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Patent 2433794 Summary

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(12) Patent Application: (11) CA 2433794
(54) English Title: USES FOR POLYCATIONIC COMPOUNDS AS VACCINE ADJUVANTS
(54) French Title: UTILISATIONS DE COMPOSES POLYCATIONIQUES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 39/00 (2006.01)
  • A61K 31/7084 (2006.01)
  • A61K 31/7125 (2006.01)
  • A61K 39/002 (2006.01)
  • A61K 39/02 (2006.01)
  • A61K 39/12 (2006.01)
  • A61K 39/39 (2006.01)
  • A61K 47/48 (2006.01)
  • A61P 35/00 (2006.01)
  • A61P 37/00 (2006.01)
(72) Inventors :
  • LINGNAU, KAREN (Austria)
  • MATTNER, FRANK (Austria)
  • SCHMIDT, WALTER (Austria)
  • BUSCHLE, MICHAEL (Austria)
(73) Owners :
  • INTERCELL AG (Austria)
(71) Applicants :
  • INTERCELL AG (Austria)
(74) Agent: FETHERSTONHAUGH & CO.
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2002-01-07
(87) Open to Public Inspection: 2002-07-11
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/EP2002/000062
(87) International Publication Number: WO2002/053184
(85) National Entry: 2003-07-04

(30) Application Priority Data:
Application No. Country/Territory Date
PCT/EP01/00087 European Patent Office (EPO) 2001-01-05
A 672/2001 Austria 2001-04-25

Abstracts

English Abstract




The invention relates to the use of a polycationic compound for the
preparation of a medicament with retarded in vivo release.


French Abstract

La présente invention concerne l'utilisation d'un composé polycationique pour la préparation d'un médicament à libération in vivo retardée.

Claims

Note: Claims are shown in the official language in which they were submitted.



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Claims:

1. Use of a polycationic compound for the preparation of a
subcutaneously, intramuscularily, intra- or transdermally admin-
istered vaccine, comprising an antigen, with retarded in.vivo
release of said antigen.

2. Use according to claim 1, characterised in that said antigen
is an antigen with side effects due to the distribution of this
antigen throughout the body of an individual, if applied without
the polycationic compound.

3. Use of a polycationic compound for the preparation of a sub-
cutaneously, intramuscularily, intra- or transdermally admin-
istered vaccine for the induction of a depot effect.

4. Use according to any one of claims 1 to 3, characterized in
that said antigen is selected from the group consisting of an
antigen from a viral or a bacterial pathogen, an antigen from an
eukaryotic pathogen, a tumor antigen, an autoimmune antigen or
mixtures thereof.

5. Use according to claims 1 to 4, characterized in that said
polycationic compound is a polycationic peptide, preferably a
basic polypeptide, an organic polycation comprising peptide
bonds or mixtures thereof.

6. Use according to any one of claims 1 to 5, characterized in
that said polycationic compound is polylysine, polyarginine, a
polypeptide containing more than 50 % of basic amino acid
residues in a range of more than 5, especially more than 10,
amino acid residues or mixtures thereof.

7. Use according to any one of claims 1 to 5, characterized in
that said polycationic compound is a synthetic peptide contain-
ing at least two KLK-motifs separated by a linker of 3 to 7 hy-
drophobic amino acids.

8. Use according to any one of claims 1 to 7, characterized in
that said vaccine further comprises a compound with an inflam-



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matory potential.

9. Use according to any one of claims 1 to 8, characterized in
that said vaccine further comprises a compound with a pharmaco-
logical half-time at the site of administration of below 10
minutes, preferably below 5 minutes, especially below 1 minute.

10. Use according to any one of claims 1 to 9, characterized in
that said vaccine further comprises immunogenic nucleic acid mo-
lecules.

11. Use according to any one of claims 1 to 10,.characterized in
that said vaccine further comprises inosine containing ODNs (I-
ODNS).

12. Use according to any one of claims 1 to 11, characterized in
that said vaccine is a locally acting vaccine.

13. Use according to any one of claims 1 to 12, characterized in
that said vaccine further comprises an active substance, said
active substance having an affinity to said polycationic com-
pound.


Description

Note: Descriptions are shown in the official language in which they were submitted.



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Uses for Polycationic Compounds
The invention relates to new uses for polycationic compounds.
Pharmaceutically used polycationic compounds, for example the
polycationic amino acid polymers poly-L-arginine and poly-L-ly-
sine, have been shown to allow very efficient charging of antigen
presenting cells (APCs) with antigens in vitro and in vivo. This
is thought to be the key event for triggering immune cascades,
eventually leading to the induction of antigen specific immune
effector cells that are able to destroy or neutralise targets. It
has been shown previously that a number of polycationic compounds
excert effects on immune cells (Buschle et al., Gene
Ther.Mol.Biol. 1 (1998), 309-321; Buschle et al.,
Proc.Natl.Acad.Sci. USA, 94 (1997), 3256-3261).
Co-injection of a mixture of poly-L-arginine and poly-L-lysine
together with an appropriate antigen as a vaccine protects ani-
mals from tumor growth in several animal models. A vaccine con-
sisting of polycationic compounds and antigens is accepted in the
art as being a very effective form of treatment (WO 97/30721).
Many pharmaceutical substances administered to an individual are
often quickly distributed throughout the body. The rapid systemic
distribution of the drug usually causes strong and harmful side
effects. The medical effect would be better if the medicament
would stay in higher amounts at the site of administration and be
gradually and continuously released to the whole body in small
amounts.
It is an object~of the present invention to provide means for
keeping a medicament which is desired to act locally at the site
of administration (depot effect). It is a further object of the
invention to prevent or ameliorate side effects of drugs which
are due to a too fast distribution of this drug throughout the
body .
These objects are solved by the use of a polycationic compound
for the preparation of a medicament with retarded in vivo re-
lease. It has surprisingly been found out in the course of the
CONFIRMATION COPY


