Note: Descriptions are shown in the official language in which they were submitted.
21%61~2
PEPTIDE ~Y~-n~SIS ON CHITOSAN
s
FIELD OF THE lNv~..lON
The present invention relates to a support for
peptide synthesis and peptide antigen amplification, and in
particular, to a chitosan support therefor.
BACRGROUND OF THE lNV~.~lON
Synthetic peptides are currently used for the
development of sequence specific antibodies and are being
incorporated into highly specific vaccines. However, the
peptides must generally be presented as part of larger
molecules in order to function as effective immunogens and
must be co-injected with an adjuvant in order to obtain an
optimum response.
In order to present peptides as larger molecules,
the peptides are often conjugated to a carrier or support.
One such support is a carrier protein, such as keyhole
limpet haemocyanin. However, the peptide is randomly
coupled to the protein, usually through free amino groups,
resulting in a non-specific peptide presentation and a
structure which is difficult to predict and to control.
Moreover, the carrier protein itself may be a multiepitope
antigen. This can be a serious disadvantage, especially in
vaccine development. In particular, the protein carrier
could lead to an adverse response, for example, by
generating an auto-immune response.
In an effort to overcome the pro~lems associated
with the use of protein carriers, researchers have
synthesized peptides onto a branching lysine core resulting
in a multiple antigenic peptide (MAP) structure (Posnett,
2126132
D.N. et al J Biol Chem 263: 4: 1719-1725; 1988). The MAP
construct provides control over the resultant structure and
peptide presentation. However, there are also disadvantages
associated with the use of MAP. In particular, it was found
S that antibodies raised against the MAP did not always cross-react with the cognate protein (Briand, J.P. et al J Immunol
Methods 156: 255-265; 1992).
Furthermore, the MAP is typically synthesized on a
solid phase resin support, from which the complex must
generally be cleaved following synthesis. Cleavage of the
complex from the support, usually with anhydrous
hydrofluoric acid, can cause side reactions. The
reattachment of side chains results in a non-uniform
complex. The side chains also represent impurities which
may cause immunology problems.
Chitin is a naturally-occurring biodegradable
polysaccharide that forms a base for the hard outer
integuments of crustaceans, insects and other invertebrates.
Chitosan is derived from chitin by deacetylation.
Chitosan has been used as an adsorbent for affinity
chromatography (Moriguchi et al, United States Patent Number
4,879,340; November 7, 1989) and as a carrier for
immobilized enzymes (Kawamura et al, United Sates Patent
Number 4,833,237; May 23, 1989). Chitosan has also been
used as a carrier for antigen or antibody (Unitika Kabushiki
Kaisha, Japanese Patent Application Number 89012280-B;
February 28, 1989). Prior to coupling the antigen or
antibody, the chitosan is treated with a reagent having at
least two functional groups reactive with the amino groups
and the hydroxyl groups of the chitosan support. However,
as discussed hereinabove with respect to carrier proteins
such as keyhole limpet haemocyanin, the antigen or antibody
is randomly coupled, resulting in a non-specific peptide
presentation and a structure which is difficult to predict
and to control.
An object of the present invention is to synthesize
a peptide on a support having little or no antigenic
21261~2
properties which can then be used directly for injection
into an animal. The sequence of the resultant structure is
specific and, as such, is predictable and controllable.
SUMMARY OF-T~E lN V ~ . ION
According to one aspect of the present invention,
there is provided a process for peptide synthesis on
chitosan, comprising the steps of: providing amino acids
having side-chain reactive groups thereof blocked with
blocking groups; protecting the ~-amino group of the amino
acids with a temporary protecting group; attaching a first
amino acid to the free amino groups of the chitosan;
releasing the temporary protecting group from the attached
amino group; sequentially attaching the desired amino acids
to the previously attached amino acid and releasing the
temporary protecting group therefrom until the desired
peptide is synthesized to form a peptide-chitosan complex;
and removing the blocking groups from the side-chain
reactive groups of the amino acids.
