U.S. patent application number 16/337886 was filed with the patent office on 2019-11-14 for methods and compositions for noninvasive detection of organ transplant rejection.
The applicant listed for this patent is Emory University, Georgia Tech Research Corporation. Invention is credited to Andrew B. Adams, Gabriel A. Kwong, Quoc Mac, David V. Mathews.
Application Number | 20190345534 16/337886 |
Document ID | / |
Family ID | 61760126 |
Filed Date | 2019-11-14 |
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United States Patent
Application |
20190345534 |
Kind Code |
A1 |
Kwong; Gabriel A. ; et
al. |
November 14, 2019 |
Methods And Compositions For Noninvasive Detection Of Organ
Transplant Rejection
Abstract
An activity-based nanosensor composition for detecting protease
activity comprising a cleavable detectable substrate and methods of
use are disclosed.
Inventors: |
Kwong; Gabriel A.; (Atlanta,
GA) ; Adams; Andrew B.; (Atlanta, GA) ; Mac;
Quoc; (Atlanta, GA) ; Mathews; David V.;
(Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georgia Tech Research Corporation
Emory University |
Atlanta
Atlanta |
GA
GA |
US
US |
|
|
Family ID: |
61760126 |
Appl. No.: |
16/337886 |
Filed: |
September 28, 2017 |
PCT Filed: |
September 28, 2017 |
PCT NO: |
PCT/US2017/054105 |
371 Date: |
March 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62400656 |
Sep 28, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/68 20130101;
G01N 33/573 20130101; G01N 2800/245 20130101; C12Q 1/48 20130101;
C12Q 1/25 20130101; G01N 33/53 20130101; C12Q 1/37 20130101 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37 |
Goverment Interests
GOVERNMENT SPONSORSHIP
[0002] This invention was made with government support under Grant
No. DP2HD091793 awarded by the National Institute of Health and
Grant No. 1451512 from the National Science Foundation
(DGE-1451512). The government has certain rights in the invention.
Claims
1. An activity-based nanosensor comprising: a scaffold; a linker
coupled to the scaffold; a peptide substrate coupled to the linker;
and a detectable reporter coupled to the peptide substrate.
2. The nanosensor of claim 1, wherein the scaffold is selected from
the group consisting of a nanoparticle, a nanostructure, a
microparticle, a protein, a sugar, a nucleic acid-based scaffold,
an imaging contrast agent, and a polymer.
3. The nanosensor of claim 1, wherein the scaffold is configured to
prevent renal clearance of the peptide substrate.
4. The nanosensor of claim 1 wherein the scaffold comprises a
nanoparticle having a diameter from about 3 nm to about 2
microns.
5. The nanosensor of claim 1, wherein the scaffold comprises an
iron oxide nanoparticle.
6. (canceled)
7. The nanosensor of claim 1, wherein the linker comprises a
cysteine residue.
8. The nanosensor of claim 1, wherein the peptide substrate
comprises a target protease cleavage sequence.
9. The nanosensor of claim 1, wherein the peptide substrate
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs 2-136.
10. The nanosensor of claim 1, wherein the peptide substrate
comprises a target protease selected from the group consisting of T
cell proteases, complement proteases, fibrosis proteases, and
inflammation-related proteases.
11. The nanosensor of claim 1, wherein the peptide substrate
comprises a target protease selected from the group consisting of
Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X,
Cls, Clr, MASP2, Factor I, Factor D, ADAMTS1, MMP2, MMP9, elastase,
cathepsin G, PR-3, thrombin, kallikrein 1, kallikrein 6, tryptase,
and chymase.
12. The nanosensor of claim 1, wherein the detectable reporter is
selected from the group consisting of a fluorophore, a luminescent
reporter, a ligand encoded reporter, a mass spectrometry tag, a
contrast agent for imaging, a PET-detectable domain, and a nucleic
acid tag.
13.-16. (canceled)
17. The nanosensor of claim 1, wherein the linker is coupled to the
scaffold via a first spacer; wherein the peptide substrate is
coupled to the linker via a second spacer; wherein the detectable
reporter is coupled to the substrate via a third spacer; and
wherein at least one of the first, second, and third spacers
comprise a GGS amino acid sequence.
18. A method of diagnosing tissue rejection comprising:
administering a nanosensor to a subject, the nanosensor comprising:
a scaffold; a linker coupled to the scaffold; a target protease
coupled to the linker; and a detectable reporter coupled to the
target protease; obtaining a sample of a bodily fluid from the
subject; detecting a level of the detectable reporter in the
sample; determining an activity of the target protease based at
least in part on the level of the detectable reporter in the
sample; comparing the activity of the target protease in the sample
to a reference activity of the target protease; and diagnosing
tissue rejection in the subject if the activity of the target
protease in the sample is greater than the reference activity of
the target protease.
19. The method of claim 18, wherein administering the nanosensor
comprises intravenously administering the nanosensor to the
subject.
20. (canceled)
21. The method of claim 18, wherein the bodily fluid is urine.
22. The method of claim 18, wherein the method is for diagnosing
acute organ rejection in a transplant recipient subject; and
wherein the reference activity of the target protease is determined
by one or both of the activity of the target protease in a sample
taken from the subject before receiving the transplanted tissue and
the activity of the target protease in a sample taken from a
control subject.
23. The method of 18, wherein the method is for diagnosing acute
organ rejection in a transplant recipient subject and further
comprises treating the acute organ rejection by administering a
therapeutic agent configured to treat acute organ rejection when
acute organ rejection is diagnosed.
24. The method of claim 18, wherein the method is for diagnosing
acute organ rejection in a transplant recipient subject; wherein
the scaffold is selected from the group consisting of a
nanoparticle, a nanostructure, a microparticle, a protein, a sugar,
a nucleic acid-based scaffold, an imaging contrast agent, and a
polymer; wherein the target protease is selected from the group
consisting of T cell proteases, complement proteases, fibrosis
proteases, and inflammation-related proteases; and wherein the
detectable reporter is selected from the group consisting of a
fluorophore, a luminescent reporter, a ligand encoded reporter, a
mass spectrometry tag, a contrast agent for imaging, a
PET-detectable domain, and a nucleic acid tag.
25. An activity-based nanosensor comprising: a scaffold selected
from the group consisting of a nanoparticle, a nanostructure, a
microparticle, a protein, a sugar, a nucleic acid-based scaffold,
an imaging contrast agent, and a polymer; a linker coupled to the
scaffold via a first spacer; a target protease coupled to the
linker via a second spacer, the target protease selected from the
group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8,
Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D,
ADAMTS1, MMP2, MMP9, elastase, cathepsin G, PR-3, thrombin,
kallikrein 1, kallikrein 6, tryptase, and chymase; and a detectable
reporter coupled to the target protease via a third spacer, the
detectable reporter selected from the group consisting of a
fluorophore, a luminescent reporter, a ligand encoded reporter, a
mass spectrometry tag, a contrast agent for imaging, a
PET-detectable domain, and a nucleic acid tag; wherein the scaffold
is configured to prevent renal clearance of the target protease;
and wherein at least one of the first, second, and third spacers
comprises a GGS amino acid sequence.
26. The nanosensor of claim 25, wherein the linker comprises a
thiol group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/400,656, filed on 28 Sep. 2016, the disclosure
of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 25, 2017, is named GTRC7411PCT_SL.txt and is 39,603 bytes
in size.
BACKGROUND OF THE DISCLOSURE
1. Field of the Disclosure
[0004] Embodiments of the present disclosure relates generally to
methods and compositions for noninvasive detection of transplant
rejection, immune conditions related to T cell cytotoxicity (such
as for example and not limitation graft versus host disease (GvHD),
autoimmune diseases, and immuno-oncology), and sensing T cell
cytotoxicity (e.g., to predict treatment efficacy in patients being
treated with cancer immune therapies such as checkpoint blockade
inhibitors or CAR T cell therapies), and more specifically to
compositions comprising scaffolds (such as for example and not
limitation, protein scaffolds, polymer scaffolds, and particles
(e.g., a microparticle or nanoparticle)) linked to
protease-specific detectable peptides that can be administered to
transplant recipients and used to detect acute and chronic
transplant rejection by cleavage of the composition via the
accumulation of the detectable peptides locally at the site of
cleavage and/or in a bodily fluid (such as for example and not
limitation, urine, lymphatic fluid, blood, plasma, and/or
saliva).
2. Background
[0005] Organ transplantation remains the single most effective
treatment for end-stage organ failure, and early detection of
transplant rejection is critical for managing immunosuppression and
the long-term survival of recipients (1, 2). During acute cellular
rejection (ACR), graft damage is mediated by recipient cytotoxic
CD8 T cells that are activated by alloantigens displayed by antigen
presenting cells (APC) and target donor cells for killing (3, 4).
Although ACR episodes may appear at any time during the life of the
graft even years after immunological quiescence (4), ACR can be
effectively treated with anti-rejection drugs that target T cells
(e.g., thymoglobulin or anti-CD3 antibodies). Therefore, the
ability to measure the level of anti-graft T cell responses at an
early stage of ACR plays an indispensable role in managing graft
health and function (5). Currently, the gold standard for
diagnosing ACR is the core tissue biopsy, but this procedure is
invasive, subject to sampling error (tissue specimen typically
represents .about.1/10,000th the volume of the organ), and
associated with patient morbidity (6). Noninvasive approaches
include measuring biomarkers that indicate organ dysfunction, such
as blood urea nitrogen (BUN) and serum creatinine for kidney
allografts (7, 8), or biomarkers associated with allograft cell
death, such as sequencing cell-free donor-derived DNA from the
blood of heart transplant patients (9). These biomarkers indicate
graft health at stages that are downstream of cytotoxic T cell
activity; noninvasive methods that directly measure anti-graft
immune activity as early biomarkers of ACR are needed.
[0006] During onset of ACR, recipient cytotoxic T cells kill
allograft cells by releasing cytolytic granules containing perforin
to permeate target cell membranes and the serine protease granzyme
B (GzmB) to induce apoptosis (4, 10). Previous work on histological
analysis of allograft tissue sections showed increases in
graft-infiltrating CD8 T cells along with elevated expression of
GzmA, GzmB and perforin during acute rejection (11, 12). Moreover,
in patients with mild ACR and low histological grades, the presence
of GzmB-expressing lymphocytes and perforin was predictive of rapid
progression to severe ACR (12). Noninvasive approaches to monitor
anti-graft lymphocyte activity include work that showed a
correlation between RNA transcript levels of GzmB, perforin, and
Fas ligand from patient urine to ACR (13, 14). However, the
activity of GzmB is highly contextual and dependent on the presence
of endogenous inhibitors; for example, previous work showed that
higher levels of the serpin protease inhibitor, PI-9, was present
in kidney allografts with mild compared to severe ACR (15).
[0007] Chronic rejection of transplanted tissue is also an issue in
successful organ transplantation. Methods of detecting, diagnosing,
and monitoring chronic rejection in transplant recipients are also
needed; preferably, such methods are noninvasive. Proteases that
are active during chronic rejection include but are not limited to
proteases involved in T cell killing (such as for example and not
limitation, Granzyme A and Granzyme B), T cell activation (such as
for example and not limitation, MALT1, Caspase 8, Calpain 2, and
Cathepsin X) (50), apoptosis (such as for example and not
limitation, Caspase 3 and Caspase 8), complement activation (such
as for example and not limitation, Cls, Clr, MASP2, Factor I, and
Factor D) (51), fibrosis (such as for example and not limitation,
ADAMTS1, MMP2, and MMP9) (52, 53), and inflammation (such as for
example and not limitation, elastase, cathepsin G, PR-3, thrombin,
kallikrein 1, kallikrein 6, tryptase, and chymase).
[0008] What is needed, therefore, is a composition and method for
detecting acute and chronic rejection in transplant recipients, as
well as detecting immune conditions related to T cell cytotoxicity
(such as for example and not limitation graft versus host disease
(GvHD), autoimmune diseases, and immuno-oncology), and sensing T
cell cytotoxicity (e.g., to predict treatment efficacy in patients
being treated with cancer immune therapies such as checkpoint
blockade inhibitors or CAR T cell therapies). The method of
detection may take advantage of the nature of the composition,
wherein the composition comprises a detectable peptide containing a
protease cleavage site linked or coupled to a scaffold, wherein the
detectable peptide accumulates locally and/or in a bodily fluid due
to the protease being active during acute or chronic rejection.
Embodiments of the present disclosure are directed to such
compositions and methods.
BRIEF SUMMARY OF THE DISCLOSURE
[0009] As specified in the Background Section, there is a great
need in the art to identify technologies for detection of
transplant rejection, immune conditions related to T cell
cytotoxicity (such as for example and not limitation graft versus
host disease (GvHD), autoimmune diseases, and immuno-oncology), and
sensing T cell cytotoxicity (e.g., to predict treatment efficacy in
patients being treated with cancer immune therapies such as
checkpoint blockade inhibitors or CAR T cell therapies) and use
this understanding to develop novel detection compositions and
methods of using such compositions to detect these conditions.
Embodiments of the present disclosure relate generally to such
methods and compositions and more specifically to compositions that
can comprise scaffolds linked to detectable protease-specific
peptides that can be administered to transplant recipients and used
to detect acute and chronic transplant rejection by cleavage of the
composition via the accumulation of the detectable peptide locally
at the site of cleavage and/or in a bodily fluid (such as for
example and not limitation, urine, lymphatic fluid, blood, plasma,
and/or saliva).
[0010] In one aspect, the disclosure provides an activity-based
nanosensor comprising: a scaffold; a linker coupled to the
scaffold; at least one peptide substrate coupled to the linker; and
a detectable reporter domain coupled to the at least one peptide
substrate.
[0011] In some embodiments, the scaffold is selected from the group
consisting of a nanoparticle, a nanostructure, a microparticle, a
protein, a sugar, a nucleic acid-based scaffold, an imaging
contrast agent, and a polymer.
[0012] In some embodiments, the scaffold is configured to prevent
renal clearance of the substrate.
[0013] In other embodiments, the scaffold is a nanoparticle having
a diameter from about 3 nm to about 2 microns. In some embodiments,
the scaffold is an iron oxide nanoparticle. In some embodiments,
the linker comprises a thiol group.
[0014] In some embodiments, the linker comprises at least one
cysteine residue.
[0015] In other embodiments, the at least one peptide substrate
comprises a target protease cleavage sequence.
[0016] In some embodiments, the at least one peptide substrate
comprises at least one amino acid sequence selected from the group
consisting of SEQ ID NOs 2-136.