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present invention that polycationic compounds, if applied to-
gether with other pharmaceutically active compounds, which are
quickly distributed in the individual, when administered without
polycationic compounds, exhibit an effect of a retarded re-
lease of the active compound from the site of administration.
The polycationic compound seems to keep the active pharmaceutical
compound in a depot which allows a retarded in vivo release of
the medicament which is often desired for an effective treatment
with the pharmaceutically active principle.
An important field, where such a retarded in vivo release is ad-
vantageous is vaccination. If an antigen is presented for an ex-
tended period of time to the immune system of an individual to be
vaccinated, the immune system has an enhanced possibility to cre-
ate an efficient immune response against such an antigen. If, on
the other hand, such an antigen is quickly distributed throughout
the body, the antigen is quickly degraded and diluted so that an
efficient immune response may not be achieved for many promising
antigens. According to the present invention polycationic com-
pounds therefore are used for providing a depot of e.g. such an
antigen, which allows a long lasting continuous and effective
presentation of this antigen to the immune system in order to
create a protective immunity. Furthermore, when the antigens are
applied in combination with immunostimulatory compounds (e. g.
CpG-ODN), the slow release of these immunostimulatory compounds
from the depot should result in a continuous stimulation of the
immune system.
The present invention is especially beneficial if the combined
medicament is administered, e.g. subcutaneously, intravenously,
intranasally, intramusculary, intradermally or transdermally.
However, other application forms, such as parenteral or topical
application, are also suitable for the present invention. How-
ever, the depot effect seems to be mostly significant if the com-
position is injected or implanted.
The present invention is preferably used in connection with all
medicaments for which a retarded in vivo release is desired, e.g.
antigens, allergens, drugs, which include cytokines, chemokines,
immunostimulatory nucleic acids, cytotoxic drugs or anti-angio-


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genic drugs or compounds needed for wound healing.
The antigen to be used within the course of the present invention
is not critical, it may preferably be selected from the group
consisting of an antigen from a viral, bacterial or a parasitic
pathogen, an antigen from an eucaryotic pathogen, a tumor anti-
gen, an autoimmune antigen or mixtures thereof. Especially pre-
ferred are negatively charged antigens or hydrophobic antigens.
Further examples of antigens are whole-killed organisms, such as
inactivated viruses or bacteria, fungi, protozoa or even cancer
cells. Antigens may also consist of subfractions of these organ-
isms/tissues, of proteins, or, in their most simple form, of pep-
tides. Antigens can also be recognised by the immune system in
form of glycosylated proteins or peptides and may also be or con-
tain polysaccharides or lipids. Short peptides can be used, since
e.g: cytotoxic T cells (CTL) recognise antigens in form of short
usually 8-11 amino acids long peptides in conjunction with major
histocompatibility complex (MHC). B cells recognise longer pep-
tides starting at around 15 amino acids. By contrast to T cell
epitopes, the three dimensional structure of B cell antigens may
also be important for recognition by antibodies.
Preferred pathogens are selected from human immune deficiency vi-
rus (HIV), hepatitis A and B viruses, hepatitis C virus (HCV),
Rous sarcoma virus (RSV), Epstein Barr virus (EBV),Influenza vi-
rus, Rotavirus, Staphylococcus aureus, Chlamydia pneumoniae,
Chlamydia trachomatis,~Mycobacterium tuberculosis, Streptococcus
pneumoniae, Bacillus anthracis, Vibrio cholerae, Plasmodium sp.
(P1. falciparum, P1. vivax, etc.), Aspergillus sp. or Candida al-
bicans. Antigens may also be molecules expressed by cancer cells
(tumor antigens). Antigens may also be derived antigens. The
derivation process may include the purification of a specific
protein from the pathogen/cancer cells, the inactivation of the
pathogen as well as the proteolytic or chemical derivatisation or
stabilisation of such a protein. In the same way also tumor anti-
gens (cancer vaccines) or autoimmune antigens may be used to-
gether with a polycationic compound according to the present
invention.
The polycationic compounds) to be used according to the present


