that
the pharmaceutical composition is not a composition comprising a
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and isRNA722A (SEQ ID No.
105) or isRNA722B (SEQ ID No. 122) as nucleic acid cargo to the
protein vaccine Ovalbumine (OVA protein).
[0700] In some embodiment, it may be preferred, provided the
polymeric carrier cargo complex comprises a polymeric carrier,
preferably a polymeric carrier formed by disulfide-crosslinked
cationic components, and at least one nucleic acid molecule, that
the pharmaceutical composition is not a composition comprising a
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and isRNA722A (SEQ ID No.
105) or isRNA722B (SEQ ID No. 122) as nucleic acid cargo to the
Ovalbumine-specific peptide vaccine SIINFEKL.
[0701] Accordingly, in some embodiments, it may be preferred that
the pharmaceutical composition is not a composition comprising a
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C (SEQ ID No. 6) as carrier and isRNA722A
(SEQ ID No. 105) or isRNA722B (SEQ ID No. 122) as nucleic acid
cargo and an antigen which is Ovalbumin (OVA protein) (SEQ ID No.
117).
[0702] In some embodiments, it may be further preferred, provided
that the pharmaceutical composition comprises a polymeric carrier
cargo complex comprising a polymeric carrier formed by
disulfide-crosslinked cationic components and at least one nucleic
acid molecule, that the at least one antigen is not an antigen
associated with a cancer or tumour disease, particularly lymphoma
or a lymphoma associated disease, wherein said antigen is an
immunoglobulin idiotype of a lymphoid blood cell or a T cell
receptor idiotype of a lymphoid blood cell or a fragment, variant
and/or derivative of such an immunoglobulin idiotype or T cell
receptor idiotype.
[0703] In this context, an immunoglobulin idiotype may be
understood to be a peptide or protein having the particular
molecular shape of the variable region of an immunoglobulin
expressed by a particular type of B-cells. Such an idiotype may be
used as antigen for eliciting an immune response directed against
this particular type of B-cells, for example against malignant
B-cells. A T cell receptor idiotype may be understood to be a
peptide or protein having the particular molecular shape of the
variable region of a T cell receptor expressed by a particular type
of T-cells. Such an idiotype may be used as antigen for eliciting
an immune response directed against this particular type of
T-cells, for example against malignant T-cells.
[0704] In some embodiments, it may be preferred, provided that the
pharmaceutical composition comprises a polymeric carrier cargo
complex comprising a polymeric carrier formed by
disulfide-crosslinked cationic components and at least one nucleic
acid molecule, that the at least one antigen associated with a
cancer or tumour disease is not an antigen associated with
lymphoma, preferably not B-cell lymphoma, T-cell lymphoma or
Non-Hodgkin's lymphoma.
[0705] In some embodiments it may be preferred, provided that the
pharmaceutical composition comprises a polymeric carrier cargo
complex comprising a polymeric carrier formed by
disulfide-crosslinked cationic components and at least one nucleic
acid molecule, that the at least one antigen associated with a
cancer or tumour disease is not an antigen derived from a malignant
cell, preferably not from a malignant B cell or a malignant T
cell.
[0706] In some further embodiments, it may be preferred that the
pharmaceutical composition may comprise no further component than
the components A) and B), preferably no other mRNA component (other
than comprised by the components A), preferably the pharmaceutical
composition may not comprise any mRNA at all.
[0707] In some further embodiments, it may be preferred, provided
the pharmaceutical composition comprises mRNA (other than nucleic
acid of component A), the mRNA may not be a mRNA encoding a peptide
or antigen according to B), further preferred the mRNA may not be a
mRNA encoding Ovalbumin, PSMA, Luciferase or STEAP.
[0708] In some further embodiments, it may be preferred, provided
the pharmaceutical composition contains a mRNA (other than nucleic
acid of component A), particularly mRNA encoding a peptide or
antigen according to B), and/or mRNA encoding Ovalbumin, PSMA,
Luciferase or STEAP, the mRNA may not be complexed with protamin,
preferably not in a ratio of 2:1 or 4:1 or between 2:1 and 4:1.
[0709] In some further embodiments, it may be preferred that the
claimed pharmaceutical composition may not be used for treatment of
pancreas carcinoma or non-small cell lung carcinoma.
[0710] In some further embodiments, it may be preferred, provided
the pharmaceutical composition comprises mRNA (other than nucleic
acid of component A), that the mRNA may not be a free mRNA.
[0711] In some further embodiments, it may be preferred, provided
the pharmaceutical composition comprises mRNA (other than nucleic
acid of component A), that the mRNA may not be complexed with
protamine.
[0712] In some further embodiments, it may be preferred, provided
the pharmaceutical composition comprises free mRNA, that the mRNA
may not encode for a therapeutically active protein and may not
encode for an antibody and may not encode for an antigen.
[0713] In some further embodiments, it may be preferred that with
respect to component A) of the inventive pharmaceutical
composition, that a) may not be protamine.
[0714] In some further embodiments, it may be preferred that with
respect to component A) of the inventive pharmaceutical
composition, that the carrier protein may not be protamine.
[0715] In some further embodiments, it may be preferred, provided
that a) of component A) is protamine, a) is not present in a ratio
of 1:2 or 1:4 or between 1:2 and 1:4, with respect to b) of
component A).
[0716] In some further embodiments, it may be preferred, provided
that the carrier protein of component A) is protamine, the carrier
protein is not present in a ratio of 1:2 or 1:4 with respect to the
nucleic acid of component A).
[0717] In some further embodiments, it may be preferred, that with
respect to component A) the nucleic acid is not an mRNA.
[0718] In some further embodiments, it may be preferred, provided
the nucleic acid of component A) is an mRNA, that the mRNA does not
encode Ovalbumin, PSMA, Luciferase or STEAP.
[0719] In some further embodiments, it may be preferred, provided
the nucleic acid, i.e. b), of the component A) is mRNA; that the
mRNA is not a free mRNA, but is exclusively complexed with the
carrier protein of a).
[0720] In some further embodiments, component (B) is not ovalbumin
or a fragment of ovalbumin. Preferably, the pharmaceutical
composition, the kit, or the pharmaceutical package according to
the present invention does not comprise ovalbumin or a fragment of
ovalbumin or a nucleic acid sequence coding for ovalbumin or coding
for a fragment of ovalbumin.
FIGURES
[0721] The following Figures are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
[0722] FIG. 1: shows the raw correlation curve of polymeric carrier
cargo complexes formed by the disulfide-crosslinked cationic
peptides CR.sub.12C and CR.sub.7C as carrier after lyophilisation
compared to complexes with non-polymerizing cationic peptides as
carrier (R.sub.12 and R.sub.7) by dynamic light scattering using a
Zetasizer Nano (Malvern Instruments, Malvern, UK). The hydrodynamic
diameters were measured with fresh prepared complexes and with
reconstituted complexes after lyophilisation The mass ratio of
peptide:RNA was 1:2. As result it can be shown that the polymeric
carrier cargo complexes comprising cystein-containing peptides as
cationic components which lead to a polymerization of the polymeric
carrier by disulfide bonds do not change in size in contrast to the
complexes formed by non-polymerizing peptides which increase in
size and therefore are not stable during the lyophilization step.
Therefore complexes with polymerized peptides as polymeric carriers
show advantageous properties for lyophilization.
[0723] FIG. 2: shows the Zeta-potential of polymeric carrier cargo
complexes formed by the disulfide-cross-linked cationic peptide
CR.sub.12C and the R722 as nucleic acid cargo at different w/w
ratios. As can be seen, the zeta potential changes from positive to
negative when the w/w ratio is changed from excess peptide to a 1:1
ratio (peptide/RNA).
[0724] FIG. 3A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w)
(CR.sub.12C/CpG 2216). As can be seen, the polymeric carrier cargo
complexes lead to an increase of hIFNa cytokine release in hPBMCs
compared to the nucleic acid cargo alone or the cationic peptide
alone.
[0725] FIG. 3B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w)
(CR.sub.12C/CpG 2216). As can be seen, the polymeric carrier cargo
complexes do not lead to an increase in hTNFa cytokine release in
hPBMCs compared to the nucleic acid cargo alone or the cationic
peptide alone.
[0726] FIG. 4A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the mRNA R491 coding for luciferase as nucleic acid cargo in a mass
ratio of 1:2 (w/w) (CR.sub.12C/R491). As can be seen, the polymeric
carrier cargo complexes lead to an increase of hIFNa cytokine
release in hPBMCs compared to the nucleic acid cargo alone or the
cationic peptide alone.
[0727] FIG. 4B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the mRNA R491 coding for luciferase as nucleic acid cargo in a mass
ratio of 1:2 (w/w) (CR.sub.12C/R491). As can be seen, the polymeric
carrier cargo complexes lead to an increase of hTNFa cytokine
release in hPBMCs compared to the nucleic acid cargo alone or the
cationic peptide alone.
[0728] FIG. 5A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid
cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich). As
can be seen, the polymeric carrier cargo complexes lead to an
increase of hIFNa cytokine release in hPBMCs compared to the
nucleic acid cargo alone or the cationic peptide alone.
[0729] FIG. 5B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid
cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich). As
can be seen, the polymeric carrier cargo complexes lead to an
increase of hTNFa cytokine release in hPBMCs compared to the
nucleic acid cargo alone or the cationic peptide alone.
[0730] FIG. 6A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.7C and
the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As
can be seen, the polymeric carrier cargo complexes (CR.sub.7C/R722)
lead to an increase of hIFNa cytokine release in hPBMCs compared to
cargo complexes (R.sub.7/R722) formed by the non-polymerized
peptide R.sub.7.
[0731] FIG. 6B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.7C and
the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As
can be seen, the polymeric carrier cargo complexes (CR.sub.7C/R722)
only leads to a weak increase of hTNFa cytokine release in hPBMCs
compared to carrier cargo complexes (R.sub.7/R722) formed by the
non-polymerized peptide R.sub.7.
[0732] FIG. 7A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.9C and
the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As
can be seen, the inventive polymeric carrier cargo complexes
(CR.sub.9C/R722) lead to an increase of hIFNa cytokine release in
hPBMCs compared to carrier cargo complexes (R.sub.9/R722) formed by
the non-polymerized peptide R.sub.9.
[0733] FIG. 7B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.9C and
the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As
can be seen, the polymeric carrier cargo complexes (CR.sub.9C/R722)
do not lead to an increase of hTNFa cytokine release in hPBMCs
compared to carrier cargo complexes (R.sub.9/R722) formed by the
non-polymerized peptide R.sub.9.
[0734] FIG. 8A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the isRNA R722 as nucleic acid cargo at different w/w ratios. As
can be seen, the polymeric carrier cargo complexes lead to an
increase in hIFNa cytokine release in hPBMCs compared to the
nucleic acid cargo alone or the cationic peptide alone.
[0735] FIG. 8B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier cargo complexes
formed by the disulfide-crosslinked cationic peptide CR.sub.12C and
the isRNA R722 as nucleic acid cargo at different w/w ratios. As
can be seen, the polymeric carrier cargo complexes lead to an
increase in hTNFa cytokine release in hPBMCs compared to the
nucleic acid cargo alone or the cationic peptide alone.
[0736] FIG. 9A: shows the secretion of hIFNa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier complexes formed by
the cationic peptides CH.sub.6R.sub.4H.sub.6C,
CH.sub.3R.sub.4H.sub.3C and CHK.sub.7HC and the isRNA R722 as
nucleic acid cargo at different N/P ratios. As can be seen, the
polymeric carrier cargo complexes lead to an increase in hIFNa
cytokine release in hPBMCs compared to the nucleic acid cargo alone
or the cationic peptide alone.
[0737] FIG. 9B: shows the secretion of hTNFa cytokine (in vitro) in
hPBMCs after stimulation with polymeric carrier complexes formed by
the disulfide-crosslinked cationic peptides
CH.sub.6R.sub.4H.sub.6C, CH.sub.3R.sub.4H.sub.3C and CHK.sub.7HC
and the isRNA R722 as nucleic acid cargo at different N/P ratios.
As can be seen, the polymeric carrier cargo complexes lead to an
increase in hTNFa cytokine release in hPBMCs compared to the
nucleic acid cargo alone or the cationic peptide alone.
Particularly polymeric cargo complexes with an N/P ratio greater or
equal 1 result in TNFalpha secretion.
[0738] FIG. 10: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the protein antigen Ovalbumine (OVA protein)
for the use as an adjuvant in tumour challenge experiments.
[0739] For this purpose 7 female C57BL/6 mice per group were
vaccinated three times in two weeks with pg 5 .mu.g Ovalbumin
protein combined with 45 .mu.g CR.sub.12C/R722 (1:2; w/w). For
comparison mice were injected without the polymeric cargo
complexes.
[0740] As can be seen, the polymeric carrier cargo complex
extremely decelaterates the tumour growth compared to the protein
antigen alone, which has no effect on tumor growth in comparison to
the buffer control.
