U.S. patent application number 17/020237 was filed with the patent office on 2022-01-13 for protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage.
This patent application is currently assigned to THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. The applicant listed for this patent is THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Richard T. LEE, Vincent SEGERS.
Application Number | 20220009984 17/020237 |
Document ID | / |
Family ID | 1000006048771 |
Filed Date | 2022-01-13 |
United States Patent
Application |
20220009984 |
Kind Code |
A9 |
LEE; Richard T. ; et
al. |
January 13, 2022 |
PROTEASE RESISTANT MUTANTS OF STROMAL CELL DERIVED FACTOR-1 IN THE
REPAIR OF TISSUE DAMAGE
Abstract
The present invention is directed stromal cell derived factor-1
peptides that have been mutated to make them resistant to digestion
by the proteases dipeptidyl peptidase IV (DPPIV) and matrix
metalloproteinase-2 (MMP-2) but which maintain the ability of
native SDF-I to attract T cells. The mutants may be attached to
membranes formed by self-assembling peptides and then implanted at
sites of tissue damage to help promote repair.
Inventors: |
LEE; Richard T.; (Weston,
MA) ; SEGERS; Vincent; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Boston |
MA |
US |
|
|
Assignee: |
THE BRIGHAM AND WOMEN'S HOSPITAL,
INC.
Boston
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20210246182 A1 |
August 12, 2021 |
|
|
Family ID: |
1000006048771 |
Appl. No.: |
17/020237 |
Filed: |
September 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15374539 |
Dec 9, 2016 |
10774124 |
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17020237 |
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14039580 |
Sep 27, 2013 |
9631005 |
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15374539 |
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13175070 |
Jul 1, 2011 |
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14039580 |
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12683253 |
Jan 6, 2010 |
7999067 |
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13175070 |
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11976032 |
Oct 19, 2007 |
7696309 |
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12683253 |
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15374539 |
Dec 9, 2016 |
10774124 |
|
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11976032 |
|
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14039580 |
Sep 27, 2013 |
9631005 |
|
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15374539 |
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11976032 |
Oct 19, 2007 |
7696309 |
|
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14039580 |
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60929353 |
Jun 22, 2007 |
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60853441 |
Oct 23, 2006 |
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60929353 |
Jun 22, 2007 |
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60853441 |
Oct 23, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/52 20130101;
C12N 5/0652 20130101; C07K 14/521 20130101; A61K 38/00
20130101 |
International
Class: |
C07K 14/52 20060101
C07K014/52; C12N 5/077 20060101 C12N005/077 |
Claims
1-20. (canceled)
21. A fusion protein comprising the formula:
A--(L).sub.n--(R).sub.q, wherein: A is an isolated mutant form of
stromal cell derived factor-1 (SDF-1) peptide comprising the
formula mSDF-1 or X.sub.p-mSDF-1, wherein mSDF-1 comprises the
amino acid sequence of at least amino acids 1-8 of SEQ ID NO:52 and
which is optionally extended at the C terminus of amino acids 1-8
of SEQ ID NO: 52 by all or any portion of the remaining sequence of
SEQ ID NO:52, shown as amino acids 9-68 of the full-length of SEQ
ID NO: 52 and the amino acids 1-8 comprises a mutation at the
fourth and/or fifth amino acids from the N terminus of the amino
acids 1-8 of SEQ ID NO:52, and wherein: a) X is a proteinogenic
amino acid or a protease protective organic group; b) p is an
integer from 1 to 4; and c) the mutant form of SDF-1 peptide has
chemoattractant activity for T cells, is inactivated by dipeptidyl
peptidase IV (DPPIV) at a rate that is less than one-half of the
rate at which native SDF-1 is inactivated, and is inactivated by
MMP-2 at a rate that is less than one-half of the rate at which
native SDF-1 is inactivated; L is a linker sequence of 3-9 amino
acids; R is a self-assembling peptide comprising the amino acid
sequence of any one of SEQ ID NOs: 1-51; n is an integer from 0-3;
and q is an integer from 1-3.
22. The fusion protein of claim 21, wherein A comprises: i) at
least amino acids 1-8 of SEQ ID NO:53 and which is optionally
extended at the C terminus of amino acids 1-8 of SEQ ID NO: 53 by
all or any portion of the remaining sequence of SEQ ID NO:53, shown
as amino acids 9-68 of the full-length of SEQ ID NO: 53; ii) at
least amino acids 1-8 of SEQ ID NO:54 and which is optionally
extended at the C terminus of amino acids 1-8 of SEQ ID NO: 54 by
all or any portion of the remaining sequence of SEQ ID NO:54, shown
as amino acids 9-68 of the full-length of SEQ ID NO: 54; iii) at
least amino acids 1-8 of SEQ ID NO:55 and which is optionally
extended at the C terminus of amino acids 1-8 of SEQ ID NO: 55 by
all or any portion of the remaining sequence of SEQ ID NO:55, shown
as amino acids 9-68 of the full-length of SEQ ID NO: 55; or iv) at
least amino acids 1-8 of SEQ ID NO:56 and which is optionally
extended at the C terminus of amino acids 1-8 of SEQ ID NO: 56 by
all or any portion of the remaining sequence of SEQ ID NO:56, shown
as amino acids 9-68 of the full-length of SEQ ID NO: 56.
23. The fusion protein of claim 21, wherein A comprises: i) at
least amino acids 1-17 of SEQ ID NO:53 and which is optionally
extended at the C terminus of amino acids 1-17 of SEQ ID NO: 53 by
all or any portion of the remaining sequence of SEQ ID NO:53, shown
as amino acids 18-68 of the full-length of SEQ ID NO: 53; ii) at
least amino acids 1-17 of SEQ ID NO:54 and which is optionally
extended at the C terminus of amino acids 1-17 of SEQ ID NO: 54 by
all or any portion of the remaining sequence of SEQ ID NO:54, shown
as amino acids 18-68 of the full-length of SEQ ID NO: 54; iii) at
least amino acids 1-17 of SEQ ID NO:55 and which is optionally
extended at the C terminus of amino acids 1-17 of SEQ ID NO: 55 by
all or any portion of the remaining sequence of SEQ ID NO:55, shown
as amino acids 18-68 of the full-length of SEQ ID NO: 55; or iv) at
least amino acids 1-17 of SEQ ID NO:56 and which is optionally
extended at the C terminus of amino acids 1-17 of SEQ ID NO: 56 by
all or any portion of the remaining sequence of SEQ ID NO:56, shown
as amino acids 18-68 of the full-length of SEQ ID NO: 56.
24. The fusion protein of claim 21, wherein A comprises the
sequence of any one of SEQ ID NOs: 53-56.
25. The fusion protein of claim 21, wherein X is serine.
26. The fusion protein of claim 25, wherein p is 1.
27. The fusion protein of claim 21, wherein R comprises the amino
acid sequence of SEQ ID NO: 35.
28. The fusion protein of claim 27, wherein q is 1.
29, The fusion protein of claim 21, wherein L comprises the amino
acid sequence of any one of SEQ ID NOs: 57-59.
30. The fusion protein of claim 29, wherein n is 1.
31. A method of treating a patient to promote the repair of damaged
tissue, the method comprising administering to the patient the
fusion protein of claim 21.
32. The method of claim 31, wherein the patient is treated for a
disease or condition selected from stroke, limb ischemia, tissue
damage due to trauma, and diabetic ulcer.
33. A biologically-compatible membrane comprising the fusion
protein of claim 21.
34. A method of treating a patient to promote the repair of damaged
tissue, the method comprising administering to the patient the
biologically-compatible membrane of claim 33.
35. The method of claim 34, wherein the patient is treated for a
disease or condition selected from stroke, limb ischemia, tissue
damage due to trauma, and diabetic ulcer.
36. The method of claim 34, wherein the biologically-compatible
membrane is injected or implanted at the site of tissue damage.
37. The method of claim 34, wherein the patient is treated for
damage to cardiac tissue and the biologically-compatible membrane
is injected or implanted into the myocardium of the patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. 120, 121,
or 365(c) of U.S. patent application Ser. No. 15/374,539 filed Dec.
