U.S. patent application number 15/144397 was filed with the patent office on 2016-08-18 for site-specific chemoenzymatic protein modifications.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is NOVARTIS AG. Invention is credited to Jennifer Cobb, Zachary Robinson, Aimee Usera.
Application Number | 20160237116 15/144397 |
Document ID | / |
Family ID | 51298955 |
Filed Date | 2016-08-18 |
United States Patent
Application |
20160237116 |
Kind Code |
A1 |
Usera; Aimee ; et
al. |
August 18, 2016 |
SITE-SPECIFIC CHEMOENZYMATIC PROTEIN MODIFICATIONS
Abstract
The present invention relates to methods and reagents for use in
site-selective modification of proteins having lysine residues with
functionalized peptides using a chemoenzymatic microbial
transglutaminase-mediated reaction. The functionalized proteins may
be used for study or therapeutic uses.
Inventors: |
Usera; Aimee; (Cambridge,
MA) ; Robinson; Zachary; (Arlington, MA) ;
Cobb; Jennifer; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVARTIS AG |
Basel |
|
CH |
|
|
Assignee: |
Novartis AG
|
Family ID: |
51298955 |
Appl. No.: |
15/144397 |
Filed: |
May 2, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14329758 |
Jul 11, 2014 |
9359400 |
|
|
15144397 |
|
|
|
|
62016044 |
Jun 23, 2014 |
|
|
|
61845273 |
Jul 11, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6415 20170801;
A61P 37/04 20180101; A61K 49/0002 20130101; C07C 271/22 20130101;
A61K 38/05 20130101; C07D 229/02 20130101; C07K 7/02 20130101; C07K
5/0808 20130101; C07C 2602/24 20170501; A61K 38/00 20130101; A61K
38/07 20130101; C07D 209/42 20130101; C07K 5/06043 20130101; C07C
317/28 20130101; C07D 257/08 20130101; C07K 5/101 20130101; C07K
1/13 20130101; A61K 38/06 20130101; C07K 5/06104 20130101; C07K
1/1075 20130101 |
International
Class: |
C07K 5/103 20060101
C07K005/103; A61K 47/48 20060101 A61K047/48; C07K 5/062 20060101
C07K005/062; A61K 49/00 20060101 A61K049/00; C07K 5/083 20060101
C07K005/083 |
Claims
1. A method for modifying a protein, comprising: providing a target
protein having at least one lysine residue; contacting the target
protein with a modifying compound having the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z in the
presence of a microbial transglutaminase to form a modified
protein; wherein x is 0 or 1; y is 0 or 1; z is 0 or 1; R.sup.1 is
selected from the group consisting of: acetyl, ##STR00135## wherein
R.sup.4 is selected from --H, --N.sub.3, and ##STR00136## W is
selected from: C.sub.1-C.sub.6 linear or branched alkyl or
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu; A is absent or selected from --O--, --NH--,
and --S--; B is absent or selected from --O--, --C(O)--, --NH--,
--C(O)NH--, --NHC(O)--, --NHC(O)O--, --OC(O)NH--, --OC(O)O--,
--C.dbd.N(OH)--, --S(O.sub.2)--, --NHS(O.sub.2)--,
--S(O.sub.2)NH--, --S(O)--, --NHS(O)--, --S(O)NH--; --C(O)O--,
--OC(O)--, --S--, .dbd.NH--O--, .dbd.NH--NH-- and
.dbd.NH--N(C.sub.1-C.sub.20alkyl)-; R.sup.2 is selected from the
group consisting of: a fatty acid, linear or branched
C.sub.1-C.sub.3 alkyl-N.sub.3, cyclooctynyl, fluorophore,
polysaccharide, --CH(OCH.sub.3).sub.2, ##STR00137## ##STR00138##
each n is an integer independently selected from 0 to 6; each Q is
selected from H and NO.sub.2.
2-97. (canceled)
98. A conjugate of a compound of the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-A-W--B--R.sup.2 wherein x is 0
or 1 is 0 or 1 z is 0 or 1; R.sup.1 is selected from the group
consisting of: acetyl, ##STR00139## wherein R.sup.4 is selected
from --H, --N.sub.3, and ##STR00140## W is selected from:
C.sub.1-C.sub.6 linear or branched alkyl or polyethylene glycol
having a molecular weight of between about 40 and about 80,000 amu;
A is absent or selected from --O--, --NH-- and --S--; B is absent
or selected from --O--, --C(O)--, --NH--, --C(O)NH--, --NHC(O)--,
--NHC(O)O--, --OC(O)NH--, --OC(O)O--, --C.dbd.N(OH)--, --S(O2)-,
--NHS(O.sub.2)--, --S(O.sub.2)NH--, --S(O)--, --NHS(O)--,
--S(O)NH--; --C(O)O--, --OC(O)--, --S--, .dbd.NH--O--,
.dbd.NH--NH-- and .dbd.NH--N(C.sub.1-C.sub.20alkyl); R.sup.2 is
selected from the group consisting of: a fatty acid, linear or
branched C.sub.1-C.sub.3 alkyl-N.sub.3, cyclooctynyl, fluorophore,
polysaccharide, --CH(OCH.sub.3).sub.2, ##STR00141## ##STR00142##
each n is an integer independently selected from 0 to 6; and each Q
is selected from H and --NO.sub.2.
99. The conjugate claim 98, wherein the conjugate protein is
CRM.sub.197.
100. The conjugate of claim 98, wherein the conjugate protein is
GBS.sub.80.
101. A vaccine comprising a conjugate of claim 98.
102. A conjugate of a compound of claim 97.
103. A vaccine comprising a conjugate of claim 99.
104. A vaccine comprising a conjugate of claim 100.
105. A therapeutic protein comprising a compound of claim 96.
106. A therapeutic protein comprising a compound of claim 97.
107. An imaging agent comprising a compound of claim 96.
108. An imaging agent comprising a compound of claim 97.
109. A labeling tool comprising a compound of claim 96.
110. A labeling tool comprising a compound of claim 97.
111. The method of claim 1, wherein R.sup.1 is selected from the
group consisting of: ##STR00143##
112. The conjugate of claim 97, wherein R.sup.1 is selected from
the group consisting of: ##STR00144##
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a novel method of
introducing modifying groups to a protein. In particular, the
present invention relates to the selective derivation of lysine
residues in proteins using a chemoenzymatic microbial
transglutaminase-mediated reaction for modifying proteins and
methods for their preparation and use.
BACKGROUND
[0002] It is well-known that the properties and characteristics of
proteins may be modified by conjugating groups to the protein. For
example, U.S. Pat. No. 4,179,337 disclosed proteins conjugated to
polyethylene or polypropylene glycols. Generally, such conjugation
generally requires some functional group in the protein to react
with another functional group in a conjugating group. Amino groups,
such as the N-terminal amino group or the .epsilon.-amino group in
lysine residues have been used in combination with suitable
acylating reagents for this purpose. It is often desired or
necessary to control the conjugation reaction, such as where the
conjugating compounds are attached to the protein and to control
how many conjugating groups are attached. This is often referred to
as specificity or selectivity.
[0003] Site-specific modification of proteins is a longstanding
challenge in the pharmaceutical and biotechnology arts. The classic
methods oftentimes lead to non-specific labeling (e.g. NHS Lys
labeling) or require engineering (e.g. maleimide Cys labeling or
unnatural amino acids). In addition, the repertoire of selective
chemical reactions, however, is very limited. One alternative is,
by recombinant methods, to introduce special unnatural amino acids
having a unique reactivity and then exploit this reactivity in the
further derivatization. Another alternative is the use of enzymes
which recognize structural and functional features of the protein
to be modified. An example of this is the use of microbial
transglutaminase (mTGase) to selectively modify Gln residues in
growth hormone. Other documents disclose the use of
transglutaminase to alter the properties of physiologically active
proteins. See e.g. EP 950 665, EP 785 276 and Sato, Adv. Drug
Delivery Rev., 54, 487-504 (2002), which disclose the direct
reaction between proteins comprising at least one Gln and
amine-functionalized PEG or similar ligands in the presence of
transglutaminase; see also Wada in Biotech. Lett., 23, 1367-1372
(2001), which discloses the direct conjugation of P-lactoglobulin
with fatty acids by means of transglutaminase. The reaction
catalyzed by the transglutaminase is a transamidation reaction in
which the primary amide of the glutamine residue is converted to a
secondary amide from a primary amine present in the reaction
mixture.
[0004] The selective derivatization of proteins remains a very
difficult task; the derivatization of lysines in a protein by
acylation is an even more inherently non-selective process. Thus,
there is at present no efficient method for the selective
derivatization of lysine residues. Accordingly, there is a need in
the art for methods of selectively derivatizing amino acid residues
such as lysine in proteins or polypeptides.
SUMMARY
[0005] In one aspect, a method for modifying a protein is
disclosed. The method permits site selective modifications. The
method includes providing a target protein having at least one
lysine residue; contacting the target protein with a modifying
compound having the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z in the
presence of a microbial transglutaminase to form a modified
protein;
wherein x is 0 or 1; y is 0 or 1; z is 0 or 1; R.sup.1 is selected
from the group consisting of: acetyl,
##STR00001##
wherein each R.sup.4 is selected from --H, --N.sub.3, and
##STR00002##
W is selected from: C.sub.1-C.sub.6 linear or branched alkyl or
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu; A is absent or selected from --O--, --NH--,
and --S--; B is absent or selected from --O--, --C(O)--, --NH--,
--C(O)NH--, --NHC(O)--, --NHC(O)O--, --OC(O)NH--, --OC(O)O--,
--C.dbd.N(OH)--, --S(O.sub.2)--, --NHS(O.sub.2)--,
--S(O.sub.2)NH--, --S(O)--, --NHS(O)--, --S(O)NH--; --C(O)O--,
--OC(O)--, --S--, .dbd.NH--O--, .dbd.NH--NH-- and
.dbd.NH--N(C.sub.1-C.sub.20alkyl)-; R.sup.2 is selected from the
group consisting of: a fatty acid, linear or branched
C.sub.1-C.sub.3 alkyl-N.sub.3, cyclooctynyl, fluorophore,
polysaccharide, --CH(OCH.sub.3).sub.2,
##STR00003## ##STR00004##
each n is an integer independently selected from 0 to 6; each Q is
selected from H and --NO.sub.2.
[0006] In some embodiments, the method also includes controlling
the pH environment of the target protein to a pH greater than 7;
and contacting the modified protein with a molecule having a
cysteine residue.
[0007] In some embodiments, the molecule having cysteine residue is
N.sup.5--((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glu-
tamine.
[0008] In some embodiments, microbial transglutaminase is Ajinomoto
microbial transgluaminase TI. In some embodiments, the protein is a
carrier protein. In some embodiments, the protein is CRM.sub.197.
In some embodiments, the protein is selected from: bacterial toxin,
bacterial toxin fragments, detoxified bacterial toxins, antibodies,
and antibody fragments.
[0009] In some embodiments, the method includes reacting the
R.sub.1 group with a biointeractive agent or an analytical
agent.
[0010] In some embodiments, the method includes reacting the
R.sup.2 group with a biointeractive agent or an analytical agent.
In some embodiments, the analytical agent is a label. In some
embodiments, the method includes detecting the label.
[0011] In another aspect, conjugate prepared by the disclosed
processes are also disclosed. In another aspect, therapeutic
proteins are disclosed prepared by the disclosed processes. In
another aspect, imaging agents are disclosed prepared by the
disclosed processes. In another aspect, labelling tools are
disclosed prepared by the disclosed processes.
[0012] In another aspect, compounds of the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z are
disclosed where R.sup.1, x, y, A, W, B, R.sup.2 and z have the
meanings described herein.
[0013] In one aspect, a method for modifying a protein is
disclosed. The method includes providing a target protein having at
least one lysine residue; contacting the target protein with a
modifying compound having the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z in the
presence of a microbial transglutaminase, wherein x is 0 or 1; y is
0 or 1; z is 0 or 1; R.sup.1 is selected from the group consisting
of: acetyl,
##STR00005##
W is selected from C.sub.1-C.sub.6 linear or branched alkyl or
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu; R.sup.2 is selected from the group consisting
of: linear or branched C.sub.1-C.sub.3 alkyl-N.sub.3, cyclooctynyl,
fluorophore, polysaccharide,
##STR00006##
to form a modified protein.
[0014] In some embodiments, x is 1. In some embodiments, R.sup.1 is
selected from acetyl,
##STR00007##
[0015] In some embodiments, R.sup.1 is acetyl. In some embodiments,
R.sup.1 is
##STR00008##
In some embodiments, R.sup.1 is
##STR00009##
[0016] In some embodiments, y is 1 and z is 0. In some embodiments,
wherein y is 1 and z is 1.
[0017] In some embodiments, W is selected from C.sub.1-C.sub.6
linear or branched alkyl. In some embodiments, W is C.sub.2 linear
alkyl. In some embodiments, R.sup.2 is
##STR00010##
[0018] In some embodiments, x is 0, y is 1, and R.sup.1 is
##STR00011##
[0019] In some embodiments, wherein x is 0 and R.sup.1 is
##STR00012##
[0020] In some embodiments, y is 0 and z is 1. In some embodiments,
y is 1 and z is 1.
[0021] In some embodiments, W is C.sub.1-C.sub.6 linear or branched
alkyl. In some embodiments, W is C.sub.2 linear alkyl. In some
embodiments, W is C.sub.5 linear alkyl. In some embodiments, W is
linear or branched polyethylene glycol having a molecular weight of
between about 40 and about 3000 amu. In some embodiments, W is
linear polyethylene glycol having a molecular weight of between
about 40 and about 80 amu.
[0022] In some embodiments, R.sup.2 is linear or branched
C.sub.1-C.sub.3 alkyl-N.sub.3. In some embodiments, R.sup.2 is
C.sub.2-alkyl-N.sub.3. In some embodiments, R.sup.2 is
cyclooctynyl. In some embodiments, R.sup.2 is
##STR00013##
In some embodiments, R.sup.2 is
##STR00014##
In some embodiments, R.sup.2 is a fluorophore. In some embodiments,
the fluorophore is selected from: alexa 647, alexa 750, alexa 488,
Cy5, Cy7, rhodamine, and fluorescein. In some embodiments the
fluorophore is of the formula
##STR00015##
where n is from 1 to 3 and each m is from 1 to 2. In some
embodiments, n is 1. In some embodiments, n is 2. In some
embodiments, n is 3. In some embodiments, one m is 1 and the other
m is 2. In some embodiments, both m's are 1. In some embodiments,
both m's are 2. In some embodiments, R.sup.2 is
##STR00016##
In some embodiments, R.sup.2 is a polysaccharide. In some
embodiments, the polysaccharide is selected from GBSII, GBSV, and
MenA. In some embodiments, R.sup.2 is GBSII. In some embodiments,
R.sup.2 is GBSV. In some embodiments, R.sup.2 is MenA.
[0023] In some embodiments, the method includes controlling the pH
environment of the target protein to a pH greater than 7; and
contacting the modified protein with a molecule having a cysteine
residue. In some embodiments, the molecule having a cysteine
residue is
N.sup.5--((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glu-
tamine.
[0024] In some embodiments, the microbial transglutaminase is
Ajinomoto microbial transgluaminase TI.
[0025] In some embodiments, the protein is a carrier protein. In
some embodiments, the protein is CRM.sub.197. In some embodiments,
the protein is selected from: bacterial toxin, bacterial toxin
fragments, detoxified bacterial toxins, antibodies, and antibody
fragments.
[0026] In some embodiments, the method also includes reacting the
R.sub.1 group with a biointeractive agent or an analytical agent.
In some embodiments, the method includes reacting the R.sup.2 group
with a biointeractive agent or an analytical agent. In some
embodiments, the analytical agent is a label. In some embodiments,
the method also includes detecting the label.
[0027] In another aspect, a conjugate is disclosed that is prepared
from the methods disclosed herein. In another aspect, a vaccine is
disclosed that is prepared with a conjugate or a modified protein
disclosed herein. In another aspect, a therapeutic protein is
disclosed having a modified protein disclosed herein. In another
aspect, an imaging agent is disclosed having a modified protein
disclosed herein. In another aspect, a labeling tool is disclosed
having modified protein disclosed herein.
[0028] In another aspect, compound of the formula
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z are
disclosed wherein x is 0 or 1; y is 0 or 1; z is 0 or 1; R.sup.1 is
selected from the group consisting of: acetyl,
##STR00017##
W is selected from C.sub.1-C.sub.6 linear or branched alkyl or
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu; R.sup.2 is selected from the group consisting
of: linear or branched C.sub.1-C.sub.3 alkyl-N.sub.3, cyclooctynyl,
fluorphore, polysaccharide,
##STR00018##
[0029] In some embodiments, the compound is selected from the group
consisting of:
##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023##
[0030] In some embodiments, a conjugate of a compound is disclosed
wherein the conjugate includes a conjugate protein and a compound
disclosed herein. In some embodiments, the conjugate protein is
CRM.sub.197. In some embodiments, the conjugate protein is
GBS.sub.80.
[0031] In some embodiments, a vaccine is disclosed comprising a
conjugate disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a synthetic scheme depicts the addition of a
protein modifying group reacting with a protein (CRM197) catalyzed
by microbial transglutaminase.
[0033] FIG. 2 is an SDS-Page gel electrophoresis characterizing the
conjugation of MenA polysaccharide with CRM.sub.197 using a
compound of the invention.
[0034] FIG. 3 is an SDS-Page gel electrophoresis characterizing the
site selective conjugation of GBS80 antigenic polysaccharide with
CRM.sub.197 using a compound of the invention, wherein lane 1 is
MW, lane 2 is GBS80-K--N.sub.3, lane 3 is GBS80-K--N.sub.3/PSV 1 mg
of protein, lane 4 is GBS80-K--N.sub.3/PSV 1 mg of protein, and
lane 5 is GBS80-K--N.sub.3/PSV 1 mg of protein.
[0035] FIG. 4 is an SDS-Page gel electrophoresis characterizing the
resulting reaction of a conjugated GBS80 antigenic polysaccharide
with CRM.sub.197 using a compound of the invention, wherein lane 1
is MW, lane 2 is GBS80-K--N.sub.3, lane 3 is GBS80-K--N.sub.3/PSII
1 mg of protein, lane 4 is GBS80-K--N.sub.3/PSII 1 mg of protein,
and lane 5 is GBS80-K--N.sub.3/PSII 1 mg of protein.
[0036] FIG. 5 shows ELISA immunoassay results for determination of
Ig titers against GBS II polysaccharide antigen, wherein ELISA anti
PSII IgG and survival results at 1.0 ug dose of PS.
[0037] FIG. 6 shows ELISA immunoassay results for determination of
Ig titers against GBS V polysaccharide antigen, wherein ELISA anti
PSV IgG and survival results at 1.0 ug dose of PS.
[0038] FIG. 7 shows opsonophagocytosis assay results for using GBS
strains.
DETAILED DESCRIPTION
[0039] All references made to patents, patent publications, and
other literature are made for their incorporation into this
disclosure to the extent permissible by law.
[0040] The present invention addresses the aforementioned needs by
providing a method of introducing modifying compounds to a target
protein in a selective manner via reaction with a modifying
compound, while using conventional chemical methods. The method is
generally depicted in FIG. 1. A lysine residue from a target
protein (for example CRM.sub.197) reacts with a glutamine residue
(Gln) from a modifying compound of formula (I):
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z or of
formula (II):
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z. The
resulting product is a protein having one or more groups capable of
further chemical functionalization.
[0041] In one aspect, a process for modifying a protein includes:
(a) forming an activated complex between an auxiliary protein and a
modifying compound by catalytic action of microbial
transglutaminase; (b) transferring the modifying compound from the
activated complex to a target protein thereby creating a modified
protein. As such, a "modified protein" as used herein, refers to a
protein or polypeptide that has been selectively modified by
addition of a modifying compound using microbial
transglutaminase.
[0042] In some embodiments, the method includes a transglutaminase
catalyzed reaction of a target protein having at least two lysines
residues with a modifying compound. The modifying compound is a
glutamine-containing protein of the formula (I):
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z or of
formula (II):
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z.
[0043] In this process, an activated acyl complex is formed by
reacting the glutamine residue in the modifying compound with
microbial transglutaminase in order to attach the modifying
compound. In one embodiment, the modifying compound is transferred
by acylation to a lysine residue in the target protein. In one
embodiment, R.sup.1 and R.sup.2 are desired substituents, where at
least one of them has a chemical group that is suitable for further
modification. Thus, the process involves a microbial
transglutaminase reaction in order to selectively modify a lysine
residue in a target protein.
[0044] As used herein, the term "amu" is an abbreviation for atomic
mass units also frequently referred to as Dalton units.
[0045] As used herein, the term "polypeptide" refers to a polymer
of amino acid residues joined by peptide bonds, whether produced
naturally or synthetically. Polypeptides of less than about 10
amino acid residues are commonly referred to as "peptides." The
term "peptide" is intended to indicate a sequence of two or more
amino acids joined by peptide bonds, wherein said amino acids may
be natural or unnatural. The term encompasses the terms
polypeptides and proteins, which may consist of two or more
peptides held together by covalent interactions, such as for
instance cysteine bridges, or non-covalent interactions.
[0046] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
nonpeptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless. A protein or
polypeptide encoded by a non-host DNA molecule is a "heterologous"
protein or polypeptide.
[0047] An "isolated polypeptide" is a polypeptide that is
essentially free from cellular components, such as carbohydrate,
lipid, or other proteinaceous impurities associated with the
polypeptide in nature. Typically, a preparation of isolated
polypeptide contains the polypeptide in a highly purified form,
i.e., at least about 80% pure, at least about 90% pure, at least
about 95% pure, greater than 95% pure, such as 96%, 97%, or 98% or
more pure, or greater than 99% pure. One way to show that a
particular protein preparation contains an isolated polypeptide is
by the appearance of a single band following sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis of the protein preparation
and Coomassie Brilliant Blue staining of the gel. However, the term
"isolated" does not exclude the presence of the same polypeptide in
alternative physical forms, such as dimers or alternatively
glycosylated or derivatized forms.
[0048] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0049] A "biointeractive agent" as used herein refers to an organic
moiety or compound that invokes a biological response when
introduced into a living tissue or cell. Example of biointeractive
agents include antigens, toxins, therapeutic proteins and the like.
Biointeractive agents may be small molecules and macro
molecules.
[0050] An "analytical agent" as used herein refers to an organic
moiety or compound that can be detected by instrumental methods for
qualitatively or quantitatively characterizing the material to
which the analytical agent is bound or otherwise associated.
Examples of such analytical agents include labels for example
fluorophores or radio labels.
[0051] As used herein, the term "alkyl" is a C.sub.1-C.sub.45 alkyl
group which is linear or branched. In some embodiments, alkyl is
from C.sub.1-C.sub.20. In some embodiments, alkyl is from
C.sub.1-C.sub.12. In some embodiments, alkyl is from
C.sub.1-C.sub.6. Where alkyl is defined as a group, such as W in
formulas I and II discussed herein, it should be understood that
the group may also be known as alkylene such that there is one
substitution with an adjoining group or two substitutions between
two adjoining groups.
[0052] As used herein, the term "polyethylene glycol" or "PEG"
refers to a polyether compound with a repeating
(O--CH.sub.2--CH.sub.2).sub.n subunit having a molecular weight of
between about 40 and about 80,000 amu where n is an integer
representing the number of repeated ether subunits. Where
polyethylene glycol is defined as a group, such as W in formulas I
and II discussed herein, it should be understood that there is one
substitution with an adjoining group in which case there may be a
free alcohol group at a terminus or two substitutions between two
adjoining groups.
Transglutaminase
[0053] As mentioned above, a catalyst must be used for covalently
linking the modifying compound to the target protein. The catalyst
must be a microbial transglutaminase (also interchangeably referred
to herein as "mTGase"). The catalyst is also known as
protein-glutamine-.gamma.-glutamyltransferase from microbial
sources and catalyzes the acyl transfer reaction between the
.gamma.-carboxyamido group of a glutamine (Gln) residue in protein
or a protein chain and the .epsilon.-amino group of a lysine (Lys)
residue or various alkylamines.
