U.S. patent application number 16/773241 was filed with the patent office on 2020-10-01 for amino acid and peptide conjugates and conjugation process.
The applicant listed for this patent is AUCKLAND UNISERVICES LIMITED. Invention is credited to Margaret Anne Brimble, Peter Roderick Dunbar, Geoffrey Martyn Williams, Thomas Hugh Wright.
Application Number | 20200306370 16/773241 |
Document ID | / |
Family ID | 1000004887283 |
Filed Date | 2020-10-01 |
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United States Patent
Application |
20200306370 |
Kind Code |
A1 |
Brimble; Margaret Anne ; et
al. |
October 1, 2020 |
AMINO ACID AND PEPTIDE CONJUGATES AND CONJUGATION PROCESS
Abstract
The present invention relates to amino acid and peptide
conjugates, methods for making amino acid and peptide conjugates,
conjugates produced by the methods, pharmaceutical compositions
comprising the conjugates, methods of eliciting immune responses in
a subject and methods of vaccinating a subject, uses of the
conjugates for the same, and uses of the conjugates in the
manufacture of medicaments for the same.
Inventors: |
Brimble; Margaret Anne;
(Auckland, NZ) ; Wright; Thomas Hugh; (Auckland,
NZ) ; Dunbar; Peter Roderick; (Auckland, NZ) ;
Williams; Geoffrey Martyn; (Auckland, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AUCKLAND UNISERVICES LIMITED |
Auckland |
|
NZ |
|
|
Family ID: |
1000004887283 |
Appl. No.: |
16/773241 |
Filed: |
January 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14392231 |
Dec 23, 2015 |
10576144 |
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PCT/IB2014/062648 |
Jun 27, 2014 |
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16773241 |
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Current U.S.
Class: |
1/1 ;
514/1.1 |
Current CPC
Class: |
A61K 2039/6018 20130101;
A61K 39/0011 20130101; C12N 2710/16134 20130101; A61K 39/12
20130101; A61K 2039/627 20130101; A61K 39/39 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/00 20060101 A61K039/00; A61K 39/12 20060101
A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2013 |
NZ |
612654 |
Claims
1-93. (canceled)
94: A compound of the formula (V): ##STR00043## wherein m is an
integer from 0 to 4; n is 1 or 2; R1 and R2 at each instance of m
are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; R3,
R4, R5, R8, and R9 are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; or R9 is an amino protecting group, L3-C(O), or A2;
R6 and R7 at each instance of n are each independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl, L1 is C5-21alkyl or C4-20heteroalkyl;
L3 is C1-6alkyl or C3-6cycloalkyl; A1 and A2 are each independently
an amino acid or a peptide; or A1 is OH or OP1, wherein P1 is a
carboxyl protecting group; wherein any alkyl, cycloalkyl or
heteroalkyl present in any of R1, R2, R4, R5, R6, R7, R8, R9, L1,
and L3 and any cycloalkyl in R3 is optionally substituted with one
or more substituents independently selected from the group
consisting of halo, CN, NO.sub.2, OH, NH.sub.2, NH(C1-6alkyl),
N(C1-6alkyl) (C1-6alkyl), C1-6haloalkyl, C1-6haloalkoxy,
C(O)NH.sub.2, C(O)NH(C1-6alkyl), C(O)N(C1-6alkyl) (C1-6alkyl),
SO.sub.2(C1-6alkyl), O(C1-6alkyl), S(C1-6alkyl), S(O) (C1-6alkyl),
C(O) (C1-6alkyl), and C1-6aliphatic, wherein 1) A1 is a peptide
comprising an epitope or 2) R9 is A2 and is a peptide comprising an
epitope; and wherein the peptide comprising the epitope comprises
at least 8 amino acids, or a pharmaceutically acceptable salt or
solvate thereof.
95: The compound of claim 94, wherein R9 is independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl; or R9 is L3-C(O) or A2; and A1 and A2
are each independently a peptide; or A1 is OH; provided that: at
least one of A1 and A2 comprises an epitope; and when R9 is not A2,
A1 is a peptide.
96: The compound of claim 94, wherein L1 is C5-21alkyl.
97: The compound of claim 94, wherein m is an integer from 0 to
2.
98: The compound of claim 94, wherein R1 and R2 at each instance of
m are each independently hydrogen.
99: The compound of claim 94, wherein R3 is hydrogen.
100: The compound of claim 94, wherein R4 and R5 are each
hydrogen.
101: The compound of claim 94, wherein R6 and R7 are each
hydrogen.
102: The compound of claim 94, wherein R8 is hydrogen and R9 is
hydrogen, an amino protecting group, L3-C(O), or A2.
103: The compound of claim 94, wherein L3 is Me.
104: The compound of claim 94, wherein the peptide comprises a
peptide epitope.
105: The compound of claim 94, wherein A1 is serine or a peptide
comprising serine as the first N-terminal amino acid residue.
106: The compound of claim 94, wherein A1 and/or A2 is a peptide
comprising a solubilising group comprising an amino acid sequence
comprising two or more hydrophilic amino acid residues in the
peptide chain.
107: The compound of claim 94 wherein the peptide comprises,
consists essentially of, or consists of an amino acid sequence
selected from the group consisting of a. 8 or more contiguous amino
acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4GARGPESRLLEFYLAMPFATPMEAELAR-
RSLAQDAPPL [SEQ ID NO:1], wherein Xaa.sub.1 is absent or is S,
Xaa.sub.2 is absent or is a hydrophilic amino acid, Xaa.sub.3 is
absent or is a hydrophilic amino acid, and Xaa.sub.4 is absent or
is one or more hydrophilic amino acids, b. 8 or more contiguous
amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL
[SEQ ID NO:2], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is
absent or is a hydrophilic amino acid, and Xaa.sub.3 is absent or
is from one to ten hydrophilic amino acids, c. 8 or more contiguous
amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID
NO:3], wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent
or is from one to four hydrophilic amino acids, d. 8 or more
contiguous amino acid residues from the sequence
SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID NO:4], e. 8 or
more contiguous amino acid residues from the sequence
GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID NO:5], f. 8 or more
contiguous amino acid residues from the sequence LAMPFATPM [SEQ ID
NO:6], g. 8 or more contiguous amino acid residues from the
sequence FATPMEAEL [SEQ ID NO:7], h. 8 or more contiguous amino
acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4VPGVLLKEFTVSGNILTIRLTAADHR [SEQ
ID NO:8], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent
or is a hydrophilic amino acid, Xaa.sub.3 is absent or is a
hydrophilic amino acid, and Xaa.sub.4 is absent or is one or more
hydrophilic amino acids, i. 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID
NO:9], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent or
is a hydrophilic amino acid, and Xaa.sub.3 is absent or is from one
to ten hydrophilic amino acids, j. 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO:10],
wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent or is
from one to four hydrophilic amino acids, k. 8 or more contiguous
amino acid residues from the sequence
SKKKKVPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO:11], l. 8 or more
contiguous amino acid residues from the sequence
VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO:12], m. 8 or more contiguous
amino acid residues from the sequence EFTVSGNIL [SEQ ID NO:13], n.
8 or more contiguous amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4LQQLSLLMWITQCFLPVFLAQPPSGQRR
[SEQ ID NO:14], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is
absent or is a hydrophilic amino acid, Xaa.sub.3 is absent or is a
hydrophilic amino acid, and Xaa.sub.4 is absent or is one or more
hydrophilic amino acids o. 8 or more contiguous amino acid residues
from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID
NO:15], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent or
is a hydrophilic amino acid, and Xaa.sub.3 is absent or is from one
to ten hydrophilic amino acids, p. 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO:16],
wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent or is
from one to four hydrophilic amino acids, q. 8 or more contiguous
amino acid residues from the sequence
SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO:17], r. 8 or more
contiguous amino acid residues from the sequence
LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO:18], s. 8 or more
contiguous amino acid residues from the sequence SLLMWITQCFLPVF
[SEQ ID NO:19], t. 8 or more contiguous amino acid residues from
the sequence SLLMWITQC [SEQ ID NO:20], u. the sequence of any one
of SEQ ID NOs: 1 to 20, v. or any combination of two or more of (a)
to (u) above.
108: A pharmaceutical composition comprising an effective amount of
a peptide conjugate of claim 94 or a pharmaceutically acceptable
salt or solvate thereof, and a pharmaceutically acceptable
carrier.
109: A method of vaccinating or eliciting an immune response in a
subject comprising administering to the subject an effective amount
of a peptide conjugate of claim 94 or a pharmaceutically acceptable
salt or solvate thereof or an effective amount of a pharmaceutical
composition comprising a peptide conjugate of claim 94 or a
pharmaceutically acceptable salt or solvate thereof, and a
pharmaceutically acceptable carrier.
110: The compound of claim 94, wherein L1 is C11-C21alkyl; m is 0;
R3 is hydrogen; R4 and R5 are each hydrogen; n is 1, R6, R7, and R8
are each hydrogen; R9 is hydrogen or L3-C(O), wherein L3 is
methyl.
111: A method for making a peptide conjugate according to claim 94,
the method comprising reacting a lipid-containing conjugation
partner, and an amino acid-comprising conjugation partner under
conditions effective to conjugate the lipid-containing conjugation
partner to the amino acid-comprising conjugation partner by the
hydrothiolation of a carbon-carbon double bond with a thiol.
Description
TECHNICAL FIELD
[0001] The present invention relates to amino acid and peptide
conjugates, methods for making amino acid and peptide conjugates,
conjugates produced by the methods, pharmaceutical compositions
comprising the conjugates, methods of eliciting immune responses in
a subject and methods of vaccinating a subject, uses of the
conjugates for the same, and uses of the conjugates in the
manufacture of medicaments for the same.
BACKGROUND ART
[0002] Synthetic peptide vaccines generally comprise a synthetic
copy of an immunogenic part of protein antigens. This approach to
vaccine development has a number of advantages, including ease of
synthesis, avoidance of potentially toxic biological by-products
and straightforward characterisation.
[0003] A key issue in the development of peptide vaccines is the
lack of immunogenicity displayed by peptides as sole vaccine
components. It is usually necessary to include in the vaccine an
adjuvant, designed to activate components of the innate immune
system (e.g. Freund's adjuvant).
[0004] An alternative strategy in peptide vaccine design is to
create self-adjuvanting vaccines in which the peptide epitope of
interest is covalently linked to an appropriate adjuvant. Such
self-adjuvanting vaccines may have enhanced antigen uptake,
presentation and dendritic cell maturation compared to simple
co-formulation of the antigen with an external adjuvant.
[0005] Several self-adjuvanting vaccines have been developed, but
preparation of the vaccines can be complicated.
[0006] There is an ongoing need for new self-adjuvanting vaccines
and new methods of making self-adjuvanting vaccines. It is an
object of the present invention to go some way towards meeting
these needs; and/or to at least provide the public with a useful
choice.
[0007] Other objects of the invention may become apparent from the
following description which is given by way of example only.
[0008] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides a method for
making an amino acid or peptide conjugate, the method comprising
reacting [0010] a lipid-containing conjugation partner, and [0011]
an amino acid-comprising conjugation partner [0012] under
conditions effective to conjugate the lipid-containing conjugation
partner to the amino acid-comprising conjugation partner by the
hydrothiolation of a carbon-carbon double bond with a thiol.
[0013] Any of the embodiments described herein relate to any of the
aspects herein.
[0014] In one embodiment, the amino acid-comprising conjuation
partner is a peptide-containing conjugation partner, and the
lipid-containing conjugation partner is coupled to the peptide of
the peptide-containing conjugation partner.
[0015] In some embodiments, the lipid-containing conjugation
partner is conjugated to the or an amino acid of the amino
acid-containing conjugation partner or the peptide of the
peptide-containing conjugation partner.
[0016] In certain embodiments, the lipid-containing conjugation
partner is conjugated to the or an amino acid of the amino
acid-containing conjugation partner.
[0017] Accordingly, in another aspect, the present invention
provides a method for making a peptide conjugate, the method
comprising reacting [0018] a lipid-containing conjugation partner,
and [0019] a peptide-containing conjugation partner [0020] under
conditions effective to conjugate the lipid-containing conjugation
partner to the peptide of the peptide-containing conjugation
partner by the hydrothiolation of a carbon-carbon double bond with
a thiol.
[0021] In one embodiment, the conjugate is a lipopeptide, such that
the method is for making a lipopeptide.
[0022] In one embodiment, the lipid-containing conjugation partner
comprises the carbon-carbon double bond, and the peptide of the
peptide-containing conjugation partner comprises the thiol.
[0023] In one embodiment, the amino acid-comprising conjugation
partner comprises an epitope. In one embodiment, the
peptide-containing conjugation partner comprises an epitope. In one
embodiment, the amino acid-comprising conjugation partner comprises
two or more epitopes. In one embodiment, the peptide-containing
conjugation partner comprises two or more epitopes. In one
embodiment, the peptide conjugate comprises two or more epitopes.
In one embodiment, the epitope is a peptide epitope. In one
embodiment, the amino acid-comprising conjugation partner consists
of a peptide. In one embodiment, the amino acid-comprising
conjugation partner consists of a peptide comprising a peptide
epitope. In one embodiment, the peptide-containing conjugation
partner consists of a peptide. In one embodiment, the
peptide-containing conjugation partner consists of a peptide
comprising a peptide epitope.
[0024] In some embodiments, the amino acid-comprising conjugation
partner comprises an epitope bound to the or an amino acid of the
conjugation partner. In some embodiments, the peptide-containing
conjugation partner comprises an epitope bound to the peptide of
the peptide containing conjugation partner. In some embodiments,
the epitope is bound to the peptide via linker group.
[0025] In some embodiments, the amino acid-comprising conjugation
partner comprises a peptide epitope bound to the or an amino acid
of the conjugation partner via a linker group. In some embodiments,
the peptide-containing conjugation partner comprises a peptide
epitope bound to the peptide via a linker group.
[0026] In some embodiments, the amino acid-comprising conjugation
partner and/or the peptide-containing conjugation partner comprises
an antigenic peptide. In some embodiments, the peptide conjugate
comprises an antigenic peptide.
[0027] In some embodiments, the method further comprises coupling
the amino acid of the amino acid conjugate to an amino acid or a
peptide to provide a peptide conjugate.
[0028] In some embodiments, coupling a peptide comprises
individually coupling one or more amino acids and/or one or more
peptides.
[0029] In some embodiments, the method further comprises coupling
the amino acid of the amino acid conjugate or an amino acid of the
peptide conjugate to an amino acid or a peptide so as to provide a
peptide conjugate comprising a linker group or one or more amino
acids thereof.
[0030] In some embodiments, the method further comprises coupling
an amino acid of the peptide conjugate comprising a linker group or
one or more amino acids thereof to an amino acid or a peptide so as
to provide a peptide conjugate comprising a peptide epitope bound
to the amino acid to which lipid-containing conjugation partner is
conjugated via a linker group.
[0031] In some embodiments, the amino acid of the peptide conjugate
to which the lipid-containing conjugate is conjugated is an
N-terminal amino acid residue.
[0032] In some embodiments, the method further comprises coupling
the amino acid of the amino acid conjugate or an amino acid of the
peptide conjugate to an amino acid or a peptide so as to provide a
peptide conjugate comprising a peptide epitope.
[0033] In some embodiments, the method further comprises coupling
an epitope to the amino acid of the amino acid conjugate or an
amino acid of the peptide conjugate. In some embodiments, the
method further comprises coupling a peptide epitope to the amino
acid of the amino acid conjugate or an amino acid of the peptide
conjugate. In some embodiments, the epitope is coupled or bound via
a linker group.
[0034] In some embodiments, the method further comprises coupling
an epitope to the peptide of the peptide conjugate. In some
embodiments, the method further comprises coupling a peptide
epitope to the peptide of the peptide conjugate. In some
embodiments, the epitope is bound to the peptide via a linker
group.
[0035] In one embodiment, the amino acid-comprising conjugation
partner consists of an amino acid. In one embodiment, the carboxyl
group of the C-terminus of the amino acid is protected with a
carboxyl protecting group and/or the Na-amino group of the amino
acid is protected with an amino protecting group.
[0036] In some embodiments, the carboxyl group of the C-terminus of
the peptide is protected with a carboxyl protecting group and/or
the Na-amino group of the peptide is protected with an amino
protecting group.
[0037] In one embodiment, the lipid-containing conjugation partner
comprises one or more optionally substituted straight or branched
aliphatic or heteroaliphatic chains each containing at least 4
chain-linked atoms. In one embodiment, the lipid-containing
conjugation partner comprises one or more optionally substituted
straight or branched aliphatic or heteroaliphatic chains each
containing at least 6 chain-linked atoms. In one specifically
contemplated embodiment, the one or more chains are aliphatic. In
one specifically contemplated embodiment, the one or more chains
are saturated.
[0038] In some embodiments, the one or more chains are optionally
substituted. In some embodiments, the one or more chains are
optionally substituted with one or more aryl groups.
[0039] In some embodiments, the one or more chains comprise at
least 4, 6, 8, 10, 12, or 14 chain-linked atoms. In some
embodiments, the one or more chains comprise from 4-22, 6-22, 8-22,
10-22, 12-22, or 14-22 chain-linked atoms.
[0040] In one embodiment, the one or more chains are covalently
bound to a moiety comprising the carbon-carbon double bond or the
thiol by a heteroatom containing functional group. Examples of
heteroatom containing functional groups include but are not limited
to ether, amine, sulfide, sulfoxide, sulfone, ester, amide,
carbonate, carbamate, and urea groups.
[0041] In exemplary embodiments, the one or more chains are
covalently bound to the moiety by ester functional groups.
[0042] In one embodiment, the lipid-containing conjugation partner
comprises one or more saturated or unsaturated fatty acid esters.
In some embodiments, the fatty acid is saturated. In one
embodiment, one or more fatty acid ester is bound to the moiety
comprising to carbon-carbon double bond or thiol. In one
embodiment, the ester is an ester of the carboxyl group of the
fatty acid and an alcohol of the moiety.
[0043] In one embodiment, the fatty acid is a C4-22 fatty acid. In
one embodiment, the fatty acid is a C6-22 fatty acid. In another
embodiment, the fatty acid is a C10-22 fatty acid. In yet another
embodiment, the fatty acid is a C12-22 fatty acid. In one exemplary
embodiment, the fatty acid is a C12, C14, C16, C18, or C20 fatty
acid.
[0044] In some embodiments, the fatty acid is lauric acid, myristic
acid, palmitic acid, stearic acid, arachic acid, palmitoleic acid,
oleic acid, elaidic acid, linoleic acid, .alpha.-linolenic acid,
and arachidonic acid. In one embodiment, the fatty acid is lauric
acid, myristic acid, palmitic acid, or stearic acid. In a
specifically contemplated embodiment, the fatty acid is palmitic
acid.
[0045] In one exemplary embodiment, the lipid-containing
conjugation partner comprises one or two fatty acid esters. In a
specifically contemplated embodiment, the lipid-containing
conjugation partner comprises one fatty acid ester.
[0046] In certain embodiments, the fatty acid ester is an ester of
an alcohol comprising the carbon-carbon double bond or thiol. In
one embodiment, the alcohol is a monohydric, dihydric, or trihydric
C2-6 aliphatic alcohol. In another embodiment, the alcohol is a
monohydric or dihydric C2-4 aliphatic alcohol. In one exemplary
embodiment, the alcohol is a monohydric C2 aliphatic or monohydric
or dihydric C3 aliphatic alcohol. In a specifically contemplated
embodiment, the alcohol is a monohydric C2 alcohol.
[0047] In certain embodiments, the lipid-containing conjugation
partner comprises the carbon-carbon double bond.
[0048] In one exemplary embodiment, the alcohol comprises the
carbon-carbon double bond. In a specifically contemplated
embodiment, the alcohol is vinyl alcohol.
[0049] In specifically contemplated embodiments, the peptide is a
synthetic peptide.
[0050] In one embodiment, the amino acid-comprising conjugation
partner and/or peptide conjugate comprises a synthetic peptide. In
some embodiments, the synthetic peptide is a peptide prepared by a
method comprising solid phase peptide synthesis (SPPS).
[0051] In some embodiments, the or an amino acid of the amino
acid-comprising conjugation partner comprises the carbon-carbon
double bond or thiol. In some embodiments, an amino acid residue of
the peptide of the peptide-containing conjugation partner comprises
the carbon-carbon double bond or thiol.
[0052] In some embodiments, the amino acid residue comprising the
carbon-carbon double bond or thiol is a terminal amino acid
residue. In some embodiments, the terminal amino acid residue is an
N-terminal residue.
[0053] In some embodiments, the Na-amino group of the amino acid
comprising the carbon-carbon double bond or thiol is acylated.
[0054] In certain embodiments, the method further comprises
acylating the Na-amino group of the amino acid of the amino acid
conjugate or the amino acid residue of the peptide conjugate to
which the lipid-containing conjugation partner is conjugated. In
certain embodiments, the method further comprises acylating the
Na-amino group with a C2-20 fatty acid.
[0055] In certain embodiments, the or an amino acid of the amino
acid-comprising conjugation partner comprises the thiol. In certain
embodiments, an amino acid residue of the peptide of the
peptide-containing conjugation partner comprises the thiol. In
certain embodiments, the thiol is the thiol of a cysteine
residue.
[0056] In certain embodiments, the cysteine residue is a terminal
residue. In certain embodiments, the cysteine residue is an
N-terminal residue.
[0057] In some embodiments, the amino group of the cysteine residue
is acylated.
[0058] In one embodiment, the amino group is acylated with a C2-20
fatty acid.
[0059] In one exemplary embodiment, the C2-20 fatty acid is acetyl
or palmitoyl. In another exemplary embodiment, the C2-20 fatty acid
is acetyl.
[0060] In some embodiments, the amino acid-comprising conjugation
partner and/or peptide conjugate comprises from 8 to 220, 8 to 200,
8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70,
8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15
amino acids. In some embodiments, the peptide-containing
conjugation partner comprises from 8 to 220, 8 to 200, 8 to 175, 8
to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8
to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino
acids.
[0061] In one exemplary embodiment, the amino acid-comprising
conjugation partner and/or peptide conjugate comprises a peptide
comprising from 8 to 60 amino acids. In one exemplary embodiment,
the peptide comprises from 8 to 60 amino acids.
[0062] In other embodiments, the amino acid-comprising conjugation
partner and/or peptide conjugate comprises from 5 to 220, 8 to 220,
5 to 175, 8 to 175, 8 to 150, 10 to 150, 15 to 125, 20 to 100, 20
to 80, 20 to 60, 25 to 100, 25 to 80, 25 to 60, 30 to 80, 40 to 60,
or 50 to 60 amino acids. In other embodiments, the
peptide-containing conjugation partner comprises from 5 to 220, 8
to 220, 5 to 175, 8 to 175, 8 to 150, 10 to 150, 15 to 125, 20 to
100, 20 to 80, 20 to 60, 25 to 100, 25 to 80, 25 to 60, 30 to 80,
40 to 60, or 50 to 60 amino acids.
[0063] In other embodiments, the amino acid comprising conjugation
partner and/or peptide conjugate comprises from 5 to 150, 5 to 125,
5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5
to 20, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8
to 40, 8 to 30, 8 to 25, or 8 to 20 amino acids. In other
embodiments, the peptide-containing conjugation partner comprises
from 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to
40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to 125, 8 to 100, 8 to
75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or 8 to 20 amino
acids.
[0064] In one embodiment, the amino acid-comprising conjugation
partner and/or peptide conjugate comprises one or more solubilising
groups. In one embodiment, the peptide-containing conjugation
partner comprises one or more solubilising groups.
[0065] In certain embodiments, the solubilising group is an amino
acid sequence comprising two or more hydrophilic amino acid
residues in the peptide chain. In certain embodiments, the
solubilising group is an amino acid sequence comprising a sequence
of two or more consecutive hydrophilic amino acid residues in the
peptide chain. In one embodiment, the hydrophilic amino acid
residues are cationic amino acid residues. In one embodiment, the
cationic amino acid residues are arginine or lysine residues. In
one specifically contemplated embodiment, the cationic amino acid
residues are lysine residues. In one embodiment, the sequence
comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino
acids. In one embodiment, the solubilising group is a tri-, tetra-,
penta-, hexa-, or hepta-lysine sequence. In one specifically
contemplated embodiment, the solubilising group is a tetralysine
sequence.
[0066] In some embodiments, the peptide conjugate and/or amino-acid
comprising conjugation partner comprises a serine residue adjacent
to the amino acid residue to which the lipid-containing conjugation
partner is conjugated. In a specifically contemplated embodiment,
the peptide of the peptide-containing conjugation partner comprises
a serine residue adjacent to the amino acid residue to which the
lipid-containing conjugation partner is conjugated. In an exemplary
embodiment, the amino acid residue to which the lipid-containing
conjugation partner is conjugated is N-terminal. In a specifically
contemplated embodiment, the peptide further comprises a
consecutive sequence of two or more hydrophilic amino acid residues
adjacent to the serine residue.
[0067] In certain embodiments, the peptide conjugate and/or
amino-acid comprising conjugation partner comprises a consecutive
sequence of two or more hydrophilic amino acid residues adjacent to
the serine residue.
[0068] In certain embodiments, the peptide conjugate and/or amino
acid-comprising conjugation partner comprises only naturally
occurring amino acids. In certain embodiments, the
peptide-containing conjugation partner comprises only naturally
occurring amino acids. In other embodiments, 75% or more, 80% or
more, 85% or more, 90% or more, 95% or more, 97% or more, or 99% or
more of the amino acid residues in the peptide are naturally
occurring amino acids.
[0069] In other embodiments, 75% or more, 80% or more, 85% or more,
90% or more, 95% or more, 97% or more, or 99% or more of the amino
acid residues in the peptide conjugate and/or amino acid-comprising
conjugation partner are naturally occurring amino acids.
[0070] In exemplary embodiments, the peptide conjugate and/or amino
acid-comprising conjugation partner comprises a peptide comprising
a peptide epitope. In exemplary embodiments, the peptide of the
peptide-containing conjugation partner comprises one or more
peptide epitopes.
[0071] In one embodiment, the peptide comprises, consists of, or
consists essentially of an amino acid sequence selected from the
group consisting of [0072] (a) 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4GARGPESRLLEFYLAMPFATPMEAELAR-
RSLAQDAPPL [SEQ ID NO: 1], wherein Xaa.sub.1 is absent or is S,
Xaa.sub.2 is absent or is a hydrophilic amino acid, Xaa.sub.3 is
absent or is a hydrophilic amino acid, and Xaa.sub.4 is absent or
is one or more hydrophilic amino acids, [0073] (b) 8 or more
contiguous amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL
[SEQ ID NO:2], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is
absent or is a hydrophilic amino acid, and Xaa.sub.3 is absent or
is from one to ten hydrophilic amino acids, [0074] (c) 8 or more
contiguous amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID
NO:3], wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent
or is from one to four hydrophilic amino acids, [0075] (d) 8 or
more contiguous amino acid residues from the sequence
TABLE-US-00001 [0075] [SEQ ID NO: 4]
SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL,
[0076] (e) the sequence of any one of SEQ ID NOs: 1 to 4, [0077]
(f) 8 or more contiguous amino acid residues from the sequence
TABLE-US-00002 [0077] [SEQ ID NO: 5]
GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL,
[0078] (g) the sequence of SEQ ID NO: 5, [0079] (h) 8 or more
contiguous amino acid residues from the sequence LAMPFATPM [SEQ ID
NO:6], [0080] (i) the sequence of SEQ ID NO: 6, [0081] (j) 8 or
more contiguous amino acid residues from the sequence FATPMEAEL
[SEQ ID NO:7], [0082] (k) the sequence of SEQ ID NO: 7, [0083] (l)
8 or more contiguous amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4VPGVLLKEFTVSGNILTIRLTAADHR [SEQ
ID NO:8], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent
or is a hydrophilic amino acid, Xaa.sub.3 is absent or is a
hydrophilic amino acid, and Xaa.sub.4 is absent or is one or more
hydrophilic amino acids, [0084] (m) 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID
NO:9], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent or
is a hydrophilic amino acid, and Xaa.sub.3 is absent or is from one
to ten hydrophilic amino acids, [0085] (n) 8 or more contiguous
amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO: 10],
wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent or is
from one to four hydrophilic amino acids, (o) 8 or more contiguous
amino acid residues from the sequence
TABLE-US-00003 [0085] [SEQ ID NO: 11]
SKKKKVPGVLLKEFTVSGNILTIRLTAADHR,
[0086] (p) the sequence of any one of SEQ ID NOs: 8 to 11, [0087]
(q) 8 or more contiguous amino acid residues from the sequence
TABLE-US-00004 [0087] [SEQ ID NO: 12]
VPGVLLKEFTVSGNILTIRLTAADHR,
[0088] (r) the sequence of SEQ ID NO: 12, [0089] (s) 8 or more
contiguous amino acid residues from the sequence EFTVSGNIL [SEQ ID
NO: 13], [0090] (t) the sequence of SEQ ID NO: 13, [0091] (u) 8 or
more contiguous amino acid residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3Xaa.sub.4LQQLSLLMWITQCFLPVFLAQPPSGQRR
[SEQ ID NO: 14], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is
absent or is a hydrophilic amino acid, Xaa.sub.3 is absent or is a
hydrophilic amino acid, and Xaa.sub.4 is absent or is one or more
hydrophilic amino acids [0092] (v) 8 or more contiguous amino acid
residues from the sequence
Xaa.sub.1Xaa.sub.2Xaa.sub.3LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO:
15], wherein Xaa.sub.1 is absent or is S, Xaa.sub.2 is absent or is
a hydrophilic amino acid, and Xaa.sub.3 is absent or is from one to
ten hydrophilic amino acids, [0093] (w) 8 or more contiguous amino
acid residues from the sequence
Xaa.sub.1Xaa.sub.2LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 16],
wherein Xaa.sub.1 is absent or is S, and Xaa.sub.2 is absent or is
from one to four hydrophilic amino acids, [0094] (x) 8 or more
contiguous amino acid residues from the sequence
TABLE-US-00005 [0094] [SEQ ID NO: 17]
SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR,
[0095] (y) the sequence of any one of SEQ ID NOs: 14 to 17, [0096]
(z) 8 or more contiguous amino acid residues from the sequence
TABLE-US-00006 [0096] [SEQ ID NO: 18]
LQQLSLLMWITQCFLPVFLAQPPSGQRR,
[0097] (aa) the sequence of SEQ ID NO: 18, [0098] (bb) 8 or more
contiguous amino acid residues from the sequence
TABLE-US-00007 [0098] [SEQ ID NO: 19] SLLMWITQCFLPVF,
[0099] (cc) the sequence of SEQ ID NO: 19, [0100] (dd) 8 or more
contiguous amino acid residues from the sequence SLLMWITQC [SEQ ID
NO:20], [0101] (ee) the sequence of SEQ ID NO: 20, [0102] (ff) or
any combination of two or more of (a) to (ee) above.
[0103] In one exemplary embodiment, the peptide epitope is derived
from NY-ESO-1. In one specifically contemplated embodiment, the
peptide comprises, consists essentially of, or consists of an amino
acid sequence selected from the group consisting of 8 or more
contiguous amino acid residues from any one of SEQ ID NO: 5, 6, 7,
12, 13, 18, 19, and 20.
[0104] In one embodiment, the peptide comprises, consists
essentially of, or consists of an amino acid sequence selected from
the group consisting of any one of SEQ ID NO: 5, 6, 7, 12, 13, 18,
19, and 20.
[0105] In one embodiment, the peptide comprises, consists
essentially of, or consists of an amino acid sequence selected from
the group consisting of any one of SEQ ID NO: 5, 12, and 18.
[0106] In one embodiment, the peptide comprises, consists
essentially of, or consists of an amino acid sequence selected from
the group consisting of any one of SEQ ID NO: 5, 12, and 18.
[0107] In one embodiment, the peptide comprises, consists
essentially of, or consists of an amino acid sequence selected from
the group consisting of any one of SEQ ID NO: 4, 11, and 17.
[0108] In one specifically contemplated embodiment, the reactive
functional groups of the amino acids of the peptide-containing
conjugation partner are unprotected.
[0109] In certain embodiments, one or more reactive functional
groups of one or more amino acids of the peptide conjugate are
unprotected.
[0110] In certain embodiments, one or more reactive functional
groups of the amino acid of the amino acid conjugate are
unprotected.
[0111] In certain embodiments, one or more reactive functional
groups of one or more amino acids of the amino acid-comprising
conjugation partner are unprotected.
[0112] In certain embodiments, the amino acid-comprising
conjugation partner comprises a peptide, wherein the reactive
functional groups of the side chains of the amino acids of the
peptide are unprotected, with the exception of any thiols other
than the thiol to be reacted.
[0113] In one specifically contemplated embodiment, the reactive
functional groups of the amino acids of the peptide of the
peptide-containing conjugation partner are unprotected. In one
specifically contemplated embodiment, the reactive functional
groups of the amino acids of the peptide of the peptide-containing
conjugation partner are unprotected, with the exception of any
thiols other than the thiol to be reacted.
[0114] In one embodiment, the method comprises making an amino acid
or peptide-conjugate comprising a structure of the formula (A):
##STR00001##
[0115] wherein [0116] Z is selected from the group consisting of
--O--, --NR--, --S--, --S(O)--, --SO.sub.2--, --C(O)O--, --OC(O)--,
--C(O)NR--, --NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and
--NRC(O)NR--; [0117] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl,
wherein the alkyl or cycloalkyl is optionally substituted; [0118] m
is an integer from 0 to 4; [0119] n is 1 or 2; [0120] R1 and R2 at
each instance of m are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; or R1 is L2-C(O)--OC1-6alkyl; [0121] R3, R4, R5,
R8, and R9 are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; or R3 is L2-C(O)--OC1-6alkyl; [0122] or R9 is an
amino protecting group, L3-C(O), or A2; [0123] R6 and R7 at each
instance of n are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; [0124] L1 and L2 are each independently C5-21alkyl
or C4-20heteroalkyl; [0125] L3 is C1-21alkyl or C4-20heteroalkyl;
[0126] A1 and A2 are each independently an amino acid or a peptide;
or A1 is OH or OP1, wherein P1 is a carboxyl protecting group;
[0127] provided that: [0128] when R3 is L2-C(O)--OC1-6alkyl, R1 is
not L2-C(O)--OC1-6alkyl; and [0129] when m is an integer from 2 to
4, no more than one R1 is L2-C(O)--OC1-6alkyl; and [0130] wherein
any alkyl, cycloalkyl or heteroalkyl present in any of R1, R2, R3,
R4, R5, R6, R7, R8, R9, L1, L2 and L3 is optionally substituted;
[0131] or a pharmaceutically acceptable salt or solvate
thereof.