[0017] In some embodiments, the target protease is selected from
the group consisting of T cell proteases, complement proteases,
fibrosis proteases, and inflammation-related proteases.
[0018] In some embodiments, the target protease is selected from
the group consisting of Granzyme B, Granzyme A, MALT1, Caspase 8,
Calpain 2, Cathepsin X, Cls, Clr, MASP2, Factor I, Factor D,
ADAMTS1, MMP2, MMP9, elastase, cathepsin G, PR-3, thrombin,
kallikrein 1, kallikrein 6, tryptase, and chymase.
[0019] In some embodiments, the reporter domain is selected from
the group consisting of a fluorophore, a luminescent reporter, a
ligand encoded reporter, a mass spectrometry tag, a contrast agent
for imaging, a PET-detectable domain, and a nucleic acid tag.
[0020] In other embodiments, the reporter domain is a fluorescent
reporter.
[0021] In some embodiments, the linker is coupled to the scaffold
domain via a first spacer.
[0022] In other embodiments, the at least one peptide substrate is
coupled to the linker via a second spacer.
[0023] In still other embodiments, the reporter domain is coupled
to the substrate via a third spacer.
[0024] In some embodiments, at least one of the first, second, and
third spacers comprise a GGS amino acid sequence.
[0025] In another aspect, the disclosure provides a method of
diagnosing acute organ rejection in a transplant recipient subject
comprising:
[0026] (a) administering a nanosensor to the subject, the
nanosensor comprising: a scaffold; a linker coupled to the scaffold
domain; at least one peptide substrate coupled to the linker, the
peptide substrate comprising a target protease cleavage sequence;
and a detectable reporter coupled to the substrate;
[0027] (b) obtaining a sample of a bodily fluid from the
subject;
[0028] (c) detecting a level of the detectable reporter in the
sample of the bodily fluid;
[0029] (d) determining an activity of the target protease based on
the level of the detectable reporter in the sample of the bodily
fluid;
[0030] (e) comparing the activity of the target protease in the
sample to a reference activity of the target protease;
[0031] (f) identifying the subject as: [0032] (i) acutely rejecting
the transplant when the activity of the target protease in the
sample is greater than the reference activity of the target
protease; or [0033] (ii) not acutely rejecting the transplant when
the activity of the target protease in the sample is less than the
reference activity of the target protease; and
[0034] (g) optionally treating the acute rejection by administering
a therapeutic agent.
[0035] In some embodiments, step (a) comprises intravenously
administering the nanosensor to the subject.
[0036] In other embodiments, the bodily fluid is selected from the
group consisting of urine, blood, lymphatic fluid, plasma, and
saliva.
[0037] In some embodiments, the bodily fluid is urine.
[0038] In some embodiments, the reference is selected from the
group consisting of a sample taken from the subject before
receiving the transplanted tissue and a sample taken from a control
subject.
[0039] In some embodiments, the therapeutic agent is configured to
treat acute organ rejection.
[0040] These and other objects, features and advantages of the
present disclosure will become more apparent upon reading the
following specification in conjunction with the accompanying
description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The accompanying Figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0042] FIG. 1. Granzyme B sensing synthetic biomarkers detect onset
of acute allograft rejection by amplifying detection signals into
urine. Nanoparticles coated with GzmB substrates are intravenously
administered and accumulate in allograft tissues. Within this local
microenvironment, GzmB secreted by alloreactive CD8 T cells cleaves
the peptide substrates on nanoparticle surface, which triggers the
pharmacokinetic switch and release of fluorescent reporters into
urine. Urinary signals are quantified as early stage biomarkers of
acute cellular rejection.
[0043] FIGS. 2A-2F. Synthetic biomarkers are sensitive and specific
to proteolytic cleavage by GzmB. (FIG. 2A) Synthetic biomarkers
consist of PEG-coated IONPs functionalized with GzmB substrates. In
the presence of GzmB, peptide substrates are cleaved and
fluorescent reporters are released into solution, increasing sample
fluorescence. (FIG. 2B) Michaelis-Menten analysis of GzmB
substrates on nanoparticle surface (n=5, R2=0.79). (FIG. 2C) In
vitro protease activity assays showing normalized fluorescence of
synthetic biomarker samples after incubation with GzmB or key
proteases. (FIG. 2D) Activity assays showing normalized
fluorescence of synthetic biomarker samples in mouse plasma upon
addition Ca2+ to initiate coagulation cascade or addition of GzmB.
(FIG. 2E) Activity assays showing normalized fluorescence of
synthetic biomarker samples in control serum after addition of heat
aggregated gamma globulin (HAGG) to initiate complement cascade or
addition of GzmB. (FIG. 2F) ELISA measurements of membrane attack
complex (MAC) in activity assay supernatants of synthetic
biomarkers with control serum and complement activator (one-way
ANOVA with Turkey's post test, n=3).
[0044] FIGS. 3A-3I. Sensing GzmB during cytotoxic activity of
alloreactive T cells. (FIG. 3A) After upregulating expressing,
activated CD8 OT1 cells secrete GzmB to mediate apoptosis of
EG7-OVA target cells. (FIG. 3B) Flow cytometry plots of GzmB
activity within EG7-OVA and EL4 target cells after co-cultured with
OT1 T cells. (FIG. 3C) Quantified plot of flow analysis showing
percent of EG7-OVA and EL4 cells having intracellular GzmB
activity. (FIG. 3D) ELISA assay measuring levels of GzmB in
co-culture supernatants of OT1 T cells with EG7-OVA or EL4 target
cells at different ratios of T cells to target cells (one-way ANOVA
and Turkey's post test, n=3; nd=not detected). (FIG. 3E) Synthetic
biomarkers sense GzmB secreted in cocultures of OT1 T cells and
EG7-OVA target cells. (FIG. 3F) T cell activity assays showing
fluorescence of synthetic biomarkers in co-culture supernatants of
OT1 T cells with EG7-OVA or EL4 target cells. (FIG. 3G) Quantified
plot of T cell activity assays showing fitted value of initial
cleavage velocities. (FIG. 3H) Synthetic biomarkers sense GzmB
secreted in during alloreactive T cell killing. (FIG. 3I) T cell
activity assays showing normalized fluorescence of synthetic
biomarkers in co-culture supernatants of T cells isolated from skin
graft mice with target cells from BALB/c donor mice.
[0045] FIGS. 4A-4G. Granzyme B is upregulated at the onset of acute
cellular rejection. (FIG. 4A) Schematic of skin graft mouse model
of acute allograft rejection. (FIG. 4B) Pictures of skin grafts
showing morphological features of rejection that begin to appear on
day 9 post-transplant. (FIG. 4C) Skin graft scores showing graft
quality between allo- and iso-grafts (two-way ANOVA and Sidak's
post test, n=8) (FIG. 4D) Immunohistochemistry staining of GzmB in
graft and healthy skin tissues from mice bearing allo- or
iso-grafts. (FIG. 4E) Quantified plot of IHC data showing percent
of GzmB staining in graft and skin tissues (two-way ANOVA and
Sidak's post test, n=4-6 fields of view). (FIG. 4F) ELISA
measurements of GzmB in plasma of skin graft mice during the course
of rejection (two-way ANOVA and Sidak's post test, n=4). LOD=limit
of detection, defined by A.sub.blank+3 SD.sub.blank. (FIG. 4G) Flow
analysis of GzmB and CD44 expression in CD8+ T cells isolated from
the spleens and draining lymph nodes of mice bearing allo- or
iso-grafts on days 5, 7, and 9 post-transplant.
[0046] FIGS. 5A-5I. Urine analysis discriminates acute cellular
rejection with high sensitivity and specificity. (FIG. 5A) Mice
bearing both allo- and iso-grafts on the same animal are given
surface-labelled synthetic biomarkers for biodistribution studies.
(FIG. 5B) Top, photograph of mice bearing both skin grafts. Bottom,
near infrared fluorescent images showing synthetic biomarkers
accumulation in skin grafts. (FIG. 5C) Quantified fluorescent
signals of skin allo- and iso-grafts from mice bearing both grafts
on the same animal (one way ANOVA and Turkey's post test, n=3).
(FIG. 5D) Whole organ fluorescent images and quantified fluorescent
signals showing biodistribution of synthetic biomarkers in graft,
skin, spleen, and draining lymph nodes of skin graft mice (two way
ANOVA and Sidak's post test, n=3). (FIG. 5E) Timeline of urinalysis
study. Mice bearing either skin allografts or isografts are
administered synthetic biomarkers 4 days before and 7 days after
transplant surgeries. (FIG. 5F) Fluorescent signals of homogenized
tissue samples from skin graft mice after administration of
synthetic biomarkers showing elevated signal in kidneys of
allograft mice (Student's t-test, n=4-6). (FIG. 5G) Whole mouse
fluorescent image after administration of GSBs showing strong
signal from the bladders of mice bearing allografts. (FIG. 5H)
Normalized urine fluorescent signals after administration of
synthetic biomarkers to naive mice, isograft mice, allograft mice,
and mice bearing allografts after depletion of CD8 T cells (one way
ANOVA and Tukey's post test, n=6-8). (FIG. 5I)
Receiver-operating-characteristic (ROC) analysis showing that
synthetic biomarkers can differentiate between accepting isografts
and rejecting allografts in skin graft mice (AUC=0.969, 95%
CI=0.892-1.045).
[0047] FIG. 6. Optimization of GzmB peptide substrate. Initial
cleavage velocities of 13 GzmB-sensing synthetic biomarkers with
recombinant GzmB. Lowercase letters are d-form amino acids.
Sequence AIEFDSGc was chosen due to high rate of cleavage and its
specificity for GzmB. (n=3-8 independent assays; nd=not
detected).
[0048] FIGS. 7A-7B. In vitro characterization of synthetic
biomarkers. (FIG. 7A) Dynamic light scattering (DLS) analysis
showing size distribution of synthetic biomarkers in PBS or mouse
plasma. (FIG. 7B) Pharmacokinetic studies showing circulation
half-life of synthetic biomarkers in control Swiss Webster mice
(n=4, R.sup.2=0.86).
[0049] FIGS. 8A-8B. Upregulation of intracellular GzmB expression
in transgenic T cells (FIG. 8A) Activated OT1 CD8 T cells
upregulated GzmB expression after coincubation with EG7-OVA target
cells but not with EL4 target cells. (FIG. 8B) Flow cytometry
staining of intracellular GzmB and T cell activation marker CD44 in
CD8 T cells after coculturing activated OT1 T cells with EG7-OVA
target cells or EL4 control cells.
[0050] FIG. 9. Morphology of skin allografts and isografts during
the course of rejection. Photographs of skin allografts and
isografts on days 7, 9, 11, and 13 post-transplant. Signs of
rejection began to appear in allografts at around day 9.
[0051] FIGS. 10A-10B. Upregulation of CD8-expressing cells in skin
allografts. (FIG. 10A) Immunohistochemistry staining of GzmB in
graft and healthy skin tissues from mice bearing allo- or
iso-grafts. (FIG. 10B) Quantified plot of IHC data showing percent
of CD8 staining (two-way ANOVA and Sidak's post test, n=3-6 fields
of view).
[0052] FIG. 11. Passive accumulation of synthetic biomarkers in
skin allografts. Top panel, photograph of excised allografts,
isografts, and healthy skin from mice bearing both grafts on the
same animal. Bottom panel, near infra-red fluorescent image showing
biodistribution of synthetic biomarkers in these tissue
samples.
[0053] FIG. 12. Biodistribution of synthetic biomarkers in major
organs of skin graft mice. Near infra-red fluorescent image and
quantified signals showing biodistribution of synthetic biomarkers
in brain, heart, kidney, lung, and liver of mice bearing either
allo- or iso-graft (two-way ANOVA and Sidak's post test, n=3).
[0054] FIGS. 13A-13D. Urine pharmacokinetics of free peptide and
bare nanoparticles. (FIG. 13A) Fluorescent image showing clearance
of free peptides in bladders of skin graft mice. (FIG. 13B)
Quantified bladder fluorescent signals after administration of
labelled free peptides (one way ANOVA and Turkey's post test, n=3)
(FIG. 13C) Fluorescent image showing clearance of bare
nanoparticles in bladders of skin graft mice. (FIG. 13D) Quantified
bladder fluorescent signals after administration of
surface-labelled nanoparticles (one way ANOVA and Turkey's post
test, n=2).
[0055] FIGS. 14A-14B. Depletion of CD8 T cells before and after
transplant surgeries. (FIG. 14A) Flow cytometry analysis showing
that CD8 depletion reduces the population of CD3+ CD8+ cells in
secondary lymphoid organs right before and after skin graft
surgeries. (FIG. 14B) Quantified plot showing significant reduction
in percent of CD3+ CD8+ T cells in spleens and draining lymph nodes
of CD8-depleted mice versus. control mice (n=2-3).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0056] As specified in the Background Section, there is a great
need in the art to identify technologies for detection of
transplant rejection, immune conditions related to T cell
cytotoxicity (such as for example and not limitation graft versus
host disease (GvHD), autoimmune diseases, and immuno-oncology), and
sensing T cell cytotoxicity (e.g., to predict treatment efficacy in
patients being treated with cancer immune therapies such as
checkpoint blockade inhibitors or CAR T cell therapies) and use
this understanding to develop novel detection compositions and
methods of using such compositions to detect these conditions The
present disclosure satisfies this and other needs. Embodiments of
the present disclosure relate generally to such methods and
compositions and more specifically to compositions comprising
scaffolds linked to detectable protease-specific peptides that can
be administered to transplant recipients and used to detect acute
and chronic transplant rejection by cleavage of the composition via
the accumulation of the detectable peptides in a bodily fluid (such
as for example and not limitation, urine, lymphatic fluid, blood,
plasma, and/or saliva), locally at the site of cleavage, and/or
downstream lymph nodes.
[0057] Detecting the onset of transplant rejection is critical for
the long-term health and survival of the organ recipient, yet the
core biopsy remains the diagnostic gold standard despite its
invasiveness, risk of morbidity, and limited predictive power. For
example, during acute cellular rejection (ACR), host CD8 T cells
damage allograft tissue by releasing the protease granzyme B (GzmB)
to trigger donor cell death. To develop a noninvasive biomarker of
early ACR, the inventors engineered activity-based nanoprobes to
sense a target protease activity, e.g., GzmB, inside the body by
producing an amplified signal in host bodily fluid, e.g., urine,
for detection. These synthetic biomarkers can comprise target
protease peptide substrates conjugated to nanoparticles,
preferentially accumulate in allografts and secondary lymphoid
organs, and are activated during antigen-specific T cell killing.