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invention may be any polycationic compound, which shows e.g. the
characteristic effect according to the WO 97/30721, or others
like cationic liposomes, poly-ethylene-amine, chitosan or poly-
cations for DNA transfer. Preferred polycationic compounds are
selected from basic polypeptides, organic polycations, basic
polyaminoacids or mixtures thereof. These polyaminoacids should
have a.chain length of at least 4 amino acid residues (see: Tuft-
sin as described in Goldman et al (1983)). Especially preferred
are substances containing peptidic bounds, like polylysine,
polyarginine and polypeptides containing more than 200, espe-
cially more than 50% of basic amino acid residues in a range of
more than 8, especially more than 20, amino acid residues or mix-
tures thereof. Other preferred polycations and their pharmaceuti-
cal compositons are described in WO 97/30721 (e. g.
polyethyleneimine) and WO 99/38528. Preferably these polypeptides
contain between 5 and 500 amino acid residues, especially between
and 200 residues.
These polycationic compounds may be produced chemically or recom-
binantly or may be derived from natural sources.
Cationic (poly)peptides may also be polycationic anti-bacterial
microbial peptides with properties as reviewed in (Ganz and Le-
hrer, 1999; Hancock, 1999). These (poly)peptides may be of pro-
karyotic or animal or plant origin or may be produced chemically
or recombinantly (Andreu and Rivas, 1998; Ganz and Lehrer, 1999;
Simmaco et al., 1998). Peptides may also belong to the class of
defensins (Ganz, 1999; Ganz and Lehrer, 1999). Sequences of such
peptides can, for example, be found in the Antimicrobial Se-
quences Database under the following Internet address:
http-/lwww bbcm univ trieste.it/~tossi/paal.html
Such host defense peptides or defensines are also a preferred
form of the polycationic polymer according to the present inven-
tion. Generally, a compound allowing for activation (or down-
regulation) of the adaptive immune system, preferably mediated by
APCs (including dendritic cells) is used as polycationic polymer.
Especially preferred for use as polycationic substance in the
present invention are cathelicidin derived anti-microbial pep-


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tides or derivatives thereof (A 1416/2000, incorporated herein by
reference), especially anti-microbial peptides derived from mam-
mal cathelicidin, preferably from human, bovine or mouse.
Polycationic compounds derived from natural sources include HIV-
REV or HIV-TAT (derived cationic peptides, antennapedia peptides,
chitosan or other derivatives of chitin) or other peptides de-
rived from these peptides or proteins by biochemical or,recombi-
nant production. Other preferred polycationic compounds are
cathelin or related or derived substances from cathelin. For ex-
ample, mouse cathelin is a peptide which has the amino acid se-
quence NHS-RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH. Related or
derived cathelin substances contain the whole or parts of the
cathelin sequence with at least 15-20 amino acid residues. Deri-
vations may include the substitution or modification of the natu-
ral amino acids by amino acids which are not among the 20
standard amino acids. Moreover, further cationic residues may be
introduced into such cathelin molecules. These cathelin molecules
are preferred to be combined with the antigen and. the immunogenic
ODNs according to the present invention. However, these cathelin
molecules surprisingly have turned out to be also effective as an
adjuvant for an antigen without the addition of further adju-
wants. It is therefore possible to use such cathelin molecules as
efficient adjuvants in vaccine formulations with or without fur-
they immunostimulatirig substances.
Another preferred polycationic substance to be used according to
the present invention is a synthetic peptide containing at least
2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino
acids (A 178912000, incorporated herein by reference).
As mentioned above polycationic compounds may according to the
present invention be preferably used together with a drug for
which side effects due to a quick spread throughout the body of
an individual are known. In general, the polycationic compound
and the drug supposed to be released slowly are administered to-
gether at the same time and at the same site. In. the combined me-
dicament according to the present invention, such drugs may be
e.g. simply mixed with the polycationic compounds or provided as
a covalently combined medicament.


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Preferred compounds with inflammatory potential to be used within
the course of the present invention are immunogenic nucleic acid
molecules. It~is known that the immune system of mammals (and
probably most if not all vertebrates) recognises DNA of lower or-
ganisms, including bacteria probably due to structural and se-
quence usage differences between pathogen and host DNA. In
particular, short stretches of DNA derived from non-vertebrates
or short form oligodeoxynucleotides (ODNs) containing non-methy-
lated cytosine-guanine dinucleotides (CpG) in a certain base con-
text, are targeted. CpG motifs are found at the expected
frequency in'bacterial DNA but are much less frequent in verte-
brate DNA. In addition, non-vertebrate (i.e. bacterial) CpG mo-
tifs are not methylated, whereas vertebrate CpG sequences are.
Such ODNs containing CpG motifs (CpG-ODNs) can directly activate
monocytes and B cells. In consequence, the activation of mono-
cytes and NK cells by CpG-ODNs promotes, the induction of a Th1-
type response and the development of cytotoxic T cells. In addi-
tion, such immunogenic ODNs are used as vaccine adjuvants to en-
hance the antibody response to specific antigens (e. g.
EP 0 4~8 520 A2, WO 96/02555, WO 98/16247, etc.).
When CpG-ODNs are applied in combination with an antigen to an
animal, the CpG-ODN molecules are quickly distributed throughout
the body without providing an effective minimum concentration at
the site of administration where the desired effect should be
initiated. It could be shown by the present animal model that the
polycationic compounds inhibit the immediate spread of these
molecules and induce the formation of a depot of CpG-ODNs at the
injection site which resulted in a strongly prolonged CpG-ODN in-
duced antigen specific immune response in vivo. This CpG-ODN
model therefore was excellent for showing the depot effect of
polycationic compounds. If CpG-ODNs are applied in combination
with an antigen via injection, the CpG-ODN molecules are quickly
distributed throughout the body without providing an effective
minimum concentration at the site of administration where the de-
sired effect should be initiated. It could be shown by the pres-
ent animal model that the polycationic compounds inhibit the
immediate spread of these molecules and induce the formation of a
depot antigen and CpG-ODNs at the injection site, which resulted