[0741] FIG. 11: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the protein antigen Ovalbumine (OVA protein)
for the use as an adjuvant on the induction of Ovalbumine-specific
IgG2a antibodies.
[0742] For this purpose 5 female C57BL/6 mice per group were
vaccinated three times in two weeks with 5 .mu.g Ovalbumin protein
combined with 45 .mu.g CR.sub.12C/R722 (1:2; w/w). For comparison
mice were injected without the polymeric cargo complexes.
[0743] As can be seen, the polymeric carrier cargo complex strongly
increases the B-cell response, which proofs the beneficial adjuvant
properties of the polymeric carrier cargo complexes, particularly
in regard to the induction of a Th1-shifted immune response.
[0744] FIG. 12: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the protein antigen Ovalbumine (OVA protein)
or the Ovalbumine-specific peptide antigen SIINFEKL for the use as
an adjuvant on the induction of Ovalbumine-specific cytotoxic T
cells.
[0745] For this purpose 5 female C57BL/6 mice per group were
vaccinated three times in two weeks with 5 .mu.g Ovalbumin protein
or 50 .mu.g SIINFEKEL peptide combined with 45 .mu.g
CR.sub.12C/R722 (1:2; w/w). For comparison mice were injected
without the polymeric cargo complexes.
[0746] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Ovalbumin-specific cytotoxic T cells
compared to the vaccination with protein or peptide alone, which
further proofs the beneficial adjuvant properties of the polymeric
carrier cargo complex, particularly in regard to the induction of a
Th1-shifted immune response.
[0747] FIG. 13: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the vaccine Rabipur.RTM. (comprising
inactivated Rabies virus) for the use as an adjuvant on the
induction of Rabies specific IgG antibodies (as represented by OD
405 nm). For this purpose 8 female BALB/c mice were injected
intramuscularly with the 0.1, 0.01, and the 0.001 fold human dose
of Rabipur.RTM. and 30 .mu.g R722 and 8.1 .mu.g CR.sub.12C (3.7:1
w/w). 21 days after the immunization blood samples were taken and
analysed for total IgG antibodies directed against the Rabies
virus.
[0748] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Rabies-specific IgG antibodies compared
to the vaccination with Rabipur.RTM. alone, which further proofs
the beneficial adjuvant properties of the polymeric carrier cargo
complex.
[0749] FIG. 14: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the vaccine Rabipur.RTM. or HDC (comprising
inactivated Rabies virus) for the use as an adjuvant on the
induction of Rabies specific cytotoxic T cells (as represented by
number of spots in the ELISPOT assay).
[0750] For this purpose 5 female BALB/c mice were injected
intramuscularly with the 0.01 fold human dose of Rabipur.RTM. or
HDC and 30 .mu.g R722 and 8.1 .mu.g CR.sub.12C (3.7:1 w/w). 5 days
after the immunization the mice were sacrificed, the spleens were
removed and the splenocytes were isolated.
[0751] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Rabies-specific cytotoxic T cells
compared to the vaccination with Rabipur.RTM. or HDC alone, which
further proofs the beneficial adjuvant properties of the polymeric
carrier cargo complex, particularly in regards to the induction of
a Th1-shifted immune response.
[0752] FIG. 15: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the vaccine Rabipur.RTM. (comprising
inactivated Rabies virus) for the use as an adjuvant on the
induction of Rabies specific IgG antibodies. Furthermore it shows
the effect of the polymeric carrier cargo complex on the induction
of antibodies with high affinity to the antigen (as represented by
% of bound IgG).
[0753] For this purpose 8 female BALB/c mice were injected
intramuscularly with the 0.01 fold human dose of Rabipur.RTM. and
30 .mu.g R722 and 8.1 .mu.g CR.sub.12C (3.7:1 w/w). 7 and 21 days
after the immunization blood samples were taken and analysed for
total IgG antibodies directed against the Rabies virus. To examine
the affinity of the generated antibodies directed against the
Rabies virus, during the performance of the ELISA the bound
antibodies were washed with an increasing concentration of
urea.
[0754] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Rabies-specific IgG antibodies with high
affinity to the antigen compared to the vaccination with
Rabipur.RTM. alone, which further proofs the beneficial adjuvant
properties of the polymeric carrier cargo complex, particularly in
regards to the induction of antibodies with high affinity.
[0755] FIG. 16: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the vaccine HDC (comprising inactivated
Rabies virus) for the use as an adjuvant on the induction of Rabies
virus neutralizing antibodies (as represented by IU/ml). For this
purpose 8 female BALB/c mice were injected intramuscularly with the
0.01 fold human dose of HDC and 30 .mu.g R722 and 15 .mu.g
CR.sub.12C (2:1 w/w). 21 days after the immunization blood samples
were taken and virus neutralization was analysed.
[0756] As can be seen, the polymeric carrier cargo complex strongly
increases the neutralizing antibody titer compared to the
vaccination with HDC alone, which further proofs the beneficial
adjuvant properties of the polymeric carrier cargo complex.
[0757] FIG. 17: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the swine flu vaccine Pandemrix.RTM.
(comprising inactivated H1N1 influenza virus) for the use as an
adjuvant on the induction of H1N1 influenza specific IgG2a
antibodies. For this purpose 5 female BALB/c mice were injected
intramuscularly with 0.1 .mu.g Pandemrix.RTM. and 30 .mu.g R722 and
15 .mu.g CR.sub.12C (2:1 w/w). 14 days after the immunization blood
samples were taken and analysed for induction of IgG2a antibodies
directed against H1N1 influenza virus (as represented by OD 405
nm).
[0758] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Influenza-specific IgG2a antibodies
compared to the vaccination with Panemrix.RTM. alone, which further
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0759] FIG. 18: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the A(H1N1)pdm09influenza vaccine
Celvapan.RTM. (comprising inactivated A(H1N1)pdm09influenza virus)
for the use as an adjuvant on the induction of A(H1N1)pdm09specific
cytotoxic T cells (as represented by number of spots in the ELISPOT
assay).
[0760] For this purpose 5 female BALB/c mice were injected
intramuscularly with the 0.1 .mu.g Celvapan.RTM. and 15 .mu.g R722
and 7.5 .mu.g CR.sub.12C (2:1 w/w). 6 days after the immunization
the mice were sacrificed, the spleens were removed and the
splenocytes were isolated.
[0761] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of A(H1N1)pdm09-specific cytotoxic T cells
compared to the vaccination with Celvapan.RTM. alone, which further
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0762] FIG. 19: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the seasonal influenza vaccine Begrivac.RTM.
(comprising inactivated seasonal influenza virus strains as
recommended by the WHO) for the use as an adjuvant on the induction
of influenza specific IgG2a antibodies (as represented by OD 405
nm). For this purpose 8 female BALB/c mice were injected
intramuscularly with 0.1 .mu.g Begrivac.RTM. and 30 .mu.g R722 and
15 .mu.g CR.sub.12C (2:1 w/w). 28 days after the immunization blood
samples were taken and analysed for IgG2a antibodies directed
against influenza virus.
[0763] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of Influenza-specific IgG2a antibodies
compared to the vaccination with Begrivac.RTM. alone, which further
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0764] FIG. 20: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the Hepatitis B vaccine Engerix.RTM.-B
(comprising recombinant Hepatitis B Surface Antigen (HBsAg)) for
the use as an adjuvant on the induction of HBsAG specific
antibodies (as represented by fluorescence).
[0765] For this purpose 8 female BALB/c mice were injected
intramuscularly with 0.5 .mu.g Engerix.RTM.-B and 6.25 .mu.g R722
and 1.7 .mu.g CR.sub.12C (3.7:1 w/w). 28 days after the
immunization blood samples were taken and analysed for IgG2a
antibodies directed against the HBsAGg
[0766] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of HBsAg-specific IgG2a antibodies compared
to the vaccination with Engerix.RTM.-B alone, which further proofs
the beneficial adjuvant properties of the polymeric carrier cargo
complex, particularly in regards to the induction of a Th1-shifted
immune response.
[0767] FIG. 21: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to a human papilloma virus 16 (HPV16) derived
peptide for the use as an adjuvant on the induction of HPV16 E7
specific cytotoxic T cells (as represented by number of spots in
the ELISPOT assay).
[0768] For this purpose 5 female C57BL/6 mice were injected
intradermally with 100 .mu.g of the HPV16 E7 derived peptide
E7aa43-77 and 50 .mu.g R722 and 25 .mu.g CR.sub.12C (2:1 w/w). 8
days after the immunization the mice were sacrificed, the spleens
were removed and the splenocytes were isolated.
[0769] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of HPV16 E7-specific cytotoxic T cells
compared to the vaccination with the peptide alone, which further
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0770] FIG. 22: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to a human papilloma virus 16 (HPV16) derived
peptide for the use as an adjuvant on the induction of HPV16 E7
specific cytotoxic T cells (as represented by number of spots in
the ELISPOT assay).
[0771] For this purpose 5 female C57BL/6 mice were injected
intradermally with 100 .mu.g of the HPV16 E7 derived peptide
E7aa43-77 and 50 .mu.g R722 and 25 .mu.g CR.sub.12C (2:1 w/w).
Furthermore mice were injected with the polymeric carrier cargo
complex additionally comprising the antigenic peptide E7aa43-77.
Seven days after the immunization the mice were sacrificed, the
spleens were removed and the splenocytes were isolated.
[0772] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of HPV16 E7-specific cytotoxic T cells
compared to the vaccination with the peptide alone. Furthermore the
results show that the inclusion of the antigenic peptide in the
polymeric carrier cargo complex further improves the induction of
HPV16 E7-specific cytotoxic T cells. Therefore also this experiment
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0773] FIG. 23: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the human NY-ESO-1 protein for the use as an
adjuvant on the induction of NY-ESO-1 specific cytotoxic T cells
(as represented by number of spots in the ELISPOT assay).
[0774] For this purpose 5 female C57BL/6 mice were injected
intramuscularly with 5 .mu.g NY-ESO-1 protein and 30 .mu.g R722 and
15 .mu.g CR.sub.12C (2:1 w/w) 2 times within 15 days. 7 days
after-the-last-immunization the mice were sacrificed, the spleens
were removed and the splenocytes were isolated.
[0775] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of NY-ESO-1-specific cytotoxic T cells
compared to the vaccination with the protein alone, which further
proofs the beneficial adjuvant properties of the polymeric carrier
cargo complex, particularly in regards to the induction of a
Th1-shifted immune response.
[0776] FIG. 24: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the vaccine Rabipur.RTM. (comprising
inactivated Rabies virus) for the use as an adjuvant for the
enhancement of protection against lethal virus challenge
infection.
[0777] For this purpose 8 female BALB/c mice were injected
intramuscularly with the 0.001 fold human dose of Rabipur.RTM. and
3 .mu.g R722 and 0.81 .mu.g CR.sub.12C (3.7:1 w/w). 37 days after
vaccination the mice were infected with a lethal dose of rabies
virus of challenge virus strain (CVS) using a 25-fold LD.sub.50
(lethal doses 50%).
[0778] As can be seen, the polymeric carrier cargo complex strongly
increases the survival of mice against lethal Rabies virus
infection compared to vaccination with Rabipur.RTM. alone, which
further proofs the beneficial adjuvant properties of the polymeric
carrier cargo complex.
[0779] FIG. 25: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the human papilloma virus 16 (HPV16) derived
long-chain peptide E7aa43-77 for the use as an adjuvant in tumour
challenge experiments.
[0780] For this purpose, 8 C57BL/6 mice per group were challenged
on day 1 with 1.times.10.sup.5 TC-1 cells which express the HPV E6
and E7 protein. Vaccination started on day 7 after tumor challenge
(median tumor volume 31-48 mm.sup.3). Mice were intradermally
vaccinated 5 times (on day 8, 12, 15, 19 and 22) with 5 .mu.g or 50
.mu.g E7 peptide combined with 50 .mu.g CR.sub.12C/R722 (1:2;
w/w).
[0781] For comparison, mice were injected with the polymeric cargo
complexes alone.
[0782] As can be seen, the polymeric carrier cargo complex combined
with HPV-16 derived E7 peptide E7aa43-77 even impairs the growth of
tumours compared to the polymeric carrier cargo complex alone.
[0783] FIG. 26: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the human papilloma virus 16 (HPV16) derived
E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge
experiments.
[0784] For this purpose, 8 C57BL/6 mice per group were challenged
on day 1 with 1.times.10.sup.5TC-1 cells. Vaccination started on
day 7 after tumor challenge (median tumor volume 31-48 mm.sup.3).
Mice were intradermally vaccinated 5 times (on day 8, 12, 15, 19
and 22) with 5 .mu.g or 50 .mu.g E7 peptide E7aa43-77 combined with
50 .mu.g CR.sub.12C/R722 (1:2; w/w). For comparison, mice were
injected with the E7 peptide or the polymeric cargo complexes
alone. Injection with PBS buffer served as negative control.
