U.S. patent application number 10/146182 was filed with the patent office on 2004-03-25 for methods of enhancing bioactivity of chemokines.
This patent application is currently assigned to SmithKline Beecham Corporation. Invention is credited to Balcarek, Joanna Maria, Bhatnagar, Pradip Kumar, King, Andrew Garrison, Pelus, Louis Martin.
Application Number | 20040057925 10/146182 |
Document ID | / |
Family ID | 22115875 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040057925 |
Kind Code |
A1 |
Pelus, Louis Martin ; et
al. |
March 25, 2004 |
Methods of enhancing bioactivity of chemokines
Abstract
The present invention provides method of increasing the
biological activity of KC, gro-.alpha., gro-.beta., and gro-.gamma.
proteins, truncated and modified proteins characterized by having
biological activity at least 1 log better than the full-length
protein, and pharmaceutical compositions containing same.
Inventors: |
Pelus, Louis Martin;
(Richboro, PA) ; Bhatnagar, Pradip Kumar; (Exton,
PA) ; King, Andrew Garrison; (Blue Bell, PA) ;
Balcarek, Joanna Maria; (Bala Cynwyd, PA) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham
Corporation
|
Family ID: |
22115875 |
Appl. No.: |
10/146182 |
Filed: |
May 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10146182 |
May 15, 2002 |
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09467160 |
Dec 20, 1999 |
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6399053 |
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09467160 |
Dec 20, 1999 |
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08557142 |
Mar 5, 1996 |
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6080398 |
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08557142 |
Mar 5, 1996 |
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PCT/US94/06264 |
Jun 3, 1994 |
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08557142 |
Mar 5, 1996 |
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08073800 |
Jun 8, 1993 |
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Current U.S.
Class: |
424/85.1 ;
435/320.1; 435/325; 435/69.5; 530/351; 536/23.5 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 7/06 20130101; A61P 7/00 20180101; C07K 5/0819 20130101; C07K
7/08 20130101; C07K 14/522 20130101; A61P 43/00 20180101; A61K
38/00 20130101 |
Class at
Publication: |
424/085.1 ;
530/351; 435/069.5; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 038/19; C07K
014/52; C07H 021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. A modified chemokine characterized by truncation of between
about 2 to about 8 amino acids at the amino terminus of a mature
chemokine and by at least a log higher biological activity than the
mature chemokine.
2. The protein according to claim 1 wherein said mature chemokine
is selected from the group consisting of KC, gro-.beta.,
gro-.gamma., and gro-.alpha., said chemokines of mammalian
origin.
3. The modified chemokine according to claim 1 comprising the amino
acid sequence of the mature KC protein having its amino terminus
truncated at a position between amino acid residues #2 through 8 of
SEQ ID NO: 1, wherein said modified chemokine is characterized by
having biological activity.
4. The modifed chemokine according to claim 3 consisting
essentially of amino acids 5-72 of SEQ ID NO: 1.
5. The modified chemokine according to claim 1 comprising the amino
acid sequence of mature gro-.beta. protein truncated at its N
terminus between amino acid positions 2 and 8 of SEQ ID NO: 3.
6. The modified chemokine according to claim 5 consisting
essentially of amino acids 5 to 73 of SEQ ID NO: 3.
7. The modified chemokine according to claim 1 comprising the amino
acid sequence of mature gro-.alpha. protein truncated at its N
terminus between amino acid positions 2 and 8 of SEQ ID NO: 2.
8. The modified chemokine according to claim 7 consisting
essentially of amino acids 5 to 73 of SEQ ID NO: 2.
9. The modified chemokine according to claim 1 comprising the amino
acid sequence of mature gro-.gamma. protein truncated at its N
terminus between amino acid positions 2 and 8 of SEQ ID NO: 4.
10. The modified chemokine according to claim 9 consisting
essentially of amino acids 5 to 73 of SEQ ID NO: 4.
11. A modified chemokine which is characterized by truncation of
between about two to about 8 amino acids at the carboxy terminus of
the mature chemokine and by at least a log higher biological
activity than the mature chemokine.
12. The modified chemokine according to claim 11 comprising the
amino acid sequence of the mature KC protein having its carboxy
terminus truncated at a position between amino acid residues #58
through 70 of SEQ ID NO: 1, wherein said modified KC is
characterized by having biological activity.
13. The modified chemokine according to claim 12 consisting
essentially of amino acids 1 to 68 of SEQ ID NO: 1.
14. A multimeric protein which comprises an association of one or
more modified chemokines selected from the group consisting of (a)
a modified chemokine characterized by truncation of between about 2
to about 8 amino acids at the amino terminus of the mature
chemokine and by at a log greater biological activity than that of
the mature chemokine; and (b) a modified chemokine which is
characterized by truncation of between about 2 to about 10 amino
acids at the carboxy terminus of the mature chemokine and by at
least a log greater biological activity than that of the mature
chemokine.
15. The protein according to claim 14 comprising multiple copies of
the same modified chemokine.
16. The protein according to claim 14 comprising at least two
different modified chemokines.
17. The protein according to claim 14 wherein said second chemokine
comprises a mature chemokine.
18. A nucleic acid sequence comprising the sequence encoding a
modified chemokine selected from the group consisting of (a) a
modified chemokine characterized by truncation of between about 2
to about 8 amino acids at the amino terminus of the mature protein
and by at least the biological activity of the mature protein; (b)
a modified chemokine which is characterized by truncation of
between about 2 to about 10 amino acids at the carboxy terminus of
the mature protein and by at least the biological activity of the
mature protein; and (c) a multimeric protein comprising at least
one of the proteins (a) or (b), optionally in association with a
second chemokine.
19. A plasmid comprising the nucleic acid sequence according to
claim 18 under the control of selected regulatory sequences capable
of directing the replication and expression thereof in a host
cell.
20. A host cell transfected with a plasmid of claim 19.
21. A method of producing a modified chemokine comprising culturing
a host cell of claim 20 and isolating the chemokine from the cell
or cell culture.
22. A method of enhancing the biological activity of a chemokine by
performing at least one of the steps comprising: (a) removing from
the amino terminus of the mature protein about 2 to about 8 of the
mature chemokine amino acid residues; and (b) removing from the
carboxy terminus of the mature protein about 2 to about 10 of the
mature chemokine amino acid residues.
23. A pharmaceutical composition comprising a modified chemokine
selected from the group consisting of (a) a modified chemokine
characterized by truncation of between about two to about 8 amino
acids at the amino terminus of the mature protein and by at least a
log greater biological activity than that the full-length mature
chemokine; (b) a modified chemokine which is characterized by
truncation of between about two to about 10 amino acids at the
carboxy terminus of the mature protein and by at least the
biological activity of the full-length mature chemokine; and (c) a
multimeric protein comprising at least one of the chemokines (a) or
(b) optionally in association with a second chemokine, in a
suitable pharmaceutical carrier.
24. A method for treating an inflammatory condition comprising
administering to a mammalian subject characterized by said
condition an effective amount of a pharmaceutical composition of
claim 23.
25. A method of stimulating the growth and/or differentiation of
bone marrow cells in a mammal, said improvement comprising:
administering to said mammal a modified chemokine according to
claim 1 or 11.
26. An improved method of stimulating maturation of hematopoietic
precursor cells in a mammal, said improvement comprising
administering to said mammal a modified chemokine according to
claim 1 or 11 .
27. An improved method of stimulating the growth and/or
differentiation of bone marrow cells in a mammal, said improvement
comprising administering to said mammal a mixture of GM-CSF and a
modified chemokine according to claim 1 or 11, wherein said mixture
is characterized by having a synergistic effect.
28. An antibody capable of selectively binding to a modified
chemokine-according to claim 1.
29. An antibody capable of selectively binding to a modified
chemokine according to claim 11.
30. A method for monitoring the circulating level of a selected
agent characterized by the ability to induce a hematopoietic
synergistic factor in a mammal comprising: contacting a blood
sample from the mammal with an antibody according to claim 28 or 29
, measuring levels of circulating HSF, and comparing said
circulating HSF levels following administration of said agent to
circulating HSF levels prior to administration of said agent.
31. The method according to claim 30 wherein said antibody is
optionally associated with a detectable label.
32. A method of inducing a hematopoietic synergistic factor in vivo
comprising administering to a selected mammal a compound of the
following formula: 5wherein: Y.sub.1 and Y.sub.2 are independently
CH.sub.2 or S; x is 0, 1, 2, 3, or 4; m is 0, 1, or 2; n is 0, 1,
or 2; A is pyroglutamic acid, proline, glutamine, tyrosine,
glutamic acid, 2-thiophene carboxylic acid, picolinic acid,
cyclohexane carboxylic acid, tetrahydro-2-furoic acid,
tetrahydro-3-furoic acid, 2-oxo-4-thiazolidine, cyclopentane,
3-thiophene carboxylic acid, 5-oxo-2-tetrahydrofurancarboxy- lic
acid, and pipecolinic acid; B is serine, glutamic acid, tyrosine or
aspartic acid; C is glutamic acid, tyrosine or aspartic acid; D is
lysine, arginine, tyrosine, N-methylarginine, aspartic acid,
ornithine or diaminohexynoic acid; or the carboxyamide, or hydroxy
methyl derivative thereof; E is glutamic acid, aspartic acid,
tyrosine or a peptide bond; F is tyrosine or a peptide bond;
provided that: when Y.sub.1 and Y.sub.2 are S, x is 2, 3 or 4 and m
and n are 1; or when Y.sub.1 and Y.sub.2 are CH.sub.2, x is 0, 1 or
2 and m and n are 0; or when Y.sub.1 is S and Y.sub.2 is CH.sub.2,
x is 0 and n is 1; or when Y.sub.2 is S and Y.sub.1 is CH.sub.2, x
is 0 and m is 1; or a pharmaceutically acceptable salt thereof.
33. The method according to claim 32 wherein the peptide is
selected from the group consisting of:
(pGlu-Glu-Asp).sub.2Sub(Lys).sub.2 SEQ ID NO: 5
(pGlu-Glu-Asp).sub.2Adp(Lys).sub.2 SEQ ID NO: 6
(pGlu-Glu-Glu).sub.2Sub(L- ys).sub.2 SEQ ID NO: 7
(pGlu-Asp-Asp).sub.2Sub(Lys).sub.2 SEQ ID NO: 8
(Pic-Glu-Asp).sub.2Sub(Lys).sub.2 SEQ ID NO: 9
(L-Ppc-Glu-Asp).sub.2Sub(L- ys).sub.2 SEQ ID NO: 10
(pGlu-Ser-Asp).sub.2Sub(Lys).sub.2 SEQ ID NO: 11
(pGlu-Ser-Asp).sub.2Adp(Lys).sub.2 SEQ ID NO: 12
(pGlu-Ser-Asp).sub.2Adp(- Lys-NH.sub.2).sub.2 SEQ ID NO: 13
(Pic-Ser-Asp).sub.2Adp(Lys).sub.2 SEQ ID NO: 14
(Pic-Ser-Asp).sub.2Adp(Lys-NH.sub.2).sub.2 SEQ ID NO: 15
(pGlu-Glu-Asp).sub.2Adp(Tyr-Lys).sub.2 SEQ ID NO: 16
(Pic-Glu-Asp).sub.2Adp(Lys).sub.2 SEQ ID NO: 17
(p-Glu-Glu-Asp).sub.2Sub(- Lys-NH.sub.2).sub.2 SEQ ID NO: 18
(Pic-Glu-Asp).sub.2Adp(Lys-NH.sub.2).sub- .2 SEQ ID NO: 19
34. The method according to claim 32 wherein said peptide is
administered in amounts of between about 0.01 ng/kg to 1 g/kg.
