U.S. patent application number 17/476971 was filed with the patent office on 2021-12-30 for bi-functional arginine-glycine-aspartic acid (rgd) peptides and methods to promote angiogenesis.
The applicant listed for this patent is Clemson University Research Foundation, MUSC Foundation for Research Development. Invention is credited to Chung-Jen James Chou, Jia Jia, Ying Mei.
Application Number | 20210405064 17/476971 |
Document ID | / |
Family ID | 1000005836175 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210405064 |
Kind Code |
A1 |
Mei; Ying ; et al. |
December 30, 2021 |
BI-FUNCTIONAL ARGININE-GLYCINE-ASPARTIC ACID (RGD) PEPTIDES AND
METHODS TO PROMOTE ANGIOGENESIS
Abstract
The present invention provides an in vitro method for
identifying a compound that promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation for the manufacture of a diagnostic
or therapeutic agent. The present invention further provides the
identified compounds and pharmaceutical compositions, and assays
and kits for identifying a compound or using a compound that
promotes endothelial cell adhesion, endothelial cell spreading,
endothelial cell migration and/or endothelial cell proliferation
and is useful for bioprinting.
Inventors: |
Mei; Ying; (Mount Pleasant,
SC) ; Jia; Jia; (Charleston, SC) ; Chou;
Chung-Jen James; (Mount Pleasant, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clemson University Research Foundation
MUSC Foundation for Research Development |
Clemson
Charleston |
SC
SC |
US
US |
|
|
Family ID: |
1000005836175 |
Appl. No.: |
17/476971 |
Filed: |
September 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15726766 |
Oct 6, 2017 |
11150251 |
|
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17476971 |
|
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62405523 |
Oct 7, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6893 20130101;
G01N 2333/70596 20130101; A61P 9/10 20180101; G01N 2333/70557
20130101; G01N 2500/04 20130101; C07K 7/08 20130101; C07K 14/71
20130101; C07K 14/755 20130101; G01N 2500/20 20130101; C07K 14/52
20130101; C07K 7/06 20130101; C07K 17/02 20130101; A61K 38/00
20130101; C07K 14/78 20130101; G01N 33/74 20130101; G01N 2800/7014
20130101; C07K 14/75 20130101; C07K 14/70546 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; A61P 9/10 20060101 A61P009/10; G01N 33/74 20060101
G01N033/74; C07K 14/78 20060101 C07K014/78; C07K 17/02 20060101
C07K017/02; C07K 14/71 20060101 C07K014/71; C07K 14/755 20060101
C07K014/755; C07K 14/52 20060101 C07K014/52; C07K 14/75 20060101
C07K014/75; C07K 14/705 20060101 C07K014/705 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers GM103444 and GM104941 awarded by The National Institutes of
Health and grant number EPS-0903795 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1-14. (canceled)
15. A peptide that binds to an endothelial cell integrin, the
peptide selected from the group consisting of a peptide having the
following amino acid sequence: TFALRGDNP (SEQ ID NO:1), TFALRADNP
(SEQ ID NO:2), DVEKRGDREEAHVP (SEQ ID NO:3), IQRGDIDAMIS (SEQ ID
NO:4), DAVKQLQAAERGDA (SEQ ID NO:5), PMQKMRGDVFSP (SEQ ID NO:6),
RSDGTG (SEQ ID NO:7), EAPRGDVYQG (SEQ ID NO:8), GLOGERGRO (SEQ ID
NO:9), GFOGERGVQ (SEQ ID NO:10), DGEA (SEQ ID NO:11), GFOGER (SEQ
ID NO:12), GLKGEN (SEQ ID NO:13), LDV (SEQ ID NO:14), REDV (SEQ ID
NO:15), PEDGIHE (SEQ ID NO:16), PHSRN (SEQ ID NO:17), ALNGR (SEQ ID
NO:18), IAFQRN (SEQ ID NO:19), IKLLI (SEQ ID NO:20), SIKVAV (SEQ ID
NO:21), AGQWHRVSVRWG (SEQ ID NO:22), TWSQKALHHRVP (SEQ ID NO:23),
SIYITRF (SEQ ID NO:24), SYWYRIEASRTG(SEQ ID NO:25) YIGSR (SEQ ID
NO:26), RDIAEIIKDI (SEQ ID NO:27), VFDNFVLK(SEQ ID NO:28) ,
AEIDGIEL (SEQ ID NO:29), SETQRGDVFVP (SEQ ID NO:30), PASYRGDSC (SEQ
ID NO:31), VTGRGDSPAS (SEQ ID NO:32), PQVTRGDVFTMP (SEQ ID NO:37),
or variants at least 90% identical thereto.
16. A pharmaceutical composition comprising the peptide of claim 15
and a pharmaceutically acceptable carrier, diluent, or
excipient.
17. A hydrogel composition comprising at least one integrin-binding
peptide of claim 15.
18. A hydrogel composition comprising: (a) at least one
integrin-binding peptide of claim 15; and (b) a biocompatible
polymer, wherein the integrin-binding peptide is linked to the
biocompatible polymer.
19. The hydrogel composition of claim 17, wherein the linkage is a
covalent or non-covalent linkage.
20. The hydrogel composition of claim 18, wherein the biocompatible
polymer is functionalized with a VEGF or a VEGF mimetic peptide and
an integrin-binding peptide that binds at least one type of
endothelial cell integrin.
21. The hydrogel composition of claim 20, wherein the VEGF or the
VEGF mimetic peptide is attached to the biocompatible polymer
through a matrix metalloproteinase (MMP) degradable peptide
linkage.
22. The hydrogel composition of claim 18, wherein the biocompatible
polymer is functionalized with a VEGF or a VEGF mimetic peptide and
an integrin binding peptide that binds to an
.alpha..sub.v.beta..sub.3 and/or VLA-6 integrin.
23. The hydrogel composition of claim 18, wherein the biocompatible
polymer comprises an agarose gel, polyethylene glycol, alginate,
hyaluronic acid, polyacryylic acid, polyacrylic amide, polyvinyl
alcohol, polyhydroxyethyl methacrylate, methacrylated dextrans,
poly(N-isopropylacrylamide), or any combination thereof.
24. The hydrogel composition of claim 18, wherein the biocompatible
polymer is oxidized alginate.
25. The hydrogel composition of claim 18, wherein the hydrogel
composition is an injectable composition.
26. A method of promoting angiogenesis in a subject in need
thereof, comprising administering to the subject a peptide of claim
15, in an amount effective to promote angiogenesis.
27. A method of promoting endothelial cell adhesion, endothelial
cell spreading, endothelial cell migration and/or endothelial cell
proliferation, comprising administering to the subject a peptide of
claim 15, in an amount effective to promote endothelial cell
adhesion, endothelial cell spreading, endothelial cell migration
and/or endothelial cell proliferation.
28. A method of treating or preventing ischemic injury in a subject
in need thereof, comprising administering to the subject a peptide
of claim 15, in an amount effective to treat or prevent ischemic
injury.
29. A method of promoting tissue regeneration in a subject in need
thereof, comprising administering to the subject a peptide of claim
15, in an amount effective to promote tissue regeneration.
30. A method of bioprinting, comprising administering a peptide of
claim 15 to a substrate in an amount effective to promote tissue
regeneration.
31. A biomaterial product for bioprinting, comprising a peptide of
claim 15.
32. A kit comprising a peptide of claim 15 and a container suitable
for delivery of the peptide, pharmaceutical composition or hydrogel
composition into an administration device, with optional
instructions for the use thereof.
33. The kit of claim 32, wherein the device is a parenteral
administration device.
34. The kit of claim 32, wherein the device is an intramyocardial
device.
35. The kit of claim 32, wherein the device is a bioprinter.
Description
STATEMENT OF PRIORITY
[0001] This application is a continuation of and claims priority to
U.S. patent application Ser. No. 15/726,766, filed Oct. 6, 2017,
which claims the benefit, under 35 U.S.C. .sctn. 119(e), of U.S.
Provisional Application Ser. No. 62/405,523, filed Oct. 7, 2016,
the entire contents of each of which are incorporated by reference
herein.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0003] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn. 1.821, entitled 9662-68CT_ST25.txt, 10,247 bytes in
size, generated on Sep. 16, 2021 and filed via EFS-Web, is provided
in lieu of a paper copy. This Sequence Listing is hereby
incorporated by reference herein into the specification for its
disclosures.
RESERVATION OF COPYRIGHT
[0004] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner, Clemson University or MUSC Foundation for Research
Development, has no objection to the reproduction by anyone of the
patent document or the patent disclosure, as it appears in the U.S.
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0005] The present invention relates to arginine-glycine-aspartic
acid (RGD) peptide compounds and methods and assays for identifying
compounds, in particular, RGD peptides, that bind to and/or
modulate a cell surface integrin and compounds and methods to
promote angiogenesis.
BACKGROUND OF THE INVENTION
[0006] Vasculature plays an essential role in maintaining normal
tissue functions through the delivery of oxygen and nutrients and
removal of the waste generated by the tissues. Ischemic diseases
that affect normal blood supply pose an enormous threat to public
health. In the heart, lack of sufficient blood supply leads to cell
death and permanent loss of heart functions. Each year, 7.2 million
people die from ischemic heart disease (IHD), which accounts for
12% of all reported deaths worldwide. In addition to cardiovascular
disease, periphery artery, carotid artery, renal artery and venous
diseases can all lead to serious illness, even death. It was
estimated that over 300 million patients would benefit from
pro-angiogenic therapies in western nations. To address this
concern, significant efforts have been devoted to developing
functional biomaterials to recreate natural vasculogenic (i.e., de
novo vessel formation) and angiogenic (i.e., new vessel formed from
pre-existing vessels) environments to promote neovascularization.
To this end, numerous pro-vascularization growth factors (e.g.,
VEGF and bFGF) have been incorporated into hydrogels to achieve
controlled, local delivery. In parallel, cell adhesive peptides
(e.g., RGDS (SEQ ID NO:35)/RGDSP (SEQ ID NO:34)) have been
conjugated onto hydrogels to improve their affinity to endothelial
cells. The RGDS (SEQ ID NO:35)/RGDSP (SEQ ID NO:34) peptides
enhance the binding affinity of the constructs to
.alpha..sub.v.beta..sub.3 integrin expressed on endothelial cells
(EC) and improve EC adhesion, spreading and proliferation. Further,
recent studies showed the hydrogels functionalized with RGDS (SEQ
ID NO:35)/RGDSP (SEQ ID NO:34) peptides initiate integrin-mediated
signaling pathway, induce up-regulation of VEGF receptor 2 (VEGFR2)
and result in improved EC proliferation and migration in vitro and
improved angiogenesis in vivo.
[0007] While RGDS (SEQ ID NO:35)/RGDSP (SEQ ID NO:34) peptides
derived from the cell adhesive domain of Fibronectin (Fn) have been
extensively used to promote EC functions (e.g., adhesion,
spreading, proliferation, migration), their affinity to EC integrin
is rather moderate, which leads to suboptimal EC functions. For
example, RGDS (SEQ ID NO:35) peptide fiinctionalization has been
repeatedly used to promote EC attachment and spreading in both 2D
and 3D environments. However, ECs undergo apoptosis by 28-72 hrs
after seeding without high concentrations of angiogenic factors.
This highlights an urgent need to identify novel RGD (SEQ ID NO:36)
peptides with high affinity to EC integrin to improve their
functions.
[0008] In nature, vascular endothelium supports the dynamic
functionality of ECs. The extracellular matrix (ECM) proteins in
the vascular endothelium, including laminin (Ln), fibronectin (Fn),
vitronectin (Vn) and collagen, have been shown to provide vital
cues for EC survival and proliferation through cell-surface
integrin. Notably, biochemical analysis has shown that Ln, Fn, and
Vn contain RGD (SEQ ID NO:36) sequences that can bind to cell
surface integrin subunits to initiate cell attachment. In addition,
the previous studies also demonstrate the amino acids surrounding
the RGD (SEQ ID NO:36) peptide influence its activities. Based on
this evidence, it was reasoned that there could be additional
RGD-containing peptide segments derived from vascular endothelium
ECM proteins (i.e., Ln, Fn and Vn) with higher affinity to EC
integrin than the widely used RGDS (SEQ ID NO:35)/RGDSP (SEQ ID
NO:34) peptides. The identification of these RGD (SEQ ID NO:36)
peptides provides potent biological ligands for the development of
vascularized tissue engineering constructs.
SUMMARY OF THE INVENTION
[0009] Embodiments of the present invention are directed to methods
for identifying a compound that promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation for the manufacture of a diagnostic
or therapeutic agent, including (a) contacting a hydrogel
functionalized with at least one integrin-binding peptide with an
endothelial cell integrin; and (b) determining the binding affinity
of the integrin-binding peptide to the endothelial cell integrin,
wherein a high binding affinity indicates that the integrin-binding
peptide is a compound that promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation.
[0010] Embodiments of the present invention also provide assays for
identifying a compound that promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation for manufacture of a diagnostic or
therapeutic agent, the assay including screening a compound of
interest for its binding effect on an endothelial cell integrin
wherein the compound of interest is conjugated to a hydrogel and
contacted to an endothelial cell integrin, wherein high affinity
binding to the endothelial cell integrin indicates that the
compound of interest promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation.
[0011] Embodiments of the present invention further provide
peptides that bind an endothelial cell integrin as well as
fragments and variants of the peptides that bind to an endothelial
cell integrin when tested under the same test conditions as the
parent peptide.
[0012] Embodiments of the present invention also provide a
pharmaceutical composition including the peptides described herein
and a pharmaceutically acceptable carrier, diluent, or
excipient.
[0013] Embodiments of the present invention also provide methods of
promoting angiogenesis.
[0014] Embodiments of the present invention further provide methods
of promoting endothelial cell adhesion, endothelial cell spreading,
endothelial cell migration and/or endothelial cell
proliferation.
[0015] Embodiments of the present invention further provide methods
of treating or preventing ischemic injury.
[0016] Embodiments of the present invention further provide methods
of promoting tissue regeneration.
[0017] Embodiments of the present invention also provide a method
for bioprinting and biomaterial products for bioprinting.
[0018] Embodiments of the present invention further provide kits
including the elements necessary to carry out the processes
described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A-1C. Schematic representation of fabrication of
peptide-functionalized PEG hydrogel microarrays. (A) The printing
solutions composed of PEGDA monomer and various methacrylated
peptides were prepared in a 384-well plate. (B) The printing
solutions were placed onto poly(HEMA) coated microscope slides with
a customized microarrayer and polymerized by UV under Argon
protection to prepare peptide-functionalized PEG hydrogel spots.
Eight hydrogel spots in a microarray were shown to present the
dimension of the hydrogel spots and the distance between the
hydrogel spots. (C) High throughput analysis of cellular activities
after cell seeding onto the microarray.
[0020] FIG. 2. The methacrylated peptides are prepared by
conjugating 2-isocyanatoethyl methacrylate with the terminal amine
of the peptides on the solid-phase.
[0021] FIG. 3. hADSCs were seeded onto an 8.times.8 hydrogel
microarray prepared from PEGDA-250, PEGDA-575, or PEGDA-700 to
identify suitable formulations to inhibit unspecific cell
adhesion.
[0022] FIG. 4. Functional validation of the peptide moieties on the
hydrogels. The representative fluorescent images of blank PEGDA-700
hydrogels (left), RDGSP (SEQ ID NO:34) functionalized PEGDA-700
hydrogels (middle) and RGDSP (SEQ ID NO:33) functionalized
PEGDA-700 hydrogels (right) after hADSC seeding (blue: DAPI, green:
phalloidin, scale bar=100 .mu.m).