[0054] The transglutaminase to be used in the methods of the
present invention can be obtained from various microbial origins
with no particular limitation. Examples of useful microbial
transglutaminases include transglutaminases, such as from
Streptomyces mobaraense, Streptomyces cinnamoneum, and Streptomyces
griseocarneum (all disclosed in U.S. Pat. No. 5,156,956, which is
incorporated herein by reference), and Streptomyces lavendulae
(disclosed in U.S. Pat. No. 5,252,469, which is incorporated herein
by reference) and Streptomyces ladakanum (JP2003199569, which is
incorporated herein by reference). It should be noted that members
of the former genus Streptoverticillium are now included in the
genus Streptomyces [Kaempfer, J. Gen. Microbiol., 137, 1831-1892,
1991]. Other useful microbial transglutaminases have been isolated
from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183, which
is incorporated herein by reference) and from various Myxomycetes.
Other examples of useful microbial transglutaminases are those
disclosed in WO 96/06931 (e.g. transglutaminase from Bacilus
lydicus) and WO 96/22366, both of which are incorporated herein by
reference.
Modifying Compounds
[0055] Modifying compounds that may be used in the disclosed
methods are glutamine-containing proteins of the general formulas:
(I): R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z or
of formula (II):
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z where
Leu refers to the amino acid leucine (for example L-leucine) that
is either present or absent (i.e. when x is 1 or 0, respectively);
Gln refers to the amino acid glutamine (for example L-glutamine);
Gly refers to the amino acid residue glycine (for example
L-glycine) that is either present or absent (i.e. when y is 1 or 0,
respectively).
[0056] In some embodiments, x is 0. In some embodiments, x is 1. In
some embodiments, y is 0. In some embodiments y is 1. In some
embodiments, z is 0. In some embodiments, z is 1.
[0057] In some embodiments, R.sup.1 is acetyl. In some embodiments,
R.sup.1 is
##STR00024##
In some embodiments, R.sup.1 is
##STR00025##
In some embodiments, Q is H. In some embodiments, Q is --NO.sub.2.
In some embodiments, n is 0. In some embodiments, n is 1. In some
embodiments, n is 2. In some embodiments, n is 3. In some
embodiments, n is 4. In some embodiments, n is 5. In some
embodiments, n is 6. In some embodiments, R.sup.4 is H. In some
embodiments, R.sup.4 is --N.sub.3. In some embodiments, R.sup.4
is
##STR00026##
[0058] In some embodiments, R.sup.1 is
##STR00027##
In some embodiments, R.sup.1 is
##STR00028##
In some embodiments, R.sup.1 is
##STR00029##
In some embodiments, R.sup.1 is
##STR00030##
In some embodiments, R.sup.1 is
##STR00031##
In some embodiments, R.sup.1 is
##STR00032##
[0059] In some embodiments, W is C.sub.1-C.sub.6 linear or branched
alkyl. In some embodiments, W is polyethylene glycol having a
molecular weight of between about 40 and about 80,000 amu.
[0060] In some embodiments, A is absent. In some embodiments, A is
--O--. In some embodiments, A is --NH--. In some embodiments, A is
--S--.
[0061] In some embodiments, B is absent. In some embodiments, B is
--O--. In some embodiments, B is --C(O)--. In some embodiments, B
is --NH--. In some embodiments, B is --C(O)NH--. In some
embodiments, B is --NHC(O)--. In some embodiments, B is,
--NHC(O)O--. In some embodiments, B is --OC(O)NH--. In some
embodiments, B is --OC(O)O--. In some embodiments, B is
--C.dbd.N(OH)--. In some embodiments, B is --S(O.sub.2)--. In some
embodiments, B is --NHS(O.sub.2)--. In some embodiments, B is
--S(O.sub.2)NH--. In some embodiments, B is --S(O)--. In some
embodiments, B is --NHS(O)--. In some embodiments, B is --S(O)NH--.
In some embodiments, B is --C(O)O--. In some embodiments, B is
--OC(O)--. In some embodiments, B is --S--. In some embodiments, B
is .dbd.NH--O--. In some embodiments, B is .dbd.NH--NH--. In some
embodiments, B is .dbd.NH--N(C.sub.1-C.sub.20 alkyl)-.
[0062] In some embodiments, R.sup.2 is a fatty acid. In some
embodiments, the fatty acid may be of the following Formulae A1, A2
or A3:
##STR00033##
where R.sup.11 is CO.sub.2H or H; R.sup.12, R.sup.13 and R.sup.14
are independently of each other H, OH, CO.sub.2H, --CH.dbd.CH.sub.2
or --C.ident.CH; Ak is a branched C.sub.6-C.sub.30 alkylene; p, q,
and r are independently of each other an integer between 6 and 30;
or an amide, an ester or a pharmaceutically acceptable salt
thereof. In some embodiments, R.sup.2 is linear or branched
C.sub.1-C.sub.3 alkyl-N.sub.3. In some embodiments, R.sup.2 is
cyclooctynyl. In some embodiments, R.sup.2 is a fluorophore. In
some embodiments the fluorophore is of the formula
##STR00034##
where n is from 1 to 3 and each m is from 1 to 2. In some
embodiments, n is 1. In some embodiments, n is 2. In some
embodiments, n is 3. In some embodiments, one m is 1 and the other
m is 2. In some embodiments, both m's are 1. In some embodiments,
both m's are 2. In some embodiments, R.sup.2 is a polysaccharide.
In some embodiments, R.sup.2 is --CH(OCH.sub.3).sub.2. In some
embodiments, R.sup.2 is
##STR00035##
In some embodiments, R.sup.2 is
##STR00036##
In some embodiments, R.sup.2 is,
##STR00037##
In some embodiments, R.sup.2 is
##STR00038##
In some embodiments, R.sup.2 is
##STR00039##
[0063] In some embodiments, R.sup.2 is
##STR00040##
In some embodiments, Q is H. In some embodiments, Q is --NO.sub.2.
In some embodiments, n is 0. In some embodiments, n is from 1 to 6.
In some embodiments, n is 1. In some embodiments, n is 2. In some
embodiments, n is 3. In some embodiments, n is 4. In some
embodiments, n is 5. In some embodiments, n is 6.
[0064] In some embodiments, R.sup.2 is
##STR00041##
In some embodiments, n is 0. In some embodiments, n is from 1 to 6.
In some embodiments, n is 1. In some embodiments, n is 2. In some
embodiments, n is 3. In some embodiments, n is 4. In some
embodiments, n is 5. In some embodiments, n is 6.
[0065] In some embodiments, R.sup.2 is
##STR00042##
In some embodiments, n is 0. In some embodiments, n is from 1 to 6.
In some embodiments, n is 1. In some embodiments, n is 2. In some
embodiments, n is 3. In some embodiments, n is 4. In some
embodiments, n is 5. In some embodiments, n is 6.
[0066] In some embodiments, R.sup.2 is
##STR00043##
In some embodiments, R.sup.2 is
##STR00044##
[0067] In compounds of formula II, the R2 groups shown below
already include some embodiments incorporating B in formula I
above.
[0068] In some embodiments, n is 0. In some embodiments, n is 1. In
some embodiments, n is 2. In some embodiments, n is 3. In some
embodiments, n is 4. In some embodiments, n is 5. In some
embodiments, n is 6.
[0069] In some embodiments, R.sup.1 is selected from acetyl,
##STR00045##
In some embodiments, R.sup.1 is selected from acetyl,
##STR00046##
In some embodiments, R.sup.1 is selected from acetyl,
##STR00047##
In some embodiments, R.sup.1 is selected from acetyl,
##STR00048##
In some embodiments, R.sup.1 is selected from
##STR00049##
[0070] In some embodiments, R.sup.1 is selected from acetyl and
##STR00050##
In some embodiments, R.sup.1 is selected from acetyl and
##STR00051##
In some embodiments, R.sup.1 is selected from acetyl and
##STR00052##
In some embodiments, R.sup.1 is selected from
##STR00053##
In some embodiments, R.sup.1 is selected from acetyl,
##STR00054##
In some embodiments, R.sup.1 is selected from
##STR00055##
[0071] In some embodiments, R.sup.1 is acetyl. In some embodiments,
R.sup.1 is
##STR00056##
In some embodiments, R.sup.1 is,
##STR00057##
In some embodiments, R.sup.1 is
##STR00058##
[0072] In addition, a multifunctional group A-W--B--R.sup.2 or
NH--W--R.sup.2 may be present or absent (i.e. when z is 1 or 0,
respectively). In embodiments having NH--W--R.sup.2, W is selected
from C.sub.1-C.sub.6 linear or branched alkyl or linear or branched
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu. In some embodiments, W is selected from
C.sub.1-C.sub.6 linear alkyl. In some embodiments, W is selected
from C.sub.1-C.sub.6 branched alkyl. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 10,000 amu. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 3,000 amu. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 80 amu. In some embodiments, W is
selected from linear or branched polyethylene glycol having a
molecular weight of between about 2,000 and about 80,000 amu. In
some embodiments, the polyethylene glycol is functionalized with a
heteroatom (such as oxygen, nitrogen, or sulfur) capable reacting
with a reagent to form a bond with another heteroatom, carbon,
carbonyl, sulfonyl, thionyl, and the like.
[0073] In embodiments having A-W--B--R.sup.2, W is selected from
C.sub.1-C.sub.6 linear or branched alkyl or linear or branched
polyethylene glycol having a molecular weight of between about 40
and about 80,000 amu. In some embodiments, W is selected from
C.sub.1-C.sub.6 linear alkyl. In some embodiments, W is selected
from C.sub.1-C.sub.6 branched alkyl. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 10,000 amu. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 3,000 amu. In some embodiments, W is
selected from linear polyethylene glycol having a molecular weight
of between about 40 and about 80 amu. In some embodiments, W is
selected from linear or branched polyethylene glycol having a
molecular weight of between about 2,000 and about 80,000 amu.
[0074] In some embodiments, A is absent. In some embodiments, A is
--O--. In some embodiments, A is --NH--. In some embodiments, A is
--S--.
[0075] In some embodiments, B is absent. In some embodiments, B is
--O--. In some embodiments, B is --C(O)--. In some embodiments, B
is --NH--. In some embodiments, B is --C(O)NH--. In some
embodiments, B is --NHC(O)--. In some embodiments, B is,
--NHC(O)O--. In some embodiments, B is --OC(O)NH--. In some
embodiments, B is --OC(O)O--. In some embodiments, B is
--C.dbd.N(OH)--. In some embodiments, B is --S(O.sub.2)--. In some
embodiments, B is --NHS(O.sub.2)--. In some embodiments, B is
--S(O.sub.2)NH--. In some embodiments, B is --S(O)--. In some
embodiments, B is --NHS(O)--. In some embodiments, B is --S(O)NH--.
In some embodiments, B is --C(O)O--. In some embodiments, B is
--OC(O)--. In some embodiments, B is --S--. In some embodiments, B
is .dbd.NH--O--. In some embodiments, B is .dbd.NH--NH--. In some
embodiments, B is .dbd.NH--N(C.sub.1-C.sub.20 alkyl)-.
[0076] In some embodiments, R.sup.2 is selected from
C.sub.1-C.sub.3 linear or branched alkyl-N.sub.3, cyclooctynyl,
fluorophore,
##STR00059##
and a polysaccharide.
[0077] In some embodiments, R.sup.2 is branched or linear
C.sub.1-C.sub.3 alkyl-N.sub.3. In some embodiments, R.sup.2 is
branched C.sub.3 alkyl-N.sub.3. In some embodiments, R.sup.2 is
linear C.sub.1-C.sub.3 alkyl-N.sub.3. In some embodiments, R.sup.2
is C.sub.1 alkyl-N.sub.3. In some embodiments, R.sup.2 is C.sub.2
alkyl-N.sub.3. In some embodiments, R.sup.2 is branched C.sub.3
alkyl-N.sub.3.
[0078] In some embodiments, R.sup.2 is cyclooctynyl. The point of
attachment relative to the alkyne functionality group may be at any
position so long as the alkyne can be subsequently reacted or
functionalized. For example, R.sup.2 can be connected to W or B at
the third position, i.e.
##STR00060##
the fourth position, i.e.
##STR00061##
or the fifth position, i.e.
##STR00062##
[0079] In some embodiments, R.sup.2 is fluorophore. Suitable
fluorophores include those that can re-emit light upon light
excitation. Typically, the fluorophore contains several conjugated
pi-bonds, such as are present in aromatic groups. Examples include
fluorescein, rhodamine, Cy dyes such as Cy5 and Cy7, Alexa dies
such as Alexa 750, Alexa 647, and Alexa 488, coumarins, and the
like.
[0080] In some embodiments, R.sup.2 is
##STR00063##
[0081] In some embodiments, R.sup.2 is
##STR00064##
[0082] In some embodiments, R.sup.2 is
##STR00065##
which corresponds to cytotoxic MMAF connected through a carbonyl to
W in NH--W--R.sup.2.
[0083] When the multifunctional group A-W--B--R.sup.2 or
NH--W--R.sup.2 is absent, then the adjacent amino acid residue
whether glutamine or glycine terminates with the carboxylic acid of
the residue (the C-terminus of the peptide backbone of the
modifying compound).
Polysaccharides
[0084] In some embodiments, R.sup.2 is polysaccharide. The
polysaccharide may be any antigenic polysaccharide, particularly a
polysaccharide from a pathogenic organism. Conjugates of these
polysaccharides may be useful for immunizing a subject against
infection caused by the pathogenic organism. Exemplary
polysaccharides are described below. In particular, the
polysaccharide may be a bacterial polysaccharide, e.g. a bacterial
capsular polysaccharide. Representative bacterial polysaccharides
are described in Table 1.
TABLE-US-00001 TABLE I Polysaccharide Repeat Unit Haemophilus
.fwdarw.3)-.beta.-D-Ribf-(I.fwdarw.1)-D-Ribitol-(5.fwdarw.OPO.sub.3.fwdar-
w. influenzae Type b (`PRP`) Neisseria meningitides Group A
.fwdarw.6)-.alpha.-D-ManpNAc(3OAc)-(I.fwdarw.OPO.sub.3.fwdarw.
Group C .fwdarw.9)-.alpha.-D-Neu5Ac(7/8OAc)-(2.fwdarw. Group W135
.fwdarw.6)-.alpha.-D-Galp-(I.fwdarw.4)-.alpha.-D-Neu5Ac(9OAc)-2-
.fwdarw. Group Y
.fwdarw.6)-.alpha.-D-Glcp-(I.fwdarw.4)-.alpha.-D-Neu5Ac(9OAc)-2.fw-
darw. Salmonella .fwdarw.-.alpha.-D-GalpNAcA(3OAc)-(I.fwdarw.
enterica Typhi Vi Streptococcus pneumoniae Type 1
.fwdarw.3)-D-AAT-.alpha.-Galp-(I.fwdarw.4)-.alpha.-D-GalpA(2/3OAc)-
(I.fwdarw.3)-.alpha.-D-GalpA-(I.fwdarw. Type 2
.fwdarw.4-.beta.-D-Glcp-(I.fwdarw.3)-[.alpha.-D-GlcpA-(I.fwdarw.6)--
.alpha.-D-Glcp-
(I.fwdarw.2)]-.alpha.-L-Rhap-(I.fwdarw.3)-.alpha.-L-Rhap-(I.fwdarw.3)-.be-
ta.-L- Rhap-(I.fwdarw. Type 3
.fwdarw.3)-.beta.-D-GlcA-(I.fwdarw.4)-.beta.-D-Glcp-(I.fwdarw. Type
4
.fwdarw.3.beta.-D-ManpNAc-(I.fwdarw.3)-.alpha.-L-FucpNAc-(I.fwdarw.-
3)-.alpha.-D- GalpNAc-(I.fwdarw.4)-.alpha.-
[0085] The polysaccharides may be used in the form of
oligosaccharides. These are conveniently formed by fragmentation of
purified polysaccharide (e.g. by hydrolysis), which will usually be
followed by purification of the fragments of the desired size.
[0086] Polysaccharides may be purified from natural sources. As an
alternative to purification, polysaccharides may be obtained by
total or partial synthesis.
N. meningitidis Capsular Polysaccharides
[0087] The polysaccharide may be a bacterial capsular
polysaccharide. Exemplary bacterial capsular polysaccharides
include those from N. meningitidis. Based on the organism's
capsular polysaccharide, various serogroups of N. meningitidis have
been identified, including A, B, C, H, I, K, L, 29E, W135, X, Y,
and Z. The polysaccharide may be from any of these serogroups.
Typically, the polysaccharide is from one of the following
meningococcal serogroups: A, C, W135 and Y.
[0088] The capsular polysaccharides will generally be used in the
form of oligosaccharides. These are conveniently formed by
fragmentation of purified capsular polysaccharide (e.g. by
hydrolysis), which will usually be followed by purification of the
fragments of the desired size. Fragmentation of polysaccharides is
typically performed to give a final average degree of
polymerization (DP) in the oligosaccharide of less than 30 (e.g.
between 10 and 20, for example around 10 for serogroup A; between
15 and 25 for serogroups W135 and Y, for example between 15 and 20;
between 12 and 22 for serogroup C; etc.). DP can conveniently be
measured by ion exchange chromatography or by colorimetric assays
(Ravenscroft et al. Vaccine 17, 2802-2816 (1999)).
[0089] If hydrolysis is performed, the hydrolysate will generally
be sized in order to remove short-length oligosaccharides
(Costantino et al. Vaccine 17, 1251-1263 (1999)). This can be
achieved in various ways, such as ultrafiltration followed by
ion-exchange chromatography. Oligosaccharides with a degree of
polymerization of less than or equal to about 6 can be removed for
serogroup A, and those less than around 4 can be removed for
serogroups W135 and Y.
[0090] Chemical hydrolysis of saccharides generally involves
treatment with either acid or base under conditions that are
standard in the art. Conditions for depolymerization of capsular
polysaccharides to their constituent monosaccharides are known in
the art. One depolymerization method involves the use of hydrogen
peroxide (see WO02/058737 which is incorporated herein by
reference).
[0091] Hydrogen peroxide is added to a saccharide (e.g. to give a
final H.sub.2O.sub.2 concentration of 1%), and the mixture is then
incubated (e.g. at around 55.degree. C.) until a desired chain
length reduction has been achieved. The reduction over time can be
followed by removing samples from the mixture and then measuring
the (average) molecular size of saccharide in the sample.
Depolymerization can then be stopped by rapid cooling once a
desired chain length has been reached
Serogroups C, W135 and Y
[0092] Techniques for preparing capsular polysaccharides from
meningococci have been known for many years, and typically involve
a process comprising the steps of polysaccharide precipitation
(e.g. using a cationic detergent), ethanol fractionation, cold
phenol extraction (to remove protein) and ultracentrifugation (to
remove LPS) (for example, see Frash, Advances in Biotechnological
Processes 13, 123-145 (1990) (eds. Mizrahi & Van Wezel).
[0093] One such process involves polysaccharide precipitation
followed by solubilization of the precipitated polysaccharide using
a lower alcohol (see WO03/007985 which is incorporated herein by
reference).
[0094] Precipitation can be achieved using a cationic detergent
such as tetrabutylammonium and cetyltrimethylammonium salts (e.g.
the bromide salts), or hexadimethrine bromide and
myristyltrimethylammonium salts. Cetyltrimethylammonium bromide
(`CTAB`) is particularly preferred (Inzana, Infect. Immun. 55,
1573-1579 (1987). Solubilization of the precipitated material can
be achieved using a lower alcohol such as methanol, propan-1-ol,
propan-2-ol, butan-1-ol, butan-2-ol, 2-methyl-propan-1-ol,
2-methyl-propan-2-ol, diols, etc., but ethanol is particularly
suitable for solubilizing CTAB-polysaccharide complexes. Ethanol
may be added to the precipitated polysaccharide to give a final
ethanol concentration (based on total content of ethanol and water)
of between 50% and 95%.
[0095] After re-solubilization, the polysaccharide may be further
treated to remove contaminants. This is particularly important in
situations where even minor contamination is not acceptable (e.g.
for human vaccine production). This will typically involve one or
more steps of filtration e.g. depth filtration, filtration through
activated carbon may be used, size filtration and/or
ultrafiltration. Once filtered to remove contaminants, the
polysaccharide may be precipitated for further treatment and/or
processing. This can be conveniently achieved by exchanging cations
(e.g. by the addition of calcium or sodium salts).
[0096] As an alternative to purification, capsular polysaccharides
of the present invention may be obtained by total or partial
synthesis e.g. Hib synthesis is disclosed in Kandil et al.
Glvcoconi J 14, 13-17. (1997), and MenA synthesis in Berkin et al.
Chemistry 8, 4424-4433 (2002).
[0097] The polysaccharide may be chemically modified, that is it
may be O-acetylated or de-O-acetylated. Any such de-O-acetylation
or hyper-acetylation may be at specific positions in the
polysaccharide. For instance, most serogroup C strains have
O-acetyl groups at position C-7 and/or C-8 of the sialic acid
residues, but about 15% of clinical isolates lack these O-acetyl
groups (Glode et al. J Infect Pis 139, 52-56 (1979); see also
WO94/05325 and U.S. Pat. No. 5,425,946 that are incorporated herein
by reference). The acetylation does not seem to affect protective
efficacy (e.g. unlike the Menjugate.TM. product, the NeisVac-C.TM.
product uses a de-O-acetylated polysaccharide, but both vaccines
are effective). The serogroup W135 polysaccharide is a polymer of
sialic acid-galactose disaccharide units. The serogroup Y
polysaccharide is similar to the serogroup W135 polysaccharide,
except that the disaccharide repeating unit includes glucose
instead of galactose. Like the serogroup C polysaccharides, the
MenW135 and MenY polysaccharides have variable O-acetylation, but
at sialic acid 7 and 9 positions (see WO2005/033148 which is
incorporated herein by reference). Any such chemical modifications
preferably take place before conjugation, but may alternatively or
additionally take place during conjugation.
[0098] Polysaccharides from different serogroups can be purified
separately, and may then be combined either before or after
conjugation.
Serogroup A
[0099] The polysaccharide may be from a serogroup A. The
polysaccharide can be purified in the same way as for serogroups C,
W135 and Y (see above), although it is structurally different,
whereas the capsules of serogroups C, W135 and Y are based around
sialic acid (N-acetyl-neuraminic acid, NeuAc), the capsule of
serogroup A is based on N-acetyl-mannosamine, which is the natural
precursor of sialic acid. The serogroup A polysaccharide is
particularly susceptible to hydrolysis, and its instability in
aqueous media means that (a) the immunogenicity of liquid vaccines
against serogroup A declines over time, and (b) quality control is
more difficult, due to release of saccharide hydrolysis products
into the vaccine.
[0100] Native MenA capsular polysaccharide is a homopolymer of
(a1.fwdarw.6)-linked N-acetyl-D-mannosamine-1-phosphate, with
partial O-acetylation at C3 and C4. The principal glycosidic bond
is a 1-6 phosphodiester bond involving the hemiacetal group of C1
and the alcohol group of C6 of the D-mannosamine. The average chain
length is 93 monomers. It has the following formula:
##STR00066##
[0101] A modified polysaccharide has been prepared which retains
the immunogenic activity of the native serogroup A polysaccharide
but which is much more stable in water. Hydroxyl groups attached at
carbons 3 and 4 of the monosaccharide units are replaced by a
blocking group (see WO03/080678 & WO2008/084411).
[0102] The number of monosaccharide units having blocking groups in
place of hydroxyls can vary. For example, all or substantially all
the monosaccharide units may have blocking groups. Alternatively,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the
monosaccharide units may have blocking groups. At least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 monosaccharide units may have
blocking groups.
[0103] Likewise, the number of blocking groups on a monosaccharide
unit may vary. For example, the number of blocking groups on any
particular monosaccharide unit may be 1 or 2.
[0104] The terminal monosaccharide unit may or may not have a
blocking group instead of its native hydroxyl. It is preferred to
retain a free anomeric hydroxyl group on a terminal monosaccharide
unit in order to provide a handle for further reactions (e.g.
conjugation). Anomeric hydroxyl groups can be converted to amino
groups (--NH.sub.2 or --NH-E, where E is a nitrogen protecting
group) by reductive amination (using, for example,
NaBH.sub.3CN/NH.sub.4CI), and can then be regenerated after other
hydroxyl groups have been converted to blocking groups.