[0132] In one embodiment, the method comprises making a
peptide-conjugate comprising a structure of the formula (A):
##STR00002##
[0133] wherein [0134] Z is selected from the group consisting of
--O--, --NR--, --S--, --S(O)--, --SO.sub.2--, --C(O)O--, --OC(O)--,
--C(O)NR--, --NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and
--NRC(O)NR--; [0135] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl,
wherein the alkyl or cycloalkyl is optionally substituted; [0136] m
is an integer from 0 to 4; [0137] n is 1 or 2; [0138] R1 and R2 at
each instance of m are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; or R1 is L2-C(O)--OC1-6alkyl; [0139] R3, R4, R5,
R8, and R9 are each independently hydrogen, C1-6alkyl, or
C3-6cycloalkyl; or R3 is L2-C(O)--OC1-6alkyl; [0140] or R9 is
L3-C(O) or A2; [0141] R6 and R7 at each instance of n are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0142] L1 and
L2 are each independently C5-21alkyl or C4-20heteroalkyl; [0143] L3
is C1-21alkyl or C4-20heteroalkyl; [0144] A1 and A2 are each
independently a peptide; or A1 is OH; [0145] provided that: [0146]
when R9 is not A2, A1 is a peptide; [0147] when R3 is
L2-C(O)--OC1-6alkyl, R1 is not L2-C(O)--OC1-6alkyl; and [0148] when
m is an integer from 2 to 4, no more than one R1 is
L2-C(O)--OC1-6alkyl; and [0149] wherein any alkyl, cycloalkyl or
heteroalkyl present in any of R1, R2, R3, R4, R5, R6, R7, R8, R9,
L1, L2 and L3 is optionally substituted; [0150] or a
pharmaceutically acceptable salt or solvate thereof.
[0151] In one embodiment, the method comprises making an amino acid
or peptide-conjugate comprising a structure of the formula (B):
##STR00003##
[0152] wherein [0153] Z is selected from the group consisting of
--O--, --NR--, --S--, --S(O)--, --SO.sub.2--, --C(O)O--, --OC(O)--,
--C(O)NR--, --NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and
--NRC(O)NR--; [0154] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl,
wherein the alkyl or cycloalkyl is optionally substituted; [0155] p
is an integer from 0 to 4; [0156] q is an integer from 0 to 2;
[0157] R11 and R22 at each instance of p are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11 is
L2-C(O)--OC1-6alkyl; [0158] R33, R44, R55, R66, R77, R8, and R9 are
each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33
is L2-C(O)--OC1-6alkyl; [0159] or R9 is an amino protecting group,
L3-C(O), or A2; [0160] Ra and Rb at each instance of q are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0161] L1 and
L2 are each independently C5-21alkyl or C4-20heteroalkyl; [0162] L3
is C1-21alkyl or C4-20heteroalkyl; [0163] A1 and A2 are each
independently an amino acid or a peptide; or A1 is OH or OP1,
wherein P1 is a carboxyl protecting group; [0164] provided that:
[0165] when R33 is L2-C(O)--OC1-6alkyl, R11 is not
L2-C(O)--OC1-6alkyl; and [0166] when p is an integer from 2 to 4,
no more than one R11 is L2-C(O)--OC1-6alkyl; and [0167] wherein any
alkyl, cycloalkyl, or heteroalkyl present in any of R11, R22, R 33,
R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and L3 is optionally
substituted; [0168] or a pharmaceutically acceptable salt or
solvate thereof.
[0169] In one embodiment, the method comprises making a
peptide-conjugate comprising a structure of the formula (B):
##STR00004##
wherein [0170] Z is selected from the group consisting of --O--,
--NR--, --S--, --S(O)--, --S02-, --C(O)O--, --OC(O)--, --C(O)NR--,
--NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and --NRC(O)NR--;
[0171] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl, wherein the
alkyl or cycloalkyl is optionally substituted; [0172] p is an
integer from 0 to 4; [0173] q is an integer from 0 to 2; [0174] R11
and R22 at each instance of p are each independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl; or R11 is L2-C(O)--OC1-6alkyl; [0175]
R33, R44, R55, R66, R77, R8, and R9 are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is
L2-C(O)--OC1-6alkyl; [0176] or R9 is L3-C(O) or A2; [0177] Ra and
Rb at each instance of q are each independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl; [0178] L1 and L2 are each
independently C5-21alkyl or C4-20heteroalkyl; [0179] L3 is
C1-21alkyl or C4-20heteroalkyl; [0180] A1 and A2 are each
independently a peptide; or A1 is OH; [0181] provided that: [0182]
when R9 is not A2, A1 is a peptide; [0183] when R33 is
L2-C(O)--OC1-6alkyl, R11 is not L2-C(O)--OC1-6alkyl; and [0184]
when p is an integer from 2 to 4, no more than one R11 is
L2-C(O)--OC1-6alkyl; and [0185] wherein any alkyl, cycloalkyl, or
heteroalkyl present in any of R11, R22, R 33, R44, R55, R66, R77,
R8, R9, Ra, Rb, L1, L2, and L3 is optionally substituted; [0186] or
a pharmaceutically acceptable salt or solvate thereof.
[0187] In one embodiment, the lipid-containing conjugation partner
is a compound of the formula (A1):
##STR00005##
wherein [0188] Z is selected from the group consisting of --O--,
--NR--, --S--, --S(O)--, --SO.sub.2--, --C(O)O--, --OC(O)--,
--C(O)NR--, --NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and
--NRC(O)NR--; [0189] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl,
wherein the alkyl or cycloalkyl is optionally substituted; [0190] m
is an integer from 0 to 4; [0191] R1 and R2 at each instance of m
are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or
R1 is L2-C(O)--OC1-6alkyl; [0192] R3, R4, and R5 are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R3 is
L2-C(O)--OC1-6alkyl; [0193] L1 and L2 are each independently
C5-21alkyl or C4-20heteroalkyl; [0194] provided that: [0195] when
R3 is L2-C(O)--OC1-6alkyl, R1 is not L2-C(O)--OC1-6alkyl; and
[0196] when m is an integer from 2 to 4, no more than one R1 is
L2-C(O)--OC1-6alkyl; and [0197] wherein any alkyl, cycloalkyl or
heteroalkyl present in any of R1, R2, R3, R4, R5, L1, and L2 is
optionally substituted, [0198] or a pharmaceutically acceptable
salt or solvate thereof.
[0199] In another embodiment, the lipid containing conjugation
partner is a compound of the formula (B1):
##STR00006##
wherein [0200] Z is selected from the group consisting of --O--,
--NR--, --S--, --S(O)--, --SO.sub.2--, --C(O)O--, --OC(O)--,
--C(O)NR--, --NRC(O)--, --OC(O)O--, --NRC(O)O--, --OC(O)NR--, and
--NRC(O)NR--; [0201] R is hydrogen, C1-6alkyl, or C3-6cycloalkyl,
wherein the alkyl or cycloalkyl is optionally substituted; [0202] p
is an integer from 0 to 4; [0203] R11 and R22 at each instance of p
are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or
R11 is L2-C(O)--OC1-6alkyl; [0204] R33 and R44 are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R33 is
L2-C(O)--OC1-6alkyl; [0205] L1 and L2 are each independently
C5-21alkyl or C4-20heteroalkyl; [0206] provided that: [0207] when
R33 is L2-C(O)--OC1-6alkyl, R11 is not L2-C(O)--OC1-6alkyl; and
[0208] when p is an integer from 2 to 4, no more than one R11 is
L2-C(O)--OC1-6alkyl; and [0209] wherein any alkyl, cycloalkyl, or
heteroalkyl present in any of R11, R22, R 33, R44, L1, and L2 is
optionally substituted; [0210] or a pharmaceutically acceptable
salt or solvate thereof.
[0211] In one embodiment, the lipid-containing conjugation partner
is a compound of the formula (II) as defined in any of the
embodiments described herein.
[0212] In one embodiment, the lipid-containing conjugation partner
is a compound of the formula (IIA) as defined in any of the
embodiments described herein.
[0213] In one embodiment, the amino acid-comprising conjugation
partner is a compound of the formula (III) as defined in any of the
embodiments described herein.
[0214] In one embodiment, the peptide-containing conjugation
partner is a compound of the formula (III) as defined in any of the
embodiments described herein.
[0215] In one embodiment, the amino acid-comprising conjugation
partner is a compound of the formula (IIIA) as defined in any of
the embodiments described herein.
[0216] In one embodiment, the peptide-containing conjugation
partner is a compound of the formula (IIIA) as defined in any of
the embodiments described herein.
[0217] In one embodiment, the method comprises making an amino acid
or peptide conjugate comprising a structure of the formula (I)
##STR00007##
[0218] wherein [0219] m is an integer from 0 to 4; [0220] n is 1 or
2; [0221] R1 and R2 at each instance of m are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R1 is
L2-C(O)--OC1-6alkyl; [0222] R3, R4, R5, R8, and R9 are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R3 is
L2-C(O)--OC1-6alkyl; [0223] or R9 is an amino protecting group,
L3-C(O), or A2; [0224] R6 and R7 at each instance of n are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0225] L1 and
L2 are each independently C5-21alkyl or C4-20heteroalkyl; [0226] L3
is C1-21alkyl or C4-20heteroalkyl; [0227] A1 and A2 are each
independently an amino acid or a peptide; or A1 is OH or OP1,
wherein P1 is a carboxyl protecting group; [0228] provided that:
[0229] when R3 is L2-C(O)--OC1-6alkyl, R1 is not
L2-C(O)--OC1-6alkyl; and [0230] when m is an integer from 2 to 4,
no more than one R1 is L2-C(O)--OC1-6alkyl; and [0231] wherein any
alkyl, cycloalkyl or heteroalkyl present in any of R1, R2, R3, R4,
R5, R6, R7, R8, R9, L1, L2 and L3 is optionally substituted; [0232]
or a pharmaceutically acceptable salt or solvate thereof;
[0233] the method comprising reacting a lipid-containing
conjugation partner of the formula (II)
##STR00008##
[0234] wherein m, R1, R2, R3, R4, R5, and L1 are as defined in the
compound of formula (I);
[0235] and a peptide-containing conjugation partner comprising a
structure of the formula (III)
##STR00009##
[0236] wherein n, R6, R7, R8, R9 and A1 are as defined in the
compound of formula (I);
[0237] under conditions effective to conjugate the compound of
formula (II) with the compound of formula (III) by hydrothiolation
of the carbon-carbon double bond in the compound of formula (II)
with the thiol in the compound of formula (III).
[0238] In one embodiment, the method comprises making a peptide
conjugate comprising a structure of the formula (I)
##STR00010##
[0239] wherein [0240] m is an integer from 0 to 4; [0241] n is 1 or
2; [0242] R1 and R2 at each instance of m are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R1 is
L2-C(O)--OC1-6alkyl; [0243] R3, R4, R5, R8, and R9 are each
independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R3 is
L2-C(O)--OC1-6alkyl; [0244] or R9 is L3-C(O) or A2; [0245] R6 and
R7 at each instance of n are each independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl; [0246] L1 and L2 are each
independently C5-21alkyl or C4-20heteroalkyl; [0247] L3 is C1-21
alkyl or C4-20heteroalkyl; [0248] A1 and A2 are each independently
a peptide; or A1 is OH; [0249] provided that: [0250] when R9 is not
A2, A1 is a peptide; [0251] when R3 is L2-C(O)--OC1-6alkyl, R1 is
not L2-C(O)--OC1-6alkyl; and [0252] when m is an integer from 2 to
4, no more than one R1 is L2-C(O)--OC1-6alkyl; and [0253] wherein
any alkyl, cycloalkyl or heteroalkyl present in any of R1, R2, R3,
R4, R5, R6, R7, R8, R9, L1, L2 and L3 is optionally substituted;
[0254] or a pharmaceutically acceptable salt or solvate
thereof;
[0255] the method comprising reacting a lipid-containing
conjugation partner of the formula (II)
##STR00011##
[0256] wherein m, R1, R2, R3, R4, R5, and L1 are as defined in the
compound of formula (I);
[0257] and a peptide-containing conjugation partner comprising a
structure of the formula (III)
##STR00012##
[0258] wherein n, R6, R7, R8, R9 and A1 are as defined in the
compound of formula (I);
[0259] under conditions effective to conjugate the compound of
formula (II) with the compound of formula (III) by hydrothiolation
of the carbon-carbon double bond in the compound of formula (II)
with the thiol in the compound of formula (III).
[0260] In one embodiment, the method comprises making an amino acid
or peptide conjugate comprising a structure of the formula
(IA),
##STR00013##
[0261] wherein [0262] p is an integer from 0 to 4; [0263] q is an
integer from 0 to 2; [0264] R11 and R22 at each instance of p are
each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11
is L2-C(O)--OC1-6alkyl; [0265] R33, R44, R55, R66, R77, R8, and R9
are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or
R33 is L2-C(O)--OC1-6alkyl; [0266] or R9 is an amino protecting
group, L3-C(O), or A2; [0267] Ra and Rb at each instance of q are
each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0268]
L1 and L2 are each independently C5-21alkyl or C4-20heteroalkyl;
[0269] L3 is C1-21 alkyl or C4-20heteroalkyl; [0270] A1 and A2 are
each independently an amino acid or a peptide; or A1 is OH or OP1,
wherein P1 is a carboxyl protecting group; [0271] provided that:
[0272] when R33 is L2-C(O)--OC1-6alkyl, R11 is not
L2-C(O)--OC1-6alkyl; and [0273] when p is an integer from 2 to 4,
no more than one R11 is L2-C(O)--OC1-6alkyl; and [0274] wherein any
alkyl, cycloalkyl, or heteroalkyl present in any of R11, R22, R 33,
R44, R55, R66, R77, R8, R9, Ra, Rb, L1, L2, and L3 is optionally
substituted; [0275] or a pharmaceutically acceptable salt or
solvate thereof;
[0276] the method comprising reacting a compound of the formula
(IIA)
##STR00014##
[0277] wherein p, R11, R22, R33, R44, and L1 are as defined in the
compound of formula (IA);
[0278] and a compound of the formula (IIIA)
##STR00015##
[0279] wherein q, R55, R66, R77, R8, R9, Ra, Rb, and A1 are as
defined in the compound of formula (IA);
[0280] under conditions effective to conjugate the compound of
formula (IIA) with the compound of formula (IIIA) by
hydrothiolation of the carbon-carbon double bond in the compound of
formula (IIIA) with the thiol in the compound of formula (IIA).
[0281] In one embodiment, the method comprises making a peptide
conjugate comprising a structure of the formula (IA),
##STR00016##
[0282] wherein [0283] p is an integer from 0 to 4; [0284] q is an
integer from 0 to 2; [0285] R11 and R22 at each instance of p are
each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or R11
is L2-C(O)--OC1-6alkyl; [0286] R33, R44, R55, R66, R77, R8, and R9
are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; or
R33 is L2-C(O)--OC1-6alkyl; [0287] or R9 is L3-C(O) or A2; [0288]
Ra and Rb at each instance of q are each independently hydrogen,
C1-6alkyl, or C3-6cycloaIkyl; [0289] L1 and L2 are each
independently C5-21alkyl or C4-20heteroalkyl; [0290] L3 is
C1-21alkyl or C4-20heteroalkyl; [0291] A1 and A2 are each
independently a peptide; or A1 is OH; [0292] provided that: [0293]
when R9 is not A2, A1 is a peptide; [0294] when R33 is
L2-C(O)--OC1-6alkyl, R11 is not L2-C(O)--OC1-6alkyl; and [0295]
when p is an integer from 2 to 4, no more than one R11 is
L2-C(O)--OC1-6alkyl; and [0296] wherein any alkyl, cycloalkyl, or
heteroalkyl present in any of R11, R22, R 33, R44, R55, R66, R77,
R8, R9, Ra, Rb, L1, L2, and L3 is optionally substituted; [0297] or
a pharmaceutically acceptable salt or solvate thereof;
[0298] the method comprising reacting a compound of the formula
(IIA)
##STR00017##
[0299] wherein p, R11, R22, R33, R44, and L1 are as defined in the
compound of formula (IA);
[0300] and a compound of the formula (IIIA)
##STR00018##
[0301] wherein q, R55, R66, R77, R8, R9, Ra, Rb, and A1 are as
defined in the compound of formula (IA);
[0302] under conditions effective to conjugate the compound of
formula (IIA) with the compound of formula (IIIA) by
hydrothiolation of the carbon-carbon double bond in the compound of
formula (IIIA) with the thiol in the compound of formula (IIA).
[0303] In one embodiment, at least one of L1 and L2 is
C5-22alkyl.
[0304] In one embodiment, p is an integer from 0 to 2. In another
embodiment, p is 0 or 1.
[0305] In some embodiments, R11 and R22 at each instance of p are
each independently hydrogen; or R11 is L2-C(O)--OCH2. In one
embodiment, R11 on the carbon adjacent to L1-C(O)--O is
L2-C(O)--OCH2.
[0306] In one specifically contemplated embodiment, R11 and R22 at
each instance of p are each independently hydrogen.
[0307] In one embodiment, R33 is hydrogen or L2-C(O)--OCH2.
[0308] In one embodiment, R33 and R44 are each hydrogen.
[0309] In one specifically contemplated embodiment, q is 0 or 1. In
one specifically contemplated embodiment, q is 0.
[0310] In one specifically contemplated embodiment, R55, R66, and
R77 are each hydrogen.
[0311] In some embodiments, Ra and Rb are at each instance of q are
each hydrogen.
[0312] In one embodiment, L1 is C11-21alkyl; p is 1; R11 is
hydrogen or L2-C(O)--OCH2; R22 is hydrogen; R33 is hydrogen or
L2-C(O)--OCH2; R44 is hydrogen; and L2 is C11-21alkyl.
[0313] In one embodiment, R55, R66, R77, Ra, Rb and R8 are each
hydrogen; and R9 is hydrogen, L3-C(O), or A2. In one embodiment,
R55, R66, R77, Ra, Rb and R8 are each hydrogen; and R9 is hydrogen
or L3-C(O).
[0314] In one embodiment, L1 is C11-21alkyl; p is 1; R11 is
hydrogen or L2-C(O)--OCH2; R22 is hydrogen; R33 is hydrogen or
L2-C(O)--OCH2; R44 is hydrogen; L2 is C11-21alkyl; R55, R66, R77,
Ra, Rb and R8 are each hydrogen; and R9 is hydrogen, L3-C(O), or
A2.
[0315] In one embodiment, L1 is C5-21alkyl. In another embodiment,
L1 is C9-21alkyl. In yet another embodiment, L1 is C11-21alkyl. In
one exemplary embodiment, L1 is C11, C13, C15, C17, or C19alkyl. In
one specifically contemplated embodiment, L1 is C15alkyl.
[0316] In one embodiment, L1 comprises a linear chain of 9-21
carbon atoms. In one specifically contemplated embodiment, L1 is
linear C15alkyl.
[0317] In one embodiment, m is an integer from 0 to 2. In another
embodiment, m is 0 or 1.
[0318] In one specifically contemplated embodiment, m is 0.
[0319] In some embodiments, R1 and R2 at each instance of m are
each independently hydrogen; or R1 is L2-C(O)--OCH2. In one
embodiment, R1 on the carbon atom adjacent to L1-C(O)--O is
L2-C(O)--OCH2.
[0320] In one specifically contemplated embodiment, R1 and R2 at
each instance of m are each independently hydrogen.
[0321] In one embodiment, R3 is hydrogen or L2-C(O)--OCH2. In one
specifically contemplated embodiment, R3 is hydrogen.
[0322] In one embodiment, L2 is C5-21alkyl. In another embodiment,
L2 is C9-21alkyl. In yet another embodiment, L2 is C11-21alkyl. In
one exemplary embodiment, L2 is C11, C13, C15, C17, or C19alkyl. In
another exemplary embodiment, L2 is C15alkyl.
[0323] In one specifically contemplated embodiment, R4 and R5 are
each hydrogen.
[0324] In one specifically contemplated embodiment, n is 1.
[0325] In one specifically contemplated embodiment, R6 and R7 are
each hydrogen.
[0326] In exemplary embodiments, R8 is hydrogen.
[0327] In one embodiment, R8 and R9 are each hydrogen; or R9 is
L3-C(O) or A2. In one exemplary embodiment R8 is hydrogen and R9 is
L3-C(O).
[0328] In some embodiments, L3 is C1-21alkyl. In one specifically
contemplated embodiment, L3 is methyl or linear C15alkyl. In
exemplary embodiments, L3 is methyl.
[0329] Those skilled in the art will appreciate that the structures
of formula (III) and (IIIA) may comprise a peptide of the
peptide-containing conjugation partner. As described herein, the
peptide may be optionally substituted, modified, or bound to
various other moieties as described herein to provide the peptide
containing conjugation partner.
[0330] In one embodiment A1 is a peptide comprising an epitope. In
one embodiment A2 is a peptide comprising an epitope.
[0331] In some embodiments, A1 is a peptide comprising a peptide
epitope. In some embodiments, A2 is a peptide comprising a peptide
epitope. In one embodiment, A1 is a peptide, wherein the peptide
comprises a peptide epitope.
[0332] In another embodiment, A2 is a peptide, wherein the peptide
comprises a peptide epitope.
[0333] In one embodiment, A1 is a peptide substituted with an
epitope. In one embodiment, A2 is a peptide substituted with an
epitope.
[0334] In one embodiment, the epitope is bound to the peptide via a
linker group.
[0335] In one embodiment, the epitope is a peptide epitope.
[0336] In some embodiments, A1 and/or A2 are each independently a
peptide comprising from about 8 to 220, 8 to 200, 8 to 175, 8 to
150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to
50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids. In
one exemplary embodiment, A1 and A2 are each independently a
peptide comprising from about 8 to 60 amino acids.
[0337] In other embodiments, A1 and/or A2 are each independently a
peptide comprising from about 8 to 220, 8 to 200, 8 to 175, 8 to
150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to
50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids.
[0338] In other embodiments, A1 and/or A2 are each independently a
peptide comprising from about 5 to 150, 5 to 125, 5 to 100, 5 to
75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150,
8 to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8
to 25, or 8 to 20 amino acids.
[0339] In some embodiments, A1 and/or A2 are each independently a
peptide, wherein the peptide comprises 8 to 60 amino acids.
[0340] In some embodiments, A1 and/or A2 are each independently a
peptide comprising or substituted with a peptide epitope, wherein
the peptide epitope comprises from 8 to 60 amino acids.
[0341] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen
or L2-C(O)--OCH2; 12 is C11-21alkyl; and R4 and R5 are each
hydrogen.
[0342] In one embodiment, n is 1; R6, R7, and R8 are each hydrogen;
and R9 is hydrogen, 13-C(O), or A2. In one embodiment, n is 1; R6,
R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O). In one
embodiment, L3 is methyl or linear C15alkyl.
[0343] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen
or L2-C(O)--OCH2; 12 is C11-21alkyl; R4 and R5 are each hydrogen; n
is 1; R6, R7, and R8 are each hydrogen; R9 is hydrogen, L3-C(O), or
A2. In one embodiment, L1 is C11-21alkyl; m is 0; R3 is hydrogen or
L2-C(O)--OCH2; L2 is C11-21alkyl; R4 and R5 are each hydrogen; n is
1; R6, R7, and R8 are each hydrogen; R9 is hydrogen or L3-C(O).
[0344] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; and R4 and R5 are each hydrogen.
[0345] In one embodiment, n is 1; R6, R7, and R8 are each hydrogen;
and R9 is hydrogen, 13-C(O), or A2. In one embodiment, n is 1; R6,
R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O). In one
embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 is
hydrogen or L3-C(O), wherein L3 is methyl.
[0346] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen, L3-C(O), or A2. In one embodiment,
L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5 are each
hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 is hydrogen
or L3-C(O).
[0347] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen or L3-C(O), wherein L3 is methyl.
[0348] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; and R4 and R5 are each hydrogen.
[0349] In one embodiment, n is 1; R6, R7, and R8 are each hydrogen;
and R9 is hydrogen, 13-C(O), or A2. In one embodiment, n is 1; R6,
R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O). In one
embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 is
hydrogen or L3-C(O), wherein L3 is methyl.
[0350] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen, L3-C(O), or A2. In one embodiment,
L1 is C11-21alkyl; m is 0; R3 is hydrogen; R4 and R5 are each
hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 is hydrogen
or L3-C(O).
[0351] In one embodiment, L1 is C11-21alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen or L3-C(O), wherein L3 is methyl.
[0352] In some embodiments, A1 is a peptide comprising serine as
the first N-terminal amino acid residue. In some embodiments, A1
and/or A2 is a peptide comprising a solubilising group. In some
embodiments, the solubilising group comprises an amino acid
sequence comprising two or more hydrophilic amino acid residues in
the peptide chain. In certain embodiments, A1 is a peptide
comprising a solubilising group comprising an amino acid sequence
comprising two or more hydrophilic amino acid residues in the
peptide chain.
[0353] In some embodiments, A1 is a peptide comprising serine as
the first N-terminal amino acid residue and a solubilising group
comprising an amino acid sequence comprising two or more
hydrophilic amino acid residues in the peptide chain adjacent to
the serine.
[0354] In some embodiments, the solubilising group comprises an
amino acid sequence comprising two or more consecutive hydrophilic
amino acid residues in the peptide chain.
[0355] In one embodiment, the hydrophilic amino acid residues are
cationic amino acid residues.
[0356] In one embodiment, the cationic amino acid residues are
arginine or lysine residues. In one specifically contemplated
embodiment, the cationic amino acid residues are lysine residues.
In one embodiment, the sequence comprises from 2 to 20, 2 to 15, 2
to 10, 3 to 7, or 3 to 5 amino acids. In one embodiment, the
solubilising group is a tri-, tetra-, penta-, hexa-, or
hepta-lysine sequence. In one specifically contemplated embodiment,
the solubilising group is a tetralysine sequence.
[0357] In some embodiments, R9 is hydrogen, an amino protecting
group or L3-C(O). In some embodiments, R9 is hydrogen or
L3-C(O).
[0358] In some embodiments, R9 is hydrogen or an amino protecting
group, and the method further comprises acylating the amino acid
conjugate or peptide conjugate so as to replace the hydrogen or
amino protecting group at R9 with L3-C(O). In some embodiments,
acylating the amino acid conjugate or peptide conjugate so as to
replace the amino protecting group at R9 with L3-C(O) comprises
removing the amino protecting group at R9 to provide a hydrogen at
R9.
[0359] In some embodiments, A1 and/or A2 is an amino acid or a
peptide. In some embodiments, A1 and/or A2 is a peptide.
[0360] In some embodiments, A1 is OH or OP1 and/or R9 is hydrogen,
an amino protecting group or L3-C(O). In some embodiments, A1 is
OP1 or OH and/or R9 is hydrogen, an amino protecting group or
L3-C(O). In some embodiments, A1 is a OP1 or OH and R9 is hydrogen,
an amino protecting group or L3-C(O).
[0361] In some embodiments, A1 is a OP1 or OH and/or R9 is
hydrogen, an amino protecting group or L3-C(O), and the method
comprises coupling an amino acid or a peptide so as to replace A1
and/or R9 with the amino acid or peptide.
[0362] In some embodiments, A1 is a OP1 or OH and R9 is hydrogen,
an amino protecting group or L3-C(O) and the method further
comprises coupling an amino acid or a peptide so as to replace A1
and/or R9 with the amino acid or peptide.
[0363] In some embodiments, coupling a peptide comprises
individually coupling one or more amino acids and/or one or more
peptides.
[0364] In some embodiments, coupling the amino acid or peptide
provides a peptide conjugate comprising a peptide epitope. In some
embodiments, the coupling the amino acid or peptide provides a
peptide conjugate comprising a linker group or one or more amino
acids thereof. In some embodiments, coupling the amino acid or
peptide provides a peptide conjugate comprising a peptide epitope
bound to the amino acid to which lipid-containing conjugation
partner is conjugated via a linker group.
[0365] In some embodiments, the amino protecting group is Boc,
Fmoc, Cbz (carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl),
Bpoc (2-(4-biphenyl)isopropoxycarbonyl) and Dde
(1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl). In some embodiments,
the amino protecting group is Boc or Fmoc.
[0366] In some embodiments, the carboxyl protecting group is
tert-butyl or benzyl. In one embodiment, the compound of the
formula (I) is a compound of the formula (IV):
##STR00019##
[0367] wherein [0368] R3 is hydrogen or L2-C(O)--OCH2; [0369] R9 is
hydrogen, an amino protecting group, L3-C(O), or A2; and [0370] L1
and L2 are each independently C5-21alkyl or C4-20heteroalkyl;
[0371] 1.3 is C1-21alkyl or C4-20heteroalkyl; [0372] A1 and A2 are
each independently an amino acid or a peptide; or A1 is OH or OP1,
wherein P1 is a carboxyl protecting group;
[0373] or a pharmaceutically acceptable salt or solvate
thereof.
[0374] In one embodiment, the compound of the formula (I) is a
compound of the formula (IV):
##STR00020##
[0375] wherein [0376] R3 is hydrogen or L2-C(O)--OCH2; [0377] R9 is
hydrogen, L3-C(O), or A2; and [0378] L1 and L2 are each
independently C5-21alkyl or C4-20heteroalkyl; [0379] L3 is
C1-21alkyl or C4-20heteroalkyl; [0380] A1 and A2 are each
independently a peptide; or A1 is OH; [0381] provided that: [0382]
when R9 is not A2, A1 is a peptide;
[0383] or a pharmaceutically acceptable salt or solvate
thereof.
[0384] In one embodiment, L1, A1, A2, L2, and L3 in the compound of
formula (IV) are each independently as defined in any of the
embodiments relating to the compound of the formula (I).
[0385] In one specifically contemplated embodiment, R3 is
hydrogen.
[0386] In another specifically contemplated embodiment, R9 is
acetyl.
[0387] In another specifically contemplated embodiment, R3 is
hydrogen and R9 is acetyl.
[0388] In some embodiments, the method is for making a compound of
the formula (IV), wherein L1 is C15 linear alkyl, R3 is hydrogen,
R9 is Fmoc, and A1 is OH, and the method comprises reacting vinyl
palmitate and Fmoc-Cys-OH.
[0389] In some embodiments, the amino protecting group is not Fmoc.
In some embodiments, the amino protecting group is Boc.
[0390] In some embodiments, the amino acid-comprising conjugation
partner is not Fmoc-Cys-OH.
[0391] In some embodiments, the peptide conjugate comprises 3 or
more, 4 or more, or 5 or more contiguous amino acids. In some
embodiments, the compound of formula (I) comprises 3 or more, 4 or
more, or 5 or more contiguous amino acids.
[0392] In one embodiment, the conditions effective to conjugate the
lipid-containing conjugation partner to the amino acid-comprising
conjugation partner comprises the generation of one or more free
radicals. In one embodiment, the conditions effective to conjugate
the lipid-containing conjugation partner to the peptide-containing
conjugation partner comprises the generation of one or more free
radicals.
[0393] In some embodiments, the generation of one or more free
radicals is initiated thermally and/or photochemically. In certain
embodiments, the generation of one or more free radicals is
initiated by the thermal and/or photochemical degradation of a free
radical initiator. In exemplary embodiments, the generation of one
or more free radicals is initiated by the thermal degradation of a
thermal initiator or the photochemical degradation of a
photochemical initiator.
[0394] In some embodiments, thermal degradation of the free radical
initiator comprises heating the reaction mixture at a suitable
temperature. In some embodiments, the reaction mixture is heated at
a temperature is from about 40.degree. C. to about 200.degree. C.,
from about 50.degree. C. to about 180.degree. C., from about
60.degree. C. to about 150.degree. C., from about 65.degree. C. to
about 120.degree. C., from about 70.degree. C. to about 115.degree.
C., from about 75.degree. C. to about 110.degree. C., or from about
80.degree. C. to about 100.degree. C. In other embodiments, the
reaction mixture is heated at a temperature of at least about
40.degree. C., at least about 50.degree. C., at least about
60.degree. C., or at least about 65.degree. C. In one specifically
contemplated embodiment, the reaction mixture is heated at a
temperature of about 90.degree. C.
[0395] In some embodiments, photochemical degradation of the free
radical initiator comprises irradiation with ultraviolet light. In
a specifically contemplated embodiment, the ultraviolet light has a
wavelength of about 365 nm. In exemplary embodiments, photochemical
degradation of the free radical initiator is carried out at about
ambient temperature.
[0396] In one specifically contemplated embodiment, the thermal
initiator is 2,2'-azobisisobutyronitrile (AIBN). In one
specifically contemplated embodiment, the photoinitiator is
2,2-dimethoxy-2-phenylacetophenone (DMPA).
[0397] In certain embodiments, the reaction is carried out in a
liquid medium. In one embodiment, the liquid medium comprises a
solvent. In one embodiment, the solvent is selected from the group
consisting of N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO),
N,N-dimethylformamide (DMF), dichloromethane (DCM),
1,2-dichloroethane, and mixtures thereof. In one specifically
contemplated embodiment, the solvent comprises NMP, DMSO, or a
mixture thereof.
[0398] In one specifically contemplated embodiment, the solvent
comprises DMSO.
[0399] In some embodiments, the reaction is carried out in the
presence of one or more additives that inhibit dimerisation,
telomerisation, or polymerisation. In some exemplary embodiments,
the additive is selected from the group consisting of reduced
glutathione (GSH), 2,2'-(ethylenedioxy)diethanethiol (DODT),
1,4-dithiothreitol (DTT), and protein.
[0400] In a specifically contemplated embodiment, the additive is
DTT. In some embodiments, the additive is DTT or tert-butyl
mercaptan.
[0401] In some embodiments, the one or more additive is selected
from the group consisting of TFA, tert-butyl mercaptan, and a
combination thereof. In certain embodiments, the one or more
additive is a combination of TFA and tert-butyl mercaptan. In some
embodiments, the reaction is carried out for a period of time from
about 5 minutes to about 48 h, 5 minutes to about 24 h, from about
5 minutes to about 12 hours, from about 5 minutes to about 6 hours,
from about 5 minutes to about 3 hours, 5 minutes to 2 hours, or
form about 5 minutes to about 1 hour. In exemplary embodiments, the
reaction is carried out for a period of time from about 5 minutes
to about 1 h. In some embodiments, the reaction is carried out
until one of the conjugation partners is at least about 70%, 80%,
90%, 95%, 97%, 99%, or 100% consumed.