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in a strongly prolonged CpG-ODN induced antigen-specific immune
response in vivo.
Therefore, a preferred embodiment of the present invention is
characterised in that the medicament is to be applied together
with the polycationic compound further comprises immunogenic oli-
godesoxy nucleic acid molecules (ODNs), especially ODNs contain-
ing CpG motifs (CpG-ODNs), inosine containing ODNs (T-ODNs) or
mixtures or combinations thereof. I-ODNs are described for exam-
ple in the Austrian patent application A 1973/2000 (incorporated
herein by reference). Mixtures of I-ODNs with CpG-ODNs may also
be provided as well as combinations of these two principles, e.g.
an I-ODN containing CpG motifs.
The induction of a depot effect according to the present inven-
tion is of course most desired for pharmaceutically active sub-
stances which are supposed to act locally at the given
administration site. Therefore, the invention is significantly
advantageous for substances, which should act locally but are
easily transported and diffused away from this site by diffusion
of transportation processes in the body. Such substances may in-
clude antigens, allergens, cytokines, chemokines, immunostimula-
tory nucleic acids, cytotoxic drugs or anti-angiogeni:c drugs or
compound needed for wound healing.
A preferred embodiment of the present invention relates to the
use of the polycationic substances in combination with substances
which otherwise rapidly diffuse from the administration site,
i.e. have a rather short pharmacological half life, especially
with respect to the site of administration. Therefore, preferred
rapidly diffusing substances with a pharmacological half life
(drop of the concentration of the substance by half), especially
at the site of administration, of below 10 minutes, more pre-
ferred below 5 minutes, especially below 1 minute.
Preferably, such substances to be applied together with polycati-
onic compounds in order to achieve a depot effect show a certain
affinity to the polycationic compound, i.e. hydrophobic interac-
tion, hydrogen bridges, electrostatic interactions, polar or
ionic interactions. Of course, the depot effect may also be


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achieved by covalent binding of the components in the combined
pharmaceutical preparation; although non-covalent interactions of
drug and polycationic compounds are preferred.
It is known (PCT/EP 01/00087) that the co-application of polyca-
tionic compounds and CpG-OI7Ns with an antigen strongly and syner-
gystically enhances the induction of an antigen specific immune
response when compared to the injection without poly-cationic
compounds. That is reflected by a high number of IFN-y-producing
cells isolated from draining lymph nodes (ELISPOT assay). As
stated above within the course of the present invention, it could
be shown that this strong local immune response (day 4/draining
lymph node cells) induced after one single injection of an anti-
gen with a mixture of polycationic compounds (as an example
polyarginine pR 60 is used) and CpG-ODNs converts to a systemic
immune response which is very long lasting. According to the pre-
sent invention, the complex formation ability of substances such
as CpG-ODNs with polycationic compounds is used for preventing a
systemic distribution and the subsequent fast resorption of such
substances, thereby providing a strong prolongation of the prop-
erties of such substances, e.g. a prolongation of the immu-
nostimulatory properties of CpG-ODNs. In addition, preventing the
systemic distribution avoids the induction of potential harmful
systemic side effects of immunostmulatory agents.
This model using CpG-ODNs and polycationic peptides is further
described and analysed in the example section. Moreover,. to pro-
vide an. analysable pharmaceutical target, an Ovalbumin-derived
peptide (OVAzs7-as4) is used as a model compound (a model antigen) .
The present invention also relates to treating a patient with a
drug, supposed to be retardedly released in vivo comprising ad-
ministering that drug together with an effective amount of a
polycationic compound inducing a depot effect of that drug.
The amounts of polycationic compound to be administered is highly
depending on the necessities of the individual composition and
optionally on the drug to be administered together with the poly-
cationic polymer. In case of poly-L-arginine and poly-L-lysine
preferred amounts of polycation are 0.001-1000 pg/administration


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unit, more preferred 0,1-10 mg/dose, especially around or beyond
0,1 mg/20g body weight (of mice) or the equivalent dose for hu
mans.
The invention will be described in more detail by way of the fol-
lowing examples and the drawing figures, yet it is not restricted
to these particular embodiments.
Fig. 1 shows that the combined application of poly-L-arginine,
CpG-ODN and antigen induces strong antigen-specific immune re-
sponses which are systemic and very long lasting. The figure
shows peripheral blood lymphocytes stimulated ex vivo with OVAZS7-
z64 peptide;
Fig. 2a shows that poly-L-arginine induces the formation of a de-
pot at the injection site. This figure shows photos from the in-
jection sites at the indicated time points after vaccination.
White lines surround the area where the fluorescence labelled
compounds of the vaccine can be detected;
Fig. 2b shows that the co-application of poly-L-arginine inhibits
the spreading of CpG-ODN-Cy5 throughout the body. This figure
shows FRCS analyses of lymphoid and non-lymphoid tissues at~day 1
after injection of CpG-ODN-Cy5 (B) or CpG-ODN-Cy5 and pR 60-FITC
(C). Untreated mice were used as a control (A);
Fig. 3 shows that poly-L-arginine induces the formation of a de-
pot at the injection site when co-injected at least with one more
molecule. This figure shows photos from the injection sites at
day 4 after vaccination;
Fig. 4 shows that co-injected poly-L-arginine prevents the CpG-
ODN-induced systemic production of TNF-(x and IL-6 in vivo. Mice
were injected into the hind footpads and one hour later serum was
prepared. The amount of TNF-oc and IL-6 in the sera was determined
by ELISA;