[0105] Blocking groups to replace hydroxyl groups may be directly
accessible via a derivatizing reaction of the hydroxyl group i.e.
by replacing the hydrogen atom of the hydroxyl group with another
group. Suitable derivatives of hydroxyl groups which act as
blocking groups are, for example, carbamates, sulfonates,
carbonates, esters, ethers (e.g. silyl ethers or alkyl ethers) and
acetals. Some specific examples of such blocking groups are allyl,
Aloe, benzyl, BOM, t-butyl, trityl, TBS, TBDPS, TES, TMS, TIPS,
PMB, MEM, MOM, MTM, THP, etc. Other blocking groups that are not
directly accessible and which completely replace the hydroxyl group
include C.sub.1-12 alkyl, C.sub.3-12 alkyl, C.sub.5-12 aryl,
C.sub.5-12 aryl C.sub.1-6 alkyl, NR.sup.21R.sup.22 (R.sup.21 and
R.sup.22 are defined in the following paragraph), H, F, CI, Br,
CO.sub.2H, CO.sub.2(C.sub.1-6 alkyl), CN, CF.sub.3, CCI.sub.3,
etc.
[0106] Typical blocking groups are of the formula: --O--X'--Y' and
--OR.sup.23 wherein: X' is C(O), S(O) or SO.sub.2; Y is C.sub.1-12
alkyl, C.sub.1-12 alkoxy, C.sub.3-12 cycloalkyl, C.sub.5-12 aryl or
C.sub.5-12 aryl-C.sub.1-6 alkyl, each of which may optionally be
substituted with 1, 2 or 3 groups independently selected from F,
CI, Br, CO.sub.2H, CO.sub.2(C.sub.1-6 alkyl), CN, CF.sub.3 or
CCI.sub.3; or Y' is NR.sup.21R.sup.22; R.sup.21 and R.sup.22 are
independently selected from H, C.sub.1-12 alkyl, C.sub.3-12
cycloalkyl, C.sub.5-12 aryl, C.sub.5-12 aryl-C.sub.1-6 alkyl; or
R.sup.21 and R.sup.22 may be joined to form a C.sub.3-12 saturated
heterocyclic group; R.sup.23 is C.sub.1-12 alkyl or C.sub.3-12
cycloalkyl, each of which may optionally be substituted with 1, 2
or 3 groups independently selected from F, Cl, Br,
CO.sub.2(C.sub.1-6 alkyl), CN, CF.sub.3 or CCl.sub.3; or R.sup.23
is C.sub.5-12 aryl or C.sub.5-12 aryl-C.sub.1-6 alkyl, each of
which may optionally be substituted with 1, 2, 3, 4 or 5 groups
selected from F, Cl, Br, CO.sub.2H, CO.sub.2(C.sub.1-6 alkyl), CN,
CF.sub.3 or CCI.sub.3. When R.sup.23 is C.sub.1-12 alkyl or
C.sub.3-12 cycloalkyl, it is typically substituted with 1, 2, or 3
groups as defined above. When R.sup.21 and R.sup.22 are joined to
form a C.sub.3-12 saturated heterocyclic group, it is meant that
R.sup.21 and R.sup.22 together with the nitrogen atom form a
saturated heterocyclic group containing any number of carbon atoms
between 3 and 12 (e.g. C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7,
C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12). The heterocyclic
group may contain 1 or 2 heteroatoms (such as N, O, or S) other
than the nitrogen atom. Examples of C.sub.3-12 saturated
heterocyclic groups are pyrrolidinyl, piperidinyl, morpholinyl,
piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.
[0107] Blocking groups --O--X--Y and --OR.sup.23 can be prepared
from --OH groups by standard derivatizing procedures, such as
reaction of the hydroxyl group with an acyl halide, alkyl halide,
sulfonyl halide, etc. Hence, the oxygen atom in --O--X'--Y' is
usually the oxygen atom of the hydroxyl group, while the --X'--Y'
group in --O--X'--Y' usually replaces the hydrogen atom of the
hydroxyl group.
[0108] Alternatively, the blocking groups may be accessible via a
substitution reaction, such as a Mitsonobu-type substitution. These
and other methods of preparing blocking groups from hydroxyl groups
are well known.
[0109] Specific blocking groups for use in the invention are
--OC(O)CF.sub.3 (Nilsson & Svensson Carbohydrate Research 69,
292-296 (1979)) and a carbamate group OC(O)NR.sup.21R.sup.22, where
R.sup.21 and R.sup.22 are independently selected from C.sub.1-6
alkyl. Typically, R.sup.21 and R.sup.22 are both methyl i.e. the
blocking group is --OC(O)NMe.sub.2. Carbamate blocking groups have
a stabilizing effect on the glycosidic bond and may be prepared
under mild conditions.
[0110] A particularly preferred blocking group is --OC(O)CH.sub.3
(see WO2008/084411). The proportion of 4- and/or 3-positions in the
modified Neisseria meningitidis serogroup A polysaccharide that
have this blocking group may vary. For example, the proportion of
4-positions that have blocking groups may be about 0%, at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or about 100%,
with at least 80% and about 100% being preferred. Similarly, the
proportion of 3-positions that have blocking groups may be about
0%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or
about 100%, with at least 80% and about 100% being preferred.
Typically, the proportion of 4- and 3-positions that have blocking
groups is about the same at each position. In other words, the
ratio of 4-positions that have blocking groups to 3-positions that
have blocking groups is about 1:1. However, in some embodiments,
the proportion of 4-positions that have blocking groups may vary
relative to the proportion of 3-positions that have blocking
groups. For example, the ratio of 4-positions that have blocking
groups to 3-positions that have blocking groups may be 1:20, 1:19,
1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8,
1:7, 1:6, 1:5, 1:4, 1:3 or 1:2. Similarly, the ratio of 3-positions
that have blocking groups to 4-positions that have blocking groups
may be 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11,
1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2.
[0111] Typical modified MenA polysaccharides contain n
monosaccharide units, where at least h % of the monosaccharide
units do not have --OH groups at both of positions 3 and 4. The
value of h is 24 or more (e.g. 25, 26, 27, 28, 29, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100) and is
usually 50 or more. The absent --OH groups are blocking groups as
defined above.
[0112] Other typical modified MenA polysaccharides comprise
monosaccharide units, wherein at least s of the monosaccharide
units do not have --OH at the 3 position and do not have --OH at
the 4 position. The value of s is at least 1 (e.g. 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90). The
absent --OH groups are blocking groups as defined above.
[0113] Suitable modified MenA polysaccharides have the formula:
##STR00067##
wherein p is an integer from 1 to 100 (particularly an integer from
5 to 25, usually 15-25); T is of the formula (A) or (B):
##STR00068##
[0114] each Z group is independently selected from OH or a blocking
group as defined above; and
[0115] each Q group is independently selected from OH or a blocking
group as defined above;
[0116] Y is selected from OH or a blocking group as defined
above;
[0117] E is H or a nitrogen protecting group; and wherein more than
about 7% (e.g. 8%, 9%, 10% or more) of the Q groups are blocking
groups. In some embodiments, the hydroxyl group attached at carbon
1 in formula (A) is replaced by a blocking group as defined above.
In some embodiments, E in formula (B) is a linker or a carrier
molecule as discussed below. When E is a linker, the linker may be
covalently bonded to a carrier molecule.
[0118] Each of the p+2 Z groups may be the same or different from
each other. Likewise, each of the n+2 Q groups may be the same or
different from each other. All the Z groups may be OH.
Alternatively, at least 10%, 20, 30%, 40%, 50% or 60% of the Z
groups may be OAc. Typically, about 70% of the Z groups are OAc,
with the remainder of the Z groups being OH or blocking groups as
defined above. At least about 7% of Q groups are blocking groups.
Typically, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
even 100% of the Q groups are blocking groups.
Glucans
[0119] The polysaccharide may be a glucan. Glucans are
glucose-containing polysaccharides found inter alia in fungal cell
walls. The oglucans include one or more a-linkages between glucose
subunits, whereas .beta.-glucans include one or more
.beta.-linkages between glucose subunits. The glucan used in
accordance with the invention includes .beta. linkages, and may
contain only .beta. linkages (i.e. no a linkages).
[0120] The glucan may comprise one or more .beta.-1,3-linkages
and/or one or more .beta.-1,6-linkages. It may also comprise one or
more .beta.-1,2-linkages and/or .beta.-1,4-linkages, but normally
its only .beta. linkages will be .beta.-1,3-linkages and/or
.beta.-1 6-linkages. The glucan may be branched or linear.
Full-length native .beta.-glucans are insoluble and have a weight
average molecular weight in the megadalton range. Thus, it is
better to use soluble glucans in conjugates. Solubilization may be
achieved by fragmenting long insoluble glucans. This may be
achieved by hydrolysis or, more conveniently, by digestion with a
glucanase (e.g. with a .beta.-1,3-glucanase or a
.beta.-1,6-glucanase). As an alternative, short glucans can be
prepared synthetically by joining monosaccharide building
blocks.
[0121] Low molecular weight glucans are preferred, particularly
those with a weight average molecular weight of less than 100 kDa
(e.g. less than 80, 70, 60, 50, 40, 30, 25, 20, or 15 kDa). It is
also possible to use oligosaccharides e.g. containing 60 or fewer
(e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,
44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4) glucose monosaccharide units. Within this
range, oligosaccharides with between 10 and 50 or between 20 and 40
monosaccharide units. The glucan may be a fungal glucan. A fungal
glucan will generally be obtained from a fungus but, where a
particular glucan structure is found in both fungi and non-fungi
(e.g. in bacteria, lower plants or algae) then the non-fungal
organism may be used as an alternative source. Thus the glucan may
be derived from the cell wall of a Candida, such as C. albicans, or
from Coccidioides immitis, Trichophyton verrucosum, Blastomyces
dermatidis, Cryptococcus neoformans, Histoplasma capsulatum,
Saccharomyces cerevisiae, Paracoccidioides brasiliensis, or
Pythiumn insidiosum.
[0122] There are various sources of fungal .beta.-glucans. For
instance, pure .beta.-glucans are commercially available e.g.
pustulan (Calbiochem) is a .beta.-1,6-glucan purified from
Umbilicaria papullosa. .beta.-glucans can be purified from fungal
cell walls in various ways. Tokunaka et al. Carbohydrate Research
316, 161-172. (1999), for instance, discloses a two-step procedure
for preparing a water-soluble .beta.-glucan extract from Candida,
free from cell-wall mannan, involving NaCIO oxidation and DMSO
extraction. The resulting product (`Candida soluble
.beta.-D-glucan` or `CSBG`) is mainly composed of a linear
.beta.-1,3-glucan with a linear .beta.-1,6-glucan moiety.
Similarly, WO03/097091 discloses the production of GG-zym from
Calbicans. Such glucans from C. albicans, include (a)
.beta.-1,6-glucans with .beta.-1,3-glucan lateral chains and an
average degree of polymerization of about 30, and (b)
.beta.-1,3-glucans with .beta.-1,6-glucan lateral chains and an
average degree of polymerization of about 4.
[0123] In some embodiments, the glucan is a .beta.-1,3 glucan with
some .beta.-1,6 branching, as seen in e.g. laminarins. Laminarins
are found in brown algae and seaweeds. The .beta.(1-3):.beta.(1-6)
ratios of laminarins vary between different sources e.g. it is as
low as 3:2 in Eisenia bicyclis laminarin, but as high as 7:1 in
Laminaria digititata laminarin (Pang et al. Biosci Biotechnol
Biochem 69, 553-8 (2005)). Thus the glucan may have a
.beta.(1-3):.beta.(1-6) ratio of between 1.5:1 and 7.5:1 e.g. about
2:1, 3:1, 4:1, 5:1, 6:1 or 7:1. Optionally, the glucan may have a
terminal mannitol subunit, e.g. a 1,1-O-linked mannitol residue
(Read et al. Carbohydr Res. 281, 187-201 (1996). The glucan may
also comprise mannose subunits.
[0124] In other embodiments, the glucan has exclusively or mainly
.beta.-1,3 linkages, as seen in curdlan. These glucans may elicit
better protection than glucans comprising other linkages,
particularly glucans comprising .beta.-1,3 linkages and a greater
proportion of .beta.-1,6 linkages. Thus the glucan may be made
solely of .beta.-1,3-linked glucose residues (e.g. linear
.beta.-D-glucopyranoses with exclusively 1,3 linkages). Optionally,
though, the glucan may include monosaccharide residues that are not
.beta.-1,3-linked glucose residues e.g. it may include
.beta.-1,6-linked glucose residues. The ratio of .beta.-1,3-linked
glucose residues to these other residues should be at least 8:1
(e.g. >9:1, >10:1, >11:1, >12:1, >13:1, >14:1,
>15:1, >16:1, >17:1, >18:1, >19:1, >20:1,
>25:1, >30:1, >35:1, >40:1, >45:1, >50:1,
>75:1, >100:1, etc.) and/or there are one or more (e.g.
>1, >2, >3, >4, >5, >6, >7, >8, >9,
>10, >11, >12, etc.) sequences of at least five (e.g.
>5, >6, >7, >8, >9, >10, >11, >12, >13,
>14, >15, >16, >17, >18, >19, >20, >30,
>40, >50, >60, etc.) adjacent non-terminal residues linked
to other residues only by .beta.-1,3 linkages. By "non-terminal" it
is meant that the residue is not present at a free end of the
glucan. In some embodiments, the adjacent non-terminal residues may
not include any residues coupled to a carrier molecule or linker.
The presence of five adjacent non-terminal residues linked to other
residues only by .beta.-1,3 linkages may provide a protective
antibody response, e.g. against C. albicans.
[0125] In further embodiments, a conjugate may include two
different glucans e.g. a first glucan having a
.beta.(1-3):.beta.(1-6) ratio of between 1.5:1 and 7.5:1, and a
second glucan having exclusively or mainly .beta.-1,3 linkages. For
instance a conjugate may include both a laminarin glucan and a
curdlan glucan. Where a .beta.-glucan includes both .beta.-1,3 and
.beta.-1,6 linkages at a desired ratio and/or sequence then this
glucan may be found in nature (e.g. a laminarin), or it may be made
artificially. For instance, it may be made by chemical synthesis,
in whole or in part.
[0126] Methods for the chemical synthesis of .beta.-1,3/3-1,6
glucans are known, for example from Takeo and Tei Carbohvdr Res.
145, 293-306 (1986), Tanaka et al. Tetrahedron Letters 44,
3053-3057 (2003), Ning et al. Tetrahedron Letters 43, 5545-5549
(2002), Geurtsen et al. Journal of Organic Chemistry 64
(21):7828-7835 (1999), Wu et al. Carbohvdr Res. 338, 2203-12
(2003), Nicolaou et al. J. Am. Chem. Soc. 119, 449-450 (1997),
Yamada et al. Tetrahedron Letters 40, 4581-4584 (1999), Yamago et
al. Org. Lett. 24, 3867-3870 (2001), Yuguo et al. Tetrahedron 60,
6345-6351 (2004), Amaya et al. Tetrahedron Letters 42:9191-9194
(2001), Mei et al. Carbohvdr Res. 340. 2345-2351 (2005).
[0127] .beta.-glucan including both .beta.-1,3 and .beta.-1,6
linkages at a desired ratio may also be made starting from an
available glucan and treating it with a .beta.-1,6-glucanase (also
known as glucan endo-1,6-.beta.-glucosidase, 1,6-.beta.-D-glucan
glucanohydrolase, etc.; EC 3.2.1.75) or a .beta.-1,3-glucanase
(such as an exo-1,3-glucanase (EC 3.2.1.58) or an
endo-1,3-glucanase (EC 3.2.1.39) until a desired ratio and/or
sequence is reached.
[0128] When a glucan containing solely .beta.-1,3-linked glucose is
desired then .beta.-1,6-glucanase treatment may be pursued to
completion, as .beta.-1,6-glucanase will eventually yield pure
.beta.-1,3 glucan. More conveniently, however, a pure
.beta.-1,3-glucan may be used. These may be made synthetically, by
chemical and/or enzymatic synthesis e.g. using a
(1.fwdarw.3)-.beta.-D-glucan synthase, of which several are known
from many organisms (including bacteria, yeasts, plants and fungi).
Methods for the chemical synthesis of .beta.-1,3 glucans are known,
for example from Takeo et al. Carbohydr Res. 245, 81-96 (1993),
Jamois et al. Glycobiology 15(4), 393-407 (2005), Lefeber et al. C
em. Eur. J. 7(20):4411-4421 (2001) and Huang et al. Carbohydr Res.
340, 603-608 (2005). As a useful alternative to synthesis, a
natural .beta.-1,3-glucan may be used, such as a curdlan (linear
.beta.-1,3-glucan from an Agrobacterium previously known as
Alcaligenes faecalis var. myxogenes; commercially available e.g.
from Sigma-Aldrich catalog C7821) or paramylon (.beta.-1,3-glucan
from Euglena). Organisms producing high levels of
.beta.-1,3-glucans are known in the art e.g. the Agrobacterium of
U.S. Pat. No. 5,508,191 or MiKyoung et al. Biochemical Engineering
Journal. 16, 163-8 (2003), or the Euglena gracilis of Barsanti et
al. J App. Phycology, 13, 59-65 (2001).
[0129] Laminarin and curdlan are typically found in nature as high
molecular weight polymers e.g. with a weight average molecular
weight of at least 100 kDa. They are often insoluble in aqueous
media. In their natural forms, therefore, they are not well suited
to immunization. Thus, in some embodiments, a shorter glucan e.g.
those containing 60 or fewer glucose monosaccharide units (e.g. 59,
58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42,
41, 40 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4). A glucan having a number of glucose residues in the
range of 2-60 may be used e.g. between about 10-50 or between about
20-40 glucose units. A glucan with 25-30 glucose residues is
particularly useful. Suitable glucans may be formed e.g. by acid
hydrolysis of a natural glucan, or by enzymatic digestion e.g. with
a glucanase, such as a .beta.-1,3-glucanase. A glucan with 11-19,
e.g. 13-19 and particularly 15 or 17, glucose monosaccharide units
is also useful. In particular, glucans with the following
structures (A) or (B) are specifically envisaged for use:
##STR00069##
wherein s+2 is in the range of 2-60, e.g. between 10-50 or between
2-40.
[0130] In some embodiments, s+2 is in the range of 25-30 or 11-19,
e.g. 13-17. In particular, s+2=15 is suitable. In addition, s+2=6
is suitable.
##STR00070##
wherein t is in the range of 0-9, e.g. between 1-7 or between 2-6.
Preferably, t is in the range of 3-4 or 1-3. In particular, t=2 is
suitable. The * and ** indicate the respective attachment points of
the polysaccharide units.
[0131] In some embodiments, the glucan contains between 5 to 7
glucose monosaccharide units (i.e. 5, 6 or 7). In particular, a
glucan having 6 glucose monosaccharide units may be preferred. For
example, the glucan may be a curdlan having 6 glucose
monosaccharide units.
[0132] In some embodiments, the glucan is a single molecular
species. In these embodiments, all of the glucan molecules are
identical in terms of sequence.
[0133] Accordingly, all of the glucan molecules are identical in
terms of their structural properties, including molecular weight
etc. Typically, this form of glucan is obtained by chemical
synthesis, e.g. using the methods described above. Alternatively,
in other embodiments, the glucan may be obtained from a natural
glucan, e.g. a glucan from L. digitata, Agrobacterium or Euglena as
described above, with the glucan being purified until the required
single molecular species is obtained. Natural glucans that have
been purified in this way are commercially available. A glucan that
is a single molecular species may be identified by measuring the
polydispersity (Mw/Mn) of the glucan sample. This parameter can
conveniently be measured by SEC-MALLS, for example as described in
Bardotti et al. Vaccine 26, 2284-96 (2008). Suitable glucans for
use in this embodiment of the invention have a polydispersity of
about 1, e.g. 1.01 or less.
[0134] Solubility of natural glucans, such as curdlan, can be
increased by introducing ionic groups (e.g. by sulfation,
particularly at 0-6 in curdlan). Such modifications may be used
with the invention, but are ideally avoided as they may alter the
glucan's antigenicity.
[0135] When the polysaccharide is a glucan, it is typically a
laminarin.
S. pneumoniae Capsular Polysaccharides
[0136] As discussed above, the polysaccharide may also be a
bacterial capsular polysaccharide. Further exemplary bacterial
capsular polysaccharides include those from S. pneumoniae. When the
polysaccharide is a capsular polysaccharide from S. pneumoniae, it
is typically from one of the following pneumococcal serotypes: 1,
2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,
18C, 19A, 19F, 20, 22F, 23F, and 33F. In some embodiments, it is
from 1, 5, 6B, 14, 19F, and 23F. Capsular polysaccharides from S.
pneumoniae comprise repeating oligosaccharide units which may
contain up to 8 sugar residues. The oligosaccharide units for the
main S. pneumoniae serotypes are described in the table above,
Jones An. Acad. Bras. Cienc, 77(2), 293-324 (2005) and Jones, J
Pharm Biomed Anal 38, 840-850 (2005).
S. agalactiae Capsular Polysaccharides
[0137] Further exemplary bacterial capsular polysaccharides include
those from Streptococcus agalactiae ("GBS"). The capsular
polysaccharide is covalently linked to the peptidoglycan backbone
of GBS, and is distinct from the group B antigen, which is another
polysaccharide that is attached to the peptidoglycan backbone.
[0138] The GBS capsular polysaccharides are chemically related, but
are antigenically very different. All GBS capsular polysaccharides
share the following trisaccharide core:
.beta.-D-GlcpNAc(1.fwdarw.3).beta.-D-Galp(1.fwdarw.4).beta.-D-Glcp
[0139] The various GBS serotypes differ by the way in which this
core is modified. The difference between serotypes 1a and III, for
instance, arises from the use of either the GlcNAc (1a) or the Gal
(III) in this core for linking consecutive trisaccharide cores.
[0140] Serotypes 1a and 1b both have a
[a-D-NeupNAc(2.fwdarw.3).beta.-D-Galp-(1.fwdarw.] disaccharide
linked to the GlcNAc in the core, but the linkage is either
1.fwdarw.4 (1a) or 1.fwdarw.3 (1b).
[0141] GBS-related disease arises primarily from serotypes Ia, Ib,
II, III, IV, V, VI, VII, and VIII, with over 85% being caused by
five serotypes: Ia, Ib, III & V. A polysaccharide from one of
these four serotypes may be used. The capsular polysaccharides of
each of these four serotypes include: (a) a terminal
N-acetyl-neuraminic acid (NeuNAc) residue (commonly referred to as
sialic acid), which in all cases is linked 2.fwdarw.3 to a
galactose residue; and (b) a N-acetyl-glucosamine residue (GlcNAc)
within the trisaccharide core.
[0142] All four polysaccharides include galactose residues within
the trisaccharide core, but serotypes Ia, Ib, II & III also
contain additional galactose residues in each repeating unit.
[0143] Polysaccharides used may be in their native form, or may
have been modified. For example, the polysaccharide may be shorter
than the native capsular polysaccharide, or may be chemically
modified. In particular, the serotype V capsular polysaccharide
used in the invention may be modified as described in WO2006/050341
and Guttormsen et al. Proc Natl Acad Sci USA. 105(15), 5903-8
(2008) Epub 2008 Mar. 31. For example, a serotype V capsular
polysaccharide that has been substantially desialylated.
Desialylated GBS serotype V capsular polysaccharide may be prepared
by treating purified GBS serotype V capsular polysaccharide under
mildly acidic conditions (e.g. 0.1 M sulphuric acid at 80.degree.
C. for 60 minutes) or by treatment with neuraminidase. Thus the
polysaccharide used according to the invention may be a
substantially full-length capsular polysaccharide, as found in
nature, or it may be shorter than the natural length. Full-length
polysaccharides may be depolymerized to give shorter fragments for
use with the invention e.g. by hydrolysis in mild acid, by heating,
by sizing chromatography, etc. In particular, the serotype II
and/or III capsular polysaccharides used in the invention may be
depolymerized as described in WO96/40795 and Michon et al. Clin
Vaccine Immunol. (2006) 13(8), 936-43.
[0144] The polysaccharide may be chemically modified relative to
the capsular polysaccharide as found in nature. For example, the
polysaccharide may be de-O-acetylated (partially or fully),
de-N-acetylated (partially or fully), N-propionated (partially or
fully), etc. De-acetylation may occur before, during or after
conjugation, but preferably occurs before conjugation. Depending on
the particular polysaccharide, de-acetylation may or may not affect
immunogenicity. The relevance of O-acetylation on GBS
polysaccharides in various serotypes is discussed in Lewis et al.
PNAS USA 101, 11123-8 (2004), and in some embodiments O-acetylation
of sialic acid residues at positions 7, 8 and/or 9 is retained
before, during and after conjugation e.g. by
protection/de-protection, by re-acetylation, etc. However,
typically the GBS polysaccharide used in the present invention has
substantially no O-acetylation of sialic acid residues at positions
7, 8 and/or 9. In particular, when the GBS polysaccharide has been
purified by base extraction as described below, then O-acetylation
is typically lost. The effect of de-acetylation etc. can be
assessed by routine assays.