[0402] In certain embodiments, the reaction is carried out under
substantially oxygen free conditions.
[0403] In some embodiments, the method comprises [0404] reacting
the lipid-containing conjugation partner and an amino
acid-comprising conjugation partner to provide an amino acid or
peptide conjugate; [0405] synthesising the amino acid sequence of a
peptide by solid phase peptide synthesis (SPPS); [0406] coupling
the amino acid of the amino acid conjugate or an amino acid of the
peptide conjugate to the solid phase bound peptide by SPPS so as to
provide a peptide conjugate comprising a peptide epitope, a peptide
conjugate comprising a linker group or one or more amino acids
thereof, or a peptide conjugate comprising a peptide epitope bound
to the amino acid to which lipid-containing conjugation partner is
conjugated via a linker group.
[0407] In some embodiments, the method further comprises acylating
the No-amino group of the amino acid of the amino acid conjugate or
the amino acid to which the lipid-containing conjugation partner is
conjugated of any one of the peptide conjugates.
[0408] In some embodiments, the method comprises cleaving the
peptide conjugate from the solid phase support.
[0409] In some embodiments, the method comprises [0410]
synthesising the amino acid sequence of the peptide of the
peptide-containing conjugation partner by solid phase peptide
synthesis (SPPS); and [0411] reacting the lipid-containing
conjugation partner and peptide-containing conjugation partner in
accordance with any of the embodiments described herein.
[0412] In exemplary embodiments, the method comprises [0413]
synthesising the amino acid sequence of the peptide of the
peptide-containing conjugation partner by SPPS, [0414] cleaving the
peptide from the solid phase support; and [0415] reacting the
lipid-containing conjugation partner and peptide-containing
conjugation partner in accordance with any of the embodiments
described herein.
[0416] In one embodiment, the peptide-containing conjugation
partner is not purified prior to reaction with the lipid-containing
conjugation partner.
[0417] In some embodiments, one or more protecting groups are
removed on cleaving the peptide from the solid phase support. In
certain embodiments, all of the protecting groups present in the
peptide are removed.
[0418] In one embodiment, the SPPS is Fmoc-SPPS.
[0419] In some embodiments, the amino acid residue in the peptide
of the peptide-containing conjugation partner bearing the
carbon-carbon double bond or thiol to be reacted is an N-terminal
amino acid residue and the method comprises acylating the
N-terminal amino group prior to cleaving the peptide from the solid
phase. In exemplary embodiments, the amino acid residue is an
N-terminal residue. In specifically contemplated embodiments, the
N-terminal residue is a cysteine residue.
[0420] In one embodiment, the method further comprises separating
the peptide conjugate from the reaction medium and optionally
purifying the peptide conjugate.
[0421] In another aspect, the present invention provides an amino
acid conjugate or peptide conjugate made by a method of the present
invention.
[0422] In another aspect, the present invention provides a peptide
conjugate made by a method of the present invention.
[0423] In another aspect, the present invention provides a compound
of the formula (V):
##STR00021##
[0424] wherein [0425] m is an integer from 0 to 4; [0426] n is 1 or
2; [0427] R1 and R2 at each instance of m are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0428] R3, R4, R5, R8, and
R9 are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl;
or R9 is an amino protecting group, L3-C(O), or A2; [0429] R6 and
R7 at each instance of n are each independently hydrogen,
C1-6alkyl, or C3-6cycloalkyl, [0430] L1 is C5-21alkyl or
C4-20heteroalkyl; [0431] L3 is C1-6alkyl or C3-6cycloalkyl; [0432]
A1 and A2 are each independently an amino acid or a peptide; or A1
is OH or OP1, wherein P1 is a carboxyl protecting group; and [0433]
wherein any alkyl, cycloalkyl or heteroalkyl present in any of R1,
R2, R3, R4, R5, R6, R7, R8, R9, L1, and L3 is optionally
substituted, and
[0434] or a pharmaceutically acceptable salt or solvate
thereof.
[0435] In another aspect, the present invention provides a compound
of the formula (V):
##STR00022##
[0436] wherein [0437] m is an integer from 0 to 4; [0438] n is 1 or
2; [0439] R1 and R2 at each instance of m are each independently
hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0440] R3, R4, R5, R8, and
R9 are each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl;
or R9 is L3-C(O) or A2; [0441] R6 and R7 at each instance of n are
each independently hydrogen, C1-6alkyl, or C3-6cycloalkyl; [0442]
L1 is C5-21alkyl or C4-20heteroalkyl; [0443] L3 is C1-6alkyl or
C3-6cycloalkyl; [0444] A1 and A2 are each independently a peptide;
or A1 is OH; [0445] provided that: [0446] at least one of A1 and A2
comprises an epitope; and [0447] when R9 is not A2, A1 is a
peptide; and [0448] wherein any alkyl, cycloalkyl or heteroalkyl
present in any of R1, R2, R3, R4, R5, R6, R7, R8, R9, L1, and L3 is
optionally substituted, and
[0449] or a pharmaceutically acceptable salt or solvate
thereof.
[0450] In one embodiment, m, n, R6, R7, A1 and A2 are each
independently as defined in any of the embodiments relating to the
compound of formula (I).
[0451] In one embodiment, L1 is C5-21alkyl. In one embodiment, L1
is C5-21alkyl. In another embodiment, L1 is C9-21alkyl. In yet
another embodiment, L1 is C11-21alkyl. In one exemplary embodiment,
L1 is C11, C13, C15, C17, or C19alkyl. In one specifically
contemplated embodiment, L1 is C15alkyl.
[0452] In one embodiment, L1 comprises a linear chain of 9-21
carbon atoms. In one specifically contemplated embodiment, L1 is
linear C15alkyl.
[0453] In one embodiment, m is an integer from 0 to 2. In another
embodiment, m is 0 or 1.
[0454] In one specifically contemplated embodiment, m is 0.
[0455] In one specifically contemplated embodiment, R1 and R2 at
each instance of m are each independently hydrogen.
[0456] In one specifically contemplated embodiment, R3 is
hydrogen.
[0457] In one specifically contemplated embodiment, R4 and R5 are
each hydrogen.
[0458] In one specifically contemplated embodiment, n is 1.
[0459] In one specifically contemplated embodiment, R6 and R7 are
each hydrogen.
[0460] In exemplary embodiments, R8 is hydrogen.
[0461] In some embodiments, R8 is hydrogen and R9 is hydrogen, an
amino protecting group, L3-C(O), or A2. In one embodiment, R8 and
R9 are each hydrogen; or R9 is L3-C(O) or A2. In one exemplary
embodiment R8 is hydrogen and R9 is L3-C(O). In one specifically
contemplated embodiment, L3 is methyl.
[0462] In some embodiments, A1 is OP1 or OH and R9 is hydrogen, an
amino protecting group or L3-C(O).
[0463] In some embodiments, A1 and/or A2 is an amino acid or a
peptide. In some embodiments, the peptide comprises an epitope.
[0464] In some embodiments, A1 is serine or a peptide comprising
serine as the first N-terminal amino acid residue.
[0465] In some embodiments, A1 and/or A2 is a peptide comprising a
solubilising group comprising an amino acid sequence comprising two
or more hydrophilic amino acid residues in the peptide chain.
[0466] In some embodiments, A1 is a peptide comprising serine as
the first N-terminal amino acid residue and a solubilising group
comprising an amino acid sequence comprising two or more
hydrophilic amino acid residues in the peptide chain adjacent to
the serine.
[0467] In one embodiment, L1 is C11-21 alkyl; m is 0; R3 is
hydrogen; and R4 and R5 are each hydrogen.
[0468] In one embodiment, n is 1; R6, R7, and R8 are each hydrogen;
and R9 is hydrogen, L3-C(O), or A2. In one embodiment, n is 1; R6,
R7, and R8 are each hydrogen; and R9 is hydrogen or L3-C(O). In one
embodiment, n is 1; R6, R7, and R8 are each hydrogen; and R9 is
hydrogen or L3-C(O), wherein L3 is methyl.
[0469] In one embodiment, L1 is C11-21 alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen, L3-C(O), or A2. In one embodiment,
L1 is C11-21 alkyl; m is 0; R3 is hydrogen; R4 and R5 are each
hydrogen; n is 1; R6, R7, and R8 are each hydrogen; R9 is hydrogen
or L3-C(O).
[0470] In one embodiment, L1 is C11-21 alkyl; m is 0; R3 is
hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are
each hydrogen; R9 is hydrogen or L3-C(O), wherein L3 is methyl.
[0471] In some embodiments, L1 is C15 linear alkyl; m is 0; n is 1;
R3, R4, R5, R6, R7, and R8 are each hydrogen; R9 is Fmoc, and A1 is
OH in the compound of the formula (V).
[0472] In some embodiments, the amino protecting group of R9 is not
Fmoc. In some embodiments, the amino protecting group of R9 is
Boc.
[0473] In some embodiments, the compound of formula (V) comprises 3
or more, 4 or more, or 5 or more contiguous amino acids.
[0474] In some embodiments, the amino and/or carboxyl protecting
groups are as defined in any of the embodiments relating to the
compound of formula (I).
[0475] Those skilled in the art will appreciate that compound of
formula (V) is a peptide conjugate and certain embodiments relating
to the peptide conjugates of the conjugation method described
herein also apply to the compounds of formula (V).
[0476] In some embodiments, the compound of formula (V) is a self
adjuvanting peptide.
[0477] In some embodiments, the compound comprises a linker or one
or more amino acids thereof. In some embodiments, the peptide
comprises a linker or one or more amino acids thereof. In some
embodiments, the peptide comprises a peptide epitope bound to via a
linker to the amino acid to which L1 is bound. In some embodiments,
the peptide comprises two or more epitopes. In some embodiments,
the peptide comprises a peptide antigen. In some embodiments, the
linker is an amino acid sequence from about 2 to 20, 2 to 18, 2 to
16, 2 to 14, 2 to 12, 2 to 10, or 2 to 8 amino acids in length.
[0478] In another aspect, the present invention provides an
isolated, purified, or recombinant peptide comprising or consisting
of 20 or more contiguous amino acids from the amino acid sequence
of any one of SEQ ID NOs 1-5, 8-12, or 14-18.
[0479] In one embodiment, the peptide comprises, consists of, or
consists essentially of an amino acid sequence selected from the
group consisting of any one of SEQ ID NOs 1-5, 8-12, or 14-18.
[0480] In one embodiment, the peptide comprises, consists of, or
consists essentially of an amino acid sequence selected from the
group consisting of any one of SEQ ID NOs 4, 5, 11, 12, 17, and
18.
[0481] In another aspect, the present invention provides a
pharmaceutical composition comprising an effective amount of a
peptide conjugate of the present invention or a pharmaceutically
acceptable salt or solvate thereof, and a pharmaceutically
acceptable carrier.
[0482] In one embodiment, the pharmaceutical composition is an
immunogenic composition.
[0483] In one embodiment, the composition does not include an
extrinsic adjuvant.
[0484] In some embodiments, the composition is a vaccine.
[0485] In one embodiment, the pharmaceutical composition comprises
an effective amount of two or more peptide conjugates of the
present invention, for example the pharmaceutical composition
comprises an effective amount of three or more peptide conjugates
of the present invention.
[0486] In another aspect, the present invention provides a
pharmaceutical composition comprising an effective amount of a
peptide of the present invention or a pharmaceutically acceptable
salt or solvate thereof, and a pharmaceutically acceptable
carrier.
[0487] In one embodiment, the pharmaceutical composition comprises
an effective amount of two or more peptides of the present
invention, for example the pharmaceutical composition comprises an
effective amount of three or more peptides of the present
invention.
[0488] In one embodiment, the pharmaceutical composition comprises
an effective amount of one or more peptide conjugates of the
present invention together with one or more peptides of the present
invention, or any combination thereof. For example, the
pharmaceutical composition comprises an effective amount of two or
more peptide conjugates of the present invention and one or more
peptides of the present invention, or an effective amount of one or
more peptide conjugates of the present invention and two or more
peptides of the present invention.
[0489] In another aspect, the present invention provides a method
of vaccinating or eliciting an immune response in a subject
comprising administering to the subject an effective amount of a
peptide conjugate or peptide of the present invention.
[0490] In another aspect, the present invention provides use of a
peptide conjugate or peptide of the invention for vaccinating or
eliciting an immune response in a subject.
[0491] In another aspect, the present invention provides use of a
peptide conjugate or a peptide of the invention in the manufacture
of a medicament for vaccinating or eliciting an immune response in
a subject.
[0492] In another aspect, the present invention provides a method
of vaccinating or eliciting an immune response in a subject
comprising administering to the subject an effective amount of the
pharmaceutical composition of the present invention.
[0493] In another aspect, the present invention provides use of a
pharmaceutical composition of the invention for vaccinating or
eliciting an immune response in a subject.
[0494] In another aspect, the present invention provides use of one
or more peptides of the present invention or one or more peptide
conjugates of the present invention in the manufacture of a
medicament for vaccinating or eliciting an immune response in a
subject.
[0495] In another aspect, the present invention provides a method
of eliciting an immune response in a subject comprising
administering to the subject an effective amount of a peptide
conjugate of the present invention or a pharmaceutically acceptable
salt or solvate thereof.
[0496] In another aspect, the present invention provides use of a
peptide conjugate of the invention or a pharmaceutically acceptable
salt or solvate thereof for eliciting an immune response in a
subject.
[0497] In another aspect, the present invention provides use of a
peptide conjugate of the invention or a pharmaceutically acceptable
salt or solvate thereof in the manufacture of a medicament for
eliciting an immune response in a subject.
[0498] In another aspect, the present invention provides a method
of vaccinating a subject comprising administering to the subject an
effective amount of a peptide conjugate of the present invention or
a pharmaceutically acceptable salt or solvate thereof.
[0499] In another aspect, the present invention provides use of a
peptide conjugate of the present invention for vaccinating a
subject or a pharmaceutically acceptable salt or solvate
thereof.
[0500] In another aspect, the present invention provides use of a
peptide conjugate of the invention or a pharmaceutically acceptable
salt or solvate thereof in the manufacture of a medicament for
vaccinating a subject.
[0501] In some embodiments, the method comprises the administration
of one or more peptides of the present invention and/or one or more
peptide conjugates of the present invention, for example one or
more peptides in combination with one or more peptide conjugates to
the subject.
[0502] In some embodiments, one or more peptides of the present
invention and/or one or more peptide conjugates of the present
invention, for example one or more peptides in combination with one
or more peptide conjugates are used for vaccinating or eliciting an
immune response in the subject or in the manufacture of a
medicament for vaccinating or eliciting an immune response in the
subject.
[0503] In some embodiment, two or more peptides, two or more
peptide conjugates, or one or more peptides and one or more peptide
conjugates are used or administered. In some embodiments the two or
more peptides, two or more peptide conjugates, or one or more
peptides and one or more peptide conjugates are used or
administered simultaneously, sequentially, or separately.
[0504] Asymmetric centers may exist in the compounds described
herein. The asymmetric centers may be designated as (R) or (S),
depending on the configuration of substituents in three dimensional
space at the chiral carbon atom. All stereochemical isomeric forms
of the compounds, including diastereomeric, enantiomeric, and
epimeric forms, as well as d-isomers and l-isomers, and mixtures
thereof, including enantiomerically enriched and diastereomerically
enriched mixtures of stereochemical isomers, are within the scope
of the invention.
[0505] Individual enantiomers can be prepared synthetically from
commercially available enantiopure starting materials or by
preparing enantiomeric mixtures and resolving the mixture into
individual enantiomers. Resolution methods include conversion of
the enantiomeric mixture into a mixture of diastereomers and
separation of the diastereomers by, for example, recrystallization
or chromatography, and any other appropriate methods known in the
art. Starting materials of defined stereochemistry may be
commercially available or made and, if necessary, resolved by
techniques well known in the art.
[0506] The compounds described herein may also exist as
conformational or geometric isomers, including cis, trans, syn,
anti, entgegen (E), and zusammen (Z) isomers. All such isomers and
any mixtures thereof are within the scope of the invention.
[0507] Also within the scope of the invention are any tautomeric
isomers or mixtures thereof of the compounds described. As would be
appreciated by those skilled in the art, a wide variety of
functional groups and other structures may exhibit tautomerism.
Examples include, but are not limited to, keto/enol, imine/enamine,
and thioketone/enethiol tautomerism.
[0508] The compounds described herein may also exist as
isotopologues and isotopomers, wherein one or more atoms in the
compounds are replaced with different isotopes. Suitable isotopes
include, for example, .sup.1H, .sup.2H (D), .sup.3H (T), .sup.12C,
.sup.13C, .sup.14C, .sup.16O, and .sup.18O. Procedures for
incorporating such isotopes into the compounds described herein
will be apparent to those skilled in the art. Isotopologues and
isotopomers of the compounds described herein are also within the
scope of the invention.
[0509] Also within the scope of the invention are pharmaceutically
acceptable salts and solvates, including hydrates of the compounds
described herein. Such salts include, acid addition salts, base
addition salts, and quaternary salts of basic nitrogen-containing
groups.
[0510] Acid addition salts can be prepared by reacting compounds,
in free base form, with inorganic or organic acids. Examples of
inorganic acids include, but are not limited to, hydrochloric,
hydrobromic, nitric, sulfuric, and phosphoric acid. Examples of
organic acids include, but are not limited to, acetic,
trifluoroacetic, propionic, succinic, glycolic, lactic, malic,
tartaric, citric, ascorbic, maleic, fumaric, pyruvic, aspartic,
glutamic, stearic, salicylic, methanesulfonic, benzenesulfonic,
isethionic, sulfanilic, adipic, butyric, and pivalic.
[0511] Base addition salts can be prepared by reacting compounds,
in free acid form, with inorganic or organic bases. Examples of
inorganic base addition salts include alkali metal salts, alkaline
earth metal salts, and other physiologically acceptable metal
salts, for example, aluminium, calcium, lithium, magnesium,
potassium, sodium, or zinc salts. Examples of organic base addition
salts include amine salts, for example, salts of trimethylamine,
diethylamine, ethanolamine, diethanolamine, and
ethylenediamine.
[0512] Quaternary salts of basic nitrogen-containing groups in the
compounds may be may be prepared by, for example, reacting the
compounds with alkyl halides such as methyl, ethyl, propyl, and
butyl chlorides, bromides, and iodides, dialkyl sulfates such as
dimethyl, diethyl, dibutyl, and diamyl sulfates, and the like.
[0513] The general chemical terms used in the formulae herein have
their usual meaning.
[0514] The term "aliphatic" is intended to include saturated and
unsaturated, nonaromatic, straight chain, branched, acyclic, and
cyclic hydrocarbons. Those skilled in the art will appreciate that
aliphatic groups include, for example, alkyl, alkenyl, alkynyl,
cycloalkyl, and cycloalkenyl groups. In some embodiments, the
aliphatic group is saturated.
[0515] The term "heteroaliphatic" is intended to include aliphatic
groups, wherein one or more chain carbon atoms are replaced with a
heteroatom. In some embodiments, the heteroaliphatic is
saturated.
[0516] The term "alkyl" is intended to include saturated or
unsaturated straight chain and branched chain hydrocarbon groups.
Examples of saturated hydrocarbon groups include methyl, ethyl,
n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and the
like. Unsaturated alkyl groups have one or more carbon-carbon
double bonds or triple bonds. Examples of unsaturated alkyl groups
include vinyl, prop-2-enyl, crotyl, isopent-2-enyl, 2-butadienyl,
penta-2,4-dienyl, penta-1,4-dienyl, ethynyl, prop-3-ynyl,
but-3-ynyl, and the like. In some embodiments, the alkyl is
saturated.
[0517] The term "heteroalkyl" is intended to include alkyl groups,
wherein one or more chain carbon atoms are replaced with a
heteroatom. In some embodiments, the heteroalkyl is saturated.
[0518] The term "cycloalkyl" is intended to include non-aromatic
cyclic alkyl groups. Examples of cycloalkyl groups include but are
not limited to cyclopentyl, cyclohexyl, cyclohex-1-enyl,
cyclohex-3-enyl, cycloheptyl. In some embodiments, the cycloalkyl
is saturated.
[0519] The term "heteroatom" is intended to include oxygen,
nitrogen, sulfur, or phosphorus. In some embodiments, the
heteroatom is selected from the group consisting of oxygen,
nitrogen, and sulfur.
[0520] The term "aryl" is intended to include aromatic radicals.
Examples include, but are not limited to, phenyl, tolyl, naphthyl,
indanyl, and the like. In some embodiments, aryl groups comprise
from 4 to 8 or from 6 to 8 carbon atoms in the aromatic ring
system.
[0521] As used herein, the term "substituted" is intended to mean
that one or more hydrogen atoms in the group indicated is replaced
with one or more independently selected suitable substituents,
provided that the normal valency of each atom to which the
substituent/s are attached is not exceeded, and that the
substitution results in a stable compound.
[0522] Examples of optional substituents for aliphatic,
heteroaliphatic, alkyl, heteroalkyl, and cycloalkyl groups in the
compounds described herein include but are not limited to halo, CN,
NO.sub.2, OH, NH.sub.2, NHR1, NR1R2, C1-6haloalkyl, C1-6haloalkoxy,
C(O)NH.sub.2, C(O)NHR1, C(O)NR1R1, SO.sub.2R1, OR1, SR1, S(O)R1,
C(O)R1, and C1-6aliphatic; wherein R1 and R2 are each independently
C1-6alkyl.
[0523] The term "carboxyl protecting group" as used herein is means
a group that is capable of readily removed to provide the OH group
of a carboxyl group and protects the carboxyl group against
undesirable reaction during synthetic procedures. Such protecting
groups are described in Protective Groups in Organic Synthesis
edited by T. W. Greene et al. (John Wiley & Sons, 1999) and
`Amino Acid-Protecting Groups` by Fernando Albericio (with Albert
Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009 (109)
2455-2504. Examples include, but are not limited to, alkyl and
silyl groups, for example methyl, ethyl, tert-butyl, methoxymethyl,
2,2,2-trichloroethyl, benzyl, diphenylmethyl, trimethylsilyl, and
tert-butyldimethylsilyl, and the like.
[0524] The term "amine protecting group" as used herein means a
group that is capable of being readily removed to provide the
NH.sub.2 group of an amine group and protects the amine group
against undesirable reaction during synthetic procedures. Such
protecting groups are described in Protective Groups in Organic
Synthesis edited by T. W. Greene et al. (John Wiley & Sons,
1999) and `Amino Acid-Protecting Groups` by Fernando Albericio
(with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews
2009 (109) 2455-2504. Examples include, but are not limited to,
acyl and acyloxy groups, for example acetyl, chloroacetyl,
trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl,
trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl,
picolinoyl, aminocaproyl, benzoyl, methoxy-carbonyl,
9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl,
2-trimethylsilylethoxy-carbonyl, tert-butyloxycarbonyl,
benzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2,4-dichloro-benzyloxycarbonyl, and the like. Further examples
include Cbz (carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl),
Bpoc (2-(4-biphenyl)isopropoxycarbonyl) and Dde
(1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl).
[0525] As used herein, the term "and/or" means "and", or "or", or
both.
[0526] The term "(s)" following a noun contemplates the singular
and plural form, or both.
[0527] The term "comprising" as used in this specification means
"consisting at least in part of".
[0528] When interpreting each statement in this specification that
includes the term "comprising", features other than that or those
prefaced by the term may also be present. Related terms such as
"comprise" and "comprises" are to be interpreted in the same
manner. The "containing" is also to be interpreted in the same
manner.
[0529] The invention may also be said broadly to consist in the
parts, elements and features referred to or indicated in the
specification of the application, individually or collectively, in
any or all combinations of two or more of said parts, elements or
features, and where specific integers are mentioned herein which
have known equivalents in the art to which the invention relates,
such known equivalents are deemed to be incorporated herein as if
individually set forth.
[0530] It is intended that reference to a range of numbers
disclosed herein (for example, 1 to 10) also incorporates reference
to all rational numbers within that range (for example, 1, 1.1, 2,
3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of
rational numbers within that range (for example, 2 to 8, 1.5 to
5.5, and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges
expressly disclosed herein are hereby expressly disclosed. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
[0531] Although the present invention is broadly as defined above,
those persons skilled in the art will appreciate that the invention
is not limited thereto and that the invention also includes
embodiments of which the following description gives examples.
BRIEF DESCRIPTION OF THE FIGURES
[0532] The invention will be described with reference to the
accompanying figures in which:
[0533] FIG. 1 is a series of graphs showing CD80 response of
Pam3CSK4, 22, 20, and 26 across five donors. MFI: mean fluorescence
intensity; UT: untreated; Pam3CSK4=Pam3Cys [comprises SEQ ID NO:
22]; 22=8 [comprises SEQ ID NO: 22]; 20=9 [comprises SEQ ID NO:
22]; 26=11 [comprises SEQ ID NO: 21].
[0534] FIGS. 2a and 2b are HPLC chromatograms at 210 nm of pure 25
(2a) [SEQ ID NO: 22] and 26 (2b) [comprises SEQ ID NO: 21].
[0535] FIG. 2c is an ESI-MS of the major peak of the HPLC
chromatogram of 26 [comprises SEQ ID NO: 21].
[0536] FIG. 3 is a graph showing the results of the T cell
activation assay using CMV pp65.sub.495-503 [SEQ ID NO: 27]
constructs and the CD8+ T cell clone 4D9 with allogeneic HLA-A2+
MoDC as APC, as described herein in Example 4. 142, 131, and 110
each comprise SEQ ID NO: 27.
[0537] FIG. 4 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.153-180 [SEQ ID NO: 18]
constructs and the CD8+ T cell clone 2F2 with autologous LCL as
APC, as described herein in Example 5. 143 [SEQ ID NO: 18]. 144
[SEQ ID NO: 17]. 112, 111, and 132 each comprise SEQ ID NO: 24.
[0538] FIG. 5 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.153-180 [SEQ ID NO: 18]
constructs and the CD8+ T cell clone 2F2 with allogeneic HLA-A2+
HLA-DP4+ MoDC as APC, as described herein in Example 5. 143 [SEQ ID
NO: 18]. 144 [SEQ ID NO: 17]. 112, 111, and 132 each comprise SEQ
ID NO: 24.
[0539] FIG. 6 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.153-180 [SEQ ID NO: 18]
constructs and the CD4+ T cell clone 1B7 with autologous LCL as
APC, as described herein in Example 6. 143 [SEQ ID NO: 18]. 144
[SEQ ID NO: 17]. 112, 111, and 132 each comprise SEQ ID NO: 24.
[0540] FIG. 7 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.153-180 [SEQ ID NO: 18]
constructs and the CD4+ T cell clone 1B7 with allogeneic
HLA-A2+HLA-DP4+ MoDC as APC, as described herein in Example 6. 143
[SEQ ID NO: 18]. 144 [SEQ ID NO: 17]. 112, 111, and 132 each
comprise SEQ ID NO: 24.
[0541] FIG. 8 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.153-180 [SEQ ID NO: 18]
constructs and the CD4+ T cell clone 1C11 with allogeneic HLA-A2+
HLA-DP4+ MoDC as APC, as described herein in Example 6. 143 [SEQ ID
NO: 18]. 144 [SEQ ID NO: 17]. 112, 111, and 132 each comprise SEQ
ID NO: 24.
[0542] FIG. 9 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.79-116 [SEQ ID NO: 5]
constructs and the CD8+ T cell clone 1D7 with autologous LCL as
APC, as described herein in Example 7. 145 [SEQ ID NO: 5]. 114 and
113 each comprise SEQ ID NO: 25.
[0543] FIG. 10 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.79-116 [SEQ ID NO: 5]
constructs and the CD8+ T cell clone 1F10 with autologous LCL as
APC, as described herein in Example 7. 145 [SEQ ID NO: 5]. 114 and
113 each comprise SEQ ID NO: 25.
[0544] FIG. 11 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.118-143 [SEQ ID NO: 12]
constructs and the CD8+ T cell clone 1C11 with autologous LCL as
APC, as described herein in Example 8. 147 [SEQ ID NO: 12]. 116 and
115 each comprise SEQ ID NO: 26.
[0545] FIG. 12 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.118-143 [SEQ ID NO: 12]
constructs and the CD4+ T cell clone 1E4 with autologous LCL as
APC, as described herein in Example 9. 147 [SEQ ID NO: 12]. 116 and
115 each comprise SEQ ID NO: 26.
[0546] FIG. 13 is a graph showing the results of the T cell
activation assay using NY-ESO-1.sub.118-143 [SEQ ID NO: 12]
constructs and the CD4+ T cell clone 1D6 with autologous LCL as
APC, as described herein in Example 9. 147 [SEQ ID NO: 12]. 116 and
115 each comprise SEQ ID NO: 26.
[0547] FIG. 14 is a graph showing the results of the TLR agonism
assay using the NY-ESO-1 constructs and HekBlue.TM., as described
herein in Example 10. 142, 131, and 110 each comprise SEQ ID NO:
27. 144 [SEQ ID NO: 17]. 112, 111, and 132 each comprise SEQ ID NO:
24. 114 and 113 each comprise SEQ ID NO: 25. 116 and 115 each
comprise SEQ ID NO: 26.
[0548] FIG. 15 is four graphs showing the results of the TLR
agonism using the NY-ESO-1 constructs and the IL-8 assay, as
described herein in Example 10. 131 and 110 each comprise SEQ ID
NO: 27. 112, 111, and 132 each comprise SEQ ID NO: 24. 114 and 113
each comprise SEQ ID NO: 25. 116 and 115 each comprise SEQ ID NO:
26.
DETAILED DESCRIPTION OF THE INVENTION
[0549] The present invention relates to a method of making amino
acid and peptide conjugates. The method comprises reacting an
lipid-containing conjugation partner and an amino acid comprising
conjugation partner under conditions effective to conjugate the
lipid-containing conjugation partner to the amino acid-comprising
conjugation partner in a thiol-ene reaction. In some embodiments,
the method comprises reacting an lipid-containing conjugation
partner and a peptide-containing conjugation partner under
conditions effective to conjugate the lipid-containing conjugation
partner to the peptide of the peptide-containing conjugation
partner in a thiol-ene reaction.
[0550] The thiol-ene reaction involves the addition of a thiol
across a non-aromatic carbon-carbon double bond (i.e.
hydrothiolation of the carbon-carbon double bond). The reaction
proceeds via a free radical mechanism. There are three distinct
phases in the reaction: initiation, polymerisation or coupling, and
termination. Radical generation gives rise to an electrophilic
thiyl radical which propagates across the ene group, forming a
carbon-centred radical. Chain transfer from an additional thiol
molecule then quenches the radical on carbon to give the final
product.
[0551] In the method the present invention, one conjugation partner
comprises the thiol and the other comprises the carbon carbon
double bond.
[0552] One or more free radicals may be generated in the method of
the present invention by any method known in the art. The free
radicals may be generated thermally and/or photochemically. One or
more free radical initiators may be used to initiate the generation
of free radicals. Suitable free radical initiators include thermal
initiators and photoinitiators.
[0553] Free radicals are generated from thermal initiators by
heating. The rate of degradation of the thermal initiator and
resulting free radical formation depends on the initiator and the
temperature at which the initiator is heated. Higher temperatures
generally result in faster decomposition. A person skilled in the
art will be able to select an appropriate temperature for heating
the initiator without undue experimentation.
[0554] Numerous thermal initiators are commercially available.
Examples of thermal initiators include but are not limited to
tert-amyl peroxybenzoate, 1,1'-azobis(cyclohexanecarbonitrile),
2,2'-azobisisobutyronitrile (AIBN), benzoyl peroxide, tert-butyl
hydroperoxide, tert-butyl peracetate, tert-butyl peroxide,
tert-butyl peroxybenzoate, tert-butylperoxy isopropyl carbonate,
lauroyl peroxide, peracetic acid, and potassium persulfate.
[0555] Free radicals may be generated from photoinitiators by
irradiation with light. The frequency of light necessary to induce
degradation of the photoinitiators and free radical formation
depends on the initiator. Many photoinitiators can be initiated
with ultraviolet light.
[0556] Light of a specific wavelength or wavelength range may be
used to selectively irradiate the initiator, where the
lipid-containing conjugation partner or amino acid-comprising
conjugation partner, for example a peptide-containing conjugation
partner, comprises photosensitive groups. In certain embodiments of
the method of the present invention, a frequency of about 365 nm is
used. Light of this frequency is generally compatible with the side
chains of naturally occurring amino acids.
[0557] A wide range of photoinitiators are commercially available.
Examples of photoinitiators include but are not limited to
acetophenone, anisoin, anthraquinone, anthraquinone-2-sulfonic
acid, benzil, benzoin, benzoin ethyl ether, benzoin isobutyl ether,
benzoin methyl ether, benzophenone,
3,3',4,4'-benzophenonetetracarboxylic dianhydride,
4-benzoylbiphenyl,
2-benzyl-2-(dimethylamino)-4'-morpholinobutyrophenone,
4'-bis(diethylamino)benzophenone, 4,4'-bis(dimethyla
mino)benzophenone, camphorquinone, 2-chlorothioxanthen-9-one,
dibenzosuberenone, 2,2-diethoxyacetophenone,
4,4'-dihydroxybenzophenone, 2,2-dimethoxy-2-phenylacetophenone
(DMPA), 4-(dimethylamino)benzophenone, 4,4'-dimethylbenzil,
2,5-dimethylbenzophenone, 3,4-dimethylbenzophenone,
4'-ethoxyacetophenone, 2-ethylanthraquinone,
3'-hydroxyacetophenone, 4'-hydroxyacetophenone,
3-hydroxybenzophenone, 4-hydroxybenzophenone, 1-hydroxycyclohexyl
phenyl ketone, 2-hydroxy-2-methylpropiophenone,
2-methylbenzophenone, 3-methylbenzophenone, methybenzoylformate,
2-methyl-4'-(methylthio)-2-morpholinopropiophenone,
phenanthrenequinone, 4'-phenoxyacetophenone, and
thioxanthen-9-one.
[0558] A person skilled in the art will be able to select
appropriate free radical initiators for use in the method having
regard to, for example, the nature of the lipid-containing
conjugation partner, amino acid-comprising conjugation partner, for
example a peptide-containing conjugation partner, and any other
components present in the reaction mixture. In some embodiments,
the initiator is present in the reaction in a stoichiometric ratio
relative to the starting material comprising the thiol of from
about 20:1 to about 0.05:1, from about 10:1 to about 0.05:1, from
about 5:1 to about 0.05:1, from about 3:1 to about 0.5:1.