According to another aspect of the present
invention, there is provided a peptide-chitosan complex
prepared by the process comprising the steps of: providing
a chitosan support; providing amino acids having side-chain
reactive groups thereof blocked with blocking groups;
protecting the ~-amino group of the amino acids with a
temporary protecting group; attaching a first amino acid to
the free amino groups of the chitosan; releasing the
temporary protecting group from the attached amino group;
sequentially attaching the desired amino acids to the
previously attached amino acid and releasing the temporary
protecting group therefrom until the desired peptide is
synthesized to form a peptide-chitosan complex; and removing
the blocking groups from the side-chain reactive groups of
the amino acids.
According to a further aspect of the present
invention, there is provided a peptide prepared by the
21~132
process comprising the steps of: providing a chitosan
support; providing amino acids having side-chain reactive
groups thereof blocked with blocking groups; protecting the
~-amino group of the amino acids with a temporary protecting
group; attaching a bifunctional cleavable linker molecule to
the free amino groups of the chitosan; attaching a first
amino acid to the free amino group of the cleavable linker
molecule; releasing the temporary protecting group from the
attached amino group; sequentially attaching the desired
amino acids to the previously attached amino acid and
: releasing the temporary protecting group therefrom until thedesired peptide is synthesized to form a peptide-chitosan
complex; cleaving the cleavable linker molecule to release
the peptide from the chitosan; and removing the blocking
groups from the side-chain reactive groups of the amino
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the
present invention:
Figure 1 is a graphical representation of an
elution pattern of the crude product of Example 1;
Figure 2 is a graphical representation of antibody
response to an hPTH-(44-68)-chitosan complex prepared in
accordance with the present invention; and
Figure 3 is a graphical representation of antibody
response to an hPTH-(1-17)-chitosan complex prepared in
accordance with the present invention.
2126132
-- DESCl~IPTION OF TXE PREFEB.RED EMBODIMENTS
In accordance with the present invention, chitosan,
having the following formula:
S Cl 120H C1120H C~120H C~20H
1~`0~0~0~0~0~l
NH2 3 OC~3 NH2
is used as a support for peptide synthesis. Chitosan is
produced from chitin, having the following formula:
C~120H C~20H CJ 120H CH20H
,"o~o~~~K~ ~'
NHCOCH 3 HCOCI 13 NHCOC~13
which is an N-acetylated polymer of 2-desoxy-2-amino
10 glucose, and is substantially inert to the immune system.
A portion of the residues of chitin are deacetylated in the
production of chitosan, leaving some amino groups free as
nuclei for peptide synthesis. The deacetylation reaction is
performed in a manner known to those s~illed in the art (for
lS example, Neugebauer et al, CarbohYd Res 189: 363-367; 1989).
In accordance with the present invention, once the
structure of a synthetic peptide is determined, solid phase
peptide synthesis is conducted by a stepwise coupling of the
desir-ed amino acids to the chitosan. The technique of solid
20 phase synthesis developed by R.B. Merrifield (Advances in
Enzymoloqy 32: 221-296; 1969) is widely and successfully
used for the synthesis of polypeptides, such as parathyroid
hormone. The strategy is based on having the carboxyl-
terminus of the amino acid attached to a solid support.
25 Successive amino acids are then added in a very high yield.
2126132
A first amino acid is prepared for solid phase
peptide synthesis by blocking reactive side groups, for
example the carboxyl group in glutamic and aspartic acids
and the side-chain amino group in lysine, in a manner known
to those skilled in the art. Suitable side-chain protecting
groups are^ methoxytrimethylphenylsulfonyl (Mtr) for
arginine; t-butyl for aspartic acid, glutamic acid and
serine; trityl for asparagine, glutamine and histidine; and
t-butyloxycarbonyl for lysine.