9, 2016, which is a divisional under 35 U.S.C. 120, 121, or 365(c)
of U.S. patent application Ser. No. 14/039,580 filed Sep. 27, 2013,
now U.S. Pat. No. 9,6131,005, issued Apr. 25, 2017, which is a
continuation under 35 U.S.C. 120, 121, or 365(c) of U.S. patent
application Ser. No. 13/175,070 filed Jul. 1, 2011, which is a
continuation under 35 U.S.C. 120, 121, or 365(c) of U.S. patent
application Ser. No. 12/683,253 filed Jan. 6, 2010, now U.S. Pat.
No. 7,999,067 which is a continuation under 35 U.S.C. 120, 121, or
365(c) of U.S. patent application Ser. No. 11/976,032 filed Oct.
19, 2007, now U.S. Pat. No. 7,696,309, which claims priority to and
benefit under 35 U.S.C. 119(e) of U.S. Provisional Application Nos.
60/929,353 filed on Jun. 22, 2007 and 60/853,441 filed on Oct. 23,
2006.
[0002] This application is a continuation under 35 U.S.C. 120, 121,
or 365(c) of U.S. patent application Ser. No. 15/374,539 filed Dec.
9, 2016, which is a divisional under 35 U.S.C. 120, 121, or 365(c)
of U.S. patent application Ser. No. 14/039,580 filed Sep. 27, 2013,
now U.S. Pat. No. 9,631,005, issued Apr. 25, 2017, which is a
divisional of U.S. patent application Ser. No. 11/976,032 filed
Oct. 19, 2007, now U.S. Pat. No. 7,696,309, which claims priority
to and benefit under 35 U.S.C. 119(e) of U.S. Provisional
Application Nos. 60/929,353 filed on Jun. 22, 2007 and 60/853,441
filed on Oct. 23, 2006. The contents of each of these applications
are hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The sequence listing of the present application has been
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name "Seq_List_Lee_app_ST25", creation date of
Dec. 9, 2016 and a size of 20,499 bytes. The sequence listing
submitted via EFS-Web is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0004] The present invention is directed to stromal cell derived
factor-1 (SDF-1) peptides that have been mutated in a manner that
preserves their ability to attract cells but which makes them
resistant to inactivation by proteases, particularly matrix
metalloproteinase-2 (MMP-2) and/or dipeptidyl peptidase IV
(DPPIV/CD26). When delivered to damaged tissue, these mutants
promote tissue repair. The peptides should also be useful in the
treatment of many conditions, including ulcers in the
gastrointestinal tract or elsewhere, wounds resulting from
accident, surgery or disease; and cardiac tissue damaged as the
result of a myocardial infarction. The peptides should also be
useful in treating diabetic patients to make them less susceptible
to damage caused by wounds, ulcers or lesions. In an especially
preferred embodiment, the mutated forms of SDF-1 are delivered to
damaged tissue using a membrane formed by self-assembling
peptides.
BACKGROUND OF THE INVENTION
[0005] Stromal cell derived factor-1 (SDF-1, or CXCL12) is a 68
amino acid member of the chemokine family which attracts resting
T-lymphocytes, monocytes and CD34+ stem cells. It is commonly found
in two different forms SDF-1.alpha. and SDF-1.beta. which are the
result of differential mRNA splicing (U.S. Pat. No. 5,563,048).
These forms are essentially the same except that SDF-1.beta. is
extended by four amino acids (--Arg--Phe--Lys--Met) at the C
terminus. Both forms of SDF-1 are initially made with a signal
peptide, 21 amino acids in length, that is cleaved to make the
active peptide (U.S. Pat. No. 5,563,048). For the purposes of the
present invention, it will be understood that the term "SDF-1"
refers to the active form of the peptide, i.e., after cleavage of
the signal peptide, and encompasses both SDF-1.alpha. and
SDF-1.beta..
[0006] It has also been shown that the full length, 68 amino acid,
SDF-1 sequence is not needed for activity. Peptides that have at
least the first eight N-terminal residues of SDF-1 maintain the
receptor binding and bioactivity of the full peptide, albeit at a
reduced potency. For example, SDF-1, 1-8, 1-9, 1-9 dimer, and 1-17
induce intracellular calcium and chemotaxis in T lymphocytes and
CEM cells and bind to CXC chemokine receptor 4 (CXCR4). However,
native SDF-1 has half-maximal chemoattractant activity at 5 nM,
whereas the 1-9 dimer requires 500 nM and is therefore 100-fold
less potent. The 1-17 and a 1-9 monomer analogs are 400- and
3600-fold, respectively, less potent than SDF-1. SDF-1 variants
with C-terminal cyclization have been described that have a higher
CXCR4 receptor binding affinity and cyclization of this type may,
if desired, be used in connection with the peptides described
herein. For the purposes of the present invention, the term SDF-1
will include forms of the peptide that have been truncated at the C
terminal end but which maintain SDF-1 biological activity, i.e.,
which are chemotactic for T lymphocytes and CEM cells and which
bind to CXC chemokine receptor 4 (CXCR4). At a minimum, these
truncated forms include the first eight amino acids at the
N-terminal end of the peptide.
[0007] SDF-1 plays a key-role in the homing of hematopoietic stem
cells to bone marrow during embryonic development (Nagasawa, et
al., Nature 382:635-638 (1996); Zou, et al., Nature 393:595-599
(1998)) and after stem cell transplantation (Lapidot, et al., Blood
106:1901-1910 (2005)). In addition to its role in stem cell homing,
SDF-1 is also important in cardiogenesis and vasculogenesis. SDF-1
deficient mice die perinatally and have defects in cardiac
ventricular septal formation, bone marrow hematopoiesis and
organ-specific vasculogenesis (Nagasawa, et al., Nature 382:635-638
(1996); Zou, et al., Nature 393:595-599 (1998)). It has also been
reported that abnormally low levels of SDF-1 are at least partially
responsible for the impaired wound healing associated with diabetic
patients and that impairment can be reversed by the administration
of this cytokine at the site of tissue damage (Gallagher, et al.,
J. Clin. Invest. 117:1249-1259 (2007)).
[0008] In the normal adult heart, SDF-1 is expressed
constitutively, but expression is upregulated within days after
myocardial infarction (Pillarisetti, et al., Inflammation
25:293-300 (2001)). Askari et al. increased SDF-1 expression 8
weeks after myocardial infarction by intramyocardial
transplantation of stably transfected cardiac fibroblasts
overexpressing SDF-1 in combination with G-CSF therapy (Lancet
362:697-703 (2003)). This was associated with higher numbers of
bone marrow stem cells (c-Kit or CD34 positive) and endothelial
cells in the heart and resulted in an increase of vascular density
and an improvement of left ventricular function. These studies
suggest that the insufficiency of the naturally-occurring
myocardial repair process may be in part due to inadequate SDF-1
availability. Hence, the delivery of SDF-1 in a controlled manner
after myocardial infarction may attract more progenitor cells and
thereby promote tissue repair (Penn, et al., Int. J. Cardiol.
95(Suppl. 1):S23-S25 (2004)). Apart from this, the administration
of SDF-1 may be used to improve the healing of wounds or ulcers in
patients, especially those with diabetes.
[0009] One way that may be used for the sustained delivery of drugs
at a site of tissue damage is through the use of biologically
compatible membranes. Certain peptides are capable of self-assembly
when incubated in the presence of a low concentration of monovalent
metal cation (U.S. Pat. No. 5,670,483; U.S. Pat. No. 6,548,630).
Assembly results in the formation of a gel-like membrane that is
non-toxic, non-immunogenic and relatively stable to proteases. Once
formed, membranes are stable in serum, aqueous solutions and cell
culture medium. They can be made under sterile conditions, are
capable of supporting the growth of cells and are slowly digested
when implanted in an animal's body. These characteristics make the
membranes well suited as devices for the delivery of therapeutic
agents (US 20060148703 and 20060088510).
SUMMARY OF THE INVENTION
[0010] The present invention is based, in part, on experiments that
had as their hypothesis that the beneficial effect of stromal cell
derived factor-1 (SDF-1) in the recovery of damaged cardiac tissue
is limited by high concentrations of the protease matrix
metalloproteinase-2 (MMP-2) present in such tissue. More
specifically, it was proposed that the MMP-2 cleaves SDF-1 and
thereby eliminates its ability to attract progenitor cells to the
site of tissue damage.