For example, in a skin graft mouse model of transplant rejection,
systemic administration of synthetic biomarkers significantly
elevate urine signals at the onset of ACR before features of
rejection appear in graft tissue. This is a non-limiting example of
a noninvasive approach and may allow routine monitoring of
allograft immune health without the risk of a biopsy.
[0058] Therefore, as a non-limiting example, the inventors sought
to develop a noninvasive diagnostic assay to measure the activity
of GzmB within allograft tissue as an early biomarker of ACR. This
assay can enable detection of ACR before tissue damage begins, and
thus can provide a way to detect ACR before a biopsy of the
transplanted tissue would indicate ACR. This assay can also enable
monitoring of ACR over time, as compositions comprising the
activity-based nanosensor may be administered repeatedly over a
desired timeframe to a transplant recipient, and the diagnostic
assay repeated after each administration.
[0059] Herein is described an activity-based nanosensor that can be
administered intravenously (i.v.) to the recipient and can be, for
example, engineered to detect elevations in GzmB activity by
shedding a reporter into host urine as a noninvasive biomarker of
early ACR. The present protease-sensing synthetic biomarkers
leverage enzyme turnover to locally amplify detection signals, but
by contrast, these signals are further enriched from blood into
other bodily fluids, for example, urine, by renal filtration.
Alternatively, these biomarkers can enable local detection of
protease activity (e.g., by imaging), and/or detection in a
downstream lymph node. These mechanisms for signal amplification
can allow synthetic biomarkers to be ultrasensitive for early stage
disease (17-21). In skin graft mouse models of ACR, synthetic
biomarkers accumulate in allografts and in secondary lymphoid
organs to produce significantly elevated urine signals in mice
bearing allografts at the onset of ACR. These protease-sensing
synthetic biomarkers are noninvasive, predictive, and interact
directly with host immune responses against allografts to produce
amplified detection signals in urine.
[0060] The compositions of the invention can comprise detectable
peptide sequences (also referred to herein as peptide substrates
and/or detectable peptide substrates) that can comprise protease
recognition/cleavage sites linked/coupled to scaffolds (such as for
example and not limitation, protein scaffolds, polymer scaffolds,
and particles (e.g., a microparticle or nanoparticle)) (e.g.,
activity-based nanosensors), wherein the peptide-scaffold conjugate
can be capable of detecting protease activity in vivo and allowing
noninvasive detection and/or monitoring of physiological processes.
The detectable peptide sequences can be released at the site of
cleavage, which can produce a localized signal (which can be
detected at the cleavage site), and can accumulate in draining
lymph nodes, blood, urine, and other bodily fluids where they can
be detected (e.g., by methods as described in US20140303014 and
US2014/0363833, each of which is incorporated herein by
reference).
Definitions
[0061] To facilitate an understanding of the principles and
features of the various embodiments of the disclosure, various
illustrative embodiments are explained below. Although exemplary
embodiments of the disclosure are explained in detail, it is to be
understood that other embodiments are contemplated. Accordingly, it
is not intended that the disclosure is limited in its scope to the
details of construction and arrangement of components set forth in
the following description or examples. The disclosure is capable of
other embodiments and of being practiced or carried out in various
ways. Also, in describing the exemplary embodiments, specific
terminology will be resorted to for the sake of clarity.
[0062] It must also be noted that, as used in the specification and
the appended claims, the singular forms "a," "an" and "the" include
plural references unless the context clearly dictates otherwise.
For example, reference to a component is intended also to include
composition of a plurality of components. References to a
composition containing "a" constituent is intended to include other
constituents in addition to the one named. In other words, the
terms "a," "an," and "the" do not denote a limitation of quantity,
but rather denote the presence of "at least one" of the referenced
item.
[0063] As used herein, the term "and/or" may mean "and," it may
mean "or," it may mean "exclusive-or," it may mean "one," it may
mean "some, but not all," it may mean "neither," and/or it may mean
"both." The term "or" is intended to mean an inclusive "or."
[0064] Also, in describing the exemplary embodiments, terminology
will be resorted to for the sake of clarity. It is intended that
each term contemplates its broadest meaning as understood by those
skilled in the art and includes all technical equivalents which
operate in a similar manner to accomplish a similar purpose. It is
to be understood that embodiments of the disclosed technology may
be practiced without these specific details. In other instances,
well-known methods, structures, and techniques have not been shown
in detail in order not to obscure an understanding of this
description. References to "one embodiment," "an embodiment,"
"example embodiment," "some embodiments," "certain embodiments,"
"various embodiments," etc., indicate that the embodiment(s) of the
disclosed technology so described may include a particular feature,
structure, or characteristic, but not every embodiment necessarily
includes the particular feature, structure, or characteristic.
Further, repeated use of the phrase "in one embodiment" does not
necessarily refer to the same embodiment, although it may.
[0065] Ranges may be expressed herein as from "about" or
"approximately" or "substantially" one particular value and/or to
"about" or "approximately" or "substantially" another particular
value. When such a range is expressed, other exemplary embodiments
include from the one particular value and/or to the other
particular value. Further, the term "about" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within an
acceptable standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to .+-.20%,
preferably up to .+-.10%, more preferably up to .+-.5%, and more
preferably still up to .+-.1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated, the term "about"
is implicit and in this context means within an acceptable error
range for the particular value.
[0066] Similarly, as used herein, "substantially free" of
something, or "substantially pure", and like characterizations, can
include both being "at least substantially free" of something, or
"at least substantially pure", and being "completely free" of
something, or "completely pure".
[0067] By "comprising" or "containing" or "including" is meant that
at least the named compound, element, particle, or method step is
present in the composition or article or method, but does not
exclude the presence of other compounds, materials, particles,
method steps, even if the other such compounds, material,
particles, method steps have the same function as what is
named.
[0068] Throughout this description, various components may be
identified having specific values or parameters, however, these
items are provided as exemplary embodiments. Indeed, the exemplary
embodiments do not limit the various aspects and concepts of the
present disclosure as many comparable parameters, sizes, ranges,
and/or values may be implemented. The terms "first," "second," and
the like, "primary," "secondary," and the like, do not denote any
order, quantity, or importance, but rather are used to distinguish
one element from another.
[0069] It is noted that terms like "specifically," "preferably,"
"typically," "generally," and "often" are not utilized herein to
limit the scope of the claimed disclosure or to imply that certain
features are critical, essential, or even important to the
structure or function of the claimed disclosure. Rather, these
terms are merely intended to highlight alternative or additional
features that may or may not be utilized in a particular embodiment
of the present disclosure. It is also noted that terms like
"substantially" and "about" are utilized herein to represent the
inherent degree of uncertainty that may be attributed to any
quantitative comparison, value, measurement, or other
representation.
[0070] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "50 mm" is intended to mean "about 50 mm."
[0071] It is also to be understood that the mention of one or more
method steps does not preclude the presence of additional method
steps or intervening method steps between those steps expressly
identified. Similarly, it is also to be understood that the mention
of one or more components in a composition does not preclude the
presence of additional components than those expressly
identified.
[0072] The materials described hereinafter as making up the various
elements of the present disclosure are intended to be illustrative
and not restrictive. Many suitable materials that would perform the
same or a similar function as the materials described herein are
intended to be embraced within the scope of the disclosure. Such
other materials not described herein can include, but are not
limited to, materials that are developed after the time of the
development of the disclosure, for example. Any dimensions listed
in the various drawings are for illustrative purposes only and are
not intended to be limiting. Other dimensions and proportions are
contemplated and intended to be included within the scope of the
disclosure.
[0073] As used herein, the term "subject" or "patient" refers to
mammals and includes, without limitation, human and veterinary
animals. In a preferred embodiment, the subject is human.
[0074] The terms "treat" or "treatment" of a state, disorder or
condition include: (1) preventing or delaying the appearance of at
least one clinical or sub-clinical symptom of the state, disorder
or condition developing in a subject that may be afflicted with or
predisposed to the state, disorder or condition but does not yet
experience or display clinical or subclinical symptoms of the
state, disorder or condition; or (2) inhibiting the state, disorder
or condition, i.e., arresting, reducing or delaying the development
of the disease or a relapse thereof (in case of maintenance
treatment) or at least one clinical or sub-clinical symptom
thereof; or (3) relieving the disease, i.e., causing regression of
the state, disorder or condition or at least one of its clinical or
sub-clinical symptoms. The benefit to a subject to be treated is
either statistically significant or at least perceptible to the
patient or to the physician.
[0075] As used herein the term "therapeutically effective" applied
to dose or amount refers to that quantity of a compound or
pharmaceutical composition that when administered to a subject for
treating (e.g., preventing or ameliorating) a state, disorder or
condition, is sufficient to effect such treatment. The
"therapeutically effective amount" will vary depending on the
compound or bacteria or analogues administered as well as the
disease and its severity and the age, weight, physical condition
and responsiveness of the mammal to be treated.
[0076] As used herein, the term "combination" of a composition
according to the present disclosure and at least a second
pharmaceutically active ingredient means at least two, but any
desired combination of compounds can be delivered simultaneously or
sequentially (e.g., within a 24 hour period).
[0077] Within the meaning of the present disclosure, the term
"conjoint administration" is used to refer to administration of a
composition according to the disclosure and another therapeutic
agent simultaneously in one composition, or simultaneously in
different compositions, or sequentially (preferably, within a 24
hour period).
[0078] The phrase "pharmaceutically acceptable", as used in
connection with compositions of the disclosure, refers to molecular
entities and other ingredients of such compositions that are
physiologically tolerable and do not typically produce untoward
reactions when administered to a mammal (e.g., a human).
Preferably, as used herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in mammals, and more particularly
in humans.
[0079] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous solution saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Alternatively, the
carrier can be a solid dosage form carrier, including but not
limited to one or more of a binder (for compressed pills), a
glidant, an encapsulating agent, a flavorant, and a colorant.
Suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0080] The term "a control level" as used herein encompasses
predetermined standards (e.g., a published value in a reference) as
well as levels determined experimentally in similarly processed
samples from control subjects (e.g., BMI-, age-, and gender-matched
subjects without asthma as determined by standard examination and
diagnostic methods) and/or from the subject prior to undergoing
transplant surgery.
[0081] In accordance with the present disclosure there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985);
Transcription and Translation (B. D. Hames & S. J. Higgins,
eds. (1984); Animal Cell Culture (R. I. Freshney, ed. (1986);
Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994); among others.
Compositions and Methods of the Disclosure
[0082] The compositions and methods of the disclosure can be used
to detect proteolytic activity of proteases associated with immune
conditions related to T cell cytotoxicity (e.g., graft versus host
disease (GvHD), autoimmune diseases), as well as with both acute
and chronic transplant rejection and with T cell cytotoxicity
(e.g., to predict treatment efficacy in patients being treated with
cancer immune therapies such as checkpoint blockade inhibitors or
CAR T cell therapies). These proteases include but are not limited
to T cell proteases (such as for example and not limitation,
Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, and Cathepsin
X), complement proteases (such as for example and not limitation,
Cls, Clr, MASP2, Factor I, Factor D), fibrosis proteases (such as
for example and not limitation, ADAMTS1, MMP2, MMP9), and
inflammatory proteases (such as for example and not limitation,
elastase, cathepsin G, PR-3, thrombin, kallikreins 1&6,
tryptase, and chymase). These proteases have activities that can
allow differentiation between acute (mediated by, e.g., T cell
cytotoxicity) from chronic (mediated by, e.g., complement proteases
and fibrosis) organ transplant rejection. These proteases are also
known to be involved in T cell killing (e.g., Granzyme B, Granzyme
A), T cell activation (e.g., MALT1, Caspase 8, Calpain 2, Cathepsin
X), apoptosis (e.g., Caspase 3, Caspase 8), complement activation
(e.g., Cls, Clr, MASP2, Factor I, Factor D), fibrosis (e.g.,
ADAMTS1, MMP2, MMP9), and inflammation (e.g., elastase, cathepsin
G, PR-3, thrombin, kallikreins 1&6, tryptase, and chymase).
[0083] In order to detect proteolytic activity, the compositions of
the present disclosure can be designed to contain one or more
detectable peptide sequences (also referred to herein as peptide
substrates and/or detectable peptide substrates) that are capable
of being recognized by the proteases and are also linked or coupled
to scaffolds (such as for example and not limitation, protein
scaffolds, polymer scaffolds, and particles (e.g., microparticles
or nanoparticles)), and the detectable peptide sequences can
accumulate in a bodily fluid (such as for example and not
limitation, urine, plasma, draining lymph, blood, saliva, etc.)
after being cleaved from the peptide-scaffold complex by the
protease of interest. The composition can further comprise an
optional spacer region located between the one or more detectable
peptide sequences and the scaffold and adjacent either side of the
linker region.
[0084] Also disclosed herein is a conjugate that can comprise one
or more peptide sequences operably linked to at least one scaffold,
wherein the conjugate can be recognized by a protease as described
herein. In some embodiments, the one or more peptide sequences
and/or the scaffold of the conjugate can be capable of generating a
detectable signal such that the conjugate can be visualized and
tracked in real time. In some embodiments, the one or more
detectable peptide sequences can comprise a reporter (also referred
to herein as a reporter domain) and a protease cleavage/recognition
site.
[0085] When the composition or conjugate is exposed to enzymes, for
instance, proteases, the at least one detectable peptide sequence
can be cleaved, such that the reporter of the detectable peptide
sequence is released. The reporter can be detected locally at the
site of cleavage (i.e., at the site of the protease activity) or in
the subject's blood or plasma (e.g., by ELISA), or can travel to
one or more nearby draining lymph nodes and be detected there, or
can travel to the kidney and be renally-cleared and detectable in
urine. The reporter thus functions as a "messenger" of enzyme
activity. In the absence of enzyme activity, the composition or
conjugate can remain uncleaved, indicating that the proteases are
not active. The reporter includes for example and not limitation, a
fluorophore (e.g., for fluorescent detection), a luminescent
reporter (e.g., for bioluminescent assays), a ligand encoded
reporter (e.g., for detection by ELISA or other antibody detection
systems), a mass tag (e.g., for detection by mass spectrometry),
and a nucleic acid tag (e.g., for detection by PCR), etc. In some
embodiments, the detectable peptide sequence can be detected by a
method such as, for example and not limitation, Sanger sequencing,
pyrosequencing, SOLID sequencing, massively parallel sequencing,
barcoded DNA sequencing, PCR, real-time PCR, quantitative PCR,
microarray analysis of the isolated nucleic acid with a gene chip,
restriction fragment length polymorphism analysis, allele specific
ligation, comparative genomic hybridization, microarray/microchip
analysis of the isolated nucleic acid, DNA/RNA in situ
hybridization, RNAse protection assay, Northern blot, reverse
transcriptase PCR, quantitative PCR, quantitative reverse
transcriptase PCR, quantitative real-time reverse transcriptase
PCR, reverse transcriptase treatment followed by direct sequencing,
flow cytometry, bead-based flow-cytometry, immunohistochemistry,
ELISA, RIA, Western blot, immunoaffinity chromatography, HPLC, mass
spectrometry, mass spectroscopy, protein microarray/microchip
analysis, PAGE analysis, isoelectric focusing, immunoturbidimetry,
rapid immunodiffusion, laser nephelometry, visual agglutination,
quantitative Western blot analysis, multiple reaction
monitoring-mass spectrometry (MRM Proteomics), Lowry assay,
Bradford assay, BCA assay, UV spectroscopic assays, fluorescent
assays, luminescent assays, and 2-D gel electrophoresis. In some
embodiments, the peptide sequences are detected by methods
described in any of WO2007/106415, US2010/0240050, US2014/0303014
and US2014/0363833, each of which is incorporated herein by
reference.