[0785] As can be seen, the polymeric carrier cargo complex combined
with HPV-16 derived E7 peptide strongly enhances the survival of
tumor bearing mice (Mean survival time of 44.5 days for 50 .mu.g E7
peptide+50 .mu.g polymeric carrier cargo complex; mean survival
time of 22 days 5 .mu.g E7 peptide+50 .mu.g polymeric carrier cargo
complex) compared to the E7 peptide or 50 polymeric carrier cargo
complex alone.
[0786] FIG. 27: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the human papilloma virus 16 (HPV16) derived
E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge
experiments.
[0787] For this purpose, 13 C57BL/6 mice per group were
intradermally vaccinated once per week for four weeks with the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo and the E7 peptide as indicated in the Figure.
Eight weeks after the fourth vaccination, 5 mice/group were
sacrificed, splenocytes were isolated and the frequency of
antigen-specific CD8.sup.+ T cells was determined by HPV-pentamer
staining and flow cytometry according to example 13.
[0788] As can be seen, the polymeric carrier cargo complex combined
with the HPV-16 derived E7 peptide E7aa43-77 results in a
statistically significant increase of antigen-specific CD8.sup.+ T
cells compared to mice vaccinated with 50 .mu.g of the E7 peptide
alone (p=0.0007 for 5 .mu.g E7 peptide and p=0.0002 50 .mu.g E7
peptide; statistical differences between groups were assessed by
unpaired t-test).Thus, the combination of the polymeric carrier
cargo complex combined with the HPV-16 derived E7 peptide induces a
potent memory CD8.sup.+ T cell response.
[0789] FIG. 28: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the human papilloma virus 16 (HPV16) derived
E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge
experiments.
[0790] For this purpose, 13 C57BL/6 mice per group were
intradermally vaccinated once per week for four weeks with the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo and the E7 peptide as indicated in the Figure.
Eight weeks after the fourth vaccination 8 mice/group were
challenged with 1.times.10.sup.5 TC-1 tumor cells and tumor growth
was monitored. As can be seen, the polymeric carrier cargo complex
combined with the HPV-16 derived E7 peptide E7aa43-77 results in a
drastic delay of tumor growth (4 complete responses for 5 .mu.g E7
peptide+50 .mu.g of 50 .mu.g polymeric carrier cargo complex; 7
complete responders for 50 .mu.g E7 peptide+50 .mu.g of 50 .mu.g
polymeric carrier cargo complex). Thus, the combination of the
polymeric carrier cargo complex combined with the HPV-16 derived E7
peptide induces a potent memory CD8.sup.+ T cell response.
[0791] FIG. 29: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the seasonal influenza vaccine Mutagrip.RTM.
(comprising inactivated seasonal influenza virus strains as
recommended by the WHO) for the use as an adjuvant on the induction
of influenza specific hemagglutinin inhibition (HI) titers (as
represented by HI titer).
[0792] For this purpose, 8 female BALB/c mice were injected
intramuscularly with 0.45 .mu.g or 0.045 .mu.g Mutagrip.RTM. and 5
.mu.g R722+1.35 .mu.g CR.sub.12C (3.7:1, w/w). 21 days after the
immunization blood samples were taken and HI titers were determined
in the sera. The HI titer is widely used as a surrogate parameter
of influenza vaccine efficacy with a HI titer of .gtoreq.1:40
commonly defined as the protective limit in humans.
[0793] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of influenza A H1N1-specific HI titers (A)
and H3N2-specific HI titers (B) compared to the vaccination with
Mutagrip.RTM. alone, which further demonstrates the beneficial
adjuvant properties of the polymeric carrier cargo complex.
[0794] FIG. 30: shows the (in vivo) effect of the addition of the
polymeric carrier cargo complex formed by the disulfide-crosslinked
cationic peptide CR.sub.12C as carrier and the isRNA R722 as
nucleic acid cargo to the seasonal influenza vaccine Mutagrip.RTM.
(comprising inactivated seasonal influenza virus strains as
recommended by the WHO) for the use as an adjuvant on the induction
of influenza specific IgG2a antibodies (as represented by IgG2a
titer).
[0795] For this purpose, 8 female BALB/c mice were injected
intramuscularly with 4.5 .mu.g or 0.045 .mu.g Mutagrip.RTM. and 5
.mu.g R722+1.35 .mu.g CR.sub.12C (3.7:1 w/w). 21 days after the
immunization blood samples were taken and analysed for IgG2a
antibodies directed against influenza A H1N1 virus.
[0796] As can be seen, the polymeric carrier cargo complex strongly
increases the induction of influenza A H1N1-specific IgG2a
antibodies compared to the vaccination with Mutagrip.RTM. alone,
which further demonstrates the beneficial adjuvant properties of
the polymeric carrier cargo complex, particularly in regards to
dose-sparing of the seasonal influenza vaccine.
EXAMPLES
[0797] The following examples are intended to illustrate the
invention further. They are not intended to limit the subject
matter of the invention thereto.
1. Reagents:
[0798] Cationic Peptides as Cationic Component of the Polymeric
Carrier:
TABLE-US-00007 R2: (SEQ ID NO. 109) Arg-Arg-Arg-Arg-Arg-Arg-Arg
(Arg.sub.7) CR2C: (SEQ ID NO. 1)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (CysArg.sub.7Cys) R9: (SEQ ID
NO. 110) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.9) R12: (SEQ
ID NO. 111) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg
(Arg.sub.12) CR9C: (SEQ ID NO. 2)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (Cys-Arg.sub.9-Cys)
CR12C: (SEQ ID NO. 6)
Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Cys
(Cys-Arg.sub.12-Cys)
[0799] Nucleic Acids as Cargo of the Polymeric Carrier Cargo
Complex:
TABLE-US-00008 (SEQ ID NO. 112) R1180: mRNA coding for luciferase
GGGAGAAAGCUUGAGGAUGGAGGACGCCAAGAACAUCAAGAAGGGCCCGGCGCCCUUCUACCCGCU
GGAGGACGGGACCGCCGGCGAGCAGCUCCACAAGGCCAUGAAGCGGUACGCCCUGGUGCCGGGCAC
GAUCGCCUUCACCGACGCCCACAUCGAGGUCGACAUCACCUACGCGGAGUACUUCGAGAUGAGCGU
GCGCCUGGCCGAGGCCAUGAAGCGGUACGGCCUGAACACCAACCACCGGAUCGUGGUGUGCUCGGA
GAACAGCCUGCAGUUCUUCAUGCCGGUGCUGGGCGCCCUCUUCAUCGGCGUGGCCGUCGCCCCGGC
GAACGACAUCUACAACGAGCGGGAGCUGCUGAACAGCAUGGGGAUCAGCCAGCCGACCGUGGUGUU
CGUGAGCAAGAAGGGCCUGCAGAAGAUCCUGAACGUGCAGAAGAAGCUGCCCAUCAUCCAGAAGAU
CAUCAUCAUGGACAGCAAGACCGACUACCAGGGCUUCCAGUCGAUGUACACGUUCGUGACCAGCCA
CCUCCCGCCGGGCUUCAACGAGUACGACUUCGUCCCGGAGAGCUUCGACCGGGACAAGACCAUCGC
CCUGAUCAUGAACAGCAGCGGCAGCACCGGCCUGCCGAAGGGGGUGGCCCUGCCGCACCGGACCGC
CUGCGUGCGCUUCUCGCACGCCCGGGACCCCAUCUUCGGCAACCAGAUCAUCCCGGACACCGCCAU
CCUGAGCGUGGUGCCGUUCCACCACGGCUUCGGCAUGUUCACGACCCUGGGCUACCUCAUCUGCGG
CUUCCGGGUGGUCCUGAUGUACCGGUUCGAGGAGGAGCUGUUCCUGCGGAGCCUGCAGGACUACAA
GAUCCAGAGCGCGCUGCUCGUGCCGACCCUGUUCAGCUUCUUCGCCAAGAGCACCCUGAUCGACAA
GUACGACCUGUCGAACCUGCACGAGAUCGCCAGCGGGGGCGCCCCGCUGAGCAAGGAGGUGGGCGA
GGCCGUGGCCAAGCGGUUCCACCUCCCGGGCAUCCGCCAGGGCUACGGCCUGACCGAGACCACGAG
CGCGAUCCUGAUCACCCCCGAGGGGGACGACAAGCCGGGCGCCGUGGGCAAGGUGGUCCCGUUCUU
CGAGGCCAAGGUGGUGGACCUGGACACCGGCAAGACCCUGGGCGUGAACCAGCGGGGCGAGCUGUG
CGUGCGGGGGCCGAUGAUCAUGAGCGGCUACGUGAACAACCCGGAGGCCACCAACGCCCUCAUCGA
CAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGU
CGACCGGCUGAAGUCGCUGAUCAAGUACAAGGGCUACCAGGUGGCGCCGGCCGAGCUGGAGAGCAU
CCUGCUCCAGCACCCCAACAUCUUCGACGCCGGCGUGGCCGGGCUGCCGGACGACGACGCCGGCGA
GCUGCCGGCCGCGGUGGUGGUGCUGGAGCACGGCAAGACCAUGACGGAGAAGGAGAUCGUCGACUA
CGUGGCCAGCCAGGUGACCACCGCCAAGAAGCUGCGGGGCGGCGUGGUGUUCGUGGACGAGGUCCC
GAAGGGCCUGACCGGGAAGCUCGACGCCCGGAAGAUCCGCGAGAUCCUGAUCAAGGCCAAGAAGGG
CGGCAAGAUCGCCGUGUAAGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCU
CCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAUAUUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCUCUAG
(R1180) (SEQ ID NO. 105) R722A: long non-coding isGU-rich RNA (SEQ
ID NO. 122) R722B: long non-coding isGU-rich RNA (SEQ ID NO. 113)
R491: mRNA coding for luciferase
GGGAGAAAGCUUGAGGAUGGAGGACGCCAAGAACAUCAAGAAGGGCCCGGCGCCCUUCUACCCGCU
GGAGGACGGGACCGCCGGCGAGCAGCUCCACAAGGCCAUGAAGCGGUACGCCCUGGUGCCGGGCAC
GAUCGCCUUCACCGACGCCCACAUCGAGGUCGACAUCACCUACGCGGAGUACUUCGAGAUGAGCGU
GCGCCUGGCCGAGGCCAUGAAGCGGUACGGCCUGAACACCAACCACCGGAUCGUGGUGUGCUCGGA
GAACAGCCUGCAGUUCUUCAUGCCGGUGCUGGGCGCCCUCUUCAUCGGCGUGGCCGUCGCCCCGGC
GAACGACAUCUACAACGAGCGGGAGCUGCUGAACAGCAUGGGGAUCAGCCAGCCGACCGUGGUGUU
CGUGAGCAAGAAGGGCCUGCAGAAGAUCCUGAACGUGCAGAAGAAGCUGCCCAUCAUCCAGAAGAU
CAUCAUCAUGGACAGCAAGACCGACUACCAGGGCUUCCAGUCGAUGUACACGUUCGUGACCAGCCA
CCUCCCGCCGGGCUUCAACGAGUACGACUUCGUCCCGGAGAGCUUCGACCGGGACAAGACCAUCGC
CCUGAUCAUGAACAGCAGCGGCAGCACCGGCCUGCCGAAGGGGGUGGCCCUGCCGCACCGGACCGC
CUGCGUGCGCUUCUCGCACGCCCGGGACCCCAUCUUCGGCAACCAGAUCAUCCCGGACACCGCCAU
CCUGAGCGUGGUGCCGUUCCACCACGGCUUCGGCAUGUUCACGACCCUGGGCUACCUCAUCUGCGG
CUUCCGGGUGGUCCUGAUGUACCGGUUCGAGGAGGAGCUGUUCCUGCGGAGCCUGCAGGACUACAA
GAUCCAGAGCGCGCUGCUCGUGCCGACCCUGUUCAGCUUCUUCGCCAAGAGCACCCUGAUCGACAA
GUACGACCUGUCGAACCUGCACGAGAUCGCCAGCGGGGGCGCCCCGCUGAGCAAGGAGGUGGGCGA
GGCCGUGGCCAAGCGGUUCCACCUCCCGGGCAUCCGCCAGGGCUACGGCCUGACCGAGACCACGAG
CGCGAUCCUGAUCACCCCCGAGGGGGACGACAAGCCGGGCGCCGUGGGCAAGGUGGUCCCGUUCUU
CGAGGCCAAGGUGGUGGACCUGGACACCGGCAAGACCCUGGGCGUGAACCAGCGGGGCGAGCUGUG
CGUGCGGGGGCCGAUGAUCAUGAGCGGCUACGUGAACAACCCGGAGGCCACCAACGCCCUCAUCGA
CAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGU
CGACCGGCUGAAGUCGCUGAUCAAGUACAAGGGCUACCAGGUGGCGCCGGCCGAGCUGGAGAGCAU
CCUGCUCCAGCACCCCAACAUCUUCGACGCCGGCGUGGCCGGGCUGCCGGACGACGACGCCGGCGA
GCUGCCGGCCGCGGUGGUGGUGCUGGAGCACGGCAAGACCAUGACGGAGAAGGAGAUCGUCGACUA
CGUGGCCAGCCAGGUGACCACCGCCAAGAAGCUGCGGGGCGGCGUGGUGUUCGUGGACGAGGUCCC
GAAGGGCCUGACCGGGAAGCUCGACGCCCGGAAGAUCCGCGAGAUCCUGAUCAAGGCCAAGAAGGG
CGGCAAGAUCGCCGUGUAAGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCU
CCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAUAUUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCUCUAGACA
AUUGGAAUU (R491) (SEQ ID NO. 114) CpG 2216: CpG oligonucleotide
GGGGGACGATCGTCGGGGGG (SEQ ID NO. 115) Short GU rich: GU-rich RNA
oligonucleotide GGUUUUUUUUUUUUUUUGGG
[0800] Experiments indicating the use of nucleic acid cargo R722
have been performed with the sequences R722A and/or R722B.