35. The method according to claim 33 wherein the peptide is
(pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2 SEQ ID NO: 5.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to certain proteins
and to methods of improving the biological activity of certain
proteins, and more specifically, chemokines.
BACKGROUND OF THE INVENTION
[0002] All the members of the intercrine or chemokine family are
basic heparin-binding polypeptides which have four cysteine
residues which form two disulfide bridges. All these proteins which
have been functionally characterized appear to be involved in
proinflammatory and/or restorative functions. As such, these
molecules are anticipated to have therapeutic potential in bone
marrow transplantation and the treatment of infections, cancer,
myelopoietic dysfunction, graft versus host disease, and autoimmune
diseases.
[0003] The chemokine family can be divided into two subfamilies
depending upon their amino acid sequence and chromosomal location.
The members of the .alpha. subfamily are termed the "C-X-C"
subfamily because the first two cysteines are separated by only one
amino acid. The human genes in this subfamily include IL-8,
GRO/MGSA, and IP-10; murine counterparts include KC and macrophage
inflammatory protein 2 (MIP-2). In the chemokine .beta. subfamily,
the first two cysteines are in an adjacent position (the "C-C"
subfamily). This subfamily includes human MCAF, LD-78, ACT-2, and
RANTES. The murine counterparts are JE, TCA-3, MIP-1.alpha., and
MIP-1.beta. [J. J. Oppenheim et al, Annu. Rev. Immunol., 9:617-648
(1991)].
[0004] The murine KC gene product [Oquendo et al, J. Biol. Chem.,
264:4233 (1989)] is induced by platelet-derived growth factor
(PDGF) and this is thought to be the murine homolog of the human
MGSA/gro.alpha. gene (63.0% amino acid identity to mMIP-2). KC has
been expressed in COS-1 cells to show that it encodes a secreted
protein [Oquendo, cited above].
[0005] Two forms of MIP have been found in cultures of macrophage
tumor cells from the mouse: MIP-1 and MIP-2. Murine MIP-2 (mMIP-2)
is an inducible protein whose cDNA also has been cloned and
sequenced [International Patent Application, Publication No. WO
90/02762 (Mar. 22, 1990)]. Murine MIP-2 has been shown to have
potent chemotactic activity for human polymorphonuclear leukocytes
(PMN), and to induce PMN degranulation of lysozyme but not of
.beta.-glucuronidase [Wolpe et al, J. Exp. Med., 167:570 (1987)].
Further, mMIP-2 has been shown to have myelopoietic enhancing
activities for CFU-GM [Broxmeyer et al, J. Exp. Med., 170:1583
(1989)]. The human counterpart of this factor was found to consist
of two species, MIP-2.alpha. and MIP-2.beta., also termed
gro-.beta. and gro-.gamma., respectively.
[0006] The cDNA and amino acid sequences of human gro-.beta. are
provided in International Patent Application, Publication No. WO
92/00327 (Jan. 9, 1992); the cDNA and amino acid sequences of human
gro-.gamma. are provided in International Patent Application,
Publication No. WO 92/00326 (Jan. 9, 1992). Each of these sequences
were predicted to encode a 73 amino acid mature protein.
[0007] MGSA or gro-.alpha. [Richmond et al, EMBO J., 7:2025 (1988)]
is an autocrine growth factor with potent mitogenic activity
secreted by human melanoma cells and is the product of the human
gro gene [Anisowicz et al, Proc. Natl. Acad. Sci., 84:7188
(1987)].
[0008] There remains a need in the art for methods of enhancing the
bioactivity of these mature proteins to enable their efficient use
as therapeutic or pharmaceutical products, and to minimize the
amounts of the proteins necessary to produce a therapeutic effect,
thereby lowering toxicity.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides a modified
chemokine, which includes KC protein, human gro-.alpha.,
gro-.beta., and gro-.gamma., which modified protein is
characterized by truncation of between about 2 to about 8 amino
acids at the amino terminus of the mature protein and by at least a
log higher biological activity than the mature protein.
[0010] In another aspect, the present invention provides a modified
chemokine which is characterized by truncation of between about 2
to about 10 amino acids at the carboxy terminus of the mature
protein and by at least a log higher biological activity than the
mature protein.
[0011] In still another aspect, the present invention provides a
multimeric protein which comprises an association of two or more
modified proteins of this invention. These multimers preferably
contain multiple copies of the same modified protein, e.g., a dimer
of truncated KC protein. However, multimeric forms of two or more
different modified proteins of this invention are also included in
this invention. Multimeric forms of a modified protein of this
invention and another known mature protein are also encompassed by
this invention.
[0012] In a further aspect, the present invention provides a method
of enhancing the biological activity of chemokines by modifying
and/or truncating the proteins as described above.
[0013] In yet another aspect, the present invention provides
pharmaceutical and diagnostic compositions containing the modified
and multimeric proteins of the invention, as well as methods for
administering same in therapeutic treatments.
[0014] In a further aspect, the present invention provides
antibodies characterized by the ability to selectively bind the
modified chemokines of the invention.
[0015] In still another aspect, the present invention provides a
method of monitoring the effect of a selected hematopoiesis
stimulating agent upon hematopoietic synergistic factor (HSF) in
vivo through use of an antibody of the invention.
[0016] In yet another aspect, the present invention provides a
method of inducing HSF in vivo by administering
(pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.- 2 [SEQ ID NO: 5].
[0017] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 provides the published amino acid sequence [SEQ ID
NO: 1] of the mature, native murine KC protein.
[0019] FIG. 2 provides the published amino acid sequence [SEQ ID
NO: 2] of the mature, gro-alpha human protein.
[0020] FIG. 3 provides the published amino acid sequence [SEQ ID
NO: 3] of the mature, human gro-.beta. protein.
[0021] FIG. 4 provides the published amino acid sequence [SEQ ID
NO: 4] of the mature, human gro-.gamma. protein.
[0022] FIG. 5 is a DNA map of the plasmid pea1-mkc38(-4), which is
described below in Example 2.
[0023] FIG. 6 is a DNA map of the plasmid pea1-hgro.beta.(-4),
which is described below in Example 6.
[0024] FIG. 7 illustrates the kinetics of HSF induction in vivo, as
described in Example 17.
[0025] FIG. 8 illustrates the dose-dependent response of serum HSF
levels following injection with
(pGlu-GlU-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 5], as described
in Example 17 .
[0026] FIG. 9 illustrates dose-dependent response of HSF at 6 hours
following intraperitoneal (i.p.) verses oral (p.o.) administration
of (pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 5], as
described in Example 17.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention provides modified proteins,
specifically chemokines, associated with inflammatory responses,
hematopoiesis and myelopoiesis, which modified proteins are
characterized by having enhanced biological activity as compared to
the corresponding unmodified or untruncated mature native
proteins.
[0028] As defined herein, "hematopoietic synergistic factor" or
"HSF" refers to a class of proteins, including the naturally
occurring chemokines and the modified chemokines of the invention,
which are characterized by having synergistic activity in
stimulating hematopoiesis when administered in vivo and in vitro
with another hematopoietic factor, such as a colony stimulating
factor (see Example 8), or combined with naturally circulating
CSFs. The term "chemokines", also known as "intercrines" include,
among others, the proteins conventionally referred to in the art as
KC protein, gro-.beta., gro-.alpha., and gro-.gamma., all of
mammalian origin. The amino acid sequences of four of the mature
chemokines [SEQ ID NOS: 1-4] identified above are illustrated in
FIGS. 1 through 4 herein. Also included by this definition are
analogs or derivatives of these proteins which share the biological
activity of the mature protein.
[0029] As defined herein, such analogs and derivatives include
modified proteins also characterized by alterations made in the
known amino sequence of mature proteins, e.g., the proteins
provided in SEQ ID NOS: 1-4. Such analogs are characterized by
having an amino acid sequence differing from that of the mature
protein by 8 or fewer amino acid residues, and preferably by about
5 or fewer residues. It may be preferred that any differences in
the amino acid sequences of the proteins involve only conservative
amino acid substitutions. Conservative amino acid substitutions
occur when an amino acid has substantially the same charge as the
amino acid for which it is substituted and the substitution has no
significant effect on the local conformation of the protein or its
biological activity. Alternatively, changes such as the elimination
or introduction of a certain amino acid in the sequence which may
alter the stability of the protein, or permit it to be expressed in
a desired host cell may be preferred.
[0030] As used herein, "enhanced biological activity" refers to
biological activity which is at least one log higher, i.e. 10 times
or 10 fold, than that observed in the full-length mature protein in
the HSF assay of Example 8. Additionally, this term refers to
biological activities not characteristic of the mature protein,
e.g., as with KC, the full-length mature protein may be inactive,
while the modified protein has significant activity.
[0031] The present invention provides modified desamino chemokines.
The term "desamino" is used to indicate chemokines or proteins of
the invention which have been modified such that between about 2 to
about 8, and preferably from about 5 to about 8, amino acids have
been removed from the amino terminus of the mature protein.
Optionally, particularly when expressed recombinantly, the desamino
chemokines of the invention may optionally contain an inserted
N-terminal Met. During expression by the host cell, this optional
Met may be cleaved. Alternatively, if so desired, this amino acid
may be cleaved through enzyme digestion or other known means.
[0032] In one embodiment of this aspect, the present invention
provides a desamino KC protein, which is characterized by having a
truncated amino (or N) terminus in comparison to the mature KC
protein. The modified protein can be truncated at a position
between amino acid residues #2 through 8 of mature KC protein of
FIG. 1 [SEQ ID NO: 1]. Preferably, this desamino KC, contains amino
acids 5-72 of the mature KC protein of SEQ ID NO: 1; i.e., the
first four amino acid residues of the amino terminus of the
illustrated KC protein are missing in this protein.
[0033] Surprisingly, the inventors have discovered that this
desamino KC is characterized by having bioactivity in a hemopoietic
synergistic factor assay (see Example 8) of at least 10.sup.14
Units/mg in contrast to the unmodified, full-length mature KC [SEQ
ID NO: 1] which is inactive (.sup.-0 Units/mg). It is anticipated
that when purified, this modified chemokine will be characterized
by an even higher bioactivity. The construction, synthesis and
assay of the desamino KC are described in detail in the examples
below.
[0034] Another modified chemokine embodied by the present invention
is a desamino gro-.beta. (also known as MIP-2.alpha.) protein. This
protein comprises the amino acid sequence of mature gro-.beta.
protein truncated at its N terminus between amino acid positions 2
and 8 of FIG. 3 [SEQ ID NO: 3]. In a preferred embodiment, the
desamino protein of the invention has a protein sequence spanning
amino acids 5 to 73 of the mature gro-.beta. of SEQ ID NO: 3.
[0035] The inventors have surprisingly discovered that this
desamino-gro-.beta. is characterized by having at least about two
logs higher biological activity than unmodified, full-length human
gro-.beta..
[0036] Another embodiment of the present invention is a desamino
gro-.gamma. (also known as MIP-2.beta.) protein. This protein
comprises the amino acid sequence of mature gro-.gamma. protein
truncated at its N terminus between amino acid positions 2 and 8 of
SEQ ID NO: 4. In a preferred embodiment, the modified protein of
the invention has a protein sequence spanning amino acids 5 to 73
of the mature gro-.gamma. [SEQ ID NO: 43]. The inventors have
discovered that this desamino gro-.gamma. has at least 2 log
greater bioactivity in the assay of Example 8 than the full-length
mature gro-.gamma.. It is anticipated, that upon further
purification, an even greater bioactivity will be observed.