[0145] Capsular polysaccharides can be purified by known
techniques, as described in Wessels et al. Infect Immun 57, 1089-94
(1989). A typical process involves base extraction, centrifugation,
filtration, RNase/DNase treatment, protease treatment,
concentration, size exclusion chromatography, ultrafiltration,
anion exchange chromatography, and further ultrafiltration.
Treatment of GBS cells with the enzyme mutanolysin, which cleaves
the bacterial cell wall to free the cell wall components, is also
useful.
[0146] As an alternative, the purification process described in
WO2006/082527 can be used. This involves base extraction,
ethanol/CaCI2 treatment, CTAB precipitation, and re-solubilisation.
A further alternative process is described in WO2009/081276.
S. aureus Capsular Polysaccharides
[0147] Further exemplary bacterial capsular polysaccharides include
those from S. aureus, particularly the capsular polysaccharides of
S. aureus type 5 and type 8. The structures of type 5 and type 8
capsular polysaccharides were described in Moreau et al.
Carbohydrate Res. 339(5), 285-91 (1990) and Fournier et al. Infect.
Immun. 45(1), 87-93 (1984) as:
Type 5
.fwdarw.4)-.beta.-D-ManNAcA(30Ac)-(1.fwdarw.4)-a-L-FucNAc(1.fwdarw.3)-.bet-
a.-D-FucNAc-(1
Type 8
.fwdarw.3)-.beta.-D-ManNAcA(40Ac)-(1.fwdarw.3)-a-L-FucNAc(1.fwdarw.3)-.bet-
a.-D-FucNAc-(1
[0148] Recent NMR spectroscopy data (Jones Carbohydrate Res.
340(6), 1097-106 (2005)) has led to a revision of these structures
to:
Type 5
.fwdarw.4)-.beta.-D-ManNAcA-(1.fwdarw.4)-a-L-FucNAc(30Ac)-(1.fwdarw.3)-.be-
ta.-D-FucNAc-(1
Type 8
.fwdarw.3)-.beta.-D-ManNAcA(40Ac)-(1.fwdarw.3)-a-L-FucNAc(1.fwdarw.3)-a-D--
FucNAc(1.fwdarw.
[0149] The polysaccharide may be chemically modified relative to
the capsular polysaccharide as found in nature.
[0150] For example, the polysaccharide may be de-O-acetylated
(partially or fully), de-N-acetylated (partially or fully),
N-propionated (partially or fully), etc. De-acetylation may occur
before, during or after conjugation, but typically occurs before
conjugation. The effect of de-acetylation etc. can be assessed by
routine assays. For example, the relevance of O-acetylation on S.
aureus type 5 or type 8 capsular polysaccharides is discussed in
Fattom et al. Infect Immun. 66(10):4588-92 (1998). The native
polysaccharides are said in this document to have 75%
O-acetylation. These polysaccharides induced antibodies to both the
polysaccharide backbone and O-acetyl groups. Polysaccharides with
0% O-acetylation still elicited antibodies to the polysaccharide
backbone. Both types of antibody were opsonic against S. aureus
strains that varied in their O-acetyl content. Accordingly, the
type 5 or type 8 capsular polysaccharides used in the present
invention may have between 0 and 100% O-acetylation.
[0151] The degree of O-acetylation of the polysaccharide can be
determined by any method known in the art, for example, by proton
NMR (e.g. as described in Lemercinier and Jones Carbohydrate Res.
296, 83-96 (1996), Jones and Lemercinier, J Pharm Biomed Anal.
30(4), 1233-47 (2002), WO05/033148 or WO 00/56357. A further method
is described in Hestrin J. Biol. Chem. 180, 249-261 (1949). Similar
methods may be used to determine the degree of N-acetylation of the
polysaccharide. O-acetyl groups may be removed by hydrolysis, for
example by treatment with a base such as anhydrous hydrazine
(Konadu et al. Infect. Immun. 62, 5048-5054 (1994)) or NaOH (Fattom
et al. Infect Immun. 66(10):4588-92 (1998)). Similar methods may be
used to remove N-acetyl groups. To maintain high levels of
O-acetylation on type 5 and/or 8 capsular polysaccharides,
treatments that lead to hydrolysis of the O-acetyl groups are
minimized, e.g. treatments at extremes of pH.
[0152] Capsular polysaccharides can be purified by known
techniques, as described in the references herein. A typical
process involves phenol-ethanol inactivation of S. aureus cells,
centrifugation, lysostaphin treatment, RNase/DNase treatment,
centrifugation, dialysis, protease treatment, further dialysis,
filtration, precipitation with ethanol/CaCI2, dialysis,
freeze-drying, anion exchange chromatography, dialysis,
freeze-drying, size exclusion chromatography, dialysis and
freeze-drying (Fattom et al. Infect Immun. 58(7), 2367-74 (1990)).
An alternative process involves autoclaving S. aureus cells,
ultrafiltration of the polysaccharide-containing supernatant,
concentration, lyophilisation, treatment with sodium metaperiodate
to remove teichoic acid, further ultrafiltration, diafiltration,
high performance size exclusion liquid chromatography, dialysis and
freeze-drying (Gilbert et al. J. Microb. Meth. 20, 39-46 (1994)).
The invention is not limited to polysaccharides purified from
natural sources, however, and the polysaccharides may be obtained
by other methods, such as total or partial synthesis.
Other Bacterial Capsular Polysaccharides
[0153] Further exemplary bacterial capsular polysaccharides include
those from Haemophilus influenzae Type b, Salmonella enterica Typhi
Vi and Clostridium difficile.
[0154] S. agalactiae carbohydrate: Non-capsular bacterial
polysaccharides may also be used. An exemplary non-capsular
bacterial polysaccharides is the S. pyogenes GAS carbohydrate (also
known as the GAS cell wall polysaccharide, or GASP). This
polysaccharide features a branched structure with an
L-rhamnopyranose (Rhap) backbone consisting of alternating
alpha-(1.fwdarw.2) and alpha-(1.fwdarw.3) links and
D-N-acetylglucosamine (GlcpNAc) residues
beta-(1.fwdarw.3)-connected to alternating rhamnose rings (Kreis et
al. Int J Biol Macromol. 17(3-4), 117-30 (1995)).
[0155] The GAS carbohydrate will generally be in its native form,
but it may have been modified. For example, the polysaccharide may
be shorter than the native GAS carbohydrate, or may be chemically
modified.
[0156] Thus the polysaccharide used according to the invention may
be a substantially full-length GAS carbohydrate, as found in
nature, or it may be shorter than the natural length. Full-length
polysaccharides may be depolymerized to give shorter fragments for
use with the invention e.g. by hydrolysis in mild acid, by heating,
by sizing chromatography, etc. A short fragment thought to
correspond to the terminal unit on the GAS carbohydrate has been
proposed for use in a vaccine (Hoog et al., Carbohydr Res.
337(21-23), 2023-36 (2002)). Accordingly, short fragments are
envisaged in the present invention. However, it is preferred to use
polysaccharides of substantially full-length. The GAS carbohydrate
typically has a weight average molecular weight of about 10 kDa, in
particular about 7.5-8.5 kDa. Molecular masses can be measured by
HPLC, for example SEC-HPLC using a TSK Gel G3000SW column (Sigma)
relative to pullulan standards, such as those available from
Polymer Standard Service (www. Polymer.de).
[0157] The polysaccharide may be chemically modified relative to
the GAS carbohydrate as found in nature. For example, the
polysaccharide may be de-N-acetylated (partially or fully),
N-propionated (partially or fully), etc. The effect of
de-acetylation etc., for example on immunogenicity, can be assessed
by routine assays.
[0158] In some embodiments, the polysaccharide is GBSII antigentic
polysaccharide having the structure shown below:
##STR00071##
[0159] In some embodiments, the polysaccharide is GBSV antigentic
polysaccharide having the structure shown below:
##STR00072##
[0160] In some embodiments, the polysaccharide is MenA antigenic
polysaccharide having the structure shown below:
##STR00073##
In another aspect, compounds of the formula (I)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z or (II)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z are
disclosed where x, y, z, R.sup.1, R.sup.2, A, B, and W are defined
above. In some embodiments, the compounds are any one of:
##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078##
[0161] In embodiments having an eight-membered cycloalkyne group,
that group can be attached to the modifying group by covalent
linkage. Typically, the eight-membered cycloalkyne group is
attached via a spacer and at a terminus of the spacer. The other
terminus of the spacer has a functional group for attachment to the
modifying group through the amino or carboxylic acid terminus of
the peptide, but not at the .epsilon.-amino group of the glutamine.
For example, if attachment will be at the amine portion of the
modifying group, then spacer can include any functional group that
allows attachment to an amine (e.g. a succinimidyl ester).
Similarly, if the attachment will be at the carboxylic acid portion
of the modifying group, then the spacer can include any functional
group that allows attachment to a carboxylic acid (e.g. an
amine).
[0162] In some embodiments, the eight-membered cycloalkyne group
includes one or more nitrogen atoms, such as 1, 2 or 3 nitrogen
atoms. In some embodiments, the eight-membered cycloalkyne group is
fused to one or more other ring systems, such as cyclopropane or
benzene. In one preferred embodiment, the eight-membered
cycloalkyne group is fused to a cyclopropane group. In another
preferred embodiment, the eight-membered cycloalkyne group is fused
to two benzene groups. In most preferred embodiments, the
eight-membered cycloalkyne group is a cyclooctyne group.
[0163] In one embodiment, the attachment is carried out using a
compound having the formula X.sup.1-L-X.sup.2, where X.sup.1 is the
eight-membered cycloalkyne group and X.sup.2-L is the spacer. In
these embodiments, X.sup.2 may be any group that can react with a
functional group on the amine group on the peptide, and L is a
linking moiety in the spacer.
[0164] In one embodiment, X.sup.2 is N-oxysuccinimide. This group
is suitable for attachment to an amine on a peptide. L may be a
straight chain alkyl with 1 to 10 carbon atoms (e.g. C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8,
C.sub.9, C.sub.10) e.g. (CH.sub.2).sub.4 or (CH.sub.2).sub.3. L
typically has formula -L.sup.3-L.sup.2-L.sup.1, in which L.sup.1 is
carbonyl, L.sup.2 is a straight chain alkyl with 1 to 10 carbon
atoms (e.g. C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6,
C.sub.7, C.sub.8, C.sub.9, C.sub.10) e.g. (CH.sub.2).sub.4 or
(CH.sub.2).sub.5 or L.sup.2 is absent, and L.sup.3 is --NHC(O)--,
carbonyl or --O(CH.sub.3)--.
[0165] In one embodiment, L.sup.1 is carbonyl, L.sup.2 is
(CH.sub.2).sub.5 and L.sup.3 is --NHC(O)--. In another embodiment,
L.sup.1 is carbonyl, L.sup.2 is (CH.sub.2).sub.4 and L.sup.3 is
carbonyl. In another embodiment, L.sup.1 is carbonyl, L.sup.2 is
absent and L.sup.3 is --O(CH3)-.
[0166] In one embodiment, X.sup.1 is
##STR00079##
[0167] In another embodiment, X.sup.1 is:
##STR00080##
[0168] In another embodiment, X.sup.1 is
##STR00081##
[0169] In one embodiment, a compound having the formula
X.sup.1-L-X.sup.2 is
##STR00082##
[0170] In one embodiment, a compound having the formula
X.sup.1-L-X.sup.2 is
##STR00083##
[0171] In one embodiment, a compound having the formula
X.sup.1-L-X.sup.2 is:
##STR00084##
[0172] When the R.sup.2 group includes a fluorophore, suitable
fluorophore groups may be prepared according to techniques well
known in the art. For example as shown in Scheme I, a general
protocol is exemplified for preparing a fluorophore functionalized
modifying group.
##STR00085##
Target Proteins
[0173] A target compound can be one that is a substrate for
microbial transglutaminase, for example proteins that are
substrates for microbial transglutaminase. In one aspect, the
target compound contains at least one Lys residue, and in some
embodiments at least two Lys residues. If a target compound is not
a transglutaminase substrate, per se, it is possible to insert one
or more Gln or Lys residues, and in particular Lys residues in the
protein to make the protein a substrate for transglutaminase.
Alternatively, a peptide sequence containing a lysine residue (a
peptidic tag) may be inserted. In principle, such Gln or Lys
residue may be inserted at any position in the sequence. Typically,
the insertion should be at an accessible portion of the protein or
in a flexible loop. It can also be inserted at a position where the
physiological, such as the therapeutic activity of the protein is
not affected to a degree where the protein is not useful anymore,
e.g. in a therapeutic intervention. Insertions of amino acid
residues in proteins can be brought about by standard techniques
known to persons skilled in the art, such as post-translational
chemical modification or transgenetic techniques.
[0174] Any target compound or protein that is a substrate to
transglutaminase can be modified by the methods disclosed herein,
such as e.g. enzymes, protein hormones, growth factors, antibodies
and antibody fragments, cytokines, receptors, lymphokines and
vaccine antigens. In some embodiments, the polypeptide is an
antigenic peptide.
[0175] In some embodiments, particularly when R is a
polysaccharide, the polypeptide is a carrier molecule. In general,
covalent conjugation of polysaccharides to carriers enhances the
immunogenicity of polysaccharides as it converts them from
T-independent antigens to T-dependent antigens, thus allowing
priming for immunological memory. Conjugation is particularly
useful for pediatric vaccines, (see for example Ramsay et al.
Lancet 357(9251): 195-196 (2001)) and is a well-known technique
(see reviews in Lindberg Vaccine 17 Suppl 2:S28-36 (1999), Buttery
& Moxon, J R Coll Physicians Lond 34, 163-168 (2000), Ahmad
& Chapnick, Infect Dis Clin North Am 13:113-33, vii (1999),
Goldblatt J. Med. Microbiol. 47, 563-567 (1998), European Patent
477 508, U.S. Pat. No. 5,306,492, WO98/42721, Dick et al. Conjugate
Vaccines (eds. Cruse et al.) Karger, Basel, 10, 48-114 (1989) and
Hermanson Bioconjugate Techniques, Academic Press, San Diego (1996)
ISBN: 0123423368.
[0176] The carrier protein may be a bacterial toxin toxoid. Useful
carrier proteins include bacterial toxins or toxoids, such as
diphtheria toxoid or tetanus toxoid, diphtheria and cholera toxins
and their subunits such as fragment C of tetanus toxoid and CRM197
mutant of diphtheria toxin. Other suitable carrier proteins include
the N. meningitidis outer membrane protein, synthetic peptides,
heat shock proteins, pertussis proteins, cytokines, lymphokines,
hormones, growth factors, human serum albumin (including
recombinant), artificial proteins comprising multiple human CD4+ T
cell epitopes from various pathogen-derived antigens, such as N19,
protein D from H. influenzae, pneumococcal surface protein PspA,
pneumolysin, iron-uptake proteins, toxin A or B from C. difficile,
recombinant Pseudomonas aeruginosa exoprotein A (rEPA), a GBS
protein, etc. In some embodiments, the target protein is a pilus
protein such as a GBS protein, for example GBS67 and GBS80.
Further Derivitization
[0177] A need for modifying the target proteins of the present
invention (i.e. proteins of interest) may arise for any number of
reasons, and this is also reflected in the kinds of compounds that
may be selectively modified according to the methods of the present
invention.
[0178] Generally, the methods of the invention comprise a microbial
transglutaminase catalyzed reaction of a protein containing at
least two lysines with a glutamine containing peptide of the
formula (I)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z or (II)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z.
[0179] In one embodiment, the method consists of the following
steps: (a) preparation by peptide synthesis of a compound of the
formula (I)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z, and
purification as known in the art; (b) mixing excess of this
compound
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(A-W--B--R.sup.2).sub.z with a
target protein containing at least one lysine, and in some
embodiments, more than one lysines in an aqueous buffer, optionally
containing an organic solvent, detergent or other modifier; (c)
addition to this mixture of a catalytic amount of microbial
transglutaminase; (d) a mTGase inhibitor can optionally be added to
the mixture; (e) the mixture is subjected to a purification
process, typically comprising unit operation such as ultra- or
dia-filtration and/or chromatography (ion exchange, size exclusion,
hydrophobic interaction, etc.). Selectively modified protein is
thereby obtained. The protein is characterized by standard protein
analytical methods, including chromatography, electrophoresis,
peptide mapping and mass spectroscopy.
[0180] In one embodiment, the method consists of the following
steps: (a) preparation by peptide synthesis of a compound of the
formula (II)
R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z, and
purification as known in the art; (b) mixing excess of this
compound R.sup.1-(Leu).sub.x-Gln-(Gly).sub.y-(NH--W--R.sup.2).sub.z
with a target protein containing at least one lysine, and in some
embodiments, more than one lysines in an aqueous buffer, optionally
containing an organic solvent, detergent or other modifier; (c)
addition to this mixture of a catalytic amount of microbial
transglutaminase; (d) a mTGase inhibitor can optionally be added to
the mixture; (e) the mixture is subjected to a purification
process, typically comprising unit operation such as ultra- or
dia-filtration and/or chromatography (ion exchange, size exclusion,
hydrophobic interaction, etc.). Selectively modified protein is
thereby obtained. The protein is characterized by standard protein
analytical methods, including chromatography, electrophoresis,
peptide mapping and mass spectroscopy.
[0181] Optionally, following steps (b) or (c), the modified protein
can be further modified via the functional groups of R.sup.1 or
R.sup.2 or both, if present, for example with a fluorophore label
(unless one is already present). If a label is added, the modified
protein may be detected using a variety of techniques depending on
the nature of the label such fluorescence or radiolabelling.
[0182] In some embodiments, the functional groups of R.sup.1 or
R.sup.2 or both can be radiolabelled. For example, iodine radio
labeling can be added to a linker as shown in Scheme II.
##STR00086##
[0183] As part of the mechanism of the mTGase-mediated
transamidation, an intermolecular thioester is formed by reaction
between a Cys in the active site of the MTGase and the Gln
substrate. The term "transamidation" is intended to indicate a
reaction where nitrogen in the side chain of glutamine is exchanged
with nitrogen from another compound, in particular nitrogen from
another nitrogen containing nucleophile. This intermediate may be
regarded as an activated Gln-residue, the active species being a
mTGase-thioester, which reacts with amines, e.g. a protein lysine
residue. The selectivity of the reaction is a consequence of 1) the
shear steric bulk of the mTGase-thioester while interacting with
the Lys-bearing protein substrate, and 2) more defined non-covalent
interactions between the mTGase-thioester and the Lys-bearing
protein substrate. An immediate consequence of this is that
proteins carrying activated acyl groups, Acyl-X-protein, where is X
is an atom or group that activates the acyl group towards
nucleophilic attack by a protein-lysine amine, is included in the
invention.
[0184] Thus, it may be desirable to modify proteins to alter the
physico-chemical properties of the protein, such as e.g. to
increase (or to decrease) solubility to modify the bioavailability
of therapeutic proteins. In another embodiment, it may be desirable
to modify the clearance rate in the body by conjugating compounds
to the protein which binds to plasma proteins, such as e.g.
albumin, or which increase the size of the protein to prevent or
delay discharge through the kidneys. Conjugation may also alter and
in particular decrease the susceptibility of a protein to
hydrolysis, such as e.g. in vivo proteolysis.
[0185] In another embodiment, it may be desirable to conjugate a
label to facilitate analysis of the protein. Examples of such
labels include radioactive isotopes, fluorescent markers such as
the fluorophores already described and enzyme substrates.
[0186] In still another embodiment, a compound is conjugated to a
protein to facilitate isolation of the protein. For example, a
compound with a specific affinity to a particular column material
may be conjugated to the protein. It may also be desirable to
modify the immunogenicity of a protein, e.g. by conjugating a
protein so as to hide, mask or eclipse one or more immunogenic
epitopes at the protein. The term "conjugate" as a noun is intended
to indicate a modified peptide, i.e. a peptide with a moiety bonded
to it to modify the properties of said peptide. As a verb, the term
is intended to indicate the process of bonding a moiety to a
peptide to modify the properties of said peptide.
[0187] In one embodiment, the invention provides a method of
improving pharmacological properties of target proteins. The
improvement is with respect to the corresponding unmodified
protein. Examples of such pharmacological properties include
functional in vivo half-life, immunogenicity, renal filtration,
protease protection and albumin binding of any specific
protein.
[0188] In one aspect, modified proteins of the invention may be
further modified thru further derivatization of R.sup.1,
(A-W--B--R.sup.2).sub.z, and/or NH--W--R.sup.2. Specifically,
R.sup.1 and/or R.sup.2 may comprise a chemical group suitable for
further modification. Examples of such further functionalization
include azide-alkyne Huisgen cycloaddition, more commonly known as
click chemistry, if R.sup.1 or R.sup.2 includes an azide or
cyclooctyne group. If R.sup.2 includes the tosyl sulfone which
eliminates to an .alpha.,.beta.-unsaturated ketone, conjugate
addition could be applied for further modification using a
nucleophile such as a thiol.
[0189] In some embodiments already described above, W may be
selected from: dendrimer, polyalkylene oxide, polyalkylene glycol
(PAG), polyethylene glycol (PEG\polypropylene glycol (PPG),
branched PEGs, polyvinyl alcohol (PVA, poly-carboxylate,
poly-vinylpyrolidone, polydhykne-co-maleic acid anhydride,
polystyrene-c-makic acid anhydride, dextrin, carboxymethyl-dextran;
serum protein binding-ligands, such as compounds which bind to
albumin, such as fatty acids, C.sub.5-C.sub.24 fatty acid,
aliphatic diacid (e.g. C.sub.5-C.sub.24), a structure (e. g. sialic
acid derivatives or mimetics) which inhibit the glycams from
binding to receptors (e.g. asialoglyco-protein receptor and mannose
receptor) a small organic molecule containing moieties that alters
physiological conditions, alters charge properties, such as
carboxylic acids or amines, or neutral substituents that prevent
glycan specific recognition such as smaller alkyl substituents (e.
g. C.sub.1-C.sub.5 alkyl), a low molecular organic charged radical
(e.g. C.sub.1-C.sub.25), which may contain one or more carboxylic
acids, amines, sulfonic, phosphonic acids, or combination thereof;
a low molecular neutral hydrophilic molecule (e.g.
C.sub.1-C.sub.25), such as cyclodextrin, or a polyethylene chain
which may optionally branched; polyethyleneglycol with an average
molecular weight of 2-40 KDa; a well-defined precision polymer such
as a dendrimer with an exact molecular mass ranging from 700 to
20,000 Da, or more preferably between 700-10.000 Da; and a
substantially non-imunogenic polypeptide such as albumin or an
antibody or part of an antibody optionally containing a
Fc-domain.
[0190] In one embodiment, W is a linear or branched polyethylene
glycol having a molecular weight of between about 40 and about
10,000 amu, also referred to as a "PEG." The term "PEG" is intended
to indicate polyethylene glycol including analogues thereof, for
example where a branching terminal OH-- group has been replaced by
an alkoxy group, such as methoxy group, an ethoxy group, or a
propoxy group.
[0191] Due to the process for producing mPEG these molecules often
have a distribution of molecular weights. This distribution is
described by the polydispersity index. The term "polydispersity
index" as used herein means the ratio between the weight average
molecular weight and the number average molecular weight, as known
in the art of polymer chemistry (see e.g. "Polymer Synthesis and
Characterization", J. A. Nairn, University of Utah, 2003). The
polydispersity index is a number which is greater than or equal to
one, and it may be estimated from Gel Permeation Chromatographic
data. When the polydispersity index is 1, the product is
monodisperse and is thus made up of compounds with a single
molecular weight. When the polydispersity index is greater than 1
it is a measure of the polydispersity of that polymer, i.e. the
breadth of the distribution of polymers with different molecular
weights.
[0192] The use of for example "mPEG2000" in formulas, compound
names or in molecular structures indicates an mPEG residue wherein
mPEG is polydisperse and has a molecular weight of approximately
2,000 Da.
[0193] The polydispersity index typically increases with the
molecular weight of the PEG or mPEG. When reference is made to
2,000 Da PEG and in particular 2,000 Da mPEG it is intended to
indicate a compound (or in fact a mixture of compounds) with a
polydisperisty index below 1.06, such as below 1.05, such as below
1.04, such as below 1.03, such as between 1.02 and 1.03. When
reference is made to 3,000 Da PEG and in particular 3,000 Da mPEG
it is intended to indicate a compound (or in fact a mixture of
compounds) with a polydispersity index below 1.06, such as below
1.05, such as below 1.04, such as below 1.03, such as between 1.02
and 1.03.