[0011] In order to test this hypothesis, the inventors developed
mutated forms of SDF-1 that retain their ability to attract T cells
but which are resistant to MMP-2 digestion. The mSDF-1 peptides
were attached to a specially designed membrane formed by
self-assembling peptides and then tested in an animal model of
cardiac damage. It was found that mSDF-1 attached to membranes and
implanted into the myocardium of test animals improved cardiac
recovery to a greater extent than either SDF-1 or mSDF-1 that was
not attached to membranes.
[0012] In addition, the inventors found that truncated forms of
SDF-1 maintain bioactivity and, as with the full length peptide,
mutations in the fourth or fifth amino acids protect the peptide
from protease digestion.
[0013] In its first aspect, the invention is directed to mutant
forms of SDF-1 (mSDF-1) which are characterized by a change in the
fourth and/or the fifth amino acid from the N-terminus of unmutated
SDF-1 (K P V S L S Y R C P C R F F E S H V A R A N V K H L K I L N
T P N C A L Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K
A L N K (SEQ ID NO:52)). Thus, the fourth amino acid is changed to
an amino acid other than S and/or the fifth amino acid is changed
to an amino acid other than L. As discussed above, truncated forms
of the full length SDF-1 peptide maintain biological activity
provided that the first eight amino acids (highlighted in the
sequence shown above) are present and these truncated forms may
also be made protease resistant by mutating the fourth and/or fifth
position. The invention includes these biologically active
truncated mutants as well. Put another way, the invention includes
peptides comprising the amino acid sequence of at least amino acids
1-8 of SEQ ID NO:52, which are optionally extended at the C
terminus by all or any portion of the remaining sequence of SEQ ID
NO:52, shown as amino acids 9-68. In all cases, the peptide will
have a sequence corresponding to that given in SEQ ID NO:52 except
that there will be a proteinogenic amino acid other than S at
position 4 and/or a proteinogenic amino other than L at position
5.
[0014] For the purposes of the present invention, all peptide
sequences are written from the N terminus (far left) to the C
terminus (far right) and unless otherwise indicated, all amino
acids are "proteinogenic" amino acids, i.e., they are the L-isomers
of: alanine (A); arginine (R); asparagine (N); aspartic acid (D);
cysteine (C); glutamic acid (E); glutamine (Q); glycine (G);
histidine (H); isoleucine (I); leucine (L); lysine (K); methionine
(M); phenylalanine (F); proline (P); serine (S); threonine (T);
tryptophan (W); tyrosine (Y); or valine (V). Mutant SDF-1 peptides
may be abbreviated herein as "mSDF-1," "mSDF" or SDF(NqN') where N
is the one letter designation of the amino acid originally present,
q is its position from the N terminus of the peptide and N' is the
amino acid that has replaced N. It will also be understood that,
although SEQ ID NO:52 shows the intact full length sequence of
SDF-1.alpha., this sequence may be extended at the C terminus by up
to four more amino acids, in particular with the sequence -R-F-K-M.
Thus, the invention includes mutant forms of both SDF-1.alpha. and
SDF-1.beta. (see U.S. Pat. No. 5,563,048). In some instances,
peptides that have been mutated by the addition of amino acids at
the N terminus are abbreviated as "Xp-R" where X is a proteinogenic
amino acid, p is an integer and R is the peptide prior to
extension. It will also be understood that, unless otherwise
indicated, all pharmaceutically acceptable forms of peptides may be
used, including all pharmaceutically acceptable salts.
[0015] The mSDF-1 peptides must maintain chemoattractant activity
with a sensitivity (as determined by, e.g., the effective
concentration needed to obtain 50% of maximal response in the
assays of Jurkat T cell migration described herein) of at least
1/10 the sensitivity of unmutated SDF-1. In addition, the mSDF-1
peptides must be resistant to loss of this chemoattractant activity
due to cleavage by matrix metalloproteinase-2 (MMP-2). Preferably
the rate of inactivation of mSDF-1 is less than 1/2 (and more
preferably, less than 1/4 or 1/10) the rate of inactivation of
SDF-1.
[0016] In one embodiment, the mSDF-1 peptide has the sequence: K P
V X L S Y R C P C R F F E S H V A R A N V K H L K I L N T P N C A L
Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K A L N K
(SEQ ID NO:53) where X is any of the 20 proteinogenic amino acids
except S. The most preferred of these peptides is SDF(S4V) which
has the sequence: K P V V L S Y R C P C R F F E S H V A R A N V K H
L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K L K W I Q
E Y L E K A L N K (SEQ ID NO:54). SEQ ID 53 and 54 show the full
sequence of SDF-1 peptides. However, it will be understood that
truncated versions of the peptides will maintain activity as long
as the first eight N-terminus amino acids are present. These are
also part of the invention and may be made protease resistant by
mutating the amino acids at positions 4 and/or 5.
[0017] In another embodiment, the mSDF-1 peptide has the sequence:
K P V S X S Y R C P C R F F E S H V A R A N V K H L K I L N T P N C
A L Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K A L N K
(SEQ ID NO:55)where X is any of the 20 proteinogenic amino acids
except L, W or E. The most preferred of these peptides is SDF(L5P)
which has the sequence: K P V S P S Y R C P C R F F E S H V A R A N
V K H L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K L K
W I Q E Y L E K A L N K (SEQ ID NO:56). Again, peptides that are
truncated and which have at least the first eight amino acids of
SEQ ID NO:55 or 56 are included in the invention. They may be
extended at the C terminus by additional amino acids from the
sequences shown above.
[0018] The longest mSDF-1 peptides presented above are 68 amino
acids in length. However, unless otherwise indicated, it will also
be understood that one additional proteinogenic amino acid may be
added to the N terminus without substantially changing
chemoattractant activity or MMP-2 resistance. Moreover, the
addition of an amino acid at the N terminus represents a preferred
embodiment since this will have the effect of making the peptide
resistant to digestion by a second common peptidase, dipeptidyl
peptidase IV (DPPIV/CD26, abbreviated herein as "DPPIV").
[0019] DPPIV is a 110-kD glycoprotein which is expressed in renal
proximal tubules, in intestinal epithelial cells, liver, placenta
and lung and which cleaves peptides that have a proline in the
second position from the N terminus (Kikawa, et al., Biochim.
Biophys. Acta 1751:45-51 (2005)). SDF-1 has a proline in the second
position (as can be seen above in SEQ ID NO:52) and is therefore
cleaved by DPPIV between this proline and the following valine
(Narducci, et al., Blood 107:1108-1115 (2006); Christopherson, Exp.
Hematol. 34:1060-1068 (2006)).
[0020] One way to eliminate the proteolytic effect of DPPIV would
be to change the proline in position 2 of SDF-1 (see SEQ ID NO:52).
However, this proline is essential for SDF-1's biological activity
and therefore cannot be replaced and maintain a therapeutically
effective peptide. However, activity can be maintained and a DPPIV
resistant peptide made by adding one to four amino acids (or an
organic group) to the N terminus of SEQ ID:52. For example, it has
been experimentally found that resistance to DPPIV cleavage can be
obtained by adding a serine to the N terminus of the peptide.
[0021] Thus, in another aspect, the invention is directed to the
peptide X.sub.p-SDF-1, where X is preferably, any proteinogenic
amino acid, p is an integer between 1 and 4, and SDF-1 is as shown
in SEQ ID NO:52. In preferred embodiments, n=1. It will be
understood that when p is greater than 1, each of the 2-4 added
amino acids may independently be chosen from any of the
proteinogenic amino acids described herein, i.e., any of these
proteinogenic amino acids may be in the first position, any in the
second position, etc.
[0022] SDF-1 may also be made resistant to DPPIV by adding a
"protease protective organic group" to the N-terminus. A "protease
protective organic group" is defined herein as an organic group,
other than a proteinogenic amino acid, that, when added to the N
terminal amino acid of SDF-1, results in a modified peptide that
maintains at least 10% (and preferably at least 50% or 80%) of the
chemoattractant activity of unmodified SDF-1(as determined by,
e.g., assays of Jurkat T cell migration described herein) and
which, in addition, is inactivated by DPPIV at a rate of less than
50% (and more preferably, at a rate of less than 25% or 10%) the
rate of inactivation of unmodified SDF-1. For example, X may be:
R.sup.1--(CH2).sub.d--, where d is an integer from 0-3, and R.sup.1
is selected from: hydrogen (with the caveat that when R.sup.1 is
hydrogen, d must be at least 1); a branched or straight
C.sub.1-C.sub.3 alkyl; a straight or branched C.sub.2-C.sub.3
alkenyl; a halogen, CF.sub.3; --CONR.sup.5R.sup.4; --COOR.sup.5;
--COR.sup.5; --(CH.sub.2).sub.qNR.sup.5R.sup.4;
--(CH.sub.2).sub.qSOR.sup.5; --(CH.sub.2).sub.qSO.sub.2R.sup.5,
--(CH.sub.2).sub.qSO.sub.2NR.sup.5R.sup.4; and OR.sup.5, where
R.sup.4 and R.sup.5 are each independently hydrogen or a straight
or branched C.sub.1-C.sub.3 alkyl. In instances where an organic
group is used for X, p should be 1. In addition, X may represent a
proteinogenic amino acid as discussed above, so that 1-4 amino
acids are added to SDF-1, and one or more of these added amino
acids may be substituted with a protease protective organic
group.