[0037] Another modified protein embodied by the present invention
is a desamino gro-.alpha. protein (also known as MGSA). This
protein comprises the amino acid sequence of mature gro-.alpha.
protein truncated at its N terminus between amino acid positions 2
and 8 of FIG. 2 [SEQ ID NO: 2]. In a preferred embodiment, the
modified protein of the invention has a protein sequence spanning
amino acids 5 to 73 of the mature gro-.alpha. of SEQ ID NO: 2. Upon
modification, an increase in activity similar to that observed with
these other gro proteins is anticipated for desamino
gro-.alpha..
[0038] The present invention also provides modified descarboxy
chemokines or other proteins. Such a descarboxy chemokine comprises
a full length mature chemokine having about 2 to about 10 amino
acids deleted from its carboxy terminus. Optionally, the N-terminal
methionine which is optionally inserted into the protein for
expression purposes, may be cleaved, either during the processing
of the protein by a host cell or synthetically, using known
techniques.
[0039] An example of this embodiment is a KC protein which has the
amino acid sequence of a mature KC protein truncated at its carboxy
terminus at a position between amino acids about 58 to about 70 of
SEQ ID NO: 1. In a preferred embodiment, the descarboxy KC protein
consists of amino acid residues 1 to 68 of the mature KC protein
[SEQ ID NO: 1], in which the carboxy terminal four amino acid
residues of the mature protein are lacking. Similar descarboxy
proteins can be constructed with the gro.alpha., gro.beta. and
gro.gamma. proteins.
[0040] It is anticipated that other desamino and descarboxy
chemokine may also exhibit enhanced biological activity as compared
to the respective unaltered mature chemokine. Examples of such
chemokines include .alpha. subfamily chemokines such as IL-8/NAP-1,
PF-4, and IP-10, .beta. subfamily chemokines such as MCAF/MCP-1,
LD-78, PAT464, GOS19-1, ACT-2, PAT744/G26, RANTES, I-309, and the
above-described proteins. These proteins are all described in the
literature and are known to those of skill in the art.
[0041] Moreover, as described in more detail below, modified
proteins of this invention include multimeric forms of the modified
and/or truncated proteins, e.g., dimers, trimers, tetramers and
other aggregated forms. Such multimeric forms can be prepared by
synthesis or recombinant expression and can contain chemokines
produced by a combination of synthetic and recombinant techniques
as detailed below. Multimers may form naturally upon expression or
may be constructed into such multiple forms.
[0042] It is anticipated that multimeric forms of the modified
chemokines of this invention may include multimers of the same
modified chemokine. Another multimer of this invention is formed by
the aggregation of different modified proteins. Still another
multimer of this invention is formed by the aggregation of a
modified chemokine of this invention and a known, full-length
mature chemokine.
[0043] Preferably, a dimer or multimer of the invention would
contain at least one of the descarboxy or desamino chemokine
proteins of the invention and at least one other chemokine or other
protein characterized by having the same type of biological
activity. This other protein may be an additional desamino or
descarboxy chemokine, or another, known protein.
[0044] For example, a desirable dimer of the invention comprises
two desamino KC proteins of the invention. Other desirable dimers
of the invention are two descarboxy-KC proteins of the invention, a
desamino-KC and a descarboxy-KC of the invention, or two desamino
gro-.beta. proteins of the invention. Alternatively, another dimer
of the invention may be a desamino KC protein of the invention or a
descarboxy-KC protein of the invention in combination with an
unmodified mature KC protein. Similarly, such combinations of
dimers may be formed with the descarboxy gro-.alpha., gro-.beta.,
gro-.gamma., or other chemokines of this invention. For example, a
desamino gro-.beta. protein of the invention may form a dimer with
an unmodified mature gro-.beta. protein of the invention. One of
skill in the art may obtain other desirable multimers using the
modified chemokines of the invention.
[0045] The multimers, as well as the other modified proteins of
this invention,. are characterized by enhanced biological activity
in contrast to the known mature proteins. Enhanced biological
activity provides a clear advantage for therapeutic use, i.e., that
less of the protein therapeutic need be administered to obtain a
desired therapeutic result. Such a lower dose may result in lower
toxicity and lower cost.
[0046] Thus, the present invention provides a method of enhancing
the biological activity of a selected chemokine, e.g., KC,
gro-.alpha., gro-.beta., and gro-.gamma.. This method involves
modifying natively or recombinantly produced mature chemokines as
described herein. This method can also involve preparing multimeric
aggregations thereof. According to another embodiment of this
method, the modified proteins or multimers may be synthesized as
described below.
[0047] Advantageously, this method involves preparing the desamino
and descarboxy chemokines of this invention following known
techniques. For example, these peptides are prepared by the solid
phase technique of Merrifield, J. Am. Chem. Soc., 85:2149 (1964),
or solution methods known to the art may be successfully employed.
The methods of peptide synthesis generally set forth in J. M.
Stewart and J. D. Young, "Solid Phase Peptide Synthesis", Pierce
Chemical Company, Rockford, Ill. (1984) or M. Bodansky, Y. A.
Klauser and M. A. Ondetti, "Peptide Synthesis", John Wiley &
Sons, Inc., New York, N.Y. (1976) may be used to produce the
peptides of this invention.
[0048] Each amino acid or peptide is suitably protected as known in
the peptide art. For example, the
.alpha.-fluoroenylmethyloxycarbonyl group (Fmoc) or
t-butoxycarbonyl (t-Boc) group are preferred for protection of the
amino group, especially at the .alpha.-position. A suitably
substituted carbobenzoxy group may be used for the .epsilon.-amino
group of lysine and benzyl group for the .beta. and .gamma. carboxy
groups of Asp and Glu respectively. Suitable substitution of the
carbobenzoxy protecting group is ortho and/or para substitution
with chloro, bromo, nitro or methyl, and is used to modify the
reactivity of the protective group. Except for the t-Boc group, the
protective groups are, most conveniently, those which are not
removed by mild acid treatment. These protective groups are removed
by such methods as catalytic hydrogenation, sodium in liquid
ammonia or HF treatment as known in the art.
[0049] If solid phase synthetic methods are used, the peptide is
built up sequentially starting from the carboxy terminus and
working toward the amino terminus of the peptide. Solid phase
synthesis is begun by covalently attaching the C terminus of a
protected amino acid to a suitable resin, such as benzhydrylamine
resin (BHA), methylbenzhydrylamine resin (MBHA) or chloromethyl
resin (CMR), as is generally set forth in U.S. Pat. No. 4,244,946
or phenyl acid amino methyl resin (PAM). A BHA or MBHA support
resin is used if the carboxy terminus of the product peptide is to
be a carboxamide. ACMR or PAM resin is generally used if the
carboxy terminus of the product peptide is to be a carboxy group,
although this may also be used to produce a carboxamide or
ester.
[0050] The protective group on the .alpha.-amino group is removed
by mild acid treatment (i.e. trifluoroacetic acid). Suitable
deprotection, neutralization and coupling cycles known in the art
are used to sequentially add amino acids without isolation of the
intermediate, until the desired peptide has been formed. The
completed peptide may then be deblocked and/or split from the
carrying resin in any order.
[0051] Treatment of a resin supported peptide with HF or HBr/acetic
acid splits the peptide from the resin and produces the carboxy
terminal amino acid as a carboxylic acid or carboxamide.
[0052] If an ester is desired, the CMR or Pam resin may be treated
with an appropriate alcohol, such as methyl, ethyl, propyl, butyl
or benzyl alcohol, in the presence of triethylamide to cleave the
peptide from the resin and product the ester directly.
[0053] Esters of the peptides of this invention may also be
prepared by conventional methods from the carboxylic acid
precursor. Typically, the carboxylic acid is treated with an
alcohol in the presence of an acid catalyst. Alternatively, the
carboxylic acid may be converted to an activated acyl intermediate,
such as an acid halide, and treated with an alcohol, preferably in
the presence of a base.
[0054] The preferred method for cleaving a peptide from the support
resin is to treat the resin supported peptide with anhydrous HF in
the presence of a suitable cation scavenger, such as anisole or
dimethoxybenzene. This method simultaneously removes all protecting
groups, except a thioalkyl group protecting sulfur, and splits the
peptide from the resin. Peptides hydrolyzed in this way from the
CMR and Pam resins are carboxylic acids, those split from the BHA
resin are obtained as carboxamides.
[0055] Modification of the terminal amino group of the peptide is
accomplished by alkylation or acylation by methods generally known
in the art. These modifications may be carried out upon the amino
acid prior to incorporation into the peptide, or upon the peptide
after it has been synthesized and the terminal amino group
liberated, but before the protecting groups have been removed.
[0056] Typically, acylation is carried out upon the free amino
group using the acyl halide, anhydride or activated ester, of the
corresponding alkyl or aryl acid, in the presence of a tertiary
amine. Monoalkylation is carried out most conveniently by reductive
alkylation of the amino group with an appropriate aliphatic
aldehyde or ketone in the presence of a mild reducing agent, such a
lithium or sodium cyanoborohydride. Dialkylation may be carried out
by treating the amino group with an excess of an alkyl halide in
the presence of a base.
[0057] Solution synthesis of peptides is accomplished using
conventional methods used for amide bonds. Typically, a protected
t-Boc amino acid which has a free carboxyl group is coupled to a
protected amino acid which has a free amino group using a suitable
coupling agent, such as N,N'-dicyclohexyl carbodiimide (DCC),
optionally in the presence of catalysts such as
1-hydroxybenzotriazole (HOBT) or dimethylamino pyridine (DMAP).
Other methods, such as the formation of activated esters,
anhydrides or acid halides, of the free carboxyl of a protected
t-Boc-amino-acid, and subsequent reaction with the free amine of a
protected amino acid, optionally in the presence of a base, are
also suitable. For example, a protected Boc-amino acid or peptide
is treated in an anhydrous solvent, such as methylene chloride or
tetrahydrofuran (THF), in the presence of a base, such as N-methyl
morpholine, DMAP (dimethylaminopyridine) or a trialkyl amine, with
isobutyl chloroformate to form the "activated anhydride", which is
subsequently reacted with the free amine of another protected amino
acid or peptide. The peptide formed by these methods may be
deprotected selectively, using conventional techniques, at the
amino or carboxy terminus and coupled to other peptides or amino
acids using similar techniques. After the peptide has been
completed, the protecting groups may be removed as hereinbefore
described, such as by hydrogenation in the presence of a palladium
or platinum catalyst, treatment with sodium in liquid ammonia,
hydrofluoric acid or alkali.
[0058] If the final peptide, after it has been deprotected,
contains a basic group, an acid addition salt may be prepared. Acid
addition salts of the peptides are prepared in a standard manner in
a suitable solvent from the parent compound and a slight excess of
an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric,
acetic, maleic, succinic or methanesulfonic. The acetate salt form
is especially useful. If the final peptide contains an acidic
group, cationic salts may be prepared. Typically the parent
compound is treated with a slight excess of an alkaline reagent,
such as a hydroxide, carbonate or alkoxide, containing the
appropriate cation. Cations such as Na.sup.+, K.sup.+, Ca.sup.++
and NH.sub.4.sup.+ are examples of cations present in
pharmaceutically acceptable salts. Na.sup.+ and NH.sub.4.sup.+ are
especially preferred.
[0059] However, in a preferred method, full-length, mature
chemokines are digested with a suitable enzyme to produce the
modified proteins of the present invention. Currently, the
preferred enzyme is DPP-IV, which is commercially available from
Enzyme Products Systems, Inc.
[0060] The desamino and descarboxy chemokines of this invention may
also be produced by other techniques known to those of skill in the
art, for example, genetic engineering techniques. See, e.g.,
Sambrook et al, in Molecular Cloning, a Laboratory Manual, 2nd
edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989). Systems for cloning and expression of a selected protein in
a desired microorganism or cell, including, e.g. E. coli, Bacillus,
Streptomyces, mammalian, insect, and yeast cells, are known and
available from private and public laboratories and depositories and
from commercial vendors.