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E X A M P L E S
In the present examples it is shown that the strong local immune
response (day 4/draining lymph node cells) induced after one sin-
gle injection of antigen with a mixture of pR60 and CpG-ODN con-
verts to a systemic immune response which is, most importantly,
very long lasting (Example 1). Even 372 days after injection (the
latest time point analysed), around 500 antigen-specific, IFN-g
producing T cells per million peripheral blood lymphocytes can be
detected. One possible explanation for this effect might be that
a complex-formation of CpG-ODN with poly-L-arginine prevents the
systemic distribution of CpG-ODN and the subsequent fast resorp-
tion of CpG-ODN. Hence, this results in a strong prolongation of
the immunostimulatory properties of CpG-ODNs.
In order to investigate this assumption, fluorescence-labeled
compounds were injected together subcutaneously into the flank of
mice. At different time points after this treatment, injection
sites were inspected for the presence of labeled compounds. In
example 2a and 2b, OVAzs7-zs4-peptide (unlabeled) , poly-L-arginine-
FITC (yellow) and CpG-ODN-Cy5 (blue) were used for injections.
After injection of OVAzs7-zs4 peptide with poly-L-arginine-FITC the
formation of a depot could be detected at the injection site. The
inj ection of OVAzS~_zs4-peptide with CpG-ODN-Cy5 resulted in the
distribution of CpG-ODN-Cy5 all over the skin (example 2a). As
simultaneously determined by FRCS, analyses (example 2b), CpG-ODN-
Cy5 is also detectable in secondary lymphoid organs (draining
lymph node, spleen) and non-lymphoid tissues (lung, liver, kid-
ney, heart) . In contrast, when OVAz57_zs4-peptide and CpG-ODN-Cy5
were injected together with poly-L-arginine-FITC, the CpG-ODN-Cy5
was restricted to the depot formed by poly-L-arginine (example
2a). FRCS analyses from these mice (example 2b) revealed that
CpG-ODN-Cy5 is not detectable in the periphery, due to the fact
that CpG-ODN-Cy5 is trapped by poly-L-arginine in the depot at
the injection site. Both, poly-L-arginine-FITC and CpG-ODN-Cy5
can be detected within this depot at least up to day 92 after in-
jection (the latest time point analysed). This observation im-
plies that the combination of peptide and poly-L-arginine with
CpG-ODN led to a far long lasting existence of the depot compared


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to the combination of peptide and poly-L-arginine. In example 3,
TRP-21s1-lss peptide-FITC (yellow), poly-L-arginine-TRITC (red-vio-
let), CpG-ODN-Cy5 (blue) were used for injections. When TRP-21s1-
~ss peptide-FITC was injected either alone or in combination with
CpG-ODN-Cy5, the peptide was not detectable at the injection site
at day 4. The injection of poly-L-arginine-TRITC alone resulted
in its distribution all over the skin. The injection of CpG-ODN-
Cy5 either alone or in combination with TRP-21st-lss peptide-FITC
resulted in the distribution of CpG-ODN-Cy5 all over the skin.
This experiment.also indicates that. poly-L-arginine only induced
the formation of a depot when it was injected with at least one
more molecule (peptide and/or CpG-ODN).
Thus, these findings imply that poly-L-arginine induces a depot
at the injection site within other compounds (antigen and/or im-
munostimulatory CpG-ODN) are kept. In the 'case of co-injection of
OVAzs7-zsa peptide, poly-L-arginine and CpG-ODN, the slow release of
both peptide and CpG-ODN from this depot is most likely responsi-
ble for the persistent activation of accessory cells and subse-
quently the persistent stimulation of T cells. In consequence,
this leads to the observed long lasting existence of high numbers
of antigen-specific T cells in the periphery after one single in-
jection.
Beside their potent immunostimulatory effects, CpG-ODNs are de-
scribed to have potentially harmful side effects in that they in-
duce the systemic release of high amounts of pro-inflammatory
cytokines such as TNF-oc and IL-6, which could induce a shock~syn-
drome (Sparwasser 1997, Lipford 1997). As described in example
2a, 2b and 3, CpG-ODNs are not systemically present when injected
in combination with poly-L-arginine. Therefore, it was investi-
gated whether the co-administration of poly-L-ar.ginine affects
the CpG-ODN-induced systemic production of TNF-oc and IL-6. Serum
levels of both cytokines were determined by ELISA one hour after
injection. Example 4 demonstrates that neither the injection of
OVAzs7-zs9-peptide alone nor in combination with poly-L-arginine led
to the induction of significant amounts of TNF-oc and IL-6 in the
serum, whereas the inj ection of OVAzs7-zsa-peptide in combination
with CpG-ODN induces high concentrations of both cytokines. How-
ever, upon co-administration of OVAzs7-zsa-peptide with poly-L-ar-