[0023] FIGS. 5A-5C. Selection of a suitable linker to ensure the
exposure of peptide moieties on PEG hydrogel surface. (A) A list of
peptides used for the linker selection: RGDSP (SEQ ID NO:33)
peptides fused with zero/two/four/six glycine linker, RDGSP (SEQ ID
NO:34) and no peptide functionalization (blank PEGDA-700 hydrogel)
have been employed as controls. (B) Effects of peptide
concentration on the average number of the attached hADSCs on the
hydrogel spots and the sigmoidal curve-fits. (C) Effects of glycine
linker length on the saturated number of attached hADSCs on the
hydrogel spots. All values are mean+SD. Asterisk denotes
significant difference between blank PEGDA-700 hydrogels,
MethG.sub.4RDGSP (SEQ ID NO:47) and MethRGDSP (SEQ ID NO:33),
MethG.sub.2RGDSP (SEQ ID NO:45). Double asterisk denotes
significant difference between MethRGDSP (SEQ ID NO:33),
MethG.sub.2RGDSP (SEQ ID NO:45) and MethG.sub.4RGDSP (SEQ ID
NO:46), MethG.sub.6RGDSP (SEQ ID NO:48).
[0024] FIGS. 6A-6E. hiPSC-CM adhesion and sarcomere formation on
hydrogel microarrays. (A) The representative pictures of hiPSC-CMs
on PEG hydrogel spots functionalized with different RGD (SEQ ID
NO:36) peptides (blue: DAPI; green: sarcomere actinin; red:
Troponin-I, scale bar=50 .mu.m): (a) PEG hydrogel spots
functionalized with RGD (SEQ ID NO:36) peptides that could not
support adhesion of hiPSC-CMs. (b) PEG hydrogel spots
functionalized with peptides that can support minimal cell
adhesion. (c) PEG hydrogel spots functionalized with peptides that
can moderately support cell adhesion (d) PEG hydrogel spots
functionalized with RGD (SEQ ID NO:36) peptides that can
effectively promote hiPSC-CM adhesion and sarcomere formation, a
critical step for cardiomyocyte maturation. (B) A list of RGD (SEQ
ID NO:36) peptides used in this experiment and their molecular
origin. (C) The average number of attached hiPSC-CMs on the
hydrogel spots functionalized with RGD (SEQ ID NO:36) peptides from
laminin .beta.4 chain, RGD (SEQ ID NO:36) peptides from Vn and two
controls (i.e., blank PEGDA-700 hydrogel and RDGSP (SEQ ID NO:34)
peptide functionalized hydrogel). All values are mean+SD. Asterisk
denotes significant difference between RGD (SEQ ID NO:36) peptide
from laminin .beta.4 chain, RGD (SEQ ID NO:36) peptide from Vn and
two control groups. Double asterisk denotes significant difference
between laminin .beta.4 RGD (SEQ ID NO:36) peptides and Vn RGD (SEQ
ID NO:36) peptide. (D) The average number of attached hiPSC-CMs on
PEG hydrogel spots functionalized with all different RGD (SEQ ID
NO:36) sequences. Asterisk denotes significant difference between
the "active" RGD (SEQ ID NO:36) peptides and "inactive" RGD (SEQ ID
NO:36) peptides plus two control groups. Double asterisk denotes
significant difference between laminin .beta.4 RGD (SEQ ID NO:36)
peptides, RGDSP (SEQ ID NO:33) and other RGD (SEQ ID NO:36)
peptides from ECM proteins. Peptides labeled with asterisk were
identified through bioinformatics screening. (E) The sarcomere
actinin expressions of hiPSC-CMs (pixels per cell) cultured on the
hydrogel spots. Asterisk denotes significant difference between RGD
(SEQ ID NO:36) peptide from laminin .beta.4 chain and RGD (SEQ ID
NO:36) peptides from Vn, Fn, .alpha.5-2, .alpha.4. Peptides labeled
with asterisk were identified through bioinformatics screening.
[0025] FIG. 7. The RGD (SEQ ID NO:36) peptide library used to
screen high affinity ligands to endothelial cell integrins.
[0026] FIGS. 8A-8F. Functional improvements of endothelial cells
(EC) by .alpha.1 peptide and related mechanistic studies. (A)
Adhesion of HUVECs to microarrayed PEG hydrogels functionalized
with (a) no peptide (PEG), (b) RGDS (SEQ ID NO:35), (c) RGDSP (SEQ
ID NO:33), (d) .alpha.1 peptide, and e: the related quantified cell
adhesion. Blue-DAPI, Green-Phalloidin, scale bar is 100 um. (B)
Adhesion of HUVECs to 2D alginates substrates hydrogels
functionalized with 10% (w/w) peptide of a: RGDS (SEQ ID NO:35), b:
RGDSP (SEQ ID NO:33), c: .alpha.1 peptide, and d: related
quantified cell adhesion. Blue-DAPI, Green-Phalloidin, scale bar is
50 um. (C) HUVEC proliferation and network formation in 3D alginate
hydrogels functionalized with 10% (w/w) peptide of (a) RGDS (SEQ ID
NO:35), (b) RGDSP (SEQ ID NO:33), (c) .alpha.1 peptide, and (d)
related quantified cell adhesion. Blue-DAPI, Green-Phalloidin,
scale bar is 25 um. (D) Adhesion of HUVECs treated with integrin
blocking antibodies (x-axis) on 2D alginate substrates
functionalized with RGDS (SEQ ID NO:35)/RGDSP (SEQ ID
NO:34)/.alpha.1 peptide. (E) mRNA expression of VEGFR2 of HUVECs
cultured in 3D alginate hydrogels functionalized with no
peptide/RGDS (SEQ ID NO:35)/RGDSP (SEQ ID NO:34)/.alpha.1 peptide.
(F) A schematic summary of the bi-functional .alpha.1 peptide
capable of binding both .alpha..sub.v.beta..sub.3 and VLA-6
integrins and enhancing cell adhesion, proliferation and VEGFR2
expression.
[0027] FIG. 9. Scheme of synergistic multi-signaling interactions
between .alpha.1 peptide, QK peptide and EC surface receptors
(i.e., .alpha..sub.v.beta..sub.3, VLA-6 integrins and VEGFR2).
[0028] FIG. 10. Modular synthetic route to prepare .alpha.1 peptide
functionalized alginates and MMP-responsive QK peptide
(GPQG.dwnarw.IAGKLTWQELYQLKYKGI, SEQ ID NO:41) functionalized
alginates.
[0029] FIGS. 11A-11D. HUVECs cultured in 3D alginate hydrogels
functionalized with (A) .alpha.1 peptide and covalently bound QK,
(B) RGDSP (SEQ ID NO:33) and MMP-responsive QK, (C) .alpha.1
peptide and MMP-responsive QK. (D) normalized total network length
of (A)-(C). Blue-DAPI, Green-Phalloidin, scale bar is 25 .mu.m.
DETAILED DESCRIPTION
[0030] The present invention is further described below in greater
detail. This description is not intended to be a detailed catalog
of all the different ways in which the invention may be
implemented, or all the features that may be added to the instant
invention. For example, features illustrated with respect to one
embodiment may be incorporated into other embodiments, and features
illustrated with respect to a particular embodiment may be deleted
from that embodiment. In addition, numerous variations and
additions to the various embodiments suggested herein will be
apparent to those skilled in the art in light of the instant
disclosure which do not depart from the instant invention. Hence,
the following specification is intended to illustrate some
particular embodiments of the invention, and not to exhaustively
specify all permutations, combinations and variations thereof.
Further, all patent and patent application references referred to
in this patent application are hereby incorporated by reference in
their entirety as if set forth fully herein. Unless otherwise
defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. The terminology used in the
description of the invention herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting of the invention.
[0031] As used herein, "a," "an" or "the" can mean one or more than
one. Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0032] The term "about," as used herein when referring to a
measurable value such as an amount of dose (e.g., an amount of a
compound) and the like, is meant to encompass variations of
.+-.20%, .+-.10%, .+-.5%, .+-.1%, .+-.0.5%, or even .+-.0.1% of the
specified amount.
[0033] As used herein, the transitional phrase "consisting
essentially of" (and grammatical variants) means that the scope of
a claim is to be interpreted to encompass the specified materials
or steps recited in the claim, "and those that do not materially
affect the basic and novel characteristic(s)" of the claimed
invention. Thus, the term "consisting essentially of" when used in
a claim of this invention is not intended to be interpreted to be
equivalent to "comprising."
[0034] The term "endothelial" cells refer to simple squamous cells;
a layer of which cells line the inside surfaces of body cavities,
blood vessels, and lymph vessels. Endothelial cells provide a
barrier between the blood and the rest of the body tissues. Other
specialized functions of endothelial cells include producing nitric
oxide (NO), blood vessel formation (angiogenesis), adhesion,
spreading, migration and/or proliferation, blood clotting,
inflammation, vasoconstriction, vasodilation, blood pressure and
water regulation.
[0035] The term "arginine-glycine-aspartic acid" or "RGD" (SEQ ID
NO:36) peptide refers to a peptide including the sequence
L-arginine, glycine, and L-aspartic acid (RGD) (SEQ ID NO:36). The
RGD (SEQ ID NO:36) sequence may function as the cell attachment
site of a number of adhesive extracellular matrix, blood, and cell
surface proteins, with various integrins recognizing the RGD (SEQ
ID NO:36) sequence in their adhesion protein ligands.
[0036] The terms "polypeptide," "protein," and "peptide" refer to a
chain of covalently linked amino acids. In general, the term
"peptide" can refer to shorter chains of amino acids (e.g., 2-50
amino acids); however, all three terms overlap with respect to the
length of the amino acid chain. Polypeptides, proteins, and
peptides may comprise naturally occurring amino acids,
non-naturally occurring amino acids, or a combination of both. The
polypeptides, proteins, and peptides may be isolated from sources
(e.g., cells or tissues) in which they naturally occur, produced
recombinantly in cells in vivo or in vitro or in a test tube in
vitro, and/or synthesized chemically. Such techniques are known to
those skilled in the art. See, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y.,
1989); Ausubel et al. Current Protocols in Molecular Biology (Green
Publishing Associates, Inc. and John Wiley & Sons, Inc., New
York).
[0037] The term "fragment," as applied to a polypeptide, will be
understood to mean an amino acid sequence of reduced length
relative to a reference polypeptide or amino acid sequence and
comprising, consisting essentially of, and/or consisting of an
amino acid sequence of contiguous amino acids identical, or
substantially identical, to the reference polypeptide or amino acid
sequence. Such a polypeptide fragment according to the invention
may be, where appropriate, included in a larger polypeptide of
which it is a constituent. In some embodiments, such fragments can
comprise, consist essentially of, and/or consist of peptides having
a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30 35, 40,
45, 50, 75, 100, 150, 200, or more consecutive amino acids of a
polypeptide or amino acid sequence according to the invention.
Moreover, as used herein, "portion" or "fragment" are used
interchangeably and refers to less than the whole of the structure
that substantially retains at least one biological activity
normally associated with that molecule, protein or polypeptide. In
particular embodiments, the "fragment" or "portion" substantially
retains all of the activities possessed by the unmodified protein.
By "substantially retains" biological activity, it is meant that
the protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%,
75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological
activity of the native protein (and can even have a higher level of
activity than the native protein).
[0038] A fragment of a polypeptide or protein of this invention can
be produced by methods well known and routine in the art. Fragments
of this invention can be produced, for example, by enzymatic or
other cleavage of naturally occurring peptides or polypeptides or
by synthetic protocols that are well known. Such fragments can be
tested for one or more of the biological activities of this
invention according to the methods described herein, which are
routine methods for testing activities of polypeptides, and/or
according to any art-known and routine methods for identifying such
activities. Such production and testing to identify biologically
active fragments of the polypeptides described herein would be well
within the scope of one of ordinary skill in the art and would be
routine.
[0039] The term "variant" refers no more than one, two, three,
four, five, six, seven, eight, nine or ten amino acid substitutions
in the sequence of interest. The variant retains at least one
biological activity normally associated with that amino acid
sequence. In particular embodiments, the functional variant retains
at least about 40%, 50%, 60%, 75%, 85%, 90%, 95% or more biological
activity normally associated with the full-length amino acid
sequence. In other embodiments, a functional variant is an amino
acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%
97% or 98% similar to the peptide sequence disclosed herein (or
fragments thereof).
[0040] As used herein, the terms "express," "expressing," or
"expression" (or grammatical variants thereof) in reference to a
gene or coding sequence can refer to transcription to produce an
RNA and, optionally translation to produce a polypeptide. Thus,
unless the context indicates otherwise, the terms "express,"
"expressing," "expression" and the like can refer to events at the
transcriptional, post-transcriptional, translational and/or
post-translational level.
[0041] "Posttranslational modification" has its usual and customary
meaning and includes but is not limited to removal of leader
sequence, .gamma.-carboxylation of glutamic acid residues,
.beta.-hydroxylation of aspartic acid residues, N-linked
glycosylation of asparagine residues, O-linked glycosylation of
serine and/or threonine residues, sulfation of tyrosine residues,
phosphorylation of serine residues and any combination thereof.
Posttranslational modifications apply to the peptides described
herein.
[0042] The term "isolated" can refer to a nucleic acid, polypeptide
or cell that is substantially free of other cellular material,
viral material, and/or culture medium (when produced by recombinant
DNA techniques), or chemical precursors or other chemicals (when
chemically synthesized). Moreover, an "isolated fragment" is a
fragment of a nucleic acid or polypeptide that is not naturally
occurring as a fragment and would not be found in the natural
state. The term "extracellular matrix" or "ECM" protein refers to
proteins found in the non-cellular macromolecular network providing
physical scaffolding and biochemical support to surrounding cells.
Exemplary ECM proteins include, but are not limited to, laminin
(Ln), fibronectin (Fn), vitronectin (Vn), gelatin or collagen
protein.
[0043] The term "integrin" refers to a cell-surface cellular
adhesion molecule that can bind to ECM structures, such as the ECM
proteins described herein.
[0044] The term "hydrogel" refers to a polymeric biomaterial having
hydrophilic properties generally having water or a biological fluid
as the continuous phase.
[0045] The term "biocompatible" refers to the condition of being
not deleterious to living cells, tissues or organisms to the extent
of not causing serious harm to the host, the host being a human or
non-human host such as the subjects described herein.
[0046] The term "biomaterial product" refers to a biocompatible
substance that has been engineered and is suitable to construct,
replace, repair or augment cells, tissues and/or organs.
[0047] The term "angiogenesis" refers to the formation of new blood
vessels derived from pre-existing blood vessels.
[0048] The terms "pro-angiogenic" or "angiogenic" growth factors
(including all genes and isoforms of each gene product) for use in
accordance with the methods of the present invention include, but
are not limited to, vascular endothelial cell growth factor (VEGF),
acidic fibroblast growth factor (aFGF), basic fibroblast growth
factor (bFGF), epidermal growth factor, transforming growth factors
.alpha. and .beta., platelet-derived endothelial growth factor,
platelet-derived growth factor, tumor necrosis factor .alpha.,
hepatocyte growth factor (scatter factor), erythropoietin, colony
stimulating factor (CSF), macrophage-CSF (M-CSF),
granulocyte/macrophage CSF (GM-CSF), angiopoietin 1 and 2, and
nitric oxide synthase (NOS). The nucleic acid and amino acid
sequences for these and other angiogenic growth factors are
available in public databases such as GenBank and in the
literature. Additionally, human VEGF 1 (VEGF A) exists in at least
four principal isoforms, VEGF.sub.121; VEGF.sub.145; VEGF.sub.165;
and VEGF.sub.189. There also exists VEGF 2 (also referred to as
VEGF C); VEGF B; and VEGF D. Pro-angiogenic growth factors are
further described below.