[0023] In the formula X.sub.p-SDF-1, SDF-1 may optionally include
any of the mutations in positions 4 and/or 5 of SEQ ID NO:52 as
described above. Thus, the invention encompasses peptides of the
form X.sub.p-mSDF-1, where X and p are as defined above and mSDF-1
is selected from: SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:55; and SEQ
ID NO:56. These doubly mutated peptides will be resistant to both
DPPIV and MMP-2.
[0024] The invention also encompasses fusion proteins in which any
of the above mSDF-1, X.sub.p-SDF-1 or X.sub.p-mSDF-1 sequences are
linked to self-assembling peptides capable of forming a
biologically compatible membrane. Membranes with attached protease
resistant SDF-1 can be implanted in a patient at a site of tissue
damage, especially cardiac tissue damage, wounds (whether
accidental, surgical or the result of disease) or ulcers and will
maintain the SDF-1 biological activity at that site for a prolonged
period of time. Fusion proteins are formed either by joining the C
terminal end of a protease resistant SDF-1 peptide directly to the
N terminal end of a self-assembling peptide or the two peptides can
be joined by a linker sequence. Thus, the invention includes fusion
proteins of the formula: A--(L).sub.n--(R).sub.q, where n is an
integer from 0-3, q is an integer from 1-3, A is one of the
protease resistant SDF-1 peptides (i.e., mSDF-1, X.sub.p-SDF-1 or
X.sub.p-mSDF-1) described above, L is a linker sequence 3-9 amino
acids long, and R is a self-assembling peptide selected from the
group consisting of:
TABLE-US-00001 AKAKAEAEAKAKAEAE,; (SEQ ID NO: 1) AKAEAKAEAKAEAKAE,;
(SEQ ID NO: 2) EAKAEAKAEAKAEAKA,; (SEQ ID NO: 3) KAEAKAEAKAEAKAEA,;
(SEQ ID NO: 4) AEAKAEAKAEAKAEAK,; (SEQ ID NO: 5) ADADARARADADARAR,;
(SEQ ID NO: 6) ARADARADARADARAD,; (SEQ ID NO: 7) DARADARADARADARA,;
(SEQ ID NO: 8) RADARADARADARADA,; (SEQ ID NO: 9) ADARADARADARADAR,;
(SEQ ID NO: 10) ARADAKAEARADAKAE,; (SEQ ID NO: 11)
AKAEARADAKAEARAD,; (SEQ ID NO: 12) ARAKADAEARAKADAE,; (SEQ ID NO:
13) AKARAEADAKARADAE,; (SEQ ID NO: 14) AQAQAQAQAQAQAQAQ,; (SEQ ID
NO: 15) VQVQVQVQVQVQVQVQ,; (SEQ ID NO: 16) YQYQYQYQYQYQYQYQ,; (SEQ
ID NO: 17) HQHQHQHQHQHQHQHQ,; (SEQ ID NO: 18) ANANANANANANANAN,;
(SEQ ID NO: 19) VNVNVNVNVNVNVNVN,; (SEQ ID NO: 20)
YNYNYNYNYNYNYNYN,; (SEQ ID NO: 21) HNHNHNHNHNHNHNHN,; (SEQ ID NO:
22) ANAQANAQANAQANAQ,; (SEQ ID NO: 23) AQANAQANAQANAQAN,; (SEQ ID
NO: 24) VNVQVNVQVNVQVNVQ,; (SEQ ID NO: 25) VQVNVQVNVQVNVQVN,; (SEQ
ID NO: 26) YNYQYNYQYNYQYNYQ,; (SEQ ID NO: 27) YQYNYQYNYQYNYQYN,;
(SEQ ID NO: 28) HNHQHNHQHNHQHNHQ,; (SEQ ID NO: 29)
HQHNHQHNHQHNHQHN,; (SEQ ID NO: 30) AKAQADAKAQADAKAQAD,; (SEQ ID NO:
31) VKVQVDVKVQVDVKVQVD,; (SEQ ID NO: 32) YKYQYDYKYQYDYKYQYD,; (SEQ
ID NO: 33) HKHQHDHKHQHDHKHQHD,; (SEQ ID NO: 34) RARADADARARADADA,;
(SEQ ID NO: 35) RADARGDARADARGDA,; (SEQ ID NO: 36)
RAEARAEARAEARAEA,; (SEQ ID NO: 37) KADAKADAKADAKADA,; (SEQ ID NO:
38) AEAEAHAHAEAEAHAH,; (SEQ ID NO: 39) FEFEFKFKFEFEFKFK,; (SEQ ID
NO: 40) LELELKLKLELELKLK,; (SEQ ID NO: 41) AEAEAKAKAEAEAKAK,; (SEQ
ID NO: 42) AEAEAEAEAKAK,; (SEQ ID NO: 43) KAKAKAKAEAEAEAEA,; (SEQ
ID NO: 44) AEAEAEAEAKAKAKAK,; (SEQ ID NO: 45) RARARARADADADADA,;
(SEQ ID NO: 46) ADADADADARARARAR,; (SEQ ID NO: 47)
DADADADARARARARA,; (SEQ ID NO: 48) HEHEHKHKHEHEEIKHK,; (SEQ ID NO:
49) VEVEVEVEVEVEVEVEVEVE,; (SEQ ID NO: 50) and
RFRFRFRFRFRFRFRFRFRF,. (SEQ ID NO: 51)
[0025] The most preferred self-assembling peptide is:
RARADADARARADADA, (SEQ ID NO:35) with q=1; and preferred protease
resistant SDF-1 peptides are SDF(S4V) and X.sub.p-SDF(S4V),
especially where p=1. When joined together, the resulting fusion
proteins are, for convenience, abbreviated as SDF(S4V)-RAD or
X.sub.p-SDF(S4V)-RAD. Preferred linker sequences occur when n=1 and
L is GGGGGG (abbreviated as "6G," SEQ ID NO:57); GIVGPL (SEQ ID
NO:58) and PVGLIG (SEQ ID NO:59). The lattermost represents an
MMP-2 cleavage site ("MCS"). GIVGPL (SEQ ID NO:58) represents a
scrambled version of MCS and is abbreviated as "SCR." Surprisingly,
this sequence was also found to undergo MMP-2 cleavage, although at
a slower rate than MCS. Preferred, fusion proteins containing
linker sequences are: SDF(S4V)-6G-RAD; X.sub.p-SDF(S4V)-6G-RAD;
SDF(S4V)-MCS-RAD; X.sub.p-SDF(S4V)-MCS-RAD; SDF(S4V)-SCR-RAD; and
XP-SDF(S4V)-SCR-RAD. Again, p is preferably 1.
[0026] In another aspect, the invention is directed to nucleic
acids comprising a nucleotide sequence encoding any of the protease
resistant peptides or fusion proteins described above, vectors in
which these nucleic acids are operably linked to a promoter
sequence and host cells transformed with the vectors. The term
"operably linked" refers to genetic elements that are joined in a
manner that enables them to carry out their normal functions. For
example, a sequence encoding a peptide is operably linked to a
promoter when its transcription is under the control of the
promoter and the transcript produced is correctly translated into
the peptide.