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- 12 -
ginine and CpG-ODN, this systemic production of TNF-a and IL-6
was totally abolished. Thus, these data in combination with the
findings demonstrated in Example 2 and 3 indicate that the local-
isation of CpG-ODN via the depot formation mediated by poly-L-ar-
ginine prevents the systemic distribution of CpG-ODN and
subsequently the systemic release of pro-inflammatory cytokines.
In parallel, in vitro studies were performed to clarify whether
the complexation of CpG-ODN by poly-L-arginine can also directly
influence the stimulation of mouse bone marrow-derived CDllc*
dendritic cells by CpG-ODN concerning the production of TNF-oc and
IL-6. For this purpose, CDllc+ dendritic cells were incubated ei-
ther with poly-L-arginine, CpG-ODN or the combination of poly-L-
arginine and CpG-ODN (example 5). The levels of TNF-oc and IL-6
were determined in the supernatants derived from these cultures.
After incubation with poly-L-arginine neither TNF-oc, nor IL-6 were
detectable, whereas after incubation with CpG-ODN significant
amounts of both cytokines are produced. Impressively, the pres-
ence of poly-L-arginine inhibited the CpG-ODN-induced production
of TNF-a, and IL-6 by these cells .
Thus, these results indicate that the complexation of CpG-ODN by
poly-L-arginine not only inhibits the systemic but also the local
release of pro-inflammatory cytokines. In consequence, these
beneficial effects of poly-L-arginine prevent probably uncon-
trolled and excessive systemic and local immune responses induced
by CpG-ODNs.
Further in vitro-experiments revealed that poly-L-arginine also
inhibits the polyinosinic-polycytidylic acid-induced production
of pro-inflammatory cytokines by human dendritic cells.
Thus, these observations imply a general anti-inflammatory effect
of poly-L-arginine. The risks of the application of immunogenic
but potential harmful substances can be probably minimised by the
co-application of poly-L-arginine. The rapid systemic distribu-
tion of such substances can be prevented by the property of poly-
L-arginine to form a depot in which alI compounds are trapped.
Furthermore, the complexation of these substances by poly-L-ar-
ginine can e.g. inhibit the release of toxic amounts of pro-in-


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 13 -
flammatory cytokines.
Example 1:
The combined application of Ovalbumin-peptide/poly-L-arginine (pR
60)/ CpG-ODN leads to the induction of strong antigen-specific
immune responses which are systemic and very long lasting.
Mice C57B1/6 (Harlan/Olac)
Peptide OVA257~-264 Peptide (SIINFEKL) , a MHC class I (H-2Kb)-
' restricted epitope of chicken Ovalbumin (Rotz-
schke, O. et al., Eur. J. Immunol. 1991 21 (11):
2891-4),synthesised by standard solid phase F-
moc synthesis, HPLC purified and ana
lysed by mass spectroscopy for purity.
Dose: 300 ~.g/mouse
Poly-L-Arginine 60 (pR60)
Poly-L-Arginine with an average degree of poly-
merisation of 60 arginine residues; SIGMA chemi
cals
Dose: 100 ug/mouse
CpG-ODN 1668 phosphothioate-modified oligodinucleotides con
taming a CpG- motif: tcc atg acg ttc ctg atg ct,
synthesised by NAPS Gottingen GmbH.
Dose: 5nmol/mouse
Exberimental aroubs (5 mice per group)
1. OVAzs7-as4 Peptide + CpG-ODN + pR 60
2. OVAZS7-~s4 Peptide + CpG-ODN
3. OVA~57_zsa-Peptide + pR 60
On day 0, mice were injected into each hind footpad with a total
volume of 100~.~.1 (501 per footpad) containing the above mentioned
compounds. Blood was taken via the tail vein at the indicated
time points and peripheral blood lymphocytes (PBLs) were isolated
using a Ficoll gradient.~PBLs were stimulated ex vivo with the
antigen used for vaccination; with medium (background) and.Conca-
navalin A (positive control). IFN-'y-ELISPOTs were carried out as
described (Miyahira et al., 1995). This method is a widely used
procedure allowing the quantification of antigen-specific T
cells. Spots representing single IFN-y producing T cells were


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 14 -
counted and the number of background spots was substracted from
all samples. There were many spots detected after the stimulation
with Con A (data not shown) indicating a good condition of the
used lymphocytes. For each experimental group of mice the number
of spots/1x106 PBLs are illustrated in Figure 1.
Example 2a:
Poly-L-arginine induces the formation of a depot at the injection
site
Mice C57B1/6 (Harlan/Olac)
Peptide OVA257-264 Peptide (SIINFEKL) , a MHC class I (H-
2Kb)-restricted epitope of chicken Ovalbumin
(Rotzschke, O.et al., Eur. J. Immunol. 1991
21(11): 2891-4), synthesised by standard solid
phase F-moc synthesis, HPLC purified and analysed
by mass spectroscopy for purity.
Dose: 300ug/mouse
Poly-L-Arginine 60-FITC (pR60-FITC)
Poly-L-Arginine with an average degree of poly-
merisation of 60 arginine residues; SIGMA chemi-
' cats
For fluorescein (FITC) labeling of poly-L-ar-
ginine, the poly-L-arginine was dissolved in
50mM HEPES pH 7,9 (l0mg/500u1). A 5-fold molar ex-
cess of FITC (Molecular Probes, Eugene, OR)
in an equal
volume of DMSO was added to the poly-L-arginine
solution. The solution was kept at room tempera-
ture in the dark for 2,5 hours. Unreacted dye was
separated by running the mixture over a PD10 col-
umn(Pharmacia, Uppsala, Sweden), using 50mM Hepes,
pH 7,9, as eluent. The solution was then dialysed
against 2 x 5 liter aqua dest., pH 7,4(0,1M HCL),
over night. After lyophilisation poly-L-arginine
FITC was dissolved in aqua bidest. with a concen-
tration of l0mg/ml.
Dose: 100 ug/mouse
CpG-ODN 1668-Cy5
phosphothioate-modified, Cy5-labeled oligodinu-