[0194] In some embodiments, the above methods also include a step
of controlling the pH environment of the protein to a pH greater
than 7 and contacting the site selective labeled protein with a
peptide having a cysteine residue. In some embodiments, the peptide
having a cysteine residue is
N.sup.5--((R)-1-((carboxymethyl)amino)-3-mercapto-1-oxopropan-2-yl)-L-glu-
tamine. In some embodiments, the peptide having a cysteine reside
can be substituted with any thiol containing molecule such as
polysaccharides with thiols, cytotoxics with thiols,
thiol-functionalized PEGs, and the like.
Pharmaceutical Compositions
[0195] In another aspect, pharmaceutical compositions comprising a
protein modified by any of the methods disclosed herein. In one
aspect, such a pharmaceutical composition comprises a modified
protein such as growth hormone (GH), which is present in a
concentration from 10-15 mg/ml to 200 mg/ml, such as e.g. 10-10
mg/ml to 5 mg/ml and wherein the composition has a pH from 2.0 to
10.0. The composition may further comprise a buffer system,
preservative(s), tonicity agent(s), chelating agent(s), stabilizers
and surfactants. In one embodiment, the pharmaceutical composition
is an aqueous composition. Such compositions typically exist as a
solution or a suspension. In a further embodiment, the
pharmaceutical composition is an aqueous solution. The term
"aqueous composition" is defined as a composition comprising at
least 50% w/w water. Likewise, the term "aqueous solution" is
defined as a solution comprising at least 50% w/w water, and the
term "aqueous suspension" is defined as a suspension comprising at
least 50% w/w water.
[0196] In another embodiment, the pharmaceutical composition is a
freeze-dried composition, to which a physician, patient, or
pharmacist adds solvents and/or diluents prior to use. In another
embodiment the pharmaceutical composition is a dried composition
(e.g. freeze-dried or spray-dried) ready for use without any prior
dissolution.
[0197] In a further aspect, a pharmaceutical composition comprising
an aqueous solution of a modified protein, such as e.g. a Modified
GH protein, and a buffer, wherein the modified protein, such as
e.g. Modified GH protein is present in a concentration from 0.1-100
mg/ml or above, and wherein said composition has a pH from about
2.0 to about 10.0.
[0198] In a another embodiment, the pH of the composition is
selected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,
3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,
9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.
[0199] In a further embodiment, the buffer is selected from
ammonium bicarbonate, sodium acetate, sodium carbonate, citrate,
glycylglycine, histidine, glycine, lysine, arginine, sodium
dihydrogen phosphate, disodium hydrogen phosphate, sodium
phosphate, and tris(hydroxymethyl)aminomethane, bicine, tricine,
malic acid, succinate, maleic acid, fumaric acid, tartaric acid,
aspartic acid, TRIS, or mixtures thereof.
[0200] In a further embodiment, the composition may also include a
pharmaceutically acceptable preservative. For example, the
preservative may be phenol, o-cresol, m-cresol, p-cresol, methyl
p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol,
butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol,
chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea,
chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl
p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol), or mixtures thereof. The
preservative may be present in a concentration from 0.1 mg/ml to 20
mg/m or 0.1 mg/ml to 5 mg/ml. In a further embodiment, the
preservative is present in a concentration from 5 mg/ml to 10 mg/ml
or from 10 mg/ml to 20 mg/ml.
[0201] In a further embodiment, the composition may include an
isotonic agent. In a further embodiment, the isotonic agent is
selected from a salt (e.g. sodium chloride), a sugar or sugar
alcohol, an amino acid (e.g. L-glycine, L-histidine, arginine,
lysine, isoleucine, aspartic acid, tryptophan, threonine), an
alditol (e.g. glycerol (glycerine), 1,2-propanediol
(propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar
such as mono-, di-, or polysaccharides, or water-soluble glucans,
including for example fructose, glucose, mannose, sorbose, xylose,
maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one embodiment the sugar
additive is sucrose. Sugar alcohol is defined as a C.sub.4-C.sub.8
hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol,
xylitol, arabitol, and mixtures of the same. In one embodiment, the
sugar alcohol additive is mannitol. In one embodiment, the sugar or
sugar alcohol concentration is between about 1 mg/ml and about 150
mg/ml, or from 1 mg/ml to 50 mg/ml. The isotonic agent is present
in a concentration from 1 mg/ml to 7 mg/ml or from 8 mg/ml to 24
mg/ml, or from 25 mg/ml to 50 mg/ml. The use of an isotonic agent
in pharmaceutical compositions is well-known to the skilled person.
For convenience, reference is made to Remington: The Science and
Practice of Pharmacy, 201h edition, 2000.
[0202] In the present context, the term "pharmaceutically
acceptable salt" is intended to indicate salts which are not
harmful to the patient. Such salts include pharmaceutically
acceptable acid addition salts, pharmaceutically acceptable metal
salts, ammonium and alkylated ammonium salts. Acid addition salts
include salts of inorganic acids as well as organic acids.
Representative examples of suitable inorganic acids include
hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric
acids and the like. Representative examples of suitable organic
acids include formic, acetic, trichloroacetic, trifluoroacetic,
propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic,
maleic, malic, malonic, mandelic, oxalic, picric, pyruvic,
salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,
ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,
gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic,
p-aminobenzoic, glutamic, benzenesulfonic, p-toluenes-ulfonic acids
and the like. Further examples of pharmaceutically acceptable
inorganic or organic acid addition salts include the
pharmaceutically acceptable salts listed in J. Phann. Sci. 1977,
66, 2, which is incorporated herein by reference. Examples of metal
salts include lithium, sodium, potassium, magnesium salts and the
like. Examples of ammonium and alkylated ammonium salts include
ammonium, methylammonium, dimethylammonium, trimethylammonium,
ethylammonium, hydroxyethylammonium, diethylammonium,
butylammonium, tetramethylammonium salts and the like.
[0203] In a further embodiment, the composition includes a
chelating agent. The chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. The chelating agent is present in a
concentration from 0.1 mg/ml to 5 mg/ml, from 0.1 mg/ml to 2 mg/ml,
or from 2 mg/ml to 5 mg/ml. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience, reference is made to Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, 2000.
[0204] In a further embodiment, the composition includes a
stabilizer. The use of a stabilizer in pharmaceutical compositions
is well-known to the skilled person. For convenience, reference is
made to Remington: The Science and Practice of Pharmacy, 20th
edition, 2000. More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a protein that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
compositions. By "aggregate formation" is intended a physical
interaction between the protein molecules that results in formation
of oligomers, which may remain soluble, or large visible aggregates
that precipitate from the solution. By "during storage" is intended
a liquid pharmaceutical composition or composition once prepared,
is not immediately administered to a subject. Rather, following
preparation, it is packaged for storage, either in a liquid form,
in a frozen state, or in a dried form for later reconstitution into
a liquid form or other form suitable for administration to a
subject. By "dried form" is intended the liquid pharmaceutical
composition or composition is dried either by freeze drying (i.e.,
lyophilization; see, for example, Williams and Polli (1984) J.
Parenteral Sci. Technol. 38:48-59), spray drying (see Masters
(1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug
Devel. Ind. Phann. 18:1169-1206; and Mumenthaler et al. (1994)
Phann. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a protein during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that protein, resulting in loss of therapeutic efficacy of the
pharmaceutical composition. Furthermore, aggregate formation may
cause other problems such as blockage of tubing, membranes, or
pumps when the protein-containing pharmaceutical composition is
administered using an infusion system.
[0205] The pharmaceutical compositions may also include an amount
of an amino acid base sufficient to decrease aggregate formation by
the protein during storage of the composition. By "amino acid base"
is intended an amino acid or a combination of amino acids, where
any given amino acid is present either in its free base form or in
its salt form. Where a combination of amino acids is used, all of
the amino acids may be present in their free base forms, all may be
present in their salt forms, or some may be present in their free
base forms while others are present in their salt forms. In one
embodiment, amino acids to use in preparing the compositions of the
invention are those carrying a charged side chain, such as
arginine, lysine, aspartic acid, and glutamic acid. Any
stereoisomer (i.e., L or D isomer, or mixtures thereof) of a
particular amino acid (methionine, histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine and mixtures
thereof) or combinations of these stereoisomers or glycine or an
organic base such as but not limited to imidazole, may be present
in the pharmaceutical compositions so long as the particular amino
acid or organic base is present either in its free base form or its
salt form. In one embodiment the L-stereoisomer of an amino acid is
used. In one embodiment the L-stereoisomer is used. Compositions of
the invention may also be formulated with analogues of these amino
acids. By "amino acid analogue" is intended a derivative of the
naturally occurring amino acid that brings about the desired effect
of decreasing aggregate formation by the protein during storage of
the liquid pharmaceutical compositions of the invention. Suitable
arginine analogues include, for example, aminoguanidine, ornithine
and N-monoethyl L-arginine, suitable methionine analogues include
ethionine and buthionine and suitable cysteine analogues include
S-methyl-L-cysteine. As with the other amino acids, the amino acid
analogues are incorporated into the compositions in either their
free base form or their salt form. In a further embodiment, the
amino acids or amino acid analogues are used in a concentration,
which is sufficient to prevent or delay aggregation of the
protein.
[0206] In a further embodiment, methionine (or other sulphuric
amino acids) or analogous amino acids, may be added to inhibit
oxidation of methionine residues to methionine sulfoxide when the
protein acting as the therapeutic agent is a protein comprising at
least one methionine residue susceptible to such oxidation. By
"inhibit" is intended minimal accumulation of methionine oxidized
species over time. Inhibiting methionine oxidation results in
greater retention of the protein in its proper molecular form. Any
stereoisomer of methionine (L or D isomer) or any combinations
thereof can be used. The amount to be added should be an amount
sufficient to inhibit oxidation of the methionine residues such
that the amount of methionine sulfoxide is acceptable to regulatory
agencies. Typically, this means that the composition contains no
more than about 10% to about 30% methionine sulfoxide. Generally,
this can be obtained by adding methionine such that the ratio of
methionine added to methionine residues ranges from about 1:1 to
about 1000:1, such as 10:1 to about 100:1.
[0207] In a further embodiment, the composition may include a
stabilizer selected from the group of high molecular weight
polymers or low molecular compounds. The stabilizer may be selected
from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PV A),
polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates
thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride).
[0208] The pharmaceutical compositions may also include additional
stabilizing agents, which further enhance stability of a
therapeutically active protein therein. Stabilizing agents include,
but are not limited to, methionine and EDTA, which protect the
protein against methionine oxidation, and a nonionic surfactant,
which protects the protein against aggregation associated with
freeze-thawing or mechanical shearing.
[0209] In a further embodiment, the composition also includes a
surfactant. The surfactant may be selected from a detergent,
ethoxylated castor oil, polyglycolyzed glycerides, acetylated
monoglycerides, sorbitan fatty acid esters,
polyoxypropylenepolyoxyethylene block polymers (e.g. poloxamers
such as Pluronic.RTM. F68, poloxamer 188 and 407, Triton X-100),
polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and
polyethylene derivatives such as alkylated and alkoxylated
derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 and
Brij-35), monoglycerides or ethoxylated derivatives thereof,
diglycerides or polyoxyethylene derivatives thereof, alcohols,
glycerol, lectins and phospholipids (e.g. phosphatidyl serine,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol and sphingomyelin), derivates of
phospholipids (e.g. dipalmitoyl phosphatidic acid) and
lysophospholipids (e.g. palmitoyl lysophosphatidyl-L-serine and
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy
(alkyl ether)-derivatives of lysophosphatidyl and
phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,
lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids (e.g. cephalins), glyceroglycolipids (e.g.
galactopyransoide), sphingoglycolipids (e.g. ceramides,
gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid derivatives--(e.g. sodium tauro-dihydrofusidate etc.),
long-chain fatty acids and salts thereof C.sub.6-C.sub.12 (e.g.
oleic acid and caprylic acid), acylcarnitines and derivatives,
N.sup..alpha.-acylated derivatives of lysine, arginine or
histidine, or side-chain acylated derivatives of lysine or
arginine, N.sup..alpha.-acylated derivatives of diproteins
comprising any combination of lysine, arginine or histidine and a
neutral or acidic amino acid, N.sup..alpha.-acylated derivative of
a triprotein comprising any combination of a neutral amino acid and
two charged amino acids, DSS (docusate sodium, CAS registry no
[577-11-7]), docusate calcium, CAS registry no [128-49-4]),
docusate potassium, CAS registry no [7491-09-0]), SDS (sodium
dodecyl sulphate or sodium lauryl sulphate), sodium caprylate,
cholic acid or derivatives thereof, bile acids and salts thereof
and glycine or taurine conjugates, ursodeoxycholic acid, sodium
cholate, sodium deoxycholate, sodium taurocholate, sodium
glycocholate,
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, amomc
(alkyl-arylsulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
nonionic surfactants (e.g. Dodecyl P-D-glucopyranoside),
poloxamines (e.g. Tetronic's), which are tetrafunctional block
copolymers derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine, or the surfactant may be
selected from the group of imidazoline derivatives, or mixtures
thereof.
[0210] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience, reference is
made to Remington: The Science and Practice of Pharmacy, 20.sup.th
edition, 2000.
[0211] It is possible that other ingredients may be present in the
pharmaceutical composition. Such additional ingredients may include
wetting agents, emulsifiers, antioxidants, bulking agents, tonicity
modifiers, chelating agents, metal ions, oleaginous vehicles,
proteins (e.g., human serum albumin, gelatin or proteins) and a
zwitterion (e.g., an amino acid such as betaine, taurine, arginine,
glycine, lysine and histidine). Such additional ingredients, of
course, should not adversely affect the overall stability of the
pharmaceutical composition.
[0212] Pharmaceutical compositions containing a modified protein,
such as e.g. a modified GH protein may be administered to a patient
in need of such treatment at several sites, for example, at topical
sites, for example, skin and mucosal sites, at sites which bypass
absorption, for example, administration in an artery, in a vein, in
the heart, and at sites which involve absorption, for example,
administration in the skin, under the skin, in a muscle or in the
abdomen.
[0213] Administration of pharmaceutical compositions may be through
several routes of administration, for example, lingual, sublingual,
buccal, in the mouth, oral, in the stomach and intestine, nasal,
pulmonary, for example, through the bronchioles and alveoli or a
combination thereof, epidermal, dermal, transdermal, vaginal,
rectal, ocular, for examples through the conjunctiva, uretal, and
parenteral to patients in need of such a treatment.
[0214] Compositions may be administered in several dosage forms,
for example, as solutions, suspensions, emulsions, microemulsions,
multiple emulsion, foams, salves, pastes, plasters, ointments,
tablets, coated tablets, rinses, capsules, for example, hard
gelatin capsules and soft gelatin capsules, suppositories, rectal
capsules, drops, gels, sprays, powder, aerosols, inhalants, eye
drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries,
vaginal rings, vaginal ointments, injection solution, in situ
transforming solutions, for example in situ gelling, in situ
setting, in situ precipitating, in situ crystallization, infusion
solution, and implants.
[0215] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the Modified GH protein, increase bioavailability,
increase solubility, decrease adverse effects, achieve
chronotherapy well known to those skilled in the art, and increase
patient compliance or any combination thereof. Examples of
carriers, drug delivery systems and advanced drug delivery systems
include, but are not limited to, polymers, for example cellulose
and derivatives, polysaccharides, for example dextran and
derivatives, starch and derivatives, poly(vinyl alcohol), acrylate
and methacrylate polymers, polylactic and polyglycolic acid and
block co-polymers thereof, polyethylene glycols, carrier proteins,
for example albumin, gels, for example, thermogelling systems, for
example block co-polymeric systems well known to those skilled in
the art, micelles, liposomes, microspheres, nanoparticulates,
liquid crystals and dispersions thereof, L2 phase and dispersions
there of, well known to those skilled in the art of phase behavior
in lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0216] Compositions are useful in the composition of solids,
semisolids, powder and solutions for pulmonary administration of a
modified protein, such as e.g. a Modified GH protein, using, for
example a metered dose inhaler, dry powder inhaler and a nebulizer,
all being devices well known to those skilled in the art.
Therapeutic Uses of the Modified Proteins
[0217] To the extent that the unmodified protein is a therapeutic
protein, the invention also relates to the use of the modified
proteins in therapy, and in particular to pharmaceutical
compositions comprising the modified proteins. Thus, as used
herein, the terms "treatment" and "treating" mean the management
and care of a patient for the purpose of combating a condition,
such as a disease or a disorder. The term is intended to include
the full spectrum of treatments for a given condition from which
the patient is suffering, such as administration of the active
compound to alleviate the symptoms or complications, to delay the
progression of the disease, disorder or condition, to alleviate or
relief the symptoms and complications, and/or to cure or eliminate
the disease, disorder or condition as well as to prevent the
condition, wherein prevention is to be understood as the management
and care of a patient for the purpose of combating the disease,
condition, or disorder and includes the administration of the
active compounds to prevent the onset of the symptoms or
complications, The patient to be treated is preferably a mammal, in
particular a human being, but it may also include animals, such as
dogs, cats, cows, sheep and pigs. Nonetheless, it should be
recognized that therapeutic regimens and prophylactic
(preventative) regimens represents separate aspects for the uses
disclosed herein and contemplated by treating physician or
veterinarian.
[0218] A "therapeutically effective amount" of a modified protein
as used herein means an amount sufficient to cure, alleviate or
partially arrest the clinical manifestations of a given disease and
its complications. An amount adequate to accomplish this is defined
as "therapeutically effective amount". Effective amounts for each
purpose will depend on e.g. the severity of the disease or injury
as well as the weight, sex, age and general state of the subject.
It will be understood that determining an appropriate dosage may be
achieved using routine experimentation, by constructing a matrix of
values and testing different points in the matrix, which is all
within the ordinary skills of a trained physician or
veterinarian.
[0219] The methods and compositions disclosed herein provide
modified proteins for use in therapy. As such, a typical parenteral
dose is in the range of 10-9 mg/kg to about 100 mg/kg body weight
per administration. Typical administration doses are from about
0.0000001 to about 10 mg/kg body weight per administration. The
exact dose will depend on e.g. indication, medicament, frequency
and mode of administration, the sex, age and general condition of
the subject to be treated, the nature and the severity of the
disease or condition to be treated, the desired effect of the
treatment and other factors evident to the person skilled in the
art. Typical dosing frequencies are twice daily, once daily,
bi-.cndot.daily, twice weekly, once weekly or with even longer
dosing intervals. Due to the prolonged half-lives of the active
compounds compared to the corresponding un-conjugated protein,
dosing regimen with long dosing intervals, such as twice weekly,
once weekly or with even longer dosing intervals is a particular
embodiment. Many diseases are treated using more than one
medicament in the treatment, either concomitantly administered or
sequentially administered. It is, therefore, contemplated that the
modified proteins in therapeutic methods for the treatment of one
of the diseases can be used in combination with one or more other
therapeutically active compound normally used in the treatment of a
disease. It is also contemplated that the use of the modified
protein in combination with other therapeutically active compounds
normally used in the treatment of a disease in the manufacture of a
medicament for that disease.
EXAMPLES
General Preparation Methods for Modifying Compounds
[0220] Unless otherwise specified, starting materials were
generally available from commercial sources such as Aldrich
Chemicals Co. (Milwaukee, Wis.), Lancaster Synthesis, Inc.
(Windham, N.H.), Acros Organics (Fairlawn, N.J.). Microbial
transglutaminase was provided by Ajinomoto North America, Inc.
(Itasca, Ill.). CRM.sub.197 (CAS Number 92092-36-9) is available
from Aldrich Chemicals Co. (Milwaukee, Wis.). Monomethyl auristatin
F was purchased from Concortis (San Diego, Calif.). Cyclooctyne
reagents were purchased from Synaffix (Nijmegen, The Netherlands).
MenA antigenic polysaccharide was supplied by Novartis
NV&D.
[0221] Modifying compounds were purified by column chromatography
(Interchim puriflash 430) and analyzed by NMR spectroscopy (400 MHz
Bruker), LCMS (Waters Acquity UPLC-UV-CAD-MS), and LCUV (Agilent
1200 series UPLC-UV). Labeled CRM.sub.197 is characterized by LCMS
(UPLC-UV-TOF-MS HRMS Waters Acquity UPLC Qtof). Labeled CRM.sub.197
is purified by amicon filters (3 kDa or 10 kDa MWCO), and/or SEC
(General Electric AKTA purifier).
[0222] Identification of the protein site of modification (site
selectivity) was characterized by protein mapping. The mTGase used
in the examples is microbial transglutaminase from Anjinomoto North
America, Inc. (Itasca, Ill.).
[0223] The following acronyms used in the examples below have the
corresponding meanings:
MMAF: monomethylauristatin F mTGase: microbial transglutaminase
MWCO: molecular weight cut off
NHS: N-hydroxysuccinimide
[0224] BCN-NHS: (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl
N-succinimidyl carbonate HATU:
(O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate) RT: room temperature Rt: retention time
Z-Q-G-NH-(PEG).sub.3-N.sub.3
##STR00087##
[0226] Commercially available ZQG (1 g, 2.96 mmol), Amine-PEG-Azide
(0.882 mL, 4.45 mmol), DIPEA (1.553 mL, 8.89 mmol), and HATU (1.127
g, 2.96 mmol) were added together in DMF and stirred at RT for 16
hrs. The solution was loaded directly onto a 55 g C-18 RP column
and purified by column chromatography 5-80% MeCN/Water. Due to the
large amount of MeOH required to load sample poor peak shape
observed. Reduced volume of the desired peak and reloaded onto the
column. The product was purified a second time by column
chromatography 5-80% MeCN/Water. Yield: 300 mg (19% yield).
[0227] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.65-1.79 (m, 1H)
1.82-1.95 (m, 1H) 2.07-2.17 (m, 2H) 3.14-3.26 (m, 2H) 3.36-3.44 (m,
4H) 3.46-3.56 (m, 8H) 3.57-3.62 (m, 2H) 3.68 (d, J=5.81 Hz, 2H)
3.91-4.04 (m, 1H) 5.03 (d, J=2.27 Hz, 2H) 6.77 (br. s., 1H)
7.23-7.34 (m, 2H) 7.34-7.39 (m, 4H) 7.55 (d, J=7.58 Hz, 1H) 7.82
(t, J=5.56 Hz, 1H) 8.16 (t, J=5.68 Hz, 1H); HRMS calculated for
(C.sub.23H.sub.35N.sub.7O.sub.8): 537.2547. observed: (M+1)
538.2623.
ZQ-NH-(PEG).sub.3N.sub.3
##STR00088##
[0229] Commercially available NH.sub.2-(PEG).sub.3-N.sub.3 (0.087
mL, 0.318 mmol), Huenig's Base (0.069 mL, 0.398 mmol), and
commercially available ZQ-NHS (100 mg, 0.265 mmol) were combined in
DMSO and mixed at RT 16 hrs. The reaction was loaded directly onto
a 35 g C-18 column for purification 10-75% MeCN/H.sub.2O. Yield: 90
mg (70% yield).
[0230] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.62-1.75 (m, 1H)
1.77-1.91 (m, 1H) 2.08 (dt, J=9.14, 5.88 Hz, 2H) 3.11-3.27 (m, 2H)
3.35-3.43 (m, 4H) 3.48-3.56 (m, 8H) 3.57-3.62 (m, 2H) 3.94 (td,
J=8.31, 5.38 Hz, 1H) 5.01 (s, 2H) 6.73 (br. s., 1H) 7.23 (br. s.,
1H) 7.28-7.41 (m, 6H) 7.88 (t, J=5.62 Hz, 1H); HRMS calculated for
(C.sub.21H.sub.32N.sub.6O.sub.7): 480.2332. observed: (M+1)
481.2440.
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-Q-G
##STR00089##
[0232] Commercially available QG (92 mg, 0.316 mmol) and Huenig's
Base (64.5 .mu.L, 0.369 mmol) were dissolved in 2 mL of 1:1
Water:DMSO and warmed to 35.degree. C. Commercially available
Click-Easy.TM. BCN N-hydroxysuccinimide ester I (50 mg, 0.246 mmol)
was dissolved in 2 mL of DMSO and slowly added to the reaction. The
reaction mixed at 40.degree. C. for 1 hr. The product was purified
by column chromatography (20 g C-18, 0-70 MeCN/Water). Product
eluted around 50% MeCN. Yield: 46 mg (49% yield).