[0027] Preferred nucleic acids encoding protease resistant SDF-1
peptides and fusion proteins include:
TABLE-US-00002 (SEQ ID NO: 60)
aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagcca
tgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaact
gtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgc
attgacccgaagctaaagtggattcaggagtacctggagaaagctttaaa caag; (SEQ ID
NO: 61) aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagcca
tgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaact
gtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgc
attgacccgaagctaaagtggattcaggagtacctggagaaagctttaaa
caagtgaggaatcgtgggacctctgcgtgcccgtgccgacgccgacgccc
gtgcccgtgccgacgccgacgcc; (SEQ ID NO: 62)
aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagcca
tgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaact
gtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgc
attgacccgaagctaaagtggattcaggagtacctggagaaagattaaac
aagcctgtgggactgatcggagtgcccgtgccgacgccgacgcccgtgcc
cgtgccgacgccgacgcc; and (SEQ ID NO: 63)
aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagcca
tgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaact
gtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgc
attgacccgaagctaaagtggattcaggagtacctggagaaagattaaac
aagggaggcgggggaggtgggcgtgcccgtgccgacgccgacgcccgtgc
ccgtgccgacgccgacgcc
[0028] In another aspect, the invention is directed to a
biologically compatible membrane formed from self-assembling
peptides as described in published US applications 20060148703 and
20060088510 which have mSDF-1, Xp-SDF-1 or Xp-mSDF-1 peptides
attached. The term "biologically compatible" indicates that the
membranes are non-toxic and can be implanted in a patient without
triggering an immune response. Between 0.1% and 10% (and preferably
0.5-5%) of the peptides that assemble into the membrane are bound
to a mutant SDF-1. Binding may be either covalent or noncovalent.
Noncovalent bonding occurs when protease resistant SDF-1 peptides
are simply trapped in the membrane matrix and when protease
resistant SDF-1 peptides are bound to self-assembling peptides in
the membrane by biotin/avidin linkages. As used herein, the term
"avidin" is intended to include streptavidin as well. The membranes
may, optionally, have other therapeutic agents, e.g., platelet
derived growth factor (PDGF) or interleukin-8, attached as
well.
[0029] The use of biotin and avidin for linking molecules is well
known in the art and standard methodology can be used for attaching
protease resistant SDF-1 peptides to self-assembling peptides
either before or after membrane formation. Specific methodology for
using biotin/avidin in connection with self-assembling membranes
has been described in US 20060088510 and this methodology can be
applied to forming membranes with attached cytokine. In order to
prevent steric interference between the biotin/avidin groups and
protease resistant peptides, a spacer may be included between the
two. The spacer can take the form of 1-15 (preferably 1-10) fatty
acids or 1-15 (preferably)-10) amino acids and should separate the
protease resistant SDF-1 peptide from the self-assembling peptide
by at least an additional 12 angstroms and by no more than an
additional 250 angstroms. Methodology for incorporating spacers of
this type is well known in the art. In a preferred embodiment,
about 1% of the self-assembling peptides used in membranes are
attached to protease resistant SDF-1. It is also preferable that
the self-assembling peptides making up membranes be homogeneous,
i.e., that all of the peptides are identical.
[0030] As an alternative, protease resistant SDF-1 peptides may be
joined to a self-assembling peptide that is part of the membrane by
a peptide bond, i.e., the protease resistant SDF-1 may be part of a
fusion protein in which it is joined to a self-assembling peptide
either directly or via an intervening linker amino acid sequence.
Any of the fusion proteins described above may be used, with
SDF(S4V)-6G-RAD; X.sub.p-SDF(S4V)-6G-RAD; SDF(S4V)-MCS-RAD;
X.sub.p-SDF(S4V)-MCS-RAD; SDF(S4V)-SCR-RAD and
X.sub.p-SDF(S4V)-SCR-RAD being particularly preferred. The
membranes are made from the fusion proteins (or from the
self-assembling peptides) by taking advantage of the fact that the
self-assembling peptides described herein do not congregate
together in water, but assemble into a membrane in the presence of
a low concentration of monovalent metal cation. Thus, for example,
fusion proteins may be made under conditions in which self-assembly
does not occur and then exposed to conditions that promote membrane
formation, e.g., low monovalent metal cation concentration. The end
result is a matrix which can be implanted into a patient and which
will maintain a high concentration of SDF-1 biological activity at
the site of implantation. Alternatively, the fusion proteins can be
incorporated into an injectable pharmaceutical composition at a
concentration of monovalent cation that is too low to induce
self-assembly and can then administered to a patient to induce
membrane formation in vivo.
[0031] The mutated SDF-1 peptides are resistant to cleavage by
MMP-2 and/or DPPIV but maintain at least a portion (at least 10%
and preferably more than 25%, 50% or 80%) of the chemoattractant
activity of native SDF-1. Thus, they are ideally suited for use at
sites, such as damaged cardiac tissue, where MMP-2 (or DPPIV) is
present at a high concentration. In addition, an MMP-2 cleavage
site can, if desired, be placed in linker regions joining the SDF-1
peptides to the self-assembling peptides. This will allow for the
protease resistant SDF-1 peptides to be released from an implanted
membrane over time.
[0032] The compositions described above should be useful in the
treatment of any disease or condition characterized by high
concentrations of MMP-2 and/or DPPIV where attraction of stem cells
might induce regeneration or healing. This would include the
treatment of inflammatory and ischemic diseases such as stroke,
limb ischemia; wound healing: and diabetic ulcers. In an especially
preferred embodiment, the invention is directed to a method of
treating damaged cardiac tissue, for example subsequent to a heart
attack, by injecting or implanting any of the biologically
compatible peptide membranes or fusion proteins described above at
or near the site of damage. Preferably, membranes will be injected
or implanted directly into the damaged tissue, e.g., myocardium, of
a patient. The membranes should be large enough to prevent the
protease resistant SDF-1 from being washed away by bodily fluids
and a sufficient amount of mSDF-1 should be present to promote the
migration of T cells to the site of injury. Guidance with regard to
these parameters is provided by the experiments described
herein.
DESCRIPTION OF THE INVENTION
[0033] The present invention is based upon the concept that the
recovery of damaged tissue, e.g., damaged cardiac tissue, is
promoted by exposing the tissue to SDF-1 that has been mutated to
make it resistant to MMP-2 and/or DPPIV cleavage and which is
delivered by means of a membrane formed by spontaneously assembling
peptides. The self-assembling peptides have been described in U.S.
Pat. Nos. 5,670,483 and 6,548,630 (hereby incorporated by reference
in their entirety). Methods of attaching factors to membranes and
the use of the membranes in delivering therapeutic agents to
cardiac tissue have also been described (see published US
applications 20060148703 and 20060088510, hereby incorporated by
reference in their entirety). The same procedures for making and
using membranes may be applied to the present invention.
Description of Self-Assembling Peptides
[0034] The peptides used for self-assembly should be at least 12
residues in length and contain alternating hydrophobic and
hydrophilic amino acids. Peptides longer than about 200 amino acids
tend to present problems with respect to solubility and membrane
stability and should therefore be avoided. Ideally, peptides should
be about 12-24 amino acids in length.
[0035] The self-assembling peptides must be complementary. This
means that the amino acids on one peptide must be capable of
forming ionic bonds or hydrogen bonds with the amino acids on
another peptide. Ionic bonds would form between acidic and basic
amino acid side chains. The hydrophilic basic amino acids include
Lys, Arg, His, and Orn. The hydrophilic acidic amino acids are Glu
and Asp. Ionic bonds would form between an acidic residue on one
peptide and a basic residue on another. Amino acids that form
hydrogen bonds are Asn and Gln. Hydrophobic amino acids that may be
incorporated into peptides include Ala, Val, Ile, Met, Phe, Tyr,
Trp, Ser, Thr, and Gly.
[0036] Self-assembling peptides must also be "structurally
compatible." This means that they must maintain an essentially
constant distance between one another when they bind. Interpeptide
distance can be calculated for each ionized or hydrogen bonding
pair by taking the sum of the number of unbranched atoms on the
side-chains of each amino acid in the pair. For example, lysine has
five and glutamic acid has four unbranched atoms on their side
chains. An interaction between these two residues on different
peptides would result in an interpeptide distance of nine atoms. In
a peptide containing only repeating units of EAK, all of the ion
pairs would involve lysine and glutamate and therefore a constant
interpeptide distance would be maintained. Thus, these peptides
would be structurally complementary. Peptides in which the
variation in interpeptide distance varies by more than one atom
(about 3-4 angstroms) will not form gels properly. For example, if
two bound peptides have ion pairs with a nine-atom spacing and
other ion pairs with a seven-atom spacing, the requirement of
structural complementarity would not have been met. A full
discussion of complementarity and structural compatibility may be
found in U.S. Pat. Nos. 5,670,483 and 6,548,630.