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 15 -
cleotides containing a CpG motif:
tcc atg aca ttc ctg atg ct, synthesised by NAPS
Gottingen GmbH.
Dose: 5nmo1/mouse
Experimental groups (1 mouse / group 1/ indicated time point, 3
mice / group 2-4 / time point)
1. untreated
2. OVAz57-264 Peptide + pR 60-FITC
3. OVAz57_zsa Peptide + CpG-ODN1668-Cy5
4. OVAz57_zs4-Peptide + pR 60-FITC + CpG-ODN 1668-Cy5
On day 0 mice were injected subcutaneously into the right flank
with a total volume of 100.1 containing the above mentioned Com-
pounds. Animals were sacrificed at the indicated time points af-
ter injection and photos were taken from the injection sites
(Fig. 2a) .
Example 2b:
Co-application of poly-L-argiaiae inhibits the distribution of
CpG-ODN-Cy5 throughout the body
Mice C57B1/6 (Harlan/Olac)
Poly-L-Arginine 60-FITC (pR60-FITC)
Poly-L-Arginine with an average degree of poly-
merisation of 60 arginine residues; SIGMA chemi-
cals
For fluorescein (FITC) labeling of poly-L-ar-
ginine, the poly-L-arginine was dissolved in
50mM HEPES pH 7,9 (l0mg/500~.~.1). A 5-fold molar ex-
cess of FITC (Molecular Probes, Eugene, OR)
in an equal volume of DMSO was added to the poly-L-
arginine solution. The solution was kept at.room
tempera ' tune in the dark for 2,5 hours. Unre-
acted dye
was separated by running the mixture over a PD10
column (Pharmacia, Uppsala, Sweden) using 50mM He
pes pH 7,9 as eluent. The solution was then dia-
lysed against ~ x 5 liter aqua dest., pH 7,4 (0,1M
HCL), over night. After lyophilisation poly-L-ar-


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 16 -
pine-FITC was dissolved in aqua bidest. with a
concentration of l0mg/ml.
Dose: 100 ug/mouse
CpG-ODN 1668-Cy5
phophothioate-modified, Cy5-labeled oligodinucleo-
tides containing a CpG motif: tcc ata aca ttc ctg
atg ct, synthesised by NAPS Gottingen GmbH.
Dose: 5nmo1/mouse
Experimental groups (1 mouse / group 1/ indicated time point, 3
mice / group 2-4 / time point)
1. untreated
2. CpG-ODN1668-Cy5
3. pR 60-FITC + CpG-ODN 1668-Cy5
Mice were injected subcutaneously into the right flank with a to-
tal volume of 100.1 containing the above mentioned compounds. One
day after injection, mice were sacrificed and FACS-analyses were
performed from secondary lymphoid organs (draining lymph node,
spleen) as well as non-lymphoid tissues (lung, liver, kidney,
heart) (Fig.. 2b).
Example 3:
Poly-L-arginine induces the formation of a depot at the injection
site when co-injected at least with one more molecule
Mice C57B1/6 (Harlan/Olac)
Peptide TRP-2-Peptide (VYDFFVWL), a MHC class I (H-2Kb)-
restricted epitope of mouse tyrosinase related
protein-2 (Bloom, M.B, et al., J Exp Med 1997 185,
453-459), synthesised by standard solid phase F-
moc synthesis, HPLC purified and analysed by mass
spectroscopy for purity. For fluorescein (FITC)
labeling, the TRP-2 -peptide was dissolved in
181-188
1M sodium borate, pH 7,9. An 8-fold molar excess
of FITC (Molecular Probes, Eugene, OR) in an equal
volume of DMF was added to the peptide solution.
The solution was kept at room temperature for four
hours. Unreacted dye was seoarated ba running the


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 17 -
mixture over a G25 gel filtration column (Pharma-
cia, Uppsala, Sweden) using 0,1o TFA in water as
eluent. Two moles of FITC were incorporated per
mol of peptide (N-terminus, side chain of lysine)
Dose: 100ug/mouse
Poly-L-Arginine 60-TRITC (pR60-TRITC) .
Poly-L-Arginine with an average degree of poly-
merisation of 60 arginine residues; SIGMA chemi-
cals. For TRITC-labeling of poly-L-arginine, the
poly-L-arginine was dissolved in 50mM HEPES pH 7,9
(l0mg/500u1). A 5-fold molar excess of FITC (Mo-
lecular Probes, Eugene, ORj in an equal volume of
DMSO was added to the poly-L-arginine solution.
The solution was kept at room temperature in the
dark for 2,5 hours. Unreacted dye was separated by
running the mixture over a PD10 column (Pharmacia,
Uppsala, Sweden), using 50mM Hepes, pH 7,9, as
eluent. The solution was then dialysed against 2 x
liter aqua dest., pH 7,4 (0,1M HCL), over night.
After lyophilisation poly-L-argine-TRITC was dis-
solved in aqua bidest. with a concentration of
l0mg/ml.
Dose: 100 ~.zg/mouse
CpG-ODN 16&8-Cy5
phosphothioate-modified, Cy5-labeled oligodinu
cleotides containing a CpG motif: tcc atg aca ttc
ctg atg ct, synthesised by NAPS Gottingen GmbH.
Dose: 5nmol/mouse
Experimental groups (1 mouse / group 1/ indicated time point, 3
mice / group 2-4 / time point) .
1. untreated
2. TRP-2 -FITC
isi-isa
3. pR60-TRITC °
4. CpG-ODN1668-Cy5
5 . TRP-2181-isa-FITC + pR6 0-TRITC
6. TRP-~laz-isa-FITC + CpG-ODN-Cy5
7. pR60-TRITC + CpG-ODN 1668-Cy5
8. TRP-~~81-lss-FITC + pR 60-TRITC + CpG-ODN 1668-Cy5