[0086] The scaffold can comprise a protein scaffold (e.g., albumin,
IgG, IgG Fc, antibody-based, antibody fragment-based, etc.), a
polymer scaffold (e.g., PEG, PLGA), a DNA scaffold (e.g., (DNA
organisms), a sugar scaffold (e.g., dextran), imaging (e.g.,
magnetic resonance imaging) contrast agents (e.g., gadolinium, iron
oxide) and a particle (e.g., a microparticle or nanoparticle, such
as for example and not limitation, nanostructures including
nanofibers, nanorods, nanotubes). In some embodiments, the scaffold
is detectable, e.g., by fluorescence, mass spectrometry, magnetic
imaging, ELISA, luminescence, etc. The scaffold can have a size
ranging from 3 nanometers to 2 micrometers, including from 10
nanometers to 1.5 micrometers, 20 nanometers to 1 micrometer, 50
nanometers to 0.1 micrometer, and 50 nanometers to 150 nanometers.
The scaffold can provide the composition with a longer circulation
half-life, such as for example and not limitation, by preventing
the composition from being trafficked to the lymph and/or being
cleared from circulation due to its small size. The circulation
half-life of the compositions of the disclosure can be at least 1
hour to 52 weeks, from 2 hours to 36 hours, from 3 hours to 24
hours, and/or 5 hours to 12 hours. The scaffold can be monovalent
or polyvalent, meaning that it can be coupled to at least one
detectable peptide sequence to 5,000 detectable peptide sequences,
including from 50-100 detectable peptide sequences.
[0087] As used herein, the term "particle" includes nanoparticles
as well as microparticles. Nanoparticles are defined as particles
of less than 1.0 .mu.m in diameter. A preparation of nanoparticles
includes particles having an average particle size of less than 1.0
.mu.m in diameter. Microparticles are particles of greater than 1.0
.mu.m in diameter but less than 1 mm. A preparation of
microparticles includes particles having an average particle size
of greater than 1.0 .mu.m in diameter. The microparticles may
therefore have a diameter of at least 5, at least 10, at least 25,
at least 50, or at least 75 microns, including sizes in ranges of
5-10 microns, 5-15 microns, 5-20 microns, 5-30 microns, 5-40
microns, or 5-50 microns. A composition of particles may have
heterogeneous size distributions ranging from 10 nm to mm sizes. In
some embodiments, the diameter is about 5 nm to about 500 nm. In
other embodiments, the diameter is about 100 nm to about 200 nm. In
other embodiments, the diameter is about 10 nm to about 100 nm. The
particles may be composed of a variety of materials including iron,
ceramic, metallic, natural polymer materials (including lipids,
sugars, chitosan, hyaluronic acid, etc.), synthetic polymer
materials (including poly-lactide-coglycolide, poly-glycerol
sebacate, etc.), and non-polymer materials, or combinations
thereof. The polymers may be biodegradable (such as for example and
not limitation, synthetic polymers such as polymers of lactic acid
and glycolic acid, polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butic acid), poly(valeric acid),
poly(caprolactone), poly(hydroxybutyrate),
poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), and
natural polymers such as algninate and other polysaccharides
including dextran and cellulose, collagen, chemical derivatives
thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art), albumin
and other hydrophilic proteins, zein and other prolamines and
hydrophobic proteins, copolymers and mixtures thereof. In general,
these materials degrade either by enzymatic hydrolysis or exposure
to water in vivo, by surface or bulk erosion. The foregoing
materials may be used alone, as physical mixtures (blends), or as
co-polymers. In some embodiments the polymers are polyesters,
polyanhydrides, polystyrenes, polylactic acid, polyglycolic acid,
and copolymers of lactic and glycoloic acid and blends thereof), or
non-biodegradable (such as for example and not limitation, ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof). The particles may be composed in whole or in
part of polymers or non-polymer materials. Non-polymer materials,
for example, may be employed in the preparation of the particles.
Exemplary materials include alumina, calcium carbonate, calcium
sulfate, calcium phosphosilicate, sodium phosphate, calcium
aluminate, calcium phosphate, hydroxyapatite, tricalcium phosphate,
dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate,
amorphous calcium phosphate, octacalcium phosphate, and silicates.
In certain embodiments, the particles may comprise a calcium salt
such as calcium carbonate, a zirconium salt such as zirconium
dioxide, a zinc salt such as zinc oxide, a magnesium salt such as
magnesium silicate, a silicon salt such as silicon dioxide or a
titanium salt such as titanium oxide or titanium dioxide.
[0088] The detectable peptide sequences may be coupled to one or
more linkers to aid in joining the one or more peptide sequences to
the scaffold. The linker may be any suitable linker for joining
peptide sequences to scaffolds. In one embodiment, the linker
comprises a small molecule linker SIA to join primary amines on
nanoparticle surface to thiols on cysteine-terminated peptides. In
another embodiment, the linker comprises small molecule linkers
such as for example and not limitation, amine to sulfhydryl linkers
(e.g., BMPS, MBS, SMCC, SMPH, SPDP, SBAP), carboxyl to amine
linkers (e.g., DCC, EDC, EDAC), and/or sulfhydryl-to-carbohydrate
linkers (e.g., BMPH, EMCH, MPBH). In one embodiment, the linker may
contain a thiol group, such as for example and not limitation, in a
cysteine residue (which may be in D or L form). Suitable linkers
are well known to those of skill in the art and include, but are
not limited to, straight or branched-chain carbon linkers,
heterocyclic carbon linkers, peptide linkers, nucleic acid
molecules, polypeptides, lipids, fatty acids, peptide nucleic
acids, aptamers, DNA, RNA, leucine zippers, oligonucleotides,
oligopeptides, biotin, avidin, streptavidin, haptene antibody bonds
or biotin avidin bonds. Linkers can also be derivatives of PEG or
other biocompatible polymers of different sizes. The linkers can be
capable of forming covalent bonds to amino groups, carboxyl groups,
or sulfhydryl groups or hydroxyl groups. Amino-binding linkers
include reactive groups such as carboxyl groups, isocyanates,
isothiocyanates, esters, haloalkyls, and the like. Carboxyl-binding
linkers are capable of forming include reactive groups such as
various amines, hydroxyls and the like. Sulfhydryl-binding linkers
include reactive groups such as sulfhydryl groups, acrylates,
isothiocyanates, isocyanates and the like. Hydroxyl binding groups
include reactive groups such as carboxyl groups, isocyanates,
isothiocyanates, esters, haloalkyls, and the like. In certain
embodiments, an end of the linker is capable of binding to a
crystalline composition formed from a Group IV metal element. The
linker may be of any suitable length and can be varied to bring the
detectable peptide sequence and the scaffold closer together or
farther apart as desired. Exemplary short linkers may be strands of
RNA, DNA, short amino acid sequences, polypeptides, fatty acids,
proteins, antibodies, or other small molecules. In some
embodiments, the detectable peptide sequences are linked/coupled to
the at least one scaffold by a thiol group, such as that of a
cysteine residue (e.g., a N-terminal and/or C-terminal and/or
internal cysteine residue). In other embodiments, the detectable
peptide sequences are linked/coupled to the scaffold by a lysine
linker or residue (e.g., a N-terminal and/or C-terminal and/or
internal lysine linker or residue). In some embodiments, the one or
more detectable peptide sequences are operably linked to the at
least one scaffold through a linker or chemical linkage comprising
at least one bond selected from the group consisting of: a covalent
bond, an electrostatic bond and a chelation bond. In some
embodiments, the bond is a covalent bond, which can be a bond
through a functional group selected from the group consisting of: a
hydroxyl, a carboxyl, a carbonyl, a sulfhydryl, an amine, an amide,
a nitrile, a nitrogen with a free lone pair of electrons, an amino
acid, a thiol, a polyethylene glycol, a sulfonic acid, a sulfonyl
halide, and an acyl halide. In some embodiments, the bond is an
amide bond formed through reaction of a carboxyl group of the at
least one scaffold and an amine group of the one or more detectable
peptide sequences. In some embodiments, the bond is a thioether
bond formed through a reaction involving the thiol group of a
natural or engineered cysteine residue of the one or more
detectable peptide sequences. In some embodiments, the one or more
peptide sequences are operably linked to at least one scaffold
through a linker or chemical linkage comprising at least one
chelation bond. The chelation bond can be formed between a metal of
the scaffold and a metal-chelating ligand attached to or otherwise
associated with the one or more peptide sequences. The
metal-chelating ligand can comprise one or more naturally occurring
or engineered histidine residues of the one or more detectable
peptide sequences. In some embodiments, the metal-chelating ligand
comprises a histidine tag fused to the N-terminus or the C-terminus
of the one or more peptide sequences. In some embodiments, the one
or more detectable peptide sequences are linked to the at least one
scaffold by enzymatic linkages, such as for example and not
limitation, by a bond formed by a ligase, sortase mediated
linkages, HaloTag linkages, SNAP-tag linkages, CLIP-tag linkages,
full-length or split intein-mediated linkages, BirA-mediated
linkages, Sfp-mediated linkages, and other bioconjugation methods.
In some embodiments, the linker is a protein of 10-100 amino acids
in length. Optionally, the linker may be 8 nm-100 nm, 6 nm-100 nm,
8 nm-80 nm, 10 nm-100 nm, 13 nm-100 nm, 15 nm-50 nm, or 10 nm-50 nm
in length.
[0089] The compositions of the invention may also comprise at least
one optional spacer region located between the one or more
detectable peptide sequences and the scaffold. The optional spacer
region(s) may be located on either side of the linker, e.g.,
between the linker and the scaffold and/or between the linker and
the one or more detectable peptide sequences. In some embodiments,
the spacer region can comprise a GGS amino acid sequence. In other
embodiments, the spacer can be a small peptide, such as for example
and not limitation, GGS, GGGS, (GGGS).sup.2, (GGGS).sup.3,
(Gly).sup.6, (Gly).sup.8, (EAAK).sup.3, PAPAP, A(EAAAK).sup.3. In
some embodiments, the linker can comprise primarily D-form amino
acids to resist proteolytic cleavage. Spacers can also be
derivatives of PEG or other biocompatible polymers of different
sizes.
[0090] The peptide sequences of the disclosure can include those
that can be recognized by proteases associated with immune
conditions (e.g., graft versus host disease (GvHD), autoimmune
diseases), as well as with both acute and chronic transplant
rejection and with T cell cytotoxicity (e.g., to predict treatment
efficacy in patients being treated with cancer immune therapies
such as checkpoint blockade inhibitors or CAR T cell therapies). In
one embodiment, the peptide sequences are recognized by Granzyme B
(GzmB), such as a recombinant GzmB (SEQ ID NO: 1). In another
embodiment, the peptide sequences are recognized by T
cell-associated proteases (such as for example and not limitation,
Granzyme B, Granzyme A, MALT1, Caspase 8, Calpain 2, and Cathepsin
X). In yet another embodiment, the peptide sequences are recognized
by complement-associated proteases (such as for example and not
limitation, Cls, Clr, MASP2, Factor I, Factor D). In one
embodiment, the peptide sequences are recognized by
fibrosis-associated proteases (such as for example and not
limitation, ADAMTS1, MMP2, MMP9). In another embodiment, the
peptide sequences are recognized by inflammation-associated
proteases (such as for example and not limitation, elastase,
cathepsin G, PR-3, thrombin, kallikreins 1&6, tryptase, and
chymase). The peptide sequences may contain amino acids in all L
form, all D form, or a mix of L and D forms (ranging from 50% D and
50% L forms to 99% D or L form and 1% L or D form).
[0091] In some embodiments, the one or more detectable peptide
sequences linked to the scaffold comprises a reporter, a GzmB
recognition/cleavage sequence comprising any of SEQ ID NOs 2 to 81,
and combinations thereof, and another peptide, protein, nucleic
acid, lipid, fatty acid, oligonucleotide, oligopeptide, oligolipid,
antibody, antibody fragment, aptamer and/or binding protein
(including binding protein fragments), which can aid in coupling
the detectable peptide to the scaffold. The peptide sequences
comprising any of SEQ ID NOs 2 to 81 can contain amino acids in all
L form, all D form, or a mix of L and D forms (ranging from 50% D
and 50% L forms to 99% D or L form and 1% L or D form). In one
specific embodiment, the one or more peptide sequences comprises a
GzmB recognition/cleavage sequence comprising any of SEQ ID NOs 2
to 81 and combinations thereof, and polyethylene glycol (e.g., one
or more polyethylene glycol molecules). The polyethylene glycol may
be of any suitable molecular weight, such as for example and not
limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000
to 50,000, and 5,000 to 20,000.
[0092] In other embodiments, the one or more detectable peptide
sequences linked to the scaffold comprises a reporter, a protease
recognition/cleavage sequence comprising any of SEQ ID NOs 82 to
136, and combinations thereof, and another peptide, protein,
nucleic acid, lipid, fatty acid, oligonucleotide, oligopeptide,
oligolipid, antibody, antibody fragment, aptamer and/or binding
protein (including binding protein fragments), which can aid in
coupling the detectable peptide to the scaffold. The peptide
sequences comprising any of SEQ ID NOs 82 to 136 can contain amino
acids in all L form, all D form, or a mix of L and D forms (ranging
from 50% D and 50% L forms to 99% D or L form and 1% L or D form).