[0559] The lipid-containing conjugation partner and amino
acid-comprising conjugation partner, for example a
peptide-containing conjugation partner, in the reaction are as
defined in any of the embodiments described herein.
[0560] The lipid-containing conjugation partner and amino
acid-comprising conjugation partner, for example a
peptide-containing conjugation partner, may be prepared using known
synthetic chemistry techniques (for example, the methods generally
described in Louis F Fieser and Mary F, Reagents for Organic
Synthesis v. 1-19, Wiley, New York (1967-1999 ed.) or Beilsteins
Handbuch der organischen Chemie, 4, Aufl. Ed. Springer-Verlag
Berlin, including supplements (also available via the Beilstein
online database)) or, in some embodiments, may be commercially
available.
[0561] Lipid-containing conjugation partner compounds of the
formula (II)
##STR00023##
[0562] wherein m, R1, R2, R3, R4, R5, and L1 are each independently
as defined in any of the embodiments described for the compound of
formula (I) may be prepared by reacting a compound of the formula
(VI)
L.sup.1-C(O)--X (VI)
[0563] wherein X is OH or a suitable leaving group with a compound
of the formula (VII):
##STR00024##
[0564] under conditions effective for esterification. Methods for
esterification are well known in the art. For example, when X is
chloro, the reaction may be carried out in the presence of a base,
such as pyridine or triethylamine, in a suitable solvent. The acid
chloride may be converted in situ to a more reactive species (e.g.
to the corresponding iodide, using sodium iodide). The temperature
at which the reaction is carried out depends on the reactivity of
the acid species and the solvent used.
[0565] Numerous compounds of formula (VI) are commercially
available. Others may be prepared using standard synthetic
chemistry techniques from commercially available precursors. For
example, compounds of formula (VI) wherein X is chloro may be
prepared treating the corresponding carboxylic acid with thionyl
chloride in a suitable solvent or mixture of solvents.
[0566] Lipid containing conjugation partner compounds of the
formula (IIA)
##STR00025##
[0567] wherein p, R11, R22, R33, R44, and L1 are as defined in the
compound of formula (IA) may be prepared by reacting a compound of
the formula (VI) as defined above with a compound of the formula
(VIII):
##STR00026##
[0568] wherein P is a suitable protecting group under conditions
effective for esterification, and then removing the protecting
group.
[0569] Alternatively, compounds of the formula (IIA) may be
prepared by reacting a compound of the formula (VI) as defined
above with a compound of the formula (IX):
##STR00027##
[0570] wherein P is a suitable protecting group under conditions
effective for esterification, removing the protecting group, and
then converting the corresponding alcohol to a thiol. Suitable
methods for converting the alcohol to a thiol will be apparent to
those skilled in the art.
[0571] Preparation of the compounds may involve the protection and
deprotection of various chemical groups. The need for protection
and deprotection, and the selection of appropriate protecting
groups, can be readily determined by a person skilled in the art.
Protecting groups and methods for protection and deprotection are
well known in the art (see e.g. T. W. Greene and P. G. M. Wuts,
Protective Groups in Organic Synthesis, 34 Ed., Wiley & Sons,
Inc., New York (1999)).
[0572] Similarly, compounds of formula (VII), (VIII), and (IX) are
also commercially available or may be prepared from commercially
available precursors using standard synthetic chemistry
techniques.
[0573] The order in which the lipid-containing conjugation partner
and amino acid-comprising conjugation partner, for example a
peptide-containing conjugation partner, and any other components
present in the reaction mixture are introduced into the reaction
vessel may vary. The reaction may be carried out as a one-pot
procedure.
[0574] The stoichiometry of the lipid-containing conjugation
partner and amino acid-comprising conjugation partner, for example
a peptide-containing conjugation partner, in the reaction may vary.
In some embodiments, the stoichiometric ratio of amino
acid-comprising conjugation partner to lipid-containing conjugation
partner is from about 1:0.5 to about 1:20, from about 1:1 to about
1:10, from about 1:1 to about 1:5, from about 1:1 to about 1:3. In
some embodiments, the stoichiometric ratio of peptide-containing
conjugation partner to lipid-containing conjugation partner is from
about 1:0.5 to about 1:20, from about 1:1 to about 1:10, from about
1:1 to about 1:5, from about 1:1 to about 1:3.
[0575] The reaction may be carried out at any suitable temperature.
In some embodiments, the reaction is carried out at a temperature
from about -25.degree. C. to about 200.degree. C., from about
-10.degree. C. to about 150.degree. C., from about 0.degree. C. to
about 125.degree. C., from about ambient temperature to about
100.degree. C. In some embodiments, the reaction is carried out at
a temperature of less than about 200.degree. C., less than about
175.degree. C., less than about 150.degree. C., less than about
125.degree. C., or less than about 100.degree. C.
[0576] In some embodiments, the reaction is carried out at a
temperature above ambient temperature. In one embodiment, the
reaction is carried out at a temperature from 40 to 200.degree. C.,
from 50 to 150.degree. C., from 60 to 100.degree. C., from 65 to
90.degree. C., or from 70 to 80.degree. C. In some embodiments, the
reaction is carried out at a temperature greater than 40 OC,
greater than 50.degree. C., greater than 75.degree. C., greater
than 100 OC, or greater than 150 OC.
[0577] The temperature at which the reaction is carried out may
depend on how free radicals are generated in the reaction. The
temperature used may be selected to control the rate of the
reaction. The temperature may be adjusted during the course of the
reaction to control the rate of the reaction. By controlling the
rate of the reaction it may be possible to minimise or obviate the
formation of undesirable by products (e.g. telomerisation or
polymerisation products).
[0578] If free radicals are generated thermally (e.g. using a
thermal initiator), the reaction will generally be carried out at a
temperature above ambient temperature. The temperature will depend
on the reactivity of the species from which free radicals are
generated.
[0579] If free radicals are generated photochemically the reaction
may be carried out, advantageously, at ambient temperature. In
certain embodiments, it may be desirable to cool the reaction
mixture to slow the rate of reaction or conversely heat the
reaction mixture to increase the rate of reaction.
[0580] A person skilled in the art will be able to select
appropriate temperatures for carrying out the method having regard
to the reactivity of the lipid-containing conjugation partner,
amino acid-comprising conjugation partner, for example a
peptide-containing conjugation partner, and free radical initiator
if used.
[0581] The temperature at which the reaction is carried out may be
controlled by heating or cooling the reaction mixture. The
temperature of the reaction mixture may be controlled by suitable
method known in the art. Heat may be applied to the reaction
mixture, for example, using a heat exchanger within the reaction
vessel, a heating jacket surrounding the reaction vessel, or by
immersing the reaction vessel in a heated liquid (e.g. an oil or
sand bath). In certain exemplary embodiments, the reaction mixture
is heated by microwave irradiation.
[0582] The progress of the reaction may be monitored by any
suitable means, for example, by thin layer chromatography (TLC) or
high performance liquid chromatography (HPLC). The reaction may be
allowed to proceed to substantial completion, as monitored by the
consumption of at least one of the starting materials. In some
embodiments, the reaction is allowed to proceed for a period of
time from 1 minute to 7 days, 5 minutes to 72 hours, 10 minutes to
48 hours, 10 minutes to 24 hours. In other embodiments, the
reaction is allowed to proceed for a period of time less than 72 h,
less than 48 h, less than 24 h, less than 12 h, less than 6 h, less
than 4 h, less than 2 h, or less than 1 h.
[0583] In some embodiments, the reaction is carried out until at
least about 50%, at least about 60%0, at least about 70%0, at least
about 75%0, at least about 80%0, at least about 850%, at least
about 90%0, at least about 95%0, at least about 97%0, at least
about 99%0 of the lipid-containing conjugation partner or amino
acid-comprising conjugation partner, whichever is
stoichiometrically less, has been consumed. In some embodiments,
the reaction is carried out until at least about 50%, at least
about 60%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 97%, at least about 99% of the lipid-containing
conjugation partner or peptide-containing conjugation partner,
whichever is stoichiometrically less, has been consumed. The
consumption of starting materials may be monitored by any suitable
method, for example, HPLC.
[0584] The reaction mixture may be mixed by any suitable method
known in the art, for example, using a magnetic or mechanical
stirrer. The method used may depend on the scale on which the
reaction is carried out.
[0585] The reaction is generally carried out in a liquid reaction
medium. The liquid reaction medium may comprise a solvent. Examples
of suitable solvents include dimethylformamide, dichloromethane,
1,2-dichloroethane, chloroform, carbon tetrachloride, water,
methanol, ethanol, dimethylsulfoxide, trifluoroacetic acid, acetic
acid, acetonitrile, and mixtures thereof.
[0586] The solvent may be selected based on the solubility of the
lipid-containing conjugation partner and amino acid-comprising
conjugation partner, for example a peptide-containing conjugation
partner, in the solvent. The solubility of the free radical
initiator may also be relevant. In some embodiments, the
lipid-containing conjugation partner is hydrophobic. The
hydrophobicity or hydrophilicity of an amino acid-comprising
conjugation partner, for example a peptide-containing conjugation
partner, may vary depending on, for example, amino acid sequence of
the peptide of a peptide-containing conjugation partner. The
presence of a solubilising group in the peptide-containing
conjugation partner may increase solubility in polar solvents, such
as water. A person skilled in the art will be able to select an
appropriate solvent without undue experimentation.
[0587] The reaction may be carried out under substantially
oxygen-free conditions. Oxygen may quench free radicals formed in
the reaction. The reaction mixture may be degassed with an inert
gas (e.g. nitrogen or argon) that is substantially oxygen-free to
remove any dissolved oxygen before free radicals are generated.
Alternatively, individual components of the reaction mixture may be
degassed with inert gas that is substantially oxygen-free prior to
being combined in the reaction vessel. The reaction may be carried
out under an atmosphere of inert gas that is substantially
oxygen-free.
[0588] The method of the present invention may be carried out at
ambient pressure.
[0589] If the rate of chain transfer relative to propagation in the
thiol-ene reaction is slow, undesirable dimerisation,
telomerisation, or polymerisation may occur.
[0590] An additive that inhibits dimerisation, telomerisation, or
polymerisation may be included in the reaction mixture in the
method of the present invention. The inventors have found that in
some embodiments the inclusion of an extraneous thiol that
facilitates chain transfer as an additive in the reaction mixture
reduces the formation of undesirable by products. The extraneous
thiol may, in some embodiments, increase the efficiency of the
desired thiol ene reaction. Examples of suitable extraneous thiols
include but are not limited to reduced glutathione, DODT, DTT,
protein, and the like. The inventors have found that in some
embodiments the inclusion of DTT resulted in no undesirable by
products.
[0591] In certain embodiments, the extraneous thiol is a sterically
hindered thiol. Non-limiting examples of a suitable sterically
hindered extraneous thiol include tert-butyl mercaptan and
1-methylpropyl mercaptan.
[0592] The inclusion of an acid in some embodiments may also
inhibit dimerisation, telomerisation, or polymerisation. The acid
may be a strong inorganic acid, for example HCl, or organic acid,
for example TFA. In certain embodiments, the additive is TFA.
[0593] The inventors have found that in some embodiments including
both tert-butyl mercaptan and TFA as additives in the reaction
mixture can reduce the the formation of oligomers, and increase the
conversion of starting material to the desired product.
Accordingly, in certain exemplary embodiments, the reaction mixture
comprises a combination of TFA and tert-butyl mercaptan.
[0594] The additive is generally used in an amount sufficient to
minimise the formation of undesirable by products without adversely
affecting the reaction or any, optional, subsequent steps in the
method. In some embodiments, the additive is present in the
reaction a stoichiometric ratio relative to the starting material
comprising the thiol of from about 20:1 to about 0.05:1, from about
10:1 to about 0.5:1, from about 5:1 to about 1:1, from about 3:1 to
about 1:1.
[0595] In some embodiments, less than about 50%, less than about
40%, less than about 30%, less than about 25%, less than about 20%,
less than about 15%, less than about 10%, less than about 5%, less
than about 3%, or less than about 1% by weight of the
lipid-containing conjugation partner and amino acid-comprising
conjugation partner starting materials used in the reaction are
undesirable by products resulting from dimerisation,
telomerisation, or polymerisation. In some embodiments, less than
about 50%, less than about 40%, less than about 30%, less than
about 25%, less than about 20%, less than about 15%, less than
about 10%, less than about 5%, less than about 3%, or less than
about 1% by weight of the lipid-containing conjugation partner and
peptide-containing conjugation partner starting materials used in
the reaction are undesirable by products resulting from
dimerisation, telomerisation, or polymerisation. The purity of the
products of the reaction may be determined by, for example,
HPLC.
[0596] The concentration of the lipid-containing conjugation
partner and amino acid-comprising conjugation partner, for example
a peptide-containing conjugation partner, respectively, in the
reaction mixture may also affect the reaction. Those skilled in the
art will be able to vary the concentration of the lipid-containing
conjugation partner and peptide-containing conjugation partner in
the reaction mixture to e.g. optimise yield and purity without
undue experimentation.
[0597] In some embodiments, the starting material comprising the
thiol is present in a concentration from about 0.05 mM to about 1
M, from about 0.5 mM to about 1 M, from about 1 mM to about 1 M. In
some embodiments, the concentration is at least about 0.05 mM, 0.5
mM, or 1 mM.
[0598] In some embodiments, the concentration of the starting
material comprising the alkene is at least about 0.05 mM, 0.5 mM,
or 1 mM.
[0599] In some embodiments, the amino acid conjugate or peptide
conjugate may be separated from the reaction medium after the
reaction and optionally purified. In some embodiments, the peptide
conjugate may be separated from the reaction medium after the
reaction and optionally purified. The conjugate may be separated
from the reaction medium using any suitable method known in the
art, for example, by precipitation.
[0600] In some embodiments, the amino acid or peptide conjugate is
purified after separating it from the reaction medium. In some
embodiments, the peptide conjugate is purified after separating it
from the reaction medium. In specifically contemplated embodiments,
the conjugate is purified by HPLC using one or more suitable
solvents.
[0601] The peptide conjugate produced by and/or the
peptide-containing conjugation partner in the method of the present
invention may comprise a synthetic peptide. Synthetic peptides may
be prepared using solid phase peptide synthesis (SPPS).
[0602] The basic principle for solid phase peptide synthesis (SPPS)
is a stepwise addition of amino acids to a growing polypeptide
chain anchored via a linker molecule to a solid phase support,
typically a resin particle, which allows for cleavage and
purification once the polypeptide chain is complete. Briefly, a
solid phase resin support and a starting amino acid are attached to
one another via a linker molecule. Such resin-linker-acid matrices
are commercially available.
[0603] The amino acid to be coupled to the resin is protected at
its No-terminus by a chemical protecting group.
[0604] The amino acid may also have a side-chain protecting group.
Such protecting groups prevent undesired or deleterious reactions
from taking place during the process of forming the new peptide
bond between the carboxyl group of the amino acid to be coupled and
the unprotected Na-amino group of the peptide chain attached to the
resin.
[0605] The amino acid to be coupled is reacted with the unprotected
Na-amino group of the N-terminal amino acid of the peptide chain,
increasing the chain length of the peptide chain by one amino acid.
The carboxyl group of the amino acid to be coupled may be activated
with a suitable chemical activating agent to promote reaction with
the Na-amino group of the peptide chain. The Na-protecting group of
N-terminal amino acid of the peptide chain is then removed in
preparation for coupling with the next amino acid residue. This
technique consists of many repetitive steps making automation
attractive whenever possible. Those skilled in the art will
appreciate that peptides may be coupled to the Na-amino group of
the solid phase bound amino acid or peptide instead of an
individual amino acid, for example where a convergent peptide
synthesis is desired.
[0606] When the desired sequence of amino acids is achieved, the
peptide is cleaved from the solid phase support at the linker
molecule.
[0607] SPPS may be carried out using a continuous flow method or a
batch flow method. Continuous flow permits real-time monitoring of
reaction progress via a spectrophotometer, but has two distinct
disadvantages--the reagents in contact with the peptide on the
resin are diluted, and scale is more limited due to physical size
constraints of the solid phase resin. Batch flow occurs in a filter
reaction vessel and is useful because reactants are accessible and
can be added manually or automatically.
[0608] The types of protecting groups are commonly used for
protecting the N-alpha-amino terminus: "Boc"
(tert-butyloxycarbonyl) and "Fmoc" (9-fluorenylmethyloxycarbonyl).
Reagents for the Boc method are relatively inexpensive, but they
are highly corrosive and require expensive equipment and more
rigorous precautions to be taken. The Fmoc method, which uses less
corrosive, although more expensive, reagents is typically
preferred.
[0609] For SPPS, a wide variety of solid support phases are
available. The solid phase support used for synthesis can be a
synthetic resin, a synthetic polymer film or a silicon or silicate
surface (e.g. controlled pore glass) suitable for synthesis
purposes. Generally, a resin is used, commonly polystyrene
suspensions, or polystyrene-polyethyleneglycol, or polymer supports
for example polyamide. Examples of resins functionalized with
linkers suitable for Boc-chemistry include PAM resin, oxime resin
SS, phenol resin, brominated Wang resin and brominated PPOA resin.
Examples of resins suitable for Fmoc chemistry include AMPB-BHA
resin, Sieber amide resin, Rink acid resin, Tentagel S AC resin,
2-chlorotrityl chloride resin, 2-chlorotrityl alcohol resin,
TentaGel S Trt-OH resin, Knorr-2-chlorotrityl resin,
hydrazine-2-chlorotrityl resin, ANP resin, Fmoc photolable resin,
HMBA-MBHA resin, TentaGel S HMB resin, Aromatic Safety Catch
resinBAl resin and Fmoc-hydroxylamine 2 chlorotrityl resin. Other
resins include PL Cl-Trt resin, PL-Oxime resin and PL-HMBA
Resin.
[0610] For each resin appropriate coupling conditions are known in
the literature for the attachment of the starting monomer or
sub-unit.
[0611] Preparation of the solid phase support includes solvating
the support in an appropriate solvent (e.g. dimethylformamide). The
solid phase typically increases in volume during solvation, which
in turn increases the surface area available to carry out peptide
synthesis.
[0612] A linker molecule is then attached to the support for
connecting the peptide chain to the solid phase support. Linker
molecules are generally designed such that eventual cleavage
provides either a free acid or amide at the C-terminus. Linkers are
generally not resin-specific. Examples of linkers include peptide
acids for example
4-hydroxymethylphenoxyacetyl-4'-methylbenzyhydrylamine (HMP), or
peptide amides for example benzhydrylamine derivatives.
[0613] The first amino acid of the peptide sequence may be attached
to the linker after the linker is attached to the solid phase
support or attached to the solid phase support using a linker that
includes the first amino acid of the peptide sequence. Linkers that
include amino acids are commercially available.
[0614] The next step is to deprotect the Na-amino group of the
first amino acid. For Fmoc SPPS, deprotection of the Na-amino group
may be carried out with a mild base treatment (piperazine or
piperidine, for example). Side-chain protecting groups may be
removed by moderate acidolysis (trifluoroacetic acid (TFA), for
example). For Boc SPPS, deprotection of the Na-amino group may be
carried out using for example TFA.
[0615] Following deprotection, the amino acid chain extension, or
coupling, proceeds by the formation of peptide bonds. This process
requires activation of the C-.alpha.-carboxyl group of the amino
acid to be coupled. This may be accomplished using, for example, in
situ reagents, preformed symmetrical anhydrides, active esters,
acid halides, or urethane-protected N-carboxyanhydrides. The in
situ method allows concurrent activation and coupling. Coupling
reagents include carbodiimide derivatives, for example
N,N'-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide.
Coupling reagents also include uronium or phosphonium salt
derivatives of benzotriazol. Examples of such uronium and
phosphonium salts include HBTU
(O-1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate), BOP
(benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate), PyBOP
(Benzotriazole-1-yl-oxy-tripyrrolidinophosphonium
hexafluorophosphate), PyAOP, HCTU
(O-(1H-6-chloro-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate), TCTU
(O-1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate), HATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate), TATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate), TOTU
(O-[cyano(ethoxycarbonyl)methyleneamino]-N,N,N',N''-tetramethyluronium
tetrafluoroborate), and HAPyU
(O-(benzotriazol-1-yl)oxybis-(pyrrolidino)-uronium
hexafluorophosphate. In some embodiments, the coupling reagent is
HBTU, HATU, BOP, or PyBOP.
[0616] After the desired amino acid sequence has been synthesized,
the peptide is cleaved from the resin. The conditions used in this
process depend on the sensitivity of the amino acid composition of
the peptide and the side-chain protecting groups. Generally,
cleavage is carried out in an environment containing a plurality of
scavenging agents to quench the reactive carbonium ions that
originate from the protective groups and linkers. Common cleaving
agents include, for example, TFA and hydrogen fluoride (HF). In
some embodiments, where the peptide is bound to the solid phase
support via a linker, the peptide chain is cleaved from the solid
phase support by cleaving the peptide from the linker.
[0617] The conditions used for cleaving the peptide from the resin
may concomitantly remove one or more side-chain protecting
groups.
[0618] The use of protective groups in SPPS is well established.
Examples of common protective groups include but are not limited to
acetamidomethyl (Acm), acetyl (Ac), adamantyloxy (AdaO), benzoyl
(Bz), benzyl (Bzl), 2-bromobenzyl, benzyloxy (BzlO),
benzyloxycarbonyl (Z), benzyloxymethyl (Bom),
2-bromobenzyloxycarbonyl (2-Br-Z), tert-butoxy (tBuO),
tert-butoxycarbonyl (Boc), tert-butoxymethyl (Bum), tert-butyl
(tBu), tert-buthylthio (tButhio), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), cyclohexyloxy (cHxO), 2,6-dichlorobenzyl (2,6-DiCl-Bzl),
4,4'-dimethoxybenzhydryl (Mbh),
1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-methyl-butyl (ivDde),
4-{N-[1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-methylbutyl]-amino)
benzyloxy (ODmab), 2,4-dinitrophenyl (Dnp),
fluorenylmethoxycarbonyl (Fmoc), formyl (For),
mesitylene-2-sulfonyl (Mts), 4-methoxybenzyl (MeOBzl),
4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), 4-methoxytrityl
(Mmt), 4-methylbenzyl (MeBzl), 4-methyltrityl (Mtt),
3-nitro-2-pyridinesulfenyl (Npys),
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),
2,2,5,7,8-pentamethyl-chromane-6-sulfonyl (Pmc), tosyl (Tos),
trifluoroacetyl (Tfa), trimethylacetamidomethyl (Tacm), trityl
(Trt) and xanthyl (Xan).
[0619] Where one or more of the side chains of the amino acids of
the peptide contains functional groups, such as for example
additional carboxylic, amino, hydroxy or thiol groups, additional
protective groups may be necessary. For example, if the Fmoc
strategy is used, Mtr, Pmc, Pbf may be used for the protection of
Arg; Trt, Tmob may be used for the protection of Asn and Gin; Boc
may be used for the protection of Trp and Lys; tBu may be used for
the protection of Asp, Glu, Ser, Thr and Tyr; and Acm, tBu,
tButhio, Trt and Mmt may be used for the protection of Cys. A
person skilled in the art will appreciate that there are numerous
other suitable combinations.
[0620] The methods for SPPS outlined above are well known in the
art. See, for example, Atherton and Sheppard, "Solid Phase Peptide
Synthesis: A Practical Approach," New York: IRL Press, 1989;
Stewart and Young: "Solid-Phase Peptide Synthesis 2nd Ed.,"
Rockford, Ill.: Pierce Chemical Co., 1984; Jones, "The Chemical
Synthesis of Peptides," Oxford: Clarendon Press, 1994; Merrifield,
J. Am. Soc. 85:2146-2149 (1963); Marglin, A. and Merrifield, R. B.
Annu. Rev. Biochem. 39:841-66 (1970); and Merrifield R. B. JAMA.
210(7):1247-54 (1969); and "Solid Phase Peptide Synthesis--A
Practical Approach" (W. C. Chan and P. D. White, eds. Oxford
University Press, 2000). Equipment for automated synthesis of
peptides or polypeptides is readily commercially available from
suppliers such as Perkin Elmer/Applied Biosystems (Foster City,
Calif.) and may be operated according to the manufacturer's
instructions.
[0621] Following cleavage from the resin, the peptide may be
separated from the reaction medium, e.g. by centrifugation or
filtration. The peptide may then be subsequently purified, e.g. by
HPLC using one or more suitable solvents.
[0622] Advantageously, the inventors have found that in some
embodiments the peptide-containing conjugation partner may be used
in the method of the present invention without purification
following cleavage of the peptide from the resin.
[0623] The inventors have also advantageously found that the method
of the present invention can be carried out using a
peptide-containing conjugation partner, wherein the peptide does
not contain an No-amino group protecting group or any side chain
protecting groups. The reaction is generally selective for reaction
of a thiol and a non-aromatic carbon-carbon double bond.
[0624] It may be necessary to protect thiol groups present in the
peptide-containing conjugation partner (e.g. in cysteine residues
of the peptide) with a protective group to prevent undesirable
competing reactions in the method of the present invention. The
thiol groups may be protected with a protective group that is not
removable under the conditions used to remove one or more other
protecting groups present in the peptide or to cleave the peptide
from the resin. Typically, the peptide will be synthesised using
amino acids bearing the appropriate protecting groups. A person
skilled in the art will be able to select appropriate protecting
groups without undue experimentation.
[0625] In certain embodiments, the amino acid-comprising
conjugation partner and lipid-containing conjugation partner
comprise one or more unsaturated carbon-carbon bonds in addition to
the carbon-carbon double bond to be reacted. In certain
embodiments, the peptide-containing conjugation partner and
lipid-containing conjugation partner comprise one or more
unsaturated carbon-carbon bonds in addition to the carbon-carbon
double bond to be reacted. Those skilled in the art will appreciate
that the selectivity of the thiol for the carbon-carbon double bond
to be reacted in such embodiments may depend on, for example, the
steric and/or electronic environment of the carbon-carbon double
bond relative to the one or more unsaturated carbon-carbon bonds.
In certain embodiments, the carbon-carbon double bond to be reacted
is activated relative to any other unsaturated carbon-carbon bonds
in the amino acid-comprising conjugation partner and
lipid-containing conjugation partner. In certain embodiments, the
carbon-carbon double bond to be reacted is activated relative to
any other unsaturated carbon-carbon bonds in the peptide-containing
conjugation partner and lipid-containing conjugation partner.
[0626] In some embodiments, the No-amino group of the amino acid of
the amino acid-comprising conjugation partner comprising the
carbon-carbon double bond or thiol is acylated, for example
acetylated. In some embodiments, the method of the present
invention may comprise acylating, for example acetylating, the
No-amino group of the amino acid of the amino acid-comprising
conjugation partner comprising the carbon-carbon double bond or
thiol to be reacted.
[0627] Where a peptide-containing conjugation partner has been
synthesised by SPPS, acylation may be carried out prior to or after
cleavage from the resin. In some embodiments, the amino acid
residue of the peptide-containing conjugation partner bearing the
carbon-carbon double bond or thiol to be reacted is an N-terminal
amino acid residue, and the method comprises acylating the
N-terminal amino group prior to cleaving the peptide.
[0628] In some embodiments, the method further comprises acylating
the No-amino group of the amino acid of the amino acid conjugate or
the amino acid residue of the peptide conjugate to which the
lipid-containing conjugation partner is conjugated.
[0629] Acylation of the No-amino group of an amino acid may be
carried out by reacting an amino acid or peptide with an acylating
agent in the presence of base in a suitable solvent, for example
DMF. Non-limiting examples of acylating agents include acid
halides, for example acid chlorides such as acetyl chloride, and
acid anhydrides, for example acetic anhydride. Such agents maybe
commercially available or may be prepared by methods well known in
the art. Non-limiting examples of suitable bases include
triethylamine, diisopropylethylamine, 4-methylmorpholine, and the
like.
[0630] In other embodiments, the synthesising the peptide of the
peptide-containing conjugation partner comprises coupling an amino
acid or a peptide comprising an amino acid that is acylated at the
Na-amino group and comprises the carbon-carbon double bond or thiol
to be reacted to one or more amino acids and/or one or more
peptides.
[0631] In some embodiments, the method comprises coupling the amino
acid of the amino acid conjugate to an amino acid or a peptide to
provide a peptide conjugate. In some embodiments, the method
comprises coupling the amino acid of the amino acid conjugate to an
amino acid or peptide bound to a solid phase resin support by SPPS.
In some embodiments, the method comprises coupling the amino acid
of the amino acid conjugate to a peptide bound to a solid phase
resin support by SPPS. The method may comprise synthesising the
peptide bound to the solid phase resin support by SPPS.
[0632] In some embodiments, the method further comprises coupling
the amino acid of the amino acid conjugate or an amino acid of the
peptide conjugate to an amino acid or a peptide so as to provide a
peptide conjugate comprising a peptide epitope. In some
embodiments, the peptide to be coupled comprises a peptide epitope.
In other embodiments, a peptide epitope is formed on coupling. The
coupling may be carried out by SPPS as described herein.
[0633] In some embodiments, the method comprises coupling the amino
acid of the amino acid conjugate to a peptide bound to a solid
phase resin support by SPPS so as to provide a peptide conjugate
comprising a peptide epitope.
[0634] In one embodiment, the peptide of the peptide conjugate to
be coupled is bound to a solid phase resin support, and the method
comprises coupling an amino acid of the peptide conjugate to be
coupled to an amino acid or a peptide so as to provide a peptide
conjugate comprising a peptide epitope.
[0635] In an alternate embodiment, the method comprises coupling an
amino acid of the peptide conjugate to an amino acid or peptide
bound to a solid phase resin support by SPPS so as to provide
peptide conjugate comprising a peptide epitope.
[0636] In some embodiments, the method further comprises coupling
an epitope, for example a peptide epitope, to the amino acid
conjugate or peptide conjugate. Where the method comprises coupling
a peptide epitope, the coupling may be carried out by SPPS as
described herein.
[0637] In certain embodiments, the epitope, for example a peptide
epitope, is coupled or bound via a linker group. In certain
embodiments, the linker group is an amino sequence, for example a
sequence of two or more, three or more, or four or more contiguous
amino acids. In certain embodiments, the linker comprises from
about 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 4 to
20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, or 4 to 10 amino acids.
[0638] It will be appreciate by those skilled in the art that
coupling an amino acid or a peptide to another amino acid or
peptide as described herein may comprise forming a peptide bond
between the No-terminus of the amino acid or an amino acid of the
peptide of one coupling partner and the C-terminus of the amino
acid or an amino acid of the peptide of the other coupling
partner.
[0639] In some embodiments, the method of the present invention
comprises synthesising the amino acid sequence of the peptide of
the peptide-containing conjugation partner by SPPS; and reacting
the lipid-containing conjugation partner with the
peptide-containing conjugation partner.
[0640] In some embodiments, synthesising the amino acid sequence of
the peptide of the peptide-containing conjugation partner by SPPS
comprises coupling an amino acid or peptide to an amino acid or
peptide bound to a solid phase resin support to provide the amino
acid sequence of the peptide or a portion thereof. In certain
embodiments, the amino acid sequence of the entire peptide of the
peptide-containing conjugation partner is synthesised by SPPS.
[0641] The peptide-containing conjugation partner may be reacted
with the lipid-containing conjugation partner while bound to a
solid phase resin support. Alternatively, the peptide may be
cleaved from the solid phase resin support, and optionally
purified, prior to reaction with the lipid-containing conjugation
partner.
[0642] The peptide conjugate and/or amino acid-comprising
conjugation partner, for example a peptide-containing conjugation
partner, may comprise one or more solubilising groups. The one or
more solubilising groups increase the solubility of, for example,
the peptide-containing conjugation partner in polar solvents, such
as water. In exemplary embodiments, the solubilising group does not
adversely affect the biological activity of the peptide
conjugate.
[0643] The presence of a solubilising group may be advantageous for
formulation and/or administration of the peptide conjugate as a
pharmaceutical composition.
[0644] In some embodiments, the solubilising group is bound to the
peptide of the peptide conjugate and/or peptide-containing
conjugation partner. In some embodiments, the solubilising group is
bound to the peptide of the peptide-containing conjugation partner.
In some embodiments, the peptide of the peptide conjugate and/or
the peptide of the peptide-containing partner comprises a
solubilising group. In some embodiments, the peptide of the
peptide-containing partner comprises a solubilising group.
[0645] In some embodiments, the solubilising group is bound to the
side chain of an amino acid in the peptide chain. In some
embodiments, the solubilising group is bound to the C- or
N-terminus of the peptide chain. In some embodiments, the
solubilising group is bound between two amino acid residues in the
peptide chain. In some embodiments, the solubilising group is bound
to the Na-amino group of one amino acid residue in the peptide
chain and the carboxyl group of another amino acid residue in the
peptide chain.
[0646] Examples of suitable solubilising groups include, but are
not limited to, hydrophilic amino acid sequences or polyethylene
glycols (PEGs).
[0647] In one embodiment, the solubilising group is a hydrophilic
amino acid sequence comprising two or more hydrophilic amino acid
residues in the peptide chain. In some embodiments, the
solubilising group is an amino acid sequence comprising a sequence
of two or more consecutive hydrophilic amino acid residues in the
peptide chain. Such solubilising groups may be formed by adding
each amino acid of the solubilising group to the peptide chain by
SPPS.
[0648] In another embodiment, the solubilising group is a
polyethylene glycol. In some embodiments, the polyethylene glycol
is bound to the Na-amino group of one amino acid residue in the
peptide chain and the carboxyl group of another amino acid residue
in the peptide chain.
[0649] In some embodiments, the polyethylene glycol comprises from
about 1 to about 100, about 1 to about 50, about 1 to about 25,
about 1 to about 20, about 1 to about 15, about 1 to about 15,
about 1 to about 10, about 2 to about 10, or about 2 to about 4
ethylene glycol monomer units. Methods for coupling polyethylene
glycols to peptides are known.
[0650] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises an antigen, for
example, an antigenic peptide. In one embodiment, the peptide of
the peptide conjugate or peptide-containing conjugation partner is
or comprises an antigen; or an antigen is bound to peptide,
optionally via a linker. In some embodiments, the
peptide-containing conjugation partner comprises an antigen, for
example, an antigenic peptide. In one embodiment, the peptide of
the peptide-containing conjugation partner is or comprises an
antigen; or an antigen is bound to peptide, optionally via a
linker.