[0049] The terms "ischemic injury" or "ischemic disease" refer to
diseases or disorders resulting from an insufficient supply of
blood to an organ, often due to an occluded blood vessel. Examples
of such include, but are not limited to, coronary artery disease,
peripheral artery disease, ischemic wounds and diabetic ulcers.
[0050] The term "tissue regeneration" refers to the process of
remodeling, renewal, growth, maintenance and/or improved function
of cells, and in particular, cells collectively forming a tissue.
The tissue may be tissue associated with the nervous system,
endocrine system, hematopoietic system, gastrointestinal tract,
renal system, cardiac system, vascular system, reproductive system,
musculoskeletal system or combinations thereof The tissue may be
tissue associated with an organ such as the appendix, bladder,
brain, ear, esophagus, eye, gall bladder, heart, kidney, large
intestine, liver, lung, mouth, muscle, nose, ovary, pancreas,
parathyroid gland, pineal gland, pituitary gland, skin, small
intestine, spleen, stomach, testes, thymus, thyroid gland, trachea,
uterus, vermiform appendix or combinations thereof Tissue
regeneration also includes wound healing.
[0051] "Bioprinting" or "3D bioprinting" refers to a process of
creating cell patterns using 3D printing technologies with cell
function, integrity and/or viability preserved during the printing
process. Bioprinting usually employs a layer-by-layer method to
create tissue-like structures that can be used in biomedical
engineering and tissue regeneration and remodeling fields.
Bioprinting can be used to print tissues and organs as well as
scaffolds of the same.
[0052] By the term "treat," "treating" or "treatment of" (and
grammatical variations thereof) it is meant that the severity of
the subject's condition is reduced, at least partially improved or
ameliorated and/or that some alleviation, mitigation or decrease in
at least one clinical symptom is achieved and/or there is a delay
in the progression of the disease or disorder. In representative
embodiments, the term "treat", "treating" or "treatment of" (and
grammatical variations thereof) refer to a reduction in the amount
and/or frequency of undesirable or uncontrolled bleeding.
[0053] A "treatment effective" amount as used herein is an amount
that is sufficient to treat (as defined herein) the subject. Those
skilled in the art will appreciate that the therapeutic effects
need not be complete or curative, as long as some benefit is
provided to the subject.
[0054] The term "prevent," "preventing" or "prevention of" (and
grammatical variations thereof) refer to prevention and/or delay of
the onset and/or progression of a disease, disorder and/or a
clinical symptom(s) in a subject and/or a reduction in the severity
of the onset and/or progression of the disease, disorder and/or
clinical symptom(s) relative to what would occur in the absence of
the methods of the invention. In representative embodiments, the
term "prevent," "preventing" or "prevention of" (and grammatical
variations thereof) refer to prevention and/or delay of the onset
and/or progression of undesirable or uncontrolled bleeding in the
subject, with or without other signs of clinical disease. The
prevention can be complete, e.g., the total absence of the disease,
disorder and/or clinical symptom(s). The prevention can also be
partial, such that the occurrence of the disease, disorder and/or
clinical symptom(s) in the subject and/or the severity of onset
and/or the progression is less than what would occur in the absence
of the present invention.
[0055] A "prevention effective" amount as used herein is an amount
that is sufficient to prevent (as defined herein) the disease,
disorder and/or clinical symptom in the subject. Those skilled in
the art will appreciate that the level of prevention need not be
complete, as long as some benefit is provided to the subject.
[0056] The efficacy of treating an injury or disorder by the
methods of the present invention can be determined by detecting a
clinical improvement as indicated by a change in the subject's
symptoms and/or clinical parameters as would be well known to one
of skill in the art or in the improved properties of the cells
and/or tissue as assessed by suitability for intended purposes of
the present invention including, but not limited to, tissue
regeneration, tissue transplants, wound healing, skin grafts,
etc.
[0057] The present invention provides methods and biomaterials that
can synergistically engage more than one type of endothelial cell
integrins to promote endothelial cell adhesion, endothelial cell
spreading, endothelial cell migration and/or endothelial cell
proliferation further providing vascular network formation for
therapeutic angiogenesis and/or vascular grafts.
[0058] The present invention also provides methods and biomaterials
that synergistically engage endothelial cell integrins and
pro-angiogenic growth factors to promote endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation further providing vascular network
formation for therapeutic angiogenesis and/or vascular grafts.
[0059] In some embodiments, the present invention provides an in
vitro method for identifying a compound that promotes endothelial
cell adhesion, endothelial cell spreading, endothelial cell
migration and/or endothelial cell proliferation for the manufacture
of a diagnostic or therapeutic agent, comprising, consisting of, or
consisting essentially of: (a) contacting a hydrogel functionalized
with at least one integrin-binding peptide with an endothelial cell
integrin; and (b) determining the binding affinity of
integrin-binding peptide to the endothelial cell integrin, wherein
a high binding affinity indicates that the integrin-binding peptide
is a compound that promotes endothelial cell adhesion, endothelial
cell spreading, endothelial cell migration and/or endothelial cell
proliferation. According to embodiments of the present invention,
the number of attached cells on each of peptide-functionalized
poly(ethylene glycol) (PEG) hydrogel spots is normalized to the
number of attached cells on RGDS (SEQ ID NO:35) peptide
functionalized PEG hydrogel spots. If the normalized cell binding
is greater than 1, high affinity binding is achieved.
[0060] In some embodiments, the integrin-binding peptide is derived
from at least one protein selected from the group consisting of
laminin (Ln), fibronectin (Fn), vitronectin (Vn), collagen,
fibrinogen, von Willebrand factor, thrombospondin, laminin,
entactin, tenascin, osteopontin, bone sialoprotein, and subunits
thereof. In some embodiments, the laminin subunit is laminin
subunit .alpha.1. In some embodiments, the integrin-binding peptide
is an arginine-glycine-aspartic acid (RGD) (SEQ ID NO:36)
peptide.
[0061] In some embodiments, a hydrogel of the present invention
includes agarose, polyethylene glycol, alginate, hyaluronic acid,
polyacryylic acid, polyacrylic amide, polyvinyl alcohol,
polyhydroxyethyl methacrylate, methacrylated dextrans,
poly(N-isopropylacrylamide), or any combination thereof In some
embodiments, the hydrogel includes polyethylene glycol.
[0062] In some embodiments, the hydrogel is functionalized (e.g.,
coupled) with more than one integrin-binding peptide, wherein the
integrin-binding peptide is the same type of integrin-binding
peptide or at least one of the integrin-binding peptides is
different from one other integrin-binding peptide.
Functionalization of hydrogels with peptides includes conjugation
of peptides to hydrogels through covalent and/or non-covalent
bonding. As shown in the examples, covalent conjugation of peptides
onto hydrogels has been accomplished using at least three different
chemistries: 1) co-polymerized methacrylated peptides with
poly(ethylene glycol) diacrylates, 2) EDC-NHS chemistry to
conjugate peptides to alginate hydrogel, and 3) click chemistry to
conjugate peptides to alginate hydrogel. In particular embodiments,
the hydrogel is functionalized to an integrin-binding peptide that
binds to .alpha.v.beta.3 integrin. In some embodiments, the
integrin-binding peptide binds to VLA-6 integrin. In some
embodiments, the integrin-binding peptide binds to both
.alpha.v.beta.3 and VLA-6 integrin.
[0063] In some embodiments, the identified compound promotes
endothelial cell adhesion, endothelial cell spreading, endothelial
cell migration and/or endothelial cell proliferation by modulating
the activity of vascular endothelial growth factor receptor 2
(VEGFR2). In yet some embodiments, the compound modulates the
activity of VEGFR2 by up-regulating VEGFR2 mRNA expression. As used
herein, "modulate," "modulates" or "modulation" refers to
enhancement (e.g., an increase) or inhibition (e.g., a reduction)
in the specified activity.
[0064] Embodiments of the present invention also provide assays for
identifying a compound that promotes endothelial cell adhesion,
endothelial cell spreading, endothelial cell migration and/or
endothelial cell proliferation for manufacture of a diagnostic or
therapeutic agent, the assay comprising, consisting essentially of,
or consisting of screening a compound of interest for its binding
effect on an endothelial cell integrin wherein the compound of
interest is conjugated to a hydrogel and contacted to an
endothelial cell integrin, wherein high affinity binding to the
endothelial cell integrin indicates that the compound of interest
promotes endothelial cell adhesion, endothelial cell spreading,
endothelial cell migration and/or endothelial cell proliferation.
In some embodiments the assay is a microarray. "Microarray" as used
herein refers to a large collection of miniaturized
peptide-functionalized hydrogel spots placed onto two-dimensional
substrates (e.g., glass slides) in a spatially numbered matrix.
[0065] In some embodiments, the endothelial cell integrin is
derived from an endothelial cell that includes endothelial cells
derived from stem cells, progenitor cells, and different organs
(such as the appendix, bladder, brain, ear, esophagus, eye, gall
bladder, heart, kidney, large intestine, liver, lung, mouth,
muscle, nose, ovary, pancreas, parathyroid gland, pineal gland,
pituitary gland, skin, small intestine, spleen, stomach, testes,
thymus, thyroid gland, trachea, uterus, or vermiform appendix) and
from different species including mouse, rat, rabbit, sheep, goat
and human. In some embodiments, the endothelial cell is a human
umbilical vein endothelial cell (HUVEC), a human induced
pluripotent stem cell-derived endothelial cell (hiPSC-EC), a human
endothelial progenitor cell (hEPC), a human microvascular
endothelial cell (hMEC), and combinations thereof The endothelial
cell integrins may be derived from or included in a sample
including the cell types described herein. In particular
embodiments, the endothelial cell integrin is derived from a human
umbilical vein endothelial cell (HUVEC).
[0066] Embodiments of the present invention also provide peptides
that bind to an endothelial cell integrin, the peptide selected
from the group comprising, consisting essentially of, or consisting
of the following sequences: TFALRGDNP (SEQ ID NO:1) (derived from
Laminin subunit .alpha.1); TFALRADNP (SEQ ID NO:2); DVEKRGDREEAHVP
(SEQ ID NO:3) (derived from Laminin subunit .alpha.1); IQRGDIDAMIS
(SEQ ID NO:4) (derived from Laminin subunit .alpha.3);
DAVKQLQAAERGDA (SEQ ID NO:5) (derived from Laminin subunit
.alpha.4); PMQKMRGDVFSP (SEQ ID NO:6) (derived from Laminin subunit
.beta.4); RSDGTG (SEQ ID NO:7) (derived from Laminin subunit
.gamma.2); and EAPRGDVYQG (SEQ ID NO:8) (derived from Laminin
subunit .gamma.3), and fragments and variants thereof that bind to
an endothelial cell integrin when tested under the same test
conditions as the parent peptide including non-naturally occurring
and/or modified peptides.
[0067] Embodiments of the present invention also provide
pharmaceutical compositions comprising, consisting essentially of
or consisting of a peptide described herein, and a pharmaceutically
acceptable carrier, diluent, or excipient. The particular choice of
carrier, diluent, or excipient and formulation will depend upon the
particular route of administration for which the composition is
intended.
[0068] The pharmaceutical compositions of the present invention may
be suitable for parenteral, oral, inhalation spray, topical,
rectal, nasal, buccal, vaginal or implanted reservoir
administration, etc. The term "parenteral" as used herein includes
subcutaneous, intradermal, intravenous, intramuscular,
intra-articular, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional, intraarterial, intramyocardial and
intracranial injection or infusion techniques.
[0069] Where the compounds described herein are to be applied in
the form of solutions or injections, the compounds may be used by
dissolving or suspending in any conventional diluent. The diluents
may include, for example, physiological saline, Ringer's solution,
an aqueous glucose solution, an aqueous dextrose solution, an
alcohol, a fatty acid ester, glycerol, a glycol, an oil derived
from plant or animal sources, a paraffin and the like. These
preparations may be prepared according to any conventional method
known to those skilled in the art.
[0070] Embodiments of the present invention further provide
hydrogel compositions comprising, consisting essentially of, or
consisting of (a) at least one integrin-binding peptide selected
from the group comprising, consisting essentially of, or consisting
of the following sequences: TFALRGDNP (SEQ ID NO:1) (derived from
Laminin subunit .alpha.1); DVEKRGDREEAHVP (SEQ ID NO:3) (derived
from Laminin subunit .alpha.1); IQRGDIDAMIS (SEQ ID NO:4) (derived
from Laminin subunit .alpha.3); DAVKQLQAAERGDA (SEQ ID NO:5)
(derived from Laminin subunit .alpha.4); PMQKMRGDVFSP (SEQ ID NO:6)
(derived from Laminin subunit .beta.4); RSDGTG (SEQ ID NO:7)
(derived from Laminin subunit .gamma.2); and EAPRGDVYQG (SEQ ID
NO:8) (derived from Laminin subunit .gamma.3), and fragments and
variants thereof that bind to an endothelial cell integrin when
tested under the same test conditions as the parent peptide and (b)
a biocompatible polymer, wherein the integrin-binding peptide is
linked to the biocompatible polymer.
[0071] Embodiments of the present invention also provide hydrogel
compositions comprising, consisting essentially of, or consisting
of (a) at least one integrin-binding peptide selected from the
group comprising, consisting essentially of, or consisting of the
following sequences: TFALRGDNP (SEQ ID NO:1) (derived from Laminin
subunit .alpha.1); DVEKRGDREEAHVP (SEQ ID NO:3) (derived from
Laminin subunit .alpha.1); IQRGDIDAMIS (SEQ ID NO:4) (derived from
Laminin subunit .alpha.3); DAVKQLQAAERGDA (SEQ ID NO:5) (derived
from Laminin subunit .alpha.4); PMQKMRGDVFSP (SEQ ID NO:6) (derived
from Laminin subunit .beta.4); RSDGTG (SEQ ID NO:7) (derived from
Laminin subunit .gamma.2); and EAPRGDVYQG (SEQ ID NO:8) (derived
from Laminin subunit .gamma.3), and fragments and variants thereof
that bind to an endothelial cell integrin when tested under the
same test conditions as the parent peptide and/or at least one
integrin-binding peptide selected from the group consisting of
GLOGERGRO (SEQ ID NO:9), GFOGERGVQ (SEQ ID NO:10), DGEA (SEQ ID
NO:11), GFOGER (SEQ ID NO:12), GLKGEN (SEQ ID NO:13), LDV (SEQ ID
NO:14), REDV (SEQ ID NO:15), PEDGIHE (SEQ ID NO:16), PHSRN (SEQ ID
NO:17), ALNGR (SEQ ID NO:18), IAFQRN (SEQ ID NO:19), IKLLI (SEQ ID
NO:20), SIKVAV (SEQ ID NO:21), AGQWHRVSVRWG (SEQ ID NO:22),
TWSQKALHHRVP (SEQ ID NO:23), SIYITRF (SEQ ID NO:24), SYWYRIEASRTG
(SEQ ID NO:25), YIGSR (SEQ ID NO:26), RDIAEIIKDI (SEQ ID NO:27),
VFDNFVLK (SEQ ID NO:28), AEIDGIEL (SEQ ID NO:29), SETQRGDVFVP (SEQ
ID NO:30), PASYRGDSC (SEQ ID NO:31), VTGRGDSPAS (SEQ ID NO:32),
RGDSP (SEQ ID NO:33), RGDS (SEQ ID NO:35), RGD (SEQ ID NO:36),
PQVTRGDVFTMP (SEQ ID NO:37), and fragments and variants thereof;
(b) pro-angiogenic growth factors that include, but are not limited
to, VEGF (vascular endothelial growth factor); Ang2 (angiopoietin
2); PDGF (platelet-derived growth factor); PLGF (placenta growth
factor); SDF-1 (stromal cell-derived factor-1); FGF (fibroblast
growth factor); Ang1 (angiopoietin 1) and fragments and variants
thereof, and the fragments and variants of pro-angiogenic growth
factors, which include pro-angiogenic growth-factor mimetic
peptides that include, but are not limited to, VEGF mimetic peptide
(KLTWQELYQLKYKGI, SEQ ID NO:38); PDGF mimetic peptide
(C*VRKIEIVRKK)2-Ahx-Ahx-Ahx-RKRKLERIAR-NH2) (SEQ ID NO:39); Ang 1
mimetic peptide (PEG-CHHHRHSF, SEQ ID NO:40) tetramer); and/or a
pro-angiogenic growth factor binding compound that include, but are
not limited to, heparin, heparin-binding peptide; and (c) a
biocompatible polymer, wherein the integrin-binding peptide and/or
the pro-angiogenic growth factors and fragments and variants
thereof are linked to the biocompatible polymer.