Antigens and Epitopes:
TABLE-US-00009 [0801] Ovalbumine-derived peptide (SEQ ID NO. 116)
SIINFEKL Ovalbumine: (SEQ ID NO. 117)
MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDST
RTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSF
SLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWV
ESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFR
VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEV
SGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMA
MGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGV
DAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP HPV16 E7aa43-77: (SEQ ID NO.
118) GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR HPV16 E7 aa48-57: (SEQ ID
NO. 119) DRAHYNIVTF HPV16 E7 aa49-57 (H-2 Db): (SEQ ID NO. 120)
RAHYNIVTF NY-ESO-1: (SEQ ID NO. 121)
MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGA
GAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPF
ATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQ
LQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR
2. Preparation of Nucleic Acid Sequences:
[0802] For the present examples nucleic acid sequences as indicated
in example 1 were prepared and used for formation of the
polymerized polymeric carrier cargo complexes or for
non-polymerized carrier cargo complexes for comparison. These
polymeric carrier cargo complexes were used for in vitro and in
vivo transaction, for in vitro immunostimulation and for particle
characterizations.
[0803] According to a first preparation, the DNA sequences, coding
for the corresponding RNA sequences R1180, R722 and R491 sequences
were prepared. The sequences of the corresponding RNAs are shown in
the sequence listing (SEQ ID NOs: 112, 105, and 113).
[0804] The short GU rich sequences and the CpG 2216
oligonucleotides were prepared by automatic solid-phase synthesis
by means of phosphoramidite chemistry. The sequences are shown in
the sequence listing (SEQ ID NOs: 115 and 114).
In Vitro Transcription:
[0805] The respective DNA plasmids prepared according to Example 2
for R1180, R722 and R491 were transcribed in vitro using
T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany)
following the manufactures instructions. Subsequently the mRNA was
purified using PureMessenger.RTM. (CureVac, Tubingen, Germany).
3. Synthesis of Polymeric Carrier Cargo Complexes:
[0806] The nucleic acid sequences defined above in Example 1 were
mixed with the cationic components as defined in Example 1.
Therefore, the indicated amount of nucleic acid sequence was mixed
with the respective cationic component in mass ratios as indicated,
thereby forming a complex. If polymerizing cationic components were
used according to the present invention polymerization of the
cationic components took place simultaneously to complexation of
the nucleic acid cargo. Afterwards the resulting solution was
adjusted with water to a final volume of 50 .mu.l and incubated for
30 min at room temperature. The different ratios of cationic
component/nucleic acid used in the experiments are shown in Table
1.
TABLE-US-00010 TABLE 1 Sample (cationic Molar peptide/nucleic acid)
Mass ratio N/P ratio ratio CR.sub.12C/R1180 1:2 0.9 44:1
CR.sub.12C/R1180 2:1 3.6 185:1 R.sub.12/R1180 1:2 0.7 48:1
R.sub.12/R1180 2:1 2.5 146:1 CR.sub.9C/R1180 2:1 0.9 55:1
R.sub.9/R1180 2:1 1.1 65:1 CR.sub.7C 1:2 0.8 70:1 R.sub.7 1:2 1.0
85:1 CR.sub.12C/CpG 1:2.5 4.9 8:1 CR.sub.12C/R491 1:2 0.9 150:1
CR.sub.12C/short GU-rich 1:2.5 4.9 8:1 CR.sub.12C/R722 5:1 9.6
444:1 CR.sub.12C/R722 4:1 7.6 355:1 CR.sub.12C/R722 3:1 5.7 266:1
CR.sub.12C/R722 2:1 3.8 177:1 CR.sub.12C/R722 1:1 1.9 88:1
CR.sub.12C/R722 1:2 0.9 44:1 CR.sub.12C/R722 1:3 0.6 29:1
CR.sub.12C/R722 1:4 0.5 22:1 CR.sub.12C/R722 1:5 0.4 17:1 N/P ratio
= is a measure of the ionic charge of the cationic component of the
polymeric carrier or of the polymeric carrier as such. In the case
that the cationic properties of the cationic component are provided
by nitrogen atoms the N/P ratio is the ratio of basic nitrogen
atoms to phosphate residues, considering that nitrogen atoms confer
to positive charges and phosphate of the phosphate backbone of the
nucleic acid confers to the negative charge.
[0807] N/P is preferably calculated by the following formula:
[0807] N / P = pmol [ RNA ] * ratio * cationic AS g RNA * 3 * 1000
##EQU00001## [0808] As an example the RNA R722 according to SEQ ID
NO: 122 was applied, which has a molecular weight of 186 kDa.
Therefore 1 .mu.g R722 RNA confers to 5.38 pmol RNA. 4. Cytokine
Stimulation in hPBMCs:
[0809] HPBMC cells from peripheral blood of healthy donors were
isolated using a Ficoll gradient and washed subsequently with
1.times.PBS (phophate-buffered saline). The cells were then seeded
on 96-well microtiter plates (200.times.10/well). The hPBMC cells
were incubated for 24 h with 10 pl of the polymeric carrier cargo
complex from Example 3 containing the indicated amount of nucleic
acid in X-VIVO 15 Medium (BioWhittaker). The immunostimulatory
effect was measured by detecting the cytokine production of the
hPBMCs (Tumour necrose factor alpha and Interferon alpha).
Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated
over night (o/n) with binding buffer (0.02% NaN.sub.3, 15 mM
Na.sub.2CO.sub.3, 15 mM NaHCO.sub.3, pH 9.7), additionally
containing a specific cytokine antibody. Cells were then blocked
with 1.times.PBS, containing 1% BSA (bovine serum albumin). The
cell supernatant was added and incubated for 4 h at 37.degree. C.
Subsequently, the microtiter plate was washed with 1.times.PBS,
containing 0.05% Tween-20 and then incubated with a Biotin-labelled
secondary antibody (BD Pharmingen, Heidelberg, Germany).
Streptavidin-coupled horseraddish peroxidase was added to the
plate. Then, the plate was again washed with 1.times.PBS,
containing 0.05% Tween-20 and ABTS
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added
as a substrate. The amount of cytokine was determined by measuring
the absorption at 405 nm (OD 405) using a standard curve with
recombinant cytokines (BD Pharmingen, Heidelberg, Germany) with the
Sunrise ELISA-Reader from Tecan (Crailsheim, Germany). The
respective results are shown in FIG. 3-9.
5. Zetapotential Measurements:
[0810] The Zeta potential of the polymeric carrier cargo complexes
was evaluated by the laser Doppler electrophoresis method using a
Zetasizer Nano (Malvern Instruments, Malvern, UK). The measurement
was performed at 25.degree. C. and a scattering angle of
173.degree. was used. The results are shown in FIG. 2.
6. Stability of Complexes after Lyophilization
[0811] The hydrodynamic diameters of polymeric carrier cargo
complexes as prepared above were measured by dynamic light
scattering using a Zetasizer Nano (Malvern Instruments, Malvern,
UK) according to the manufacturer's instructions. The measurements
were performed at 25.degree. C. in buffer analysed by a cumulant
method to obtain the hydrodynamic diameters and polydispersity
indices of the polymeric carrier cargo complexes. Polymeric carrier
cargo complexes were formed as indicated in Example 3 and the
hydrodynamic diameters were measured with fresh prepared complexes
and with reconstituted complexes after lyophilization. The
respective results of the experiment are shown in FIG. 1.
7. Immunization Experiments:
[0812] a) Immunization with Ovalbumine or SIINFEKL:
[0813] For immunization the vaccines Ovalbumine protein (OVA) (5
.mu.g) or Ovalbumin-specific peptide SIINFEKL (50 .mu.g) were
combined with the polymeric cargo complexes R722/CR.sub.12C (in a
ratio of 2:1 w/w) (30 .mu.g R722/15 .mu.g CR.sub.12C) as adjuvant
and injected intradermally into female C57BL/6 mice (7 mice per
group for tumour challenge and 5 mice per group for detection of an
immune response). The vaccination was repeated 2 times in 2 weeks.
For comparison mice were injected alone with the antigens.
[0814] b) Immunization with Rabies Vaccine:
[0815] For immunization the vaccine Rabipur.RTM. or HDC (both
comprise inactivated Rabies virus) (0.1, 0.01 and 0.001 fold human
dose) was combined with the polymeric cargo complexes
R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (30 .mu.g R722/8.1 .mu.g
CR.sub.12C) as adjuvant and injected intramuscularly into female
Balb/c mice (5 or 8 mice per group; as indicated). For comparison
mice were injected with Rabipur.RTM. or HDC alone.
[0816] c) Immunization with Influenza a(H1N1)Pdm09 (Swine Flu)
Vaccine:
[0817] For immunization the vaccine Pandemrix.RTM. or Celvapan.RTM.
(both comprise inactivated A(H1N1)pdm09 influenza virus) (0.1
.mu.g/dose) was combined with the polymeric cargo complexes
R722/CR.sub.12C (in a ratio of 2:1 w/w) (15 .mu.g R722/7.5 .mu.g
CR.sub.12C for Celvapan.RTM. and 30 .mu.g R722/15 .mu.g CR.sub.12C
for Pandemrix.RTM.) as adjuvant and injected intramuscularly into
female Balb/c mice (5 mice per group). For comparison mice were
injected with Pandemrix.RTM. or Celvapan.RTM. alone.
[0818] d) Immunization with Seasonal Influenza Vaccine:
[0819] For immunization the seasonal influenza vaccine
Begrivac.RTM. (comprises inactivated influenza virus strains as
recommended by the WHO; season 2009/2010) (0.1 .mu.g/dose) was
combined with the polymeric cargo complexes R722/CR.sub.12C (in a
ratio of 2:1 w/w) (30 .mu.g R722/15 .mu.g CR.sub.12C) as adjuvant
and injected intramuscularly into female Balb/c mice (8 mice per
group). For comparison mice were injected with Begrivac.RTM.
alone.
[0820] e) Immunization with Hepatitis B vaccine:
[0821] For immunization the Hepatitis B vaccine Engerix.RTM.-B
(comprises recombinant Hepatitis B surface antigen) (0.5
.mu.g/dose) was combined with the polymeric cargo complexes
R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (6.25 .mu.g R722/1.7
.mu.g CR.sub.12C) as adjuvant and injected intramuscularly into
female Balb/c mice (8 mice per group). For comparison mice were
injected with Engerix.RTM.-B alone.
[0822] Immunization with human papilloma virus 16 (HPV16)
E7-derived peptide:
[0823] For immunization the HPV16-derived peptide E7 aa43-77 (100
.mu.g/dose) was combined with the polymeric cargo complexes
R722/C.sub.12C (in a ratio of 2:1 w/w) (50 .mu.g R722/25 .mu.g
CR.sub.12C) as adjuvant and injected intradermally into female
C57BL/6 mice (5 mice per group). For comparison mice were injected
with peptide alone.
[0824] In a further experiment (FIG. 22) the HPV-derived peptide E7
aa43-77 (100 .mu.g/dose) was combined with the polymeric cargo
complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (50 .mu.g R722/25
.mu.g CR.sub.12C) during the polymerization step c) of the method
of preparing the polymeric carrier cargo complexed as defined
above. Therefore the HPV-derived peptide is part of the polymeric
carrier cargo complex and is indicated as E7
aa43-77/R722/CR.sub.12C. For comparision in this further
experiment, mice were injected with peptide alone (E7 aa43-77) and
the inventive pharmaceutical composition comprising the E7 aa43-77
peptide as antigen and the polymeric carrier cargo complex as
adjuvant, wherein the polymeric carrier cargo complex does not
comprise the antigen (E7 aa43-77+R722/CR.sub.12C).
[0825] g) Immunization with NY-ESO-1 protein:
[0826] For immunization the tumour antigen NY-ESO-1 protein (5
.mu.g/dose) was combined with the polymeric cargo complexes
R722/CR.sub.12C (in a ratio of 2:1 w/w) (30 .mu.g R722 15 .mu.g
CR.sub.12C) as adjuvant and injected 2 times within 15 days
intramuscularly into female C57BL/6 mice (5 mice per group). For
comparison mice were injected with protein alone.