[0651] In one embodiment, the antigen comprises a peptide
comprising an epitope. In one embodiment, the peptide comprising an
epitope is a glycopeptide comprising an epitope. In one embodiment,
the antigen comprises a glycopeptide comprising an epitope.
[0652] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises an epitope. In
some embodiments, the peptide of the peptide conjugate and/or
peptide-containing conjugation partner comprises an epitope. In
some embodiments, the peptide-containing conjugation partner
comprises an epitope. In some embodiments, the peptide of the
peptide-containing conjugation partner comprises an epitope.
[0653] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises two or more
epitopes, for example, the peptide of the peptide conjugate and/or
peptide-containing conjugation partner comprises two or more
epitopes.
[0654] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner is or comprises a
glycopeptide comprising an epitope. In some embodiments, the
peptide of the peptide conjugate and/or peptide-containing
conjugation partner is a glycopeptide. In some embodiments, the
peptide conjugate and/or peptide-containing conjugation partner
comprises a glycopeptide comprising an epitope bound to the peptide
of the peptide conjugate and/or peptide-containing conjugation
partner. In some embodiments, the peptide-containing conjugation
partner is or comprises a glycopeptide comprising an epitope. In
some embodiments, the peptide of the peptide-containing conjugation
partner is a glycopeptide. In some embodiments, the
peptide-containing conjugation partner comprises a glycopeptide
comprising an epitope bound to the peptide of the
peptide-containing conjugation partner.
[0655] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises a proteolytic
cleavage site. In some embodiments, the peptide of the peptide
conjugate and/or peptide-containing conjugation partner comprises a
proteolytic cleavage site. In some embodiments, the
peptide-containing conjugation partner comprises a proteolytic
cleavage site. In some embodiments, the peptide of the
peptide-containing conjugation partner comprises a proteolytic
cleavage site.
[0656] In some embodiments, the peptide of the peptide conjugate
and/or peptide-containing conjugation partner comprises one or more
linker groups. In some embodiments, the peptide of the
peptide-containing conjugation partner comprises one or more linker
groups.
[0657] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises a linker group. In
some embodiments, the peptide-containing conjugation partner
comprises a linker group.
[0658] In some embodiments, the peptide conjugate and/or
peptide-containing conjugation partner comprises an epitope bound
to the peptide of the peptide conjugate and/or peptide-containing
conjugation partner via a linker group. In some embodiments, the
peptide-containing conjugation partner comprises an epitope bound
to the peptide of the peptide-containing conjugation partner via a
linker group.
[0659] Examples of linker groups include but are not limited to
amino acid sequences (for example, a peptide), polyethylene glycol,
alkyl amino acids, and the like. In some embodiments, the linker is
or comprises a proteolytic cleavage site. In some embodiments, the
linker is or comprises a solubilising group.
[0660] In some embodiments, the linker is bound between two amino
acid residues in the peptide chain.
[0661] In some embodiments, the linker group is bound to the
Na-amino group of one amino acid residue in the peptide conjugate
and/or peptide-containing conjugation partner and the carboxyl
group of another amino acid residue in the peptide-containing
conjugation partner. In some embodiments, the linker group is bound
to the Na-amino group of one amino acid residue in the
peptide-containing conjugation partner and the carboxyl group of
another amino acid residue in the peptide-containing conjugation
partner.
[0662] In certain embodiments, the linker group is cleavable in
vivo from the amino acids to which it is bound. In certain
embodiments, the linker group is cleavable by hydrolysis in vivo.
In certain embodiments, the linker group is cleavable by enzymatic
hydrolysis in vivo. Linker groups may be introduced by any suitable
method known in the art.
[0663] The method may further comprise coupling an epitope to the
amino acid of the amino acid conjugate or the peptide of the
peptide conjugate. The epitope may be bound via a linker group, as
described above. In some embodiments, the epitope is a peptide
epitope. In some embodiments, the method comprises coupling a
glycopeptide comprising an epitope.
[0664] It will be appreciated that in certain desirable
embodiments, the peptide conjugates of the invention maintain
appropriate uptake, processing, and presentation by antigen
presenting cells. Desirably, the lipid-containing conjugate does
not interfere with presentation of any antigenic peptide present in
the conjugate by antigen presenting cells. The examples presented
herein establish that conjugates of the invention are presented by
antigen presenting cells comparably with non-conjugated, related
peptides.
[0665] Confirmation of the identity of the peptides synthesized may
be conveniently achieved by, for example, amino acid analysis, mass
spectrometry, Edman degradation, and the like.
[0666] The method of the present invention may further comprise
separating the amino acid conjugate from the liquid reaction
medium. Alternatively, the method of the present invention may
further comprise separating the peptide conjugate from the liquid
reaction medium. Any suitable separation methods known in the art
may be used, for example, precipitation and filtration. The
conjugate may be subsequently purified, for example, by HPLC using
one or more suitable solvents.
[0667] The present invention also relates to amino acid conjugates
and peptide conjugates made by the method of the present invention.
The conjugates are as defined in any of the embodiments described
herein.
[0668] The present invention also relates to a compound of the
formula (V), which is an amino acid conjugate.
[0669] The present invention also relates to a compound of the
formula (V), which is a peptide conjugate.
[0670] The peptide conjugates may be pure or purified, or
substantially pure.
[0671] As used herein "purified" does not require absolute purity;
rather, it is intended as a relative term where the material in
question is more pure than in the environment it was in previously.
In practice the material has typically, for example, been subjected
to fractionation to remove various other components, and the
resultant material has substantially retained its desired
biological activity or activities. The term "substantially
purified" refers to materials that are at least about 60% free,
preferably at least about 75% free, and most preferably at least
about 90% free, at least about 95% free, at least about 98% free,
or more, from other components with which they may be associated
during manufacture.
[0672] The term ".alpha.-amino acid" or "amino acid" refers to a
molecule containing both an amino group and a carboxyl group bound
to a carbon which is designated the .alpha.-carbon. Suitable amino
acids include, without limitation, both the D- and L-isomers of the
naturally-occurring amino acids, as well as non-naturally occurring
amino acids prepared by organic synthesis or other metabolic
routes. Unless the context specifically indicates otherwise, the
term amino acid, as used herein, is intended to include amino acid
analogs.
[0673] In certain embodiments the peptide-containing conjugation
partner comprises only natural amino acids. The term "naturally
occurring amino acid" refers to any one of the twenty amino acids
commonly found in peptides synthesized in nature, and known by the
one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F,
P, S, T, W, Y and V.
[0674] The term "amino acid analog" or "non-naturally occurring
amino acid" refers to a molecule which is structurally similar to
an amino acid and which can be substituted for an amino acid. Amino
acid analogs include, without limitation, compounds which are
structurally identical to an amino acid, as defined herein, except
for the inclusion of one or more additional methylene groups
between the amino and carboxyl group (e.g., a-amino .beta.-carboxy
acids), or for the substitution of the amino or carboxy group by a
similarly reactive group (e.g., substitution of the primary amine
with a secondary or tertiary amine, or substitution or the carboxy
group with an ester).
[0675] Unless otherwise indicated, conventional techniques of
molecular biology, microbiology, cell biology, biochemistry and
immunology, which are within the skill of the art may be employed
in practicing the methods described herein. Such techniques are
explained fully in the literature, such as, Molecular Cloning: A
Laboratory Manual, second edition (Sambrook et al., 1989);
Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell
Culture (R. I. Freshney, ed., 1987); Handbook of Experimental
Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer
Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987); Current Protocols in Molecular Biology (F. M. Ausubel et
al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et
al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et
al., eds., 1991); The Immunoassay Handbook (David Wild, ed.,
Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow
et al., eds., 1987); and Methods of Immunological Analysis (R.
Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH
Verlags gesellschaft mbH, 1993).
[0676] The term "peptide" and the like is used herein to refer to
any polymer of amino acid residues of any length. The polymer can
be linear or non-linear (e.g., branched), it can comprise modified
amino acids or amino acid analogs. The term also encompasses amino
acid polymers that have been modified naturally or by intervention,
for example, by disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other modification
or manipulation, for example conjugation with labeling or bioactive
component.
[0677] The inventors have found that the certain peptide conjugates
of the present invention have immunological activity.
[0678] Cell-mediated immunity is primarily mediated by
T-lymphocytes. Pathogenic antigens are expressed on the surface of
antigen presenting cells (such as macrophages, B-lymphocytes, and
dendritic cells), bound to either major histocompatibility MHC
Class I or MHC Class II molecules. Presentation of pathogenic
antigen coupled to MHC Class II activates a helper (CD4+) T-cell
response. Upon binding of the T-cell to the antigen-MHC II complex,
CD4+ T-cells, release cytokines and proliferate.
[0679] Presentation of pathogenic antigens bound to MHC Class I
molecules activates a cytotoxic (CD8+) T-cell response. Upon
binding of the T-cell to the antigen-MHC I complex, CD8+ cells
secrete perforin and other mediators, resulting in target cell
death. Without wishing to be bound by any theory, the applicants
believe that in certain embodiments an enhanced response by CD8+
cells is achieved in the presence of one or more epitopes
recognised by CD4+ cells.
[0680] Methods to assess and monitor the onset or progression of a
cell-mediated response in a subject are well known in the art.
Convenient exemplary methods include those in which the presence of
or the level of one or more cytokines associated with a
cell-mediated response, such as those identified herein, is
assessed. Similarly, cell-based methods to assess or monitor the
onset and progression of a cell-mediated response are amenable to
use in the present invention, and may include cell proliferation or
activation assays, including assays targeted at identifying
activation or expansion of one or more populations of immune cells,
such as T-lymphocytes.
[0681] In certain embodiments, methods of the invention elicit both
a cell-mediated immune response and a humoral response.
[0682] The humoral immune response is mediated by secreted
antibodies produced by B cells. The secreted antibodies bind to
antigens presented on the surface of invading pathogens, flagging
them for destruction.
[0683] Again, methods to assess and monitor the onset or
progression of a humoral response are well known in the art. These
include antibody binding assays, ELISA, skin-prick tests and the
like.
[0684] Without wishing to be bound by theory, the inventors believe
that the peptide conjugates in some embodiments stimulate Toll like
receptors (TLRs).
[0685] Toll-like receptors (TLRs) are highly conserved pattern
recognition receptors (PRRs) that recognise pathogen-associated
molecular patterns and transmit danger signals to the cell (Kawai,
T., Akira, S., Immunity 2011, 34, 637-650). TLR2 is a cell-surface
receptor expressed on a range of different cell types, including
dendritic cells, macrophages and lymphocytes (Coffman, R. L., Sher,
A., Seder, R. A., Immunity 2010, 33, 492-503).
[0686] TLR2 recognises a wide range of microbial components
including lipopolysaccharides, peptidoglycans and lipoteichoic
acid. It is unique amongst TLRs in that it forms heterodimers, with
either TLR1 or TLR6; the ability to form complexes with other PRRs
may explain the wide range of agonists for TLR2 (Feldmann, M.,
Steinman, L., Nature 2005, 435, 612-619). Upon ligand binding and
heterodimerisation, signalling takes place via the MyD88 pathway,
leading to NF.kappa.B activation and consequent production of
inflammatory and effector cytokines.
[0687] Di- and triacylated lipopeptides derived from bacterial
cell-wall components have been extensively studied as TLR2 agonists
(Eriksson, E. M. Y., Jackson, D. C., Curr. Prot. and Pept. Sci.
2007, 8, 412-417). Lipopeptides have been reported to promote
dendritic cell maturation, causing the up-regulation of
co-stimulatory molecules on the cell surface and enhanced
antigen-presentation. Lipopeptides have also been reported to
stimulate macrophages to release cytokines and promote the
activation of lymphocytes including B cells and CD8+ T cells.
[0688] In some embodiments, the peptide conjugate has TLR2 agonist
activity. In some embodiments, the peptide conjugate has TLR2
agonist activity comparable to Pam3CSK4 [comprises SEQ ID NO: 22].
In some embodiments, the peptide conjugate has TLR2 agonist
activity at least about 50%, about 60%, about 70%, about 80%, about
90% that of Pam3CSK4 [comprises SEQ ID NO: 22]. In some
embodiments, for example in embodiments where a modulated immune
response is desirable, the peptide conjugate has TLR2 agonist
activity less that that of Pam3CSK4 [comprises SEQ ID NO: 22]. For
example, the peptide conjugate has TLR2 agonist activity less than
about 50%, less than about 40%, less than about 30%, less than
about 20%, or less than about 10% that of Pam3CSK4 [comprises SEQ
ID NO: 22].
[0689] In some embodiments, the peptide of the peptide conjugate
and/or peptide-containing conjugation partner comprises a serine
amino acid residue adjacent to the amino acid through which the
lipid-containing conjugation partner is conjugated to the peptide.
In some embodiments, the peptide of the peptide containing
conjugation partner comprises a serine amino acid residue adjacent
to the amino acid through which the lipid-containing conjugation
partner is conjugated to the peptide. The presence of the serine
amino acid residue in this position may enhance TLR2 binding. In
some embodiments, the serine amino acid residue is bound to the
C-termini of the amino acid through which the lipid-containing
conjugation partner is conjugated to the peptide.
[0690] As will be appreciated by those skilled in the art on
reading this disclosure, the peptide conjugate may comprise an
epitope, including, for example two or more epitopes. In some
embodiments, the epitope is a peptide epitope. A person skilled in
the art will appreciate that a wide range of peptide epitopes may
be employed in the present invention.
[0691] Antigens
[0692] It will be appreciated that a great many antigens, for
example tumour antigens or antigens from various pathogenic
organisms, have been characterised and are suitable for use in the
present invention. All antigens, whether or not presently
characterized, that are capable of eliciting an immune response are
contemplated.
[0693] Accordingly, depending on the choice of antigen the
conjugates of the present invention find application in a wide
range of immunotherapies, including but not limited to the
treatment and prevention of infectious disease, the treatment and
prevention of cancer, and the treatment of viral re-activation
during or following immunosuppression, for example in patients who
have had bone marrow transplants or haematopoietic stem cell
transplants.
[0694] Also contemplated are antigens comprising one or more amino
acid substitutions, such as one or more conservative amino acid
substitutions.
[0695] A "conservative amino acid substitution" is one in which an
amino acid residue is replaced with another residue having a
chemically similar or derivatised side chain. Families of amino
acid residues having similar side chains, for example, have been
defined in the art. These families include, for example, amino
acids with basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Amino acid analogs
(e.g., phosphorylated or glycosylated amino acids) are also
contemplated in the present invention, as are peptides substituted
with non-naturally occurring amino acids, including but not limited
to N-alkylated amino acids (e.g. N-methyl amino acids), D-amino
acids, 1-amino acids, and .gamma.-amino acids.
[0696] Fragments and variants of antigens are also specifically
contemplated.
[0697] A "fragment" of a peptide, is a subsequence of the peptide
that performs a function that is required for the enzymatic or
binding activity and/or provides three dimensional structure of the
peptide, such as the three dimensional structure of a
polypeptide.
[0698] The term "variant" as used herein refers to peptide
sequences, including for example peptide sequences different from
the specifically identified sequences, wherein one or more amino
acid residues is deleted, substituted, or added. Variants are
naturally-occurring variants, or non-naturally occurring variants.
Variants are from the same or from other species and may encompass
homologues, paralogues and orthologues. In certain embodiments,
variants of peptides including peptides possess biological
activities that are the same or similar to those of the wild type
peptides. The term "variant" with reference to peptides encompasses
all forms of peptides as defined herein.
[0699] Those of skill in the art will appreciate that the
conjugates of the present invention are in certain embodiments
particularly suited for stimulating T-cell responses, for example
in the treatment of neoplastic diseases, including cancer.
Conjugates of the present invention comprising one or more tumour
antigens are specifically contemplated. It will be appreciated that
tumour antigens contemplated for use in the preparation of peptide
conjugates of the invention will generally comprise one or more
peptides. In certain embodiments of the invention, including for
example pharmaceutical compositions of the invention, one or more
additional tumour antigens may be present, wherein the one or more
tumour antigens does not comprise peptide. Tumour antigens are
typically classified as either unique antigens, or shared antigens,
with the latter group including differentiation antigens,
cancer-specific antigens, and over-expressed antigens. Examples of
each class of antigens are amenable to use in the present
invention. Representative tumour antigens for use in the treatment,
for example immunotherapeutic treatment, or vaccination against
neoplastic diseases including cancer, are discussed below.
Compounds, vaccines and compositions comprising one or more
antigens prepared using those methods of immunisation are
specifically contemplated.
[0700] In certain embodiments, the tumour antigen is a
peptide-containing tumour antigen, such as a polypeptide tumour
antigen or glycoprotein tumour antigens. In certain embodiments,
the tumour antigen is a saccharide-containing tumour antigen, such
as a glycolipid tumour antigen or a ganglioside tumour antigen. In
certain embodiments, the tumour antigen is a
polynucleotide-containing tumour antigen that expresses a
polypeptide-containing tumour antigen, for instance, an RNA vector
construct or a DNA vector construct, such as plasmid DNA.
[0701] Tumour antigens appropriate for the use in the present
invention encompass a wide variety of molecules, such as (a)
peptide-containing tumour antigens, including peptide epitopes
(which can range, for example, from 8-20 amino acids in length,
although lengths outside this range are also common),
lipopolypeptides and glycoproteins, (b) saccharide-containing
tumour antigens, including poly-saccharides, mucins, gangliosides,
glycolipids and glycoproteins, including and (c) polynucleotides
that express antigenic polypeptides. Again, those skilled in the
art will recognise that a tumour antigen present in a conjugate or
composition of the present invention will typically comprise
peptide. However, embodiments of the invention where one or more
conjugates comprises a tumour antigen that does not itself comprise
peptide, but for example is bound to the amino acid-comprising or
peptide-containing conjugation partner, are contemplated.
Similarly, compositions of the invention in which one or more
tumour antigens that does not itself comprise peptide is present
are contemplated.
[0702] In certain embodiments, the tumour antigens are, for
example, (a) full length molecules associated with cancer cells,
(b) homologues and modified forms of the same, including molecules
with deleted, added and/or substituted portions, and (c) fragments
of the same, provided said fragments remain antigenic or
immunogenic. In certain embodiments, the tumour antigens are
provided in recombinant form. In certain embodiments, the tumour
antigens include, for example, class I-restricted antigens
recognized by CD8+ lymphocytes or class II-restricted antigens
recognized by CD4+ lymphocytes.
[0703] Shared tumour antigens are generally considered to be
native, unmutated sequences that are expressed by tumours due to
epigenetic changes that allow de-repression of
developmentally-repressed genes. Accordingly, shared antigens are
typically considered preferable to over-expressed or
differentiation-associated antigens because there is no expression
in normal tissues. Also, the same antigens can be targeted in a
number of cancer patients. For example, the cancer-testis antigen
NY-ESO-1 is present in the majority of patients with many tumours,
and a sizeable minority of patients with other tumours. In another
example, breast differentiation tumour antigens NYBR-1 and NYBR-1.1
are found in a proportion of breast cancer sufferers. Shared tumour
antigens thus represent an attractive target for development.
[0704] The use of shared tumour antigens, such cancer-testis
antigens including NY-ESO-1, CTSP-1, CTSP-2, CTSP-3, CTSP-4, SSX2,
and SCP1, and breast cancer antigens NYBR-1 and NYBR-1.1, in
conjugates of the present invention is specifically contemplated
herein.
[0705] In one exemplary embodiment, the peptide of the
peptide-containing conjugation partner or of the peptide conjugate
comprises one or more epitopes derived from NY-ESO-1. In one
embodiment, the peptide comprises one or more epitopes derived from
NY-ESO-1 residues 79-116 [SEQ ID NO: 5]. In one embodiment, the
peptide comprises one or more epitopes derived from NY-ESO-1
residues 118-143 [SEQ ID NO: 12]. In one embodiment, the peptide
comprises one or more epitopes derived from NY-ESO-1 residues
153-180 [SEQ ID NO: 18].
[0706] In one specifically contemplated embodiment, the peptide of
the peptide-containing conjugation partner or of the peptide
conjugate, comprises, consists essentially of, or consists of an
amino acid sequence selected from the group consisting of 8 or more
contiguous, 10 or more contiguous, 12 or more contiguous, 15 or
more contiguous, 20 or more contiguous, or 25 or more contiguous
amino acids from any one of SEQ ID NOs: 1 to 20.
[0707] In various embodiments, the peptide comprises more that one
amino acid sequence selected from the group consisting of any one
of SEQ ID NOs: 1 to 20. In one embodiment, the peptide comprises
one or more amino acid sequences selected from the group consisting
of SEQ ID NOs: 4-7, 12, 13, and 18-20.
[0708] Similarly, the prostate vaccine Sipuleucel-T (APC8015,
Provengem), which comprises the antigen prostatic acid phosphatase
(PAP), is present in 95% of prostate cancer cells. At least in part
due to this potential for efficacy in a significant proportion of
prostate cancer sufferers, Sipuleucel-T was approved by the FDA in
2010 for use in the treatment of asymptomatic, hormone-refractory
prostate cancer. The use of PAP antigen in conjugates of the
present invention is specifically contemplated in the present
invention.
[0709] Unique antigens are considered to be those antigens that are
unique to an individual or are shared by a small proportion of
cancer patients, and typically result from mutations leading to
unique protein sequences. Representative examples of unique tumour
antigens include mutated Ras antigens, and mutated p53 antigens. As
will be appreciated by those skilled in the art having read this
specification, the methods of the present invention enable the
ready preparation of conjugates comprising one or more unique
tumour antigens, for example to elicit specific T-cell responses to
one or more unique tumour antigens, for example in the preparation
of patient-specific therapies.
[0710] Accordingly, representative tumour antigens include, but are
not limited to, (a) antigens such as RAGE, BAGE, GAGE and MAGE
family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2,
MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for
example, to address melanoma, lung, head and neck, NSCLC, breast,
gastrointestinal, and bladder tumours), (b) mutated antigens, for
example, p53 (associated with various solid tumours, for example,
colorectal, lung, head and neck cancer), p21/Ras (associated with,
for example, melanoma, pancreatic cancer and colorectal cancer),
CDK4 (associated with, for example, melanoma), MUM1 (associated
with, for example, melanoma), caspase-8 (associated with, for
example, head and neck cancer), CIA 0205 (associated with, for
example, bladder cancer), HLA-A2-R1701, beta catenin (associated
with, for example, melanoma), TCR (associated with, for example,
T-cell non-Hodgkins lymphoma), BCR-abl (associated with, for
example, chronic myelogenous leukemia), triosephosphate isomerase,
MA 0205, CDC-27, and LDLR-FUT, (c) over-expressed antigens, for
example, Galectin 4 (associated with, for example, colorectal
cancer), Galectin 9 (associated with, for example, Hodgkin's
disease), proteinase 3 (associated with, for example, chronic
myelogenous leukemia), Wilm's tumour antigen-1 (WT 1, associated
with, for example, various leukemias), carbonic anhydrase
(associated with, for example, renal cancer), aldolase A
(associated with, for example, lung cancer), PRAME (associated
with, for example, melanoma), HER-2/neu (associated with, for
example, breast, colon, lung and ovarian cancer), alpha-fetoprotein
(associated with, for example, hepatoma), KSA (associated with, for
example, colorectal cancer), gastrin (associated with, for example,
pancreatic and gastric cancer), telomerase catalytic protein, MUC-1
(associated with, for example, breast and ovarian cancer), G-250
(associated with, for example, renal cell carcinoma), p53
(associated with, for example, breast, colon cancer), and
carcinoembryonic antigen (associated with, for example, breast
cancer, lung cancer, and cancers of the gastrointestinal tract such
as colorectal cancer), (d) shared antigens, for example,
melanoma-melanocyte differentiation antigens such as MART-1/Melan
A, gp100, MC1R, melanocyte-stimulating hormone receptor,
tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase
related protein-2/TRP2 (associated with, for example, melanoma),
(e) prostate associated antigens such as PAP, prostatic serum
antigen (PSA), PSMA, PSH-P1, PSM-P1, PSM-P2, associated with for
example, prostate cancer, (f) immunoglobulin idiotypes (associated
with myeloma and B cell lymphomas, for example), and (g) other
tumour antigens, such as polypeptide- and saccharide-containing
antigens including (i) glycoproteins such as sialyl Tn and sialyl
Le.sup.x (associated with, for example, breast and colorectal
cancer) as well as various mucins; glycoproteins are coupled to a
carrier protein (for example, MUC-1 are coupled to KLH); (ii)
lipopolypeptides (for example, MUC-1 linked to a lipid moiety);
(iii) polysaccharides (for example, Globo H synthetic
hexasaccharide), which are coupled to a carrier proteins (for
example, to KLH), (iv) gangliosides such as GM2, GM12, GD2, GD3
(associated with, for example, brain, lung cancer, melanoma), which
also are coupled to carrier proteins (for example, KLH).
[0711] Other representative tumour antigens amenable to use in the
present invention include TAG-72, (See, e.g., U.S. Pat. No.
5,892,020; human carcinoma antigen (See, e.g., U.S. Pat. No.
5,808,005); TP1 and TP3 antigens from osteocarcinoma cells (See,
e.g., U.S. Pat. No. 5,855,866); Thomsen-Friedenreich (TF) antigen
from adenocarcinoma cells (See, e.g., U.S. Pat. No. 5,110,911);
KC-4 antigen from human prostrate adenocarcinoma (See, e.g., U.S.
Pat. No. 4,743,543); a human colorectal cancer antigen (See, e.g.,
U.S. Pat. No. 4,921,789); CA125 antigen from cystadenocarcinoma
(See, e.g., U.S. Pat. No. 4,921,790); DF3 antigen from human breast
carcinoma (See, e.g., U.S. Pat. Nos. 4,963,484 and 5,053,489); a
human breast tumour antigen (See, e.g., U.S. Pat. No. 4,939,240);
p97 antigen of human melanoma (See, e.g., U.S. Pat. No. 4,918,164);
carcinoma or orosomucoid-related antigen (CORA) (See, e.g., U.S.
Pat. No. 4,914,021); T and Tn haptens in glycoproteins of human
breast carcinoma, MSA breast carcinoma glycoprotein; MFGM breast
carcinoma antigen; DU-PAN-2 pancreatic carcinoma antigen; CA125
ovarian carcinoma antigen; YH206 lung carcinoma antigen,
Alphafetoprotein (AFP) hepatocellular carcinoma antigen;
Carcinoembryonic antigen (CEA) bowel cancer antigen; Epithelial
tumour antigen (ETA) breast cancer antigen; Tyrosinase; the raf
oncogene product; gp75; gp100; EBV-LMP 1 & 2; EBV-EBNA 1, 2
& 3C; HPV-E4, 6, 7; C017-1A; GA733; gp72; p53; proteinase 3;
telomerase; and melanoma gangliosides. These and other tumour
antigens, whether or not presently characterized, are contemplated
for use in the present invention.
[0712] In certain embodiments, the tumour antigens are derived from
mutated or altered cellular components. Representative examples of
altered cellular components include, but are not limited to ras,
p53, Rb, altered protein encoded by the Wilms' tumour gene,
ubiquitin, mucin, protein encoded by the DCC, APC, and MCC genes,
as well as receptors or receptor-like structures such as neu,
thyroid hormone receptor, platelet derived growth factor (PDGF)
receptor, insulin receptor, epidermal growth factor (EGF) receptor,
and the colony stimulating factor (CSF) receptor.
[0713] Polynucleotide-containing antigens used in the present
invention include polynucleotides that encode polypeptide tumour
antigens such as those listed above. In certain embodiments, the
polynucleotide-containing antigens include, but are not limited to,
DNA or RNA vector constructs, such as plasmid vectors (e.g., pCMV),
which are capable of expressing polypeptide tumour antigens in
vivo.
[0714] The present invention also contemplates the preparation of
conjugates comprising viral antigens that are capable of
stimulating T-cell to elicit effective anti-viral immunity in
patients who are or have been immunosuppressed, for example
patients who have had bone marrow transplants, haematopoietic stem
cell transplants, or are otherwise undergoing
immunosuppression.
[0715] Similarly, antigens derived from viruses associated with
increased incidence of cancer, or that are reported to be
cancer-causing, such as human papillomavirus, hepatitis A virus,
and hepatitis B virus, are contemplated for use in the present
invention.
[0716] For example, in certain embodiments, the tumour antigens
include, but are not limited to, p15, Hom/Mel-40, H-Ras, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, human
papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and
C virus antigens, human T-cell lymphotropic virus antigens,
TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9,
CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,
791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA),
CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733
(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,
RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.
[0717] Representative antigens for use in vaccination against
pathogenic organisms are discussed below. Compounds, vaccines and
compositions comprising one or more antigens prepared using those
methods of immunisation are specifically contemplated.
[0718] Tuberculosis Antigens
[0719] It will be appreciated that a great many M. tuberculosis
antigens have been characterised and are suitable for use in the
present invention. All M. tuberculosis antigens, whether or not
presently characterized, that are capable of eliciting an immune
response are contemplated.
[0720] Exemplary M. tuberculosis antigens suitable for use include
early secretary antigen target (ESAT)-6, Ag85A, Ag85B (MPT59),
Ag85B, Ag85C, MPT32, MPT51, MPT59, MPT63, MPT64, MPT83, MPB5,
MPB59, MPB64, MTC28, Mtb2, Mtb8.4, Mtb9.9, Mtb32A, Mtb39, Mtb41,
TB10.4, TB10C, TB11B, TB12.5, TB13A, TB14, TB15, TB15A, TB16,
TB16A, TB17, TB18, TB21, TB20.6, TB24, TB27B, TB32, TB32A, TB33,
TB38, TB40.8, TB51, TB54, TB64, CFP6, CFP7, CFP7A, CFP7B, CFP8A,
CFP8B, CFP9, CFP10, CFP11, CFP16, CFP17, CFP19, CFP19A, CFP19B,
CFP20, CFP21, CFP22, CFP22A, CFP23, CFP23A, CFP23B, CFP25, CFP25A,
CFP27, CFP28, CFP28B, CFP29, CFP30A, CFP30B, CFP50, CWP32, hspX
(alpha-crystalline), APA, Tuberculin purified protein derivative
(PPD), ST-CF, PPE68, LppX, PstS-1, PstS-2, PstS-3, HBHA, GroEL,
GroEL2, GrpES, LHP, 19 kDa lipoprotein, 71 kDa, RD1-ORF2, RD1-ORF3,
RD1-ORF4, RD1-ORF5, RD1-ORF8, RD1-ORF9A, RD1-ORF9B, Rv1984c,
Rv0577, Rv1827, BfrB, Tpx. Rv1352, Rv1810, PpiA, Cut2, FbpB, FbpA,
FbpC, DnaK, FecB, Ssb, RplL, FixA, FixB, AhpC2, Rv2626c, Rv1211,
Mdh, Rv1626, Adk, CIpP, SucD (Belisle et al, 2005; U.S. Pat. No.
7,037,510; US 2004/0057963; US 2008/0199493; US 2008/0267990), or
at least one antigenic portion or T-cell epitope of any of the
above mentioned antigens.
[0721] Hepatitis Antigens
[0722] A number of hepatitis antigens have been characterised and
are suitable for use in the present invention. Exemplary hepatitis
C antigens include C--p22, E1--gp35, E2--gp70, NS1--p7, NS2--p23,
NS3--p70, NS4A--p8, NS4B--p27, NS5A--p56/58, and NS5B--p68, and
together with one or more antigenic portions or epitopes derived
therefrom are each (whether alone or in combination) suitable for
application in the present invention. All hepatitis antigens,
whether or not presently characterized, that are capable of
eliciting an immune response are contemplated.
[0723] Influenza Antigens
[0724] Many influenza antigens have been characterised and are
suitable for use in the present invention. Exemplary influenza
antigens suitable for use in the present invention include PB, PB2,
PA, any of the hemagglutinin (HA) or neuramimidase (NA) proteins,
NP, M, and NS, and together with one or more antigenic portions or
epitopes derived therefrom are each (whether alone or in
combination) suitable for application in the present invention. All
influenza antigens, whether or not presently characterized, that
are capable of eliciting an immune response are contemplated.
[0725] Anthrax Antigens
[0726] A number of B. anthracis antigens have been identified as
potential candidates for vaccine development and are useful in the
present invention. For example, PA83 is one such antigen for
vaccine development. Currently, only one FDA licensed vaccine for
anthrax is available called "Anthrax Vaccine Adsorbed" (AVA) or
BioThrax.RTM.. This vaccine is derived from the cell-free
supernatant of a non-encapsulated strain of B. anthracis adsorbed
to aluminum adjuvant. PA is the primary immunogen in AVA. Other
exemplary anthrax antigens suitable for use in the present
invention include Protective antigen (PA or PA63), LF and EF
(proteins), poly-gamma-(D-glutamate) capsule, spore antigen
(endospore specific components), BcIA (exosporium specific
protein), BxpB (spore-associated protein), and secreted proteins.
All anthrax antigens together with one or more antigenic portions
or epitopes derived therefrom, whether or not presently
characterized, that are capable of eliciting an immune response are
contemplated.
[0727] Tularemia Antigens
[0728] A number of F. tularensis antigens have been identified as
potential candidates for vaccine development and are useful in the
present invention. For example, AcpA and IgIC are antigens suitable
for vaccine development. Other exemplary Tularemia antigens
suitable for use in the present invention include O-antigen, CPS,
outer membrane proteins (e.g. FopA), lipoproteins (e.g. Tul4),
secreted proteins and lipopolysaccharide. All tularemia antigens
together with one or more antigenic portions or epitopes derived
therefrom, whether or not presently characterized, that are capable
of eliciting an immune response are contemplated.
[0729] Brucellosis Antigens
[0730] A number of B. abortusis antigens have been identified as
potential candidates for vaccine development and are useful in the
present invention. For example, Omp16 is one such antigen for
vaccine development. Other exemplary Brucellosis antigens suitable
for use in the present invention include O-antigen,
lipopolysaccharide, outer membrane proteins (e.g. Omp16), secreted
proteins, ribosomal proteins (e.g. L7 and L12), bacterioferritin,
p39 (a putative periplasmic binding protein), groEL (heat-shock
protein), lumazine synthase, BCSP31 surface protein, PAL16.5 OM
lipoprotein, catalase, 26 kDa periplasmic protein, 31 kDa Omp31, 28
kDa Omp, 25 kDa Omp, and 10 kDA Om lipoprotein. All brucellosis
antigens together with one or more antigenic portions or epitopes
derived therefrom, whether or not presently characterized, that are
capable of eliciting an immune response are contemplated.