In one specific embodiment, the one or more peptide sequences
comprises a reporter, a protease recognition/cleavage sequence
comprising any of SEQ ID NOs 82 to 136 and combinations thereof,
and polyethylene glycol (e.g., one or more polyethylene glycol
molecules). The polyethylene glycol may be of any suitable
molecular weight, such as for example and not limitation, 100 to
200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and
5,000 to 20,000.
[0093] In specific embodiments, the one or more detectable peptide
sequences linked to the scaffold may comprise a
recognition/cleavage site for GzmB. In some embodiments, the
peptide may comprise an amino acid sequence comprising at least one
of AIEPDGSC (SEQ ID NO: 2), ASGIEPDSGGSC (SEQ ID NO: 3),
AKSKIEFDFGVKKC (SEQ ID NO: 4), AIEPDSGC (SEQ ID NO: 5), AIEPDGSSKC
(SEQ ID NO: 6), AIEPDSGSKC (SEQ ID NO: 7), AKSIEPDGSSKC (SEQ ID NO:
8), AKSIEPDSGSKC (SEQ ID NO: 9), AIEFDGSC (SEQ ID NO: 10), AIEFDSGC
(SEQ ID NO: 11), AIEFDSGSKC (SEQ ID NO: 12), AKSIEFDSGSKC (SEQ ID
NO: 13), AIEFDSGVSKC (SEQ ID NO: 14), and combinations thereof,
including combinations with any peptide disclosed herein. Suitable
linkers may be added to the N-terminus and/or C-terminus of the
peptide, and/or to residues within the one or more peptide
sequences. The amino acids can be present in all L form, all D
form, or a mix of L and D forms (ranging from 50% D and 50% L forms
to 99% D or L form and 1% L or D form). The disclosure also
contemplates nucleic acids (e.g., DNA and RNA) encoding each of SEQ
ID NOs 2-14, and modifications thereof (e.g., linkers) as well as
vectors comprising such nucleic acids, and host cells comprising
such vectors. The peptides comprising any of the amino acid
sequences comprising SEQ ID NOs 2-14 may further comprise one or
more of polyethylene glycol (of any suitable molecular weight, such
as for example and not limitation, 100 to 200,000, 1000 to 100,000,
2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an
internal lysine residue for linking, and/or a cysteine residue at
the N-terminus and/or C-terminus. The disclosure further
contemplates methods of making said vectors and host cells
according to cloning methods well known in the art.
[0094] In some embodiments, the one or more detectable peptide
sequences linked to the scaffold may comprise an amino acid
sequence comprising one or more D-form amino acids, such as for
example and not limitation, at least one of AIEPDGSc (SEQ ID NO:
15), AsGIEPDSGGsc (SEQ ID NO: 16), AksKIEFDFGVKkc (SEQ ID NO: 17),
AIEPDSGc (SEQ ID NO: 18), AIEPDGSskc (SEQ ID NO: 19), AIEPDSGskc
(SEQ ID NO: 20), AksIEPDGSskc (SEQ ID NO: 21), AksIEPDSGskc (SEQ ID
NO: 22), AIEFDGSc (SEQ ID NO: 23), AIEFDSGc (SEQ ID NO: 24),
AIEFDSGskc (SEQ ID NO: 25), AksIEFDSGskc (SEQ ID NO: 26),
AIEFDSGVskc (SEQ ID NO: 27), and combinations thereof, including
combinations with any peptide disclosed herein. Suitable linkers
may be added to the N-terminus and/or C-terminus of the peptide,
and/or to residues within the one or more peptide sequences. The
disclosure also contemplates nucleic acids (e.g., DNA and RNA)
encoding each of SEQ ID NOs 15-27, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NOs 15-27 may further
comprise one or more of polyethylene glycol (of any suitable
molecular weight, such as for example and not limitation, 100 to
200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and
5,000 to 20,000), Glufib, an internal lysine residue for linking,
and/or a cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0095] In other embodiments, the one or more detectable peptide
sequences linked to the scaffold comprises a GzmB
recognition/cleavage sequence and a Glu-1-Fibrinopeptide B peptide
EGVNDNEEGFFSAR (Glufib peptide; SEQ ID NO: 28). In one embodiment,
the Glufib peptide comprises one or more D-form amino acids, such
as for example and not limitation, eGvndneeGffsar (lowercase
letters denote D-form amino acids; SEQ ID NO: 29). In some
embodiments, the peptide comprises an amino acid sequence
comprising at least one of AIEPDGSC (SEQ ID NO: 30), ASGIEPDSGGSC
(SEQ ID NO: 31), AKSKIEFDFGVKKC (SEQ ID NO: 32), AIEPDSGC (SEQ ID
NO: 33), AIEPDGSSKC (SEQ ID NO: 34), AIEPDSGSKC (SEQ ID NO: 35),
AKSIEPDGSSKC (SEQ ID NO: 36), AKSIEPDSGSKC (SEQ ID NO: 37),
AIEFDGSC (SEQ ID NO: 38), AIEFDSGC (SEQ ID NO: 39), AIEFDSGSKC (SEQ
ID NO: 40), AKSIEFDSGSKC (SEQ ID NO: 41), AIEFDSGVSKC (SEQ ID NO:
42), aIEPDGSc (SEQ ID NO: 43), asGIEPDSGGsc (SEQ ID NO: 44),
aksKIEFDFGVKkc (SEQ ID NO: 45), aIEPDSGc (SEQ ID NO: 46),
aIEPDGSskc (SEQ ID NO: 47), aIEPDSGskc (SEQ ID NO: 48),
aksIEPDGSskc (SEQ ID NO: 49), aksIEPDSGskc (SEQ ID NO: 50),
aIEFDGSc (SEQ ID NO: 51), aIEFDSGc (SEQ ID NO: 52), aIEFDSGskc (SEQ
ID NO: 53), aksIEFDSGskc (SEQ ID NO: 54), aIEFDSGVskc (SEQ ID NO:
55), and combinations thereof, which are operatively (e.g.,
transcriptionally and/or translationally) linked/coupled to
EGVNDNEEGFFSAR (SEQ ID NO: 28) and/or eGvndneeGffsar (SEQ ID NO:
29). In specific embodiments, the peptide comprises an amino acid
sequence comprising one or more of EGVNDNEEGFFSARKAIEPDGSC (SEQ ID
NO: 56), EGVNDNEEGFFSARKASGIEPDSGGSC (SEQ ID NO: 57),
EGVNDNEEGFFSARKAKSKIEFDFGVKKC (SEQ ID NO: 58),
EGVNDNEEGFFSARKAIEPDSGC (SEQ ID NO: 59), EGVNDNEEGFFSARKAIEPDGSSKC
(SEQ ID NO: 60), EGVNDNEEGFFSARKAIEPDSGSKC (SEQ ID NO: 61),
EGVNDNEEGFFSARKAKSIEPDGSSKC (SEQ ID NO: 62),
EGVNDNEEGFFSARKAKSIEPDSGSKC (SEQ ID NO: 63),
EGVNDNEEGFFSARKAIEFDGSC (SEQ ID NO: 64), EGVNDNEEGFFSARKAIEFDSGC
(SEQ ID NO: 65), EGVNDNEEGFFSARKAIEFDSGSKC (SEQ ID NO: 66),
EGVNDNEEGFFSARKAKSIEFDSGSKC (SEQ ID NO: 67),
EGVNDNEEGFFSARKAIEFDSGVSKC (SEQ ID NO: 68), eGvndneeGffsarKaIEPDGSc
(SEQ ID NO: 69), eGvndneeGffsarKasGIEPDSGGsc (SEQ ID NO: 70),
eGvndneeGffsarKaksKIEFDFGVKkc (SEQ ID NO: 71),
eGvndneeGffsarKaIEPDSGc (SEQ ID NO: 72), eGvndneeGffsarKaIEPDGSskc
(SEQ ID NO: 73), eGvndneeGffsarKaIEPDSGskc (SEQ ID NO: 74),
eGvndneeGffsarKaksIEPDGSskc (SEQ ID NO: 75),
eGvndneeGffsarKaksIEPDSGskc (SEQ ID NO: 76),
eGvndneeGffsarKaIEFDGSc (SEQ ID NO: 77), eGvndneeGffsarKaIEFDSGc
(SEQ ID NO: 78), eGvndneeGffsarKaIEFDSGskc (SEQ ID NO: 79),
eGvndneeGffsarKaksIEFDSGskc (SEQ ID NO: 80),
eGvndneeGffsarKaIEFDSGVskc (SEQ ID NO: 81), and combinations
thereof, including combinations with any peptide disclosed herein.
Suitable linkers may be added to the N-terminus and/or C-terminus
of the one or more peptide sequences, and/or to residues within the
one or more peptide sequences. The amino acids can be present in
all L form, all D form, or a mix of L and D forms (ranging from 50%
D and 50% L forms to 99% D or L form and 1% L or D form). The
disclosure also contemplates nucleic acids (e.g., DNA and RNA)
encoding each of SEQ ID NOs 28-81, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NOs 30-81 may further
comprise one or more of polyethylene glycol (of any suitable
molecular weight, such as for example and not limitation, 100 to
200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and
5,000 to 20,000), Glufib, an internal lysine residue for linking,
and/or a cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0096] In other embodiments, the one or more detectable peptide
sequences linked to the scaffold comprises a recognition/cleavage
site that can be recognized by another T cell-related protease,
e.g., a protease involved in T cell killing and/or a protease
involved in T cell activation. Non-limiting examples of proteases
involved in T cell killing include Granzyme B and Granzyme A.
Non-limiting examples of proteases involved in T cell activation
include MALT1, Caspase 8, Calpain 2, and Cathepsin X.
[0097] In an embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Granzyme A
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of AAPVRSL (SEQ ID
NO: 82), ALDPRSF (SEQ ID NO: 83), ATQNKAS (SEQ ID NO: 84) and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding each of SEQ ID NOs 82-84, and modifications
thereof (e.g., linkers) as well as vectors comprising such nucleic
acids, and host cells comprising such vectors. The peptides
comprising any of the amino acid sequences comprising SEQ ID NOs
82-84 may further comprise one or more of polyethylene glycol (of
any suitable molecular weight, such as for example and not
limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000
to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue
for linking, and/or a cysteine residue at the N-terminus and/or
C-terminus. The disclosure further contemplates methods of making
said vectors and host cells according to cloning methods well known
in the art.
[0098] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a MALT1
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising ACYLD (SEQ ID NO: 85) and
combinations including combinations with any peptide disclosed
herein. Suitable linkers may be added to the N-terminus and/or
C-terminus of the one or more peptide sequences, and/or to residues
within the one or more peptide sequences. The amino acids can be
present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NO: 85, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NO: 85 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0099] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Caspase 8
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of AIETDGS (SEQ ID
NO: 86) and ALEVDCY (SEQ ID NO: 87) and combinations thereof,
including combinations with any peptide disclosed herein. Suitable
linkers may be added to the N-terminus and/or C-terminus of the one
or more peptide sequences, and/or to residues within the one or
more peptide sequences. The amino acids can be present in all L
form, all D form, or a mix of L and D forms (ranging from 50% D and
50% L forms to 99% D or L form and 1% L or D form). The disclosure
also contemplates nucleic acids (e.g., DNA and RNA) encoding each
of SEQ ID NOs 86-87, and modifications thereof (e.g., linkers) as
well as vectors comprising such nucleic acids, and host cells
comprising such vectors. The peptides comprising any of the amino
acid sequences comprising SEQ ID NOs 86-87 may further comprise one
or more of polyethylene glycol (of any suitable molecular weight,
such as for example and not limitation, 100 to 200,000, 1000 to
100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000),
Glufib, an internal lysine residue for linking, and/or a cysteine
residue at the N-terminus and/or C-terminus. The disclosure further
contemplates methods of making said vectors and host cells
according to cloning methods well known in the art.
[0100] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Caspase 3
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of ADEVDNK (SEQ ID
NO: 116), ADEVDGV (SEQ ID NO: 117), ADEVDRD (SEQ ID NO: 118),
ADEVDGV (SEQ ID NO: 119) and ALEVDCY (SEQ ID NO: 120) and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding each of SEQ ID NOs 86-87, and modifications
thereof (e.g., linkers) as well as vectors comprising such nucleic
acids, and host cells comprising such vectors. The peptides
comprising any of the amino acid sequences comprising SEQ ID NOs
86-87 may further comprise one or more of polyethylene glycol (of
any suitable molecular weight, such as for example and not
limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000
to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue
for linking, and/or a cysteine residue at the N-terminus and/or
C-terminus. The disclosure further contemplates methods of making
said vectors and host cells according to cloning methods well known
in the art.
[0101] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Calpain2
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising AEPLFAERK (SEQ ID NO: 88) and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NO: 88, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NO: 88 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0102] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Cathepsin X
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising AMNPKFA (SEQ ID NO: 89) and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NO: 89, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NO: 89 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0103] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a Cls, Clr, MASP2,
Factor I, and/or Factor D recognition/cleavage site, such as for
example and not limitation, an amino acid sequence comprising at
least one of ALQRIYKC (SEQ ID NO: 90), AKSVARTLLVKC (SEQ ID NO:
91), AEEKQRIIGC (SEQ ID NO: 92), AQRQRIIGGC (SEQ ID NO: 93),
ALGRGGSC (SEQ ID NO: 94), AKYLGRSYKVC (SEQ ID NO: 95), ARALERGLQDC
(SEQ ID NO: 96), ASLGRKIQIC (SEQ ID NO: 97), AGLQRALEIC (SEQ ID NO:
98), AKVFMGRVYDPC (SEQ ID NO: 99), ASSTGRNGFKC (SEQ ID NO: 100),
AKTTGGRIYGGC (SEQ ID NO: 101), ADPRGGSC (SEQ ID NO: 102), AVPRGGSC
(SEQ ID NO: 103), ALPSRSSKIC (SEQ ID NO: 104), AHRGRTLEIC (SEQ ID
NO: 105), ASTGRNGFKC(SEQ ID NO: 106), AQQKRKIVLC (SEQ ID NO: 107),
AQARKIVLC (SEQ ID NO: 108), AQARGGSC (SEQ ID NO: 109), and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding each of SEQ ID NOs 90-109, and modifications
thereof (e.g., linkers) as well as vectors comprising such nucleic
acids, and host cells comprising such vectors. The peptides
comprising any of the amino acid sequences comprising SEQ ID NOs
90-109 may further comprise one or more of polyethylene glycol (of
any suitable molecular weight, such as for example and not
limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000
to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue
for linking, and/or a cysteine residue at the N-terminus and/or
C-terminus. The disclosure further contemplates methods of making
said vectors and host cells according to cloning methods well known
in the art.