[0072] In some embodiments, the hydrogel compositions include
covalent or non-covalent linkages. In some embodiments, the
biocompatible polymer of the hydrogel is functionalized with VEGF
mimetic peptide (KLTWQELYQLKYKGI, SEQ ID NO:38) and an
integrin-binding peptide that binds at least one type of
endothelial cell integrin. In some embodiments, the VEGF mimetic
peptide is attached to the biocompatible polymer through a matrix
metalloproteinase (MMP) degradable peptide linkage (for example,
GPQG.dwnarw.IAGKLTWQELYQLKYKGI (SEQ ID NO:41), PES.dwnarw.LRAG (SEQ
ID NO:42), GPQG.dwnarw.IWGQ (SEQ ID NO:43), VPLS.dwnarw.LYSG (SEQ
ID NO:44)). In some embodiments, the biocompatible polymer of the
hydrogel composition is functionalized with a VEGF mimetic peptide
and an integrin binding peptide that binds to an
.alpha..sub.v.beta.3 integrin. In some embodiments, the
biocompatible polymer of the hydrogel composition is functionalized
with a VEGF mimetic peptide and an integrin binding peptide that
binds to a VLA-6 integrin. In some embodiments, the biocompatible
polymer of the hydrogel composition is functionalized with a VEGF
mimetic peptide and an integrin binding peptide that binds to an
.alpha..sub.v.beta.3 integrin and a VLA-6 integrin. In some
embodiments, the biocompatible polymer of the hydrogel composition
is functionalized with an integrin binding peptide that binds to an
.alpha..sub.v.beta.3 integrin and a VLA-6 integrin.
[0073] In some embodiments, the biocompatible polymer comprises,
consists essentially of, consists of agarose, polyethylene glycol,
alginate, hyaluronic acid, polyacrylic acid, polyacrylic amide,
polyvinyl alcohol, polyhydroxyethyl methacrylate, methacrylated
dextrans, poly(N-isopropylacrylamide), or any combination thereof
In particular embodiments, the biocompatible polymer is 5% oxidized
alginate.
[0074] In particular embodiments, the hydrogel composition is an
injectable and/or extrudable composition. In some embodiments, the
injectable hydrogel may be 1% w/w alginates with 5% oxidation. In
some embodiments, the extrudable hydrogel is applicable for
bioprinting. In some embodiments, the extrudable hydrogel for
bioprinting may be 1% w/w alginates with 5% oxidation.
[0075] Embodiments of the present invention further provide methods
of promoting angiogenesis in a subject in need thereof comprising,
consisting essentially of or consisting of administering to the
subject a peptide described herein, a pharmaceutical composition
described herein, a hydrogel composition described herein and/or a
biomaterial product described herein, in an amount effective to
promote angiogenesis.
[0076] Embodiments of the present invention provide methods of
promoting endothelial cell adhesion, endothelial cell spreading,
endothelial cell migration and/or endothelial cell proliferation
comprising, consisting essentially of or consisting of
administering a peptide described herein, a pharmaceutical
composition described herein, a hydrogel composition described
herein and/or a biomaterial product described herein in an amount
effective to promote endothelial cell adhesion, endothelial cell
spreading, endothelial cell migration and/or endothelial cell
proliferation.
[0077] Embodiments of the present invention provide methods of
treating or preventing ischemic injury in a subject in need thereof
comprising, consisting essentially of or consisting of
administering to the subject a peptide described herein, a
pharmaceutical composition described herein, a hydrogel composition
and/or a biomaterial product described herein, in an amount
effective to treat or prevent ischemic injury. The ischemic injury
or ischemic disease refers to diseases or disorders resulting from
an insufficient supply of blood to an organ, often due to an
occluded blood vessel. Examples of such include, but are not
limited to, coronary artery disease, peripheral artery disease,
ischemic wounds and diabetic ulcers.
[0078] Embodiments of the present invention also provide methods of
promoting tissue regeneration in a subject in need thereof
comprising, consisting essentially of or consisting of
administering to the subject a peptide described herein, a
pharmaceutical composition described herein and/or a hydrogel
composition described herein, in an amount effective to promote
tissue regeneration. The tissue regeneration refers to the process
of remodeling, renewal, growth, maintenance and/or improved
function of cells, and in particular, cells collectively forming a
tissue. The tissue may be tissue associated with the nervous
system, endocrine system, hematopoietic system, gastrointestinal
tract, renal system, cardiac system, vascular system, reproductive
system, musculoskeletal system or combinations thereof The tissue
may be tissue associate with an organ such as the appendix,
bladder, brain, ear, esophagus, eye, gall bladder, heart, kidney,
large intestine, liver, lung, mouth, muscle, nose, ovary, pancreas,
parathyroid gland, pineal gland, pituitary gland, skin, small
intestine, spleen, stomach, testes, thymus, thyroid gland, trachea,
uterus, vermiform appendix or combinations thereof. Tissue
regeneration also includes wound healing.
[0079] The subjects to be treated according to the present
invention include any subject in whom promotion of angiogenesis,
prevention and/or treatment of ischemic injury and/or tissue
regeneration is desired or needed, as well as any subject prone to
such. In some embodiments, the subject is a human; however, a
subject of this invention can include an animal subject,
particularly mammalian subjects such as canines, felines, bovines,
caprines, equines, ovines, porcines, rodents (e.g. rats and mice),
lagomorphs, primates (including non-human primates), etc.,
including domesticated animals, companion animals and wild animals
for veterinary medicine or treatment or pharmaceutical drug
development or biomedical research purposes.
[0080] The subjects relevant to this invention may be male or
female and may be any species and of any race or ethnicity,
including, but not limited to, Caucasian, African-American,
African, Asian, Hispanic, Indian, etc., and combined backgrounds.
The subjects may be of any age, including newborn, neonate, infant,
child, adolescent, adult, and geriatric.
[0081] Embodiments of the present invention also provide kits
including the elements necessary to carry out the processes
described above. Such a kit may comprise a carrier being
compartmentalized to receive in close confinement therein one or
more containers, such as tubes or vials. One or more of the
containers may contain a peptide or composition described herein.
One or more containers may contain one or more enzymes or reagents
to be utilized in desired reactions. These enzymes may be present
by themselves or in admixtures, in lyophilized form or in
appropriate buffers. The kit may contain all of the additional
elements necessary to carry out techniques of the invention, such
as buffers, control plasmid, oligonucleotides, extraction reagents,
fixation agents, permeability agents, enzymes, pipettes, plates,
nucleic acids, gel materials, transfer materials, autoradiography
supplies, instructions and the like. In particular, embodiments of
the present invention provide kits comprising, consisting
essentially of or consisting of a peptide described herein, a
pharmaceutical composition described herein, a hydrogel composition
described herein and/or a biomaterial product described herein, and
a container suitable for delivery of the peptide, pharmaceutical
composition or hydrogel composition into an administration device,
with optional instructions for the use thereof. In some
embodiments, the administration device is a parenteral
administration device. In some embodiments, the administration
device is an intramyocardial device. In some embodiments, the kit
is not limited by size and includes a biomaterial product and/or a
bioprinter.
[0082] The general procedure for implementing the methods and
assays of the present invention can be readily understood and
appreciated by one skilled in the art. Some aspects of the present
invention are described in more detail in the following
non-limiting Examples. These are not intended to restrict the
present invention, and may be modified within the range not
deviating from the scope of this invention.
EXAMPLES
Example 1: Development of Peptide-Functionalized Synthetic Hydrogel
Microarrays for Stem Cell and Tissue Engineering Applications
[0083] This experimental section describes the development of a
platform technology based on light-assisted co-polymerization of
poly(ethylene glycol) diacrylates (PEGDA) and
methacrylated-peptides to fabricate peptide-functionalized hydrogel
microarrays. To this end, the high efficiency of solid-phase
peptide synthesis and isocyanation chemistry was leveraged to
develop a robust synthetic route for preparing
methacrylated-peptides. Due to their high solubility in DMF and
high miscibility with low molecular PEGDA, methacrylated-peptides
can be effectively incorporated into PEG hydrogels in a ratiometric
and homogenous manner In addition, several parameters were
optimized, including the length of the linker between methacrylate
functional groups and cell-binding peptide moieties to ensure high
accessibility of the peptide functional groups to the cell-surface
receptors. To apply the peptide-functionalized hydrogel technology,
we constructed a library composed of 12 different RGD (SEQ ID
NO:36) peptides to develop synthetic culture substrates for human
induced pluripotent stein cell-derived cardiomyocytes (hiPSC-CMs),
a cell type known for poor adhesion to synthetic substrates. While
6 of the 12 peptides were found through reported literature,
bioinformatic screening of ECM proteins led to the identification
of 6 unexplored RGD (SEQ ID NO:36) peptides. Notably, 2 out of 6
unexplored RGD (SEQ ID NO:36) peptides showed substantial affinity
to hiPSC-CMs. One of them, PMQKMRGDVFSP (SEQ ID NO:6) from laminin
.beta.4 subunit, was found to have the highest affinity to
hiPSC-CMs. With the support of bioinformatic screening,
peptide-functionalized hydrogel microarrays are shown here to be a
promising strategy to rapidly identify novel biological ligands for
the development of functional biomaterials for stein cell and
tissue engineering applications.
[0084] Materials and Instruments. All chemicals used for this study
were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise
stated. Microarray spotting pins (946MP9B) were purchased from
Arrayit Corporation (Sunnyvale, Calif.). A custom designed
microarrayer was assembled and produced by BioDot (Irvine, Calif.).
The liquid chromatography-mass spectrometer (LC-MS) system used is
Thermo Fisher LCQ Fleet.TM. Ion Trap Mass Spectrometer.
[0085] Bioinformatics-assisted ECM protein screening.
Bioinformatics-assisted ECM protein screening for highly conserved
sequences was performed using the following database: UniProt
database, which is supported by European Bioinformatics Institute
(EMBI-EBI), the SIB Swiss Institute of Bioinformatics, and the
Protein Information Resource (PIR). The specific sequence of each
ECM protein//ECM protein subunit was collected from mammalian
species, including human, mouse, rat, chimpanzee, horse, sheep,
rabbit, bovine, guinea pig, cat and dog. The protein alignment was
achieved by using the tool of Clustal Omega from EMBL-EBI. The
algorithm is described by J. Soding.
Monomer Preparation and Array Fabrication
[0086] Synthesis and characterization of methacrylated peptides.
Peptides used in this work were synthesized by solid phase peptide
synthesis (SPPS). The SPPS was conducted using the standard
procedure described in Novabiochem peptide synthesis manual. To
prepare methyacrylated peptides, 2-isocyanatoethyl methacrylate (3
equivalent (eq) dissolved in DMF) was used to react with the
terminal amine group of the peptide chain (1 eq) before they were
cleaved from the resin. This solid-phase isocyanation chemistry was
first reported by Lee Ayres et al. All the methacrylated peptides
prepared in this study were purified by using a Combiflash.RTM.
purification system (RediSep Rf) in Reversed Phase format using C18
Columns (Teledyne Isco, Lincoln, Nebr.) running a solvent gradient
from 100% H.sub.2O to 100% acetonitrile in 15.about.20 minutes. The
peptides were eluted from the column at approximately 70%
acetonitrile/30% H.sub.2O. The purified peptides were subsequently
characterized by LC-MS.
[0087] Microarray Fabrication. Methacrylated peptides were
dissolved in DMF at pre-designated ratios and mixed with PEGDA
(containing 1% DMPA as initiator) (DMF solution of methacrylated
peptide: PEGDA=1:1 (v/v)) and then transferred into a 384 well
plate for microarray fabrication. The microarrays were printed in a
humid Ar-atmosphere on epoxy monolayer-coated glass slides
(Xenopore XENOSLIDE E, Hawthorne, N.J.) that were first dip-coated
in 4 v/v% poly(hydroxyethyl methacrylate) (i.e., poly(HEMA)) using
a customized microarrayer (Biodot). Spots were polymerized via 10 s
exposure to long wave UV using a XX-15L UV bench lamp (365 nm) (UVP
LLC, Upland, Calif.), dried at <50 mtorr for at least 7 days.
Before use, the chips were sterilized by UV for 30 min for each
side, and then washed with PBS twice for 15 min to remove residual
monomer or solvent. Additional information to prepare the
microarrays for different applications is provided below.
[0088] PEGDA selection. Three commercially available PEGDA (M.
W.=250, 575, 700) were selected and mixed at the designated ratios
to produce the hydrogel microarrays (FIG. 3). To determine their
abilities to inhibit unspecific cell adhesion, human
adipose-derived stem cells (hADSCs) were seeded on the array and
cultured for 12 hours. They were then fixed and stained with DAPI
(1:1000 in DPBS) for cell number counting and phalloidin (1:200 in
DPBS) for F-actin to estimate cell spreading.
[0089] The effects of glycine linker length. The methacrylated
peptides used in these experiments are shown in FIG. 5A. PEGDA and
methacrylated peptides were mixed at varied peptide concentrations
(i.e., 0.5, 1, 3, 6, 9, 12 and 15 mM) to prepare microarrayed
hydrogels with different peptide concentrations. hADSCs were seeded
onto the array and cultured for 12 hours. They were then fixed and
stained with DAPI (1:1000 in DPBS) for cell number counting and
phalloidin (1:200 in DPBS) for F-actin to estimate cell
spreading.
[0090] Screening RGD peptides for hiPSC-CM adhesion and
quantification of sarcomere formation. The methacrylate peptides
used in this experiment are shown in FIG. 6B. PEGDA and
methacrylated peptides were mixed at one fixed peptide
concentration (15 mM) to prepare microarrayed hydrogels with a
constant peptide concentration. hiPSC-CMs (human induced
pluripotent stem cell-derived cardiomyocytes from Cellular Dynamics
International, Madison, WI, USA) were seeded onto the microarray
and cultured for 3 days to facilitate the formation of sarcomere
structures. hiPSC-CMs were stained with DAPI to approximate cell
number and phalloidin for F-actin to estimate cell spreading.
Sarcomere structure was examined by using immunofluorecence
microscopy.