[0061] Currently, the most preferred method of producing the
descarboxy and desamino chemokines of the invention is through
direct recombinant expression of the modified chemokine. For
example, the descarboxy or desamino murine KC protein can be
recombinantly expressed by inserting its DNA coding sequence into a
conventional plasmid expression vector under the control of
regulatory sequences capable of directing the replication and
expression of the protein in a selected host cell. See, for
example, the description in Examples 2 and 3 below.
[0062] The recombinant expression of the desamino and/or descarboxy
gro.alpha., gro.beta., and gro.gamma. can be obtained using
analogous techniques. Among the preferred expression systems for
these chemokines also includes eukaryotic cells, including insect
and yeast cells. However, the most preferred expression system, as
with the KC, is a bacterial system. Because these proteins are not
believed to be glycosylated, there appears to be no conformational
problems associated with translation and expression in
bacteria.
[0063] The modified chemokines of the invention which are produced
in the cell or secreted into the medium can then be purified
therefrom using conventional techniques such as cell lysis and gel
chromatography.
[0064] Desirably, these desamino and descarboxy chemokines of the
invention naturally combine, including those synthetically produced
in monomeric form, into dimers, trimers, and other aggregates. For
example, a monomeric descarboxy-KC protein of the invention may be
co-expressed in a selected host cell which has been co-transfected
with one or more vectors containing the coding sequences of the
modified chemokine of the invention. Alternatively, two or more
copies of the monomeric descarboxy-KC protein of the invention may
be on a single vector, or incorporated into a chromosome of a host
cell. Preferably, this host cell is bacterial or mammalian.
[0065] Thus, as another embodiment of the invention, nucleic acid
sequence encoding the modified chemokines of the invention may be
obtained conventionally or designed by one of skill in the art with
knowledge of the amino acid sequences of the chemokine. Such
nucleic acid sequences can be designed with preferred codons for
the expression system selected, e.g., bacterial, yeast, etc.
Similarly, the nucleotide sequences encoding the expression
plasmids may be obtained conventionally depending upon the
selection of the plasmid and the selection of the modified
chemokine protein to be expressed therein. The nucleotide sequences
encoding these proteins may also contain an optional initiating Met
codon.
[0066] In another aspect, the present invention further provides
pharmaceutical compositions useful in the treatment of
inflammation, fever, viral, fungal, and bacterial infections,
cancer, myelopoietic dysfunction, hematopoiesis disorders and
autoimmune diseases. These compositions contain a therapeutically
effective amount of a modified chemokine of this invention and an
acceptable pharmaceutical carrier. As used herein, the term
"pharmaceutical" includes veterinary applications of the
invention.
[0067] Modified chemokines of the invention for therapeutic use
include, without limitation, a desamino KC, a descarboxy-KC, a
desamino gro.beta., gro-.alpha., or gro-.gamma. or a descarboxy
gro.beta., gro-.alpha., or gro-.gamma., multimers containing them,
and combinations thereof.
[0068] The modified chemokines of the invention can be formulated
into pharmaceutical compositions and administered in the same
manner as described for the mature proteins [see, e.g.,
International Patent Application, Publication No. WO 90/02762 (Mar.
22, 1990)]. The difference in the preparation and use of the
pharmaceutical compositions of this invention is the ability to
provide lesser amounts of protein to accomplish the same
therapeutic effect for which the mature protein is used. The term
"therapeutically effective amount" refers to that amount of a
modified chemokine, whether in monomeric or, preferably, multimeric
form, which is useful for alleviating a selected condition.
[0069] Generally, a desamino or descarboxy chemokine of the
invention is administered in an amount between about 0.01 ng/kg
body weight to about 1 g/kg and preferably about 0.01 ng/kg to 100
.mu.g/kg per dose. Preferably, these pharmaceutical compositions
are administered to human or other mammalian subjects by injection.
However, administration may be by any appropriate internal route,
and may be repeated as needed, e.g. one to three times daily for
between 1 day to about three weeks.
[0070] Suitable pharmaceutical carriers are well known to those of
skill in the art and may be readily selected. Currently, the
preferred carrier is saline. optionally, the pharmaceutical assays
of the invention may contain other active ingredients or be
administered in conjunction with other therapeutics. Suitable
optional ingredients or other therapeutics include those
conventional for treating conditions of this nature, e.g. other
anti-inflammatories, diuretics, and immune suppressants, among
others. Desirably, these modified chemokines of the invention are
particularly well suited for administration in conjunction with
colony stimulating factor.
[0071] Thus, the invention also provides improved methods of
treating inflammation, autoimmune disorders, and conditions
characterized by low production and/or differentiation of
hematopoietic and/or bone marrow cells. This method involves
administering to a selected mammal a pharmaceutical composition of
the invention. Preferably, this composition is administered
together with or contains a colony stimulating factor. Suitable
sources of colony stimulating factor are well known and include,
e.g., natural, synthetic and recombinant GM-CSF, M-CSF, G-CSF and
IL-3. In another preferred embodiment, a descarboxy or desamino
chemokine of the invention can be administered in vivo, and
permitted to act in synergy with the natural colony stimulating
factors found in a selected patient.
[0072] In one preferred embodiment, the descarboxy and desamino
chemokines of the invention are particularly well suited for
internal use in conjunction with GM-CSF, an approved treatment for
such conditions which is unfortunately extremely toxic. The use of
a modified chemokine of the invention, such as a desamino
gro-.beta., in combination with CSF, which combination has been
observed to have synergy, permits lower doses of CSF to be
administered to a patient, resulting in a lower toxicity of the
GM-CSF.
[0073] In another aspect, the present invention provides antibodies
to the modified chemokines of the invention. Such antibodies are
characterized by binding preferentially to the modified chemokines
of the invention, i.e. they are capable of discriminating against
the unmodified or full-length chemokines. These antibodies may be
generated using conventional techniques for production of
monoclonal [W. D. Huse et al, Science, 246:1275-1281 (1989); Kohler
and Milstein] or polyclonal antibodies. The antibodies of the
invention are anticipated to be useful as diagnostic reagents for
measuring levels of hematopoietic synergistic factor (HSF) in a
mammal's blood stream.
[0074] The present invention further provides a method for
monitoring the circulating level and/or efficacy of a selected
agent characterized by the ability to induce HSF in a mammal. Such
HSF-inducing agents include, without limitation,
hematopoiesis-inducing compounds such as those disclosed in
co-owned application Ser. No. 07/819,024, corresponding to issued
Canadian patent No. 2,020,838, and in U.S. Pat. No. 4,987,122.
[0075] These peptides are illustrated by the formula (I): 1
[0076] wherein:
[0077] Y.sub.1 and Y.sub.2 are independently CH.sub.2 or S;
[0078] x is 0, 1, 2, 3, or 4;
[0079] m is 0, 1, or 2;
[0080] n is 0, 1, or 2;
[0081] A is pyroglutamic acid, proline, glutamine, tyrosine,
glutamic acid, 2-thiophene carboxylic acid, picolinic acid,
cyclohexane carboxylic acid, tetrahydro-2-furoic acid,
tetrahydro-3-furoic acid, 2-oxo-4-thiazolidine, cyclopentane,
3-thiophene carboxylic acid,
(S)-(+)-5-oxo-2-tetrahydrofuran-carboxylic acid, and pipecolinic
acid;
[0082] B is serine, glutamic acid, tyrosine or aspartic acid;
[0083] C is glutamic acid, tyrosine or aspartic acid;
[0084] D is lysine, arginine, tyrosine, N-methylarginine, aspartic
acid, ornithine or diaminohexynoic acid; or the carboxyamide, or
hydroxy methyl derivative thereof;
[0085] E is glutamic acid, aspartic acid, tyrosine or a peptide
bond;
[0086] F is tyrosine or a peptide bond;
[0087] provided that:
[0088] when Y.sub.1 and Y.sub.2 are S, x is 2, 3 or 4 and m and n
are 1; or
[0089] when Y.sub.1 and Y.sub.2 are CH.sub.2, x is 0, 1 or 2 and m
and n are 0;
[0090] or
[0091] when Y.sub.1 is S and Y.sub.2 is CH.sub.2, x is 0 and n is
1; or
[0092] when Y.sub.2 is S and Y.sub.1 is CH.sub.2, x is 0 and m is
1; or a pharmaceutically acceptable salt thereof.
[0093] Also included in this invention are pharmaceutically
acceptable salt complexes of the compounds of this invention. It
should be noted in formula (I) that A comprises the terminal amino
group of the amino acid residue corresponding to pyroglutamic acid,
proline, glutamine, tyrosine or glutamic acid. Similarly, D
comprises the terminal carboxyl group of amino acid residue
corresponding to lysine, arginine, tyrosine, N-methyl arginine,
diamino hexynoic acid or the carboxamide or hydroxy methyl
derivative thereof.
[0094] The abbreviations and symbols commonly used in the art are
used herein to describe the peptides. Amino acids are abbreviated
by their conventional three-letter designations.
[0095] pGlu=pyroglutamic acid
[0096] Pic=picolinic acid
[0097] Ppc=pipecolinic acid
[0098] Ppg=propargyl glycine
[0099] Orn=ornithine
[0100] p-(NH.sub.2)Phe=para-aminophenylalanine
[0101] Hna=2,6-diamino-4-hexynoic acid 2
[0102] Chc=cyclohexane carboxylic acid
[0103] Tfc=tetrahydro-2-furoic acid
[0104] Otz=2-oxo-4-thiazolidine
[0105] Cpa=cyclopentane
[0106] Tpc=3-thiophene carboxylic acid
[0107] S-Otf=(S)-(+)-5-oxo-2-tetrahydrofurane-carboxylic acid
[0108] t-BOC=tert. butyloxy carbonyl
[0109] Bz=benzyl
[0110] Cl-Z=p-chloro carbabenzyloxy carbonyl
[0111] (Z=carbobenzyloxy carbonyl)
[0112] DCC=dicylohexyl carbodiimide
[0113] DIEA=diisopropylethyl amine
[0114] EDC=(N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide
[0115] Hna=diaminohexynoic acid
[0116] HOBT=hydroxybenzotriazole
[0117] NMP=N-methyl-2-pyrrolidinone
[0118] N-MeArg=N-methyl arginine
[0119] Prc=bis BOC-S,S'=1,3-propanediylcysteine
[0120] Etc=bis BOC-S,S'-1,2-ethanediylcysteine
[0121] Buc=bis BOC-S,S'-1,4-butanediylcysteine
[0122] R-Otf=(R)-(-)-S-oxo-2-tetrahydrofurane-carboxylic acid
[0123] In accordance with conventional representation, the amino
terminus is on the left and the carboxy terminus is on the right.
All chiral amino acids may be in the D or L absolute configuration.
All optical isomers are contemplated.
[0124] The amino terminus may be protected by acylation. Examples
of such protecting groups are, t-butoxycarbonyl (t-Boc) ,
CH.sub.3CO and Ar--CO (Ar=benzyl, or phenyl).
[0125] The C-terminus may be carboxy as in the case of the natural
amino acid or the carboxamide --C(O)NH.sub.2 or hydroxymethyl
(--CH.sub.2--OH).