[0037] It should also be recognized that membranes may be formed
from either a homogeneous mixture of peptides or a heterogeneous
mixture of peptides. The term "homogeneous" in this context means
peptides that are identical with one another. "Heterogeneous"
indicates peptides that bind to one another but which are
structurally different. Regardless of whether homogenous or
heterogeneous peptides are used, the requirements with respect to
the arrangement of amino acids, length, complementarity, and
structural compatibility apply. In addition, it should be
recognized that the carboxyl and amino groups of the terminal
residues of peptides can either be protected or not protected using
standard groups.
Making of Peptides
[0038] The self-assembling and protease resistant SDF-1 peptides of
the present invention can be made by solid-phase peptide synthesis
using standard N-tert-butyoxycarbonyl (t-Boc) chemistry and cycles
using n-methylpyrolidone chemistry. Once peptides have been
synthesized, they can be purified using procedures such as high
pressure liquid chromatography on reverse-phase columns. Purity may
also be assessed by HPLC and the presence of a correct composition
can be determined by amino acid analysis. A purification procedure
suitable for mSDF-1 peptides is described in the Examples
section.
[0039] Fusion proteins may either be chemically synthesized or made
using recombinant DNA techniques. The full sequences of these
proteins are described herein and examples are provided of DNA
sequences that can be used in producing them.
Binding of SDF-1 to Self-Assembling Peptides
[0040] Several strategies may be used for attaching protease
resistant SDF-1 to self-assembling peptides. One strategy is
non-covalent binding which has previously been shown to be
effective in delivering PDGF-BB, a growth factor, to tissues
(Hsieh, et al., J. Clin. Invest. 116:237-248 (2006)).
[0041] A second attachment strategy is the biotin-sandwich method
(Davis, et al., Proc. Nat'l Acad. Sci. USA 103:8155-8160 (2006)) in
which a protease resistant SDF-1 is biotinylated and bound to
biotinylated peptides using tetravalent streptavidin as a linker.
To accomplish this, the protease resistant SDF-1 may be coupled to
the 15 amino acid sequence of an acceptor peptide for biotinylation
(referred as AP; Chen, et al., Nat. Methods 2:99-104 (2005)).
Because the active site of SDF-1 is situated near the amino
terminus, fusion proteins should be made by incorporating the extra
sequences at the C-terminus. The acceptor peptide sequence allows
site-specific biotinylation by the E. coli enzyme biotin ligase
(BirA; Chen, et al., Nat. Methods 2:99-104 (2005)). Many commercial
kits are available for biotinylating proteins. However, many of
these kits biotinylate lysine residues in a nonspecific manner, and
this may reduce mSDF-1 activity as it has been shown that the
N-terminal lysine of SDF-1 is crucial for receptor binding and
activity (Crump, et al, EMBO 1 16:6996-7007 (1997)). Biotinylated
self-assembling peptides are made by MIT Biopolymers laboratory and
when mixed in a 1 to 100 ratio with native self-assembling
peptides, self-assembly of nanofibers should not be disturbed
(Davis, et al., Proc. Nat'l Acad. Sci. USA 103:8155-8160
(2006)).
[0042] A third targeting strategy is direct incorporation of
protease resistant SDF-1 peptides into self-assembling nanofibers
by construction of a fusion protein of mutated SDF-1 with a
self-assembling peptide. For example an mSDF-1 may be coupled to
the 16 amino acid sequence of SEQ ID NO:35. This "RAD" portion of
the fusion protein will incorporate into the nanofiber scaffold
while assembling.
Formation of Membranes
[0043] The self-assembling peptides and fusion proteins described
herein will not form membranes in water, but will assemble in the
presence of a low concentration of monovalent metal cation. The
order of effectiveness of these cations is
Li.sup.+>Na.sup.+>K.sup.+>Cs.sup.+ (U.S. Pat. No.
6,548,630). A concentration of monovalent cation of 5 mM should be
sufficient for peptides to assemble and concentrations as high as 5
M should still be effective. The anion associated with the
monovalent cation is not critical to the invention and can be
acetate, chloride, sulfate, phosphate, etc.
[0044] The initial concentration of self-assembling peptide will
influence the final size and thickness of membranes formed. In
general, the higher the peptide concentration, the higher the
extent of membrane formation. Formation can take place at peptide
concentrations as low as 0.5 mM or 1 mg/ml. However, membranes are
preferably formed at higher initial peptide concentrations, e.g.,
10 mg/ml, to promote better handling characteristics. Overall, it
is generally better to form membranes by adding peptides to a salt
solution rather than adding salt to a peptide solution.
[0045] The formation of membranes is relatively unaffected by pH or
by temperature. Nevertheless, pH should be maintained below 12 and
temperatures should generally be in the range of 4-90.degree. C.
Divalent metal cations at concentrations equal to or above 100 mM
result in improper membrane formation and should be avoided.
Similarly, a concentration of sodium dodecyl sulfate of 0.1% or
higher should be avoided.
[0046] Membrane formation may be observed by simple visual
inspection and this can be aided, if desired, with stains such as
Congo Red. The integrity of membranes can also be observed
microscopically, with or without stain.
Pharmaceutical Compositions and Dosages
[0047] Membranes with attached protease resistant SDF-1 peptides or
fusion proteins may be incorporated into a pharmaceutical
composition containing a carrier such as saline, water, Ringer's
solution and other agents or excipients. The dosage form will
generally be designed for implantation or injection, particularly
into cardiac tissue but topical treatments will also be useful,
e.g., in the treatment of wounds. All dosage forms may be prepared
using methods that are standard in the art (see e.g., Remington's
Pharmaceutical Sciences, 16th ed. A. Oslo. ed., Easton, Pa.
(1980)).
[0048] It is expected that the skilled practitioner will adjust
dosages on a case by case basis using methods well established in
clinical medicine. The optimal dosage will be determined by methods
known in the art and will be influenced by factors such as the age
of the patient, disease state and other clinically relevant
factors.
EXAMPLES
Example 1
Biological Effects and Protease Resistance of SDF-1 Mutants
SDF-1 Purification and Expression
[0049] The DNA sequence of mature SDF-1.alpha. may be cloned from
human cDNA into pET-Sumo vector and an extra N-terminal serine
residue may be incorporated to facilitate cleavage by Sumo protease
(yielding an SDF-1 form of 69 AA). Fusion proteins may be made by
incorporating RAD or AP sequences in reverse primers. Sumo-SDF-1
fusion proteins are expressed in Rosetta DE3 E coli and grown to an
optical density of 1.5 (600 nm) at 37.degree. C. Cells are induced
with 0.25 mM isopropyl .beta.-D-thiogalactoside for 4 h and
harvested by centrifugation. As described below, SDF-1.alpha. may
be purified by a 3-step procedure; all steps being performed at
21.degree. C.
[0050] Cells from a 4-L growth were lysed in 300 ml lysis buffer
(6M Guanidine, 20 mM phosphate (pH 7.8), 500 mM NaCl) and
homogenized. Debris is collected by centrifugation at 3000 g. The
first purification step consisted of capture of the poly-histidine
tag present in the SUMO-SDF-1.alpha. fusion protein with
Nickel-NTA. Nickel-NTA resin was washed with wash buffer (8M Urea,
500 mM NaCl, 20 mM phosphate (pH 6.2)) and the bound protein was
eluted at pH 4. Further purification and oxidative refolding were
performed on a Cation Exchange HPLC column. The sample was adjusted
to binding buffer (8M Urea, 30 mM 2-mercaptoethanol, 1 mM EDTA, 50
mM Tris pH8) and loaded on the HPLC column. Refolding of Sumo-SDF-1
was performed on the column with a 2 h run of refolding buffer (50
mM Tris pH8, 75 mM NaCl, 0.1 mM reduced Glutathione and 0.1 mM
oxidized Glutathione). Sumo-SDF-1 was eluted with a step gradient
(0.5 to 1M NaCl) and concentrated. The SUMO-SDF-1 fusion protein
was cleaved by Sumo Protease 1 (1U/50 .mu.g protein) in 50 mM Tris
pH 8.0, 500 mM NaCl. The sample was adjusted to 0.1%
trifluoroacetic acid (TFA) and loaded on a C18 Reversed Phase HPLC
column for the final purification step. The column was subjected to
a linear gradient from 30 to 40% acetonitrile in 0.1% TFA. The
fractions containing SDF-1 were lyophilized and resuspended.