[0091] Briefly, hiPSC-CMs on the microarray were fixed with 4% PFA
solution and blocked by 10% goat serum. After incubated with mouse
anti-alpha sarcomeric actinin antibody (Abeam, Cambridge, UK) and
rabbit anti-troponin I antibody (Santa Cruz, Dallas, Tex.) at a
dilution ratio of 1:200 in PBS (with 0.1% Triton-100X) at room
temperature for 1 hr, the microarrays were stained with the
secondary antibodies (Alexa-488 goat anti-mouse IgG and Alexa-647
goat anti-rabbit IgG) at a dilution ratio of 1:200 in PBS (with
0.1% Triton-100.times.). Subsequently, the microarrays are stained
with DAPI (1:1000 in DPBS) for nuclear counting. The fluorescently
stained microarrays were imaged with a TCS SP5 AOBS laser scanning
confocal microscope (Leica Microsystems, Inc., Exton, Pa.).
Z-stacked Images collected from the microarray were analyzed by
using the ImageJ (National Institutes of Health) for
semi-quantitative analysis of the expression level of alpha
sarcomeric actinin of hiPSC-CMs on the microarrays. The sarcomeric
actinin expression level of hiPSC-CMs on each hydrogel spot was
determined by the total fluorescence intensities of sarcomeric
actinin staining divided by the total cell number on the hydrogel
spot, which was then normalized to the blank PEG-700 hydrogel
spots. The fluorescence intensities of sarcomeric actinin staining
on each hydrogel spot were obtained by taking the sum of the green
(sarcomeric actinin staining) pixels (i.e., fluorescence area
coverage) through the total thickness of the Z-stacked images.
Cell Culture
[0092] hADSC culture. hADSCs (Lonza, Basel, Switzerland) were used
to study cell attachment for the hydrogel array. The cells were
cultured in low glucose Dulbecco's modified Eagle's medium with 10%
fetal bovine serum and 1% penicillin-streptomycin, 1% glutamine and
1% antimycin (Gibco Life Technologies, Grand Island, N.Y.). At
>80% confluency, cells were detached using trypLE Express (Gibco
Life Technologies) and passaged. All experiments were conducted
using passage 5 (P5) hADSCs. The cells were seeded along with
culture media onto the hydrogel microarrays. After 12 hours
culture, the cells were fixed and stained to examine the cell
attachments on each spot. hADSCs were stained with DAPI (1:1000 in
DPBS) in order to approximate cell number. Cell spreading was
visualized using phalloidin (1:200 in DPBS) staining.
[0093] hiPSC-CMs culture. hiPSC-derived cardiomyocytes (iCell
Cardiomyocytes, Cellular Dynamics International, Madison, Wis.,
USA) were cultured according to the manufacturer's protocol.
Briefly, hiPSC-derived cardiomyocytes were plated on 0.1% gelatin
coated 6-well plates in iCell Cardiomyocytes Plating Medium
(Cellular Dynamics International) at a density of about
3.times.10.sup.5 to 4.0.times.10.sup.5 cells/well and incubated at
37.degree. C. in 5% CO.sub.2 for 4 days. Two days after plating,
the plating medium was removed and replaced with 4 mL of iCell
Cardiomyocytes Maintenance Medium (Cellular Dynamics
International). After 4 days of monolayer pre-culture, cells were
detached using trypLE Express (Gibco Life Technologies, Grand
Island, NY) and seeded along with culture media on the hydrogel
microarrays. Cells were culture for 3 days to allow the hiPSC-CMs
to develop sarcomere structures. hiPSC-CMs were stained with DAPI
(1:1000 in DPBS) to approximate cell attachment number and
phalloidin (1:200 in DPBS) for F-actin to estimate cell spreading.
Sarcomere structures were visualized using sarcomere actinin and
troponin-I staining as described above.
Statistical Analysis
[0094] The results were shown in the mean+standard derivation (SD)
and analyzed using Sigmaplot and Excel statistical software.
[0095] FIG. 1 shows a general strategy for the fabrication of
peptide-functionalized PEG microarrays for stem cell and tissue
engineering applications. To fabricate the microarrays, nanoliters
of PEGDA and methacrylated-peptides have been robotically deposited
onto poly(HEMA) coated glass slides and photo-polymerized in situ.
This approach is chosen due to the high polymerization rate of
photopolymerization and the high solubility of
methacrylated-peptides in DMF. In addition, peptide-functionalized
PEG hydrogels have been extensively employed in stem cell and
tissue engineering applications. This makes it possible to quickly
translate the screening results into design principles for the
improved fabrication of 2D culture substrates and 3D scaffolds.
[0096] FIG. 2 demonstrates a general procedure to prepare
methacrylated-peptides. After solid-phase peptide synthesis,
2-isocyanatoethyl methacrylate was used to react with the terminal
amine of the peptides in order to conjugate methacrylate groups. As
the conjugation reaction step was right after peptide synthesis on
the solid-phase, this route allows for the preparation of
methacrylated-peptides from virtually any peptides. Further, this
solid-phase conjugation reaction has been proven very effective and
efficient.
[0097] To provide a low cell adhesion background for peptide
screening, PEGDA of different molecular weights were screened to
generate the non-fouling PEG hydrogel substrates. To this end,
three commercially available low molecular weight PEGDA: PEGDA-250
(molecular weight, M. W.=250), PEGDA-575 (M. W.=575), PEGDA-700 (M.
W.=700), have been used to fabricate an 8.times.8 microarray to
screen for formulations that can resist non-specific cell adhesion.
After seeding human adipose-derived stein cells (hADSCs) onto the
hydrogel microarray, every spot composed of PEGDA-250 showed
extensive cell adhesion. While the spots made by PEGDA-575 were
right at the threshold to resist cell attachment (only 1 or 2
cells/spot), no cell attachment was recorded for those made of
PEGDA-700 (FIG. 3). The differences in cell adhesion can be
attributed to the ethylene glycol chain length of the PEGDA, as the
longer ethyl glycol chain provides significantly enhanced chain
flexibility to resist protein adsorption and cell adhesion. Since
the spots prepared from the PEGDA-700 showed high resistance to
non-specific cell adhesion, PEGDA-700 was selected to co-polymerize
with methacrylated-peptides to prepare peptide-functionalized PEG
hydrogel microarrays.
[0098] To validate the functions of the peptide moieties on the
hydrogels, we synthesized a methacrylated-peptide containing a cell
adhesive moiety (G.sub.4RGDSP) (SEQ ID NO:46) and its scrambled
sequence (G.sub.4RDGSP) (SEQ ID NO:47). The methacrylated-peptides
were then co-polymerized with PEGDA-700 to prepare PEG hydrogel
spots functionalized with cell adhesive RGD (SEQ ID NO:36) peptides
or the scrambled RDG (SEQ ID NO:36) peptide. As shown in the FIG.
4, PEG hydrogels modified with a high concentration of (15 mM)
RGD-peptide were able to effectively promote adhesion of hADSCs
(FIG. 4, right), while no cell adhesion was found on the scrambled
peptide (RDG) (SEQ ID NO:36) functionalized PEG hydrogels (FIG. 4,
middle). These results indicate the function of peptides is
retained during the microarray fabrication process.
[0099] The length of the linker between peptide moiety and hydrogel
surface has been shown to significantly influence peptide
activities and further affect cell behavior. While there is one
ethylene glycol group between methacrylate and the cell-binding
peptide moiety, it may not be sufficient to ensure the exposure of
the peptides on the hydrogel surface for cell recognition.
Enlightened by the idea of using a 4-glycine linker to extend the
RGD (SEQ ID NO:36) peptide from hydrogel surface, we designed and
synthesized methacrylated-RGDSP-peptides with no glycine linker
(MethRGDSP) (SEQ ID NO:33), 2 glycine linker (MethG.sub.2RGDSP)
(SEQ ID NO:45), 4 glycine linker (MethG.sub.4RGDSP) (SEQ ID NO:46)
and 6 glycine linker (MethG.sub.6RGDSP) (SEQ ID NO:48), as listed
in FIG. 5A. The microarrays composed with these peptides have been
fabricated and hADSCs were seeded onto the array. Sigmoidal
relationships between the number of attached cells and peptide
concentration were found for all of these RGD (SEQ ID NO:36)
peptides (FIG. 5B). Given the sigmoidal relationship, small changes
in peptide concentration can result in large shifts in cell
attachment numbers at lower peptide concentrations. To reduce
variation in the high throughput analysis, the saturated (maximum)
number of attached cells has been used to examine the effects of
changing glycine linker length (FIG. 5C). MethG.sub.4RGDSP (SEQ ID
NO:46) and MethG.sub.6RGDSP (SEQ ID NO:48) showed similar
saturation numbers for attached cells (FIGS. 5B and 5C). They are
about one-fold higher than the saturation number of attached cells
of MethRGDSP (SEQ ID NO:33) and MethG.sub.2RGDSP (SEQ ID NO:45).
These results indicate that longer linkers improved the exposure of
the peptides on the hydrogel surface. Also, the similar cell
attachment between MethG.sub.4RGDSP (SEQ ID NO:46) and
MethG.sub.6RGDSP (SEQ ID NO:48) suggested that 4 glycine provides
sufficient length as a linker between the methacrylate and peptide
components for our system. Therefore, further experiments were
performed using peptides modified with the four-glycine
[0100] The newly developed peptide-functionalized hydrogel
microarray will allow us to rapidly identify novel peptides to
functionalize biomaterials for numerous stein cell and tissue
engineering applications. To this end, we used this technology to
screen adhesion peptides for human induced pluripotent stem
cell-derived cardiomyocytes (hiPSC-CMs). While hiPSC-CMs hold
remarkable promise as a cell source to treat cardiovascular
diseases, they have been reported as having poor adhesion on
synthetic substrates. hiPSC-CMs express integrin .alpha..sub.3,
.alpha..sub.5, .alpha..sub.6, .alpha..sub.7, .alpha..sub.v,
.beta..sub.1 and .beta..sub.5. Given the high affinity of RGD (SEQ
ID NO:36) peptides to integrin .alpha..sub.v.beta..sub.5, we
reasoned that RGD (SEQ ID NO:36) peptide functionalization can
improve the binding affinity of PEG hydrogel substrates to
hiPSC-CMs. To rapidly identify RGD (SEQ ID NO:36) peptide
candidates with the potential of high affinity to hiPSC-CMs, we
utilized an online bioinformatics tool (UniProtKB database) to
screen and align the whole sequence of fibronectin, vitronectin and
laminin through multiple species. We selected 12 different RGD (SEQ
ID NO:36) peptides (FIG. 6B) to construct a PEG hydrogel microarray
functionalized with these peptides. The candidates include: 1) 6
RGD (SEQ ID NO:36) peptides that have been reported to improve cell
adhesion, such as those selected from laminin-.alpha.1,
laminin-.alpha.5, fibronectin and vitronectin, and 2) 6 RGD (SEQ ID
NO:36) peptides that have not been studied, but have been shown to
be highly conserved sequences among the different mammalian
species. The highly conserved RGD (SEQ ID NO:36) sequences among
different mammalian species indicate their importance for certain
fundamental functions (e.g. cell adhesion/integrin binding). One
RGD (SEQ ID NO:36) peptide (PQVTRGDVFTMP, SEQ ID NO:37) from
vitronectin, has been included in the microarrays as a control as
they were shown to support adhesion of hiPSC-CMs.
[0101] hiPSC-CMs were seeded onto the RGD (SEQ ID NO:36) peptides
functionalized PEG hydrogel microarrays to examine the abilities of
different RGD (SEQ ID NO:36) peptides for the enhanced cell
adhesion. The cell adhesion response varied among the hydrogels:
.about.50% of RGD (SEQ ID NO:36) peptides could not support
adhesion of hiPSC-CMs (FIGS. 6A-a, 6A-b), some RGD (SEQ ID NO:36)
peptides (e.g., PQVTRGDVFTMP, SEQ ID NO:37 and SETQRGDVFVP, SEQ ID
NO:30) support moderate cell adhesion (FIG. 6A-c), and PMQKMRGDVFSP
(SEQ ID NO:6) (laminin .beta.4 chain) showed the greatest ability
to promote hiPSC-CM adhesion and sarcomere formation, a critical
step for cardiomyocytes maturation (FIGS. 6A-d). The screening
results have been validated with 18 replicates. It is worthwhile to
note 2 out of 6 unexplored RGD (SEQ ID NO:36) peptides
(PMQKMRGDVFSP, SEQ ID NO:6, DAVKQLQAAERGDA, SEQ ID NO:5) have shown
substantial activities to support hiPSC-CM adhesion. This supports
our hypothesis that highly conserved RGD (SEQ ID NO:36) peptide
sequences among different species indicate their importance in
functions (e.g., cell adhesion/integrin binding). This highlights
the power of the combination of the microarray technology we
developed here and the bioinformatics tool we utilized to rapidly
identify novel biological ligands for the development of functional
biomaterials for stem cell and tissue engineering applications. To
the best of our knowledge, the highest cell adhesive peptide
identified in this study, PMQKMRGDVFSP (SEQ ID NO:6) from laminin
.beta.4 subunit, has not been recognized as being a cell-adhesive
peptide. Our current research includes the utilization of this
novel RGD (SEQ ID NO:36) peptide from laminin .beta.4 subunit to
prepare 3D scaffolds for cardiac tissue engineering applications.
Notably, the RGD (SEQ ID NO:36) peptide from vitronectin
(PQVTRGDVFTMP, SEQ ID NO:37) showed moderate binding affinity for
hiPSC-CMs (FIG. 6C), which could explain a previous report that
hiPSC-CMs detach from synthetic substrates during the cardiac
differentiation process.
[0102] We also examined the effects of the peptide sequences on
sarcomere formation of hiPSC-CMs using sarcomeric actinin staining
(FIGS. 6A, 6E), as sarcomeres are structural and functional units
for cardiomyocytes contractions. The trend of alpha sarcomeric
actinin expression per cell was found similar to that of cell
adhesion (i.e., the affinities of peptide ligands). This can be
attributed to that the high affinity peptide ligands can provide
sufficient support for cardiomyocyte contractions and facilitate
sarcomere formation. Consistent with the cell adhesion results, the
RGD (SEQ ID NO:36) peptide from laminin .beta.4 subunit supported
hiPSC-CMs with the highest sarcomeric actinin expression. With the
assistance from confocal microscope, the detailed sarcomere
structures were revealed. This data suggests the RGD (SEQ ID NO:36)
peptide from laminin .beta.4 subunit can effectively support
hiPSC-CM attachment, spreading and contractile structure
development. These results are in agreement with a recent report
that showed integrin binding is essential for hiPSC-CM
maturation.
[0103] Recent advances in stem cell and tissue engineering
strategies highlight an unmet need to rapidly identify suitable
biomaterials for cell-specific applications. Here we developed a
peptide-functionalized PEG hydrogel microarray based on
light-assisted, co-polymerizations between poly(ethylene glycol)
diacrylates (PEGDA) and methacrylated-peptides. By leveraging
solid-phase peptide/organic synthesis, methacrylate-peptides can be
synthesized from virtually any peptide sequences. When combined
with a cell-adhesion resistant hydrogel derived from PEGDA-700, we
have developed a framework for fabricating peptide-functionalized
hydrogel microarrays. In addition, we identified a linker composed
of 4 glycines that can ensure sufficient exposure for the peptide
moieties on the hydrogel surface. Lastly, we combined
peptide-functionalized microarray technology with bioinformatics to
identify novel biological ligands with high affinity to hiPSC-CMs,
a cell type known for poor adhesion to synthetic substrates. Among
6 unexplored RGD (SEQ ID NO:36) peptides, 2 peptides showed
substantial affinity to hiPSC-CMs. PMQKMRGDVFSP (SEQ ID NO:6) from
laminin .beta.4 subunit, a peptide that had not previously been
recognized as being cell adhesive, was found to have the highest
affinity to hiPSC-CMs and the most developed sarcomere
structures.