8. Detection of an Antigen-Specific Immune Response (B-Cell Immune
Response):
[0827] a) Detection of Antibodies Directed Against Ovalbumine:
[0828] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting antigen specific
antibodies. Therefore, blood samples Were taken from vaccinated
mice 5 days after the last vaccination and sera were prepared.
MaxiSorb plates (Nalgene Nunc International) were coated with
Gallus gallus ovalbumine protein. After blocking with 1.times.PBS
containing 0.05% Tween-20 and 1% BSA the plates were incubated with
diluted mouse serum. Subsequently a biotin-coupled secondary
antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing,
the plate was incubated with Horseradish peroxidase-streptavidin
and subsequently the conversion of the ABTS substrate
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was
measured. The results of this induction of antibodies upon
vaccination with an inventive pharmaceutical composition are shown
in FIG. 11.
[0829] b) Detection of Antibodies Directed Against Rabies
Virus:
[0830] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting Rabies virus specific
total IgG antibodies. Therefore, blood samples were taken from
vaccinated mice 7 and 21 days after vaccination and sera were
prepared. MaxiSorb plates (Nalgene Nunc International) were coated
with the commercially available rabies vaccine containing
inactivated virus (HDC; 1:10000). After blocking with 1.times.PBS
containing 0.05% Tween-20 and 1% BSA the plates were incubated with
diluted mouse serum. Subsequently a Horseradish peroxidase-coupled
secondary antibody (Anti-mouse-IgG Pharmingen) was added. After
washing, the plate was developed using ABTS and subsequently the
conversion of the ABTS substrate
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was
measured. The results of this induction of antibodies upon
vaccination with an inventive pharmaceutical composition are shown
in FIG. 13.
[0831] c) Determination of the Affinity of Antibodies Directed
Against Rabies Virus:
[0832] Detection of the total IgG antibodies directed against
Rabies virus was carried out as disclosed under b) with the
differences that mouse sera were only tested at a dilution of 1:40.
Furthermore after incubation with the mouse serum the plates were
washed with an increasing concentration of urea (6, 7 and 8 M
urea). By washing with urea only antibodies with a high affinity to
the antigen can be detected. The results of this induction of
antibodies upon vaccination with an inventive pharmaceutical
composition are shown in FIG. 15.
[0833] d) Detection of Antibodies Directed Against A(H1N1)pdm09
Influenza Virus (Swine Flu):
[0834] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting A(H1N1)pdm09
influenza virus specific IgG2a antibodies. Therefore, blood samples
were taken from vaccinated mice 14 days after vaccination and sera
were prepared. MaxiSorb plates (Nalgene Nunc International) were
coated with Influenza A/California/7/09 .ANG.(H1N1)pdm09
inactivated virus (NIBSC, UK) (at 1 .mu.g/ml). After blocking with
1.times.PBS containing 0.05% Tween-20 and 1% BSA the plates were
incubated with diluted mouse serum. Subsequently a biotin-coupled
secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After
washing, the plate was incubated with Horseradish
peroxidase-streptavidin and subsequently the conversion of the ABTS
substrate (2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid)
was measured to determine the induction of IgG2a antibodies. The
results of this induction of antibodies upon vaccination with an
inventive pharmaceutical composition are shown in FIG. 17.
[0835] e) Detection of Antibodies Directed Against Seasonal
Influenza Virus Strains:
[0836] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting influenza virus
specific IgG2a antibodies. Therefore, blood samples were taken from
vaccinated mice 28 days after vaccination and sera were prepared.
MaxiSorb plates (Nalgene Nunc International) were coated with
Influvac 2009/10.RTM. (at 5 .mu.g/ml) containing the same viral
Influenza antigens as the Influenza vaccine used for vaccination.
After blocking with 1.times.PBS containing 0.05% Tween-20 and 1%
BSA the plates were incubated with diluted mouse serum.
Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a
Pharmingen) was added. After washing, the plate was incubated with
Horseradish peroxidase-streptavidin and subsequently the conversion
of the ABTS substrate
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was
measured to determine the induction of IgG2a antibodies. The
results of this induction of antibodies upon vaccination with an
inventive pharmaceutical composition are shown in FIG. 19.
[0837] f) Detection of Antibodies Directed Against Hepatitis B
Surface Antigen (HBsAg):
[0838] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting HBsAG specific IgG2a
antibodies. Therefore, blood samples were taken from vaccinated
mice 28 days after vaccination and sera were prepared. MaxiSorb
plates (Nalgene Nunc International) were coated with recombinant
Hepatitis B Surface Antigen (HBsAG) (Aldevron, USA) (1
.mu.g/ml).
[0839] After blocking with 1.times.PBS containing 0.05% Tween-20
and 1% BSA the plates were incubated with diluted mouse serum.
Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a
Pharmingen) was added. After washing, the plate was incubated with
Horseradish peroxidase-streptavidin and subsequently the conversion
of the ABTS substrate
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was
measured to determine the induction of IgG2a antibodies. The
results of this induction of antibodies upon vaccination with an
inventive pharmaceutical composition are shown in FIG. 20.
9. Detection of an Antigen Specific Cellular Immune Response by
ELISPOT:
[0840] a) Detection of Cytotoxic T Cell Response Directed Against
Ovalbumine:
[0841] 5 days after the last vaccination mice were sacrificed, the
spleens were removed and the splenocytes were isolated. For
detection of INFgamma a coat multiscreen plate (Millipore) was
incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat
Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3)
comprising antibody against INF.gamma. (BD Pharmingen, Heidelberg,
Germany). The next day 1.times.10.sup.6 cells/well were added and
re-stimulated with 1 .mu.g/well of relevant peptide (SIINFEKL of
ovalbumin); irrelevant peptide (Connexin=control peptide) or buffer
without peptide. Afterwards the cells are incubated for 24 h at
37.degree. C. The next day the plates were washed 3 times with PBS,
once with water and once with PBS/0.05% Tween-20 and afterwards
incubated with a biotin-coupled secondary antibody for 11-24 h at
4.degree. C. Then the plates were washed with PBS/0.05% Tween-20
and incubated for 2 h at room temperature with alkaline phosphatase
coupled to streptavidin in blocking buffer. After washing with
PBS/0.05% Tween-20 the substrate (5-Bromo-4-Cloro-3-Indolyl
Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma
Aldrich, Taufkirchen, Germany) was added to the plate and the
conversion of the substrate could be detected visually. The
reaction was then stopped by washing the plates with water. The
dried plates were then read out by an ELISPOT plate reader. For
visualization of the spot levels the numbers were corrected by
background subtraction. The results of this induction of specific
cytotoxic T-cells upon vaccination with an inventive pharmaceutical
composition are shown in FIG. 12.
[0842] b) Detection of a Cytotoxic T Cell Response Directed Against
Rabies Virus:
[0843] 5 days after vaccination mice were sacrificed, the spleens
were removed and the splenocytes were isolated. For detection of
INFgamma a coat multiscreen plate (Millipore) was incubated
overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH
9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising
antibody against INF.gamma. (BD Pharmingen, Heidelberg, Germany).
The next day 5.times.10.sup.5 cells/well were added and
re-stimulated with inactivated Rabies virus (Rabipur.RTM. 1:100 or
HDC 1:100)) or buffer without peptide (BSA). Afterwards the cells
are incubated for 24 h at 37.degree. C. The next day the plates
were washed 3 times with PBS, for 5 minutes with water and once
with PBS/0.05% Tween-20 and afterwards incubated with a
biotin-coupled secondary antibody for 11-24 h at 4.degree. C. Then
the plates were washed with PBS/0.05% Tween-20 and incubated for 2
h at room temperature with alkaline phosphatase coupled to
streptavidin in blocking buffer. After washing with PBS/0.05%
Tween-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro
Blue Tetrazolium Liquid Substrate System from Sigma Aldrich,
Taufkirchen, Germany) was added to the plate and the conversion of
the substrate could be detected visually. The reaction was then
stopped by washing the plates with water. The dried plates were
then read out by an ELISPOT plate reader. For visualization of the
spot levels the numbers were corrected by background subtraction.
The results of this induction of specific cytotoxic T-cells upon
vaccination with an inventive pharmaceutical composition are shown
in FIG. 14.
[0844] c) Detection of a Cytotoxic T Cell Response Directed Against
Swine Flu (A(H1N1)pdm09):
[0845] 6 days after vaccination mice were sacrificed, the spleens
were removed and the splenocytes were isolated. For detection of
INFgamma a coat multiscreen plate (Millipore) was incubated
overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH
9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising
antibody against INF.gamma. (BD Pharmingen, Heidelberg, Germany).
The next day 5.times.10.sup.5 cells/well were added and
re-stimulated with Influenza A/California/7/09 A(H1N1)pdm09
inactivated virus (NIBSC, UK) (10 .mu.g/ml?) or buffer without
peptide (BSA). Afterwards the cells are incubated for 24 h at
37.degree. C. The next day the plates were washed 3 times with PBS,
for 5 minutes with water and once with PBS/0.05% Tween-20 and
afterwards incubated with a biotin-coupled secondary antibody for
11-24 h at 4.degree. C. Then the plates were washed with PBS/0.05%
Tween-20 and incubated for 2 h at room temperature with alkaline
phosphatase coupled to streptavidin in blocking buffer. After
washing with PBS/0.05% Tween-20 the substrate
(5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid
Substrate System from Sigma Aldrich, Taufkirchen, Germany) was
added to the plate and the conversion of the substrate could be
detected visually. The reaction was then stopped by washing the
plates with water. The dried plates were then read out by an
ELISPOT plate reader. For visualization of the spot levels the
numbers were corrected by background subtraction. The results of
this induction of specific cytotoxic T-cells upon vaccination with
an inventive pharmaceutical composition are shown in FIG. 18.
[0846] d) Detection of a Cytotoxic T Cell Response Directed Against
E7 Protein of Human Papilloma Virus 16 (HPV16):
[0847] 8 days after vaccination mice were sacrificed, the spleens
were removed and the splenocytes were isolated. For detection of
INFgamma a coat multiscreen plate (Millipore) was incubated
overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH
9.6, 10.59 WI Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising
antibody against INF.gamma. (BD Pharmingen, Heidelberg, Germany).
The next day 5.times.10.sup.5 cells/well were added and
re-stimulated with different E7 derived peptides (E7 aa43-77,
E748-57, E7 aa49-57) (1 .mu.g/ml), an irrelevant peptide (LacZ
peptide H-2 Ld) or buffer without peptide (DMSO). Afterwards the
cells are incubated for 24 h at 37.degree. C. The next day the
plates were washed 3 times with PBS, for 5 minutes with water and
once with PBS/0.05% Tween-20 and afterwards incubated with a
biotin-coupled secondary antibody for 11-24 h at 4.degree. C. Then
the plates were washed with PBS/0.05% Tween-20 and incubated for 2
h at room temperature with alkaline phosphatase coupled to
streptavidin in blocking buffer. After washing with PBS/0.05%
Tween-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro
Blue Tetrazolium Liquid Substrate System from Sigma Aldrich,
Taufkirchen, Germany) was added to the plate and the conversion of
the substrate could be detected visually. The reaction was then
stopped by washing the plates with water. The dried plates were
then read out by an ELISPOT plate reader. For visualization of the
spot levels the numbers were corrected by background subtraction.
The results of this induction of specific cytotoxic T-cells upon
vaccination with an inventive pharmaceutical composition including
a peptide antigen from a pathogen associated with infectious
disease are shown in FIG. 21 for the E7aa43-77 peptide antigen not
included in the polymeric cargo complex, and additionally for the
E7aa43-77 peptide antigen when included in the polymeric cargo
complex in FIG. 22.
[0848] e) Detection of a Cytotoxic T Cell Response Directed Against
the Tumour Antigen NY-ESO-1:
[0849] 7 days after vaccination mice were sacrificed, the spleens
were removed and the splenocytes were isolated. For detection of
INFgamma a coat multiscreen plate (Millipore) was incubated
overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH
9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising
antibody against INF.gamma. (BD Pharmingen, Heidelberg, Germany).
The next day 1.times.10.sup.6 cells/well were added and
re-stimulated with an epitope library of NY-ESO-1 comprising
predicted MHC I and MHC II epitopes. Afterwards the cells are
incubated for 24 h at 37.degree. C. The next day the plates were
washed 3 times with PBS, once with water and once with PBS/0.05%
Tween-20 and afterwards incubated with a biotin-coupled secondary
antibody for 11-24 h at 4.degree. C. Then the plates were washed
with PBS/0.05% Tween-20 and incubated for 2 h at room temperature
with alkaline phosphatase coupled to streptavidin in blocking
buffer. After washing with PBS/0.05% Tween-20 the substrate
(5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid
Substrate System from Sigma Aldrich, Taufkirchen, Germany) was
added to the plate and the conversion of the substrate could be
detected visually. The reaction was then stopped by washing the
plates with water. The dried plates were then read out by an
ELISPOT plate reader. For visualization of the spot levels the
numbers were corrected by background subtraction. The results of
this induction of specific cytotoxic T-cells upon vaccination with
an inventive pharmaceutical composition including at least one
tumour antigen are shown in FIG. 23.