The ~-amino group of the amino acid is then
protected by a fluorenylmethyloxycarbonyl (Fmoc) group and
the carboxyl-terminus of the Fmoc-amino acid is activated
for coupling to the free amino groups of the chitosan with,
for example, a TBTU/HOBT/NMM (2-(lH-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate/hydroxy-
benzotriazole/N-methylmorpholine) coupling procedure. The
Fmoc group acts as a temporary protecting group on the ~-
amino groups of the amino acid and can be removed under mild
alkaline conditions without affecting the alkali-stable
side-chain protecting groups and the link to the support
(Atherton, E. and Sheppard, R.C. Solid Phase Peptide
SYnthesis: A Practical APProach IRL Press, New York, N.Y.;
1989).
Alternatively, a linker or spacer molecule is first
attached to the free amino groups of the chitosan support.
Suitable linkers are linear or branched molecules having at
least two functional groups. The linker has a first
functional group for coupling to a free amino group of the
chitosan and a second functional group for coupling to the
carboxyl-terminal of the amino acid of the peptide antigen.
An example of a suitable linker is ~-aminocaproic acid.
The linker may also be a cleavable linker, such as
benzhydrylamine (Bernatowicz et al Tetrahedron Letters 30:
4645; 1989) which yields a peptide amide after cleavage or
4-(4-hydroxymethyl-3-methoxyphenyl)-butyric acid
(Florsheimer and Riniker, European Peptide Symposium,
Barcelona; 1990) which yields a peptide with a carboxy-
2I26t 32
terminus after cleavage. The linker is cleaved afterpeptide synthesis to release the peptide from the chitosan
support. The synthesis of a peptide-chitosan complex using
a cleavable linker is useful in determining the quality of
a peptide-chitosan complex synthesized with a non-cleavable
linker (as will be shown in Example 1). The cleaved peptide
may also be used for any desired application.
The linker serves to distance the first amino acid
from the chitosan support to alleviate steric interference.
Furthermore, by providing a linker molecule having a third
functional group, immunostimulatory structures can also be
introduced by using the third functional group as the point
of attachment. Thus, the potential exists for building an
adjuvant property into the peptide-chitosan complex.
After addition of the first amino acid or linker,
any remaining free amino groups on the chitosan are then
acylated with a C2-C20 fatty acid derivative, for example with
acetyl or palmityl groups. This provides a further means
for altering the biological properties of the resultant
structure.
In the case wherein a linker is first attached to
the chitosan support, the carboxyl-terminus of the first
Fmoc-amino acid of the desired peptide sequence is then
coupled to the free amino groups of the linker.
The Fmoc group of the first amino acid, coupled
either directly to the chitosan or via a linker molecule, is
then released from the ~-amino group of the first amino acid
in a manner known to those skilled in the art, for example,
by addition of a base. Stepwise synthesis of the peptide
then continues by coupling of additional Fmoc-amino acids
and subsequent releasing of the Fmoc group to expose the ~-
amino groups for coupling of the next desired amino acid.
The synthesis may be conducted in an automatic continuous
flow peptide synthesizer, such as, for example, the Milligen
9050 PlusTM (Millipore Corp., Milford, Massachusetts,
U.S.A.).
2l26i32
Once the desired peptide has been synthesized, the
amino acid side-chain protecting groups are removed in a
manner known to those skilled in the art, for example, by
treatment with 95% trifluoroacetic acid (TFA)/water. The
structure is then washed free of unreacted chemicals and the
peptide-chitosan complex is used successfully as a peptide
antigen presenter with no further purification required.
The resultant peptide-chitosan complex is insoluble
at neutral pH. Accordingly, after completion of the
synthesis, the chitosan can be fragmented to soluble
oligomers using chitinase or chitosanase or by carrying out
a partial hydrolysis with anhydrous hydrofluoric acid.
Alternatively, the peptide-chitosan complex may be
fragmented, using chitinase or chitosanase, prior to removal
of the protecting groups from the amino acids.