[0126] Preferred compounds are those in which:
[0127] A is pyroglutamic acid, picolinic acid, proline, tyrosine,
or pipecolinic acid;
[0128] B is glutamic acid, serine, aspartic acid or tyrosine;
[0129] C is aspartic acid, glutamic acid, tyrosine or lysine;
[0130] D is lysine, or the carboxyamide derivative thereof,
arginine, N-methylarginine, 2,6-diamino-4-hexynoic acid, aspartic
acid or ornithine;
[0131] E is a bond;
[0132] Y.sub.1 and Y.sub.2 are CH.sub.2;
[0133] x is 0 or 2;
[0134] m and n are 0.
[0135] More preferred are compounds wherein:
[0136] A is pyroglutamic acid, proline or picolinic acid;
[0137] B is glutamic acid, aspartic and or serine;
[0138] C is aspartic acid or glutamic acid;
[0139] D is lysine or the carboxyamide derivative thereof;
[0140] E is a bond;
[0141] Y.sub.1 and Y.sub.2 are CH.sub.2; and
[0142] x is 0 or 2
[0143] and the chiral amino acids are in the L absolute
configuration.
[0144] Especially preferred are:
[0145] (pGlu-Glu-Asp).sub.2Sub(Lys).sub.2 [SEQ ID NO: 5]
[0146] (pGlu-Glu-Asp).sub.2Adp(Lys).sub.2 [SEQ ID NO: 6]
[0147] (pGlu-Glu-Glu)..sub.2Sub(Lys).sub.2 [SEQ ID NO: 7]
[0148] (pGlu-Asp-Asp).sub.2Sub(Lys).sub.2 [SEQ ID NO: 8]
[0149] (Pic-Glu-Asp).sub.2Sub(Lys).sub.2 [SEQ ID NO: 9]
[0150] (L-Ppc-Glu-Asp).sub.2Sub(Lys).sub.2 [SEQ ID NO: 10]
[0151] (pGlu-Ser-Asp).sub.2Sub(Lys).sub.2 [SEQ ID NO: 11]
[0152] (pGlu-Ser-Asp).sub.2Adp(Lys).sub.2 [SEQ ID NO: 12]
[0153] (pGlu-Ser-Asp).sub.2Adp(Lys-NH.sub.2).sub.2 [SEQ ID NO:
13]
[0154] (Pic-Ser-Asp).sub.2Adp(Lys).sub.2 [SEQ ID NO: 14]
[0155] (Pic-Ser-Asp).sub.2Adp(Lys-NH.sub.2).sub.2 [SEQ ID NO:
15]
[0156] (pGlu-Glu-Asp).sub.2Adp(Tyr-Lys).sub.2 [SEQ ID NO: 16]
[0157] (Pic-Glu-Asp).sub.2Adp(Lys).sub.2 [SEQ ID NO: 17]
[0158] (p-Glu-Glu-Asp).sub.2Sub(Lys-NH.sub.2).sub.2 [SEQ ID NO:
18]
[0159] (Pic-Glu-Asp).sub.2Adp(Lys-NH-.sub.2).sub.2 [SEQ ID NO:
19]
[0160] Of the above-described peptides, the most preferred is
(pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 5]. These peptides
may be prepared by the solid phase or solution phase techniques
described above.
[0161] This method of the invention involves contacting a sample of
body fluids from a mammal to which has been previously administered
the HSF inducing agent with an antibody of the invention. The
preferred body fluids are blood, plasma and serum. However, other
suitable samples can be readily determined. The antibody is used to
measure the levels of HSF. The circulating HSF levels following
administration of the HSF-inducing agent are compared to a
base-line level or the circulating HSF levels prior to
administration of the HSF-inducing agent. From the measurement of
induced HSF levels, the therapeutic efficacy of the selected agent
can be determined, and treatment can be monitored and adjusted as
necessary.
[0162] For example, a monoclonal or polyclonal
anti-desamino-gro.alpha. antibody may desirably be used as a
reagent in an assay for detecting the levels of HSF induced by,
e.g., (pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2. Suitable assay formats
are well known in the art. Currently, however, the preferred format
is an enzyme-linked immunosorbent assay (ELISA).
[0163] The antibodies of the invention may be associated with
individual labels, and where more than one antibody is employed in
a diagnostic method, the labels are desirably interactive to
produce a detectable signal. Most desirably, the label is
detectable visually, e.g. colorimetrically. Detectable labels for
attachment to antibodies useful in the diagnostic assays of this
invention may also be easily selected by one skilled in the art of
diagnostic assays. Labels detectable visually are preferred for use
in clinical applications due to the rapidity of the signal and its
easy readability. For colorimetric detection, a variety of enzyme
systems have been described in the art which will operate
appropriately. Colorimetric enzyme systems include, e.g.,
horseradish peroxidase (HRP) or alkaline phosphatase (AP). Other
proximal enzyme systems are known to those of skill in the art,
including hexokinase in conjunction with glucose-6-phosphate
dehydrogenase. Also, bioluminescence or chemiluminescence can be
detected using, respectively, NAD oxidoreductase with luciferase
and substrates NADH and FMN or peroxidase with luminol and
substrate peroxide. Other conventional label systems that may be
employed include fluorescent compounds, radioactive compounds or
elements, or immunoelectrodes. These and other appropriate label
systems and methods for coupling them to antibodies or peptides are
known to those of skill in the art.
[0164] The present invention also provides a diagnostic kit which
enables the monitoring of circulating levels of HSF in the blood
stream. Such a kit may contain a sufficient amount of at least one
modified chemokine of the invention or at least one antibody of the
invention and such components as are necessary to practice the
assay. Such assays are conventional, and the necessary reagents and
other components of such a kit are well known to those of skill in
the art.
[0165] Also provided by the present inventions are methods of
inducing HSF in vivo by administering, e.g.,
(pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 5]. In general,
this or another selected peptide described herein may be
administered to human patients by injection in the dose range of
0.5 ng to 1 mg, preferably 5-500 ng, or orally in the dose range of
50 ng to 5 mg, for example, 0.01 mg to 1 mg per 70 kg body weight
per day; if administered by infusion or similar techniques, the
dose may be in the range 0.005 ng to 1 mg per 70 kg body weight,
for example, about 0.03 ng over six days. It is desirable to
produce a concentration of peptide of about 10.sup.-15M to
10.sup.-5M in the extracellular fluid of the patient.
[0166] Advantageously, the peptide may be administered as the
active ingredient in a pharmaceutical composition or a
physiologically compatible salt thereof. The peptide or salt may be
in association with a pharmaceutical carrier or excipient. The
compositions according to the invention may be presented for
example, in a form suitable for oral, nasal, parenteral or rectal
administration. Suitable carriers and excipients are well known and
can be readily determined by one of skill in the art. Dosage units
containing the peptide preferably contain 0.1-100 mg, for example
1-50 mg of the peptide of formula (I) or salt thereof.
[0167] The following examples illustrate the preferred methods for
preparing the modified and truncated chemokines of the invention.
Also provided are comparative examples demonstrating the enhanced
bioactivity of these chemokines as compared to the chemokines from
which they are derived. Examples 9 through 16 provides are
illustrations of how the peptides used in the methods of the
invention are synthesized. In these examples, all temperatures are
in degrees Centigrade. Amino acid analysis were performed upon a
Dionex Autoion 100. Analysis for peptide content is based upon
Amino Acid Analysis. FAB mass spectra were performed upon a VG ZAB
mass spectrometer using fast atom bombardment. These examples are
illustrative only and do not limit the scope of the invention.
EXAMPLE 1
Synthesis of Desamino KC
[0168] The procedure used for synthesis of modified murine KC
protein, amino acid sequence #5-72 of the mature KC protein of SEQ
ID NO: 1, follows.
[0169] The KC protein sequence was synthesized using solid-phase
methods adapted to a fully automated protein synthesizer (Applied
Biosystems Model 430 A). Boc-Lys(Cl-Z)-PAM resin (0.75 g, 0.5 mmol)
was charged in a reaction vessel and the target protein was
synthesized according to the manufacturer's suggested protocols
using 2 mmol of each N.sup..alpha.-t-Boc amino acid. Each coupling
reaction was carried out twice followed by N-capping with acetic
anhydride. Sterically hindered residues were coupled a third time.
The following side chain protecting groups were used: benzyl (Thr,
Ser); 4-methylbenzyl (Cys); toluenesulfonyl (Arg);
2-chlorobenzyloxycarbonyl (Lys); dinitrophenyl (His); cyclohexyl
(Asp, Glu). The coupling yields of certain difficult sequences were
monitored by the Kaiser test. After completion of the synthesis the
protein-resin was washed with DCM and dried.
[0170] A portion of the protein-resin (0.2 g, 0.017 mmol) was
treated with DMF-BME-DIEA (75:20:5) for 4.times.30 min. to
deprotect the His residues and, after extensive washing with DMF
and DCM, the N.sup..alpha.-t-Boc group was removed by treatment
with 50% TFA in DCM for 20 min. The protein was further deprotected
and cleaved from the resin using HF (5 ml) at -5.degree. C. for one
hour in the presence of anisole (0.5 ml), dimethylsulfide (0.5 ml)
and p-thiocresole (0.1 g). The HF was evaporated and the
protein-resin mixture was washed with ether containing 5%
.beta.-mercaptoethanol (BME). The protein was extracted from the
resin with 6 M guanidine HCL/0.1 M HOAc (30 ml). A portion of the
crude protein (1.75 ml, -6.6 mg) was first purified on a
preparative size exclusion column (Beckman TSK 3000SW) using
phosphate buffered saline (PBS) buffer at a flow rate of 2 ml/min,
and then on a C-18 Vydac preparative column using
acetonitrile-water (0.1% TFA) buffer system at a flow rate of 5
ml/min. 0.36 mg + of pure protein was obtained. Amino acid analysis
and fast atom bombardment-mass spectrometry (FABS-MS) confirmed the
structure.
[0171] FAB-MS: [MH+] at mz: 7457.4 a.m.u., where MH+ represents a
positively charged mass ion, m/z is mass/charge and a.m.u. is
atomic mass units. Amino acid analysis:
1 Asp 4.00 (4) Val 5.53 (6) Thr 3.69 (4) Met 2.44 (2) Ser 2.19 (2)
Ile 3.18 (4) Glu 8.45 (10) Leu 8.01 (9) Gly 4.09 (4) Pro 4.02 (5)
Ala 4.64 (4) Arg 2.09 (2) His 1.99 (2) Lys 6.96 (7)
[0172] Chemically synthesized and native purified full-length KC
are both inactive as synergistic factors. Chemically synthesized
desamino KC prepared according to this invention is a potent
synergistic factor.
EXAMPLE 2
Plasmid Construction for Expression of Murine Desamino KC
[0173] A partial cDNA clone encoding full-length murine KC was
obtained from the American Type Culture Collection in Rockville,
Md. (ATCC No. 37591). A 422 bp TfiI-HincII fragment (bp 125-553)
was isolated from this cDNA clone. (All base pair and amino acid
numbers refer to the sequence available in Genbank.RTM., accession
number J04596.)
[0174] A linker comprising bp 74-124 was synthesized with a TfiI
overhang at the 3' end and a HindIII overhang at the 5' end using
conventional technology. An NdeI site was incorporated at the 5'
end to generate a Met codon in-frame with the KC product.
[0175] The linker and KC fragment were ligated to pUC18 [ATCC
15752-B1] which had been cut with HindIII and HincII, to generate
pMKC. pMKC encodes the entire mature KC gene product (aa 25-96)
with the addition of an N-terminal Met.
[0176] All E. coli expression of KC and its derivatives was done
using plasmid pEA191kn [SmithKline Beecham]. pEA191kn is a
derivative of pSKF301 [Shatzman et al, "Expression Using Vectors
with Phage .lambda. Regulatory Sequences" in Current Protocols in
Molecular Biology, ed. F. A. Ausubel et al, pp. 16.3.1-16.3.11
(1990)] created by 1) removing a portion of the Amp resistance gene
and replacing it with a kanamycin resistance gene and 2)inserting
the .lambda.rexB gene, which was engineered to remove endogenous
NdeI sites, upstream of the P.sub.L promoter.