[0731] Meningitis Antigens
[0732] A number of N. meningitidis antigens have been identified as
potential candidates for vaccine development and are useful in the
present invention. For example, Cys6, PorA, PorB, FetA, and ZnuD
are antigens suitable for vaccine development. Other exemplary
Meningitis antigens suitable for use in the present invention
include O-antigen, factor H binding protein (fHbp), TbpB, NspA,
NadA, outer membrane proteins, group B CPS, secreted proteins and
lipopolysaccharide. All menigitis antigens together with one or
more antigenic portions or epitopes derived therefrom, whether or
not presently characterized, that are capable of eliciting an
immune response are contemplated.
[0733] Dengue Antigens
[0734] A number of Flavivirus antigens have been identified as
potential candidates for vaccine development to treat dengue fever
and are useful in the present invention. For example, dengue virus
envelope proteins E1-E4 and the membrane proteins M1-M4 are
antigens suitable for vaccine development. Other exemplary dengue
antigens suitable for use in the present invention include C, preM,
1, 2A, 2B, 3, 4A, 4B and 5. All dengue antigens together with one
or more antigenic portions or epitopes derived therefrom, whether
or not presently characterized, that are capable of eliciting an
immune response are contemplated.
[0735] Ebola Antigens
[0736] A number of ebola virus antigens have been identified as
potential candidates for vaccine development to treat ebola
infection and are useful in the present invention. For example,
Filoviridae Zaire ebolavirus and Sudan ebolavirus virion spike
glycoprotein precursor antigens ZEBOV-GP, and SEBOV-GP,
respectively, are suitable for vaccine development. Other exemplary
ebola antigens suitable for use in the present invention include
NP, vp35, vp40, GP, vp30, vp24 and L. All ebola antigens together
with one or more antigenic portions or epitopes derived therefrom,
whether or not presently characterized, that are capable of
eliciting an immune response are contemplated.
[0737] West Nile Antigens
[0738] A number of West Nile virus antigens have been identified as
potential candidates for vaccine development to treat infection and
are useful in the present invention. For example, Flavivirus
envelope antigen (E) from West Nile virus (WNV) is a non-toxic
protein expressed on the surface of WNV virions (WNVE) and are
suitable for vaccine development. Other exemplary WNV antigens
suitable for use in the present invention include Cp, Prm, NS1,
NS2A, NS2B, NS3, NS4A, NS4B and NS5.
[0739] All West Nile antigens together with one or more antigenic
portions or epitopes derived therefrom, whether or not presently
characterized, that are capable of eliciting an immune response are
contemplated.
[0740] The above-listed or referenced antigens are exemplary, not
limiting, of the present invention.
[0741] The present invention also relates to pharmaceutical
composition comprising an effective amount of a peptide conjugate
of the present invention or a pharmaceutically acceptable salt or
solvent thereof, and a pharmaceutically acceptable carrier.
[0742] The present invention relates to a pharmaceutical
composition comprising an effective amount of a peptide of the
present invention or a pharmaceutically acceptable salt or solvate
thereof, and a pharmaceutically acceptable carrier.
[0743] The pharmaceutical compositions may comprise an effective
amount of two or more peptides of the invention, two or more
peptide conjugates of the invention, or one more peptides of the
invention and one or more peptide conjugates of the invention in
combination.
[0744] The term "pharmaceutically acceptable carrier" refers to a
carrier (adjuvant or vehicle) that may be administered to a subject
together with the peptide or peptide conjugate of the present
invention, or a pharmaceutically acceptable salt or solvent
thereof, and a pharmaceutically acceptable carrier.
[0745] Pharmaceutically acceptable carriers that may be used in the
compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, self-emulsifying drug
delivery systems (SEDDS) such as d-.alpha.-tocopherol
polyethyleneglycol 1000 succinate, surfactants used in
pharmaceutical dosage forms such as Tweens or other similar
polymeric delivery matrices, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-.beta.-cyclodextrins, or other solubilized
derivatives may also be advantageously used to enhance delivery.
Oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant, or carboxymethyl cellulose or similar
dispersing agents, which are commonly used in the formulation of
pharmaceutically acceptable dosage forms such as emulsions and or
suspensions.
[0746] The compositions are formulated to allow for administration
to a subject by any chosen route, including but not limited to oral
or parenteral (including topical, subcutaneous, intramuscular and
intravenous) administration.
[0747] For example, the compositions may be formulated with an
appropriate pharmaceutically acceptable carrier (including
excipients, diluents, auxiliaries, and combinations thereof)
selected with regard to the intended route of administration and
standard pharmaceutical practice. For example, the compositions may
be administered orally as a powder, liquid, tablet or capsule, or
topically as an ointment, cream or lotion. Suitable formulations
may contain additional agents as required, including emulsifying,
antioxidant, flavouring or colouring agents, and may be adapted for
immediate-, delayed-, modified-, sustained-, pulsed- or
controlled-release.
[0748] The compositions may be formulated to optimize
bioavailability, immunogenicity, or to maintain plasma, blood, or
tissue concentrations within the immunogenic or therapeutic range,
including for extended periods. Controlled delivery preparations
may also be used to optimize the antigen concentration at the site
of action, for example.
[0749] The compositions may be formulated for periodic
administration, for example to provide continued exposure.
Strategies to elicit a beneficial immunological response, for
example those that employ one or more "booster" vaccinations, are
well known in the art, and such strategies may be adopted.
[0750] The compositions may be administered via the parenteral
route. Examples of parenteral dosage forms include aqueous
solutions, isotonic saline or 5% glucose of the active agent, or
other well-known pharmaceutically acceptable excipients.
Cyclodextrins, for example, or other solubilising agents well-known
to those familiar with the art, can be utilized as pharmaceutical
excipients for delivery of the therapeutic agent.
[0751] Examples of dosage forms suitable for oral administration
include, but are not limited to tablets, capsules, lozenges, or
like forms, or any liquid forms such as syrups, aqueous solutions,
emulsions and the like, capable of providing a therapeutically
effective amount of the composition. Capsules can contain any
standard pharmaceutically acceptable materials such as gelatin or
cellulose. Tablets can be formulated in accordance with
conventional procedures by compressing mixtures of the active
ingredients with a solid carrier and a lubricant. Examples of solid
carriers include starch and sugar bentonite.
[0752] Active ingredients can also be administered in a form of a
hard shell tablet or a capsule containing a binder, e.g., lactose
or mannitol, a conventional filler, and a tabletting agent.
[0753] Examples of dosage forms suitable for transdermal
administration include, but are not limited, to transdermal
patches, transdermal bandages, and the like.
[0754] Examples of dosage forms suitable for topical administration
of the compositions include any lotion, stick, spray, ointment,
paste, cream, gel, etc., whether applied directly to the skin or
via an intermediary such as a pad, patch or the like.
[0755] Examples of dosage forms suitable for suppository
administration of the compositions include any solid dosage form
inserted into a bodily orifice particularly those inserted
rectally, vaginally and urethrally.
[0756] Examples of dosage of forms suitable for injection of the
compositions include delivery via bolus such as single or multiple
administrations by intravenous injection, subcutaneous, subdermal,
and intramuscular administration or oral administration.
[0757] Examples of dosage forms suitable for depot administration
of the compositions and include pellets of the peptides or peptide
conjugates or solid forms wherein the peptides or peptide
conjugates are entrapped in a matrix of biodegradable polymers,
microemulsions, liposomes or are microencapsulated.
[0758] Examples of infusion devices for the compositions include
infusion pumps for providing a desired number of doses or steady
state administration, and include implantable drug pumps.
[0759] Examples of implantable infusion devices for compositions
include any solid form in which the peptides or peptide conjugates
are encapsulated within or dispersed throughout a biodegradable
polymer or synthetic, polymer such as silicone, silicone rubber,
silastic or similar polymer.
[0760] Examples of dosage forms suitable for transmucosal delivery
of the compositions include depositories solutions for enemas,
pessaries, tampons, creams, gels, pastes, foams, nebulised
solutions, powders and similar formulations containing in addition
to the active ingredients such carriers as are known in the art to
be appropriate. Such dosage forms include forms suitable for
inhalation or insufflation of the compositions, including
compositions comprising solutions and/or suspensions in
pharmaceutically acceptable, aqueous, or organic solvents, or
mixture thereof and/or powders. Transmucosal administration of the
compositions may utilize any mucosal membrane but commonly utilizes
the nasal, buccal, vaginal and rectal tissues. Formulations
suitable for nasal administration of the compositions may be
administered in a liquid form, for example, nasal spray, nasal
drops, or by aerosol administration by nebulizer, including aqueous
or oily solutions of the polymer particles. Formulations may be
prepared as aqueous solutions for example in saline, solutions
employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bio-availability, fluorocarbons,
and/or other solubilising or dispersing agents known in the
art.
[0761] Examples of dosage forms suitable for buccal or sublingual
administration of the compositions include lozenges, tablets and
the like. Examples of dosage forms suitable for opthalmic
administration of the compositions include inserts and/or
compositions comprising solutions and/or suspensions in
pharmaceutically acceptable, aqueous, or organic solvents.
[0762] Examples of formulations of compositions, including
vaccines, may be found in, for example, Sweetman, S. C. (Ed.).
Martindale. The Complete Drug Reference, 33rd Edition,
Pharmaceutical Press, Chicago, 2002, 2483 pp.; Aulton, M. E. (Ed.)
Pharmaceutics. The Science of Dosage Form Design. Churchill
Livingstone, Edinburgh, 2000, 734 pp.; and, Ansel, H. C., Allen, L.
V. and Popovich, N. G. Pharmaceutical Dosage Forms and Drug
Delivery Systems, 7th Ed., Lippincott 1999, 676 pp. Excipients
employed in the manufacture of drug delivery systems are described
in various publications known to those skilled in the art
including, for example, Kibbe, E. H. Handbook of Pharmaceutical
Excipients, 3rd Ed., American Pharmaceutical Association,
Washington, 2000, 665 pp. The USP also provides examples of
modified-release oral dosage forms, including those formulated as
tablets or capsules. See, for example, The United States
Pharmacopeia 23/National Formulary 18, The United States
Pharmacopeial Convention, Inc., Rockville Md., 1995 (hereinafter
"the USP"), which also describes specific tests to determine the
drug release capabilities of extended-release and delayed-release
tablets and capsules. The USP test for drug release for
extended-release and delayed-release articles is based on drug
dissolution from the dosage unit against elapsed test time.
Descriptions of various test apparatus and procedures may be found
in the USP. Further guidance concerning the analysis of extended
release dosage forms has been provided by the F.D.A. (See Guidance
for Industry. Extended release oral dosage forms: development,
evaluation, and application of in vitro/in vivo correlations.
Rockville, Md.: Center for Drug Evaluation and Research, Food and
Drug Administration, 1997).
[0763] While the composition may comprise one or more extrinsic
adjuvants, advantageously in some embodiments this is not
necessary. In some embodiments, the peptide conjugate comprises an
epitope and is self adjuvanting.
[0764] The present invention provides a method of vaccinating or
eliciting an immune response in a subject comprising administering
to the subject an effective amount of a peptide conjugate or
peptide of the present invention. The present invention also
relates to use of a peptide conjugate or peptide of the invention
for vaccinating or eliciting an immune response in a subject, and
to use of a peptide conjugate or a peptide of the invention in the
manufacture of a medicament for vaccinating or eliciting an immune
response in a subject.
[0765] The present invention also provides a method of vaccinating
or eliciting an immune response in a subject comprising
administering to the subject an effective amount of the
pharmaceutical composition of the present invention. The present
invention also relates to use of a pharmaceutical composition of
the invention for vaccinating or eliciting an immune response in a
subject, and to the use of one or more peptides of the present
invention or one or more peptide conjugates of the present
invention in the manufacture of a medicament for vaccinating or
eliciting an immune response in a subject.
[0766] The present invention provides a method of eliciting an
immune response in a subject comprising administering to the
subject an effective amount of a peptide of the present invention.
The present invention also relates to use of a conjugate of the
invention for eliciting an immune response, and to use of a peptide
conjugate of the invention in the manufacture of a medicament for
eliciting an immune response in a subject.
[0767] The present invention provides a method of vaccinating a
subject comprising administering to the subject an effective amount
of a peptide of the present invention. The present invention also
relates to use of a conjugate of the invention for eliciting an
immune response, and to use of a peptide conjugate of the invention
in the manufacture of a medicament for eliciting an immune response
in a subject.
[0768] The administration or use of one or more peptides of the
present invention and/or one or more peptide conjugates of the
present invention, for example one or more peptide in together with
one or more peptide conjugates, for vaccinating or eliciting an
immune response in the subject is contemplated herein.
[0769] Where two or more peptides, two or more peptide conjugates,
or one or more peptides and one or more peptide conjugates are
administered or used, the two or more peptides, two or more peptide
conjugates, or one or more peptides and one or more peptide
conjugates may be administered or used simultaneously,
sequentially, or separately.
[0770] A "subject" refers to a vertebrate that is a mammal, for
example, a human. Mammals include, but are not limited to, humans,
farm animals, sport animals, pets, primates, mice and rats.
[0771] An "effective amount" is an amount sufficient to effect
beneficial or desired results including clinical results. An
effective amount can be administered in one or more administrations
by various routes of administration.
[0772] The effective amount will vary depending on, among other
factors, the disease indicated, the severity of the disease, the
age and relative health of the subject, the potency of the compound
administered, the mode of administration and the treatment desired.
A person skilled in the art will be able to determine appropriate
dosages having regard to these any other relevant factors.
[0773] The efficacy of a composition can be evaluated both in vitro
and in vivo. For example, the composition can be tested in vitro or
in vivo for its ability to induce a cell-mediated immune response.
For in vivo studies, the composition can be fed to or injected into
an animal (e.g., a mouse) and its effects on eliciting an immune
response are then assessed. Based on the results, an appropriate
dosage range and administration route can be determined.
[0774] The composition may be administered as a single dose or a
multiple dose schedule. Multiple doses may be used in a primary
immunisation schedule and/or in a booster immunisation
schedule.
[0775] In certain embodiments, eliciting an immune response
comprises raising or enhancing an immune response. In exemplary
embodiments, eliciting an immune response comprises eliciting a
humoral and a cell mediated response.
[0776] In certain embodiments, eliciting an immune response
provides immunity.
[0777] The immune response is elicited for treating a disease or
condition. A person skilled in the art will appreciate that the
peptides and peptide conjugates described herein are useful for
treating a variety of diseases and conditions, depending, for
example, on the nature of epitope.
[0778] In some embodiments, the diseases or conditions are selected
from those associated with the various antigens described
herein.
[0779] In some embodiments an infectious disease, cancer, or viral
re-activation post-bone marrow transplant or following induction of
profound immunosuppression for any other reason.
[0780] The term "treatment", and related terms such as "treating"
and "treat", as used herein relates generally to treatment, of a
human or a non-human subject, in which some desired therapeutic
effect is achieved. The therapeutic effect may, for example, be
inhibition, reduction, amelioration, halt, or prevention of a
disease or condition.
[0781] The compositions may be used to elicit systemic and/or
mucosal immunity. Enhanced systemic and/or mucosal immunity may be
reflected in an enhanced TH1 and/or TH2 immune response. The
enhanced immune response may include an increase in the production
of IgG1 and/or IgG2a and/or IgA.
EXAMPLES
Example 1. Preparation of Conjugates 200, 20 [Comprises SEQ ID NO:
22], 22 [Comprises SEQ ID NO: 22], and 26 [Comprises SEQ ID NO:
21]
[0782] 1.1 General Details
[0783] Commercially available starting materials were purchased
from Acros Organics, Ajax Finechem, Alfa Aesar, CEM, GL-Biochem,
Merck, NOVA Biochem, Sigma Aldrich and TCI and were used as
supplied. Dried solvents were prepared through distillation under
N2 or argon atmosphere. Tetrahydrofuran (THF) was freshly distilled
over sodium/benzophenone ketyl. Methanol (MeOH) and toluene were
freshly distilled over calcium hydride. Yields refer to
chromatographically and spectroscopically (.sup.1H NMR) homogenous
materials unless otherwise stated.
[0784] Thin layer chromatography (TLC) was performed on Merck
Kieselgel F.sub.254 200 .mu.m silica plates. Ultraviolet light was
used as a visualising agent and the general developing agents of
potassium permanganate in an aqueous basic solution and vanillin in
an ethanolic solution. Specific developing agents used were
ethanolic solutions of ninhydrin with acid for the identification
of primary amines. Heating was applied when using any developing
agent. Silica gel (0.063-0.100 mm) was used for flash column
chromatography.
[0785] Nuclear magnetic resonance (NMR) spectra were acquired at
room temperature in CDCl.sub.3 or D.sub.2O on a Bruker DRX400
spectrometer operating at 400 MHz for .sup.1H nuclei and 100 MHz
for .sup.13C nuclei. Reference peaks for 1H and .sup.13C spectra
were respectively set to 50.00 and .delta. 77.0 for CDCl.sub.3 and
.delta.4.79 for .sup.1H spectra in D.sub.2O. NMR data were reported
in values of chemical shift as parts per million (ppm) on the
.delta. scale, and coupling constants in hertz (Hz). Multiplicities
were reported as s=singlet, d=doublet, t=triplet, q=quartet,
dd=doublet of doublets, dt=doublet of triplets, tt=triplet of
triplets, dq=doublet of quartets, dqn=doublet of quintets,
sx=sextet, br s=broad singlet, and m=multiplet. The assignment of
C.sub.q was used to denote a quaternary carbon.
[0786] High resolution mass spectra were obtained on a Bruker
microOTOF-Q II mass spectrometer at a nominal resolution of 5000.
Analytical high-performance liquid chromatography (HPLC) and liquid
chromatography-mass spectrometry (LC-MS) chromatograms were
acquired on either a Dionex UltiMate 3000 HPLC system with a
Finnigan Surveyor MSQ Plus mass spectrometer or an Agilent 1120
Compact LC system with a Hewlett Packard Series 1100 MSD mass
spectrometer. Analytical reverse phase (RP) HPLC was performed
using the MeCN/H.sub.2O+0.1% TFA solvent system. Semipreparative RP
HPLC was performed on a Dionex UltiMate 3000 HPLC system using the
MeCN/H.sub.2O+0.1% TFA solvent system. Microwave reactions were
performed using a CEM Liberty Automated Microwave system.
[0787] 1.2 General Method for Peptide Chain Elongation
[0788] Manual Synthesis Method
[0789] Swelled peptide-resin was treated with 20% v/v/piperidine in
DMF (5.0 mL) and shaken for 20 min at r.t. The solution was drained
and the resin washed with DMF (.times.2) and DCM (.times.2). A
coupling mixture of Fmoc-AA-OH (2.0 eq.), HBTU (2.0 eq) and
iPr.sub.2NEt (4.0 eq.) in DMF (1 mL) was added and the resin shaken
for 1 hr. Resin was drained and washed again. The procedure was
repeated for the remaining residues in the sequence.
[0790] Automated Synthesis Method (Standard, 0.2 Mmol Scale)
[0791] Peptido-resin was transferred to the reaction vessel of a
Tribute automated peptide synthesiser. Automated synthesis was
undertaken with cycles of Fmoc deprotection and Fmoc-AA-OH coupling
steps. Deprotection was undertaken by addition of 20% v/v
piperidine in DMF (6.0 mL) and agitation (2.times.7 min). Following
resin drainage and DMF washing (4 mL.times.3), a coupling step was
performed with 5 eq. Fmoc-AA-OH dissolved in HBTU (0.24 mM, in DMF,
4 mL). 2 M N-methylmorpholine (NMM) in DMF (4 mL) was utilised in
the base-addition step. Coupling proceeded for 1 hr. After DMF
washing steps, the next cycle of deprotection and coupling
commenced, repeating until all amino acids were coupled.
[0792] Procedure for Coupling of Cysteine Derivatives (0.1 Mmol
Scale)
[0793] Peptido-resin was swelled in 1:1 CH.sub.2Cl.sub.2: DMF for
30 min, then drained. A coupling mixture of a Cys amino acid (0.2
mmol, 2 eq.), BOP (0.4 mmol, 4 eq.) and HOBt.H.sub.2O (0.4 mmol, 4
eq.) was dissolved in 1:1 CH.sub.2Cl.sub.2: DMF (2 mL).
2,4,6-collidine (0.4 mmol, 4 eq.) was then added and the resultant
solution added to the peptido-resin. The resin was agitated for 1
hr, or until ninhydrin test indicated no free amines. The resin was
then drained, washed with DMF (2.times.) and CH.sub.2Cl.sub.2
(2.times.), and dried.
[0794] Ninhydrin Test Procedure
[0795] A small portion of resin was taken, washed with
CH.sub.2Cl.sub.2 and allowed to dry. 1 drop each of solutions of 5%
v/v ninhydrin in EtOH, 80% w/v phenol in EtOH and 2% v/v KCN in
pyridine were added to the resin and the mixture heated at
90.degree. C. for 2 minutes. Blue-coloured beads and solution
indicated the presence of free primary amines, while a yellow
colour indicated no free amino groups present.
[0796] 1.3 Preparation of Amino Add Conjugate 200
[0797] N-Fluorenylmethoxycarbonyl-[R]-cysteine
##STR00028##
[0798] Fmoc-Cys(Trt)-OH (1.0 g, 1.7 mmol) was dissolved in
CH.sub.2Cl.sub.2 (50 mL). TFA (1.5 mL, 19.6 mmol) and iPr.sub.3SiH
(0.75 mL) were added, causing the solution to turn yellow. The
solution was agitated for 2 hrs at room temperature, at which point
the solution had turned colourless. The mixture was basified to pH
9 by addition of Na.sub.2CO.sub.3.H.sub.2O and washed with EtOAc.
The solution was acidified with 10M HCl, extracted with EtOAc and
concentrated in vacuo to give a white powder and a pink residue.
The powder and residue were dissolved in 4:1 MeCN: H.sub.2O and
lyophilised, giving a crude pink-white powder (424 mg, crude yield
73.1%). This crude product was carried through to the thiol-ene
reactions described below.
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((2-(palmitoyloxy)ethy-
l)thio)propanoic Acid (200)
##STR00029##
[0800] Thermal Initiation (Li, J. Dong, S. et al. Chemistry as an
Expanding Resource in Protein Science: Fully Synthetic and Fully
Active Human Parathyroid Hormone-Related Protein (1-141).
Angewandte Chemie International Edition 2012, 51 (49),
12263-12267).
[0801] Fmoc-Cys-OH (100 mg, 0.29 mmol), vinyl palmitate (476 .mu.L,
1.5 mmol) and AIBN (9.6 mg, 59 .mu.mol) were dissolved in degassed
1,2-dichloroethane (3 mL). The reaction mixture was then heated
under reflux (90.degree. C.) for 24 hr, after which TLC indicated
complete consumption of Fmoc-Cys-OH. The solution was then allowed
to cool to r.t. The solvent was removed under reduced pressure.
Presence of the desired product 200 in the crude reaction mixture
was confirmed by mass spectrometry.
[0802] MS (ESI.sub.-): For C.sub.36H.sub.51NO.sub.6S.sup.- [M-H]-
m/z calcd. 625.96 found 626.0
[0803] Photo-Initiation
[0804] Fmoc-Cys-OH (100 mg, 0.29 mmol) was dissolved in degassed,
anhydrous DMF (500 .mu.L). Vinyl palmitate (90 .mu.L, 0.3 mmol) and
DMPA (5.0 mg, 20 .mu.mol) were dissolved in degassed
CH.sub.2Cl.sub.2 (200 .mu.L). The two solutions were combined and
the resultant mixture irradiated for 6 hr (365 nm UV) in a standard
photochemical apparatus. When no further change in the reaction
mixture could be observed by TLC, solvent was removed under reduced
pressure. The crude product was purified by silica gel flash
chromatography (3:1 EtOAc:n-hexanes+2% AcOH), followed by
lyophilization from 1:1 H2O:MeCN+0.1% TFA to afford the title
compound as a powdery white solid (24 mg, 13%). Structure of the
desired product 200 was confirmed by mass spectrometry.
[0805] MS (ESI.sup.-): For C.sub.36H.sub.51NO.sub.6S.sup.- [M-H]-
m/z calcd. 625.96 found 626.0
[0806] 1.4 Preparation of Peptide Conjugates 20, 22, and 26
[0807] Peptides
##STR00030##
[0808] To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g
loading, 0.2 mmol scale) pre-swelled in 1:1 CH.sub.2Cl.sub.2: DMF
was added a coupling mixture of
Fmoc-L-Val-O--CH.sub.2-phi-OCH2-CH.sub.2--COOH (155.2 mg, 0.3
mmol), HBTU (113.8 mg, 0.3 mmol) and iPr.sub.2NEt (104 .mu.L, 0.6
mmol) in DMF (3 mL). The resin was shaken for 2 hrs at r.t., after
which a ninhydrin test indicated complete coupling. The Fmoc-Val
amino group was then deprotected by treatment of the resin with 20%
v/v piperidine in DMF (5 mL) for 20 mins at r.t. The resin was
transferred to a Tribute automated peptide synthesiser. Chain
elongation up to and including the Ser residue was performed using
the general automated coupling method. Coupling of the
Fmoc-Cys(Trt)-OH residue was performed manually with addition of a
mixture of Fmoc-Cys(Trt)-OH (235 mg, 0.4 mmol), BOP (360 mg, 0.8
mmol), HOBt.H.sub.2O (120 mg, 0.8 mmol) and 2,4,6-collidine (120
.mu.L, 0.8 mmol) in 1:1 CH.sub.2Cl.sub.2: DMF (2 mL). The resin was
shaken for 1 hr at r.t., after which a ninhydrin test indicated
complete coupling. Final Fmoc deprotection was accomplished by
treatment of the resin with 20% v/v piperidine in DMF (5 mL) for 20
mins at r.t.
[0809] After Fmoc deprotection, N-acetylation was performed by
adding acetic anhydride (50 .mu.L) and iPr.sub.2NEt (50 .mu.L) in
DMF (3 mL) to the resin. The resin was then shaken for 30 min at
r.t., after which a ninhydrin test indicated no remaining free
amines. The resin was drained, washed with DMF and CH.sub.2Cl.sub.2
and air dried. A cleavage cocktail of
TFA:H.sub.2O:DODT:iPr.sub.3SiH (94:2.5:2.5:1% v/v, 10.0 mL) was
added to the dry resin and the mixture shaken for 4 hr at r.t. The
cleavage cocktail was then treated with cold diethyl ether to
precipitate the crude peptide, which was centrifuged at 4000 rpm
for 5 minutes. The supernatant was discarded and the pellet washed
with diethyl ether, before repeating the spinning step. The ether
phase was then discarded and the peptide dried with N.sub.2 flow.
The crude peptide was then lyophilised from H.sub.2O+0.1% TFA. The
crude product was carried through to the thiol-ene reaction step
outlined below.
[0810] MS (ESI+): For C.sub.74H.sub.14N.sub.20O.sub.20S.sub.2.sup.+
[M+H].sup.+ m/z calcd. 1688.11 found 1688.8
##STR00031##
[0811] To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g
loading, 0.2 mmol scale) pre-swelled in 1:1 CH.sub.2Cl.sub.2: DMF
was added a coupling mixture of Fmoc-Rink-Amide-OH (216 mg, 0.4
mmol), HBTU (151.8 mg, 0.4 mmol) and iPr.sub.2NEt (140 .mu.L, 0.8
mmol) in DMF (2 mL). The resin was shaken for 1 hr at r.t., after
which a ninhydrin test indicated complete coupling. The linker
amino group was then deprotected by treatment of the resin with 20%
v/v piperidine in DMF (5 mL) for 20 mins at r.t. The resin was
transferred to a Tribute automated peptide synthesiser. Chain
elongation up to and including the Ser residue was performed using
the general automated coupling method. Coupling of the Cys residue
was performed manually with addition of a mixture of
Fmoc-Cys(Trt)-OH (235 mg, 0.4 mmol), BOP (360 mg, 0.8 mmol),
HOBt.H.sub.2O (120 mg, 0.8 mmol) and 2,4,6-collidine (120 .mu.L,
0.8 mmol) in 1:1 CH.sub.2Cl.sub.2: DMF (2 mL). The resin was shaken
for 1 hr at r.t., after which a ninhydrin test indicated complete
coupling. Final Fmoc deprotection was accomplished by treatment of
the resin with 20% v/v piperidine in DMF (5 mL) for 20 mins at r.t.
The resin was drained, washed with DMF and CH.sub.2Cl.sub.2 and air
dried. A cleavage cocktail of TFA:H.sub.2O:DODT:iPr.sub.3SiH
(94:2.5:2.5:1% v/v, 10.0 mL) was added to the dry resin and the
mixture shaken for 2 hr at r.t. The cleavage cocktail was then
treated with cold diethyl ether to precipitate the crude peptide,
which was centrifuged at 4000 rpm for 5 minutes. The supernatant
was discarded and the pellet washed with diethyl ether, before
repeating the spinning step. The ether phase was then discarded and
the peptide dried with N.sub.2 flow. The crude peptide was then
lyophilised from H.sub.2O+0.1% TFA. The crude product was carried
through to the thiol-ene reaction step outlined below.
[0812] MS (ESI+): For C.sub.30H.sub.61N.sub.11O.sub.7S.sup.+
[M+H].sup.+ m/z calcd. 719.94 found 720.0
##STR00032##
[0813] To aminomethyl polystyrene (PS) resin (0.20 g, 1.0 mmol/g
loading, 0.2 mmol scale) pre-swelled in 1:1 CH.sub.2Cl.sub.2: DMF
was added a coupling mixture of Fmoc-Rink-Amide-OH (216 mg, 0.4
mmol), HBTU (151.8 mg, 0.4 mmol) and iPr.sub.2NEt (140 .mu.L, 0.8
mmol) in DMF (2 mL). The resin was shaken for 1 hr at r.t., after
which a ninhydrin test indicated complete coupling. The linker
amino group was then deprotected by treatment of the resin with 20%
v/v piperidine in DMF (5 mL) for 20 mins at r.t. The resin was
transferred to a Tribute automated peptide synthesiser. Chain
elongation up to and including the Ser(Trt) residue was performed
using the general automated coupling method. Coupling of the Cys
residue was performed manually with addition of a mixture of
Fmoc-Cys(Trt)-OH (235 mg, 0.4 mmol), BOP (360 mg, 0.8 mmol),
HOBt.H.sub.2O (120 mg, 0.8 mmol) and 2,4,6-collidine (120 .mu.L,
0.8 mmol) in 1:1 CH.sub.2Cl.sub.2: DMF (2 mL). The resin was shaken
for 1 hr at r.t., after which a ninhydrin test indicated complete
coupling. After Fmoc deprotection, N-acetylation was performed by
adding acetic anhydride (50 .mu.L) and iPr.sub.2NEt (50 .mu.L) in
DMF (3 mL) to the resin. The resin was then shaken for 30 min at
r.t., after which a ninhydrin test indicated no remaining free
amines. The resin was drained, washed with DMF and CH.sub.2Cl.sub.2
and air dried. A cleavage cocktail of
TFA:H.sub.2O:DODT:iPr.sub.3SiH (94:2.5:2.5:1% v/v, 10.0 mL) was
added to the dry resin and the mixture shaken for 2 hr at r.t. The
cleavage cocktail was then treated with cold diethyl ether to
precipitate the crude peptide, which was centrifuged at 4000 rpm
for 5 minutes. The supernatant was discarded and the pellet washed
with diethyl ether, before repeating the spinning step. The ether
phase was then discarded and the peptide dried with N.sub.2 flow.
The crude peptide was then lyophilised from H.sub.2O+0.1% TFA. The
crude product was carried through to the thiol-ene reaction step
outlined below.
[0814] MS (ESI+): For C.sub.32H.sub.63N.sub.11O.sub.8S.sup.+
[M+H].sup.+ m/z calcd. 761.98 found 762.0
[0815] Peptide Conjugates
[0816] Thiol-ene Reaction Product of
Ac-Cys-Ser-Lys-Lys-Lys-Lys-NH.sub.2 24 [SEQ ID NO: 22] and Vinyl
Palmitate 22
##STR00033##
[0817] To crude peptide 24 [SEQ ID NO: 22] (25 mg, 32.6 .mu.mol)
and DMPA (3.3 mg, 13.1 .mu.mol) in a solution of NMP (4 mL) was
added vinyl palmitate (52.9 .mu.L, 0.16 mmol). The resultant
mixture was irradiated, with agitation, at 365 nm for 1 hr in a
standard UV photochemical apparatus. The desired product 22
[comprises SEQ ID NO: 22] was detected by mass analysis. The crude
product 22 [comprises SEQ ID NO: 22] was purified via
semi-preparative RP HPLC on a Phenomenex Gemini C18 column running
a gradient of 5-65% MeCN:H.sub.2O+0.1% TFA (3% MeCN per min,
50.degree. C.). Mass spectrometry confirmed the structure of the
desired product 22 [comprises SEQ ID NO: 22] (5.1 mg, 14.94% `from
crude`).
[0818] R.sub.t=11.50 min on a Phenomenex Gemini C18 3.mu. 110 .ANG.
2.0.times.50 mm column using a 5-95% MeCN:H.sub.2O+0.1% TFA, 3%
MeCN per min gradient; MS (ESI+): For
C.sub.50H.sub.97N.sub.11O.sub.10S.sup.+ [M+H].sup.+ m/z calcd.
1044.4 found 1044.9
[0819] Thiol-ene Reaction Product of
Cys-Ser-Lys-Lys-Lys-Lys-NH.sub.2 [SEQ ID NO: 22] and Vinyl
Palmitate 20
##STR00034##
[0820] The thiol-ene reaction of crude
Cys-Ser-Lys-Lys-Lys-Lys-NH.sub.2 [SEQ ID NO: 22] with 5 eq. vinyl
palmitate, 0.4 eq. DMPA in NMP, 1 hr irradiation at 365 nm gave the
desired product 20 (Pam-CSK4) [comprises SEQ ID NO: 22] by MS
analysis.