Activity of purified SDF-1 was tested by migration of Jurkat
T-lymphocyte cell line.
Modification of SDF-1 Constructs
[0051] SDF-1 fusion constructs were modified by insertional
mutagenesis with one of three sequences: one sequence is
susceptible to MMP-2 cleavage (MMP cleavage site or MCS), another
sequence contains the same amino acids but in a random order
(scrambled sequence, or SCR), and the third sequence contains 6
glycines as a linker.
Mutations of the MMP Cleavage Sites in Chemokines
[0052] SDF-1 is cleaved by MMP-2 in its active site at the
N-terminus, leaving an N-terminal tetrapeptide and inactive
SDF-1(5-68). Specific mutagenesis of 4 different amino acids was
performed in order to render SDF-1 resistant to MMP-2 cleavage,
based on substrate sequences of MMP-2 described by Netzel-Arnett et
al (Biochemistry 32:6427-6432 (1993)). The four different
constructs were expressed and purified as described for SDF-1. Of
the 4 different mutations, SDF-1(L5W) and SDF-1(L5E) showed minimal
activity on T-cell migration. In contrast, SDF-1(S4V) and
SDF-1(L5P) showed bioactivity comparable to native SDF-1. Because
SDF-1(L5P) was more difficult to purify, SDF-1(S4V) was selected
for further experiments.
Effect of Mutations on Protease Susceptibility and Chemoattractant
Activity
[0053] The mutated forms of SDF-1 were examined in an assay of
migration of Jurkat T cells at a concentration of 100 nM. This
assay indicated that both SDF-1(S4V) and SDF-1(L5P) retained most
of the activity of unmutated SDF-1 in promoting T cell migration.
This activity was greatly reduced in SDF-1(L5W) mutants and
SDF-1(L5E) mutants.
[0054] The susceptibility of the peptides to cleavage by MMP-2 was
determined by incubating the mutants with the enzyme for one hour
and then examining the incubation product by SDS-PAGE. This
revealed that, unlike SDF-1, the mutants did not undergo a
positional shift indicative of cleavage. MMP-2 incubation was also
found to reduce the chemoattractant activity of SDF-1 but not
SDF-1(S4V) as shown by a Jurkat T-cell migration assay. These
results suggest that the S4V variant of SDF-1 retains chemokine
bioactivity but is resistant to activation by MMP-2.
In Vivo Data
[0055] A blinded and randomized study was performed to evaluate the
effect of different SDF-1 forms on cardiac function after
myocardial infarction in rats. Ejection fraction was measured with
a Millar catheter system for measurement of intraventricular
pressures and ventricular volumes. Both SDF-1(S4V)-6G-RAD and
SDF-1(S4V)-MCS-RAD significantly increased cardiac function 4 weeks
after myocardial infarction in rats compared to MI only group. This
indicates that both MMP-2 resistance (SDF-1(S4V)) and attachment to
membranes are necessary for successful cardiac repair therapy.
Example 2
Experiments with Truncated Forms of SDF-1
[0056] 3 truncated forms of SDF-1 were synthesized commercially;
all include the first 17 amino acids of native SDF-1. Two variants
of SDF-1 17AA were designed to be more resistant to MMP-2, based on
our prior work with the entire SDF-1 protein:
TABLE-US-00003 SDF-1 17AA: KPVSLSYRCPCRFFESH (SEQ ID 64) SDF-1(S4V)
17AA: KPVVLSYRCPCRFFESH (SEQ ID 65) SDF-1(L5P) 17AA:
KPVSPSYRCPCRFFESH (SEQ ID 66)
[0057] Migration experiments were performed with the Jurkat
T-lymphocyte cell line. Truncated SDF-1 17AA was 500 times less
potent than native SDF-1 but maximal migration induced was similar
to native SDF-1. Therefore, if 500 times higher concentrations were
used compared to full-length protein, the same migratory response
of T-lymphocytes should be observed. The mutated SDF-1(S4V) 17AA
and SDF-1(L5P) 17AA were three times less potent than SDF-1 17AA
without mutation. This is a similar shift to that seen between
native SDF-1 and SDF-1(S4V).
[0058] Cleavage experiments of the peptides with MMP-2 were
performed: 2 nmole of SDF-1 17AA, SDF-1(S4V) 17AA, and SDF-1(L5P)
17AA were incubated with MMP-2 for 1 h at RT. Proteins were run on
an SDS-PAGE showing cleavage of SDF-1 17AA, but not of SDF-1(S4V)
17AA or SDF-1(L5P) 17AA. Thus, these truncated proteins may be
useful therapeutically, as they are still bioactive and also MMP-2
resistant.
[0059] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by those of skill in the art that the invention may be
practiced within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
Sequence CWU 1
1
66116PRTArtificialsynthetic sequence designed for self assembly
1Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu1 5
10 15216PRTArtificialsynthetic sequence designed for self assembly
2Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu1 5
10 15316PRTArtificialsynthetic sequence designed for self assembly
3Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala1 5
10 15416PRTArtificialsynthetic sequence designed for self assembly
4Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala1 5
10 15516PRTArtificialsynthetic sequence designed for self assembly
5Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys1 5
10 15616PRTArtificialsynthetic sequence designed for self assembly
6Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg1 5
10 15716PRTArtificialsynthetic sequence designed for self assembly
7Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp1 5
10 15816PRTArtificialsynthetic sequence designed for self assembly
8Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala1 5
10 15916PRTArtificialsynthetic sequence designed for self assembly
9Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala1 5
10 151016PRTArtificialsynthetic sequence designed for self assembly
10Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg1
5 10 151116PRTArtificialsynthetic sequence designed for self
assembly 11Ala Arg Ala Asp Ala Lys Ala Glu Ala Arg Ala Asp Ala Lys
Ala Glu1 5 10 151216PRTArtificialsynthetic sequence designed for
self assembly 12Ala Lys Ala Glu Ala Arg Ala Asp Ala Lys Ala Glu Ala
Arg Ala Asp1 5 10 151316PRTArtificialsynthetic sequence designed
for self assembly 13Ala Arg Ala Lys Ala Asp Ala Glu Ala Arg Ala Lys
Ala Asp Ala Glu1 5 10 151416PRTArtificialsynthetic sequence
designed for self assembly 14Ala Lys Ala Arg Ala Glu Ala Asp Ala
Lys Ala Arg Ala Asp Ala Glu1 5 10 151516PRTArtificialsynthetic
sequence designed for self assembly 15Ala Gln Ala Gln Ala Gln Ala
Gln Ala Gln Ala Gln Ala Gln Ala Gln1 5 10
151616PRTArtificialsynthetic sequence designed for self assembly
16Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln1
5 10 151716PRTArtificialsynthetic sequence designed for self
assembly 17Tyr Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln Tyr Gln
Tyr Gln1 5 10 151816PRTArtificialsynthetic sequence designed for
self assembly 18His Gln His Gln His Gln His Gln His Gln His Gln His
Gln His Gln1 5 10 151916PRTArtificialsynthetic sequence designed
for self assembly 19Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn
Ala Asn Ala Asn1 5 10 152016PRTArtificialsynthetic sequence
designed for self assembly 20Val Asn Val Asn Val Asn Val Asn Val
Asn Val Asn Val Asn Val Asn1 5 10 152116PRTArtificialsynthetic
sequence designed for self assembly 21Tyr Asn Tyr Asn Tyr Asn Tyr
Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn1 5 10
152216PRTArtificialsynthetic sequence designed for self assembly
22His Asn His Asn His Asn His Asn His Asn His Asn His Asn His Asn1
5 10 152316PRTArtificialsynthetic sequence designed for self
assembly 23Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn
Ala Gln1 5 10 152416PRTArtificialsynthetic sequence designed for