Fragmenting the fully protected peptide-chitosan
complex provides the opportunity to derivatize the peptide-
chitosan complex fragments. For example, the fragments may
be derivatized with acetic anhydride and dimethylamino
pyridine in dimethylformamide (DMF) to produce esters of the
sugar hydroxyl groups. Any fatty acid anhydride can be used
in place of the acetic anhydride, including for example
palmityl anhydride. The fragments may also be derivatized
by the formation of O-ether derivatives of the sugar
hydroxyl groups, for example, by the treatment of the
peptide-chitosan complex with methyliiodide plus silver
oxide in DMF. In both cases, the Fmoc and side-chain
protecting groups on the peptide are finally removed by
treatment with organic base followed by 95~ TFA in DCM. The
fragments have adjuvant properties which increase the
immunogenicity of the peptide sequence.
The following Examples illustrate the present
invention. Two sequences from human parathyroid hormone
(hPTH) were synthesized on chitosan. The use of linkers and
cleavable linkers is also illustrated.
2126132
Example 1
-
Chitosan, derived from squid pen chitin (Sea
Fisheries Institute, Gdynia, Poland), was dissolved in 1 M
acetic acid and freeze-dried to give a white soft material.
The chitosan was about 79% deacetylated, as determined
according to the method of Neugebauer et al (Carbohyd Res
189: 363-367; 1989).
A cleavable linker was conjugated to the free amino
groups of 150 mg of the chitosan preparation. The cleavable
linker was Fmoc-2,4-dimethoxy-4'-(carboxymethyloxy)-
benzhydrylamine trialkoxy~enhydrylamine (Bernatowicz et al
Tetrahedron Letters 30: 4645; 1989). The cleavable linker-
chitosan complex had the following formula:
Me~OM
NH - C H
OCH2C-chit~n
The cleavable linker-chitosan complex and 150 mg of
unconjugated chitosan preparation were placed in a column of
a continuous-flow peptide synthesizer (Milligen 9050 Plus~).
The two types of chitosan were physically separated in the
column by a frit. The packed column was washed with
dichloromethane (DCM), 20% diisopropylethylamine (DIPEA) in
DCM, and DMF. Synthesis of the following peptide sequence
was carried out automatically:
Val-Arg-Ala-Tyr-Asn-Gln-Pro-Ala-Gly-Asp-Val-Arg
The side-chains of the amino acids were protected
in the following manner: (1) the guanido group of arginine
was protected as the Pmc (2r2~5~7~8-pentamethyl-chroman-6-
2126132
sulfonyl) derivative thereof; (2) the carboxyl group of
aspartic acid was protected as the t-butyl ester thereof;
(3) the amide nitrogens of glutamine and asparagine were
protected as the trityl derivatives thereof; and (4) the
hydroxyl group of tyrosine was protected as the t-butyl
ester thereof. Amino acid derivatives were purchased from
Bachem Chemicals, California, U.S.A. The ~-amino groups of
the amino acids were protected with an Fmoc group during
coupling.
Couplings were performed by preactivation of the
protected amino acids ln situ with a mixture of
TBTU/HOBT/NMM in DMF. A four-fold excess of activated amino
acids was used with double coupling throughout the
synthesis. After each coupling step, unreacted amino acids
were "capped" by reaction with acetic anhydride in the
presence of HOBT to prevent the formation of deletion
analogues, which would be formed if the following amino acid
residue were added without the capping step and the previous
coupling had not gone to completion. The coupling times for
arginine and glycine additions were increased from 30
minutes to 60 minutes.
Fmoc deprotection was accomplished by a 6 minute
flow at 6 ml/min of 20% piperidine in DMF. The release was
monitored at 300 nm.
After attachment of the first amino acid residue,
the chitosan was acetylated with acetic anhydride to
acetylate any unreacted amino groups on the chitosan.