[0104] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises an ADAMTS1
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of AIPENFF (SEQ ID
NO: 110), AKEEEGL (SEQ ID NO: 111), ANLVYMV (SEQ ID NO: 112), and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding each of SEQ ID NOs 110-112, and modifications
thereof (e.g., linkers) as well as vectors comprising such nucleic
acids, and host cells comprising such vectors. The peptides
comprising any of the amino acid sequences comprising SEQ ID NOs
110-112 may further comprise one or more of polyethylene glycol (of
any suitable molecular weight, such as for example and not
limitation, 100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000
to 50,000, and 5,000 to 20,000), Glufib, an internal lysine residue
for linking, and/or a cysteine residue at the N-terminus and/or
C-terminus. The disclosure further contemplates methods of making
said vectors and host cells according to cloning methods well known
in the art.
[0105] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a MMP2
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of ARLAAIT (SEQ ID
NO: 113), ASLSRLT (SEQ ID NO: 114), and combinations thereof,
including combinations with any peptide disclosed herein. Suitable
linkers may be added to the N-terminus and/or C-terminus of the one
or more peptide sequences, and/or to residues within the one or
more peptide sequences. The amino acids can be present in all L
form, all D form, or a mix of L and D forms (ranging from 50% D and
50% L forms to 99% D or L form and 1% L or D form). The disclosure
also contemplates nucleic acids (e.g., DNA and RNA) encoding each
of SEQ ID NOs 113-114, and modifications thereof (e.g., linkers) as
well as vectors comprising such nucleic acids, and host cells
comprising such vectors. The peptides comprising any of the amino
acid sequences comprising SEQ ID NOs 113-114 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0106] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a MMP9
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising APLGVRGK (SEQ ID NO: 115), and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NO: 115, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NO: 115 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0107] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises an elastane, cathepsin
G, and/or PR-3 recognition/cleavage site, such as for example and
not limitation, an amino acid sequence comprising at least one of
AAAPVc (SEQ ID NO: 121), AAAPAc (SEQ ID NO: 122), AAAPLc (SEQ ID
NO: 123), AAAPMc (SEQ ID NO: 124), AAAPFc (SEQ ID NO: 125), and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NOs 121-125, and modifications thereof
(e.g., linkers) as well as vectors comprising such nucleic acids,
and host cells comprising such vectors. The peptides comprising any
of the amino acid sequences comprising SEQ ID NOs 121-125 may
further comprise one or more of polyethylene glycol (of any
suitable molecular weight, such as for example and not limitation,
100 to 200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000,
and 5,000 to 20,000), Glufib, an internal lysine residue for
linking, and/or a cysteine residue at the N-terminus and/or
C-terminus. The disclosure further contemplates methods of making
said vectors and host cells according to cloning methods well known
in the art.
[0108] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a thrombin
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of GGFPRSGGGc (SEQ
ID NO: 126), AGFPRSGGGc, (SEQ ID NO: 127), and combinations
thereof, including combinations with any peptide disclosed herein.
Suitable linkers may be added to the N-terminus and/or C-terminus
of the one or more peptide sequences, and/or to residues within the
one or more peptide sequences. The amino acids can be present in
all L form, all D form, or a mix of L and D forms (ranging from 50%
D and 50% L forms to 99% D or L form and 1% L or D form). The
disclosure also contemplates nucleic acids (e.g., DNA and RNA)
encoding SEQ ID NOs 126-127, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NOs 126-127 may further
comprise one or more of polyethylene glycol (of any suitable
molecular weight, such as for example and not limitation, 100 to
200,000, 1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and
5,000 to 20,000), Glufib, an internal lysine residue for linking,
and/or a cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0109] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a kallikrein 1
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising AIKFFSAc (SEQ ID NO: 128), and
combinations thereof, including combinations with any peptide
disclosed herein. Suitable linkers may be added to the N-terminus
and/or C-terminus of the one or more peptide sequences, and/or to
residues within the one or more peptide sequences. The amino acids
can be present in all L form, all D form, or a mix of L and D forms
(ranging from 50% D and 50% L forms to 99% D or L form and 1% L or
D form). The disclosure also contemplates nucleic acids (e.g., DNA
and RNA) encoding SEQ ID NO: 128, and modifications thereof (e.g.,
linkers) as well as vectors comprising such nucleic acids, and host
cells comprising such vectors. The peptides comprising any of the
amino acid sequences comprising SEQ ID NO: 128 may further comprise
one or more of polyethylene glycol (of any suitable molecular
weight, such as for example and not limitation, 100 to 200,000,
1000 to 100,000, 2,500 to 75,000, 4,000 to 50,000, and 5,000 to
20,000), Glufib, an internal lysine residue for linking, and/or a
cysteine residue at the N-terminus and/or C-terminus. The
disclosure further contemplates methods of making said vectors and
host cells according to cloning methods well known in the art.
[0110] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a kallikrein 6
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of ALRQRESc (SEQ ID
NO: 129), AAEFRHDc (SEQ ID NO: 130), and combinations thereof,
including combinations with any peptide disclosed herein. Suitable
linkers may be added to the N-terminus and/or C-terminus of the one
or more peptide sequences, and/or to residues within the one or
more peptide sequences. The amino acids can be present in all L
form, all D form, or a mix of L and D forms (ranging from 50% D and
50% L forms to 99% D or L form and 1% L or D form). The disclosure
also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID
NOs 129-130, and modifications thereof (e.g., linkers) as well as
vectors comprising such nucleic acids, and host cells comprising
such vectors. The peptides comprising any of the amino acid
sequences comprising SEQ ID NOs 129-130 may further comprise one or
more of polyethylene glycol (of any suitable molecular weight, such
as for example and not limitation, 100 to 200,000, 1000 to 100,000,
2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an
internal lysine residue for linking, and/or a cysteine residue at
the N-terminus and/or C-terminus. The disclosure further
contemplates methods of making said vectors and host cells
according to cloning methods well known in the art.
[0111] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a chymase
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of AAAPFc (SEQ ID
NO: 131), AQFVLTEc (SEQ ID NO: 132), ARETYGEc (SEQ ID NO: 133),
AATVYVDc (SEQ ID NO: 134), and combinations thereof, including
combinations with any peptide disclosed herein. Suitable linkers
may be added to the N-terminus and/or C-terminus of the one or more
peptide sequences, and/or to residues within the one or more
peptide sequences. The amino acids can be present in all L form,
all D form, or a mix of L and D forms (ranging from 50% D and 50% L
forms to 99% D or L form and 1% L or D form). The disclosure also
contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID NOs
131-134, and modifications thereof (e.g., linkers) as well as
vectors comprising such nucleic acids, and host cells comprising
such vectors. The peptides comprising any of the amino acid
sequences comprising SEQ ID NOs 131-134 may further comprise one or
more of polyethylene glycol (of any suitable molecular weight, such
as for example and not limitation, 100 to 200,000, 1000 to 100,000,
2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an
internal lysine residue for linking, and/or a cysteine residue at
the N-terminus and/or C-terminus. The disclosure further
contemplates methods of making said vectors and host cells
according to cloning methods well known in the art.
[0112] In another embodiment, the one or more detectable peptide
sequences linked to the scaffold comprises a tryptase
recognition/cleavage site, such as for example and not limitation,
an amino acid sequence comprising at least one of APLDKKRc (SEQ ID
NO: 135), ADKVKAQc (SEQ ID NO: 136), and combinations thereof,
including combinations with any peptide disclosed herein. Suitable
linkers may be added to the N-terminus and/or C-terminus of the one
or more peptide sequences, and/or to residues within the one or
more peptide sequences. The amino acids can be present in all L
form, all D form, or a mix of L and D forms (ranging from 50% D and
50% L forms to 99% D or L form and 1% L or D form). The disclosure
also contemplates nucleic acids (e.g., DNA and RNA) encoding SEQ ID
NOs 135-136, and modifications thereof (e.g., linkers) as well as
vectors comprising such nucleic acids, and host cells comprising
such vectors. The peptides comprising any of the amino acid
sequences comprising SEQ ID NOs 135-136 may further comprise one or
more of polyethylene glycol (of any suitable molecular weight, such
as for example and not limitation, 100 to 200,000, 1000 to 100,000,
2,500 to 75,000, 4,000 to 50,000, and 5,000 to 20,000), Glufib, an
internal lysine residue for linking, and/or a cysteine residue at
the N-terminus and/or C-terminus. The disclosure further
contemplates methods of making said vectors and host cells
according to cloning methods well known in the art.
[0113] The disclosure in some aspects involves administering to a
subject, such as for example and not limitation a transplant
recipient, a composition comprising at least one detectable peptide
sequence coupled/linked to a scaffold as described herein,
identifying a biological sample from the subject in which to detect
the detectable peptide sequence and optionally collecting the
sample; and, subjecting the biological sample to an analysis method
in order to detect the presence of the peptide sequence. The
presence of the detectable marker in the biological sample is
indicative of an active enzyme or a substrate within the subject,
and allows further diagnosis of a condition, detection of a
condition, prediction of a condition, classification of the
patient, and/or selection of the patient, discussed in further
detail hereinbelow. Optionally, the patient is treated with an
appropriate therapeutic composition and/or method based on the
results of the analysis method.
[0114] The disclosure also provides methods of ex vivo analysis of
protease activity. In such embodiments, a biological sample (such
as for example and not limitation, a bodily fluid containing T
cells, urine, blood, lymphatic fluid, plasma, saliva, etc.) is
collected from a subject and contacted with a composition
comprising at least one detectable peptide sequence linked/coupled
to a scaffold as described herein. The sample is subjected to
similar analyses as discussed herein in order to detect the
presence of the peptide sequence. The presence of the detectable
marker in the biological sample is indicative of an active enzyme
or a substrate within the subject, and allows further diagnosis of
a condition, detection of a condition, prediction of a condition,
classification of the patient, and/or selection of the patient,
discussed in further detail hereinbelow.
[0115] The present disclosure also provides methods of diagnosing
acute rejection in a patient who is a transplant recipient by using
the compositions described herein, as well as methods of detecting
acute rejection in such a patient, predicting acute rejection in
such a patient, classifying a transplant recipient as having acute
rejection of the transplanted tissue, monitoring acute rejection in
a transplant recipient, selecting a transplant recipient for a
clinical trial for acute rejection-related therapeutic compositions
and/or methods, and methods of treating acute rejection in a
transplant recipient. The detectable peptide sequences, as
discussed herein, are released at the site of protease cleavage,
producing a localized detectable signal (that can be detected at
the site of cleavage or downstream of the site of cleavage), and
can accumulate in draining lymph nodes, blood, urine, and other
bodily fluids where they can be detected (e.g., by methods as
described in US2014/0303014 and US2014/0363833, each of which is
incorporated herein by reference).
[0116] In one embodiment, the disclosure provides a method of
diagnosing acute organ rejection in a transplant recipient subject
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) acutely
rejecting the transplant when the activity of the target protease
in the sample is greater than the reference activity of the target
protease; or (ii) not acutely rejecting the transplant when the
activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) optionally
treating the acute rejection via a therapeutic composition and/or
method and/or preventing the acute rejection via a prophylactic
composition and/or method.
[0117] In another embodiment, the disclosure provides a method of
detecting acute organ rejection in a transplant recipient subject
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) acutely
rejecting the transplant when the activity of the target protease
in the sample is greater than the reference activity of the target
protease; or (ii) not acutely rejecting the transplant when the
activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) optionally
treating the acute rejection via a therapeutic composition and/or
method and/or preventing the acute rejection via a prophylactic
composition and/or method.
[0118] In another embodiment, the disclosure provides a method of
predicting acute organ rejection in a transplant recipient subject
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) at risk of or
likely to acutely reject the transplant when the activity of the
target protease in the sample is greater than the reference
activity of the target protease; or (ii) not at risk of or not
likely to acutely reject the transplant when the activity of the
target protease in the sample is less than the reference activity
of the target protease; and (g) optionally treating the acute
rejection via a therapeutic composition and/or method and/or
preventing the acute rejection via a prophylactic composition
and/or method.
[0119] In another embodiment, the disclosure provides a method of
classifying a transplant recipient subject as having or likely to
have acute organ rejection comprising: (a) administering a
composition comprising a nanosensor to the subject, the nanosensor
comprising a scaffold; a linker coupled to the scaffold domain; at
least one peptide substrate coupled to the linker, the peptide
substrate comprising a target protease cleavage sequence; and a
detectable reporter coupled to the peptide substrate; (b) obtaining
a sample of a bodily fluid from the subject; (c) detecting a level
of the detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of or likely to acutely reject the
transplant when the activity of the target protease in the sample
is greater than the reference activity of the target protease; or
(ii) not having, not at risk of, or not likely to acutely reject
the transplant when the activity of the target protease in the
sample is less than the reference activity of the target protease;
and (g) optionally treating the acute rejection via a therapeutic
composition and/or method and/or preventing the acute rejection via
a prophylactic composition and/or method.
[0120] In another embodiment, the disclosure provides a method of
monitoring acute organ rejection in a transplant recipient subject
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) having, at
risk of or likely to acutely reject the transplant when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of, or not likely to acutely reject the transplant when the
activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) optionally
treating the acute rejection via a therapeutic composition and/or
method and/or preventing the acute rejection via a prophylactic
composition and/or method. In some embodiments, steps (a)-(g) can
be repeated over a period of hours to weeks after the subject has
received the transplanted tissue, for example, 3 hours to three
weeks after receiving the transplanted tissue. In other
embodiments, steps (a)-(g) can be repeated over a period of hours
to weeks after the subject has clinical signs of graft functional
decline, for example, 3 hours to three weeks after the first sign
of graft functional decline.
[0121] In another embodiment, the disclosure provides a method of
selecting a transplant recipient subject for a clinical trial for
an acute organ rejection therapeutic and/or prophylactic
compositions and/or methods comprising: (a) administering a
composition comprising a nanosensor to the subject, the nanosensor
comprising a scaffold; a linker coupled to the scaffold domain; at
least one peptide substrate coupled to the linker, the peptide
substrate comprising a target protease cleavage sequence; and a
detectable reporter coupled to the peptide substrate; (b) obtaining
a sample of a bodily fluid from the subject; (c) detecting a level
of the detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) suitable for the trial when the activity of the
target protease in the sample is greater than the reference
activity of the target protease; or (ii) not suitable for the trial
when the activity of the target protease in the sample is less than
the reference activity of the target protease.