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 18 -
On day 0 mice were injected subcutaneously into the right flank
with a total volume of 1001 containing the above mentioned Com-
pounds. Animals were sacrificed at day 4 after injection and pho-
tos were taken from the injection sites (Fig. 3).
Example 4
The co-injection of poly-L-arginine prevents the CpG-ODN-induced
systemic production of TNF-oG and IL-6 in vivo
Mice C57B1/6 (Harlan/Olac)
Peptide OVA257-264 (SIINFEKL) , an MHC class I (H-2Kb) -re-
stricted epitope of chicken ovalbumin (Rotzschke
et al., 1991), was synthesised using standard
solid phase F-moc synthesis, HPLC-purified and
analysed by mass spectroscopy for purity
Dos a : 3 0 0~~.g/mouse
Poly-L-arginine 60 (pR60)
Poly-L-arginine with an average degree of poly-
merisation of 60 arginine residues; SIGMA Chemi-
cals
Dose: 100ug/mouse
CpG-ODN 1668 phosphothioate-modified oligodeoxynucleotides con-
taining a CpG motif: TCC ATG ACG TTC CTG ATG CT,
synthesised by NAPS GmbH, Gottingen.
Dose: 5 nmol/mouse
Experimental aroups: 4 mice per.group
1 . OVA
257-264
2. pR60
3 . CpG 16 6 8 "~' OVA257-264
4 . CpG 16 6 8 + pR6 0 + OVA257-264
Mice were injected into each hind footpad with a total volume of
100.1 (501 per footpad), containing the above mentioned com-
pounds. One hour after injection blood was taken from the tail-
vein and serum was prepared. The amount of the pro-inflammatory
cytokines TNF-a, and IL-6 in the sera was determined by cytokine-


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 19 -
specific ELISAs according to the manufacturer's instructions (R&D
Systems, Inc., Minneapolis, MN).
This experiment shows that inj ection of OVAzs~-as4 alone or in com-
bination with poly-L-arginine does not induce the production of
detectable amounts of TNF-oc or IL-6 (Figure 4). In contrast, the
injection of OVAZS7-264 peptide with CpG-ODN 1668 induces the sys-
temic production of TNF-oc and IL-6. When peptide and CpG-ODN were
co-injected with poly-L-arginine, the CpG-ODN induced production
of pro-inflammatory cytokines was inhibited.


CA 02433794 2003-07-04
WO 02/053184 PCT/EP02/00062
- 20 -
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Andreu, D., and Rivas, L. (1998). Animal antimicrobial peptides:
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Ganz, T. (1999). Defensins and host defense [comment]. Science
286, 420-421.
Ganz, T., and Lehrer, R. I. (1999). Antibiotic peptides from
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Goldman, R., Bar-Shavit, Z. (1983). On the mechanism of the aug-
mentation of the phagocytic capability of phagocytic cells by
Tuftsin, substance P, neurotensin, and kentsin and the interrela-
tionship between their receptors. Ann N Y Aca. Sci. 419:143-55.
Inaba et al. (1992). Generation of large numbers of dendritic
cells from mouse bone marrow cultures supplemented with granulo-
cyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693
Hancock, R. E. (1999). Host defence (cationic) peptides: what is
their future clinical potential? Drugs 57, 469-473.
Lipford, G.B., T. Sparwasser, M. Bauer, S. Zimmermann, E. Koch,
K. Heeg, H. Wagner. 1997. Immunostimulatory DNA: sequence-depend-
ent production of potentially harmful or useful cytokines. Eur.
J. Immunol. 27:3420
Sparwasser,T. T. Miethke, G. Lipford, A. Erdmann. H. Hacker, K.
Heeg, H. Wagner. 1997. Macrophages sense pathogens via DNA mo-
tifs: induction of tumor necrosis factor-a-mediated shock. Eur J
Immunol. 27:1671
Verdijk, R.M., T. Mutis, B. Esendam, J. Kamp, C.J. Melief, A.
Brand, E. Goulmy. 1999. Polyriboinosinic polyribocytidylic acid
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man dendritic cells. J Immunol. 163:57

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Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2002-01-07
(87) PCT Publication Date 2002-07-11
(85) National Entry 2003-07-04
Dead Application 2008-01-07

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Owners on Record

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Current Owners on Record
INTERCELL AG
Past Owners on Record
BUSCHLE, MICHAEL
LINGNAU, KAREN
MATTNER, FRANK
SCHMIDT, WALTER
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Description 2003-07-04 20 1,088
Abstract 2003-07-04 1 52
Claims 2003-07-04 2 71
Drawings 2003-07-04 5 318
Cover Page 2003-08-29 1 27
Claims 2003-07-05 2 77
PCT 2003-07-04 11 423
Assignment 2003-07-04 2 82
Prosecution-Amendment 2003-07-04 1 17
Correspondence 2003-08-26 1 24
Assignment 2003-08-20 3 80
Correspondence 2003-09-08 2 114
PCT 2003-07-05 11 476
Assignment 2003-09-10 1 32
PCT 2003-07-04 1 38