10. Tumour Challenge:
[0850] One week after the last vaccination 1.times.10.sup.6
E.G7-OVA cells (tumour cells which stably express ovalbumine) were
implanted subcutaneously in the vaccinated mice. Tumour growth was
monitored by measuring the tumour size in 3 dimensions using a
calliper. The results of the induction of an anti-tumoural response
upon vaccination with an inventive pharmaceutical composition are
shown in FIG. 10.
11. Virus Neutralization Test:
[0851] Detection of the virus neutralizing antibody response
(specific B-cell immune response) was carried out by the mean of
virus neutralisation assay. Therefore, blood samples were taken
from vaccinated mice 21 days after vaccination and sera were
prepared. These sera were used in fluorescent antibody virus
neutralisation (FAVN) test using the cell culture adapted challenge
virus strain (CVS) of rabies virus as recommended by the OIE (World
Organisation for Animal Health) and first described in Cliquet F.,
Aubert M. & Sagne L. (1998); J. Immunol. Methods, 212, 79-87.
Shortly, heat inactivated sera will be tested as quadruplicates in
serial two-fold dilutions as quadruplicates for there potential to
neutralise 100 TCID.sub.50 (tissue culture infectious doses 50%) of
CVS in 50 .mu.l of volume. Therefore sera dilutions are incubated
with virus for 1 hour at 37.degree. C. (in humid incubator with 5%
CO.sub.2) and subsequently trypsinized BHK-21 cells are added
(4.times.10.sup.5 cells/ml; 50 .mu.l per well). Infected cell
cultures are incubated for 48 hours in humid incubator at
37.degree. C. and 5% CO.sub.2. Infection of cells is analysed after
fixation of cells using 80% acetone at room temperature using FITC
anti-rabies conjugate. Plates were washed twice using PBS and
excess of PBS was removed. Cell cultures are scored positive or
negative for the presence of rabies virus. Negative scored cells in
sera treated wells represent neutralization of rabies virus. Each
FAVN tests includes WHO or OIE standard serum (positive reference
serum) that serves as reference for standardisation of the assay.
Neutralization activity of test sera is calculated with reference
to the standard serum and displayed as International Units/ml
(IU/ml). The results of this experiment are shown in FIG. 16.
12. Rabies Virus Challenge Infection of Mice:
[0852] 37 days after single intramuscular immunization of mice
using 0.001 fold human dose of Rabipur.RTM. and 3 .mu.g R722 and
0.81 .mu.g CR.sub.12C (3.7:1 w/w) all mice in the experiment were
infected using 25-fold LD.sub.50 of CVS strain of Rabies virus
intracranially (i.c.). Mice were monitored for specific symptoms of
Rabies disease and body weight development. The results of this
experiment are shown in FIG. 24.
13. Tumour Challenge with TC-1 Cells (Measurement of Tumour Growth
and Animal Survival in a Therapeutic Setting):
[0853] Eight C57BL/6 mice per group were challenged on day 1 with
1.times.10.sup.5 TC-1 cells which express the HPV E6 and E7
protein. Vaccination started on day 7 after tumor challenge (median
tumor volume 31-48 mm.sup.3). Mice were intradermally vaccinated 5
times (on day 8, 12, 15, 19 and 22) with 5 .mu.g or 50 .mu.g E7
peptide combined with 50 .mu.g CR.sub.12C/R722 (1:2; w/w). For
comparison, mice were injected with the polymeric cargo complexes
alone.
[0854] The polymeric carrier cargo complex combined with HPV-16
derived E7 peptide E7aa43-77 even impairs the growth of tumours
compared to the polymeric carrier cargo complex alone (FIG.
25).
[0855] The polymeric carrier cargo complex combined with HPV-16
derived E7 peptide strongly enhances the survival of tumor bearing
mice (Mean survival time of 44.5 days for 50 .mu.g E7 peptide+50
.mu.g polymeric carrier cargo complex; mean survival time of 22
days for 5 .mu.g E7 peptide+50 .mu.g polymeric carrier cargo
complex) compared to the E7 peptide or 50 polymeric carrier cargo
complex alone (FIG. 26).
14. Tumour Challenge with TC-1 Cells (Induction of a T Cell Memory
Response):
[0856] Thirteen C5713116 mice per group were intradermally
vaccinated once per week for four weeks (on days 0, 7, 14 and 21)
with the polymeric carrier cargo complex formed by the
disulfide-crosslinked cationic peptide CR12C as carrier and the
isRNA R722 as nucleic acid cargo and the E7 peptide.
[0857] Eight weeks after the fourth vaccination, 5 mice/group were
sacrificed, splenocytes were isolated and the frequency of
antigen-specific CD8.sup.+ T cells was determined by HPV-pentamer
staining and flow cytometry according to example 15.
[0858] The polymeric carrier cargo complex combined with the HPV-16
derived E7 peptide E7aa43-77 results in a statistically significant
increase of antigen-specific CD8.sup.+ T cells compared to mice
vaccinated with 50 .mu.g of the E7 peptide alone (p=0.0007 for 5
.mu.g E7 peptide and p=0.0002 50 .mu.g E7 peptide; statistical
differences between groups were assessed by unpaired t-test). Thus,
the combination of the polymeric carrier cargo complex combined
with the HPV-16 derived E7 peptide induces a potent memory
CD8.sup.+ T cell response (FIG. 27).
[0859] Eight weeks after the fourth vaccination 8 mice/group were
challenged with 1.times.10.sup.5 TC-1 tumor cells and tumor growth
was monitored.
[0860] The polymeric carrier cargo complex combined with the HPV-16
derived E7 peptide E7aa43-77 results in a drastic delay of tumor
growth (4 complete responses for 5 .mu.g E7 peptide+50 .mu.g of 50
.mu.g polymeric carrier cargo complex; 7 complete responders for 50
.mu.g E7 peptide+50 .mu.g of 50 .mu.g polymeric carrier cargo
complex). Thus, the combination of the polymeric carrier cargo
complex combined with the HPV-16 derived E7 peptide induces a
potent memory CD8.sup.+ T cell response (FIG. 28).
15. Detection of Antigen Specific Cellular Immune Responses by
Pentamer Staining:
[0861] Freshly isolated splenocytes were seeded into 96-well plates
(2.times.10.sup.6 cells/well) and stained with Fc-Block (1:100,
anti-CD16/CD32; BD Biosciences). After a 20 minute incubation, the
H-2Db-RAHYNIVTF-Pentamer (HPV 16 E7 49-57)-Pentamer-PE (10
.mu.l/well) was added and cells were incubated for an additional 30
minutes at 4.degree. C. After washing cells were stained with the
following antibodies: CD19-FITC (1:200), CD8-PerCP-Cy5.5 (1:200),
KLRG1-PECy7 (1:200), CD44-APC (1:100), CD127-eFluor450 (1:100)
(eBioscience) and CD3-APC-Cy7 (1:200) (BD Biosciences). Aqua Dye
was used to distinguish live/dead cells (Invitrogen). Cells were
collected using a Canto II flow cytometer (Beckton Dickinson). Flow
cytometry data were analysed using FlowJo software (Tree Star,
Inc.). Statistical analysis was performed using GraphPad Prism
software, Version 5.01. Statistical differences between groups were
assessed by unpaired t test with Welch's correction.
16. Immunization with Seasonal Influenza and Detection of
Antibodies:
[0862] For immunization the seasonal influenza vaccine
Mutagrip.RTM. (comprises inactivated influenza virus strains as
recommended by the WHO; season 2011/2012) (4.5, 0.45 and 0.045
.mu.g) was combined with the polymeric cargo complexes
R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (5 .mu.g R722/1.35 .mu.g
CR.sub.12C) as adjuvant and injected intramuscularly into female
Balb/c mice (8 mice per group). For comparison mice were injected
with Mutagrip.RTM. alone.
[0863] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting influenza virus
hemagglutinin inhibition (HI) titers. Therefore, blood samples were
taken from vaccinated mice 21 days after vaccination and sera were
heat inactivated, incubated with kaolin, and pre-adsorbed to
chicken red blood cells. For the HI assay, 50 .mu.l of 2-fold
dilutions of pre-treated sera were incubated with inactivated
influenza A/California/7/2009 H1N1 or influenza A/Victoria/210/2009
H3N2 (both NIBSC) and 50 pl 0.5% chicken red blood cells were
added. The results of this induction of HI titers upon vaccination
with an inventive pharmaceutical composition are shown in FIG.
29.
[0864] Detection of an antigen specific immune response (B-cell
immune response) was carried out by detecting influenza virus
specific IgG2a antibodies. Therefore, blood samples were taken from
vaccinated mice 21 days after vaccination and sera were prepared.
MaxiSorb plates (Nalgene Nunc International) were coated with
inactivated influenza A/California/7/2009 H1N1 (NIBSC, Potters Bar,
UK) at 1 .mu.g/ml. After blocking with 1.times.PBS containing 0.05%
Tween-20 and 1% BSA the plates were incubated with diluted mouse
serum. Subsequently, a biotin-coupled secondary antibody
(Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate
was incubated with Horseradish peroxidase-streptavidin and
subsequently the conversion of the ABTS substrate
(2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was
measured to determine the induction of IgG2a antibodies. The
results of this induction of antibodies upon vaccination with an
inventive pharmaceutical composition are shown in FIG. 30.