[0104] The technology we developed here can allow for the rapid
identification of biological ligands for stem cell and tissue
engineering application. As peptide-functionalized PEG hydrogels
are widely used in stem cell and tissue engineering applications,
the screening results could be quickly translated to 2D substrates
and 3D scaffold fabrication. Although PEGDA-700 was used to
fabricate hydrogel to resist non-specific cell adhesion in this
study, clearly, PEGDA-700 can be replaced with another non-fouling
hydrogel-precursors (e.g., PEGDA 3400, methacrylated hyaluronic
acids) to vary the bulk properties (e.g., stiffness) of the
hydrogel substrates. Our next step is to fabricate hydrogel
microarrays that can cover the entire physiological/pathological
range of stiffnesses. The ability to rapidly screen the combined
effects of biological ligands and mechanical properties on (stem)
cells can dramatically accelerate the advancement of the
fundamental understanding of the interaction (stem) cell activity
and biomaterials. This would further contribute to the development
of biomaterial genomics through Big Data analytics.
[0105] Finally, the peptide-functionalized hydrogel microarrays
developed here can find many applications in biomedical-related
fields beyond stem cell and tissue engineering. We can envision
that peptide-functionalized hydrogel microarrays will be used to
develop anti-infectious substrates, given the wide application of
the peptides and hydrogels for designing anti-infectious materials.
Thus, the present invention provides products and methods of
treating infections caused by bacteria, fungi, viruses, and
parasites.
[0106] Bacterial infections that can be affected using the present
invention can be caused by bacteria such as gram-negative bacteria.
Examples of gram-negative bacteria include, but are not limited to,
bacteria of the genera Salmonella, Escherichia, Kiebsiella,
Haemophilus, Pseudomonas, Proteus, Neisseria, Vibro, Helicobacter,
Brucella, Bordetella, Legionella, Campylobacter, Francisella,
Pasteurella, Yersinia, Bartonella, Bacteroides, Streptobacillus,
Spirillum and Shigella. Furthermore, bacterial infections that can
be treated using the sanitizing compositions of the present
invention can be caused by gram-negative bacteria including, but
not limited to, Escherichia coli, Pseudomonas aeruginosa, Neisseria
meningitides, Neisseria gonorrhoeae, Salmonella typhimurium,
Salmonella entertidis, Klebsiella pneumoniae, Haemophilus
influenzae, Haemophilia ducreyi, Proteus mirabilis, Vibro cholera,
Helicobacter pylori, Brucella abortis, Brucella melitensis,
Brucella suis, Bordetella pertussis, Bordetella parapertussis,
Legionella pneumophila, Campylobacter fetus, Campylobacter jejuni,
Francisella tularensis, Pasteurella multocida, Yersinia pestis,
Bartonella bacillitbrmis, Bacteroides fragilis, Bartonella
henselae, Streptobacillus moniliformis, Spirillum minus and
Shigella dysenteriae.
[0107] Bacterial infections that can be affected using the present
invention can also be caused by bacteria such as gram-positive
bacteria. Examples of gram-positive bacteria include, but are not
limited to, bacteria of the genera Listeria, Staphylococcus,
Streptococcus, Bacillus, Corynebacterium, Peptostreptococcus, and
Clostridium. Furthermore, bacterial infections that can be treated
using the sanitizing compositions of the present invention can be
caused by gram-positive bacteria including, but not limited to,
Listeria monocytogenes, Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Bacillus cereus. Bacillus
anthracia, Clostridium botulinum, Clostridium perfringens,
Clostridirrna difficile, Clostridium tetani, Corynebacterium
diphtheriae and Peptostreptococcus anaerobius. In some embodiments,
the gram-positive bacterium is methicillin-resistant Staphylococcus
aureus.
[0108] Additional bacterial infections that can be affected using
the present invention can be caused by bacteria in the genera
including, but not limited to, Actinomyces, Propionibacterium,
Nocardia and Streptomyces. Furthermore, bacterial infections that
can be treated using the sanitizing compositions of the present
invention can be caused by bacteria including, but not limited to,
Actinomyces israeli, Actinomyces gerencseriae, Actinomyces
viscosus, Actinomyces naeslundii, Propionibacterium propionicus,
Nocardia asteroides, Nocardia brasiliensis, Nocardia
otitidiscaviamm and Streptomyces somaliensis.
[0109] The effect on bacterial infections described herein can be
bacteriocidal or bacteriostatic.
[0110] Mycobacterial infections that can be affected by the present
invention can be caused by mycobacteria belonging to the
mycobacteria families including, but not limited to,
Mycobacteriaceae. Additionally, mycobacterial infections that can
be treated by the sanitizing compositions of the present invention
can be caused by mycobacteria including, but not limited to,
Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium
avium-intracellulare, Mycobacterium kansasii, and Mycobacterium
ulcerans.
[0111] Fungal infections that can be affected by the present
invention can be caused by fungi belonging to the genera including,
but not limited to, Aspergillus, Candida, Cryptococcus,
Coccidioides, Tinea, Sporothrix, Blastomyces, Histoplasma, and
Pneumocystis. Additionally, fungal infections that can be treated
using the sanitizing compositions of the present invention can be
caused by fungi including, but not limited to, Aspergillus
fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus
terreus, Aspergillus nidulans, Candida Coccidioides immitis,
Cryptococcus neoformans, Tinea unguium, Tinea corporis, Tinea
cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma
capsulatum, and Histoplasma duboisii.
[0112] Viral infections that can be treated using the sanitizing
compositions of the present invention can be caused by viruses
belonging to the viral families including, but not limited to,
Flaviviridae, Arenaviradae, Bitnyaviridae, Filoviridae, Poxviridae,
Togaviridae, Paramyxoviridae, Herpesviridae, Picornaviridae,
Caliciviridae, Reoviridae, Rhabdoviridae, Papovaviridae,
Parvoviridae, Adenoviridae, Hepadnaviridae, Coronaviridae,
Retroviridae, and Orthomyxoviridae. Furthermore, viral infections
that can be treated using the sanitizing compositions of the
present invention can be caused by the viruses including, but not
limited to, Yellow fever virus, St. Louis encephalitis virus,
Dengue virus, Hepatitis G virus, Hepatitis C virus, Bovine diarrhea
virus, West Nile virus, Japanese B encephalitis virus, Murray
Valley encephalitis virus, Central European tick-borne encephalitis
virus, Far eastern tick-born encephalitis virus, Kyasanur forest
virus, Louping ill virus, Powassan virus, Omsk hemorrhagic fever
virus, Kumilinge virus, Absetarov anzalova hypr virus, Ilheus
virus, Rocio encephalitis virus, Langat virus, Lymphocytic
choriomeningitis virus, Junin virus, Bolivian hemorrhagic fever
virus, Lassa fever virus, California encephalitis virus, Hantaan
virus, Nairobi sheep disease virus, Bunyamwera virus, Sandfly fever
virus, Rift valley fever virus, Crimean-Congo hemorrhagic fever
virus, Marburg virus, Ebola virus, Variola virus, Monkeypox virus,
Vaccinia virus, Cowpox virus, Orf virus, Pseudocowpox virus,
Molluscum contagiosum virus, Yaba monkey tumor virus, Tanapox
virus, Raccoonpox virus, Camelpox virus, Mousepox virus, Tanterapox
virus, Volepox virus, Buffalopox virus, Rabbitpox virus, Uasin
gishu disease vines, Sealpox virus, Bovine papular stomatitis
virus, Camel contagious ecthyma virus, Chamios contagious ecthyma
virus, Red squirrel parapox virus, Juncopox virus, Pigeonpox virus,
Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox
virus, Peacockpox virus, Penguinpox virus, Mynahpox virus, Sheeppox
virus, Goatpox virus, Lumpy skin disease virus, Myxoma virus, Hare
fibroma virus, Fibroma virus, Squirrel fibroma virus, Malignant
rabbit fibroma virus, Swinepox virus, Yaba-like disease virus,
Albatrosspox virus, Cotia virus, Embu virus, Marmosetpox virus,
Marsupialpox virus, Mule deer poxvirus virus, Volepox virus,
Skunkpox virus, Rubella virus, Eastern equine encephalitis virus,
Western equine encephalitis virus, Venezuelan equine encephalitis
virus, Sindbis virus, Semliki Forest virus, Chikungunya virus,
O'nyong-nyong virus, Ross River virus, parainfluenza virus, mumps
virus, measles virus (rubeola virus), respiratory syncytial virus,
Herpes simplex virus type 1, Herpes simplex virus type 2, Varicella
zoster virus, Epstein-Barr virus, Cytomegalovirus, human
b-lymphotrophic virus, human herpesvirus 7, human herpesvirus 8,
poliovirus, Coxsackie A virus, Coxsackie B virus, ECHOvirus,
rhinovirus, Hepatitis A virus, mengovirus, ME virus,
encephalomyocarditis (EMC) virus, MM virus, Columbia SK virus,
Norwalk agent, Hepatitis E virus, Colorado tick fever virus,
rotavirus, vesicular stomatitis virus, rabies virus, papilloma
virus, BK virus, JC virus, B19 virus, adeno-associated virus,
adenovirus (including serotypes 3, 7, 14, 21), Hepatitis B virus,
coronavirus, human T-cell lymphotrophic virus, human
immunodeficiency virus, human foamy virus, influenza viruses, types
A, B, C, and thogotovirus.
[0113] Parasitic infections that can be affected by the present
invention can be caused by parasites belonging to the genera
including, but not limited to, Entamoeba, Dientamoeba, Giardia,
Balantidium, Trichomonas, Cryptosporidium, Isospora, Plasmodium,
Leishmania, Trypanosoma, Babesia, Naegleria, Acanthamoeba,
Balamuthia, Enterobius, Strongyloides, Ascaradia, Trichuris,
Necator, Ancylostoma, Uncinaria, Onchocerca, Mesocestoides,
Echinococcus, Taenia, Diphylobothrium, Hymenolepsis, Moniezia,
Dicytocaulus, Dirofilaria, Wuchereria, Brugia, Toxocara,
Rhabditida, Spirurida, Dicrocoelium, Clonorchis, Echinostoma,
Fasciola, Fascioloides, Opisthorchis, Paragonimus, and Schistosoma.
Additionally, parasitic infections that can be treated using the
sanitizing compositions of the present invention can be caused by
parasites including, but not limited to, Entamoeba histolytica,
Dientamoeba fragilis, Giardia lamblia, Balantidium coli,
Trichomonas vaginalis, Cryptosporidium parvum, Isospora belli,
Plasmodium malariae, Plasmodium ovate, Plasmodium falciparum,
Plasmodium vivax, Leishmania braziliensis, Leishmania donovani,
Leishmania tropica, Trypanosoma cruzi, Trypanosoma brucei, Babesia
divergens, Babesia microti, Naegleria fowleri, Acanthamoeba
culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii,
Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba
rhysodes, Balamuthia mandrillaris, Enterobius vermicularis,
Strongyloides stercoralis, Strongyloides Ascaris lumbricoides,
Trichuris trichiura, Necator americanus, Ancylostoma duodenale,
Ancylostoma ceylanicum, Ancylostoma braziliense, Ancylostoma
caninum, Uncinaria stenocephala, Onchocerca volvulus, Mesocestoides
variabilis, Echinococcus granulosus, Taenia solium, Diphylobothrium
latum, Hymenolepis nana, Hymenolepis diminuta, Moniezia expansa,
Moniezia benedeni, Dicytocaulus viviparous, Dicytocaulus filarial,
Dicytocaulus arnfieldi, Dirofilaria repens, Dirofilaria immitis,
Wuchereria bancrofti, Brugia malayi, Toxocara canis, Toxocara cati,
Dicrocoelium dendriticum, Clonorchis sinensis, Echinostoma,
Echinostoma ilocanum, Echinostoma jassyenese, Echinostoma
malayanum, Echinostoma caproni, Fasciola hepatica, Fasciola
gigantica, Fascioloides magna, Opisthorchis viverrini, Opisthorchis
felineus, Opisthorchis sinensis, Paragonimus westermani,
Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium
and Schistosoma haematobium.
Example 2: Polymer Microarray Technology Enabled Discovery of a
Bi-Functional RGD Peptide That Promotes Endothelial Cell Adhesion,
Spreading and Proliferation
[0114] We used the peptide-functionalized hydrogel microarray
technology described in the Example 1 to screen the RGD (SEQ ID
NO:36) peptide library for the high affinity ligands to endothelial
cell (EC) integrin. This enabled the identification of a novel RGD
(SEQ ID NO:36) peptide (.alpha.1) derived from laminin-.alpha.1
domain with dramatically enhanced ability to promote EC adhesion,
spreading and proliferation in comparison with the currently used
RGDS (SEQ ID NO:35)/RGDS (SEQ ID NO:35) peptide. The mechanistic
studies revealed the .alpha.1 peptide binds to both .alpha.v.beta.3
and VLA-6 integrin that lead to the synergistic up-regulation of
VEGFR2 with the improved EC functions.
[0115] Materials and instruments. All chemicals used for this study
were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise
stated. Microarray spotting pins (946MP9B) were purchased from
Arrayit Corporation (Sunnyvale, Calif.). A custom designed
microarrayer was assembled by BioDot (Irvine, Calif.). The LC-MS
system used is Thermo Fisher LCQ Fleet.TM. Ion Trap Mass
Spectrometer. Primary and secondary antibodies were purchased from
Abcam (Cambridge, UK). The primers for RT-PCR were purchased from
ThermoFisher Scientific Inc. (Waltham, Mass.). Sodium alginate was
purchased from FMC BioPolymer (Philadelphia, Pa.).
[0116] Cell culture. Human umbilical vein endothelial cells
(HUVECs) (Lonza, Basel, Switzerland) were cultured in EGMTM-2
BulletKit.TM. Medium (Lonza, Basel, Switzerland). The medium
supplements contained 2% bovine serum albumin, hFBF-B, VEGF,
R3-IGF-1, ascorbic acid, heparin, FBS, hEGF and GA-1000. Growth
medium was changed every other day and cells were passaged every 6
days. All experiments were conducted using passage 4 (P4)
HUVECs.
[0117] Synthesis and characterization of N-terminal unmodified
peptides and methacrylated peptides. Peptides used in this work
were synthesized by solid phase peptide synthesis (SPPS). The SPPS
was conducted using the standard procedure as described in
Novabiochem peptide synthesis manual. N-terminal unmodified
peptides were cleaved from resin right after the deprotection once
reaching the designed sequences. To prepare methyacrylated
peptides, 2-isocyanatoethyl methacrylate (3 equivalent (eq)
dissolved in DMF) was used to react with the terminal amine group
of the peptide chain (1 eq) before they were cleaved from the resin
as described in the Example 1. All the peptides were purified by
flash column and characterized by LC-MS.