self assembly 24Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn Ala
Gln Ala Asn1 5 10 152516PRTArtificialsynthetic sequence designed
for self assembly 25Val Asn Val Gln Val Asn Val Gln Val Asn Val Gln
Val Asn Val Gln1 5 10 152616PRTArtificialsynthetic sequence
designed for self assembly 26Val Gln Val Asn Val Gln Val Asn Val
Gln Val Asn Val Gln Val Asn1 5 10 152716PRTArtificialsynthetic
sequence designed for self assembly 27Tyr Asn Tyr Gln Tyr Asn Tyr
Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln1 5 10
152816PRTArtificialsynthetic sequence designed for self assembly
28Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn1
5 10 152916PRTArtificialsynthetic sequence designed for self
assembly 29His Asn His Gln His Asn His Gln His Asn His Gln His Asn
His Gln1 5 10 153016PRTArtificialsynthetic sequence designed for
self assembly 30His Gln His Asn His Gln His Asn His Gln His Asn His
Gln His Asn1 5 10 153118PRTArtificialsynthetic sequence designed
for self assembly 31Ala Lys Ala Gln Ala Asp Ala Lys Ala Gln Ala Asp
Ala Lys Ala Gln1 5 10 15Ala Asp3218PRTArtificialsynthetic sequence
designed for self assembly 32Val Lys Val Gln Val Asp Val Lys Val
Gln Val Asp Val Lys Val Gln1 5 10 15Val
Asp3318PRTArtificialsynthetic sequence designed for self assembly
33Tyr Lys Tyr Gln Tyr Asp Tyr Lys Tyr Gln Tyr Asp Tyr Lys Tyr Gln1
5 10 15Tyr Asp3418PRTArtificialsynthetic sequence designed for self
assembly 34His Lys His Gln His Asp His Lys His Gln His Asp His Lys
His Gln1 5 10 15His Asp3516PRTArtificialsynthetic sequence designed
for self assembly 35Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala
Asp Ala Asp Ala1 5 10 153616PRTArtificialsynthetic sequence
designed for self assembly 36Arg Ala Asp Ala Arg Gly Asp Ala Arg
Ala Asp Ala Arg Gly Asp Ala1 5 10 153716PRTArtificialsynthetic
sequence designed for self assembly 37Arg Ala Glu Ala Arg Ala Glu
Ala Arg Ala Glu Ala Arg Ala Glu Ala1 5 10
153816PRTArtificialsynthetic sequence designed for self assembly
38Lys Ala Asp Ala Lys Ala Asp Ala Lys Ala Asp Ala Lys Ala Asp Ala1
5 10 153916PRTArtificialsynthetic sequence designed for self
assembly 39Ala Glu Ala Glu Ala His Ala His Ala Glu Ala Glu Ala His
Ala His1 5 10 154016PRTArtificialsynthetic sequence designed for
self assembly 40Phe Glu Phe Glu Phe Lys Phe Lys Phe Glu Phe Glu Phe
Lys Phe Lys1 5 10 154116PRTArtificialsynthetic sequence designed
for self assembly 41Leu Glu Leu Glu Leu Lys Leu Lys Leu Glu Leu Glu
Leu Lys Leu Lys1 5 10 154216PRTArtificialsynthetic sequence
designed for self assembly 42Ala Glu Ala Glu Ala Lys Ala Lys Ala
Glu Ala Glu Ala Lys Ala Lys1 5 10 154312PRTArtificialsynthetic
sequence designed for self assembly 43Ala Glu Ala Glu Ala Glu Ala
Glu Ala Lys Ala Lys1 5 104416PRTArtificialsynthetic sequence
designed for self assembly 44Lys Ala Lys Ala Lys Ala Lys Ala Glu
Ala Glu Ala Glu Ala Glu Ala1 5 10 154516PRTArtificialsynthetic
sequence designed for self assembly 45Ala Glu Ala Glu Ala Glu Ala
Glu Ala Lys Ala Lys Ala Lys Ala Lys1 5 10
154616PRTArtificialsynthetic sequence designed for self assembly
46Arg Ala Arg Ala Arg Ala Arg Ala Asp Ala Asp Ala Asp Ala Asp Ala1
5 10 154716PRTArtificialsynthetic sequence designed for self
assembly 47Ala Asp Ala Asp Ala Asp Ala Asp Ala Arg Ala Arg Ala Arg
Ala Arg1 5 10 154816PRTArtificialsynthetic sequence designed for
self assembly 48Asp Ala Asp Ala Asp Ala Asp Ala Arg Ala Arg Ala Arg
Ala Arg Ala1 5 10 154916PRTArtificialsynthetic sequence designed
for self assembly 49His Glu His Glu His Lys His Lys His Glu His Glu
His Lys His Lys1 5 10 155020PRTArtificialsynthetic sequence
designed for self assembly 50Val Glu Val Glu Val Glu Val Glu Val
Glu Val Glu Val Glu Val Glu1 5 10 15Val Glu Val Glu
205120PRTArtificialsynthetic sequence designed for self assembly
51Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe1
5 10 15Arg Phe Arg Phe 205268PRTHomo sapiens 52Lys Pro Val Ser Leu
Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1 5 10 15His Val Ala Arg
Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30Asn Cys Ala
Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45Val Cys
Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60Ala
Leu Asn Lys655368PRTHomo sapiensmisc_feature(4)..(4)X is ala, arg,
asx, cys, glx, gly, his, ile, leu, lys, met, phe, pro, thr, trp,
tyr, or val 53Lys Pro Val Xaa Leu Ser Tyr Arg Cys Pro Cys Arg Phe
Phe Glu Ser1 5 10 15His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile
Leu Asn Thr Pro 20 25 30Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys
Asn Asn Asn Arg Gln 35 40 45Val Cys Ile Asp Pro Lys Leu Lys Trp Ile
Gln Glu Tyr Leu Glu Lys 50 55 60Ala Leu Asn Lys655468PRTHomo
sapiens 54Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe
Glu Ser1 5 10 15His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu
Asn Thr Pro 20 25 30Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn
Asn Asn Arg Gln 35 40 45Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln
Glu Tyr Leu Glu Lys 50 55 60Ala Leu Asn Lys655568PRTHomo
sapiensmisc_feature(5)..(5)X is ala, arg, asx, cys, gln, gly, his,
ile, lys, met, phe, pro, ser, thr, tyr, or val 55Lys Pro Val Ser
Xaa Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1 5 10 15His Val Ala
Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30Asn Cys
Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45Val
Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55
60Ala Leu Asn Lys655668PRTHomo sapiens 56Lys Pro Val Ser Pro Ser
Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1 5 10 15His Val Ala Arg Ala
Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30Asn Cys Ala Leu
Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45Val Cys Ile
Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60Ala Leu
Asn Lys65576PRTHomo sapiens 57Gly Gly Gly Gly Gly Gly1 5586PRTHomo
sapiens 58Gly Ile Val Gly Pro Leu1 5596PRTHomo sapiens 59Pro Val
Gly Leu Ile Gly1 560204DNAHomo sapiens 60aagcccgtcg tcctgagcta
cagatgccca tgccgattct tcgaaagcca tgttgccaga 60gccaacgtca agcatctcaa
aattctcaac actccaaact gtgcccttca gattgtagcc 120cggctgaaga
acaacaacag acaagtgtgc attgacccga agctaaagtg gattcaggag
180tacctggaga aagctttaaa caag 20461273DNAHomo sapiens 61aagcccgtcg
tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga 60gccaacgtca
agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc
120cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg
gattcaggag 180tacctggaga aagctttaaa caagtgagga atcgtgggac
ctctgcgtgc ccgtgccgac 240gccgacgccc gtgcccgtgc cgacgccgac gcc
27362269DNAHomo sapiens 62aagcccgtcg tcctgagcta cagatgccca
tgccgattct tcgaaagcca tgttgccaga 60gccaacgtca agcatctcaa aattctcaac
actccaaact gtgcccttca gattgtagcc 120cggctgaaga acaacaacag
acaagtgtgc attgacccga agctaaagtg gattcaggag 180tacctggaga
aagctttaaa caagcctgtg ggactgatcg gagtgcccgt gccgacgccg
240acgcccgtgc ccgtgccgac gccgacgcc 26963270DNAHomo sapiens
63aagcccgtcg tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga
60gccaacgtca agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc
120cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg
gattcaggag 180tacctggaga aagctttaaa caagggaggc gggggaggtg
ggcgtgcccg tgccgacgcc 240gacgcccgtg cccgtgccga cgccgacgcc
2706417PRTHomo sapiens 64Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro
Cys Arg Phe Phe Glu Ser1 5 10 15His6517PRTHomo sapiens 65Lys Pro
Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1 5 10
15His6617PRTHomo sapiens 66Lys Pro Val Ser Pro Ser Tyr Arg Cys Pro
Cys Arg Phe Phe Glu Ser1 5 10 15His
* * * * *