[0177] For expression of the full-length mature KC (with the
addition of an N-terminal Met) an NdeI-SspI fragment encoding amino
acids 25-termination codon with the addition of an N-terminal Met
was isolated from pMKC and subcloned into pEA181kn which had been
cut with NdeI and HpaI. The resulting clone was designated
pEA1/mkc19 (wt).
[0178] To generate the desamino form of KC, a 322 bp PstI-SspI
fragment (bp 112-434) was isolated from pMKC (described above). A
linker comprising bp 86-111 was synthesized with a PstI compatible
overhang at the 3' end and an NcoI overhang at the 5' end. An NdeI
site was incorporated at the 5' end to provide an N-terminal Met in
frame with the KC gene product. The fragment and linker were
ligated to pEA181kn, cut with NcoI and HpaI.
[0179] The resulting clone, in which the desamino KC gene is fused
to the NS1 gene product, was designated pEA1-NS1/mkc21(-4). To
delete the NS1 portion of the fusion, pEA1-NS1/mkc21(-4) was cut
with NdeI and religated. The resulting clone was designated
pEA/mkc38(-4).
[0180] pEA/mkc18(wt) and pEA/mkc39(-4) have been induced with
nalidixic acid to express the full-length (amino acids 25-term with
the addition of an N-terminal Met) and the desamino (amino acids
29-term with N-terminal Met), respectively. For induction, E. coli
AR120 cells [SmithKline Beecham] transformed with the plasmids
construct were grown at 37.degree. C. in a gyrotory shaker at
250-300 rpm until AD.sub.650=0.4-0.6. One one-thousandth of the
volume of 60 mg/mL nalidixic acid (made up in 1 N NaOH) was added
to give a final concentration of 60 .mu.g/mL. Cultures were grown
for 4-5 hours and then harvested. Cells were lysed and crude
lysates tested for hematopoietic synergistic factor (HSF) activity
as described in Example 8 below.
EXAMPLE 3
Expression of Murine Desamino KC in E. coli
[0181] Murine full-length and desamino KC were subcloned to
pEA181kn, as described in Example 2 above, where they were
expressed under the control of the inducible P.sub.L promoter from
bacteriophage .lambda.. Induction of the desamino construct,
pEA1/mkc38(-4) with nalidixic acid resulted in production of a 69
amino acid protein (amino acids 29-96 of the mature KC with the
addition of a methionine at the amino-terminus). The desamino KC
product is soluble and shows synergistic activity when assayed as
crude bacterial lysate in the hematopoietic synergistic activity
assay described below in Example 8. The 73 amino acid protein
produced by nalidixic acid induction of pEA1/mkc18(wt) is also
soluble, but showed no synergistic activity unless digested with
DPP IV to produce the truncated form of the protein.
[0182] The desamino-KC combined with GM-CSF (20 U) resulted in
approximately 40-50 colonies of cells, which translated to an
activity of approximately 10.sup.14 U.
EXAMPLE 4
Synthesis of Descarboxy-KC
[0183] The native full-length and desamino KC forms were purified
from murine fibroblastic cell line, C6 [SmithKline Beecham]
supernatants by Heparin-agarose affinity chromatography with
subsequent reverse phase high performance liquid chromatography
(HPLC).
[0184] To prepare descarboxy-KC, native full length KC purified as
described above, was digested with carboxypeptidase Y to form
descarboxy-KC which is theoretically amino acid 1 to 68 of the full
length KC [SEQ ID NO: 1]. In contrast to native, full-length KC,
this descarboxy-KC is active as a synergistic factor.
EXAMPLE 5
Preparation of Desamino-gro-.beta.
[0185] Recombinant, full-length gro-.beta., which can optionally be
obtained from commercial sources, was digested with DPP IV enzyme
[Enzyme Product Systems, Inc.] to form desamino gro-.beta., which
spans amino acid 5 to 68 of the full length human gro-.beta. [SEQ
ID NO: 3].
[0186] Full-length gro.beta. shows no synergistic activity in the
hematopoietic synergistic activity assay (0 Units/mg). Digestion of
the lysate with DPP IV to produce the truncated form of the
protein, results in the appearance of signficant levels of
synergistic activity in the lysate; specifically this activity is
about 10.sup.8 Units in the assay of Example 8 when digested for a
short-term only. Even higher activities are anticipated with
increased duration of digestion.
EXAMPLE 6
Plasmid Construction for Expression of Human Desamino GRO.beta.
[0187] In the following description, all amino acid and base pair
numbers for human gro .beta. refer to the sequence as obtained in
Genbank, accession number M57731. A gene encoding the full-length
mature human gro.beta. protein (amino acids 34-106, bp 172-393,
with the addition of an N-terminal methionine corresponding to SEQ
ID NO: 3) was synthesized and subcloned to pCR2000 [SmithKline
Beecham]. The resulting clone was designated pHgro.beta.. 5' NdeI
and 3' HpaI sites were included in the synthesized gene to simplify
subsequent subcloning.
[0188] For E. coli expression, the NdeI-HpaI fragment encoding the
entire mature protein (with the addition of an N-terminal Met) was
subcloned into pEA181Kn, described above, which had been cut with
NdeI and HpaI. The resulting clone was designated
pEA1/hgro.beta.5(wt). This clone can be induced with nalidixic acid
as described above to produce a 74 amino acid product corresponding
to the full-length mature gro.beta. protein (with the added
N-terminal Met).
[0189] The desamino form of human gro.beta. was constructed as
follows. A 176 bp PstI-HpaI fragment was isolated from pHgro.beta.
and ligated with an NdeI-PstI linker (encoding amino acids 39-49,
with the addition of an N-terminal methionine) into NdeI/HpaI cut
pEA181kn. The resulting clone is designated pEA1-Hgro.beta.(-4).
FIG. 6 is a DNA map of this plasmid.
[0190] pEA1-hgro.beta.(-4) was induced with nalidixic acid as
described above to produce a 70 amino acid gro.beta. protein which
is truncated at the amino terminus of the active protein by 4 amino
acids (aa 39-106 with the addition of an N-terminal Met).
EXAMPLE 7
Expression of Human Desamino gro-.beta. in E. coli
[0191] The human gro.beta. (MIP2.alpha.) gene was synthesized and
subcdoned to E. coli expression vector pEA181kn, described in
Example 6 above, where it is expressed under the control of the
inducible P.sub.L promoter derived from bacteriophage .lambda..
Induction with nalidixic acid resulted in the production of a 74
amino acid protein (amino acids 35-termination codon with the
addition of an N-terminal methionine).
[0192] This recombinant gro.beta. (amino acids 5-73 of SEQ ID NO:
3) was found to have an activity of about 10.sup.14 Units in the
assay of Example 8.
EXAMPLE 8
Hematopoietic Synergistic Factor Activity Assay
[0193] All modified chemokines are screened in the following
conventional assay to determine whether or not the modified
chemokine is characterized by synergistic activity with colony
stimulating factor (CSF), i.e. the combination of the chemokine and
the colony stimulating factor exceeds the additive effect of the
two proteins.
[0194] Murine bone marrow cells harvested and suspended in RPMI
1649 with 10% fetal bovine serum (FBS). The bone marrow cells are
cultured with suboptimal concentrations of recombinant murine
Granulocyte-Macrophage (GM)-CSF (20 U/ml) and dilutions of test
compounds in a standard murine CFU-GM soft agar assay. Optionally,
G-CSF, M-CSF, and IL-3 (low O.sub.2 conditions) can be used as an
alternate CSF source. Marrow cells cultured with 20 units of GM-CSF
results in the growth of approximately 20-30 colonies of cells
(background). As is conventional, 1 Unit (U) is equivalent to
approximately 1 colony above background.
[0195] For example, desamino-KC combined with GM-CSF (20 U/ml)
resulted in approximately 40-50 colonies of cells, which translated
to a specific activity of approximately 10.sup.14 U/mg. Synthetic
gro.alpha. (amino acids 5-73) are also characterized by having a
specific activity of 10.sup.14 U/mg.
[0196] Even when using commercially obtained chemokines, which may
contain impurities, the method of the invention results in
increased activity. For example the following results were obtained
using shortened digestion periods.
2 Commercial1 Gro.beta. + 2 .times. 10.sup.4 U DPPIV digestion 1
.times. 10.sup.6 U Commercial gro.gamma. + 2 .times. 10.sup.3 U
DPPIV digestion 5 .times. 10.sup.5 U More dramatic increases in
activity are anticipated with increased duration of digestion, and
with synthetically produced cytokines.
EXAMPLE 9
Preparation of (p-Glu-Glu-Asp).sub.2-Pim-(Lvs).sub.2 [SEQ ID NO:
20]
[0197] 3
[0198] A half gram of t-TOC-Lys(Cl-Z)-O CH.sub.2-Pam Resin (0.63
mmol/gm) was loaded in the reaction vessel of a Beckman 990 B
synthesizer. In the deprotection step, the t-Boc group was removed
using 40% trifluoroacetic acid (TFA) in methylene chloride
(CH.sub.2Cl.sub.2) and rinsed with CH.sub.2Cl.sub.2. The
trifluoroacetate salt was neutralized by 10% DIEA/CH.sub.2Cl.sub.2.
Two mM (780 mgs) of Di-BOC-2,6-diaminopimelic acid was coupled
using 2 mM of DCC and HOBT. The coupling was done in the mixture of
15 ml of CH.sub.2Cl.sub.2 and 10 ml of DMF at room temperature for
two hours. Kaiser's test was used to monitor the coupling. Any
remaining free carboxyl groups were amidated twice by using 3 mM
(1.65 gms) of H-Lys(Z)-OBz.HCl and 3 mM of DCC and 3 mM of HOBT in
25 ml of CH.sub.2Cl.sub.2/DMF (15/10).
[0199] After two hours of coupling, the resin was washed twice with
15 ml of CH.sub.2Cl.sub.2, twice with 15 ml of DMF, twice with 15
ml of MeOH/CH.sub.2Cl.sub.2 (1:1), and finally twice with 15 ml of
CH.sub.2Cl.sub.2. After the deprotection of t-Boc using 40%
TFA/CH.sub.2Cl.sub.2 and the neutralization using 10%
DIEA/CH.sub.2Cl.sub.2, 2 mM (0.646 gm) of Boc-Asp(Bzl) and 2 mM of
DCC and 2 mM of HOBT were added and coupled for 2 hours in 25 ml of
CH.sub.2Cl.sub.2/DMF (15/10). The resin was then subjected to a
washing step as described earlier. The deprotection step and the
neutralization step were repeated before 2 mM (0.674 gm) of Boc-Glu
(Bzl), 2 mM DCC and 2 mM of HOBT were coupled in 25 ml of
CH.sub.2Cl.sub.2/DMF (15/10). After washing, deprotection and
neutralization steps, 2 mM (0.258 gm) of pGlu, 2 mM of DCC and 2 mM
of HOBT were coupled in 25 ml of CH.sub.2Cl.sub.2/DMF (15/10) for 2
hours before the resin was subjected to a washing step. Completion
of the coupling was monitored by Kaiser's test and only single
coupling was needed at each step. After the completion of the
synthesis the resin was dried and weighed. Yield: 1.2 g.
[0200] The peptide resin (1.2 gm) was charged in a cleavage
apparatus and cleaved using 10 ml of hydrofluoric acid (HF) and 1
ml anisole at -15.degree. C. for two hours. After removal of HF
under vacuum the mixture of resin and peptide was extensively
washed with ether and the peptide was extracted in glacial acetic
acid (30 ml). Most of the acid was removed from the extracts on a
rotavap and the residue was diluted in water and lyophilized. The
acetic acid extract had 810 mgs of crude peptide.