[0821] Thiol-ene Reaction Product of
Ac-Cys-Ser-Lys-Lys-Lys-Lys-Asn-Leu-Val-Pro-Met-Val-Ala-Thr-Val-OH
25 [SEQ ID NO: 21] and Vinyl Palmitate 26
##STR00035##
[0822] To crude peptide 25 [SEQ ID NO: 21] (20 mg, 11.9 .mu.mol)
and DMPA (1.2 mg, 4.74 .mu.mol) in a solution of NMP (3 mL) was
added vinyl palmitate (19.2 .mu.L, 59.3 .mu.mol). The resultant
mixture was irradiated, with agitation, at 365 nm for 1 hr in a
standard photochemical apparatus. The desired product 26 [comprises
SEQ ID NO: 21] was detected by mass analysis. The crude product 26
[comprises SEQ ID NO: 21] was purified via semi-preparative RP HPLC
on a Phenomenex Gemini C18 column running a gradient of 5-65%
MeCN:H.sub.2O+0.1% TFA (3% MeCN per min, 50.degree. C.). Mass
spectrometry confirmed the structure of the desired product 26
[comprises SEQ ID NO: 21] and the oxidised Met(O) by-product (1.67
mg, 7.15% `from crude`--including Met(O) product).
[0823] R.sub.t=11.90 min on a Phenomenex Gemini C18 3.mu. 110 .ANG.
2.0.times.50 mm column using a 5-95% MeCN:H.sub.2O+0.1% TFA, 3%
MeCN per min gradient; MS (ESI+): For
C.sub.92H.sub.168N.sub.20O.sub.2S2.sup.+ [M+2H].sup.2+ m/z calcd.
985.1 found 993.6 (Met(O))
[0824] 1.5 General Method for Thiol-Ene Reaction on Peptides
[0825] To crude or purified peptide (10 mM), DTT (30 mM) and DMPA
(4 mM) in a solution of DMSO was added vinyl palmitate (50 mM). The
resultant mixture was irradiated, with agitation, at 365 nm for 15
min in a standard UV photochemical apparatus. The desired product
was detected by ESI mass analysis. To achieve full conversion,
further addition of DMPA photoinitiator was sometimes required. The
crude product was purified via semi-preparative RP HPLC on a
Phenomenex Gemini C18 column running a gradient of 1-65%
MeCN:H2O+0.1% TFA (3% MeCN per min). Pooled fractions were
lyophilised to afford the pure products as white powders.
[0826] 1.6 Discussion
[0827] The thermal reaction of Fmoc-Cys-OH with vinyl palmitate was
conducted in 1,2-dichloroethane, using 5 equivalents of alkene and
0.2 eq. of AIBN as radical initiator.
[0828] The reaction was performed under reflux (90 degrees) for 24
hrs. Microwave heating (100W, 1 hr) produced the same result. The
desired product was detected by TLC. A number of by-products were
also formed.
[0829] Photo-initiation of the reaction was conducted with 1 eq.
vinyl palmitate and 0.2 eq. DMPA as the photo-initiator. Reactions
were conducted in a degassed DMF:DCM solvent mixture, irradiated
for 1 hr with 365 nm UV light in a standard photo-chemical
apparatus. Near complete conversion of the Fmoc-Cys-OH was observed
by TLC. Minimal by-products were formed. Purification provided the
product 200 in about 15% yield. Using 2 eq. vinyl palmitate
provided 200 in 44% yield after purification.
[0830] The thiol-ene reaction was carried out using NAc-CSK.sub.4
[comprises SEQ ID NO: 22]. The required peptide motif 24 [SEQ ID
NO: 22] was synthesised as described above. Following attachment of
Rink-Amide linker to aminomethyl resin, the SK.sub.4 sequence was
built up using automated Fmoc-SPPS (standard coupling conditions).
Fmoc-Cys(Trt)-OH was then coupled manually using conditions to
reduce epimerisation. N-acetylation was then carried out.
[0831] Mass analysis indicated that that by-product formation was
occurring upon cleavage of the peptide from the resin, due to
tert-butylation (+56) of cysteine. Repeating the synthesis of
NAc-CSK4 [comprises SEQ ID NO: 22] utilising Fmoc-Ser(Trt)-OH,
instead of Fmoc-Ser(t-Bu)-OH, resulted in a product free of the
cysteine-alkylation product. The peptide was cleaved and then
lyophilised.
[0832] The thiol-ene reaction of crude peptide 24 [SEQ ID NO: 22]
with vinyl palmitate was then carried out. N-methylpyrrolidone
(NMP) effectively solvated both the hydrophilic CSK4 peptide [SEQ
ID NO: 22] and the hydrophobic vinyl palmitate molecule.
[0833] Thermal initiation using AIBN and microwave heating was
carried out on both crude and purified peptide using excess of
vinyl palmitate (up to 20 eq.). Photo-initiation of the reaction
provided better results. Using crude peptide with DMPA as
photo-initiator, the reaction proceeded to completion following 1
hr of irradiation (5 eq. vinyl palmitate, 0.4 eq. DMPA in 2 mL
NMP). The desired product was confirmed by MS (>90% conversion,
60% purity by HPLC).
[0834] Advantageously, no purification after cleavage was required
before the thiol-ene coupling. Purification by RP-HPLC is typically
inefficient, with >50% loss of material being common. Generally,
it is advantageous to reduce the number of HPLC purification steps
required wherever possible.
[0835] Purification of the N-acetylated monoacyl lipopeptide 22
[comprises SEQ ID NO: 22] was achieved by semi-preparative RP-HPLC
using a Phenomenex C18 column, running a gradient of 5-95%
MeCN:H.sub.2O+0.1% TFA, 3% MeCN per min. The purified peptide was
then lyophilised to afford the desired product as a white powder
(5.1 mg, 14.9% from crude peptide). The relatively low yield may be
due to by-product formation in the thiol-ene reaction.
[0836] Increasing the peptide concentration to 25 mM led to a small
decrease in by-product formation (>90% conversion, 80% purity by
HPLC). Decreasing the concentration to 5 mM had the opposite
effect.
[0837] Carrying out the reaction in a mixture of NMP:H.sub.2O:DMSO
(4:2:1) in the presence of glutathione (GSH) (3 eq.) with a peptide
concentration of 5 mM resulted in mixed disulfide formation (50%
conversion, 75% purity by HPLC). Using
2,2'-(ethylenedioxy)diethanethiol (DODT) (3 eq.) in NMP with a
peptide concentration of 5 mM led to a complex mixture of products
(80% conversion by HPLC).
[0838] Advantageously, upon addition of 3 eq. DTT to the reaction
mixture (10 mM peptide in NMP) no by-products resulting from vinyl
palmitate telomerisation, or mixed disulfides, were observed and
the reaction proceeded with high conversion (>90%0 conversion,
85% purity by HPLC). Using DTT it was also possible to conduct the
reaction (25 mM peptide) in DMSO, a benign and more versatile
solvent (90% conversion, >95% purity by HPLC).
[0839] The thiol-ene reaction was also carried out using
non-acetylated analogue CSK.sub.4 [SEQ ID NO: 22]. Synthesis of the
CSK.sub.4 [SEQ ID NO: 22] motif was carried out utilising the
procedure described above. The peptide was then cleaved from resin
and lyophilised. The thiol-ene reaction of the crude product with
vinyl palmitate proceeded smoothly using 5 eq. vinyl palmitate, 0.4
eq. DMPA in NMP, 1 hr irradiation at 365 nm to give the desired
product 20 (Pam-CSK4) [comprises SEQ ID NO: 22] by MS analysis.
[0840] The thiol-ene reaction of vinyl palmitate with an
immunodominant A*0200 restricted epitope derived from the
cytomeglovirus (CMV) ppUL83 protein (`NLV peptide`) (Kopycinski, J.
et al., Sequence flexibility of the immunodominant HLA A* 0201
restricted ppUL83 CD8 T-cell of human cytomegalovirus. Journal of
medical virology 2009, 82 (1) 94-103) was also carried out.
[0841] The NLV sequence was built up by automated Fmoc-SPPS, using
standard conditions. A K.sub.4 tag and a serine residue were then
coupled to the N-terminus of the sequence. The peptidyl resin was
then removed from the synthesiser and the cysteine residue coupled
manually, using standard conditions. N-acetylation was then carried
out. The peptide was then cleaved from the resin and lyophilised to
give a white powder in good yield.
[0842] The thiol-ene reaction of the unprotected peptide 25 [SEQ ID
NO: 21] and vinyl palmitate was carried out using photo-initiation,
as described for peptides 20 [comprises SEQ ID NO: 22] and 22
[comprises SEQ ID NO: 22]. Mass analysis indicated conversion to
the palmitoylated product 26 [comprises SEQ ID NO: 21].
Purification was accomplished by semi-preparative RP-HPLC using a
Phenomenex C18 column, running a gradient of 5-95% MeCN:H.sub.2O
with 0.1% TFA. The purified peptide was lyophilised to provide the
desired product as a white powder (7.5%), along with the
corresponding Met(O) product (.about.30%).
[0843] The thiol-ene reaction of crude 25 [SEQ ID NO: 21] with
vinyl palmitate was also carried out following the general
procedure described above. ESI-MS and HPLC analysis indicated good
conversion to the palmitoylated product 26 [comprises SEQ ID NO:
21](FIGS. 2a and 2b). Purification was accomplished by
semi-preparative RP-HPLC, to give the desired product in >95%
purity (FIGS. 2b and 2c).
Example 2. Biological Activity of Peptide Conjugates 20 [Comprises
SEQ ID NO: 22], 22 [Comprises SEQ ID NO: 22], and 26 [Comprises SEQ
ID NO: 21]
[0844] 2.1 Procedures
[0845] Activation of Human Monocytes in Whole Blood
[0846] 100 .mu.l of heparinised whole blood (WB) was incubated with
100 nM, 1 .mu.M and 10 .mu.M of each compound, in duplicate, and
incubated overnight at 37.degree. C. in a 5% CO.sub.2 humidified
incubator. Pam.sub.3CSK.sub.4 [comprises SEQ ID NO: 22] (10 .mu.M;
EMC Microcollections) was used as a positive control. To detect
activation of monocytes, WB samples were stained with
anti-CD14-FITC, anti-HLA-DR-Alexa700, anti-CD80-PE-Cy7,
anti-CD40-PE, anti-CD86-APC, anti-CD16-APC-Cy7 (all from Biolegend)
for 20 mins at RT, protected from light. Following incubation, 2 ml
of BD FACS lyse (BD Biosciences) was added, incubated for 15 mins
at RT, then washed twice with ice cold wash buffer (PBS, 1% Human
Serum). Data acquisition was performed on a BD FACS Aria II (Becton
Dickinson) and analysed using FlowJo software version 7.6.5
(TreeStar). CD80 receptor expression on monocytes was detected by
gating on CD14+ HLADR+ cells.
[0847] 2.2 Discussion
[0848] The bioactivity of lipopeptides 20 [comprises SEQ ID NO:
22], 22 [comprises SEQ ID NO: 22], and 26 [comprises SEQ ID NO:
21], was assessed by flow cytometry to measure up-regulation of the
co-stimulatory molecule CD80 on human monocytes in fresh blood
samples (FIG. 1).
[0849] Monocytes were identified in five donor samples by
characteristic cell surface markers, and the expression of CD80
determined before and after exposure to each compound at three
dosages, with commercially available Pam3CSK4 [comprises SEQ ID NO:
22] (10 .mu.M) serving as a positive control.
[0850] 22 [comprises SEQ ID NO: 22] and 26 [comprises SEQ ID NO:
21] both strongly upregulated expression of CD80 at all doses
tested, demonstrating equivalent potency to Pam3CSK4 [comprises SEQ
ID NO: 22] at the 10 .mu.M dose in most donors.
[0851] In three donors, 20 [comprises SEQ ID NO: 22] showed lower
potency than 22 [comprises SEQ ID NO: 22] and 26 [comprises SEQ ID
NO: 21], consistent with acetylation of the cysteine amino group
improving potency of TLR2 agonism in human cells. The potency of 26
[comprises SEQ ID NO: 21] demonstrates that conjugation of
antigenic peptides does not affect TLR2 agonism.
Example 3. Preparation of Conjugates 200, 120, 121, 110 [Comprises
SEQ ID NO: 27], 111 [Comprises SEQ ID NO: 24], 112 [Comprises SEQ
ID NO: 24], 112A [Comprises SEQ ID NO: 24], 113 [Comprises SEQ ID
NO: 25], 114 [Comprises SEQ ID NO: 25], 115 [Comprises SEQ ID NO:
26] and 116 [Comprises SEQ ID NO: 26]
[0852] 3.1 General Details
[0853] Protected amino acids and coupling reagents were purchased
from GL-Biochem (Shanghai). The resins used in the solid-supported
syntheses were preloaded tentagel resins from Rapp Polymere GmbH
(Tuebingen) and other solvents and reagents were obtained from
Sigma (St Louis, Mo.) and Novabiochem.
[0854] The peptide syntheses described below were carried out using
standard iterative Fmoc Solid-Phase Peptide Synthesis techniques on
a Tribute peptide synthesiser (Protein Technologies International,
Tucson, Ariz.). A typical deprotection and coupling cycle carried
out on a 0.1 mmol scale entailed removal of the Fmoc protecting
group from the resin-bound amino-acid using two treatments of 20%
piperidine in DMF (4 mL.times.5 min) then washing the resin with
DMF. In a separate vessel the Fmoc amino acid (0.5 mmol) and
coupling agent
(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU), 0.45 mmol) were dissolved in DMF
(1.5 mL) and base (4-methylmorpholine, 1 mmol) added. After mixing
for 1 minute, this solution was transferred to the resin, which was
agitated at RT for 1 hour, drained and washed.
[0855] Cleavage of the peptide (0.1 mmol scale) was achieved by
suspending the resin in 5 mL trifluoroacetic acid (TFA) containing
5% (v/v) ethanedithiol (EDT) and agitating at room room temperature
for 3 hours. Triisopropylsilane (TIPS) was then added to 1% (v/v)
and agitation continued for a further five minutes before draining
the TFA into chilled diethyl ether (40 mL). The precipitated
material was pelleted by centrifugation, the ether discarded, the
pellet washed once with ether (25 mL) and air-dried or
lyophilised.
[0856] Reverse phase (RP)-HPLC was carried out using a Dionex
Ultimate 3000 HPLC system. For semi-preparative purifications, a
peptide sample was injected into a reverse-phase Phenomenex Gemini
C18 column (5.mu., 110 .ANG.; 10.times.250 mm) equilibrated in a
suitable mixture of eluent A (water/0.1% TFA) and eluent B
(MeCN/0.1% TFA) then an increasing gradient of eluent B was
generated to elute the constituent components. Analytical HPLC was
performed similarly, using a Phenomenex Gemini C18 column (3.mu.,
110 .ANG.; 4.6.times.150 mm).
[0857] Low-resolution mass spectra were obtained using an Agilent
Technologies 6120 Quadrapole mass spectrometer.
[0858] NMR spectra were obtained using a Bruker BRX400 spectrometer
operating at 400 MHz for .sup.1H NMR and at 100 MHz for .sup.13C
NMR.
[0859] In the amino acid conjugates and peptide conjugates
described below the abbreviations AcN-C(Pam-1)- and
H.sub.2N--C(Pam-1)-means
##STR00036##
wherein R is Ac or H as appropriate.
[0860] 3.2 Preparation of Peptide Conjugates by Direct
Conjugation
[0861] Peptides
[0862] Peptides 100 [SEQ ID NO: 23], 102 [SEQ ID NO: 24], 103 [SEQ
ID NO: 25], 104 [SEQ ID NO: 25], 105 [SEQ ID NO: 26] and 106 [SEQ
ID NO: 26] (Table 1) were synthesised as described and depicted
below (Scheme 1).
##STR00037##
[0863] Following synthesis of the peptide sequence up to the
penultimate amino acid using iterative Fmoc-SPPS, Fmoc-cysteine was
introduced as the N-terminal residue of the on-resin peptide by
reaction with Fmoc-Cys(Trt)-OH, HATU, and 4-methylmorpholine in
DMF. The Fmoc group was removed using 20% piperidine in DMF. As
required, the resulting amine group was converted to an acetamide
by treatment with a mixture of 20% acetic anhydride in DMF (2 mL)
and 4-methylmorpholine (1 mmol).
[0864] Following cleavage of the peptide from resin with TFA/EDT
and its precipitation in ether, the solid was dissolved in 1:1
water/MeCN and lyophilised. If the peptide contained a methionine
residue the solution was heated at 60.degree. C. for 1 hour prior
to freeze-drying to reverse any S-alkylation that may have occurred
during cleavage. The peptides were then purified by RP-HPLC to give
material of >95%.
[0865] Peptide 102 [SEQ ID NO: 24] was synthesised with the
side-chain of the non-terminal cysteine residue as a tert-butyl
thioether so as to avoid unwanted side reactions at this
location.
TABLE-US-00008 TABLE 1 Peptide SEQ ID m/z Sequence NO [M +
3H.sup.+] 100 AcHN-CSKKKVKNLVPMVATVK(Ac)-C(O)NH2 23 619.7 102 AcHN-
24 1357.8 CSKKKKLQQLSLLMWITQC(tBu)FLPVFLAQPPSGQRR- OH 103 H2N- 25
1625.5 CSKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- OH 104 AcHN-
25 1639.9 CSKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- OH 105
H2N-CSKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 1174.8 106
AcHN-CSKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 1188.8
[0866] Peptide Conjugates
[0867] The thiol-ene reaction was then performed on peptides 100
[SEQ ID NO: 24], 102 [SEQ ID NO: 24], 103 [SEQ ID NO: 25], 104 [SEQ
ID NO: 25], 105 [SEQ ID NO: 26] and 106 [SEQ ID NO: 26] to generate
the corresponding peptide conjugates 110 [comprises SEQ ID NO: 27],
112A [comprises SEQ ID NO: 24], 113 [comprises SEQ ID NO: 25], 114
[comprises SEQ ID NO: 25], 115 [comprises SEQ ID NO: 26] and 116
[comprises SEQ ID NO: 26] (Table 2).
[0868] DMPA (2.6 mg), dithiothreitol (9.2 mg), and vinyl palmitate
(40 mg, mmol) were dissolved in degassed NMP (2 mL). 100 .mu.L of
this solution was then added to 1 .mu.mol of the peptide weighed
into a small polypropylene vessel to give a solution containing 10
mM peptide, 5 mM DMPA, 30 mM DTT and 50 mM vinyl palmitate. NMP was
compatible with the reaction conditions and effectively solvated
all of the components of the reaction mixture.
[0869] The reaction vessel was flushed with nitrogen and the
vigorously stirred mixture irridiated with a hand-held 6 watt UV
lamp (Spectronics, NY) operating at 365 nm. After 30 minutes the
reaction was analysed by HPLC and showed conversion to the desired
product. The product was then isolated by RP-HPLC and unreacted
starting material recovered.
[0870] Following purification of 112A [comprises SEQ ID NO: 24] the
tert-butyl protecting group of cysteine was removed by treatment
with a mixture of triflic acid and trifluoroacetic acid (1:16 v/v)
for 3 minutes to cleanly give the fully deprotected lipopeptide 112
[comprises SEQ ID NO: 24] (Table 2A).
[0871] The peptides with non-acetylated N-terminal cysteine formed
significant amounts of the disulfide dimer, despite the presence of
the reducing agent DTT. This was not observed with the
corresponding N-acetylated peptides.
[0872] Peptide conjugates 110 [comprises SEQ ID NO: 27] and 113
[comprises SEQ ID NO: 25], 114 [comprises SEQ ID NO: 25], 115
[comprises SEQ ID NO: 26] and 116 [comprises SEQ ID NO: 26] were
also prepared from peptides 100 [SEQ ID NO: 24], and 103 [SEQ ID
NO: 25], 104 [SEQ ID NO: 25], 105 [SEQ ID NO: 26] and 106 [SEQ ID
NO: 26] by the following alternative procedure (Table 2B).
[0873] In this procedure, tert-butyl mercaptan (tBuSH) thiol was
used in place of DTT. This resulted in increased and cleaner
conversion of the substrate peptides to the desired peptide
conjugate.
[0874] Trifluoroacetic acid (TFA) was also introduced to the
reaction mixture. This further improved the reaction profile. The
formation of oligomers, minor-by-products formed by reaction of the
product peptide conjugate with a second molecule of vinyl palmitate
to give a bis-palmitoylated species, was largely suppressed by the
addition of TFA.
[0875] Due to the apparent propensity of methionine to oxidise to
the corresponding sulfoxide under these conditions, the crude
product mixtures of those peptides possessing methionine groups
were lyophilised, dissolved in TFA and treated with
tetrabutylammonium iodide to reduce methionine oxide back to
methionine.
[0876] A typical procedure was as follows. DMPA (6.5 mg) was
dissolved in degassed NMP (0.5 mL) and tert-butyl mercaptan (17
.mu.L) added and in a separate vessel vinyl palmitate (11.3 mg) was
dissolved in degassed N-methylpyrrolidinone (NMP) (0.5 mL). The
peptide (1 .mu.mol) was weighed into a small polypropylene vessel
equipped with a small stirrer and 10 .mu.L of the DMPA/tBuSH
solution added followed by 100 .mu.L of the vinyl palmitate
solution, to give a solution of approximately 10 mM peptide, 5 mM
DMPA, 30 mM DTT and 80 mM vinyl palmitate. TFA (5.5 .mu.L) was then
added, to give a 5% solution. The reaction vessel was flushed with
nitrogen and the vigorously stirred mixture irradiated with a
hand-held 6 watt UV lamp (Spectronics, NY) operating at 365 nm.
After 20 minutes further DMPA (10 .mu.L) and vinyl palmitate (50
.mu.L) were added and irradiation continued for 20 min.
[0877] For those peptides containing methionine, water (0.5 mL) and
MeCN (0.5 mL) were added and the mixture lyophilised. The resultant
solid was dissolved in neat TFA (150 .mu.L), cooled to 0.degree. C.
and tetra-n-butylammonium iodide (3.7 mg, 10 .mu.mol) in 25 .mu.L
TFA was added. After 1 minute chilled diethyl ether (0.5 mL) was
added to precipitate the reduced lipopeptide, which was pelleted by
centrifugation and lyophilised.
[0878] The reactions were analysed by HPLC to show conversion to
the desired products (Table 2B), which were then isolated by
RP-HPLC.
TABLE-US-00009 TABLE 2 Peptide m/z Sequence SEQ ID NO Conversion [M
+ 4H+] 110 AcHN-C(Pam-1)SKKKVKNLVPMVATVK(Ac)-C(O)NH2 23 54% 535.6
112A AcHN-C(Pam-1)SKKKKLQQLSLLMWITQC(tBu)FLPVFLAQPPSGQRR-OH.sup.a
24 44% 1089.1 113
H2N-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- 25 26%
1289.9 OH 114
AcHN-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- 25 42%
1300.4 OH 115 H2N-C(Pam-1)SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 20%
952.0 116 AcHN-C(Pam-1)SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 47%
962.5 .sup.aSubsequent deprotection of the Cys(tBu) residue
afforded fully deprotected lipopeptide 112
TABLE-US-00010 TABLE 2A Peptide SEQ ID m/z Sequence NO [M +4H+] 112
AcHN-C(Pam-1)SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR- 24 1075.0 OH
TABLE-US-00011 TABLE 2B Peptide SEQ ID m/z Sequence NO Conversion
[M + 4H+] 110 AcHN-C(Pam-1)SKKKVKNLVPMVATVK(Ac)-C(O)NH2 23 75%
535.6 113 H2N-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-
25 35% 1289.9 OH 114
AcHN-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- 25 41%
1300.4 OH 116 AcHN-C(Pam-1)SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26
74% 962.5
[0879] 3.3 Preparation of Amino Add Conjugates
[0880] Amino acid conjugates 200, 120, and 121 were prepared from
N-.alpha.-Fmoc-, N-.alpha.-acetyl-, and N-.alpha.-Boc-protected
cysteine, respectively, as described and depicted below (Scheme
2).
##STR00038##
[0881] (i) Radical initiator, hu (365 nm) or heat PG-71,C3
[0882] Solid N-.alpha.-protected cysteine was dissolved or
suspended to a concentration of 100 mg/mL in the indicated solvent
(Table 3) and vinyl palmitate (1.5 molar equivalents) added
followed by the indicated quantity of initiator. For reactions
conducted under photolytic conditions the solution was prepared in
a polypropylene vessel, DMPA added in the indicated molar
proportions (Table 3) and the stirred mixture then irradiated at
365 nm. For reactions carried out under thermal conditions, the
solution was prepared in a glass tube, the indicated quantity of
AIBN (azobisisobutyronitrile) added and the stirred mixture heated
either in an oil bath or in a microwave oven.
[0883] Reaction progress was monitored using thin-layer
chromatography and was allowed to proceed to completion based on
consumption of the cysteine starting material. The solvent was then
removed and the residue purified by flash column chromatography on
silica gel, eluting with hexane/ethyl acetate mixtures. The
identities of Fmoc-Cys(Pam-1)-OH (200), Ac-Cys(Pam-1)-OH (120) and
Boc-Cys(Pam-1)-OH (121), were confirmed by .sup.1H and .sup.13C NMR
and by mass spectrometry.
[0884] N-Fmoc-Cys(Pam-1)-OH (200)
##STR00039##
[0885] 1H NMR (400 MHz, CDCl.sub.3): .delta.7.76 (d, J 7.61 Hz,
2H), 7.60 (br s, J 6.80 Hz, 2H), 7.39 (t, J 7.50 Hz, 2H), 7.31 (dt,
J.sub.1 0.77 J.sub.2 7.50 Hz, 2H), 6.92-6.42 (br s, 1H, COOH), 5.72
(d, J 7.66 Hz, 1H, FmocNH), 4.70-4.62 (m, 1H), 4.45-4.38 (m, 2H),
4.26-4.18 (m, 3H), 3.14 (dd, J.sub.1 4.67 J.sub.2 13.66 Hz, 1H),
3.06 (dd, J.sub.1 5.36, J.sub.2 13.84 Hz, 1H), 2.78 (t, J 6.40 Hz,
2H), 2.29 (t, J 7.50 Hz, 2H), 1.59 (m, 2H), 1.28-1.21 (m, 24H),
0.88 (t, J 6.87 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta.174.1, 173.9, 155.9, 143.69, 143.62, 143.5, 141.3, 127.7,
127.0, 125.0, 119.9, 67.3, 63.0, 53.5, 47.0, 34.3, 34.1, 31.9,
31.2, 29.67, 29.64, 29.60, 29.4, 29.3, 29.2, 29.1, 24.8, 22.6,
14.0; HRMS Found (ESI): [M+Na].sup.+ 648.3328,
C.sub.36H.sub.51NO.sub.6SNa requires 648.3329.
[0886] N--Ac-Cys(Pam-1)-OH (120)
##STR00040##
[0887] 1H NMR (400 MHz, CDCl.sub.3): .delta. 8.21-7.70 (br s, 1H,
COOH), 6.29 (d, J 7.51 Hz, 1H, NHAc), 4.79 (ddd, J.sub.1 5.06
J.sub.2 5.49 J.sub.3 6.94 Hz, 1H), 4.22 (t, J 6.72 Hz, 1H), 4.21
(t, J 6.53 Hz, 1H), 3.12 (dd, J.sub.1 4.72 J.sub.2 14.08 Hz, 1H),
3.05 (dd, J.sub.1 5.80 J.sub.2 13.93 Hz, 1H), 2.78 (t, J 6.60 Hz,
1H), 2.77 (t, J 6.76 Hz, 1H), 2.33 (t, J 7.50 Hz, 2H), 2.09 (s,
3H), 1.65-1.55 (m, 2H), 1.27-1.22 (s, 24H), 0.87 (t, J 6.92 Hz,
3H); .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 174.0, 172.7,
171.4, 62.9, 52.1, 34.2, 33.8, 31.8, 31.2, 29.6, 29.63, 29.60,
29.4, 29.3, 29.2, 29.1, 24.8, 22.8, 22.6, 14.0; HRMS Found (ESI):
[M+Na].sup.+ 468.2750, C.sub.23H.sub.43NO.sub.5SNa requires
468.2754.
[0888] N-Boc-Cys(Pam-1)-OH (121)
##STR00041##
[0889] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.71-8.84 (br s,
1H, COOH), 5.43-5.36 (m, 1H, BocNH), 4.60-4.49 (m, 1H), 4.21 (t, J
6.80 Hz, 2H), 3.14-2.93 (m, 2H), 2.78 (t, J 6.73 Hz, 2H), 2.30 (t,
J 7.46 Hz, 2H), 1.64-1.56 (m, 2H), 1.44 (s, 9H), 1.24 (s, 24H),
0.56 (t, J 6.82 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta. 174.7, 173.8, 155.4, 63.1, 53.2, 34.4, 34.2, 31.9, 31.2,
29.69, 29.66, 29.62, 29.4, 29.3, 29.2, 29.1, 28.2, 28.1, 24.9,
22.6, 14.1; HRMS Found (ESI): [M+Na].sup.+ 526.3176,
C.sub.26H.sub.49NO.sub.6SNa requires 526.3173.
[0890] The conjugation reaction was carried out under a variety of
conditions, which are summarised in Table 3.
TABLE-US-00012 TABLE 3 N-.alpha.- Product protecting Initiator Time
(% Entry group (mol eq.) Solvent Conditions .sup.a (min)
yield).sup.b 1 Fmoc DMPA DCM hu (365 nm) 60 200 (67) (0.2) 2 Fmoc
DMPA DCM hu (365 nm) 60 200 (82) (1) 3 Fmoc AIBN DCM Microwave 80
200 (41).sup.c (1) (70.degree. C.) 4 Ac DMPA DCM hu (365 nm) 60 120
(87) (0.2) 5 Ac DMPA DCM hu (365 nm), 60 120 (74) (0.2) DTT 6 Ac
DMPA DCM hu (365 nm) 60 120 (88) (1) 7 Ac AIBN DCM Microwave 80 120
(100) (1) (70.degree. C.) 8 Boc DMPA DCM hu (365 nm) 60 121 (78)
(0.2) 9 Boc DMPA DCM hu (365 nm) 60 121 (82) (1) 10 Boc AIBN DCM
Microwave 80 121 (77) (1) (70.degree. C.) .sup.a UV irradiation
used a hand-held Spectronics 6 watt lamp operating at 365 nm;
microwave reactions were carried out using a CEM Discover microwave
reactor operating at 100 w and 70.degree. C. .sup.bYield is based
on isolated material after chromatography. .sup.cReaction
incomplete after this time, based on residual Fmoc-cysteine
substrate.
[0891] As can be seen from Table 3, the use of photolytic
conditions generated the desired products with relatively
consistent yields, regardless of the N-protecting group of the
cysteine substrate and the relative concentration of the
photoinitiator.
[0892] Under thermal conditions using AIBN as the initiator yields
varied considerably. The N-acetylated substrate afforded superior
yields of the desired product 120 (entry 9). Using microwave
heating was particularly effective, giving a quantitative yield of
acetylated product 120 (entry 7) and a high yield of the Boc
product 121 (entry 10).
[0893] 3.4 Preparation of Peptide Conjugates Via Coupling of Amino
Add Conjugates
[0894] Peptide conjugates 110 [comprises SEQ ID NO: 27], 111
[comprises SEQ ID NO: 24], 112 [comprises SEQ ID NO: 24], 113
[comprises SEQ ID NO: 25], 114 [comprises SEQ ID NO: 25], 115
[comprises SEQ ID NO: 26] and 116 [comprises SEQ ID NO: 26](Table
4) were prepared as described and depicted below (Scheme 3).
##STR00042##
[0895] The desired peptide sequence was synthesised using standard
iterative Fmoc SPPS techniques using a Tribute peptide synthesiser
as previously described. After coupling the penultimate amino acid
residue, the resin-bound peptide chain was then derivatised with
the amino acid conjugate N-Fmoc-Cys(Pam-1)-OH 200 using PyBOP and
collidine in DMF. The Fmoc group was then removed using 20%
piperidine in DMF.
[0896] The resulting peptide was then cleaved from resin using
TFA/EDT, with concomitant removal of protecting groups, to afford
peptide conjugates 111 [comprises SEQ ID NO: 24], 113 [comprises
SEQ ID NO: 25] and 115 [comprises SEQ ID NO: 26].
[0897] Alternatively, the resulting peptide was converted to the
corresponding acetamide by treatment with a mixture of 20% acetic
anhydride in DMF (2 mL) and 4-methylmorpholine (1 mmol) and then
cleaved from resin to afford peptide conjugates 110 [comprises SEQ
ID NO: 27], 112 [comprises SEQ ID NO: 24], 114 [comprises SEQ ID
NO: 25] and 116 [comprises SEQ ID NO: 26].
[0898] Alternatively, the resin-bound peptides were derivatised
with either the amino acid conjugate N-Boc-Cys(Pam-1)-OH 121 or
N--Ac-Cys(Pam-1)-OH 120. On cleavage from resin this afforded the
peptide conjugates 110 [comprises SEQ ID NO: 27], 111 [comprises
SEQ ID NO: 24], 112 [comprises SEQ ID NO: 24], 113 [comprises SEQ
ID NO: 25], 114 [comprises SEQ ID NO: 25], 115 [comprises SEQ ID
NO: 26] and 116 [comprises SEQ ID NO: 26] directly, without the
additional manipulations necessary due to the Fmoc group.
[0899] The conditions for coupling of the amino acid conjugate
reduced the propensity of the .alpha.-carbon of the amino acid to
racemise on activation. The amino acid conjugate (0.075 mmol) and
PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate) (0.1 mmol) were combined and dissolved in DMF
(0.3 mL). Neat 2,4,6-trimethylpyridine (0.1 mmol) was added and
after mixing for 30 seconds the solution transferred to 0.025 mmol
of resin, which was then agitated for 90 minutes, drained and
washed (DMF).
[0900] The peptide was then cleaved by agitating 0.015 mmol of the
resin in 1 mL of trifluoroacetic acid containing 5% (v/v)
ethanedithiol at room temperature for 3 hours. The supernatant was
then drained through a sinter into chilled diethyl ether (10 mL)
and the resin washed with a further 1 mL of TFA, which was also
added to the ether.
[0901] The precipitated material was pelleted by centrifugation and
the pellet washed once with ether (5 mL) before being dissolved in
1:1 MeCN/Water (+0.1% tfa) and lyophilised. If the peptide
contained a methionine residue the solution was heated at
60.degree. C. for 1 hour prior to freeze-drying. The peptides were
then purified (>95%) by RP-HPLC and their identities confirmed
by analytical RP-HPLC and mass spectrometry.