[0118] Microarray fabrication and screening. Methacrylated peptides
(shown in FIG. 7) were dissolved in DMF at pre-designated ratios (1
mM, 3 mM, 6 mM, 9 mM, 12 mM, 15 mM and 20 mM) and mixed with PEGDA
(containing 1% DMPA as initiator) (DMF solution of methacrylated
peptide: PEGDA=1:1 (v/v)) and then transferred into a 384 well
plate for microarray fabrication. The microarrays were printed in a
humid Ar-atmosphere on epoxy monolayer-coated glass slides
(Xenopore XENOSLIDE E, Hawthorne, N.J.) which were first dip-coated
in 4 v/v% poly(hydroxyethyl methacrylate) (i.e., poly(HEMA)) using
a customized microarrayer (Biodot). Spots were polymerized via 10 s
exposure to long wave UV (365 nm), dried at <50 mtorr for at
least 7 days. Before use, the chips were sterilized by UV for 30
min for each side, and then washed with PBS twice for 15 min to
remove residual monomer or solvent. HUVECs (human umbilical vein
endothelial cells from Lonza, Basel, Switzerland) were seeded onto
the array and cultured for 12 hours. They were then fixed and
stained with DAPI for cell number counting and phalloidin for
F-actin to estimate cell spreading for the lead peptide
identification.
[0119] Alginate synthesis and oxidation. Sodium alginate was
prepared using the method established by Bouhadir et al. The final
oxidation of the alginate was 5%.
[0120] Peptides conjugation on alginate hydrogel. The peptides were
conjugated onto the oxidized alginate by the EDC-NHS chemistry as
reported by Rowley: using aqueous carbodiimide reacted with
peptides (RGDS (SEQ ID NO:35), RGDSP (SEQ ID NO:33) and
.alpha.1-peptide) onto the oxidized alginate. In order to secure
sufficient cell attachment, 10% (w/w) peptide modification was
performed into the alginate hydrogel.
[0121] Alginate surface culture experiments. The Ca2+ containing
substrates for alginate crosslinking was prepared according to the
literature. 50 .mu.l, 1% alginate modified with peptides (RGDS (SEQ
ID NO:35), RGDSP (SEQ ID NO:33) and .alpha.1-peptide, respectively)
aqueous solution was transferred into each well in 96 well plates
(3 replicates for each peptide). The alginate was left gelling for
40 min. Then the well plates were transferred into incubator for 10
min to melt the Ca2+ containing substrates. After Ca2+ containing
substrates get removed, HUVECs were seeded onto the hydrogel layers
and cultured for 12 hours. Then they were fixed and stained with
DAPI for cell number counting and phalloidin for F-actin to
estimate cell spreading.
[0122] Antibody blocking experiments on alginate surface. The
integrin antibodies and their combinations (no antibody, anti VLA-6
only, anti-integrin .alpha.v.beta.3 only and anti
VLA-6+anti-integrin .alpha.v.beta.3,) were aliquot into predesigned
ratio (final ratios in the mixture: 1:20 for anti VLA-6, 1:100 for
anti-integrin .alpha.v.beta.3) with PBS, respectively. The HUVECs
were incubated with these antibodies for 15 min, sedimented by low
speed centrithgation, suspended in 100 .mu.l of serum-free MEM plus
0.02% BSA and then added into the 1% alginate gel coated wells. The
wells were transferred into incubator for 6 hours. After that,
unattached cells were rinsed from the wells and the attached cells
were then fixed and stained with DAPI for cell number counting and
phalloidin for F-actin.
[0123] Alginate gel culture experiments. The Ca2+ containing
substrates for alginate crosslinking was prepared as the previous
literature mentioned. Alginate modified with these peptides (RGDS
(SEQ ID NO:35), RGDSP (SEQ ID NO:33) and .alpha.1-peptide,
respectively) was dissolved into HUVECs culturing media to prepare
1% alginate solution. The solution was mixed with HUVECs at the
density of 10 million cells/ml. 250 .mu.l mixture of the alginate
and HUVECs was transferred into each well in 96 well plates (3
replicates for each peptide). The alginate was left gelling for 40
min. Then the well plates were transferred into incubator for 10
min to melt the Ca2+ containing substrates. After Ca2+ containing
substrates get removed, HUVEC culturing media was added into each
well and continue culture for additional two days. Media were
changed every day. The cells were then fixed and stained with DAPI
for cell number counting and phalloidin for F-actin to estimate
cell spreading at day 0 and day 2. The fluorescent pictures were
taken by a Leica TCS SP5 AOBS confocal microscope system. The total
tubule length and the proliferation rates were measured.
[0124] RT-PCR. Before PCR experiments, HUVECs were seeded in three
different peptides (RGDS (SEQ ID NO:35), RGDSP (SEQ ID NO:33) and
.alpha.1-peptide, respectively) modified 1% alginate gel. The cells
were cultured for 6 hrs and then harvest for RNA isolation. Total
RNA was isolated according to the kit and protocol of an RNeasy
Micro Kit (Qiagen, Vinlo, Netherlands) with the addition of the
QIAShredder (Qiagen) during the homogenization step for HUVECs. For
each group, around 0.1 million cells were used for RNA isolation.
At least 25 ng of total RNA for each group was used for cDNA
synthesis by the Bio-Rad (Hercules, USA) iScript cDNA synthesis
kit. qRT-PCR was executed with "best coverage" validated Taqman
primers (Life Technologies, Carlsbad, USA) in 10 .mu.l reactions
for the following genes: KDR (VEGFR2), ACTB, GAPDH. qRT-PCR Data
was normalized as the change in cycle threshold (dCt) from the
geometric mean of ACTB and GAPDH expression. Expression was
analyzed using mRNA expression=2{circumflex over ( )}(-(dCt)) and
then normalized to the gene expression from RGDS (SEQ ID NO:35)
samples.
[0125] Statistical analysis. The results were shown in the
mean+standard derivation (SD) and analyzed using Excel statistical
software
[0126] To rapidly screen the peptide library for the high affinity
RGD (SEQ ID NO:36) peptides to EC integrin, we fabricated
microarrayed PEG (poly(ethylene glycol)) hydrogels that are
functionalized with different RGD (SEQ ID NO:36) peptides as
described the Example 1. We then seeded HUVECs (human umbilical
vein endothelial cells) onto the microarrays. As shown in the FIG.
8A, a variety of different cell adhesions were found on the RGD
(SEQ ID NO:36) peptide-functionalized PEG hydrogel microarray,
which indicate different affinity of these RGD (SEQ ID NO:36)
peptides to EC integrin. Hydrogel spots were used to quantify the
peptide's affinity to EC integrin in this study.
[0127] RGD (SEQ ID NO:36) peptides derived from Fn, namely RGDS
(SEQ ID NO:35) and RGDSP (SEQ ID NO:33), have been extensively used
to promote EC adhesion, proliferation and network formation. Our
data showed the RGDSP (SEQ ID NO:33) has significantly higher
affinity to HUVECs than RGDS (SEQ ID NO:35) (FIG. 8A), which can be
attributed to the additional proline at the chain end that reduces
the flexibility of the RGDS (SEQ ID NO:35) peptide and increase its
affinity towards integrin. These results are in agreement with the
previous reports of the activities of these peptides to promote
fibroblast attachment.
[0128] Among all the peptides in the library, a RGD (SEQ ID NO:36)
peptide derived from Ln .alpha.1 subunit (al peptide, TFALRGDNP,
SEQ ID NO:1) showed the highest activities to promote HUVEC
adhesion (FIGS. 8A-D, 8E). The sequence of the .alpha.1 peptide was
determined through the comparison of Laminin-.alpha.1 gene between
human and chimpanzee, which demonstrates the powerful of the
Bioinformatics tool we employed here. The saturated number of
attached HUVECs of .alpha.1 peptide is .about.200% higher than
RGDSP (SEQ ID NO:33) and .about.500% higher than RGDS (SEQ ID
NO:35).
[0129] To prove the effectiveness of .alpha.1 peptide as a
biological ligand, .alpha.1 peptide was conjugated onto alginate
hydrogel. The number of HUVECs attached to 2D surface of .alpha.1
peptide-modified alginate hydrogel are .about.60% higher than that
of RGDSP (SEQ ID NO:33) and .about.150% higher than that of RGDS
(SEQ ID NO:35) (FIG. 8B). This data demonstrates the ability of
.alpha.1 peptide to promote HUVEC adhesion and spreading on the
surface of different hydrogel systems. As alginate has been widely
used as a 3D cell culture system, we prepared RGDS (SEQ ID NO:35),
RGDSP (SEQ ID NO:33) and .alpha.1 peptide functionalized 3D
alginate hydrogels to examine their effects on HUVEC functions.
Specifically, we used a 1% alginate with 10% (w/w) peptide
functionalization to prepare 3D hydrogels contains 6 million
HUVECs/ml. While F-actin staining of HUVECs in RGDS- and
RGDSP-functionalized alginate hydrogels showed limited cell
spreading and no visible network formation, the .alpha.1
peptide-functionalized alginate hydrogel showed significantly
enhanced cell proliferation and network formation after 2 days
culture (FIG. 8C). The extended culture of HUVECs in the hydrogels
confirmed the enhanced proliferation of HUVECs in the .alpha.1
peptide functionalized alginate when compared with RGDS- and
RGDSP-peptide functionalized alginates (FIG. 8C).
[0130] As described above, the .alpha.1 peptide was identified
through the bioinformatics tool. To understand the mechanisms
underpinning the enhanced adhesion, spreading and proliferation of
HUVECs on/in the .alpha.1 peptide functionalized hydrogels, we
conducted integrin-blocking experiments as a similar Ln derived
peptide A99 (CQAGTFALRGDNPQG) (SEQ ID NO:49) was reported to bind
to both VLA-6 (.alpha.6) and .alpha.v.beta.3 integrin subunits. As
shown in the FIG. 8D, blocking VLA-6 integrin does not
significantly affect cell adhesion for RGDS- and
RGDSP-functionalized alginates, while it led to .about.60% decrease
in cell adhesion on the .alpha.1 peptide functionalized alginate.
In addition, blocking .alpha.v.beta.3 integrin resulted in the
.about.35% decrease in cell adhesion for RGDS- and RGDSP-peptides
functionalized alginates and .about.60% decrease in cell adhesion
for .alpha.1 peptide functionalized alginate. When both VLA-6 and
.alpha.v.beta.3 integrin are blocked, .about.35% decrease in the
cell adhesion were found on the RGDS- and RGDSP-functionalized
alginates and .about.70% decrease in cell adhesion was found on
.alpha.1 peptide functionalized alginate. These results clearly
showed the RGDS- and RGDSP-peptides bind to .alpha.v.beta.3
integrin while the .alpha.1 peptide binds to both VLA-6 and
.alpha.v.beta.3 integrin.
[0131] mRNA expression of VEGFR2 was significantly up-regulated in
the HUVECs cultivated in .alpha.1 peptide functionalized 3D
alginate hydrogels when compared with RGDSP (SEQ ID NO:33) and RGDS
(SEQ ID NO:35) samples after 6 hours culture (FIG. 8E).
Interestingly, significant higher amount of VEGFR2 mRNA expression
was found in HUVECs cultivated in RGDSP-peptide functionalized
alginate hydrogels than those cultivated in RGDS-peptide
functionalized alginate hydrogel, which was attributed to higher
affinity of RGDSP (SEQ ID NO:33) peptide to .alpha.v.beta.3
integrin than RGDS (SEQ ID NO:35) peptide (FIGS. 8B and 8C).
[0132] To further confirm the co-signaling between .alpha.v.beta.3
and VLA-6 integrin binding, we prepared a co-signaling alginate
hydrogel functionalized with both a .alpha.v.beta.3 binding peptide
(RGDSP, SEQ ID NO:33) and a VLA-6 integrin binding peptide
(LPSHYRARNI, SEQ ID NO:50). The synergy between RGDSP (SEQ ID
NO:33) and LPSHYRARNI (SEQ ID NO:50) peptides led to significant
improvement in VEGFR2 mRNA expression when compared with
RGDSP-peptide functionalized alginate. Importantly, the combination
of RGDSP (SEQ ID NO:33) and LPSHYRARNI (SEQ ID NO:50) peptides
showed a similar capacity to improve HUVEC functions as the
.alpha.1 peptide.
[0133] These results support our hypothesis that it is the ability
of .alpha.1 peptide to engage both VLA-6 and .alpha.v.beta.3
integrin that leads to the synergistic up-regulation of VEGFR2 and
results in enhanced HUVEC adhesion, spreading and proliferation.
Co-signaling between integrin and growth factors has been utilized
to develop functional hydrogels to improve EC vascularization and
angiogenesis. To the best of our knowledge, this study, for the
first time, demonstrates the co-signaling between two integrin
binding ligands promotes EC adhesion, spreading and proliferation.
We expect this co-signaling mechanism will allow for the
development of next generation of biomaterials for the fabrication
of vascularized tissue engineering constructs and 3D bioprinting
applications. Further, integrin blocking has been used as a therapy
for cancer treatments as integrin activation have been shown to
play an essential role to promote angiogenesis in tumor growth. As
the efficacy of the .alpha.v.beta.3 integrin binding peptides in
suppressing pathological angiogenesis has been found to be rather
moderate in clinical trials, the co-signaling mechanism identified
here may provide an attractive means to develop next generation
cancer therapeutics. Thus, the present invention provides products
and methods of treating a tumor or cancer, wherein the tumor or
cancer includes, prostate cancer, breast cancer, ovarian cancer,
uterine cancer, pancreatic cancer, skin cancer, melanoma, lymphoma,
sarcoma, lung cancer, colon cancer, leukemia, renal cancer, brain
cancer, CNS cancer, neuroblastoma, oral cancer, throat cancer,
esophageal cancer, head and neck cancer and combinations
thereof.
[0134] In this study, we demonstrated the combination of modern
bioinformatics and a newly established peptide-functionalized PEG
hydrogel microarray technology enabled the identification of a
novel RGD (SEQ ID NO:36) (.alpha.1) peptide that promotes EC
functions through co-activation of .alpha.v.beta.3 and VLA-6
integrin. To the best of our knowledge, this is the first report
that shows the synergy between .alpha.v.beta.3 and VLA-6 integrin
binding promotes VEGFR2 expression and EC adhesion, spreading and
proliferation. The sequence of the al peptide was defined through
the comparison of Laminin-.alpha.1 gene between human and
chimpanzee, which demonstrates the power of the bioinformatics tool
we used to identify potential candidates to construct peptide
library. Further, the high affinity of .alpha.1 peptide to HUVEC
integrin was identified through the use of peptide-functionalized
hydrogel microarrays and further validated by using 2D/3D hydrogels
functionalized with the .alpha.1 peptide. This demonstrates the
effectiveness of the newly established peptide-functionalized
hydrogel microarray to identify novel biological ligands to control
(stem) cell behavior.
[0135] The moderate success of the tissue engineering approaches
has been attributed to the insufficient vascularization within the
scaffolds. Although numerous materials-based strategies have been
explored to improve vascularization, few previous studies have been
focused on the identification of novel biological ligands to
improve the functions of ECs. While RGDS (SEQ ID NO:35)/RGDSP (SEQ
ID NO:34) peptides have been routinely used to promote EC functions
in vascularized tissue fabrication, our data demonstrates the
dramatically improved performance of the .alpha.1 peptide when
compared with RGDS (SEQ ID NO:35)/RGDSP (SEQ ID NO:34) peptides.
This highlights the power of the bioinformatics and the newly
established peptide-functionalized hydrogel microarray technology
in identifying novel biological ligands to regulate the functions
of various (stein) cells.
Example 3: Synergistic Effect of Integrin-Binding Peptide and
Angiogenic Factors
[0136] We hypothesized that spatiotemporal distribution of VEGF
mimetic QK peptide can significantly affect the crosstalk between
.alpha.1 peptide initiated integrin signaling and QK peptide
initiated VEGFR2 signaling, which is important to EC morphogenesis
and angiogenesis.