After completion of the synthesis, the peptide-
chitosan complexes were washed, in the column, with DMF, DCM
and DMF. Both the cleavable linker-chitosan complex and the
peptide-chitosan complex were treated with 95% TFA/water
plus 3% each of phenol and thioanisole and 1.5%
ethanedithiol ("scavengers") in DCM to cleave the cleavable
linker and to remove the side-chain protecting groups. The
peptide product was precipitated by dropping into a large
excess of diethylether and the precipitate was collected by
centrifugation and freeze-dried.
-
~I26I32
The product was analyzed by HPLC on a SupelcoTM C18
silica column (4.6 x 25 mm, 5 ~m particle size). The column
was eluted with a 0 - 50% gradient of 0.1% TFA/acetonitrile
in 0.1% TFA/water. The elution pattern of the crude
product, monitored at 214 nm, is shown in Figure 1. The
correct product is indicated with an asterisk and was
identified by FABMS (Fast Atomic Bombardment Mass
Spectroscopy) as having a mass of 1344.3 (expected mass:
1344.55). The peptide amide product had the following
sequence:
Val-Arg-Ala-Tyr-Asn-Gln-Pro-Ala-Gly-Asp-Val-Arg-NH2
The remaining peptide-chitosan complex is suitable
for injection into an animal for the generation of an immune
response.
Example 2
Chitosan, derived from squid pen chitin (Sea
Fisheries Institute, Gdynia, Poland), was dissolved in 1 M
acetic acid and freeze-dried to give a white soft material.
The chitosan was about 79% deacetylated, as determined
according to the method of Neugebauer et al (Carbohyd Res
189: 363-367; 1989).
150 mg of the chitosan preparation were placed in
a column of a continuous-flow peptide synthesizer (Milligen
9050 PlusTM). The packed column was washed with DCM, 20%
DIPEA in DCM, and DMF. Synthesis of hPTH-(44-68), having
the following sequence:
Arg-Asp-Ala-Gly-Ser-Gln-Arg-Pro-
Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-
Val-Glu-Ser-His-Glu-Lys-Ser-Leu-Gly
was carried out automatically.
212~132
The side-chains were protected using amino acid
derivatives, including the Mtr derivative of arginine; the
t-butyl ester of aspartic acid, glutamic acid and serine;
the trityl derivative of asparagine, glutamine and
S histidine; and the t-butyloxycarbonyl derivative of lysine.
The ~-amino group of the amino acids were protected by Fmoc
groups.
Couplings were performed by preactivation of the
protected amino acids ln situ with a mixture of 0.3 M
TBTU/HOBT in DMF and 0.6 M DIPEA in DMF.
A four-fold excess of activated amino acids was
used, based on the total amount of free amino groups
theoretically available upon initiation of the synthesis.
Double couplings were used for isoleucine and valine, with
a preceding solvent exchange into DCM/DMF (1:1 v/v). Fmoc
deprotection was accomplished by a 6 minute flow at 6 ml/min
of 20% piperidine in DMF. The release of the Fmoc group was
monitored at 300 nm.
The peptide was synthesized directly on the free
amino groups of the chitosan. After attachment of the first
amino acid residue, the chitosan was acylated with palmityl
anhydride followed by acetyl anhydride, in 50% pyridine/DCM
overnight. After completion of the synthesis, side-chain
deprotection, without cleavage from the support, was
accomplished with 10 ml of 95% TFA/water in the presence of
appropriate scavengers (thioanisole, phenol, and 1,2-
ethanedithiol) by stirring for 2 hours to dissolution.
Under these conditions, cleavage of the glycosidic bond of
the chitosan is expected to be minimal (Otvos et al,
Tetrahedron Letters 31: 5889-5892; 1990). The product was
precipitated from the deprotection solution with 100 ml of
diethyl ether. The final precipitated product was filtered,
washed with diethyl ether, suspended in 20% acetic acid, and
lyophilized.
Amino acid analysis indicated that about 2% of the
hPTH-(44-68)-chitosan complex consisted of peptide. The
12
212gl32
hPTH-(44-68)-chitosan complex was washed extensively prior
to injection into rabbits (Example 3).
Example 3
.