TABLE-US-00013 TABLE 4 Peptide SEQ ID m/z Yield Sequence NO [M +
4H+] (%) 110 AcHN-C(Pam-1)SKKKKNLVPMVATVK(Ac)-(CO)NH2 27 535.6 27
111 H2N-C(Pam-1)SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 24 1064.6 15
112 AcHN-C(Pam-1)SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 24 1075.0 20
113 H2N-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- 25
1289.9 21 OH 114
AcHN-C(Pam-1)SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL- 25 1300.4
23 OH 115 H2N-C(Pam-1)SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 952.0
16 116 AcHN-C(Pam-1)SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 962.5
18
Examples 4. 10. Biological Activity of Peptides and Peptide
Conjugates
[0902] These examples describes the assessment of the efficacy of
the peptides and peptide conjugates of the present invention in
supporting the processing and presentation of various antigenic
peptide constructs to a various T cell clones by by antigen
presenting cells (APC).
[0903] Materials
[0904] The peptide s and peptide conjugate constructs used in the
following examples are outlined in Table 5 below.
TABLE-US-00014 TABLE 5 Peptide and peptide conjugate constructs
Peptide SEQ ID NO Peptide epitope CMV pp65 residues 495-503 [SEQ ID
NO: 27] Peptide construct Sequence 110
Pam1Cys(Ac)-SKKKKNLVPMVATVK(Ac)-NH2 27 131
Pam2Cys-SKKKKNLVPMVATVK(Ac)-NH2 27 142
Ac-Cys-SKKKKNLVPMVATVK(Ac)-NH2 27 Peptide epitope NY-ESO-1 residues
153-180 [SEQ ID NO: 18] Peptide construct Sequence 143
LQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 18 144
SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 17 132
Pam2Cys-SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 24 111
PamlCys(NH2)-SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 24 112
Pam1Cys(Ac)-SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR-OH 24 Peptide epitope
NY-ESO-1 residues 79-116 [SEQ ID NO: 5] Peptide construct Sequence
145 GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 5 133
Pam2Cys-SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 25 113
Pam1Cys(NH2)-SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 25 114
Pam1Cys(Ac)-SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 25
Peptide epitope NY-ESO-1 residues 118-143 [SEQ ID NO: 12] Patent
Designation Sequence 147 VPGVLLKEFTVSGNILTIRLTAADHR-OH 12 134
Pam2Cys-SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 115
Pam1Cys(NH2)-SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26 116
Pam1Cys(Ac)-SKKKKVPGVLLKEFTVSGNILTIRLTAADHR-OH 26
[0905] General Methods
[0906] T Cell Clone Maintenance
[0907] Human CD4+ and CD8+ T cell clones were maintained at a
concentration of 1-3.times.10.sup.6/ml in RPMI 1640 supplemented
with Glutamax, Penicillin/Streptomycin and 5% v/v human serum
("RS5", all reagents from Life Technologie). Media was refreshed
50% v/v twice weekly. RS5 for CD4+ T cell clones was supplemented
with IL-7 and IL-21, and RS5 for CD8+ T cell clones was
supplemented with IL-7+IL-15 (all cytokines from PeproTech, used at
a final concentration of 5 ng/ml).
[0908] Lymphoblastoid Cell Line ("LCL") Generation and
Maintenance
[0909] Epstein-Barr Virus ("EBV")-producing B95-8 MM cells were
grown in RPMI 1640 supplemented with Glutamax,
Penicillin/Streptomycin and 10% v/v fetal bovine serum ("RF10", all
reagents from Invitrogen) in T75 flasks (BD Biosciences).
EBV-containing medium was collected and centrifuged to pellet
non-adherent cells, and the EBV-containing supernatant then
filter-sterilised (0.22 .mu.m). 10.times.10.sup.6 donor Peripheral
Blood Mononuclear Cells ("PBMC") were suspended in 1.5 ml RF10 in
aT25 flask (BD Biosciences). 2.5 ml B95-8 cell culture supernatant
was added and cells incubated for 2 hours. 1 ml fresh RF10
containing 5% v/v Phytohemaglutanin ("PHA", Gibco) was then added
(1% v/v final concentration). Flasks were monitored for outgrowth
of B cell LCL, typically over 4-6 weeks, with transfer to a T75
flask (BD Biosciences) when required. LCL lines were cryopreserved
and working stocks maintained in RF10 at
0.5-1.5.times.10.sup.6/ml.
[0910] Culture of Monocyte-Derived Dendritic Cells ("MoDC")
[0911] Blood was taken from healthy donors with informed consent.
PBMC were purified by density centrifugation using Lymphoprep (Axis
Shield). Monocytes were isolated from PBMC using the Monocyte
Isolation Kit II (Miltenyi Biotec) and MoDC were generated by
culture in RF10 supplemented with 100 ng/ml GM-CSF and 50 ng/ml
IL-4 (both Peprotech) for 6 days. Medium was refreshed 50% v/v
every two days.
[0912] CD8+ T Cell Done Activation Assays
[0913] Donor LCL or MoDC (also referred to herein as antigen
presenting cells or "APC") were incubated for 16h in RF10+
peptide/construct at desired concentration. In samples where
Pam3Cys ("P3C") and peptide moieties were both present but
unconjugated, LCL were pre-treated with P3C for 30 minutes, and
media then refreshed with RF10 containing both P3C and peptide.
Untreated APC were incubated in RF10 only. APC/construct incubation
was performed in 96well plates (U-bottom, BD Biosciences) or in
48wp (flat bottom, BD Biosciences) depending of the nature of the
assay and the numbers of APC required per treatment. Following
incubation, APC were washed with RPMI 1640 4 times, to remove
unbound construct/P3C.
[0914] To enable flow cytometric detection, CD8+ T cell clones were
pre-stained with 0.5 .mu.M CellTrace.TM. Violet ("CTV") (Life
Technologies) following manufacturer's protocols prior to seeding
into APC wells, or were stained with anti-CD8 antibody following
co-incubation as described below.
[0915] Pulsed/washed APC and CTV-stained T cell clones were seeded
in 96well plate wells (U-bottom) at a ratio of 4:1 (APC:T cell) in
duplicate (typical numbers of cells used were 1.25.times.10.sup.4
cells/ml T cells and 5.times.10.sup.4 cells/ml APC). Following
seeding, APC/T cell plates were gently centrifuged
(.ltoreq.300.times.g, 3 minutes) to allow immediate interaction,
and co-incubated for 26 hours in a standard cell culture
incubator.
[0916] To detect T cell activation, samples were stained with the
antibodies shown in Table 6 below (anti-CD8 only used if cells not
pre-stained with CTV) in 50 .mu.l total volume of wash buffer
(PBS/FCS (1% v/v).
TABLE-US-00015 TABLE 6 Antibodies for CD8+ assay DOSE ANTI- FLUORO-
(.mu.L PER CD PHORE CLONE 50 .mu.L) SUPPLIER CATALOG# 8 FITC RPA-T8
0.5 BIOLEGEND 301006 137 APC 4B4.1 1.25 BIOLEGEND 309810
[0917] Samples were incubated on ice for 30 minutes in the dark,
and then washed twice with wash buffer to remove unbound antibody.
DAPI (1 .mu.g/ml final concentration) was added to each sample
immediately prior to acquisition to allow live/dead exclusion.
[0918] Data acquisition was performed using a BD FACSAria II with
FACSDiva software, and data analysis was performed using FlowJo
software (Treestar). Data were presented as the mean %+SD of live
clonal cells positive for CD137 expression.
[0919] CD4+ T Cell Done Activation Assays
[0920] All APC pulsing/washing/seeding; T cell clone CTV staining;
and APC/T cell co-incubation setup steps were performed as
described for "CD8+ T cell clone activation assays" above.
[0921] To detect T cell activation, samples were stained with the
antibodies shown in Table 7 below (anti-CD4 only used if cells not
pre-stained with CTV) in 50 .mu.l total volume of wash buffer
(PBS/FCS (1% v/v).
TABLE-US-00016 TABLE 7 Antibodies for CD4+ assay DOSE FLUORO-
(.mu.L PER CD PHORE CLONE 50 .mu.L) SUPPLIER CATALOG# 4 FITC RPA-T4
0.5 BIOLEGEND 300506 25 APC- BC96 2.5 BIOLEGEND 302614 OR CY7 OR
302610 APC 134 PE ACT35 1.25 BIOLEGEND 350004
[0922] Samples were incubated on ice for 30 minutes in the dark,
and then washed twice with wash buffer to remove unbound antibody.
DAPI (1 pg/ml final concentration) was added to each sample
immediately prior to acquisition to allow live/dead exclusion.
[0923] Data acquisition was performed using a BD FACSAria II with
FACSDiva software, and data analysis was performed using FlowJo
software (Treestar). Data were presented as the median fluorescence
intensity (MFI)+SD of CD25 and CD134 expression on live clonal
cells.
Example 4
[0924] These examples describe an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of various antigenic
peptide constructs to various T cell clone by antigen presenting
cells (APC).
[0925] Materials
[0926] The peptide epitope recognised by the CD8+ T cell clone
(clone 4D9) used in this example, p495-503 [SEQ ID NO: 27], is
shown within the construct sequence in bold below:
TABLE-US-00017 142: [SEQ ID NO: 27] Ac-Cys-SKKKK-NLVPMVATVK(Ac)-NH2
131: [SEQ ID NO: 27] Pam2Cys-SKKKK-NLVPMVATVK(Ac)-NH2 110: [SEQ ID
NO: 27] Pam1Cys(Ac)-SKKKK-NLVPMVATVK(Ac)-NH2
[0927] MoDC preparation and CD8+ T cell clone activation assay
carried out as described in the methods below.
[0928] Results
[0929] As shown in FIG. 3, T cell activation was detected for all
peptide constructs at 10 .mu.M, but only for peptide constructs 131
[comprises SEQ ID NO: 27] and 110 [comprises SEQ ID NO: 27] at 100
nM.
[0930] At both concentrations, constructs 131 [comprises SEQ ID NO:
27] and 110 [comprises SEQ ID NO: 27] elicited greater T cell
activation than construct 142 [SEQ ID NO: 27]. These results
support the efficacy of the peptide conjugates of the invention
to--maintain and possibly improve presentation of this epitope when
conjugated to a TLR-agonist using the methods of the invention.
Example 5
[0931] This example describes an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of NY-ESO-1.sub.153-180
[SEQ ID NO: 18] constructs to a CD8+ T cell clone by autologous LCL
and by allogeneic HLA-A2+ HLA-DP4+ MoDC.
[0932] Materials
[0933] The peptide epitope recognised by the CD8+ T cell clone
(clone 2F2) used in this example, p157-165 [SEQ ID NO: 20], is
shown within the construct sequence in bold below:
TABLE-US-00018 143: [SEQ ID NO: 18 LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
144: [SEQ ID NO: 17] SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH 112:
[SEQ ID NO: 24] Pam1Cys(Ac)-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
111: [SEQ ID NO: 24]
Pam1Cys(NH2)-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH 132: [SEQ ID NO:
24] Pam2Cys-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
[0934] LCL/MoDC preparation and CD8+ T cell clone activation assay
carried out using the methods as described in Example 4.
[0935] Results
[0936] 100% T cell activation was elicited with all constructs
(+/-P3C) at 10 .mu.M using LCL (see FIG. 4). 50-100% T cell
activation was observed at 100 nM using constructs 143 [SEQ ID NO:
18] and 144 [SEQ ID NO: 17] (+/-exogenous P3C). Only peptide
construct 143 [SEQ ID NO: 18] elicited 100% T cell activation at
100 nM (FIG. 4, first columns), suggesting that the presence of the
N-terminal SKKKK [amino acids 2-6 of SEQ ID NO: 22] motif may have
impaired processing of epitope p157-165 [SEQ ID NO: 20]. Peptide
constructs 112 [comprises SEQ ID NO: 24], 111 [comprises SEQ ID NO:
24] and 132 [comprises SEQ ID NO: 24] elicited <20% T cell
activation at 100 nM, suggesting that although TLR agonist
conjugation still allowed presentation of the p157-165 epitope [SEQ
ID NO: 20] to CD8+ T cells, it did not improve the epitope's
processing and presentation.
[0937] As shown in FIG. 5,T cell activation was also detectable
when MoDC were used as APC, reaching levels of 55-95% at 10
.mu.M.
Example 6
[0938] This example describes an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of NY-ESO-1.sub.153-180
[SEQ ID NO: 18] antigenic peptide constructs to CD4+ T cell clones
by autologous LCL and by allogeneic HLA-A2+ HLA-DP4+ MoDC.
[0939] Materials
[0940] The peptide epitope recognised by the CD4+ T cell clones 1B7
and 1C11 used in this example, p157-170 [SEQ ID NO: 19], is shown
within the construct sequence in bold below:
TABLE-US-00019 143: [SEQ ID NO: 18] LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
144: [SEQ ID NO: 17] SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH 112:
[SEQ ID NO: 24] Pam1Cys(Ac)-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
111: [SEQ ID NO: 24]
Pam1Cys(NH2)-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH 132: [SEQ ID NO:
24] Pam2Cys-SKKKK-LQQLSLLMWITOCFLPVFLAQPPSGQRR-OH
[0941] LCL/MoDC preparation and CD4+ T cell clone activation assays
were carried out as described in the methods of Example 4.
[0942] Results
[0943] As can be seen in FIGS. 6-8, T cell activation (as
determined by an increase in CD25 expression over untreated
background) was detectable for all constructs at both 10 .mu.M and
100 nM when LCL (clone 1B7 only, FIG. 6) or MoDC (clone 1B7, FIG. 7
and clone 1C11, FIG. 8) were used. Autologous LCL appeared to
elicit higher levels of CD25 upregulation than allogeneic MoDC, as
shown by a comparison of FIG. 6 compared to each of FIGS. 7 and
8.
[0944] TLR agonist conjugation gave equivalent epitope processing
and presentation on MHC class II when compared to free peptide for
this epitope (p157-170 [SEQ ID NO: 20]). These results confirm that
conjugation with a TLR agonist using the methods of the invention
maintains antigen processing and presentation to CD4+ T cells.
[0945] The combined results of FIGS. 4-8 show that conjugation with
a TLR agonist using the methods of the invention retains processing
and presentation of epitopes within the peptide antigen
NY-ESO-1153-180 [SEQ ID NO: 18] to both CD4+ and CD8+ human T
cells.
Example 7
[0946] This example describes an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of
NY-NY-ESO-1.sub.79-116 [SEQ ID NO: 5] constructs to CD8+ T cell
clones by autologous LCL.
[0947] Materials
[0948] The peptide epitope recognised by the CD8+ T cell clone 1D7
used in this example, p92-100 [SEQ ID NO: 6], is shown within the
construct sequence in bold below:
TABLE-US-00020 145: [SEQ ID NO: 5]
GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 114: [SEQ ID NO: 25]
Pam1Cys(Ac)-SKKKK-GARGPESRLLEFYLAMPFATPMEAELARRS LAQDAPPL-OH 113:
[SEQ ID NO: 25] Pam1Cys(NH2)-SKKKK-GARGPESRLLEFYLAMPFATPMEAELARR
SLAQDAPPL-OH
[0949] The peptide epitope recognised by the CD8+ T cell clone 1F10
used in this example, p96-104 [SEQ ID NO: 7], is shown within the
construct sequence in bold below:
TABLE-US-00021 145: [SEQ ID NO: 5]
GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL-OH 114: [SEQ ID NO: 25]
Pam1Cys(Ac)-SKKKK-GARGPESRLLEFYLAMPFATPMEAELARRS LAQDAPPL-OH 113:
[SEQ ID NO: 25] Pam1Cys(NH2)-SKKKK-GARGPESRLLEFYLAMPFATPMEAELARR
SLAQDAPPL-OH
[0950] LCL preparation and CD8+ T cell activation assays were
carried out as described in the methods of Example 4. LCL pulsed
with 100 nM minimal peptide epitope acted as positive control (data
not shown).
[0951] Results
[0952] Clone 1D7 (p92-100) [SEQ ID NO: 6] responded to all
constructs at all concentrations tested (see FIG. 9). Responses to
peptide constructs 114 [comprises SEQ ID NO: 25] and 113 [comprises
SEQ ID NO: 25] were slightly inferior to those with peptide
construct 145 [SEQ ID NO: 5]+/-P3C at matched concentrations,
particularly at lpM/100 nM.
[0953] Clone 1F10 (p96-104) [SEQ ID NO: 7] responded to all
constructs at all concentrations tested (FIG. 10). Responses to
peptide constructs 114 [comprises SEQ ID NO: 25] and 113 [comprises
SEQ ID NO: 25] were equivalent to, or slightly better than those
with peptide construct 145 [SEQ ID NO: 5]+/-P3C, especially at 10
.mu.M (best demonstrated by an increase in CD137 MFI; data not
shown).
[0954] These results establish that after conjugation with a TLR
agonist using the methods of the invention, peptide NY-ESO-17-116
[SEQ ID NO: 5] retains its ability to be processed and presented to
two different CD8+ T cell lines recognizing two different epitopes
(p92-100 [SEQ ID NO: 6] and p96-104 [SEQ ID NO: 7]).
Example 8
[0955] This example describes an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of
NY-NY-ESO-1.sub.118-143 [SEQ ID NO: 12] constructs to the CD8+ T
cell clone 1C11 by autologous LCL.
[0956] Materials
[0957] The peptide epitope recognised by the CD8+ T cell clone 1C11
used in this example, p125-133 [SEQ ID NO: 13], is shown within the
construct sequence in bold below:
TABLE-US-00022 147: [SEQ ID NO: 12] VPGVLLKEFTVSGNILTIRLTAADHR-OH
116: [SEQ ID NO: 26]
Pam1Cys(Ac)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH 115: [SEQ ID NO:
26] Pam1Cys(NH2)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH
[0958] LCL preparation and CD8+ T cell activation assays were
carried out as described in the methods of Example 4 above. LCL
pulsed with 1 .mu.M NY-ESO-1.sub.121-138 [amino acids 4-21 of SEQ
ID NO: 12] acted as positive control (data not shown).
[0959] Results
[0960] As shown in FIG. 11, greater than 95% T cell activation was
elicited with all constructs (+/-P3C) at 10 .mu.M. At 1 .mu.M, T
cell activation by peptide construct 147 [SEQ ID NO:
12](+/-exogenous P3C) dropped to 75%/60% respectively, but remained
at >90% for peptide constructs 116 [comprises SEQ ID NO:26] and
115 [comprises SEQ ID NO:26]. No or negligible T cell activation
was observed for peptide construct 147 [SEQ ID NO: 12]at 100 nM,
whereas peptide constructs 116 [comprises SEQ ID NO:26] and 115
[comprises SEQ ID NO:26] elicited >30% and 20% respectively.
[0961] The superiority of peptide constructs 116 [comprises SEQ ID
NO:26] and 115 [comprises SEQ ID NO:26] at inducing T cell
activation suggests that conjugation to a TLR-agonist improves
processing and -presentation of this epitope (p125-133) [SEQ ID NO:
13].
Example 9
[0962] This example describes an assessment of the efficacy of the
peptides and peptide conjugates of the present invention in
supporting the processing and presentation of
NY-NY-ESO-1.sub.118-143 [SEQ ID NO: 12] constructs to CD4+ T cell
clones 1E4 and 1D6 by autologous LCL.
[0963] Materials
[0964] The peptide constructs used in this example are shown
below:
TABLE-US-00023 147: [SEQ ID NO: 12] VPGVLLKEFTVSGNILTIRLTAADHR-OH
116: [SEQ ID NO: 26]
Pam1Cys(Ac)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH 115: [SEQ ID NO:
26] Pam1Cys(NH2)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH
[0965] The putative peptide epitope recognised by the CD4+ clone
1E4 used in this example, p127-138 [amino acids 10-21 of SEQ ID NO:
12], is shown within the construct sequence in bold below:
TABLE-US-00024 147: [SEQ ID NO: 12] VPGVLLKEFTVSGNILTIRLTAADHR-OH
116: [SEQ ID NO: 26]
Pam1Cys(Ac)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH 115: [SEQ ID NO:
26] Pam1Cys(NH2)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH
[0966] The putative peptide epitope recognised by the CD4+ clone
1D6 used in this example, p121-132 [amino acids 4-15 of SEQ ID NO:
12], is shown within the construct sequence in bold below:
TABLE-US-00025 147: [SEQ ID NO: 12] VPGVLLKEFTVSGNILTIRLTAADHR-OH
116: [SEQ ID NO: 26]
Pam1Cys(Ac)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH 115: [SEQ ID NO:
26] Pam1Cys(NH2)-SKKKK-VPGVLLKEFTVSGNILTIRLTAADHR-OH
[0967] LCL preparation and CD4+ T cell activation assays were
carried out as described in the methods of Example 4 above. LCL
pulsed with 1 .mu.M NY-ESO-1.sub.121-138 [amino acids 4-21 of SEQ
ID NO: 12] acted as positive control (data not shown).
[0968] Results
[0969] Clone 1E4 responded to all constructs (+/-exogenous P3C) at
all concentrations tested, as can clearly be seen in FIG. 12. These
results suggest that the processing of the cognate epitope for this
clone from p118-143 [SEQ ID NO: 12] is efficient, and is not
dependent on or influenced by TLR agonist ligation.
[0970] As shown in FIG. 13, clone 1D6 responds well to LCL pulsed
with p121-138 [amino acids 4-21 of SEQ ID NO: 12] (data not shown),
but does not respond to peptide construct 147 (p118-143 [SEQ ID NO:
12])+/-P3C, suggesting impairment in processing of its cognate
epitope. It is proposed that this may be either due to the presence
of N-terminal VPG or C-terminal TAADHR, in the absence of TLR
agonist conjugation. This processing defect is not rescued by
concurrent TLR ligation in trans.
[0971] However, clone 1D6 does respond to peptide constructs 116
[comprises SEQ ID NO:26] and 115 [comprises SEQ ID NO:26] (with 116
[comprises SEQ ID NO:26] eliciting a greater response than 115
[comprises SEQ ID NO:26]) in a titratable fashion (see FIG. 13).
This suggests that efficient targeting of the peptide moiety to a
late endosomal/lysosomal pathway through conjugation to a TLR
agonist alleviates the processing block observed with the
non-conjugated peptide construct 147 [SEQ ID NO: 12]. Without
wishing to be bound by any theory, applicants propose this may be
at least partly by exposure to a late endosome or lysosome-specific
protease or processing pathway.
Example 10
[0972] The example investigates TLR agonism by the peptide
constructs of the present invention.
[0973] Methods
[0974] The TLR assays were carried out as described below, using
the constructs set out in the preceding Examples.
[0975] Toll-Like Receptor 2 (TLR2) Agonism Using HekBlue Cells
[0976] HEK-Blue.TM.-hTLR2 and HEK-Blue.TM.-mTLR2 were purchased
from Invivogen. These HEK-Blue cells were produced by
co-transfection of both reporter gene SEAP (secreted embryonic
alkaline phosphatase) and either human or murine TLR2,
respectively. The SEAP reporter gene is under the control of the
IFN-B minimal promoter fused to five AP-1 and five NFkB binding
sites. Cells were cultured according to manufacturer's
instructions. On the day of the assay, the constructs were added at
the indicated concentrations in 20 .mu.l volume of endotoxin free
water in a 96-well plate. HEK-Blue.TM.-hTLR2 or HEK-Blue.TM.-hTLR2
cells were resuspended at .about.2.83.times.10.sup.4 cells/ml in
HEK-Blue.TM. Detection medium and immediately add 180 ml of the
cell suspension (.about.5.times.10.sup.4 cells per well.) The cells
were incubated overnight at 37.degree. C. in 5% CO.sub.2. SEAP
expression was quantified using an EnSpire plate reader
(PerkinElmer) at 635 nM.
[0977] Method for Detection of IL-8 Secretion from TLR2-Transiently
Transfected Hek293 Cells
[0978] Hek-293 cells were plated 3.times.10.sup.4 cells in 50 .mu.l
per well in 96-well plate with DMEM containing 10% FBS (the medium
was not supplemented with antibiotics). Cells were transfected with
either a combination of pFLAG-TLR2 plasmid and pcDNA3.1 (a kind
gift from Shimizu, as reported in Shimizu, T., Y. Kida and K.
Kuwano (2005). "A dipalmitoylated lipoprotein from Mycoplasma
pneumoniae activates NF-kappa B through TLR1, TLR2, and TLR6." J
Immunol 175(7): 4641-4646), or the control plasmid only (pcDNA3.1).
Master mix of Lipofectamine/DNA complexes were constituted in
Opti-MEM at 100ngDNA in 0.3 .mu.l Lipofectamine in a volume of 50p1
per sample. Following an incubation of 20 mins, the plasmid mix was
added to the cells. Protein expression was induced for 24 hours
prior to the addition of constructs.
[0979] The constructs were added to the wells at the indicated
concentrations to make a final volume of 200p1 per well. Following
18-hours of stimulation, the supernatant was harvested from each
sample and stored at -20.degree. C. until required. IL-8 secretion
was determined by Cytometric Bead Array (BD Biosciences) according
to manufacturer's protocol with only one modification: 25p1 of
conditioned medium was used instead of 50p1. To accurately
determine the concentration of secreted IL-8, an 11-point standard
curve (1-5000 ng/ml) was performed. Samples were analysed using a
BD-FACS Aria II (BD Biosciences) and the data analysed using FCAP
ARRAY Software and (version 1.0.1)
[0980] Result
[0981] As shown in FIGS. 14 and 15, all constructs tested
demonstrated TLR agonism in both HekBlue.TM. (FIG. 14) and IL-8
(FIG. 15) reporter systems. TLR agonism was titratable, and was
detectable above background at both concentrations tested.
[0982] In both assay systems, Pam1C(Ac) constructs elicited
stronger responses than matched Pam1C(NH.sub.2) constructs.
[0983] It is not the intention to limit the scope of the invention
to the abovementioned examples only. As would be appreciated by a
skilled person in the art, many variations are possible without
departing from the scope of the invention.
Sequence CWU 1
1
27158PRTArtificialSynthetic peptidesMISC_FEATURE(1)..(1)X1 is
absent or SMISC_FEATURE(2)..(2)X2 is absent or a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or a hydrophilic
residueMISC_FEATURE(13)..(13)X13 is absent or a hydrophilic
residueMISC_FEATURE(14)..(14)X14 is absent or a hydrophilic
residueMISC_FEATURE(15)..(15)X15 is absent or a hydrophilic
residueMISC_FEATURE(16)..(16)X16 is absent or a hydrophilic
residueMISC_FEATURE(17)..(17)X17 is absent or a hydrophilic
residueMISC_FEATURE(18)..(18)X18 is absent or a hydrophilic
residueMISC_FEATURE(19)..(19)X19 is absent or a hydrophilic
residueMISC_FEATURE(20)..(20)X20 is absent or a hydrophilic residue
1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
10 15Xaa Xaa Xaa Xaa Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu
Phe 20 25 30Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu
Ala Arg 35 40 45Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu 50
55250PRTArtificialSynthetic peptidesMISC_FEATURE(1)..(1)X1 is
absent or SMISC_FEATURE(2)..(2)X2 is absent or a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or a hydrophilic residue
2Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ala Arg Gly1 5
10 15Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala
Thr 20 25 30Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp
Ala Pro 35 40 45Pro Leu 50343PRTArtificialSynthetic
peptidesMISC_FEATURE(1)..(1)X1 is absent or is
SMISC_FEATURE(2)..(2)X2 is absent or a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or a hydrophilic residue
3Xaa Xaa Xaa Xaa Xaa Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu1 5
10 15Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu
Ala 20 25 30Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu 35
40443PRTArtificialSynthetic peptides 4Ser Lys Lys Lys Lys Gly Ala
Arg Gly Pro Glu Ser Arg Leu Leu Glu1 5 10 15Phe Tyr Leu Ala Met Pro
Phe Ala Thr Pro Met Glu Ala Glu Leu Ala 20 25 30Arg Arg Ser Leu Ala
Gln Asp Ala Pro Pro Leu 35 40538PRTArtificialSynthetic peptides
5Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met1 5
10 15Pro Phe Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu
Ala 20 25 30Gln Asp Ala Pro Pro Leu 3569PRTArtificialSynthetic
peptides 6Leu Ala Met Pro Phe Ala Thr Pro Met1
579PRTArtificialSynthetic peptides 7Phe Ala Thr Pro Met Glu Ala Glu
Leu1 5846PRTArtificialSynthetic peptidesMISC_FEATURE(1)..(1)X1 is
absent or is SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or is a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or is a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or is a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or is a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or is a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or is a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or is a hydrophilic
residueMISC_FEATURE(13)..(13)X13 is absent or is a hydrophilic
residueMISC_FEATURE(14)..(14)X14 is absent or is a hydrophilic
residueMISC_FEATURE(15)..(15)X15 is absent or is a hydrophilic
residueMISC_FEATURE(16)..(16)X16 is absent or is a hydrophilic
residueMISC_FEATURE(17)..(17)X17 is absent or is a hydrophilic
residueMISC_FEATURE(18)..(18)X18 is absent or is a hydrophilic
residueMISC_FEATURE(19)..(19)X19 is absent or is a hydrophilic
residueMISC_FEATURE(20)..(20)X20 is absent or is a hydrophilic
residue 8Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Val Pro Gly Val Leu Leu Lys Glu Phe
Thr Val Ser 20 25 30Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp
His Arg 35 40 45938PRTArtificialSynthetic
peptidesMISC_FEATURE(1)..(1)X1 is absent or is
SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or is a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or is a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or is a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or is a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or is a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or is a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or is a hydrophilic
residue 9Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Pro
Gly Val1 5 10 15Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr
Ile Arg Leu 20 25 30Thr Ala Ala Asp His Arg
351031PRTArtificialSynthetic peptidesMISC_FEATURE(1)..(1)X1 is
absent or is SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic residue
10Xaa Xaa Xaa Xaa Xaa Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val1
5 10 15Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg
20 25 301131PRTArtificialSynthetic peptides 11Ser Lys Lys Lys Lys
Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val1 5 10 15Ser Gly Asn Ile
Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg 20 25
301226PRTArtificialSynthetic peptides 12Val Pro Gly Val Leu Leu Lys
Glu Phe Thr Val Ser Gly Asn Ile Leu1 5 10 15Thr Ile Arg Leu Thr Ala
Ala Asp His Arg 20 25139PRTArtificialSynthetic peptides 13Glu Phe
Thr Val Ser Gly Asn Ile Leu1 51448PRTArtificialSynthetic
peptidesMISC_FEATURE(1)..(1)X1 is absent or is
SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or is a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or is a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or is a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or is a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or is a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or is a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or is a hydrophilic
residueMISC_FEATURE(13)..(13)X13 is absent or is a hydrophilic
residueMISC_FEATURE(14)..(14)X14 is absent or is a hydrophilic
residueMISC_FEATURE(15)..(15)X15 is absent or is a hydrophilic
residueMISC_FEATURE(16)..(16)X16 is absent or is a hydrophilic
residueMISC_FEATURE(17)..(17)X17 is absent or is a hydrophilic
residueMISC_FEATURE(18)..(18)X18 is absent or is a hydrophilic
residueMISC_FEATURE(19)..(19)X19 is absent or is a hydrophilic
residueMISC_FEATURE(20)..(20)X20 is absent or is a hydrophilic
residue 14Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Leu Gln Gln Leu Ser Leu Leu Met Trp
Ile Thr Gln 20 25 30Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser
Gly Gln Arg Arg 35 40 451540PRTArtificialSynthetic
peptidesMISC_FEATURE(1)..(1)X1 is absent or is
SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic
residueMISC_FEATURE(6)..(6)X6 is absent or is a hydrophilic
residueMISC_FEATURE(7)..(7)X7 is absent or is a hydrophilic
residueMISC_FEATURE(8)..(8)X8 is absent or is a hydrophilic
residueMISC_FEATURE(9)..(9)X9 is absent or is a hydrophilic
residueMISC_FEATURE(10)..(10)X10 is absent or is a hydrophilic
residueMISC_FEATURE(11)..(11)X11 is absent or is a hydrophilic
residueMISC_FEATURE(12)..(12)X12 is absent or is a hydrophilic
residue 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Gln
Gln Leu1 5 10 15Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu Pro Val
Phe Leu Ala 20 25 30Gln Pro Pro Ser Gly Gln Arg Arg 35
401633PRTArtificialSynthetic peptidesMISC_FEATURE(1)..(1)X1 is
absent or is SMISC_FEATURE(2)..(2)X2 is absent or is a hydrophilic
residueMISC_FEATURE(3)..(3)X3 is absent or is a hydrophilic
residueMISC_FEATURE(4)..(4)X4 is absent or is a hydrophilic
residueMISC_FEATURE(5)..(5)X5 is absent or is a hydrophilic residue
16Xaa Xaa Xaa Xaa Xaa Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr1
5 10 15Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln
Arg 20 25 30Arg1733PRTArtificialSynthetic peptides 17Ser Lys Lys
Lys Lys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr1 5 10 15Gln Cys
Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln Arg 20 25
30Arg1828PRTArtificialSynthetic peptides 18Leu Gln Gln Leu Ser Leu
Leu Met Trp Ile Thr Gln Cys Phe Leu Pro1 5 10 15Val Phe Leu Ala Gln
Pro Pro Ser Gly Gln Arg Arg 20 251914PRTArtificialSynthetic
peptides 19Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu Pro Val Phe1
5 10209PRTArtificialSynthetic peptides 20Ser Leu Leu Met Trp Ile
Thr Gln Cys1 52115PRTArtificialSynthetic peptides 21Cys Ser Lys Lys
Lys Lys Asn Leu Val Pro Met Val Ala Thr Val1 5 10
15226PRTArtificialSynthetic peptides 22Cys Ser Lys Lys Lys Lys1
52317PRTArtificialSynthetic peptides 23Cys Ser Lys Lys Lys Val Lys
Asn Leu Val Pro Met Val Ala Thr Val1 5 10
15Lys2434PRTArtificialSynthetic peptides 24Cys Ser Lys Lys Lys Lys
Leu Gln Gln Leu Ser Leu Leu Met Trp Ile1 5 10 15Thr Gln Cys Phe Leu
Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln 20 25 30Arg
Arg2544PRTArtificialSynthetic peptides 25Cys Ser Lys Lys Lys Lys
Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu1 5 10 15Glu Phe Tyr Leu Ala
Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu 20 25 30Ala Arg Arg Ser
Leu Ala Gln Asp Ala Pro Pro Leu 35 402632PRTArtificialSynthetic
peptides 26Cys Ser Lys Lys Lys Lys Val Pro Gly Val Leu Leu Lys Glu
Phe Thr1 5 10 15Val Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala
Asp His Arg 20 25 302716PRTArtificialSynthetic peptides 27Cys Ser
Lys Lys Lys Lys Asn Leu Val Pro Met Val Ala Thr Val Lys1 5 10
15
* * * * *