[0201] The crude peptide (80 mgs), obtained from acetic acid
extraction, was further purified using a preparative C-18 column.
It was passed through a pre-equilibrated (in 0.1% TFA/H.sub.2O)
column. The peptide was eluted using a linear gradient of 80%
acetonitrile, 20% H.sub.2O and 0.1% TFA.
[0202] Three isomers co-eluted (8.52 min). These were separated on
a C-18 column using a gradient of 30% (0.1% TFA in CH.sub.3CN), 70%
(0.1% TFA in H.sub.2O) to 80% (0.1% TFA in CH.sub.3CN), 20% (0.1%
TFA in H.sub.2O) over 35 minutes at a flow rate of 1.5 ml/min. The
following fractions were eluted:
[0203] fraction 1: 18.69 min
[0204] fraction 2: 19.68 min
[0205] fraction 3: 22.95 min
[0206] Amino acid analysis gave the following results:
3 Amino Acid Analysis Observed Glu 1.99 Asp 1.0 Lys 1.05 Bis amino
pimelic acid N.D. mass spec = 1157.5 (M + H).sup.+
EXAMPLE 10
Preparation of (pGlu-Glu-Asp).sub.2-Lan-(Lys).sub.2 [SEQ ID NO:
21]
[0207] [Lan=Lanthionine(SCH.sub.2CH(NH.sub.2)COOH)]
[0208] A half gram of t-BOC-Lys(Cl-Z)-CH.sub.2 PAM (0.63 m. m/g) is
charged in the reaction vessel of a Beckman 990 synthesizer. The
t-BOC group is removed using 40% TFA in methylene chloride. The
trifluoroacetic acid salt is neutralized by 10%
DIEA/CH.sub.2Cl.sub.2. Two mM of Bis BOC lanthionine is coupled
using 4 mM of DCC and HOBT in 15 ml of CH.sub.2Cl.sub.2 and 10 ml
of DMF at room temperature. The Kaiser test is used to monitor the
coupling. Any free remaining carboxyl groups are amidated using 3
mM of H-Lys (Z)-OBz.HCl; and 3 mM of DCC and HOBT in 25 ml of
CH.sub.2Cl.sub.2/DMF (15/10). After the coupling resin is
extensively washed with CH.sub.2Cl.sub.2, 30%
MeOH-CH.sub.2Cl.sub.2, and CH.sub.2Cl.sub.2 (25 ml.times.3), the
cycles of deprotection, neutralization and coupling are repeated
with the remaining amino acids in the target peptide (Asp, Glu,
pGlu). Four mM of each amino acid, DCC and HOBT are used for each
coupling. Each coupling is monitored using Kaiser test. After
completion of the synthesis, the resin is dried and weighed.
[0209] The peptide resin is charged in cleavage apparatus and
cleaved using 10 ml of hydrofluoric acid (HF) and one ml of anisole
at -15.degree. C. for two hours. After removal of the HF, the resin
is extensively washed with ether and the peptide is extracted with
glacial acetic acid (30 ml). Most of the acetic acid is removed on
a rotavap and the residue is diluted in water and lyophilized.
After purification of HPLC, the peptide is obtained.
EXAMPLE 11
Preparation of (pGlu-Glu-Asp).sub.2-Pim-(Arg-CONH.sub.2).sub.2 [SEQ
ID NO: 22]
[0210] A half gram of BOC-Tos Arg-BHA resin (0.5 m. M/g) is charged
in the reaction vessel of a Beckman 990 synthesizer. The BOC group
is removed using 40% TFA in methylene chloride. The trifluroacetic
acid salt is neutralized by 10% DIEA/CH.sub.2Cl.sub.2. One mM of
Bis BOC pimelic acid is coupled using 2 mM of DCC and HOBT in 15 ml
of CH.sub.2Cl.sub.2 and 10 ml of DMF at room temperature. The
Kaiser test is used to monitor the coupling. Any free remaining
carboxyl groups are amidated using 3 mM of H-Lys (Z)-OBz.HCl; and 3
mM of DCC and HOBT in 25 ml of CH.sub.2Cl.sub.2/DMF (15/10). After
coupling, the resin is extensively washed with CH.sub.2Cl.sub.2,
30% MeOH-CH.sub.2Cl.sub.2, and CH.sub.2Cl.sub.2 (25 ml.times.3).
The cycles of deprotection, neutralization and coupling are
repeated with the remaining amino acids in the target peptide (Asp,
Glu and p-Glu). 3 mM of amino acid, DCC and HOBT are used for each
coupling. Each coupling is monitored using the Kaiser test. After
completion of the synthesis, the resin was dried and weighed.
[0211] The peptide resin is charged in a cleavage apparatus and
cleaved using 10 ml of hydrofluoric acid (HF) and one ml of anisole
at -15.degree. C. for two hours. After removal of the HF, the resin
is extensively washed with ether and the peptide is extracted with
glacial acetic acid (30 ml). Most of the acetic acid is removed on
a rotavap and the residue is diluted in water and lyophilized.
After purification by HPLC the peptide is obtained.
EXAMPLE 12
Synthesis of Tyrosine Containing Analogs
[0212] (Tyr-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 23];
[0213] (pGlu-Tyr-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO:
24];
[0214] (pGlu-Glu-Tyr-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO:
25];
[0215] (pGlu-Glu-Asp-Tyr).sub.2-Sub-(Lys).sub.2 [SEQ ID NO:
26].
[0216] Two grams of BOC-Lys(Cl-Z)-0-Resin (Peninsula Labs.RTM.,
substitution 0.49 mM/g) was charged in a manual shaker vessel.
After deprotection and neutralization steps, 2 mM (808 mg) of
di-BOC diaminosuberic acid was coupled to the resin using 4 mM (824
mg) of dicyclohexylcarbodiimide (DCC) and 4 mM (612 mg) of
1-hydroxybenzotriazole hydrate (HOBT) in 25 ml of 50%
N-methyl-2-pyrrolidinone (NMP) and dichloromethane (DCM). The
reaction was allowed to proceed overnight followed by the addition
of 10 mM (4.06 g) H-Lys (Z)-OBz.HCl, 10 mM (1.29 g)
diisopropylethylamine (DIEA), 10 mM (2.06 g) DCC and 10 mM (1.53 g)
HOBT. After two hours, the unreacted amino groups were capped using
10% acetic anhydride in NMP/DCM (1:1).
[0217] Approximately one third of the resulting BOC-Sub-Lys-resin
was transferred to another reaction vessel. The major fraction of
the resin is called fraction I, and minor fraction is called
fraction II. The standard deprotection, neutralization and coupling
cycles were used to couple BOC-Tyr (Br-Z), BOC-Asp(OBz),
BOC-Glu(OBz), and p-Glu to the resin in fraction II. Five mM of
amino acid, DCC and HOBT were used. Coupling was performed in 2 5
ml NMP/DCM (1/1) and was monitored for completion using the Kaiser
test. Five mM of BOC-Asp(OBz) were coupled to the resin in fraction
I. One fourth of the resulting BOC-Asp-(OBz)Sub-Lys(Cl-Z) resin was
transferred to another vessel (fraction III). The remaining resin
is called fraction IV. Standard deprotection, neutralization and
coupling cycles were used to couple BOC-Tyr (Br-Z), BOC-Glu(OBz),
and p-Glu to resin in fraction III. Five mM of amino acid, DCC and
HOBT were used. Coupling was performed in 25 ml NMP/DCM (1/1) and
was monitored for completion using Kaiser test. Five mM of
BOC-Glu(OBz) were coupled to the resin in the major fraction
(fraction IV). One third of this resin was transferred to another
vessel and 5 mM of p-Glu were coupled to this fraction (fraction
VI) resulting in the synthesis of pGlu-Glu-Asp-Sub-Lys-Resin. Five
mM of BOC Tyr(Br-Z) were coupled to the major fraction (fraction V)
and the resin was further split in halves. One half of the resin
was saved as is and to the other half of the resin (fraction VII) 5
mM of p-Glu were coupled resulting in the synthesis of
p-Glu-Tyr-Glu-Asp-Sub-Lys-Resin. These resin peptides were
deprotected and cleaved using HF/anisole at 0.degree. C. for one
hour. The crude peptides (approx. 100 mg) were purified on a C-18
VYDAC 2.5 cm.times.30 cm preparative column using water/0.1%
trifluroacetic acid (TFA), and acetonitrile/0.01% TFA buffer
system.
EXAMPLE 13
Preparation of (pGlu-Glu-Asp).sub.2Prc(Lvs).sub.2 [SEQ ID NO:
27]
[0218] a. Synthesis of Bis BOC-S,S'-1-3-propanediylcysteine
[0219] Three ml of methanol were saturated with dry ammonia and 0.5
g BOC-cysteine in 0.5 ml methanol was added, followed by the
addition of 0.35 ml of 1,3 dibromopropane. Ten minutes later,
additional 0.5 g of BOC-cysteine in 0.5 ml methanol was added.
After 4.5 hours, the solvent was evaporated and the oily residue
dissolved in water. The pH of the solution was adjusted to 9, and
the solution was extracted with ether. The aqueous layer was
acidified to pH 2 and extracted with ethylacetate. The organic
layer was dried and evaporated to yield 1.12 g of Bis
BOC-S,S-1,3-propanediylcysteine. The amino acid was used without
any further purification, FAB/MS M+H=469.
[0220] b. Preparation of (pGlu-Glu-Asp).sub.2Prc(Lys).sub.2 [SEQ ID
NO: 27]
[0221] BOC-Lys resin (0.53 g, substitution 0.63 mM/g) was charged
in a manual shaker and after deprotection and neutralization
cycles, bis BOC-S,S'-1,3-propanediylcysteine (290 mg, 0.6 mM) was
coupled using 1 mM (206 mg) DCC and 1 mM (153 mg) HOBT in 10 ml
NMP/DCM (1/1). After two hours, the resin was washed with NMP and
DCM and 2 mM (765 mg) of H-Lys (Z)-OBz.HCl was added followed by
the addition of 1.5 mM (390 mg) DCC and 1.5 mM (230 mg) of HOBT in
4 ml of NMP/DCM (1/1). After 18 hours, the resin was washed using
20 ml NMP and DCM. Normal deprotection and neutralization and
coupling cycles were repeated for the coupling of BOC-Asp(OBz),
BOC-Glu(OBz) and p-Glu. One mM of amino acid, DCC and HOBT were
used. Coupling was done in 5 ml of NMP/DCM (1/1). Completion of the
coupling was monitored using Kaiser's test. The resulting resin
peptide (416 mg) was deprotected and cleaved using 0.5 ml anisole
and 8 ml of HF at 0.degree. C. for 2 hours. HF was evaporated and
the peptide resin mixture was washed with ether and extracted with
glacial acetic acid. After lyophilization, 130 mg of the crude
peptide was obtained. The crude peptide (61.5 mg) was purified on a
C18 VYDAC preparative column using acetonitrile-water (0.1% TFA)
buffer system. 16.5 mg of pure peptide was obtained.