Sequence CWU 1
1
12319PRTartificialSynthetic peptide 1Cys Arg Arg Arg Arg Arg Arg
Arg Cys 1 5 210PRTartificialSynthetic peptide 2Cys Arg Arg Arg Arg
Arg Arg Arg Arg Cys 1 5 10 311PRTartificialSynthetic peptide 3Cys
Arg Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10
412PRTartificialSynthetic peptide 4Cys Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Cys 1 5 10 513PRTartificialSynthetic peptide 5Cys Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10
614PRTartificialSynthetic peptide 6Cys Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Cys 1 5 10 715PRTartificialSynthetic peptide
7Cys Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10
15 816PRTartificialSynthetic peptide 8Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10 15
917PRTartificialSynthetic peptide 9Cys Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Cys
1018PRTartificialSynthetic peptide 10Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Cys
1119PRTartificialSynthetic peptide 11Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Cys
1220PRTartificialSynthetic peptide 12Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Cys 20
1321PRTartificialSynthetic peptide 13Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Cys
20 1422PRTartificialSynthetic peptide 14Cys Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg
Cys 20 1520RNAartificialSynthetic oligonucleotide 15gguuuuuuuu
uuuuuuuggg 201620RNAartificialSynthetic oligonucleotide
16ggggguuuuu uuuuuggggg 201740RNAartificialSynthetic
oligonucleotide 17ggggguuuuu uuuuuuuuuu uuuuuuuuuu uuuuuggggg
401839RNAartificialSynthetic oligonucleotide 18gugugugugu
guuuuuuuuu uuuuuuugug ugugugugu 391939RNAartificialSynthetic
oligonucleotide 19gguugguugg uuuuuuuuuu uuuuuuuggu ugguugguu
392020RNAartificialSynthetic oligonucleotide 20gggggggggu
uugggggggg 202120RNAartificialSynthetic oligonucleotide
21gggggggguu uugggggggg 202220RNAartificialSynthetic
oligonucleotide 22ggggggguuu uuuggggggg
202320RNAartificialSynthetic oligonucleotide 23ggggggguuu
uuuugggggg 202420RNAartificialSynthetic oligonucleotide
24gggggguuuu uuuugggggg 202520RNAartificialSynthetic
oligonucleotide 25gggggguuuu uuuuuggggg
202620RNAartificialSynthetic oligonucleotide 26gggggguuuu
uuuuuugggg 202720RNAartificialSynthetic oligonucleotide
27ggggguuuuu uuuuuugggg 202820RNAartificialSynthetic
oligonucleotide 28ggggguuuuu uuuuuuuggg
202920RNAartificialSynthetic oligonucleotide 29gggguuuuuu
uuuuuuuggg 203020RNAartificialSynthetic oligonucleotide
30gggguuuuuu uuuuuuuugg 203120RNAartificialSynthetic
oligonucleotide 31gguuuuuuuu uuuuuuuugg
203220RNAartificialSynthetic oligonucleotide 32guuuuuuuuu
uuuuuuuuug 203322RNAartificialSynthetic oligonucleotide
33gggggggggg uuuggggggg gg 223422RNAartificialSynthetic
oligonucleotide 34gggggggggu uuuggggggg gg
223522RNAartificialSynthetic oligonucleotide 35gggggggguu
uuuugggggg gg 223622RNAartificialSynthetic oligonucleotide
36gggggggguu uuuuuggggg gg 223722RNAartificialSynthetic
oligonucleotide 37ggggggguuu uuuuuggggg gg
223822RNAartificialSynthetic oligonucleotide 38ggggggguuu
uuuuuugggg gg 223922RNAartificialSynthetic oligonucleotide
39ggggggguuu uuuuuuuggg gg 224022RNAartificialSynthetic
oligonucleotide 40gggggguuuu uuuuuuuggg gg
224122RNAartificialSynthetic oligonucleotide 41gggggguuuu
uuuuuuuugg gg 224222RNAartificialSynthetic oligonucleotide
42ggggguuuuu uuuuuuuugg gg 224322RNAartificialSynthetic
oligonucleotide 43ggggguuuuu uuuuuuuuug gg
224422RNAartificialSynthetic oligonucleotide 44ggguuuuuuu
uuuuuuuuug gg 224522RNAartificialSynthetic oligonucleotide
45gguuuuuuuu uuuuuuuuuu gg 224624RNAartificialSynthetic
oligonucleotide 46gggggggggg guuugggggg gggg
244724RNAartificialSynthetic oligonucleotide 47gggggggggg
uuuugggggg gggg 244824RNAartificialSynthetic oligonucleotide
48gggggggggu uuuuuggggg gggg 244924RNAartificialSynthetic
oligonucleotide 49gggggggggu uuuuuugggg gggg
245024RNAartificialSynthetic oligonucleotide 50gggggggguu
uuuuuugggg gggg 245124RNAartificialSynthetic oligonucleotide
51gggggggguu uuuuuuuggg gggg 245224RNAartificialSynthetic
oligonucleotide 52gggggggguu uuuuuuuugg gggg
245324RNAartificialSynthetic oligonucleotide 53ggggggguuu
uuuuuuuugg gggg 245424RNAartificialSynthetic oligonucleotide
54ggggggguuu uuuuuuuuug gggg 245523RNAartificialSynthetic
oligonucleotide 55gggggguuuu uuuuuuuuug ggg
235624RNAartificialSynthetic oligonucleotide 56gggggguuuu
uuuuuuuuuu gggg 245724RNAartificialSynthetic oligonucleotide
57gggguuuuuu uuuuuuuuuu gggg 245824RNAartificialSynthetic
oligonucleotide 58ggguuuuuuu uuuuuuuuuu uggg
245932RNAartificialSynthetic oligonucleotide 59guuuuuuuuu
uuuuuuuuuu uuuuuuuuuu ug 326034RNAartificialSynthetic
oligonucleotide 60gguuuuuuuu uuuuuuuuuu uuuuuuuuuu uugg
346136RNAartificialSynthetic oligonucleotide 61ggguuuuuuu
uuuuuuuuuu uuuuuuuuuu uuuggg 366237RNAartificialSynthetic
oligonucleotide 62gggguuuuuu uuuuuuuuuu uuuuuuuuuu uuuuggg
376339RNAartificialSynthetic oligonucleotide 63ggggguuuuu
uuuuuuuuuu uuuuuuuuuu uuuuugggg 396441RNAartificialSynthetic
oligonucleotide 64gggggguuuu uuuuuuuuuu uuuuuuuuuu uuuuuugggg g
416543RNAartificialSynthetic oligonucleotide 65ggggggguuu
uuuuuuuuuu uuuuuuuuuu uuuuuuuggg ggg 436645RNAartificialSynthetic
oligonucleotide 66gggggggguu uuuuuuuuuu uuuuuuuuuu uuuuuuuugg ggggg
456747RNAartificialSynthetic oligonucleotide 67gggggggggu
uuuuuuuuuu uuuuuuuuuu uuuuuuuuug ggggggg
47687RNAartificialSynthetic oligonucleotide 68gguuugg
7698RNAartificialSynthetic oligonucleotide 69gguuuugg
8709RNAartificialSynthetic oligonucleotide 70gguuuuugg
97110RNAartificialSynthetic oligonucleotide 71gguuuuuugg
107211RNAartificialSynthetic oligonucleotide 72gguuuuuuug g
117312RNAartificialSynthetic oligonucleotide 73gguuuuuuuu gg
127413RNAartificialSynthetic oligonucleotide 74gguuuuuuuu ugg
137514RNAartificialSynthetic oligonucleotide 75gguuuuuuuu uugg
147615RNAartificialSynthetic oligonucleotide 76gguuuuuuuu uuugg
157716RNAartificialSynthetic oligonucleotide 77gguuuuuuuu uuuugg
167817RNAartificialSynthetic oligonucleotide 78gguuuuuuuu uuuuugg
177918RNAartificialSynthetic oligonucleotide 79gguuuuuuuu uuuuuugg
188019RNAartificialSynthetic oligonucleotide 80gguuuuuuuu uuuuuuugg
19819RNAartificialSynthetic oligonucleotide 81ggguuuggg
98210RNAartificialSynthetic oligonucleotide 82ggguuuuggg
108311RNAartificialSynthetic oligonucleotide 83ggguuuuugg g
118412RNAartificialSynthetic oligonucleotide 84ggguuuuuug gg
128513RNAartificialSynthetic oligonucleotide 85ggguuuuuuu ggg
138614RNAartificialSynthetic oligonucleotide 86ggguuuuuuu uggg
148715RNAartificialSynthetic oligonucleotide 87ggguuuuuuu uuggg
158816RNAartificialSynthetic oligonucleotide 88ggguuuuuuu uuuggg
168917RNAartificialSynthetic oligonucleotide 89ggguuuuuuu uuuuggg
179018RNAartificialSynthetic oligonucleotide 90ggguuuuuuu uuuuuggg
189119RNAartificialSynthetic oligonucleotide 91ggguuuuuuu uuuuuuggg
199257RNAartificialSynthetic oligonucleotide 92ggguuuuuuu
uuuuuuuugg guuuuuuuuu uuuuuugggu uuuuuuuuuu uuuuggg
579342RNAartificialSynthetic oligonucleotide 93ggguuuuuuu
uuuuuuuugg gggguuuuuu uuuuuuuuug gg 429451RNAartificialSynthetic
oligonucleotide 94ggguuugggu uuggguuugg guuuggguuu ggguuugggu
uuggguuugg g 519520RNAartificialSynthetic oligonucleotide
95gguuuuuuuu uuuuuuuggg 209657RNAartificialSynthetic
oligonucleotide 96cccuuuuuuu uuuuuuuucc cuuuuuuuuu uuuuuucccu
uuuuuuuuuu uuuuccc 579751RNAartificialSynthetic oligonucleotide
97cccuuucccu uucccuuucc cuuucccuuu cccuuucccu uucccuuucc c
519842RNAartificialSynthetic oligonucleotide 98cccuuuuuuu
uuuuuuuucc ccccuuuuuu uuuuuuuuuc cc 429960RNAartificialSynthetic
oligonucleotide 99uagcgaagcu cuuggaccua gguuuuuuuu uuuuuuuggg
ugcguuccua gaaguacacg 60100120RNAartificialSynthetic
oligonucleotide 100uagcgaagcu cuuggaccua gguuuuuuuu uuuuuuuggg
ugcguuccua gaaguacacg 60aucgcuucga gaaccuggau ccaaaaaaaa aaaaaaaccc
acgcaaggau cuucaugugc 120101229RNAartificialSynthetic
oligonucleotide 101gggagaaagc ucaagcuugg agcaaugccc gcacauugag
gaaaccgagu ugcauaucuc 60agaguauugg cccccgugua gguuauucuu gacagacagu
ggagcuuauu cacucccagg 120auccgagucg cauacuacgg uacuggugac
agaccuaggu cgucaguuga ccaguccgcc 180acuagacgug aguccgucaa
agcaguuaga uguuacacuc uauuagauc 229102547RNAartificialSynthetic
oligonucleotide 102gggagaaagc ucaagcuugg agcaaugccc gcacauugag
gaaaccgagu ugcauaucuc 60agaguauugg cccccgugua gguuauucuu gacagacagu
ggagcuuauu cacucccagg 120auccgagucg cauacuacgg uacuggugac
agaccuaggu cgucaguuga ccaguccgcc 180acuagacgug aguccgucaa
agcaguuaga uguuacacuc uauuagaucu cggauuacag 240cuggaaggag
caggaguagu guucuugcuc uaaguaccga gugugcccaa uacccgauca
300gcuuauuaac gaacggcucc uccucuuaga cugcagcgua agugcggaau
cuggggauca 360aauuacugac ugccuggauu acccucggac auauaaccuu
guagcacgcu guugcuguau 420aggugaccaa cgcccacucg aguagaccag
cucucuuagu ccggacaaug auaggaggcg 480cggucaaucu acuucuggcu
aguuaagaau aggcugcacc gaccucuaua aguagcgugu 540ccucuag
5471031083RNAartificialSynthetic oligonucleotide 103gggagaaagc
ucaagcuugg agcaaugccc gcacauugag gaaaccgagu ugcauaucuc 60agaguauugg
cccccgugua gguuauucuu gacagacagu ggagcuuauu cacucccagg
120auccgagucg cauacuacgg uacuggugac agaccuaggu cgucaguuga
ccaguccgcc 180acuagacgug aguccgucaa agcaguuaga uguuacacuc
uauuagaucu cggauuacag 240cuggaaggag caggaguagu guucuugcuc
uaaguaccga gugugcccaa uacccgauca 300gcuuauuaac gaacggcucc
uccucuuaga cugcagcgua agugcggaau cuggggauca 360aauuacugac
ugccuggauu acccucggac auauaaccuu guagcacgcu guugcuguau
420aggugaccaa cgcccacucg aguagaccag cucucuuagu ccggacaaug
auaggaggcg 480cggucaaucu acuucuggcu aguuaagaau aggcugcacc
gaccucuaua aguagcgugu 540ccucuagagc uacgcagguu cgcaauaaaa
gcguugauua gugugcauag aacagaccuc 600uuauucggug aaacgccaga
augcuaaauu ccaauaacuc uucccaaaac gcguacggcc 660gaagacgcgc
gcuuaucuug uguacguucu cgcacaugga agaaucagcg ggcauggugg
720uagggcaaua ggggagcugg guagcagcga aaaagggccc cugcgcacgu
agcuucgcug 780uucgucugaa acaacccggc auccguugua gcgaucccgu
uaucaguguu auucuugugc 840gcacuaagau ucauggugua gucgacaaua
acagcgucuu ggcagauucu ggucacgugc 900ccuaugcccg ggcuugugcc
ucucaggugc acagcgauac uuaaagccuu caagguacuc 960gacgugggua
ccgauucgug acacuuccua agauuauucc acuguguuag ccccgcaccg
1020ccgaccuaaa cugguccaau guauacgcau ucgcugagcg gaucgauaau
aaaagcuuga 1080auu 1083104229RNAartificialSynthetic oligonucleotide
104gggagaaagc ucaagcuuau ccaaguaggc uggucaccug uacaacguag
ccgguauuuu 60uuuuuuuuuu uuuuuuuuga ccgucucaag guccaaguua gucugccuau
aaaggugcgg 120auccacagcu gaugaaagac uugugcggua cgguuaaucu
ccccuuuuuu uuuuuuuuuu 180uuuuuaguaa augcgucuac ugaauccagc
gaugaugcug gcccagauc 229105546RNAartificialSynthetic
oligonucleotide 105gggagaaagc ucaagcuuau ccaaguaggc uggucaccug
uacaacguag ccgguauuuu 60uuuuuuuuuu uuuuuuuuga ccgucucaag guccaaguua
gucugccuau aaaggugcgg 120auccacagcu gaugaaagac uugugcggua
cgguuaaucu ccccuuuuuu uuuuuuuuuu 180uuuuuaguaa augcgucuac