[0233] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 0.87 (t, J=9.66
Hz, 2H) 1.27 (quin, J=8.53 Hz, 1H) 1.52 (d, J=10.88 Hz, 2H)
1.62-1.75 (m, 1H) 1.81-1.95 (m, 1H) 2.04-2.25 (m, 8H) 3.14 (br. s.,
1H) 3.49-3.73 (m, 2H) 3.89-3.99 (m, 1H) 4.05 (d, J=7.95 Hz, 2H)
6.72 (br. s., 1H) 7.21-7.31 (m, 2H) 7.88 (br. s., 1H); HRMS
calculated for (C.sub.18H.sub.25N.sub.3O.sub.6): 379.1743.
observed: (M+1) 380.1811.
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G
##STR00090##
[0235] Leucine-glutamine-glycine peptide was prepared using a
peptide synthesizer. The synthesizer was programmed for the
leucine-glutamine-glycine sequence. The resin was recovered and
peptide was removed with TFA. The solution was slowly added to
ether (cold) and to precipitate the product. The solution was
centrifuged and ether was decanted. The solid was dissolved in
water and purified by column chromatography (35 g RP C-18 column)
Product eluted with 100% water in 84% yield.
[0236] Leucine glutamine glycine peptide (30 mg, 0.07 mmol),
Huenig's Base (0.030 mL, 0.174 mmol), and commercially available
Click-Easy.TM. BCN N-hydroxysuccinimide ester I (20 mg, 0.070 mmol)
were combined in DMF and stirred for five hours at room temp. The
reaction was loaded directly onto 20 g C-18 column (0-20%
MeCN/Water) and purified over 10 CV to yield 20 mg (58% yield) of
the product. HRMS calculated for (C.sub.211H.sub.32N.sub.6O.sub.7):
492.2584. observed: (M+1) 493.2682.
Z-Q-NH-(PEG).sub.2-NHC(O)O--CH.sub.2-cyclopropylcycloctyne
##STR00091##
[0238] Commercial BocNH-(PEG)3-amine (0.308 mL, 0.994 mmol) in DMSO
was added to commercially available ZQ-NHS (250 mg, 0.663 mmol) and
stirred at RT for 3 hours. The reaction was loaded directly on to a
30 g C-18 column for purification (0-50% MeCN/Water). The product
eluted at 45% MeCN. Solvent was removed to yield a white residue
(200 mg, 59.1% yield). LCMS calculated for
(C.sub.21H.sub.32N.sub.6O.sub.7): 510.27. observed: (M+1)
511.4.
[0239] The Boc-protected product (200 mg, 0.392 mmol) was treated
with TFA (3 mL, 38.9 mmol) and shaken at RT for 10 minutes. The
reaction was dried on high vacuum overnight and crude
ZQ-NH-(PEG).sub.2-NH.sub.2 was used directly in next reaction.
Huenig's Base (2 mL, 11.45 mmol) was added to
ZQ-NH-(PEG).sub.2-NH.sub.2 (150 mg, 0.365 mmol) in 1 mL of DMSO.
Commercially available BCN-NHS (106 mg, 0.365 mmol) was then added
and reaction stirred for several hours. Product was purified by
column chromatography (35 g C-18 column 15-75% MeCN/Water). Product
eluted under DMSO peak as well as at .about.60% MeCN. Combined
fractions and concentrated to 10 mL. Ran second column at 20-50%
MeCN/water. Product eluted at 45% MeCN. The solvent was removed
under reduced pressure to yield 50 mg (23% yield) of product.
[0240] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 0.78-0.91 (m, 2H)
1.26 (quin, J=8.53 Hz, 1H) 1.41-1.59 (m, 2H) 1.62-1.75 (m, 1H)
1.77-1.90 (m, 1H) 1.95-2.30 (m, 8H) 3.11 (q, J=5.95 Hz, 2H) 3.20
(td, J=12.87, 6.66 Hz, 2H) 3.36-3.42 (m, 4H) 3.49 (s, 4H) 3.88-3.98
(m, 1H) 4.02 (d, J=8.07 Hz, 2H) 5.01 (s, 2H) 6.73 (br. s., 1H) 7.07
(t, J=5.44 Hz, 1H) 7.23 (br. s., 1H) 7.29-7.37 (m, 6H) 7.88 (t,
J=5.62 Hz, 1H). HRMS calculated for
(C.sub.30H.sub.42N.sub.4O.sub.8): 586.3003. observed: (M+1)
587.3092.
Z-Q-NH--(CH.sub.2).sub.3-dimethylacetal
##STR00092##
[0242] ZQNHS (100 mg, 0.265 mmol), 4,4-dimethoxybutan-1-amine
(0.049 mL, 0.292 mmol), and Huenig's Base (0.046 mL, 0.265 mmol)
were dissolved in DMF and mixed at RT for 1 hr. The reaction was
loaded directly onto a 35 g C-18 column for purification (0-40%
MeCN/H.sub.2O). The solvent was removed under reduced pressure to
yield 50 mg (48% yield) of the product.
[0243] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.30-1.43 (m, 2H)
1.43-1.54 (m, 2H) 1.58-1.75 (m, 1H) 1.76-1.89 (m, 1H) 2.01-2.16 (m,
2H) 3.04 (dt, J=12.57, 6.47 Hz, 2H) 3.19 (s, 6H) 3.89 (td, J=8.46,
5.31 Hz, 1H) 4.32 (t, J=5.56 Hz, 1H) 5.00 (s, 2H) 6.76 (br. s., 1H)
7.26 (br. s., 1H) 7.28-7.42 (m, 6H) 7.86 (t, J=5.68 Hz, 1H) LCMS
calculated for (C.sub.19H.sub.29N.sub.3O.sub.6): 395.21. observed:
(M+1) 396.5.
Z-Q-NH--(CH.sub.2).sub.2--NH--C(O)--CH.sub.2-Alexafluor647
##STR00093##
[0245] Commercially available ZQ-NHS (286 mg, 0.758 mmol), Huenig's
Base (0.5 mL, 2.86 mmol) and tert-butyl (2-aminoethyl)carbamate
(0.3 mL, 1.498 mmol) were combined in DCM and sonicated. The
solution stirred 16 hours. The reaction was filtered and washed
with DCMm followed by evaporation. The residue was dissolved in
MeOH/DCM 1:10, and passed through a HCO.sub.3 catch and release
column to remove the acid side product. The resulting product was
treated with TFA (1 mL, 12.98 mmol) in 15 mL of DCM were added.
Reaction mixed for 30 minutes and then evaporated on rotovap. The
residue was dissolved in MeOH/DCM 1:10 and passed through a
HCO.sub.3 catch and release column to remove acid side product. The
solvent was removed to yield 300 mg (91% yield) of the product.
[0246] The resulting amine linker (5.1 mg, 0.016 mmol) was
dissolved in DMSO (0.5 mL) and Huenig's Base (3.66 .mu.l, 0.021
mmol) was added followed by Alexflour647 (5 mg, 5.24 .mu.mol). The
reaction stirred at room temp for X. The reaction was loaded
directly onto a 35 g C-18 column for column chromatography
purification (5-35% MeCN/Water). Product eluted .about.10% MeCN.
The product was lyophilized to yield a dark purple powder. Yield:
3.5 mg (57% yield). HRMS calculated for
(C.sub.51H.sub.67N.sub.6O.sub.17S.sub.4.sup.+): mass expected:
1163.34 mass observed: M+1, 1164.
Z-Q-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00094##
[0248] 3-((tert-butoxycarbonyl)amino)propanoic acid (2 g), HATU
(4.42 g), Huenig's Base (4.62 mL) and N,O-dimethylhydroxylamine
hydrochloride (1.134 g) were combined in DCM and stirred at RT for
two hours. The reaction was poured into water and the organics were
extracted. The organic layer was evaporated to yield crude yellow
oil. The product was purified by column chromatography (50 g C-18,
10-70% MeCN/Water). Fractions collect and concentrated to yield a
yellow oil Yield: 1.3 g, 53% yield. Expected Mass: 232, Observed
M+1: 233.
[0249] tert-Butyl (3-(methoxy(methyl)amino)-3-oxopropyl)carbamate
(1.3 g, 5.60 mmol) was dissolved in dry THF and the flask was
purged with nitrogen. The solution was cooled to 0.degree. C. then
vinylmagnesium bromide (20 ml, 20.00 mmol) in THF was slowly added.
The reaction warmed to RT ON. The reaction was poured into cold
sat. NH.sub.4Cl and extracted with ethyl acetate. The organics were
dried and the solvent was removed on rotovap. The crude material
was carried on.
[0250] tert-Butyl (3-oxopent-4-en-1-yl)carbamate and
4-methylbenzenethiol were combined in MeOH and stirred at RT 3
days. Methanol was removed by rotovap to yield a thick oily yellow
residue. The residue was dissolved in DCM and washed with water
2.times.. The organic layer was dried and the solvent removed on
rotovap to yield a yellow oil. This product (1 g, 3.09 mmol) was
dissolved in DCM and cooled to 0.degree. C. TFA (3 mL, 38.9 mmol)
was slowly added then warmed to RT and stirred for 1 hour. Solvent
was removed and residue was dissolved in DCM and passed through a
carbonate catch and release column followed by a carboxylic acid
catch and release. Product went through both. Columns were washed,
the solvent was combined and then solvent by rotovap. Product was
purified by column chromatography (50 g C-18 10-70% MeCN/Water).
The product was collected solvent removed by evaporation to yield a
yellow oil. The product was collected and the solvent was removed
by evaporation to yield a yellow oil. The product was carried on
without any further purification. 850 mg, quantitative yield.
[0251] 1-Amino-5-(p-tolylthio)pentan-3-one (100 mg, 0.448 mmol) and
Huenig's Base (0.130 mL, 0.746 mmol) were dissolved in DMF then
commercially available ZQ-NHS (141 mg, 0.373 mmol) was added and
mixed at RT. The product was purified by column chromatography (25
g C-18 column 10-75% MeCN/Water). The product was collected and
organics evaporated by rotovap. MeOH was added to the sulfide
linker in water. Oxone (229 mg, 0.373 mmol) was added and the
reaction stirred at RT ON. The reaction was filtered and purified
by column chromatography (25 g C-18 column 10-75% MeCN/Water) and
product was lyophilized to 75 mg (39% yield).
[0252] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.59-1.72 (m, 1H)
1.75-1.89 (m, 1H) 2.06 (dt, J=8.99, 5.90 Hz, 1H) 2.42 (s, 3H) 2.60
(t, J=6.60 Hz, 2H) 2.77 (t, J=7.40 Hz, 2H) 3.11-3.26 (m, 2H)
3.39-3.48 (m, 2H) 3.86 (td, J=8.28, 5.32 Hz, 1H) 5.01 (d, J=1.83
Hz, 3H) 6.74 (br. s., 1H) 7.23 (br. s., 1H) 7.28-7.42 (m, 6H) 7.47
(d, J=8.19 Hz, 2H) 7.78 (d, J=8.19 Hz, 2H) 7.83 (t, J=5.50 Hz, 1H).
HRMS calculated for (C.sub.25H.sub.31N.sub.3O.sub.7S): mass
expected: 517.1883 mass observed: M+1, 518.1974.
Ac-L-Q-G-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00095##
[0254] 1-Amino-5-(p-tolylthio)pentan-3-one was prepared as
described above in the synthesis of
Z-Q-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol. LQG
was prepared as described above in the synthesis of
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G. LQG was acelated
at the N-terminus by combining LQG (27 mg, 0.085 mmol), Ac2O (9.66
.mu.l, 0.102 mmol) and Huenig's Base (0.045 mL, 0.256 mmol) in DCM
and stirring at RT for several hours. The solvent was evaporated
and water was added. The product was lyophilized and used directly
in the next reaction. LCMS Observed Mass: (M+1) 359.2; Desired
Mass: 358.4
[0255] Ac-LQG (38 mg, 0.106 mmol),
1-Amino-5-(p-tolylthio)pentan-3-one (47.4 mg, 0.212 mmol), and
Huenig's Base (0.074 mL, 0.424 mmol) were combined in DMF. HATU
(40.3 mg, 0.106 mmol) was added and the reaction stirred at RT ON.
Product was purified by column chromatography (35 g C-18 column
10-50% MeCN/Water) to yield.
[0256] The sulfide (13.5 mg, 0.024 mmol) was dissolved in 1:1
Water:MeOH. Oxone (44.2 mg, 0.072 mmol) was added and the reaction
stirred at RT ON. The product was purified by column chromatography
(25 g C-18 column 10-60% MeCN/Water).
[0257] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 0.85 (dd,
J=16.08, 6.54 Hz, 6H) 1.36-1.45 (m, 2H) 1.59 (dt, J=13.27, 6.57 Hz,
1H) 1.69-1.79 (m, 1H) 1.83 (s, 3H) 2.00-2.14 (m, 2H) 2.41 (s, 3H)
2.57-2.64 (m, 2H) 2.75 (t, J=7.40 Hz, 2H) 3.18 (q, J=6.52 Hz, 1H)
3.27 (s, 2H) 3.41 (t, J=7.40 Hz, 2H) 3.59 (dd, J=5.75, 3.18 Hz, 2H)
4.07-4.17 (m, 1H) 4.21-4.30 (m, 1H) 6.74 (br. s., 1H) 7.24 (br. s.,
1H) 7.46 (d, J=7.95 Hz, 2H) 7.68 (t, J=5.50 Hz, 1H) 7.76 (d, J=8.31
Hz, 2H) 7.93-8.05 (m, 2H) 8.11 (d, J=7.09 Hz, 1H) HRMS calculated
for (C.sub.27H.sub.41N.sub.5O.sub.8S) Desired Mass: 595.2676
Observed Mass: (M+1) 596.2755.
Z-Q-NH--(CH.sub.2).sub.4--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00096##
[0259] 6-((tert-Butoxycarbonyl)amino)hexanoic acid (1 g, 4.32 mmol)
was dissolved in DCM then N,O-dimethylhydroxylamine hydrochloride
(0.464 g, 4.76 mmol), HATU (1.808 g, 4.76 mmol), and TEA (0.723 mL,
5.19 mmol) were added. The reaction stirred at RT for several
hours, reaction not complete, so additional amine, HATU, and TEA
added. The reaction stirred 16 hrs. The reaction was filtered and
solvent was evaporated. Ether was added and the reaction was
filtered again. The product was purified by column chromatography
(25 g column 0-45% EtOAc/Hep) to 385 mg (32.5% yield).
[0260] Tert-Butyl (6-(methoxy(methyl)amino)-6-oxohexyl)carbamate
(385 mg, 1.403 mmol) was dissolved in dry THF and cooled to
0.degree. C. Vinylmagnesium bromide (4.210 mL, 4.21 mmol) was
slowly added. The reaction was allowed to warm to RT overnight. The
reaction was poured into sat. NH.sub.4Cl and organics were
extracted with EtOAc. The organic layer was washed with water and
brine, dried and evaporated. The product was purified by column
chromatography (Sunfire RP HPLC 10-40% MeCN/Water 0.1% TFA over 10
minutes 226 nm UV) to give 280 mg (83% yield).
[0261] Tert-Butyl (6-oxooct-7-en-1-yl)carbamate (280 mg, 1.160
mmol) and 4-methylbenzenethiol (173 mg, 1.392 mmol) were dissolved
in MeOH and stirred at RT for 16 hrs. Methanol was removed and the
product was purified by chromatography (25 g column 0-30%
EtOAc/Hep) to yield 160 mg (38% yield).
[0262] Tert-Butyl (6-oxo-8-(p-tolylthio)octyl)carbamate (160 mg,
0.438 mmol) and ozone (807 mg, 1.313 mmol) were combined together
in MeOH/Water 50/50 and stirred at RT for 16 hours. The reaction
was poured into water and extracted with DCM. The organic layer was
dried by evaporation and 4 Molar HCl in dioxane was added and
stirred at RT for several hours. The solvent was removed by
evaporation to yield an off-white residue. The product was purified
by a Sunfire RP HPLC (10-40% MeCN/Water 0.1% TFA) to yield 33 mg
(25% yield).
[0263] 8-amino-1-tosyloctan-3-one (20 mg, 0.067 mmol) and
commercially available ZQNHS (23.07 mg, 0.061 mmol) were combined
in DMSO and mixed at 37.degree. C. for an hour. Reaction not
complete and no progress was observed after several hours. Huenig's
Base (5.34 .mu.l, 0.031 mmol) was added and the reaction advanced.
The reaction was loaded directly onto column (6 g C-18 0-75%
MeCN/Water) for purification. Column ran twice to 2.4 mg (7%
yield).
[0264] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 1.17 (q, J=7.58
Hz, 2H) 1.28-1.44 (m, 4H) 1.61-1.75 (m, 1H) 1.77-1.90 (m, 1H)
2.00-2.16 (m, 2H) 2.37-2.43 (m, 5H) 2.74 (t, J=7.34 Hz, 2H)
2.92-3.08 (m, 2H) 3.43 (t, J=7.34 Hz, 2H) 3.90 (td, J=8.31, 5.50
Hz, 1H) 5.01 (s, 2H) 6.74 (br. s., 1H) 7.25 (br. s., 1H) 7.28-7.40
(m, 6H) 7.46 (d, J=7.82 Hz, 2H) 7.77 (d, J=8.31 Hz, 2H) 7.78-7.84
(m, 1H). HRMS calculated for (C.sub.28H.sub.37N.sub.3O.sub.7S):
559.2352. observed: (M+1) 560.2426.
Z-Q-NH-MenA Polysaccharide
##STR00097##
[0266] Amine functionalized MenA antigenic polysaccharide (10 mg)
was added to commercially available ZQNHS (10 mg 26.4 umol) in DMSO
with base and stirred for several hours. The reaction was
lyophilized, dissolved in water, and purified by a 10 KD Amicon
filter 4.times.. The flow through was then passed through a 3 KD
Amicon to recover additional product. This was carried on crude
with an estimated yield of .about.50%.
Z-Q-NH--(CH.sub.2).sub.5--C(O)-monomethylauristatin F
##STR00098##
[0268] 6-((tert-butoxycarbonyl)amino)hexanoic acid (46.5 mg, 0.201
mmol) was dissolved in DMSO (1 mL) and Huenig's Base (0.08 mL,
0.458 mmol) and HATU (76 mg, 0.201 mmol) were added. The reaction
stirred at RT for 30 minutes before adding the reaction mixture to
MMAF (50 mg, 0.067 mmol). The reaction then stirred at RT for 4
hours. The reaction mixture was loaded directly onto 35 g C-18
column for purification by column chromatography (5-75% MeCN/Water
0.1% formic acid). The solvent was removed on rotovap and placed on
high vac overnight.
[0269] The resulting product was treated with TFA (2 mL) at
0.degree. C. and brought to room temp to stir for 5 min. The
reaction was loaded directly onto a 35 g C-18 column for column
purification. The solvent was removed under reduced pressure to
yield 30 mg (52% yield).
[0270] The deprotected amine (20 mg, 0.023 mmol) was dissolved in
THF then LiOH (1 mL, 4.00 mmol) was added. The reaction stirred at
RT for 30 minutes was then loaded directly onto column (35 g C-18
column) for purification (0% MeCN followed by 20-40% MeCN/Water).
Product eluted with THF and column rerun under above conditions to
yield 10 mgs in 51% yield.
[0271] The resulting product (5 mg, 5.92 .mu.mol) was dissolved in
DMSO, added to commercially available ZQNHS (4.4 mg, 0.012 mmol),
and stirred at 37.degree. C. for 20 minutes. The reaction mixture
was loaded directly onto a 25 g C-18 column for column
chromatography purification (20-75% MeCN/Water). The solvent was
removed under reduced pressure to yield 3 mg in 46% yield. LCMS
calculated for (C.sub.21H.sub.32N.sub.6O.sub.7): 1106.6627.
observed: (M+1) 1107.9.
Phenol-(CH.sub.2).sub.2--C(O)-L-Q-G
##STR00099##
[0273] LQG was prepared as described in "Synthesis of
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G" above.
Commercially available 2,5-dioxopyrrolidin-1-yl
3-(4-hydroxyphenyl)propanoate (14.65 mg, 55.6 .mu.mol) was added to
LQG (16 mg, 50.6 .mu.mol) in DMSO with Huenig's Base (18 uL, 101
.mu.mol) and reaction stirred at room temp overnight. Reaction
loaded directly on 20 g column for purification (10-50% MeCN/Water)
to yield 13 mg of product for a 55% yield. HRMS calculated for
(C.sub.21H.sub.32N.sub.6O.sub.7): 464.2271. observed: (M+1)
465.2356.
ZQ(PEG).sub.2azidobenzylamide
##STR00100##
[0275] 4-Azidophenylacetic acid N-succinimido ester (ChemPacific,
26.7 mg. 0.073 mmol) was dissolved in DMF (1 mL) and combined with
a solution of benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopent-
an-2-yl)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121
mL, 0.585 mmol) was added and the reaction was mixed at r.t. for 4
hours at which point DIPEA (61 .mu.L, 6 eq.) and
4-azidophenylacetic acid N-succinimido ester (9 mg, 0.024 mmol)
were added. The reaction was mixed at r.t. for 2 days.
4-azidophenylacetic acid N-succinimido ester (89 mg, 0.24 mmol) was
added and the reaction was stirred at r.t. for 2 hours. The
solution was purified via MS-triggered HPLC (100-Prep3; Acid_Method
3; Sunfire 30.times.50 mm 5 .mu.m column ACN/H.sub.2O w/0.1% TFA 75
ml/min, A: Water (0.1% formic acid); B: ACN gradient 0 min 5% B; 5%
to 95% B in 1.70 min; 2.0 min 95% B; 2.1 min 5% B flow rate 2
ml/min
[0276] 1.5 ml injection; Tube Trigger M=570). Fractions with
desired product were pooled and lyophilized to give 3.6 mg of light
yellow powder (13%) of ZQ(PEG2)azido-benzylamide (benzyl
(17-amino-1-(4-azidophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazaheptadecan-
-14-yl)carbamates). LCMS SQ2; Product Analysis-Acidic;
R.sub.t=1.79: MS [M+H] observed: 570.3. calculated: 569.6.
ZQ(PEG).sub.2amidoethylmethyldiazirin
##STR00101##
[0278] Sulfo-NHS-diazirine (Thermo Scientific, 23.92 mg, 0.073
mmol) was dissolved in DMF (1 mL) and combined with a solution of
benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl-
)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). The reactants were
stirred to combine and DIPEA (0.121 mL, 0.585 mmol) was added. The
reaction was stirred at r.t. for 2 hours at which point the
reaction became cloudy and 0.5 mL DMF was added. The reaction was
stirred for an additional 2 hours and DIPEA (61 .mu.L) and
sulfo-NHS-diazirine (8 mg) were added. The reaction was mixed at
r.t. for 16 hours at which point the consumption of starting
material was observed by LCMS analysis. The solution was purified
via MH-triggered HPLC (100-Prep3; Acid_Method 3; Sunfire
30.times.50 mm Sum column ACN/H.sub.2O w/0.1% TFA 75 ml/min, 1.5 ml
injection; Tube Trigger M=521). Fractions with the desired product,
ZQ(PEG).sub.2amidoethylmethyldiazirin (benzyl
(18-amino-1-(3-methyl-3H-diazirin-3-yl)-3,14,18-trioxo-7,10-dioxa-4,13-di-
azaoctadecan-15-yl)carbamate) were pooled and lyophilized to give 2
mg of the desired compound as a white powder (8%). LCMS SQ2;
Product Analysis-Acidic; R.sub.t=1.49: MS [M+H] observed: 521.4.
calculated: 520.6.
2-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanedioic
acid
[0279] A solution of DCC (126 mg, 0.610 mmol) in DCM (1.57 mL) was
added to a solution of intermediate 4 and N-hydroxysuccinimide in
DCM (5 mL) and THF (5 mL) under N.sub.2. After 3.5 hours, the
solvent was evaporated and the residue purified by supercritical
fluid chromatography (SFC; Princeton 2-ethyl-pyridine, 20.times.150
mm, 20-30% MeOH/CO.sub.2), yielding the title compound as a
colorless oil (138 mg, 0.256 mmol, 50%): LCMS method B Rt=1.21 min,
M+H 540.5; .sup.1H NMR (600 MHz, ACETONITRILE-d3) .delta. ppm 0.91
(t, J=7.20 Hz, 3H) 1.22-1.42 (m, 34H) 1.57 (quin, J=7.34 Hz, 2H)
1.93-1.96 (m, 2H) 2.28 (t, J=7.47 Hz, 2H) 2.79 (br. d, J=6.30 Hz,
4H).