[0122] In another embodiment, the disclosure provides a method of
treating a transplant recipient subject for acute organ rejection
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) having, at
risk of or likely to acutely reject the transplant when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of, or not likely to acutely reject the transplant when the
activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) utilizing
appropriate therapeutic and/or prophylactic compositions and/or
methods if the subject has acute organ rejection as identified in
step f).
[0123] In a related aspect, the disclosure provides methods of
diagnosing chronic rejection in a patient who is a transplant
recipient by using the compositions described herein, as well as
methods of detecting chronic rejection in such a patient,
predicting chronic rejection in such a patient, classifying a
transplant recipient as having chronic rejection of the
transplanted tissue, monitoring chronic rejection in a transplant
recipient, selecting a transplant recipient for a clinical trial
for chronic rejection-related therapeutic compositions and/or
methods, and methods of treating chronic rejection in a transplant
recipient. The detectable peptide sequences, as discussed herein,
are released at the site of protease cleavage, producing a
localized detectable signal (that can be detected at the site of
cleavage or downstream of the site of cleavage), and can accumulate
in draining lymph nodes, blood, urine, and other bodily fluids
where they can be detected (e.g., by methods as described in
US2014/0303014 and US2014/0363833, each of which is incorporated
herein by reference).
[0124] In one embodiment, the disclosure provides a method of
diagnosing chronic organ rejection in a transplant recipient
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) chronically rejecting the transplant when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not chronically
rejecting the transplant when the activity of the target protease
in the sample is less than the reference activity of the target
protease; and (g) optionally treating the chronic rejection via a
therapeutic composition and/or method and/or preventing the chronic
rejection via a prophylactic composition and/or method.
[0125] In another embodiment, the disclosure provides a method of
detecting chronic organ rejection in a transplant recipient subject
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) chronically
rejecting the transplant when the activity of the target protease
in the sample is greater than the reference activity of the target
protease; or (ii) not chronically rejecting the transplant when the
activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) optionally
treating the chronic rejection via a therapeutic composition and/or
method and/or preventing the chronic rejection via a prophylactic
composition and/or method.
[0126] In another embodiment, the disclosure provides a method of
predicting chronic organ rejection in a transplant recipient
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) at risk of or likely to chronically reject the
transplant when the activity of the target protease in the sample
is greater than the reference activity of the target protease; or
(ii) not at risk of or not likely to chronically reject the
transplant when the activity of the target protease in the sample
is less than the reference activity of the target protease; and (g)
optionally treating the chronic rejection via a therapeutic
composition and/or method and/or preventing the chronic rejection
via a prophylactic composition and/or method.
[0127] In another embodiment, the disclosure provides a method of
classifying a transplant recipient subject as having or likely to
have chronic organ rejection comprising: (a) administering a
composition comprising a nanosensor to the subject, the nanosensor
comprising a scaffold; a linker coupled to the scaffold domain; at
least one peptide substrate coupled to the linker, the peptide
substrate comprising a target protease cleavage sequence; and a
detectable reporter coupled to the peptide substrate; (b) obtaining
a sample of a bodily fluid from the subject; (c) detecting a level
of the detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of or likely to chronically reject
the transplant when the activity of the target protease in the
sample is greater than the reference activity of the target
protease; or (ii) not having, not at risk of, or not likely to
chronically reject the transplant when the activity of the target
protease in the sample is less than the reference activity of the
target protease; and (g) optionally treating the chronic rejection
via a therapeutic composition and/or method and/or preventing the
chronic rejection via a prophylactic composition and/or method.
[0128] In another embodiment, the disclosure provides a method of
monitoring chronic organ rejection in a transplant recipient
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of or likely to chronically reject
the transplant when the activity of the target protease in the
sample is greater than the reference activity of the target
protease; or (ii) not having, not at risk of, or not likely to
chronically reject the transplant when the activity of the target
protease in the sample is less than the reference activity of the
target protease; and (g) optionally treating the chronic rejection
via a therapeutic composition and/or method and/or preventing the
chronic rejection via a prophylactic composition and/or method.
Steps (a)-(g) can be repeated over a period of hours to months to
years after the subject has received the transplanted tissue, for
example, 3 hours to three weeks after receiving the transplanted
tissue. In some embodiments, steps (a)-(g) are repeated every few
months beginning one to thirty years after the subject has received
the transplanted tissue. In other embodiments, steps (a)-(g) are
repeated over a period of hours to weeks after the subject has
clinical signs of graft functional decline, for example, 3 hours to
three weeks after the first sign of graft functional decline.
[0129] In another embodiment, the disclosure provides a method of
selecting a transplant recipient subject for a clinical trial for a
chronic organ rejection therapeutic and/or prophylactic
compositions and/or methods comprising: (a) administering a
composition comprising a nanosensor to the subject, the nanosensor
comprising a scaffold; a linker coupled to the scaffold domain; at
least one peptide substrate coupled to the linker, the peptide
substrate comprising a target protease cleavage sequence; and a
detectable reporter coupled to the peptide substrate; (b) obtaining
a sample of a bodily fluid from the subject; (c) detecting a level
of the detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) suitable for the trial when the activity of the
target protease in the sample is greater than the reference
activity of the target protease; or (ii) not suitable for the trial
when the activity of the target protease in the sample is less than
the reference activity of the target protease.
[0130] In another embodiment, the disclosure provides a method of
treating a transplant recipient subject for chronic organ rejection
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) having, at
risk of or likely to chronically reject the transplant when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of, or not likely to chronically reject the transplant when
the activity of the target protease in the sample is less than the
reference activity of the target protease; and (g) utilizing
appropriate therapeutic and/or prophylactic compositions and/or
methods if the subject has chronic organ rejection as identified in
step f).
[0131] In another related aspect, the disclosure provides methods
of diagnosing immune conditions related to T cell cytotoxicity
(e.g., graft versus host disease (GvHD) and autoimmune diseases).
The disclosure also provides methods of detecting such immune
conditions in a patient, predicting such immune conditions in a
patient, classifying a patient as having such immune conditions,
monitoring such immune conditions in a patient, selecting a patient
for a clinical trial for such immune condition-related therapeutic
compositions and/or methods, and methods of treating such immune
conditions in a patient. The detectable peptide sequences, as
discussed herein, are released at the site of protease cleavage,
producing a localized detectable signal (that can be detected at
the site of cleavage or downstream of the site of cleavage), and
can accumulate in draining lymph nodes, blood, urine, and other
bodily fluids where they can be detected (e.g., by methods as
described in US2014/0303014 and US2014/0363833, each of which is
incorporated herein by reference).
[0132] In one embodiment, the disclosure provides a method of
diagnosing immune conditions related to T cell cytotoxicity in a
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having the immune condition when the transplant
when the activity of the target protease in the sample is greater
than the reference activity of the target protease; or (ii) not
having the immune condition when the activity of the target
protease in the sample is less than the reference activity of the
target protease; and (g) optionally treating the immune condition
via a therapeutic composition and/or method and/or preventing the
immune condition via a prophylactic composition and/or method.
[0133] In another embodiment, the disclosure provides a method of
detecting immune conditions related to T cell cytotoxicity in a
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having the immune condition when the activity of
the target protease in the sample is greater than the reference
activity of the target protease; or (ii) not having the immune
condition when the activity of the target protease in the sample is
less than the reference activity of the target protease; and (g)
optionally treating the immune condition via a therapeutic
composition and/or method and/or preventing the immune condition
via a prophylactic composition and/or method.
[0134] In another embodiment, the disclosure provides a method of
predicting immune conditions related to T cell cytotoxicity in a
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) at risk of developing or likely to develop the
immune condition when the activity of the target protease in the
sample is greater than the reference activity of the target
protease; or (ii) not at risk of developing or not likely to
develop the immune condition when the activity of the target
protease in the sample is less than the reference activity of the
target protease; and (g) optionally treating the immune condition
via a therapeutic composition and/or method and/or preventing the
immune condition via a prophylactic composition and/or method.
[0135] In another embodiment, the disclosure provides a method of
classifying a subject as having or likely to have immune conditions
related to T cell cytotoxicity comprising: (a) administering a
composition comprising a nanosensor to the subject, the nanosensor
comprising a scaffold; a linker coupled to the scaffold domain; at
least one peptide substrate coupled to the linker, the peptide
substrate comprising a target protease cleavage sequence; and a
detectable reporter coupled to the peptide substrate; (b) obtaining
a sample of a bodily fluid from the subject; (c) detecting a level
of the detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of developing, or likely to develop
the immune condition when the activity of the target protease in
the sample is greater than the reference activity of the target
protease; or (ii) not having, not at risk of developing, or not
likely to develop the immune condition when the activity of the
target protease in the sample is less than the reference activity
of the target protease; and (g) optionally treating the immune
condition via a therapeutic composition and/or method and/or
preventing the immune condition via a prophylactic composition
and/or method.
[0136] In another embodiment, the disclosure provides a method of
monitoring immune conditions related to T cell cytotoxicity in a
subject comprising: (a) administering a composition comprising a
nanosensor to the subject, the nanosensor comprising a scaffold; a
linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of developing, or likely to develop
the immune condition when the activity of the target protease in
the sample is greater than the reference activity of the target
protease; or (ii) not having, not at risk of developing, or not
likely to develop the immune condition when the activity of the
target protease in the sample is less than the reference activity
of the target protease; and (g) optionally treating the immune
condition via a therapeutic composition and/or method and/or
preventing the immune condition via a prophylactic composition
and/or method. Steps (a)-(g) can be repeated over a period of hours
to months to years to decades, for example for a period of 3 hours
to 3 months, and the monitoring period can vary depending on the
immune condition.
[0137] In another embodiment, the disclosure provides a method of
selecting a transplant recipient subject for a clinical trial for a
therapeutic and/or prophylactic compositions and/or methods for a
T-cell toxicity-related immune disorder comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i) suitable for the
trial when the activity of the target protease in the sample is
greater than the reference activity of the target protease; or (ii)
not suitable for the trial when the activity of the target protease
in the sample is less than the reference activity of the target
protease.
[0138] In another embodiment, the disclosure provides a method of
treating a subject for a T-cell toxicity-related immune disorder
comprising: (a) administering a composition comprising a nanosensor
to the subject, the nanosensor comprising a scaffold; a linker
coupled to the scaffold domain; at least one peptide substrate
coupled to the linker, the peptide substrate comprising a target
protease cleavage sequence; and a detectable reporter coupled to
the peptide substrate; (b) obtaining a sample of a bodily fluid
from the subject; (c) detecting a level of the detectable reporter
in the sample of the bodily fluid; (d) determining an activity of
the target protease based on the level of the detectable reporter
in the sample of the bodily fluid; (e) comparing the activity of
the target protease in the sample to a reference activity of the
target protease; (f) identifying the subject as: (i) having, at
risk of developing, or likely to develop the immune condition when
the activity of the target protease in the sample is greater than
the reference activity of the target protease; or (ii) not having,
not at risk of developing, or not likely to develop the immune
condition when the activity of the target protease in the sample is
less than the reference activity of the target protease; and (g)
utilizing appropriate therapeutic and/or prophylactic compositions
and/or methods if the subject has an immune disorder as identified
in step f).
[0139] In another related aspect, the disclosure provides methods
of diagnosing T cell cytotoxicity (e.g., to predict treatment
efficacy in patients being treated with cancer immune therapies
such as checkpoint blockade inhibitors or CAR T cell therapies).
The disclosure also provides methods of detecting T cell
cytotoxicity in a patient, predicting T cell cytotoxicity in a
patient, classifying a patient as having T cell cytotoxicity,
monitoring T cell cytotoxicity in a patient, selecting a patient
for a clinical trial for T cell cytotoxicity-related therapeutic
compositions and/or methods, and methods of treating T cell
cytotoxicity in a patient. The detectable peptide sequences, as
discussed herein, are released at the site of protease cleavage,
producing a localized detectable signal (that can be detected at
the site of cleavage or downstream of the site of cleavage), and
can accumulate in draining lymph nodes, blood, urine, and other
bodily fluids where they can be detected (e.g., by methods as
described in US2014/0303014 and US2014/0363833, each of which is
incorporated herein by reference).
[0140] In one embodiment, the disclosure provides a method of
diagnosing T cell cytotoxicity in a subject comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i) having, at risk of
developing, or likely to develop T cell cytotoxicity when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of developing, or not likely to develop T cell cytotoxicity
when the activity of the target protease in the sample is less than
the reference activity of the target protease; and (g) optionally
treating the T cell cytotoxicity via a therapeutic composition
and/or method and/or preventing the T cell cytotoxicity via a
prophylactic composition and/or method.
[0141] In another embodiment, the disclosure provides a method of
detecting T cell cytotoxicity in a subject comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i) having, at risk of
developing, or likely to develop T cell cytotoxicity when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of developing, or not likely to develop T cell cytotoxicity
when the activity of the target protease in the sample is less than
the reference activity of the target protease; and (g) optionally
treating the T cell cytotoxicity via a therapeutic composition
and/or method and/or preventing the T cell cytotoxicity via a
prophylactic composition and/or method.
[0142] In another embodiment, the disclosure provides a method of
predicting T cell cytotoxicity in a subject comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i) having, at risk of
developing, or likely to develop T cell cytotoxicity when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of developing, or not likely to develop T cell cytotoxicity
when the activity of the target protease in the sample is less than
the reference activity of the target protease; and (g) optionally
treating the T cell cytotoxicity via a therapeutic composition
and/or method and/or preventing the T cell cytotoxicity via a
prophylactic composition and/or method.
[0143] In another embodiment, the disclosure provides a method of
classifying a subject as having or likely to have T cell
cytotoxicity comprising: (a) administering a composition comprising
a nanosensor to the subject, the nanosensor comprising a scaffold;
a linker coupled to the scaffold domain; at least one peptide
substrate coupled to the linker, the peptide substrate comprising a
target protease cleavage sequence; and a detectable reporter
coupled to the peptide substrate; (b) obtaining a sample of a
bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) having, at risk of developing, or likely to develop
T cell cytotoxicity when the activity of the target protease in the
sample is greater than the reference activity of the target
protease; or (ii) not having, not at risk of developing, or not
likely to develop T cell cytotoxicity when the activity of the
target protease in the sample is less than the reference activity
of the target protease; and (g) optionally treating the T cell
cytotoxicity via a therapeutic composition and/or method and/or
preventing the T cell cytotoxicity via a prophylactic composition
and/or method.