[0137] To synergize with the .alpha.1 peptide, we used the .alpha.1
peptide and an MMP-responsive QK peptide (i.e.,
GPQG.dwnarw.IAGKLTWQELYQLKYKGI, SEQ ID NO:41) to prepare an
alginate-based injectable, multi-signaling hydrogels (FIG. 9). The
MMP-responsive QK peptide was selected to introduce cell-dictated
local release of angiogenic factors to recapitulate the
pro-angiogenic microenvironment in vivo, where matrix bound VEGF is
released by MMPs. Alginates was selected because it has been used
as a biocompatible, injectable hydrogel-forming material to treat
ischemic diseases.
[0138] We developed a modular approach to prepare the
multi-signaling hydrogels (FIG. 10). Briefly, we have prepared
alginates functionalized with .alpha.1 peptide (10% (w/w)) as well
as alginates functionalized with MMP-responsive QK peptide (10%
(w/w)) using click chemistry. We then mixed these two alginates at
a ratio 1:1 (w/w) to prepare multi-signaling hydrogels to
simultaneously engage .alpha..sub.v.beta.3, VLA-6 integrins and
VEGFR2. This modular approach allowed us to rapidly prepare
alginates with different peptide functionalization to screen for an
optimal formulation to support EC morphogenesis.
[0139] When compared with hydrogels functionalized with .alpha.1
peptide and covalently bound QK and hydrogels functionalized with
RGDSP (SEQ ID NO:33) peptide and MMP-responsive QK, the synergy
between .alpha.1 peptide and MMP-responsive QK peptide leads to the
significant improvement in the EC vascular network formation (FIG.
11). These results clearly demonstrated the benefits of the
combination of .alpha.1 peptide and MMP-responsive QK peptide,
which can collaboratively activate .alpha..sub.v.beta.3 and VLA-6
integrins and controlled release VEGF mimetic peptide on cellular
demand.
[0140] Our preliminary data clearly showed the synergy between
.alpha.1 peptide and MMP-responsive QK peptide leads to the
significant improvement in the EC vascular network formation.
[0141] To prepare alginate hydrogel that can release QK peptide in
a cellular demanded, temporally controlled manner, we will
synthesize QK peptide with four different MMP sensitive linkers (QK
(FL): QK with fast linker, QK (ML): QK with moderate linker, QK
(SL): QK with slow linker, QK (NL): QK with non-degradable linker
based on a recent report from Benoit and coworkers (Table 1).
Notably, it was demonstrated that the activity of QK peptide is not
affected by the presence of residue amino acids after MMP cleavage.
These QK peptides will be conjugated to alginates using click
chemistry as shown in the FIG. 10. In addition, we will mix
alginates functionalized with QK (FL) and alginates functionalized
with QK (SL) in a 50:50 (w/w) ratio to prepare the QK (PL) sample
to create pulsatile release profile, which is a rapid release at
the beginning followed by a sustained release at a lower rate. The
pulsatile release profile was proposed as it was reported to
optimally support EC sprouting.
TABLE-US-00001 TABLE 1 MMP-responsive QK peptides with different
protease sensitivity Abbreviation Full Sequences QK (FL) QK with
"QK"-PES.dwnarw.LRAG fast (SEQ ID linker NO: 51) QK (ML) QK with
"QK"-GPQG.dwnarw.IWGQ moderate (SEQ ID NO: 52) linker QK (SL) QK
with "QK"-VPLS.dwnarw.LYSG slow (SEQ ID linker NO: 53) QK (PL) 50%
QK 50% "QK"-PES.dwnarw.LRAG with (SEQ ID fast NO: 51) linker, and
50% QK 50% "QK"-VPLS.dwnarw.LYSG with (SEQ ID slow NO: 53) linker
QK (NL) QK with "QK"-GGGG non- (SEQ ID NO:54) degradable linker
[0142] To examine the effects of the MMP-degradable linkage of the
QK peptide on the EC morphogenesis, we will seed 6 million/ml
HUVECs in 5% oxidized, 1% (w/w) alginates functionalized with 5%
(w/w) .alpha.1 peptide and 5% (w/w) QK peptide with different
MMP-degradable linkages (FL, ML, SL, PL, NL). After 2 days, the
HUVECs will be fixed and stained with DAPI and phalloidin to
quantify total network length per unit area. The vascular
morphogenesis of the HUVECs will also further examined with
immunofluorescence staining of CD31 and VE-Cadherin. This set of
experiments will allow for the identification of optimal
MMP-degradable linkage for the improved EC morphogenesis. Further,
it would allow us to establish a relationship between the release
profile of QK peptide and EC vascular network formation.
[0143] In addition to the temporal release profile, the
concentrations of QK peptides can significantly affect EC
functions. While our preliminary data demonstrated 5% (w/w)
MMP-responsive QK peptides is sufficient to promote EC vascular
network formation, additional experiments are necessary to identify
the QK peptide concentration for the optimal EC morphogenesis. To
this end, we will prepare alginate functionalized with different
concentrations of the QK peptides with the optimized MMP-responsive
linkage (i.e., 6%, 10%, 14%, 18% (w/w)) to examine their effects on
EC functions. Briefly, we will vary the ratio between the
11-Azido-3,6,9-trioxaundecan-1-amine to alginate to prepare
alginate with different azide-functionalization (--N.sub.3), a
linker for click chemistry conjugation (FIG. 10). These alginates
will be reacted with alkyne-modified MMP-QK to prepare alginate
functionalized with different concentrations of MMP-responsive QK
peptides (i.e., 6%, 10%, 14%, 18% (w/w)). These alginates will be
mixed with 10% (w/w) .alpha.1 peptide functionalized alginates at
1:1 (w/w) ration to prepare alginates functionalized with 5% (w/w)
.alpha.1 peptide and different MMP-responsive QK concentration
(i.e., 3%, 5%, 7% and 9% (w/w)). As described above, 6 million/ml
HUVECs will be seeded into these alginates, cultured for 2 day and
examined for the total network length. These experiments will allow
for the identification of optimal concentration of MMP-responsive
QK peptides for the in vivo tests.
Example 4: In Vivo Pro-Angiogenic Potential of the Acellular,
Injectable Multi-Signaling Alginate Hydrogels
[0144] We propose to inject alginate hydrogel functionalized with
.alpha.1 peptide and optimized MMP-responsive QK peptide into the
ischemic hindlimb of mouse to examine their pro-angiogenic
potential. To prepare an injectable alginate hydrogel, 0.05 mM Ca
gluconate solution will be mixed with 5% oxidized, 2% (w/w)
alginate solution at the ratio 1:1 (v/v) to prepare the 1%
injectable alginate hydrogels. High biocompatibility of this
injectable hydrogel has been supported by the high viability of the
HUVECs encapsulated in the gel (data not shown).
[0145] To induce unilateral hindlimb ischemia, a 10-week-old
C57BL/6 mouse will be anesthetized and prepared for surgery. With a
dissection microscope, we will dissect and separate the femoral
artery from the femoral vein at the proximal location near the
groin, pass a strand of 7-0 silk suture underneath the proximal end
of the femoral artery and occlude the proximal femoral artery using
double knots. Similarly, we will separate the femoral artery from
the surrounding tissues at the distal location close to the knee,
pass a strand of 7-0 suture underneath the distal end of the
femoral artery proximal to the popliteal artery and occlude the
vessel using double knots. We will then use spring scissors to
transect the segment of femoral artery between the distal and
proximal knots. 50 .mu.L of 5% oxidized, 1% (w/w) alginate hydrogel
functionalized with .alpha.1 peptide (5% w/w) and QK peptide with
optimized MMP-degradable linkage and concentration will be injected
into the quadricep (25 uL) and gastrocnemius (25 .mu.L) muscles of
the ischemic hind limb. Controls will include 50 .mu.L of 5%
oxidized, 1% (w/w) alginate functionalized with (1) no peptide, (2)
5% (w/w) .alpha.1 peptide (no QK), (3) the optimized QK peptide (no
.alpha.1), (4) 5% (w/w) .alpha.1 peptide and QK peptide with a
non-degradable linkage at the optimized concentration, (5) 5% (w/w)
.alpha.1 peptide and QK peptide with an un-optimal MMP-degradable
linkage at the optimized concentration, and (6) 5% (w/w) .alpha.1
peptide and QK peptide with an optimal MMP-degradable linkage at an
un-optimized concentration, and (7) decellularized skeletal muscle
extracellular matrix, a potential gold standard material to treat
peripheral artery disease.
[0146] To examine the pro-angiogenic potential of the
multi-signaling alginate hydrogel, the blood flow ratio between
ischemic/normal limb will be measured before surgery as well as 1,
3, 5, 7, 14, 28 and 42 days post-surgery by using a Laser Doppler
blood flow imaging system (Moor Instruments Ltd., Devon, UK). In
addition, the hindlimb muscle tissues (n=7/condition, 42 days
post-surgery) will be harvested, fixed, paraffin embedded and
stained for CD31 (Abeam) to measure capillary densities. 30
randomly chosen fields of the tissue will be analyzed, and the
total number of the blood vessels will be manually countered and
normalized to the tissue area.
Sequence CWU 1
1
5419PRTArtificialpeptide 1Thr Phe Ala Leu Arg Gly Asp Asn Pro1
529PRTArtificialpeptide 2Thr Phe Ala Leu Arg Ala Asp Asn Pro1
5314PRTArtificialpeptide 3Asp Val Glu Lys Arg Gly Asp Arg Glu Glu
Ala His Val Pro1 5 10411PRTArtificialpeptide 4Ile Gln Arg Gly Asp
Ile Asp Ala Met Ile Ser1 5 10514PRTArtificialpeptide 5Asp Ala Val
Lys Gln Leu Gln Ala Ala Glu Arg Gly Asp Ala1 5
10612PRTArtificialpeptide 6Pro Met Gln Lys Met Arg Gly Asp Val Phe
Ser Pro1 5 1076PRTArtificialpeptide 7Arg Ser Asp Gly Thr Gly1
5810PRTArtificialpeptide 8Glu Ala Pro Arg Gly Asp Val Tyr Gln Gly1
5 1099PRTArtificialpeptideMISC_FEATURE(3)..(3)Xaa is
pyrrolysineMISC_FEATURE(9)..(9)Xaa is pyrrolysine 9Gly Leu Xaa Gly
Glu Arg Gly Arg Xaa1
5109PRTArtificialpeptideMISC_FEATURE(3)..(3)Xaa is pyrrolysine
10Gly Phe Xaa Gly Glu Arg Gly Val Gln1 5114PRTArtificialpeptide
11Asp Gly Glu Ala1126PRTArtificialpeptideMISC_FEATURE(3)..(3)Xaa is
pyrrolysine 12Gly Phe Xaa Gly Glu Arg1 5136PRTArtificialpeptide
13Gly Leu Lys Gly Glu Asn1 5143PRTArtificialpeptide 14Leu Asp
Val1154PRTArtificialpeptide 15Arg Glu Asp
Val1167PRTArtificialpeptide 16Pro Glu Asp Gly Ile His Glu1
5175PRTArtificialpeptide 17Pro His Ser Arg Asn1
5185PRTArtificialpeptide 18Ala Leu Asn Gly Arg1
5196PRTArtificialpeptide 19Ile Ala Phe Gln Arg Asn1
5205PRTArtificialpeptide 20Ile Lys Leu Leu Ile1
5216PRTArtificialpeptide 21Ser Ile Lys Val Ala Val1
52212PRTArtificialpeptide 22Ala Gly Gln Trp His Arg Val Ser Val Arg
Trp Gly1 5 102312PRTArtificialpeptide 23Thr Trp Ser Gln Lys Ala Leu
His His Arg Val Pro1 5 10247PRTArtificialpeptide 24Ser Ile Tyr Ile
Thr Arg Phe1 52512PRTArtificialpeptide 25Ser Tyr Trp Tyr Arg Ile
Glu Ala Ser Arg Thr Gly1 5 10265PRTArtificialpeptide 26Tyr Ile Gly
Ser Arg1 52710PRTArtificialpeptide 27Arg Asp Ile Ala Glu Ile Ile
Lys Asp Ile1 5 10288PRTArtificialpeptide 28Val Phe Asp Asn Phe Val
Leu Lys1 5298PRTArtificialpeptide 29Ala Glu Ile Asp Gly Ile Glu
Leu1 53011PRTArtificialpeptide 30Ser Glu Thr Gln Arg Gly Asp Val
Phe Val Pro1 5 10319PRTArtificialpeptide 31Pro Ala Ser Tyr Arg Gly
Asp Ser Cys1 53210PRTArtificialpeptide 32Val Thr Gly Arg Gly Asp
Ser Pro Ala Ser1 5 10335PRTArtificialpeptide 33Arg Gly Asp Ser Pro1
5345PRTArtificialpeptide 34Arg Asp Gly Ser Pro1
5354PRTArtificialpeptide 35Arg Gly Asp Ser1363PRTArtificialpeptide
36Arg Gly Asp13712PRTArtificialpeptide 37Pro Gln Val Thr Arg Gly
Asp Val Phe Thr Met Pro1 5 103815PRTArtificialpeptide 38Lys Leu Thr
Trp Gln Glu Leu Tyr Gln Leu Lys Tyr Lys Gly Ile1 5 10
153935PRTArtificialpeptideMISC_FEATURE(23)..(25)Xaa is
aminohexanoic acid 39Cys Val Arg Lys Ile Glu Ile Val Arg Lys Lys
Cys Val Arg Lys Ile1 5 10 15Glu Ile Val Arg Lys Lys Xaa Xaa Xaa Arg
Lys Arg Lys Leu Glu Arg 20 25 30Ile Ala Arg
35408PRTArtificialpeptide 40Cys His His His Arg His Ser Phe1
54122PRTArtificialpeptide 41Gly Pro Gln Gly Ile Ala Gly Lys Leu Thr
Trp Gln Glu Leu Tyr Gln1 5 10 15Leu Lys Tyr Lys Gly Ile
20427PRTArtificialpeptide 42Pro Glu Ser Leu Arg Ala Gly1
5438PRTArtificialpeptide 43Gly Pro Gln Gly Ile Trp Gly Gln1
5448PRTArtificialpeptide 44Val Pro Leu Ser Leu Tyr Ser Gly1
5457PRTArtificialpeptide 45Gly Gly Arg Gly Asp Ser Pro1
5469PRTArtificialpeptide 46Gly Gly Gly Gly Arg Gly Asp Ser Pro1
5479PRTArtificialpeptide 47Gly Gly Gly Gly Arg Asp Gly Ser Pro1
54811PRTArtificialpeptide 48Gly Gly Gly Gly Gly Gly Arg Gly Asp Ser
Pro1 5 104915PRTArtificialpeptide 49Cys Gln Ala Gly Thr Phe Ala Leu
Arg Gly Asp Asn Pro Gln Gly1 5 10 155010PRTArtificialpeptide 50Leu
Pro Ser His Tyr Arg Ala Arg Asn Ile1 5 105111PRTArtificialpeptide
51Gly Gly Gly Gly Pro Glu Ser Leu Arg Ala Gly1 5
105212PRTArtificialpeptide 52Gly Gly Gly Gly Gly Pro Gln Gly Ile
Trp Gly Gln1 5 105312PRTArtificialpeptide 53Gly Gly Gly Gly Val Pro
Leu Ser Leu Tyr Ser Gly1 5 10548PRTArtificialpeptide 54Gly Gly Gly
Gly Gly Gly Gly Gly1 5
* * * * *