[0222] FAB/MS: M+H 1249.3
[0223] Amino Acid Analysis
[0224] Asp 2.0(2)
[0225] Glu 4.28(4)
[0226] Dpc 1.14
[0227] Lys 1.96(2)
EXAMPLE 14
Synthesis of:
[0228] ((d)-pGlu-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 28]
[0229] (pGlu-Glu-Glu).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 7]
[0230] (pGlu-(d)-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 29]
[0231] (pGlu-Asp-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO: 8]
[0232] A half gram (0.5 g) of t-BOC-Lys (Cl-Z)-O CH.sub.2-Pam Resin
(0.63 m mol/gm) war loaded in a manual shaker vessel. In the
deprotection step, the BOC group was removed using 40%
Trifluoroacetic acid (TFA) in melthylene chloride
(CH.sub.2Cl.sub.2). Trifluoroacetate salt was neutralized by 10%
DIEA/CH.sub.2Cl.sub.2. After the deprotection and neutralization
steps, Di-BOC-2,6-diaminosuberic acid (0.16 mM, 66.2 mgs) was
coupled using 0.315 mM of DCC and HOBT. The coupling was done in
the mixture of 5 ml of CH.sub.2Cl.sub.2 and 5 ml of DMF at room
temperature for four days. Kaiser's test was used to monitor the
coupling. The unreacted amino groups were capped using a 10% acetic
anhydride/DMF solution. Standard deprotection, neutralization and
coupling cycles were then followed and the targeted sequence was
assembled. Three mM of amino acid, DCC and HOBT were used. Coupling
time was four hours. Completion of the coupling was monitored by
Kaiser's test and only single coupling was needed at each step. The
peptide resin was loaded in a cleavage apparatus and cleaved using
10 ml of hydrofluoric acid (HF) and 1 ml anisole at -15.degree. C.
for two hours. After removal of HF under vacuum, the mixture of
resin and peptide was extensively washed with ether and the
peptides were extracted in 0.1% TFA and lyophilized. Purification
and characterization: The crude peptides were purified using
preparative C-18 columns. The column was pre-equlibrated in 99.9%
water and 0.1% TFA and the peptide was eluted using a linear
gradient of 80% acetonitrile, 20% water and 0.1% TFA. The peptides
were analyzed for amino acid composition. The molecular weight was
determined usign FAB MS. FAB and amino acid analysis:
[0233] FAB MS (M+H 1171.5)
[0234] Amino acid analysis: Asp 1.98(2), Glu 4.6(4) and Lys
2(2)
[0235] HPLC purity >95%
EXAMPLE 15
Preparation of (Pic-Ser-Asp).sub.2-Adp-(Lys).sub.2 [SEQ ID NO:
14]
[0236] a. Synthesis of Boc-Lvs(Cl-Z)-Resin
[0237] Boc-Lys(Cl-Z) [16.6 g, 40 mmol] was dissolved in 150 ml of
10% H.sub.2O in MeOH. The solution was neutralized using 40 ml of
1M CsCO.sub.3 solution. The neutralized solution was concentrated
using a rotary evaporator. The resulting cesium salt was diluted
with DMF and the DMF was removed using a rotary evaporator. This
step was repeated two more times. The salt was dried in vacuo for
four hours.
[0238] The cesium salt of BOc-Lys(cl-Z) was diluted with 120 ml
DMF. To this, was added chloromethyl resin. The reaction mixture
was swirled at 40.degree. C. for 48 hours.
[0239] The resin was cooled to room temperature and filtered
through a fritted funnel and washed sequentially as follows: 500 ml
DMF; 500 ml DMF: H.sub.2O (1:1); 500 ml DMF; 500 ml MeOH and 1000
ml CH.sub.2Cl.sub.2. The resin was dried in vacuum. The desired
Boc-Lys(Cl-Z)-resin was recovered.
[0240] b. Synthesis of
Boc-Asp(OBz)).sub.2-Adp-[Lys(Cl-Z)].sub.2-resin
[0241] The Boc-Lys(Cl-Z)-resin was transferred into a reaction
vessel and was suspended in CH.sub.2Cl.sub.2.
[0242] The Boc protection of the Boc-Lys(Cl-Z) resin was removed by
washing the resin with 40 ml 50% TFA in CH.sub.2Cl.sub.2 for 5
minutes followed by another 40 ml of 50% TFA wash for 30 minutes.
The resin was then washed three (3) times with 40 ml
CH.sub.2Cl.sub.2.
[0243] The resulting TFA salt was treated twice with 40 ml 10% DIEA
in CH.sub.2Cl.sub.2 for 1 minute, washed once with 40 ml
CH.sub.2Cl.sub.2 and once more with 40 ml of 10% DIEA in
CH.sub.2Cl.sub.2. The resin was thoroughly washed with
CH.sub.2Cl.sub.2 (6.times.40 ml).
[0244] For coupling N,N' di-Boc diaminoadipic acid (Boc-Adp-OH) to
the NH2-Lys(Cl-Z)-resin, the resin was suspended in 100 ml
methylene chloride (CH.sub.2Cl.sub.2) in a manual shaker vessel. In
another 50 ml flask, Boc-Adp-OH and HOBT were dissolved in 5 ml
N-methyl pyrrolidone (NMP) and 20 ml CH.sub.2Cl.sub.2. The solution
was cooled to 0.degree. C. using an ice bath. To this solution was
added DCC and the reaction mixture was stirred at 0.degree. C. for
15 minutes.
[0245] The resulting precipitates of dicyclihexylurea were filtered
off and the pre-formed active ester was added to the
NH.sub.2-Lys(2-Cl-Z)-res- in suspension. The reaction was monitored
using the Kaiser test (Ninhydrin). After 96 hours, the
dipeptide-resin was washed with CH.sub.2Cl.sub.2 (3.times.40 ml),
(3.times.40 ml) and CH.sub.2Cl.sub.2 (6.times.40 ml). Ninhydrin
test after work-up gave a slight blue tint solution compared to the
negative control. The resin was then washed once with 10% acetic
anhydride in CH.sub.2Cl.sub.2 for 10 minutes and finally with
CH.sub.2Cl.sub.2 (6.times.40 ml).
[0246] c. Synthesis of
[Boc-Asp(OBz)].sub.2-Adp-[Lys(Cl-Z)].sub.2-resin
[0247] The TFA deprotection and neutralization steps were repeated
to prepare the dipeptide-resin for the Boc-Asp(OBz) coupling. For
coupling this amino acid, Boc-Asp(OBz), and HOBT were used. The
coupling was complete after 24 hours as indicated by negative
ninhydrin test. This tripeptide-resin was also used to make other
peptides with different amino acids at position 2 (B).
[0248] A half gram of
[BOC-Asp(OBZ)].sub.2-Adp-[Lys(Cl-Z)].sub.2-resin was used for the
synthesis of (Pic-Ser-Asp).sub.2-Adp-(Lys).sub.2 [SEQ ID NO: 14].
The deprotection, neutralization and coupling cycles were repeated
with BOC-Ser(Bz)-OH and Picolinic acid. 1.8 mM of amino acids, DCC
and HOBT were used in coupling. The couplings were done in 10 ml
DMF/CH.sub.2Cl.sub.2. The peptide-resin was cleaved and crude
peptide obtained. The crude peptide was purified on a C-18 VYDAC
preparative column, using acetonitrile/water TFA buffer system.
Thirteen milligrams of pure peptide were obtained.
[0249] FAB MS (M+H 1047.4)
[0250] Amino acid analysis: Asp 2.00(2), Ser 1.62(2), Lys 1.81(2)
(Pic and Adp were not analyzed).
[0251] HPLC purity >95%
EXAMPLE 16
Preparation of (Glu-Glu-Asp).sub.2-Sub-(Lys).sub.2 [SEQ ID NO:
51]
[0252] 4
[0253] a. Synthesis of BOC-SUB-Lys-(.epsilon.-Z)COOBz
[0254] Bis-BOC (1,1) diaminosuberic acid (Sub) was synthesized
using R. Nutt's method [J. Org. Chem., 45:3078 (1980)].
[0255] Two mM of Boc-Sub (808 mg), 4 mM of
Lys-(.epsilon.-Z)-COOBz.HCl (1.56 g) and 4 mM of HOBT (0.613 g)
were dissolved in 10 ml of methylene chloride (CH.sub.2Cl.sub.2)
and the solution was chilled to -15.degree. C. using an ice/acetone
bath. Four mM (0.692 ml) of diisopropyl ethyl amine (DIEA) were
added followed by the addition of 0.772 g (4 mM) water soluble
carbodiimide (EDC). After stirring for one hour the mixture was
allowed to warm to room temperature. After three hours the
methylene chloride was evaporated and the residue was dissolved in
200 ml of ethyl acetate. The solution was washed first with 1N HCl
, then 1N NaOH, saturated NaCl solution and water. The washes were
repeated three times and each was was about 100 ml. The organic
layer was dried over MgSO.sub.4 and evaporated. 1.86 g of
BOC-Sub-(.epsilon.-Z)Lys-COOBz (79% yield) was obtained and used
further without any purification.
[0256] b. Synthesis of BOC Asp-(.beta.-OBz)Sub
Lys-(.epsilon.-Z)-COOBz
[0257] BOC-Sub-Lys(.epsilon.-Z)-COOBz (1.8 g) was dissolved in 4N
HCl-dioxane for a half hour and then evaporated to dryness. The
residue was washed with ether and dried overnight. The
hydrochloride salt was dissolved in 30 ml of CH.sub.2Cl.sub.2 and
BOCAsp-(.beta.-OBz) (1.292 g) was added. The solution was chilled
to -15.degree. C and 0.613 g HOBT, 0.554 ml DIEA and 0.772 g of EDC
were added. After stirring for two hours the mixture was allowed to
warm up to room temperature. After 18 hours (overnight) the
reaction mixture was worked up. CH.sub.2Cl.sub.2 was evaporated and
the residue was dissolved in 200 ml of ethyl acetate. The solution
was washed with 1N HCl, 1N HaOH, saturated NaCl solution and water
(washes were repeated three times and each was was about 100 ml).
The organic layer was dried over MgSO.sub.4 and evaporated.
BOC-Asp-(.beta.-OBz)-Sub-Lys-(.epsilon.-Z)COOBz Z) COOBz 1.9 g
(yield 73%). This peptide was used without any further
purification.
[0258] c. Synthesis of BOC-Glu-(.gamma.-OBz)
Asp-(.beta.-OBz)Sub-Lys-(.eps- ilon.-Z)COOBz
[0259] BOC-(.beta.-OBz) Asp-Sub-(.epsilon.-Z) Lys-COOBz, 1.8 g, was
dissolved in 15 ml of 4N HCl dioxane. After fifteen minutes the
solvent was removed and the residue was washed with ether and
dried. The hydrochloride salt was dissolved in 15 ml of N-methyl
pyrrolidone (NMP). The solution is chilled to -15.degree. C. and 4
mM (1.338) of BOC-Glu (.gamma.-OBz), 0.204 ml DIEA, 0.772 g EDC and
0.612 g of HOBT was added. The mixture was stirred overnight while
gradually warming up to room temperature. The reaction mixture was
added to a flask containing one liter of chilled 10%
Na.sub.2CO.sub.3 in saturated NaCl solution. The precipitates were
filtered, washed with water, and dried under vacuum.
BOC-(.gamma.-OBz )Glu-(.beta.-OBz)Asp-Sub-(.epsilon.-Z)Lys-COOBz
(1.3 g) was obtained and used without any further purification.
Yield: 68%.
[0260] d. Synthesis of pGlu-(.gamma.-OBz) Glu-(.beta.-OBz)
Asp-Sub-(.epsilon.-Z) Lys-COOBz
[0261] BOC-(.gamma.-OBz) Glu-(.beta.-OBz) Asp-Sub-(.epsilon.-Z)
Lys-COOBz 1.2 g was dissolved in 15 ml of 4N HCl dioxane. After
fifteen minutes, the solvent was removed and the residue was washed
with ether and dried. The hydrochloride salt
Sequence CWU 1
1
* * * * *