##STR00102##
2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-5-azat-
etracontan-40-yl)carbamoyl)-2-undecyltridecanedioic acid
[0280] t-Boc-N-amido-dPEG.RTM..sub.11-amine (100 mg, 0.155 mmol,
Quanta Biodesign) and
2#(2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2-undecyltridecanedioic
acid (80 mg, 0.148 mmol) were dissolved in THF (3 mL) and stirred
at room temperature under nitrogen nitrogen. After 30 minutes,
DIPEA (0.05 mL, 0.286 mmol) was added and the reaction mixture
stirred at room temperature overnight. Complete conversion was
observed by LCMS (Acidic Eluent A: Water+0.05% Trifluoroacetic
Acid, Eluent B: ACN, column Sunfire C18 3.5 .mu.m 3.0.times.30
mm-40.degree. C., 5-95% gradient 2 minutes, retention time 1.92
min). The reaction mixture was concentrated under reduced pressure,
then dissolved in about 1.5 mL of acetonitrile. Purified on a
MS-triggered HPLC (Sunfire 30.times.50 mm Sum column ACN/H2O w/0.1%
TFA 75 ml/min 1.5 ml injection, 65-95% ACN 3.5 min gradient,
retention time 3.23 minutes) and the fractions pooled and
lyophilized to give 85 mg clean product in 54% yield. Clear oil.
LCMS: SQ4, RXNMON-Acidic-NonPolar Rt=1.18 min, M+H 1070.1; .sup.1H
NMR (400 MHz, ACETONITRILE-d.sub.3) .delta. ppm 0.82-1.03 (m, 1H)
1.11-1.37 (m, 10H) 1.37-1.51 (m, 2H) 1.51-1.64 (m, 1H) 1.69-1.82
(m, 1H) 1.90-2.04 (m, 66H) 2.05-2.21 (m, 8H) 2.21-2.42 (m, 1H)
3.17-3.28 (m, 1H) 3.40-3.68 (m, 13H).
2-((35-Amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)carbam-
oyl)-2-undecyltridecanedioic acid
##STR00103##
[0282]
2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa--
5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioic acid (5
mg, 4.68 .mu.mol) was dissolved in DCM (Volume: 2 mL), then
trifluoroacetic acid (25 .mu.l, 0.324 mmol) was added. The reaction
mixture was stirred at room temperature under nitrogen atmosphere
for about 2 hours. Complete conversion was observed by LCMS (Acidic
Eluent A: Water+0.05% Trifluoroacetic Acid, Eluent B: ACN, column
Sunfire C18 3.5 .mu.m 3.0.times.30 mm-40.degree. C., 5-95% gradient
2 minutes, retention time 1.45 min) The reaction mixture was
concentrated under reduced pressure, then rinsed with DCM and
concentrated again 3 times. Dissolved in a mixture of acetonitrile
and DMSO. Purified on a MS-triggered HPLC (Sunfire 30.times.50 mm
Sum column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection, 45-70%
ACN 3.5 min gradient, retention time 2.50 minutes) and the
fractions pooled and lyophilized to give 2.5 mg clean product in
55% yield. Clear oil.
[0283] LCMS ZQ1 RXNMON_Acidic Rt=1.45 min, M+H 969.9; .sup.1H NMR
(400 MHz, ACETONITRILE-d.sub.3) .delta. ppm 0.62-0.91 (m, 2H)
0.91-1.10 (m, 3H) 1.10-1.31 (m, 18H) 1.46 (quin, J=7.21 Hz, 2H)
1.59-1.89 (m, 35H) 1.94-2.09 (m, 1H) 2.16 (t, J=7.40 Hz, 2H)
2.97-3.11 (m, 1H) 3.24-3.37 (m, 1H) 3.37-3.61 (m, 28H) 3.61-3.89
(m, 2H) 7.85 (br. s., 1H).
2-(((S)-5-(3-Amino-3-oxopropyl)-3,6-dioxo-1-phenyl-2,10,13,16,19,22,25,28,-
31,34,37,40-dodecaoxa-4,7-diazadotetracontan-42-yl)carbamoyl)-2-undecyltri-
decanedioic acid (ZQ-FA)
##STR00104##
[0285] A solution of
2-((2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,
35,38-dodecaoxa-5-azatetracontan-40-yl)carbamoyl)-2-undecyltridecanedioic
acid (20 mg, 0.018 mmol) in THF (Volume: 2 mL) was added to
Z-L-Gln-Osu (Santa Cruz Biotechnology, CAS 34078-85-8, 11 mg, 0.029
mmol), then DIPEA (75 .mu.l, 0.429 mmol) was added. Stirred at room
temperature under a nitrogen atmosphere over weekend. Complete
conversion was observed by LCMS (Acidic Eluent A: Water+0.05%
Trifluoroacetic Acid, Eluent B: ACN, column Sunfire C18 3.5 .mu.m
3.0.times.30 mm-40.degree. C., 5-95% gradient 2 minutes, retention
time 1.77 min) The reaction mixture was concentrated under reduced
pressure and then dissolved in acetonitrile. Purified on a
MS-triggered HPLC (Sunfire 30.times.50 mm Sum column ACN/H2O w/0.1%
TFA 75 ml/min 1.5 ml injection, 55-80% ACN 3.5 min gradient,
retention time 2.70 minutes) and the fractions pooled and
lyophilized to give 10.5 mg clean product ZQ-FA in 46% yield as a
clear colorless oil.
[0286] LCMS SQ4 RXNMON_Acidic Rt=1.60 min, M+H 1232.4; .sup.1H NMR
(400 MHz, ACETONITRILE-d.sub.3) .delta. ppm 0.67-0.93 (m, 2H)
0.93-1.10 (m, 2H) 1.10-1.32 (m, 15H) 1.45 (quin, J=7.24 Hz, 1H)
1.59-1.69 (m, 1H) 1.75-1.93 (m, 30H) 1.94-2.21 (m, 20H) 3.23 (quin,
J=5.26 Hz, 1H) 3.28-3.51 (m, 23H) 3.95 (td, J=7.73, 5.44 Hz, 1H)
4.92-5.22 (m, 1H) 5.78 (br. s., 1H) 6.13-6.42 (m, 1H) 6.88 (br. s.,
1H) 7.20-7.36 (m, 2H) 7.42 (t, J=5.07 Hz, 1H).
Azido-nitrophenyl-glutamine-glycine
##STR00105##
[0288] QG (30 mg, 0.148 mmol) was dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and sodium
1-((4-azido-2-nitrobenzoyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate
(60.1 mg, 0.148 mmol) was added in H.sub.2O (Volume: 1.000 mL,
Ratio: 1.000) followed by addition of DIPEA (0.177 mmol). The
reaction stirred for 16 hours at which time the product was
purified by HPLC (Sunfire 30.times.50 mm 5 um column ACN/H2O w/0.1%
TFA 75 ml/min 1.5 ml injection, rt=1.53) to give the desired
product in 62% yield. LCMS SQ4 RXNMON_Acidic Rt=0.68 min, M+H
394.3; .sup.1H NMR (400 MHz, methanol-d.sub.4) .delta. ppm 1H NMR
(METHANOL-d4, 400 MHz): 8.12-8.26 (m, 1H), 7.21-7.40 (m, 2H), 4.60
(dd, J=8.3, 5.7 Hz, 1H), 3.83-4.15 (m, 2H), 2.36-2.54 (m, 2H), 2.21
(d, J=7.5 Hz, 1H), 2.07 (d, J=6.6 Hz, 1H).
Diazirine-QG
##STR00106##
[0289] QG (30 mg, 0.148 mmol) was dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and sodium
1-((3-(3-methyl-3H-diazirin-3-yl)propanoyl)oxy)-2,5-dioxopyrrolidine-3-su-
lfonate (50 mg, 0.153 mmol) in H.sub.2O (Volume: 1.000 mL, Ratio:
1.000) was added followed by DIPEA (0.031 mL, 0.177 mmol). Reaction
stirred 16 hours at which time product was directly purified by
HPLC (Sunfire 30.times.50 mm Sum column 15-20% gradient ACN/H2O
w/0.1% TFA 75 ml/min 1.5 ml injection, rt=2.46) to give the desired
product in 52% yield. LCMS SQ4 RXNMON_Acidic Rt=0.61 min, M+H
314.2; .sup.1H NMR (400 MHz, methanol-d.sub.4) .delta. ppm 1H NMR
(METHANOL-d4, 400 MHz): 4.39 (dd, J=8.3, 5.7 Hz, 1H), 3.76-4.04 (m,
2H), 2.34 (m, 2H), 2.11-2.19 (m, 2H), 2.07-2.11 (m, 1H), 1.89-2.00
(m, 1H), 1.62-1.74 ppm (m, 2H), 1.01 (s, 3H).
ZQ(PEG).sub.3 Biotin
##STR00107##
[0290] ZQ NHS (45.1 mg, 0.119 mmol), biotin amine (Pierce cat
#21347, 50 mg, 0.119 mmol), and DIPEA (23 uL, 0.131 mmol) were
combined in DMF (2 mL) and stirred at room temp for 2 hours at
which time LCMS shows predominantly product. Reaction loaded on
HPLC (Sunfire 30.times.50 mm Sum column 15-20% gradient ACN/H2O
w/0.1% TFA 75 ml/min 1.5 ml injection, rt=2.46) to give the desired
product in 43% yield. LCMS SQ4 RXNMON_Acidic Rt=0.79 min, M+H
681.4; 1H NMR (METHANOL-d4, 400 MHz): d=7.33-7.41 (m, 4H),
7.27-7.33 (m, 1H), 5.08 (s, 2H), 4.48 (dd, J=7.8, 4.4 Hz, 1H), 4.29
(dd, J=7.9, 4.5 Hz, 1H), 4.12 (m, 1H), 3.57-3.67 (m, 8H), 3.49-3.57
(m, 4H), 3.34-3.43 (m, 4H), 3.15-3.23 (m, 1H), 2.92 (dd, J=12.8,
5.1 Hz, 1H), 2.70 (d, J=12.8 Hz, 1H), 2.26-2.36 (m, 2H), 2.21 (t,
J=7.4 Hz, 2H), 2.04 (m, 1H), 1.92 (m, 1H), 1.53-1.77 (m, 4H), 1.44
ppm (m, 2H).
Conjugation of Modifying Compounds to CRM.sub.197
Z-Q-G-NH-(PEG).sub.3-N.sub.3
##STR00108##
[0292] 32 .mu.L of CRM197 (32 mg/mL) is added to 1000 .mu.L of
Z-Q-G-NH-(PEG).sub.3-N.sub.3 (2 mg/mL) in 100 mM pH 8 Tris buffer
and 100 .mu.L of microbial transglutaminase (stock of 50 mg/mL in
PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 25.degree. C.
for 30 minutes. Reaction purified via SEC with a running buffer of
PBS 1.times. over 1.5 CV. One addition of the linker is observed by
Mass Spectrum. LCMS calculated: 58929. observed: (M+1) 58930.
Yield: 700 ug, 68% yield.
[0293] 32 .mu.L of CRM197 (32 mg/mL) is added to 1000 .mu.L of
Z-Q-G-NH-(PEG).sub.3-N.sub.3 (2 mg/mL) in 100 mM pH 8 Tris buffer
and 100 .mu.L of microbial transglutaminase (stock of 50 mg/mL in
PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 25.degree. C.
for 18 hours. Reaction purified via SEC with a running buffer of
PBS 1.times. over 1.5 CV. Two additions of the linker are observed
by Mass Spectrum. LCMS calculated: 59450. observed: (M+1) 59451.
Yield: 700 ug, 68% yield.
[0294] 32 uL of CRM197 (32 mg/mL) is added to 1000 .mu.L of
Z-Q-G-NH-(PEG).sub.3-N.sub.3 (2 mg/mL) in 100 mM pH 6 Sodium
acetate buffer and 100 uL of microbial transglutaminase (stock of
50 mg/mL in PBS 1.times. prepared from commercial 1% mTGase in
maltodextrin) is added. Reaction incubated at 25.degree. C. for 3
days. Reaction purified via SEC with a running buffer of PBS
1.times. over 1.5 CV. Three and four additions are observed by Mass
Spectrum. LCMS calculated: 59971, 60492; observed: (M+1) 59972,
60493. Yield: 700 ug, 68% yield.
ZQ-NH-(PEG).sub.3N.sub.3
##STR00109##
[0296] 63 .mu.L of CRM197 (32 mg/mL) is added to 18000 .mu.L of
ZQ-NH-(PEG).sub.3N.sub.3 (2 mg/mL) in 100 mM pH 8 Tris buffer and
150 uL of microbial transglutaminase (stock of 50 mg/mL in PBS
1.times. prepared from commercial 1% mTGase in maltocyclodextrin)
are added. Reaction incubated at 25.degree. C. for 1 hour. Reaction
purified via SEC with a running buffer of PBS 1.times. over 1.5 CV.
One addition of the linker is observed by Mass Spectrum. LCMS
calculated: 58872. observed: (M+1) 58875. Yield: 1.3 mg (67%).
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-Q-G
##STR00110##
[0298] 32 .mu.L of CRM197 (32 mg/mL) is added to 1000 .mu.L of
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-Q-G (2 mg/mL) in 100 mM
pH 8 Tris buffer and 100 .mu.L of microbial transglutaminase (stock
of 50 mg/mL in PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 25.degree. C.
for 3 hours. Reaction purified via SEC with a running buffer of PBS
1.times. over 1.5 CV
[0299] One addition of the linker is observed by Mass Spectrum.
LCMS calculated: 58771. observed: (M+1) 58771. Yield: 0.475 mg, 50%
yield.
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G
##STR00111##
[0301] 1 .mu.L of CRM197 (32 mg/mL) is added to 30 .mu.L of
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G (2 mg/mL) in 100 mM
pH 8 Tris buffer and 3 .mu.L of microbial transglutaminase (stock
of 50 mg/mL in PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 25.degree. C.
for 1 hour. One addition of the linker is observed by Mass
Spectrum. LCMS calculated: 58884. observed: (M+1) 58885.
[0302] 1 .mu.L of CRM197 (32 mg/mL) is added to 30 .mu.L of
Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-L-Q-G (2 mg/mL) in 100 mM
pH 8 Tris buffer and 3 .mu.L of microbial transglutaminase (stock
of 50 mg/mL in PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 25.degree. C.
for 24 hours. Two additions of the linker are observed by Mass
Spectrum. LCMS calculated: 59360. observed: (M+1) 59361.
Z-Q-NH--(CH.sub.2).sub.3-dimethylacetal
##STR00112##
[0304] 1 .mu.L of CRM197 (32 mg/mL) is added to 50 .mu.L of
Z-Q-NH--(CH.sub.2).sub.3-dimethylacetal (8 mg/mL) in 100 mM pH 8
Tris buffer and 3 uL of microbial transglutaminase (stock of 50
mg/mL in PBS 1.times. prepared from commercial 1% mTGase in
maltocyclodextrin) are added. Reaction incubated at 22.degree. C.
for 1 hour. One addition of the linker is observed by Mass
Spectrum. LCMS calculated: 58787; observed: (M+1) 58788.
Z-Q-NH--(CH.sub.2).sub.2--NH--C(O)--CH.sub.2-Alexafluor647
##STR00113##
[0306] 100 .mu.L of CRM197 (32 mg/mL) is added to 3000 .mu.L of
Z-Q-NH--(CH.sub.2).sub.2--NH--C(O)--CH.sub.2-Alexafluor647 (1
mg/mL) in 100 mM pH 8 Tris buffer and 300 uL of microbial
transglutaminase (stock of 50 mg/mL in PBS 1.times. prepared from
commercial 1% mTGase in maltocyclodextrin) are added. Reaction
incubated at 25.degree. C. for 18 hours. Reaction purified via SEC
with a running buffer of PBS 1.times. over 1.5 CV. One addition of
the Fluorophore is observed by Mass Spectrum. LCMS calculated:
59554; observed: (M+1) 59556 Yield: 2.6 mg, 81% yield.
Ac-L-Q-G-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00114##
[0308] To a solution of
Ac-L-Q-G-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
(50 .mu.L, 0.084 .mu.mol) (roughly 1 mg/mL) was added CRM (0.5
.mu.L, 0.00027 .mu.mol) and TGase (1.5 .mu.L, 1.97E-05 .mu.mol) and
mixed at 25.degree. C. for 1 hr. Reaction .about.60% complete. LCMS
calculated: 58987. observed: (M+1) 58988. Reaction taken directly
onto further modification with glutathione (procedure below).
Z-Q-NH--(CH.sub.2).sub.4--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00115##
[0310] 1 .mu.L of CRM197 (32 mg/mL) is added to 30 .mu.L of
Z-Q-NH--(CH.sub.2).sub.4--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol (1
mg/mL) in 100 mM pH 6 Sodium Acetate buffer and 3 .mu.L of
microbial transglutaminase (stock of 50 mg/mL in PBS 1.times.
prepared from commercial 1% mTGase in maltocyclodextrin) are added.
Reaction is incubated at 25.degree. C. for 3 hours. Reaction is
purified via a 10 kDa Amicon filter. One addition of the linker was
observed by Mass Spec. LCMS calculated: 58951. observed: (M+1)
58953.
Z-Q-NH-MenA Polysaccharide
##STR00116##
[0312] 156 .mu.L of CRM197 (32 mg/mL) is added to 5000 .mu.L of
Z-Q-NH-MenA Polysaccharide (1 mg/mL) in 100 mM pH 8 Tris buffer and
488 .mu.L of microbial transglutaminase (stock of 50 mg/mL in PBS
1.times. prepared from commercial 1% mTGase in maltocyclodextrin)
are added. Reaction incubated at 25.degree. C. for 18 hours.
Reaction purified via 50 kDa Amicon filter which resulted in a
final yield of 2 mg of product (38% yield). Product was confirmed
by SDS page (in above drawings paragraph 006) as the product is a
heterogeneous mixture due to the heterogeneity of the
polysaccharide.
Z-Q-NH--(CH.sub.2).sub.5--C(O)-monomethylauristatin F
##STR00117##
[0314] 50 .mu.L of CRM197 (32 mg/mL) is added to 1500 .mu.L of
Z-Q-NH--(CH.sub.2).sub.5--C(O)-monomethylauristatin F (1 mg/mL) in
100 mM pH 8 Tris buffer and 150 .mu.L of microbial transglutaminase
(stock of 50 mg/mL in PBS 1.times. prepared from commercial 1%
mTGase in maltocyclodextrin) are added. Reaction incubated at
25.degree. C. for 45 minutes. Reaction purified via SEC with a
running buffer of PBS 1.times. over 1.5 CV. One addition of MMAF is
observed on the Mass Spec. LCMS calculated: 59499 observed: 59498
Yield: 659 ug, 38% yield.
[0315] 60 .mu.L of CRM197 (32 mg/mL) is added to 1500 .mu.L of
Z-Q-NH--(CH.sub.2).sub.5--C(O)-monomethylauristatin F (1 mg/mL) in
100 mM pH 8 Tris buffer and 170 .mu.L of microbial transglutaminase
(stock of 50 mg/mL in PBS 1.times. prepared from commercial 1%
mTGase in maltocyclodextrin) are added. Reaction incubated at
25.degree. C. for 24 hours. An additional 60 .mu.L of mTGase was
added, and the reaction was incubated for another 18 hours.
Reaction purified via SEC with a running buffer of PBS 1.times.
over 1.5 CV. Two additions of MMAF are observed by the Mass
Spectrum. LCMS calculated: 60590. observed: 60589 Yield: 700 ug,
36% yield.
Phenol-(CH2)2-C(O)-L-Q-G
##STR00118##
[0317] 1 .mu.L of CRM197 (32 mg/mL) is added to 22 .mu.L of
Phenol-(CH2)2-C(O)-L-Q-G (0.5 mg/mL) in 100 mM pH 8 Tris buffer and
3 .mu.L of microbial transglutaminase (stock of 50 mg/mL in PBS
1.times. prepared from commercial 1% mTGase in maltocyclodextrin)
are added. Reaction incubated at 25.degree. C. for 1 hour. The
addition of one small molecule is observed by Mass Spec. Expected
Mass: 58856. Observed Mass: 58857.
Modification of GBS80 with Z-Q-G-NH-(PEG).sub.3-N.sub.3
##STR00119##
[0319] 2.32 mL GBS80 protein (3.49 mg/mL) was added to 14 mL
Z-Q-G-NH-(PEG).sub.3-N.sub.3 (8 mg/mL) in 100 mM sod acetate pH 6
and 50 .mu.L of mTGase (50 mg/ml in PBS) was added. Reaction was
incubated overnight at 37.degree. C. LCMS shows addition of 1 and 2
adducts and a small amount of +3. Reaction was quenched with 0.8 mL
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (10 mg/mL)
and incubated at rt for 1 hr. Reaction was then passed through zeba
spin column 3.times.. Recovered material was analyzed by LCMS
giving modified GBS80 in 78% overall yield. LCMS calculated: 53355,
53880, 54405. observed: (M+1) 53355, 53877, 54398.
mTGase-Mediated Labelling of CRM197+ZQ(PEG2)azidobenzylamide
##STR00120##
[0321] To a solution of ZQ(PEG2)azidobenzylamide (benzyl
(17-amino-1-(4-azidophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazaheptadecan-
-14-yl)carbamates) in tris buffer pH 8 (3.5 mg/mL, 86 .mu.L, 0.527
.mu.mol) was added CRM197 (33 mg/mL, 7.55 .mu.L, 0.0043 .mu.mol)
followed by a solution of transglutaminase enzyme in PBS (50 mg/mL,
7.61 .mu.L, 0.0100 .mu.mol). The reaction was stirred at 37.degree.
C. for 16 hours at which point LCMS analysis showed conversion to
+1, +2 and +3 products. LCMS QT2; Protein_35-70 kDa_3 min:
R.sub.t=1.48 min; MS [M+linker]: observed: 58958. calculated:
58962; MS [M+(2 linkers)]: observed: 59513. calculated: 59514; MS
[M+(3 linkers)]: observed: 60067. calculated: 60066.
TABLE-US-00002 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 58408 19 1.48 CRM197 + 1 ZQ-linker 58962 58958
36 1.48 CRM197 + 2 ZQ-linker 59514 59513 25 1.48 CRM197 + 3
ZQ-linker 60066 60067 20 1.48
mTGase-Mediated Labelling of
CRM197+ZQ(PEG).sub.2amidoethylmethyldiazirin
##STR00121##
[0323] To a solution of benzyl
(18-amino-1-(3-methyl-3H-diazirin-3-yl)-3,14,18-trioxo-7,10-dioxa-4,13-di-
azaoctadecan-15-yl)carbamate in tris buffer pH 8 (3.5 mg/mL, 50
.mu.L, 0.336 .mu.mol) was added CRM197 (33 mg/mL, 4.82 .mu.L,
0.0027 .mu.mol) followed by a solution of transglutaminase enzyme
in PBS (50 mg/mL, 4.85 .mu.L, 0.0064 .mu.mol). The reaction was
stirred at r.t. for two days at which point LCMS analysis showed
conversion to +1, +2 and +3 product. LCMS QT2; Protein_35-70 kDa_3
min: R.sub.t=1.69 min; MS [M+linker]: observed: 58912. calculated:
58913; MS [M+(2.times. linker)]: observed: 59415. calculated:
59416; MS [M+(3.times. linker)]: observed: 59918. calculated:
59919.
TABLE-US-00003 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 58408 11 1.69 CRM197 + 1 ZQ-linker 58913 58912
45 1.69 CRM197 + 2 ZQ-linker 59416 59415 32 1.69 CRM197 + 3
ZQ-linker 59919 59918 12 1.69
mTGase-Mediated Labelling of CRM197+ZQ-FA
##STR00122##
[0325] To a solution of ZQ-FA in 100 mM tris buffer pH 8 (8 mg/mL,
203 .mu.L, 1.316 .mu.mol) was added CRM197 (33 mg/mL, 1.515 .mu.L,
0.00086 .mu.mol) followed by a solution of transglutaminase enzyme
in PBS (50 mg/mL, 0.455 .mu.L, 0.00060 .mu.mol). The reaction was
stirred at r.t. for 16 hours. The reaction mixture was exchanged
into 100 mM tris buffer pH 8 using 10 kDa MWCO Amicon centrifugal
filter by diluting and concentrating the reaction 5 times to a
volume of 100 pt. LCMS analysis showed conversion to +1, +2, +3 and
+4 products. LCMS QT2; Protein_35-70 kDa_3 min: R.sub.t=1.45 min;
MS [M+ZQ-FA]: observed: 59625. calculated: 59624; MS [M+(2.times.