ugaauccagc gaugaugcug gcccagaucu ucgaccacaa 240gugcauauag
uagucaucga gggucgccuu uuuuuuuuuu uuuuuuuuuu uggcccaguu
300cugagacuuc gcuagagacu acaguuacag cugcaguagu aaccacugcg
gcuauugcag 360gaaaucccgu ucagguuuuu uuuuuuuuuu uuuuuuccgc
ucacuaugau uaagaaccag 420guggaguguc acugcucucg aggucucacg
agagcgcucg auacaguccu uggaagaauc 480uuuuuuuuuu uuuuuuuuuu
uugugcgacg aucacagaga acuucuauuc augcaggucu 540gcucua
5461061083RNAartificialSynthetic oligonucleotide 106gggagaaagc
ucaagcuuau ccaaguaggc uggucaccug uacaacguag ccgguauuuu 60uuuuuuuuuu
uuuuuuuuga ccgucucaag guccaaguua gucugccuau aaaggugcgg
120auccacagcu gaugaaagac uugugcggua cgguuaaucu ccccuuuuuu
uuuuuuuuuu 180uuuuuaguaa augcgucuac ugaauccagc gaugaugcug
gcccagaucu ucgaccacaa 240gugcauauag uagucaucga gggucgccuu
uuuuuuuuuu uuuuuuuuuu uggcccaguu 300cugagacuuc gcuagagacu
acaguuacag cugcaguagu aaccacugcg gcuauugcag 360gaaaucccgu
ucagguuuuu uuuuuuuuuu uuuuuuccgc ucacuaugau uaagaaccag
420guggaguguc acugcucucg aggucucacg agagcgcucg auacaguccu
uggaagaauc 480uuuuuuuuuu uuuuuuuuuu uugugcgacg aucacagaga
acuucuauuc augcaggucu 540gcucuagaac gaacugaccu gacgccugaa
cuuaugagcg ugcguauuuu uuuuuuuuuu 600uuuuuuuuuc cucccaacaa
augucgauca auagcugggc uguuggagac gcgucagcaa 660augccguggc
uccauaggac guguagacuu cuauuuuuuu uuuuuuuuuu uuuucccggg
720accacaaaua auauucuugc uugguugggc gcaagggccc cguaucaggu
cauaaacggg 780uacauguugc acaggcuccu uuuuuuuuuu uuuuuuuuuu
uucgcugagu uauuccgguc 840ucaaaagacg gcagacguca gucgacaaca
cggucuaaag cagugcuaca aucugccgug 900uucguguuuu uuuuuuuuuu
uuuuuuguga accuacacgg cgugcacugu aguucgcaau 960ucauagggua
ccggcucaga
guuaugccuu gguugaaaac ugcccagcau acuuuuuuuu 1020uuuuuuuuuu
uucauauucc caugcuaagc aagggaugcc gcgagucaug uuaagcuuga 1080auu
108310759RNAartificialSynthetic oligonucleotide 107uagcgaagcu
cuuggaccua ccuuuuuuuu uuuuuucccu gcguuccuag aaguacacg
59108120RNAartificialSynthetic oligonucleotide 108uagcgaagcu
cuuggaccua ccuuuuuuuu uuuuuuuccc ugcguuccua gaaguacacg 60aucgcuucga
gaaccuggau ggaaaaaaaa aaaaaaaggg acgcaaggau cuucaugugc
1201097PRTartificialSynthetic peptide 109Arg Arg Arg Arg Arg Arg
Arg 1 5 1109PRTartificialSynthetic peptide 110Arg Arg Arg Arg Arg
Arg Arg Arg Arg 1 5 11112PRTartificialSynthetic peptide 111Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 1121845RNAArtificial
SequenceSynthetic oligonucleotide 112gggagaaagc uugaggaugg
aggacgccaa gaacaucaag aagggcccgg cgcccuucua 60cccgcuggag gacgggaccg
ccggcgagca gcuccacaag gccaugaagc gguacgcccu 120ggugccgggc
acgaucgccu ucaccgacgc ccacaucgag gucgacauca ccuacgcgga
180guacuucgag augagcgugc gccuggccga ggccaugaag cgguacggcc
ugaacaccaa 240ccaccggauc guggugugcu cggagaacag ccugcaguuc
uucaugccgg ugcugggcgc 300ccucuucauc ggcguggccg ucgccccggc
gaacgacauc uacaacgagc gggagcugcu 360gaacagcaug gggaucagcc
agccgaccgu gguguucgug agcaagaagg gccugcagaa 420gauccugaac
gugcagaaga agcugcccau cauccagaag aucaucauca uggacagcaa
480gaccgacuac cagggcuucc agucgaugua cacguucgug accagccacc
ucccgccggg 540cuucaacgag uacgacuucg ucccggagag cuucgaccgg
gacaagacca ucgcccugau 600caugaacagc agcggcagca ccggccugcc
gaagggggug gcccugccgc accggaccgc 660cugcgugcgc uucucgcacg
cccgggaccc caucuucggc aaccagauca ucccggacac 720cgccauccug
agcguggugc cguuccacca cggcuucggc auguucacga cccugggcua
780ccucaucugc ggcuuccggg ugguccugau guaccgguuc gaggaggagc
uguuccugcg 840gagccugcag gacuacaaga uccagagcgc gcugcucgug
ccgacccugu ucagcuucuu 900cgccaagagc acccugaucg acaaguacga
ccugucgaac cugcacgaga ucgccagcgg 960gggcgccccg cugagcaagg
aggugggcga ggccguggcc aagcgguucc accucccggg 1020cauccgccag
ggcuacggcc ugaccgagac cacgagcgcg auccugauca cccccgaggg
1080ggacgacaag ccgggcgccg ugggcaaggu ggucccguuc uucgaggcca
agguggugga 1140ccuggacacc ggcaagaccc ugggcgugaa ccagcggggc
gagcugugcg ugcgggggcc 1200gaugaucaug agcggcuacg ugaacaaccc
ggaggccacc aacgcccuca ucgacaagga 1260cggcuggcug cacagcggcg
acaucgccua cugggacgag gacgagcacu ucuucaucgu 1320cgaccggcug
aagucgcuga ucaaguacaa gggcuaccag guggcgccgg ccgagcugga
1380gagcauccug cuccagcacc ccaacaucuu cgacgccggc guggccgggc
ugccggacga 1440cgacgccggc gagcugccgg ccgcgguggu ggugcuggag
cacggcaaga ccaugacgga 1500gaaggagauc gucgacuacg uggccagcca
ggugaccacc gccaagaagc ugcggggcgg 1560cgugguguuc guggacgagg
ucccgaaggg ccugaccggg aagcucgacg cccggaagau 1620ccgcgagauc
cugaucaagg ccaagaaggg cggcaagauc gccguguaag acuaguuaua
1680agacugacua gcccgauggg ccucccaacg ggcccuccuc cccuccuugc
accgagauua 1740auaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1800aaaaaauauu cccccccccc cccccccccc
cccccccccc ucuag 18451131857RNAArtificial SequenceSynthetic
oligonucleotide 113gggagaaagc uugaggaugg aggacgccaa gaacaucaag
aagggcccgg cgcccuucua 60cccgcuggag gacgggaccg ccggcgagca gcuccacaag
gccaugaagc gguacgcccu 120ggugccgggc acgaucgccu ucaccgacgc
ccacaucgag gucgacauca ccuacgcgga 180guacuucgag augagcgugc
gccuggccga ggccaugaag cgguacggcc ugaacaccaa 240ccaccggauc
guggugugcu cggagaacag ccugcaguuc uucaugccgg ugcugggcgc
300ccucuucauc ggcguggccg ucgccccggc gaacgacauc uacaacgagc
gggagcugcu 360gaacagcaug gggaucagcc agccgaccgu gguguucgug
agcaagaagg gccugcagaa 420gauccugaac gugcagaaga agcugcccau
cauccagaag aucaucauca uggacagcaa 480gaccgacuac cagggcuucc
agucgaugua cacguucgug accagccacc ucccgccggg 540cuucaacgag
uacgacuucg ucccggagag cuucgaccgg gacaagacca ucgcccugau
600caugaacagc agcggcagca ccggccugcc gaagggggug gcccugccgc
accggaccgc 660cugcgugcgc uucucgcacg cccgggaccc caucuucggc
aaccagauca ucccggacac 720cgccauccug agcguggugc cguuccacca
cggcuucggc auguucacga cccugggcua 780ccucaucugc ggcuuccggg
ugguccugau guaccgguuc gaggaggagc uguuccugcg 840gagccugcag
gacuacaaga uccagagcgc gcugcucgug ccgacccugu ucagcuucuu
900cgccaagagc acccugaucg acaaguacga ccugucgaac cugcacgaga
ucgccagcgg 960gggcgccccg cugagcaagg aggugggcga ggccguggcc
aagcgguucc accucccggg 1020cauccgccag ggcuacggcc ugaccgagac
cacgagcgcg auccugauca cccccgaggg 1080ggacgacaag ccgggcgccg
ugggcaaggu ggucccguuc uucgaggcca agguggugga 1140ccuggacacc
ggcaagaccc ugggcgugaa ccagcggggc gagcugugcg ugcgggggcc
1200gaugaucaug agcggcuacg ugaacaaccc ggaggccacc aacgcccuca
ucgacaagga 1260cggcuggcug cacagcggcg acaucgccua cugggacgag
gacgagcacu ucuucaucgu 1320cgaccggcug aagucgcuga ucaaguacaa
gggcuaccag guggcgccgg ccgagcugga 1380gagcauccug cuccagcacc
ccaacaucuu cgacgccggc guggccgggc ugccggacga 1440cgacgccggc
gagcugccgg ccgcgguggu ggugcuggag cacggcaaga ccaugacgga
1500gaaggagauc gucgacuacg uggccagcca ggugaccacc gccaagaagc
ugcggggcgg 1560cgugguguuc guggacgagg ucccgaaggg ccugaccggg
aagcucgacg cccggaagau 1620ccgcgagauc cugaucaagg ccaagaaggg
cggcaagauc gccguguaag acuaguuaua 1680agacugacua gcccgauggg
ccucccaacg ggcccuccuc cccuccuugc accgagauua 1740auaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1800aaaaaauauu cccccccccc cccccccccc cccccccccc ucuagacaau uggaauu
185711420DNAArtificial SequenceSynthetic oligonucleotide
114gggggacgat cgtcgggggg 2011520RNAArtificial SequenceSynthetic
oligonucleotide 115gguuuuuuuu uuuuuuuggg
201168PRTartificialSynthetic peptide 116Ser Ile Ile Asn Phe Glu Lys
Leu 1 5 117386PRTGallus gallus 117Met Gly Ser Ile Gly Ala Ala Ser
Met Glu Phe Cys Phe Asp Val Phe 1 5 10 15 Lys Glu Leu Lys Val His
His Ala Asn Glu Asn Ile Phe Tyr Cys Pro 20 25 30 Ile Ala Ile Met
Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 35 40 45 Ser Thr
Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro 50 55 60
Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val 65
70 75 80 His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro
Asn Asp 85 90 95 Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala
Glu Glu Arg Tyr 100 105 110 Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val
Lys Glu Leu Tyr Arg Gly 115 120 125 Gly Leu Glu Pro Ile Asn Phe Gln
Thr Ala Ala Asp Gln Ala Arg Glu 130 135 140 Leu Ile Asn Ser Trp Val
Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn 145 150 155 160 Val Leu Gln
Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val 165 170 175 Asn
Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu 180 185
190 Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro
195 200 205 Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser
Met Ala 210 215 220 Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala
Ser Gly Thr Met 225 230 235 240 Ser Met Leu Val Leu Leu Pro Asp Glu
Val Ser Gly Leu Glu Gln Leu 245 250 255 Glu Ser Ile Ile Asn Phe Glu
Lys Leu Thr Glu Trp Thr Ser Ser Asn 260 265 270 Val Met Glu Glu Arg
Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 275 280 285 Glu Glu Lys
Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 290 295 300 Asp
Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu 305 310
315 320 Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
Asn 325 330 335 Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly
Val Asp Ala 340 345 350 Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His
Pro Phe Leu Phe Cys 355 360 365 Ile Lys His Ile Ala Thr Asn Ala Val
Leu Phe Phe Gly Arg Cys Val 370 375 380 Ser Pro 385
11835PRTartificialSynthetic peptide 118Gly Gln Ala Glu Pro Asp Arg
Ala His Tyr Asn Ile Val Thr Phe Cys 1 5 10 15 Cys Lys Cys Asp Ser
Thr Leu Arg Leu Cys Val Gln Ser Thr His Val 20 25 30 Asp Ile Arg 35
11910PRTartificialSynthetic peptide 119Asp Arg Ala His Tyr Asn Ile
Val Thr Phe 1 5 10 1209PRTartificialSynthetic peptide 120Arg Ala
His Tyr Asn Ile Val Thr Phe 1 5 121180PRTartificialSynthetic
peptide 121Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp
Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly
Gly Asn Ala Gly 20 25 30 Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly
Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro
Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser
Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 Arg Gly Pro Glu
Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 Ala Thr
Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110
Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115
120 125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg
Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser
Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe
Leu Ala Gln Pro Pro Ser 165 170 175 Gly Gln Arg Arg 180
122547RNAartificialSynthetic oligonucleotide 122gggagaaagc
ucaagcuuau ccaaguaggc uggucaccug uacaacguag ccgguauuuu 60uuuuuuuuuu
uuuuuuuuga ccgucucaag guccaaguua gucugccuau aaaggugcgg
120auccacagcu gaugaaagac uugugcggua cgguuaaucu ccccuuuuuu
uuuuuuuuuu 180uuuuuaguaa augcgucuac ugaauccagc gaugaugcug
gcccagaucu ucgaccacaa 240gugcauauag uagucaucga gggucgccuu
uuuuuuuuuu uuuuuuuuuu uggcccaguu 300cugagacuuc gcuagagacu
acaguuacag cugcaguagu aaccacugcg gcuauugcag 360gaaaucccgu
ucagguuuuu uuuuuuuuuu uuuuuuccgc ucacuaugau uaagaaccag
420guggaguguc acugcucucg aggucucacg agagcgcucg auacaguccu
uggaagaauc 480uuuuuuuuuu uuuuuuuuuu uugugcgacg aucacagaga
acuucuauuc augcaggucu 540gcucuag 54712320DNAartificialSynthetic
oligonucleotide 123tccatgacgt tcctgacgtt 20
* * * * *