Four New Zealand white rabbits were each initially
injected subcutaneously with 0.25 mg of a suspension of the
hPTH-(44-68)-chitosan complex of Example 2 in 10 mM sodium
phosphate, pH 7.2, 150 mM sodium chloride (phosphate
buffered saline, PBS). Three of the rabbits were co-
injected with Freund's incomplete adjuvant tFIA). Booster
injections, at 6 and 8 weeks after initial injection,
contained 2 mg of the complex in 1 ml of PBS so that the
total peptide in each injection of the hPTH-(44-68)-chitosan
complex was from about 20 to 40 ~g.
Antibody responses were measured by enzyme-linked
immunosorbent assays (ELISA). ELISA assays were performed
using hPTH prepared as described in Sung et al (J Biol Chem
266: 2831-2835; 1991). Titer plates were coated with hPTH
by incubating each well for 16 hours at 4C with 100 ~1 of
5 ~g/ml hPTH in 50 mM sodium carbonate, pH 9.6. After
washing three times with 0.02 M Tris, 0.15 M sodium
chloride, pH 7.4 (TBS), the wells were saturated by further
incubation (1 hour, 22C) with 1% skim milk powder in 0.02
M Tris, pH 7.4. The plate was then washed three times with
TBS, 0.01 M ethylenediamine tetraacetate (EDTA), and 100 ~1
of antisera to the hPTH-(44-68)-chitosan complex diluted
into TBS was added to each well.
- After incubation at 22C for 2 hours, the wells
were washed three times with TBS, and incubated with 100 ~1
of a 1:500 dilution of alkaline phosphatase conjugated
affinity-purified goat anti-rabbit IgG in TBS (2 hours,
22C). The wells were then washed three times with 1% skim
milk powder in TBS, once with diethanolamine buffer (10 mM
diethanolamine, pH 9.8, 5 mM MgCl2), and 100 ~1 of 1 mg/ml p-
nitrophenylphosphate in ethanolamine buffer was added.
2126~32
After incubation for 30 minutes at 22C, the reaction was
terminated by the addition of 50 ~l of 4 M NaOH and the
plate was read at 405 nm. A control consisting of an
equivalent dilution of pre-immune antisera was subtracted
from each reading.
The results of the ELISA assays are shown in Figure
2. Three of the four rabbits were injected with both the
hPTH-(44-68)-chitosan complex and FIA. The results are
represented by the o, and ~ curves. The fourth rabbit was
injected without added FIA (- curve). Antisera were tested
nine weeks after initial injection, i.e. one week after the
final injection.
Example 4
Chitosan was prepared as described in Example 2.
150 mg of the chitosan preparation were placed in a column
of a continuous-flow peptide synthesizer (Milligen 9050
PlusTM). The packed column was washed with DCM, 20% DIPEA in
DCM, and DMF. A linker residue (N-Fmoc-~-aminocaproic acid)
was coupled 3 times, each time using a four-fold molar
excess of benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate (PyBOP)/DIPEA (1:2.5) to the
free amino groups of the chitosan. After washing with DMF,
the unreacted amino groups of the chitosan were palmitylated
with palmityl anhydride in dry pyridine (1:1 v/v) followed
by acetylation in the same manner.
Synthesis of hPTH-(1-17), having the following
sequence:
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-
His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser
was carried out automatically, as described in Example 2.
In the synthesis of the hPTH-(1-17) analogue, N-
methylmorpholine was used in place of DIPEA.
212fil32
`~ Example S
The hPTH-(1-17)-chitosan complex of Example 4 was
tested as an antigen in rabbits, as described in Example 3.
The results of the ELISA assays are shown in Figure 3. No
adjuvant was used in several tests of the palmitylated
derivatives, and the results were observed to be similar to
that obtained in the presence of added Freund's incomplete
adjuvant to the injected material.
1~ Figure 3 illustrates the ELISA results of antisera
from two rabbits injected with the hPTH-(1-17)-chitosan
complex with no added FIA.