ZQ-FA)]: observed: 60839. calculated: 60838; MS
[M+(3.times.ZQ-FA)]: observed: 62054. calculated: 62052; MS
[M+(4.times.ZQ-FA)]: observed: 63270. calculated: 63266.
TABLE-US-00004 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 n/a 0 n/a CRM197 + 1 ZQ-FA 59624 59625 14 1.45
CRM197 + 2 ZQ-FA 60838 60839 23 1.45 CRM197 + 3 ZQ-FA 62052 62054
35 1.45 CRM197 + 4 ZQ-FA 63266 63270 28 1.45
mTGase-Mediated Labelling of CRM197+Azido Nitrophenyl QG
[0326] To a solution of azidonitrophenyl-QG in 100 mM tris buffer
pH 8 (8 mg/mL, 100 .mu.L) was added CRM197 (33 mg/mL, 1.0 .mu.L, 33
ug) followed by a solution of transglutaminase enzyme in PBS (50
mg/mL, 1.0 .mu.L, 0.1% TGase in maltocyclodextrin). The reaction
was incubated at r.t. for 16 hours. LCMS analysis showed conversion
to +1, +2, and +3 product. LCMS QT1; Protein_20-70 kDa_3 min:
R.sub.t=1.67 min; MS [M+1 azido nitrophenyl QG]: observed: 58815.
calculated: 58803; MS [M+2 azido nitrophenyl QG]: observed: 59191.
calculated: 59196; MS [M+3 azido nitrophenyl QG]: observed: 59585.
calculated: 59589.
TABLE-US-00005 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 58428 5 n/a CRM197 + 1 58803 58815 50 1.67
azidonitrophenylQG CRM197 + 2 59196 59191 40 1.67
azidonitrophenylQG CRM197 + 3 59589 59585 5 1.67
azidonitrophenylQG
mTGase-Mediated Labelling of CRM197+Diazirine-QG
[0327] To a solution of diazirine-QG in 100 mM tris buffer pH 8 (8
mg/mL, 100 .mu.L) was added CRM197 (33 mg/mL, 1.0 .mu.L, 33 ug)
followed by a solution of transglutaminase enzyme in PBS (50 mg/mL,
1.0 .mu.L, 0.1% TGase in maltocyclodextrin). The reaction was
incubated at r.t. for 2 hours. LCMS analysis showed conversion to
+1 product. LCMS QT1; Protein_20-70 kDa_3 min: R.sub.t=1.67 min; MS
[M+diazirine-QG]: observed: 58705. calculated: 59706.
TABLE-US-00006 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 n/a 0 n/a CRM197 + 1 diazirine-QG 58706 58705
100 1.67
mTGase-Mediated Labelling of CRM197+ZQ(PEG).sub.3Biotin
[0328] To a solution of ZQ(PEG).sub.3Biotin in 100 mM tris buffer
pH 8 (8 mg/mL, 100 .mu.L) was added CRM197 (33 mg/mL, 1.0 .mu.L, 33
ug) followed by a solution of transglutaminase enzyme in PBS (50
mg/mL, 1.0 .mu.L, 0.1% TGase in maltocyclodextrin). The reaction
was incubated at r.t. for 16 hours. LCMS analysis showed conversion
to +1 product. LCMS QT1; Protein_20-70 kDa_3 min: R.sub.t=1.67 min;
MS [M+1 ZQ(PEG).sub.3Biotin]: observed: 59073. calculated: 59074;
MS [M+2 ZQ(PEG).sub.3Biotin]: observed: 59737. calculated: 59738;
MS [M+3 ZQ(PEG).sub.3Biotin]: observed: 60403. calculated:
60402.
TABLE-US-00007 Degree of Labelling Calculated Observed % R.sub.t
(min) CRM197 58410 none 0 n/a CRM197 + 1 ZQ(PEG).sub.3Biotin 59074
59073 40 1.75 CRM197 + 2 ZQ(PEG).sub.3Biotin 59738 59737 55 1.75
CRM197 + 3 ZQ(PEG).sub.3Biotin 60402 60403 5 1.75
Examples of functionalization of labeled mTGase catalyzed selective
lysine labeling of proteins:
Z-Q-G-NH-(PEG).sub.3-N.sub.3
##STR00123##
[0330] 3.2 mg of azido labeled protein was combined with
polysaccharide with a conjugation ratio of PS/Prot 6:1 w/w. Product
purified by 2.times. HA column. First run (BLOCK 1=NaPi 2 mM pH
7.2, BLOCK 2=NaPi 400 mM pH 7.2) removes free protein. Second run
(BLOCK 1=NaPi 2 mM/NaCl 550 mM pH 7.2, BLOCK 2=NaPi 10 mM pH 7.2,
BLOCK 3=NaPi 35 mM pH 7.2, BLOCK 4=NaPi 400 mM pH 7.2) removes free
polysaccharide.
##STR00124##
[0331] 3.3 mg of azido labeled protein was combined with
polysaccharide with a conjugation ratio PS/Prot 6:1 w/w. Product
purified by 2.times. HA column. First run (BLOCK 1=NaPi 2 mM pH
7.2, BLOCK 2=NaPi 400 mM pH 7.2) removes free protein. Second run
(BLOCK 1=NaPi 2 mM/NaCl 550 mM pH 7.2, BLOCK 2=NaPi 10 mM pH 7.2,
BLOCK 3=NaPi 35 mM pH 7.2, BLOCK 4=NaPi 400 mM pH 7.2) removes free
polysaccharide.
[0332] As shown in FIGS. 2-4, SDS page gel characterization of
products of these experiments were obtained. The respective yields
are also shown in Table 3 below.
TABLE-US-00008 TABLE 3 Saccharide/protein Sacch/ Free used for
Yield Conjugation Prot saccharide Protein conjugation (% final
Sample Protein chemistry (w/w) % (dionex) TOT mg (w/w) protein) GBS
PSV(alk)- X CFCC 4.3 6.7 570.7 6:1 20.6 GBS80(K-N3) GBS PSII(alk)-
X CFCC 1.5 <3.3 1685.7 6:1 24.0 GBS80(K-N3)
[0333] Each of these clicked GBS80 conjugates obtained through the
mTGase labeling method were tested biologically assays discussed
below.
ELISA Immuno Assay for Determination of Ig Titers Against GBS II or
V Polysaccharide Antigens
[0334] IgG titers against GBS polysaccharides II or V in the sera
from immunized animals were measured as follows.
[0335] Microtiter plates (Nunc Maxisorp) were coated with 100 .mu.l
of 1.0 .mu.g/mL HSA-adh (Human Serum Albumin-adipic acid
dihydrazide) conjugated polysaccharides II or V in Phosphate
Buffered Saline (PBS). The plate was incubated overnight at room
temperature and then washed three times in washing buffer (0.05%
Tween 20 in PBS). After dispensing 250 .mu.l of PBS, 2% BSA, 0.05%
Tween 20 per well, plates were incubated 90 minutes at 37.degree.
C. and then aspirated to remove the post-coating solution. Test
sera were diluted 1:400 in PBS, 2% BSA, 0.05% Tween 20. Standard
serum was prepared by pooling hyper immune sera and initial
dilutions of standard pools were chosen to obtain an optical
density (OD) of about 2.000 at 405 nm. The plates were incubated
for 1 hour at 37.degree. C. and then washed with washing buffer and
100 .mu.L of Alkaline Phosphatase-Conjugated anti-mouse IgG 1:1000
in dilution buffer were dispensed in each well. The plates were
incubated 90 minutes at 37.degree. C. and then washed with washing
buffer. 100 .mu.L of a solution of p-NitroPhenylPhosphate (p-NPP)
4.0 mg/mL in substrate buffer were dispensed in each well. The
plates were incubated 30 minutes at room temperature and then 100
.mu.L of a solution of EDTA 7% (w/v) disodium salt plus
Na.sub.2HPO.sub.4 3.5% pH 8.0, were added to each well to stop the
enzymatic reaction. The optical density (OD) at 405 nm was
measured. Total IgG titres against GBS polysaccharide antigens (II
or V) were calculated by using the Reference Line Assay Method and
results were expressed as arbitrary ELISA Units/mL (EU/mL). For
each of the three antigens, the standard serum IgG titer was
arbitrarily assigned a value of 1.0 EU/mL. The IgG titer of each
serum was estimated by interpolating the obtained ODs with the
titration curve (bias and slope) of the standard pool. Results are
displayed in FIGS. 5 and 6.
Mouse Active Maternal Immunization Model
[0336] Groups of eight CD-1 female mice (age, 6-8 weeks) were
immunized on days 1, 21, and 35 with 20 .mu.g of antigen or buffer
(PBS) formulated in alum adjuvant. Mice were then mated, and their
offspring were challenged intraperitoneally with a GBS dose
calculated to induce dead in 90% of the pups. Protection values
were calculated as [(% dead in control-% dead in vaccine)/% dead in
control].times.100. Mice were monitored on a daily basis and killed
when they exhibited defined humane endpoints that had been
pre-established for the study in agreement with Novartis Animal
Welfare Policies. Statistical analysis was performed using Fisher's
exact test. Results are displayed in Tables 4 and 5 below.
TABLE-US-00009 TABLE 4 Antigens Protected\Treated % Protection PBS
18/60 30 CRM-II 32/50 64 TT-II 19/30 63 GBS80-II 37/70 53
GBS59-1523-II 59/70 84 -- GBS80-K-N3/PSII 58/69 84 challenge strain
type II 5401
TABLE-US-00010 TABLE 5 Antigens Protected\Treated % Protection PBS
19/40 47 CRM-V 61/70 87 TT-V -- -- GBS80-V 54/57 95 GBS59-1523-V
69/79 87 GBS80-K- 53/60 88 N3/PSV challenge strain type V
CJB111
Opsonophagocytosis Assay
[0337] The opsonophagocytosis assay was performed using GBS strains
as target cells and HL-60 cell line (ATCC; CCL-240), differentiated
into granulocyte-like cells, by adding 100 mM N, N
dimethylformamide (Sigma) to the growth medium for 4 d.
Mid-exponential bacterial cells were incubated at 37.degree. C. for
1 h in the presence of phagocytic cells, 10% baby rabbit complement
(Cedarlane), and heat-inactivated mouse antisera. Negative controls
consisted of reactions either with preimmune sera, or without
HL-60, or with heat-inactivated complement. The amount of
opsonophagocytic killing was determined by subtracting the log of
the number of colonies surviving the 1-h assay from the log of the
number of CFU at the zero time point.
[0338] Results of the experiments are shown in FIG. 7.
GBS80-K-N.sub.3/PSII OPKA and IgG titers are statistically
comparable to GBS80-II conjugate made by random K conjugation. OPKA
and IgG titers show good correlation with % of survival in
challenge animal model.
Ac-L-Q-G-NH--(CH.sub.2).sub.2--C(O)--(CH.sub.2).sub.2--SO.sub.2-Tol
##STR00125##
[0340] L-Glutathione (5 .mu.L, 0.813 umol) was added followed by 50
.mu.L of 250 mM Tris HCl buffer pH 8, raising the reaction pH to 8.
After 4 hours, all of CRM was labeled with one linker as confirmed
by mass spectrometry characterization. After another 16 hours at
25.degree. C., all of CRM was labeled with L-Glutathione. Addition
of the L-Glutathione: Expected Mass: 59138, Observed Mass:
59139.
Peptide Mapping Experimental Summary:
[0341] Peptide Mapping Digestion: 5 .mu.g modified CRM197 and
positive control CRM197 samples were reduced with 20 mM DTT and
digested with 1/30 (w/w) enzyme/protein at 26.degree. C. overnight
with trypsin. An aliquot of trypsin digested protein was further
digested with GluC enzyme at 1/20 enzyme/protein ratio for 4 hr at
26.degree. C.; note all enzymes purchased from Roche Diagnostics
(Gmbh, Germany).
[0342] Reverse Phase LC-MS/MS Analysis: Resulting digested peptides
were analyzed by liquid chromatography electrospray tandem mass
spectrometry (LC-ESI MS/MS) on a Thermo LTQ Orbitrap Discovery
(Thermo Fisher Scientific Inc., Waltham, Mass.) coupled to Agilent
CapLC (Santa Clara, Calif.). Loaded .about.10-15 pmole of CRM
control and modified CRM197 digests on column at 40.degree. C.
(Waters Acuity BEH C18, 1.7 .mu.m, 1.times.100 mm column) Ran 80
min total gradient at 10 .mu.L/min stating at 0-1 min, 4% B,
increased to 7% B at 1.1 min, 45% B at 55 min, then 95% B at 63
min, followed by washing and column equilibration. Mass
spectrometer parameters included a full scan event using the FTMS
analyzer at 30000 resolution from m/z 300-2000 for 30 ms. Collision
Induced Dissociation MS/MS was conducted on the top seven intense
ions (excluding 1+ ions) in the ion trap analyzer, activated at 500
(for all events) signal intensity threshold counts for 30 ms.
[0343] Data Analysis and Database Searching: All mass spectra were
processed in Qual Browser V 2.0.7 (Thermo Scientific). Mascot
generic files (mgf) were generated with MS DeconTools (R.D. Smith
Lab, PPNL) and searched using Mascot V2.3.01 (Matrix Science Inc.,
Boston, Mass.) database search against the provided protein
sequence added to an in-house custom database and the SwissProt
database (V57 with 513,877 sequences) for contaminating proteins.
Search parameters included: enzyme: semitrypsin or trypsin/Glu-C,
allowed up to three missed cleavage; variable modifications: added
expected masses of small molecules (362.147787 Da and 463.206698
Da) to database called "CRM Tgase+alkyne 362 Da mod (CKR), CRM
Tgase+alkyne 362 Da mod (N-term), CRM Tgase+azide 463 Da mod (CKR),
CRM Tgase+azide 463 Da mod (N-term)"; peptide tolerance: .+-.20
ppm; MS/MS tolerance: .+-.0.6 Da. Sequence coverage and small
molecule modification assessments were done on ions scores with
>95% confidence. High-scoring peptide ions were then selected
for manual MS/MS analysis using Qual Browser.
[0344] Results for
CRM+Cyclooctyne-cyclopropyl-CH.sub.2--OC(O)NH-Q-G
[0345] Trypsin digest: 83% sequence coverage; No modification
detected at this ion score threshold. Trypsin/GluC digest: 97%
sequence coverage; Modification detected on Lys37 or Lys39.
TABLE-US-00011 CRM Exp095 Trypsin/GluC Digestion Sequence Coverage:
91%, Matched peptides shown in Bold Text K37 or K39 Modified with
CRM Tgase + azide 463 Da mod (CKR) SEQ ID NO: 1 1
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQ P SGTQGNYDDDW 51
KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAE 101
TIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI 151
NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLS 201
CINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEF 251
HQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKT 301
TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL 351
VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT 401
VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHI 451
SVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH 501
SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS
[0346] Results for CRM+ZQ-NH-(PEG).sub.3N.sub.3
[0347] Trypsin digest: 69% sequence coverage; No modification
detected at this ion score threshold. Trypsin/GluC digest: 91%
sequence coverage; Modification detected on Lys37 or Lys39.
TABLE-US-00012 CRM Exp083 Trypsin/GluC Digestion Sequence Coverage:
97%, Matched peptides thown in Bold Text K37 or K39 Modified with
CRM Tgase + alkyne 362 Da mod (CKR) SEQ ID NO: 2 1
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQ P SGTQGNYDDDW 51
KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAE 101
TIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI 151
NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLS 201
CINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEF 251
HQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKT 301
TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL 351
VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT 401
VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHI 451
SVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH 501
SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS
CRM Control
[0348] Trypsin digest: 85% sequence coverage.
Trypsin/GluC digest: 79% sequence coverage.
[0349] It is understood that the invention is not limited to the
embodiments set forth herein for illustration, but embraces all
such forms thereof as come within the scope of the above
disclosure.
Prophetic Examples
[0350] Benzyl
(17-amino-1-(4-azido2-nitrophenyl)-2,13,17-trioxo-6,9-dioxa-3,12-diazahep-
tadecan-14-yl)carbamate
##STR00126##
[0351] 4-azido-2-nitrophenylacetic acid N-succinimido ester (0.073
mmol) is dissolved in DMF (1 mL) and combined with a solution of
benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl-
)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL,
0.585 mmol) is added and the reaction is mixed at r.t. for 4 hours.
The solution is purified via MS-triggered HPLC (100-Prep3;
Acid_Method 3; Sunfire 30.times.50 mm Sum column ACN/H.sub.2O
w/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570).
Fractions with desired product are pooled and lyophilized.
ZQ-(PEG2) phenyl trifluoromethyldiazirine
##STR00127##
[0353] 4-trifluoromethyl diazirine phenylacetic acid N-succinimido
ester (0.073 mmol) is dissolved in DMF (1 mL) and combined with a
solution of benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopent-
an-2-yl)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121
mL, 0.585 mmol) is added and the reaction is mixed at r.t. for 4
hours. The solution is purified via MS-triggered HPLC (100-Prep3;
Acid_Method 3; Sunfire 30.times.50 mm Sum column ACN/H.sub.2O
w/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570).
Fractions with desired product are pooled and lyophilized.
ZQ-(PEG2)-tetrazine
##STR00128##
[0355] Diazirine N-succinimido ester (0.073 mmol) is dissolved in
DMF (1 mL) and combined with a solution of benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl-
)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL,
0.585 mmol) is added and the reaction is mixed at r.t. for 4 hours.
The solution is purified via MS-triggered HPLC (100-Prep3;
Acid_Method 3; Sunfire 30.times.50 mm Sum column ACN/H.sub.2O
w/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570).
Fractions with desired product are pooled and lyophilized.
ZQ-(PEG2)-tetrazine
##STR00129##
[0357] Tetrazine (PEG)N-succinimido ester (0.073 mmol) is dissolved
in DMF (1 mL) and combined with a solution of benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl-
)carbamate (20 mg, 0.049 mmol) in DMF (2.3 mL). DIPEA (0.121 mL,
0.585 mmol) is added and the reaction is mixed at r.t. for 4 hours.
The solution is purified via MS-triggered HPLC (100-Prep3;
Acid_Method 3; Sunfire 30.times.50 mm Sum column ACN/H.sub.2O
w/0.1% TFA 75 ml/min, 1.5 ml injection; Tube Trigger M=570).
Fractions with desired product are pooled and lyophilized.
ZQ(PEG2)-(3aR,4S,7R)-3a,4,7,7a-tetrahydro-4,7-methanoisobenzofuran-1,3-dio-
ne
##STR00130##
[0359]
(3aR,4S,7R)-3a,4,7,7a-Tetrahydro-4,7-methanoisobenzofuran-1,3-dione
(0.162 mmol) (0.073 mmol) is dissolved in DMF (1 mL) and combined
with a solution of benzyl
(5-amino-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1,5-dioxopentan-2-yl-
)carbamate (0.049 mmol) in DMF (2.3 mL). DIPEA (0.585 mmol) is
added and the reaction is mixed at r.t. for 4 hours. The solution
is purified via MS-triggered HPLC (100-Prep3; Acid_Method 3;
Sunfire 30.times.50 mm Sum column ACN/H.sub.2O w/0.1% TFA 75
ml/min, 1.5 ml injection; Tube Trigger M=570). Fractions with
desired product are pooled and lyophilized.
Tetrazine-QG
##STR00131##
[0361] QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and NHS tetrazine (0.148 mmol) is added in H.sub.2O
(Volume: 1.000 mL, Ratio: 1.000) followed by addition of DIPEA
(0.177 mmol). The reaction stirs for 16 hours at which time the
product is purified by HPLC (Sunfire 30.times.50 mm 5 um column
ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) to give the desired
product. Fractions are pooled and lyophilized.
Tetrazine(PEG).sub.nQG
##STR00132##
[0363] QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and NHS PEG.sub.n tetrazine (0.148 mmol) is added in
H.sub.2O (Volume: 1.000 mL, Ratio: 1.000) followed by addition of
DIPEA (0.177 mmol). The reaction stirs for 16 hours at which time
the product is purified by HPLC (Sunfire 30.times.50 mm 5 um column
ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) to give the desired
product. Fractions are pooled and lyophilized.
[0364] QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and NHS tetrazine (0.148 mmol) is added in H.sub.2O
(Volume: 1.000 mL, Ratio: 1.000) followed by addition of DIPEA
(0.177 mmol). The reaction stirs for 16 hours at which time the
product is purified by HPLC (Sunfire 30.times.50 mm Sum column
ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml injection) to give the desired
product. Fractions are pooled and lyophilized.
4-Azido-phenyl-glutamine-glycine
##STR00133##
[0366] QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and 4-azido-phenylacetic acid N-succinimido ester
(0.148 mmol) is added in H.sub.2O (Volume: 1.000 mL, Ratio: 1.000)
followed by addition of DIPEA (0.177 mmol). The reaction stirs for
16 hours at which time the product is purified by HPLC (Sunfire
30.times.50 mm Sum column ACN/H2O w/0.1% TFA 75 ml/min 1.5 ml
injection) to give the desired product. Desired fractions are
pooled and lyophilized.
Trifluoromethyldiazirine-benzyl-glutamine-glycine
##STR00134##
[0368] QG (30 mg, 0.148 mmol) is dissolved in DMF (Volume: 1 mL,
Ratio: 1.000) and 4-trifluoromethyl-diazirine-phenylacetic acid
N-succinimido ester (0.148 mmol) is added in H.sub.2O (Volume:
1.000 mL, Ratio: 1.000) followed by addition of DIPEA (0.177 mmol).
The reaction stirs for 16 hours at which time the product is
purified by HPLC (Sunfire 30.times.50 mm Sum column ACN/H2O w/0.1%
TFA 75 ml/min 1.5 ml injection) to give the desired product.
Desired fractions are pooled and lyophilized.
General Procedure for mTGase-Mediated Labelling of CRM197
[0369] To a solution of linker in tris buffer pH 8 (3.5 mg/mL,
0.527 .mu.mol or other amount and relative micromolar concentration
depending on the linker identified above) is added CRM197 (33
mg/mL, 7.55 .mu.L, 0.0043 .mu.mol) followed by a solution of
transglutaminase enzyme in PBS (50 mg/mL, 7.61 .mu.L, 0.0100
.mu.mol). The reaction is stirred at rt or 37.degree. C. for 16
hours.
[0370] Having thus described exemplary embodiments of the present
invention, it should be noted by those of ordinary skill in the art
that the within disclosures are exemplary only and that various
other alternatives, adaptations, and modifications may be made
within the scope of the present invention. Accordingly, the present
invention is not limited to the specific embodiments as illustrated
therein.
Sequence CWU 1
1
21535PRTCorynebacterium diphtheriae 1Gly Ala Asp Asp Val Val Asp
Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser Tyr His
Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys Gly Ile
Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40 45 Asp
Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 50 55
60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val
65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu
Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu
Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr Glu Glu
Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val Val Leu
Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu Tyr Ile
Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val Glu Leu
Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170 175 Ala
Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 180 185
190 Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val
195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu
His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys
Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu
Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu Leu Ser Glu Leu
Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe Ala Gly Ala Asn
Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285 Ile Asp Ser
Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295 300 Ser
Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 305 310
315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu
Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu
Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser
Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn Ser Tyr Asn Arg
Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln Pro Phe Leu His
Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400 Val Glu Asp Ser
Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410 415 Asp Ile
Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 420 425 430
Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr 435
440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala
Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val
Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn Leu His Val Ala
Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His Ser Asn Glu Ile
Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr Gln Lys Thr Val
Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525 Leu Phe Phe Glu
Ile Lys Ser 530 535 2535PRTCorynebacterium diphtheriae 2Gly Ala Asp
Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe
Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25
30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp
35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala
Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys
Ala Gly Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys
Val Leu Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys
Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val
Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser
Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser
Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155
160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp
165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg
Val Arg 180 185 190 Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu
Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu
Ser Leu Lys Glu His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu
Ser Pro Asn Lys Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln
Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu
Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe
Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280
285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp
Gly Ala 305 310 315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln
Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro
Leu Val Gly Glu Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn
Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn
Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln
Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400
Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405
410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly
Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys
Ser Lys Thr 435 440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met
Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro
Lys Ser Pro Val Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn
Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His
Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr
Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525
Leu Phe Phe Glu Ile Lys Ser 530 535
* * * * *