[0144] In another embodiment, the disclosure provides a method of
monitoring T cell cytotoxicity in a subject comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i)) having, at risk of
developing, or likely to develop T cell cytotoxicity when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of developing, or not likely to develop T cell cytotoxicity
when the activity of the target protease in the sample is less than
the reference activity of the target protease; and (g) optionally
treating the T cell cytotoxicity via a therapeutic composition
and/or method and/or preventing the T cell cytotoxicity via a
prophylactic composition and/or method. Steps (a)-(g) can be
repeated over a period of hours to months to years to decades, and
the monitoring period can vary depending on the immune condition,
for example 3 hours to 3 months.
[0145] In another embodiment, the disclosure provides a method of
selecting a subject for a clinical trial for T cell
cytotoxicity-related therapeutic and/or prophylactic compositions
and/or methods comprising: (a) administering a composition
comprising a nanosensor to the subject, the nanosensor comprising a
scaffold; a linker coupled to the scaffold domain; at least one
peptide substrate coupled to the linker, the peptide substrate
comprising a target protease cleavage sequence; and a detectable
reporter coupled to the peptide substrate; (b) obtaining a sample
of a bodily fluid from the subject; (c) detecting a level of the
detectable reporter in the sample of the bodily fluid; (d)
determining an activity of the target protease based on the level
of the detectable reporter in the sample of the bodily fluid; (e)
comparing the activity of the target protease in the sample to a
reference activity of the target protease; (f) identifying the
subject as: (i) suitable for the trial when the activity of the
target protease in the sample is greater than the reference
activity of the target protease; or (ii) not suitable for the trial
when the activity of the target protease in the sample is less than
the reference activity of the target protease.
[0146] In another embodiment, the disclosure provides a method of
treating a subject for T cell cytotoxicity comprising: (a)
administering a composition comprising a nanosensor to the subject,
the nanosensor comprising a scaffold; a linker coupled to the
scaffold domain; at least one peptide substrate coupled to the
linker, the peptide substrate comprising a target protease cleavage
sequence; and a detectable reporter coupled to the peptide
substrate; (b) obtaining a sample of a bodily fluid from the
subject; (c) detecting a level of the detectable reporter in the
sample of the bodily fluid; (d) determining an activity of the
target protease based on the level of the detectable reporter in
the sample of the bodily fluid; (e) comparing the activity of the
target protease in the sample to a reference activity of the target
protease; (f) identifying the subject as: (i) having, at risk of
developing, or likely to develop T cell cytotoxicity when the
activity of the target protease in the sample is greater than the
reference activity of the target protease; or (ii) not having, not
at risk of developing, or not likely to develop T cell cytotoxicity
when the activity of the target protease in the sample is less than
the reference activity of the target protease; and (g) utilizing
appropriate therapeutic and/or prophylactic compositions and/or
methods if the subject has T cell cytotoxicity as identified in
step f).
[0147] In any of the foregoing embodiments of any of the disclosed
methods, the composition comprising the nanosensor is administered
intravenously. In any of the foregoing embodiments, the composition
comprising the nanosensor further comprises a pharmaceutically
acceptable carrier and/or adjuvant. In any of the foregoing
embodiments, the peptide substrate comprises a detectable peptide
sequence as discussed herein, e.g., a detectable peptide sequence
comprising an amino acid sequence comprising one or more of SEQ ID
NOs 2-136. In any of the foregoing embodiments, the detectable
reporter can be, for example and not limitation, a fluorophore, a
mass spectrometry bar code, and/or an imaging agent (e.g., a
contrast imaging agent or a PET imaging agent). In any of the
foregoing embodiments, the bodily fluid can be, for example and not
limitation, urine, blood, lymphatic fluid, plasma, and/or saliva.
In any of the foregoing embodiments, a sample of a bodily fluid is
obtained 30 minutes to 48 hours, including but not limited to 30
minutes to 6 hours, after administration of the composition
comprising the nanosensor. In any of the foregoing embodiments, the
target protease is selected from the group consisting of Granzyme
B, Granzyme A, MALT1, Caspase 8, Calpain 2, Cathepsin X, Cls, Clr,
MASP2, Factor I, Factor D, ADAMTS1, MMP2, and MMP9. In any of the
foregoing embodiments, the reporter domain enables detection by
light spectroscopy, near-infrared imaging, fluorescent imaging,
bioluminescent imaging, ELISA, PCR (for DNA barcodes),
spectrophotometry, mass spectrometry (e.g., bar code mass
spectrometry), and/or imaging (e.g., CAT, MRI, PET), and/or by
methods described in any of WO2007/106415, US2010/0240050,
US2014/0303014 and US2014/0363833, each of which is incorporated
herein by reference.
[0148] In any of the foregoing embodiments, a single sample of
bodily fluid is taken from the subject for analysis. In any of the
foregoing embodiments, multiple samples of bodily fluid are taken
in order to monitor the protease activity, and thus acute and/or
chronic rejection, over time. Such samples can be taken over a
period of 30 minutes to 4 weeks after the transplant occurs in
order to monitor development of acute and/or chronic rejection. In
such an embodiment, the composition comprising the nanosensor can
be administered multiple times to the subject before each bodily
fluid sample is taken for analysis. In other embodiments, acute
and/or chronic rejection can be monitored over the patient's
lifetime after receiving the transplanted tissue, and thus may
occur months to years to decades after receiving the transplanted
tissue. In some embodiments, the monitoring may be triggered by a
clinical change or symptom development, such as clinical signs of
graft function decline. In such embodiments, the monitoring may
occur for hours to weeks to months after onset of the clinical
change or symptom development.
[0149] In any of the foregoing embodiments, the reference sample is
a sample taken from the same subject prior to the transplant, while
in other embodiments the reference sample is a sample taken from a
person of similar physical characteristics as the subject (e.g.,
BMI-, age-, and gender-matched person who is not undergoing a
transplant, does not require transplant surgery, and/or does not
have an immune disorder as determined by standard examination and
diagnostic methods). In some embodiments, the reference sample
provides a control level of the target protease activity. If the
subject is determined to be acutely and/or chronically rejecting
the transplanted tissue, therapeutic and/or prophylactic
compositions and/or methods can be employed to treat or prevent the
acute and/or chronic rejection. Such therapeutic compositions
and/or methods include, for example and not limitation, salvage
therapy with thymoglobulin and steroids, immunosuppression
therapies (such as for example and not limitation,
anti-inflammatory drugs (cortisol, prednisone, dexamethasone,
fludrocortisone acetate), anti-proliferatives (cyclophosphamide,
methotrexate, azathioprine, mytomycin C)), and T cell targeted
therapies (cyclosporine, tacrolimus, sirolimus, thymoglobulin,
OKT3, antithymocyte globulin, Basiliximab, Belatacept, Abatacept).
If the subject is determined to have or be likely to have an immune
condition related to T cell cytotoxicity, therapeutic and/or
prophylactic compositions and/or methods can be employed to treat
or prevent the immune condition, such as for example and not
limitation, immunosuppression therapies (such as for example and
not limitation, anti-inflammatory drugs (cortisol, prednisone,
dexamethasone, fludrocortisone acetate), anti-proliferatives
(cyclophosphamide, methotrexate, azathioprine, mytomycin C)), and T
cell targeted therapies (cyclosporine, tacrolimus, sirolimus,
thymoglobulin, OKT3, antithymocyte globulin, Basiliximab,
Belatacept, Abatacept).
[0150] In any of the foregoing embodiments, steps (d) and/or (e)
and/or (f) (and repetitions of such steps) are performed by a
computer, as described further herein. In certain embodiments, it
may be convenient to prepare a report of results of the patient's
identification. Thus, certain embodiments of the methods of the
disclosure comprise a further step of preparing a report containing
results from the identification, wherein said report is written in
a computer readable medium, printed on paper, or displayed on a
visual display. In certain embodiments, it may be convenient to
report results of the determination to at least one entity selected
from the group consisting of the subject, a guardian of the
subject, a physician, a medical organization, and a medical
insurer. In other embodiments, it may be convenient to report
prognosis, results of monitoring, and/or efficacy of treatment
and/or prophylactic methods to such entity.
[0151] In any of the foregoing methods, the method may further
comprise treatment and/or prophylaxis of patients determined to
have, or be at risk for having, acute or chronic rejection or
immune conditions related to T cell cytotoxicity (e.g., autoimmune
diseases, GVHD, etc). Alternatively, the method of treatment and/or
prophylaxis may comprise detection of the biomarkers and their use
in the described algorithm to identify patients having or being at
risk of having severe or lethal GVHD, and initiating treatment
and/or prophylaxis based on the identification, or being suitable
for undergoing such therapies.
[0152] In some embodiments, the treatment and/or prophylaxis
comprises administration of appropriate therapeutically effective
pharmaceutical compositions and/or use of appropriate
therapeutically effective methods.
[0153] Non-limiting examples of the inflammatory and autoimmune
diseases treatable by the methods of the present disclosure
include, e.g., inflammatory bowel disease (IBD), graft versus host
disease (GVHD), ulcerative colitis (UC), Crohn's disease, diabetes
(e.g., diabetes mellitus type 1), multiple sclerosis, arthritis
(e.g., rheumatoid arthritis), Graves' disease, lupus erythematosus,
systemic lupus erythematosus (SLE), rheumatoid arthritis (RA),
autoimmune myopathies, Sjogren's syndrome (SS), vasculitis,
scleroderma, ankylosing spondylitis, psoriasis, Behcet's disease,
autistic enterocolitis, Guillain-Barre Syndrome, myasthenia gravis,
pemphigus vulgaris, acute disseminated encephalomyelitis (ADEM),
transverse myelitis autoimmune cardiomyopathy, Celiac disease,
dermatomyositis, Wegener's granulomatosis, allergy, asthma, contact
dermatitis, atherosclerosis (or any other inflammatory condition
affecting the heart or vascular system), autoimmune uveitis, as
well as other autoimmune skin conditions, autoimmune kidney, lung,
or liver conditions, autoimmune neuropathies, etc.
[0154] It is contemplated that when used to treat various diseases,
the compositions and methods of the present disclosure can be
combined with other therapeutic agents suitable for the same or
similar diseases. Also, two or more embodiments of the disclosure
may be also co-administered to generate additive or synergistic
effects. When co-administered with a second therapeutic agent, the
embodiment of the disclosure and the second therapeutic agent may
be simultaneously or sequentially (in any order). Suitable
therapeutically effective dosages for each agent may be lowered due
to the additive action or synergy.
[0155] As a non-limiting example, the disclosure can be combined
with other therapies that block inflammation (e.g., via blockage of
ILL INF.alpha./.beta., IL6, TNF, IL13, IL23, etc.).
[0156] The compositions and methods of the disclosure can be also
administered in combination with an anti-tumor antibody or an
antibody directed at a pathogenic antigen or allergen.
[0157] The compositions and methods of the disclosure can be
combined with other immunomodulatory treatments such as, e.g.,
therapeutic vaccines (including but not limited to GVAX, DC-based
vaccines, etc.), checkpoint inhibitors (including but not limited
to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators
(including but not limited to agents that enhance 41BB, OX40,
etc.). The inhibitory treatments of the disclosure can be also
combined with other treatments that possess the ability to modulate
NKT function or stability, including but not limited to CD1d,
CD1d-fusion proteins, CD1d dimers or larger polymers of CD1d either
unloaded or loaded with antigens, CD1d-chimeric antigen receptors
(CD1d-CAR), or any other of the five known CD1 isomers existing in
humans (CD1a, CD1b, CD1c, CD1e), in any of the aforementioned forms
or formulations, alone or in combination with each other or other
agents.
[0158] Therapeutic methods of the disclosure can be combined with
additional immunotherapies and therapies. For example, when used to
aid in treating cancer, the methods and compositions of the
disclosure can be used in combination with conventional cancer
therapies, such as, e.g., surgery, radiotherapy, chemotherapy or
combinations thereof, depending on type of the tumor, patient
condition, other health issues, and a variety of factors. In
certain aspects, other therapeutic agents useful for combination
cancer therapy with the inhibitors of the disclosure include
anti-angiogenic agents. Many anti-angiogenic agents have been
identified and are known in the art, including, e.g., TNP-470,
platelet factor 4, thrombospondin-1, tissue inhibitors of
metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment),
angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF
soluble receptor, transforming growth factor beta, interferon
alpha, soluble KDR and FLT-1 receptors, placental
proliferin-related protein, as well as those listed by Carmeliet
and Jain (2000). In one embodiment, the methods and compositions of
the disclosure can be used in combination with a VEGF antagonist or
a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF
variants, soluble VEGF receptor fragments, aptamers capable of
blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies,
inhibitors of VEGFR tyrosine kinases and any combinations thereof
(e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).
[0159] The compositions of the disclosure can comprise a carrier
and/or excipient. While it is possible to use a compound of the
present disclosure for therapy as is, it may be preferable to
administer it in a pharmaceutical formulation, e.g., in admixture
with a suitable pharmaceutical excipient and/or carrier selected
with regard to the intended route of administration and standard
pharmaceutical practice. The excipient and/or carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof. Acceptable excipients and carriers for therapeutic use are
well known in the pharmaceutical art, and are described, for
example, in Remington: The Science and Practice of Pharmacy.
Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The
choice of pharmaceutical excipient and carrier can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. Oral formulations readily accommodate
additional mixtures, such as, e.g., milk, yogurt, and infant
formula. Solid dosage forms for oral administration can also be
used and can include, e.g., capsules, tablets, caplets, pills,
troches, lozenges, powders, and granules. Non-limiting examples of
suitable excipients include, e.g., diluents, buffering agents
(e.g., sodium bicarbonate, infant formula, sterilized human milk,
or other agents which allow bacteria to survive and grow [e.g.,
survive in the acidic environment of the stomach and to grow in the
intestinal environment]), preservatives, stabilizers, binders,
compaction agents, lubricants, dispersion enhancers, disintegration
agents, antioxidants, flavoring agents, sweeteners, and coloring
agents. Those of relevant skill in the art are well able to prepare
suitable solutions.
[0160] In one embodiment of any of the compositions of the
disclosure, the composition is formulated for delivery by a route
such as, e.g., oral, topical, rectal, mucosal, sublingual, nasal,
naso/oro-gastric gavage, parenteral, intraperitoneal, intravenous,
intradermal, transdermal, intrathecal, nasal, and intracheal
administration. In one embodiment of any of the compositions of the
disclosure, the composition is in a form of a liquid, foam, cream,
spray, powder, or gel. In one embodiment of any of the compositions
of the disclosure, the composition comprises a buffering agent
(e.g., sodium bicarbonate, infant formula or sterilized human
milk). In one embodiment of any of the compositions of the
disclosure, the composition is formulated for intravenous
administration.
[0161] Administration of the compounds and compositions in the
methods of the disclosure can be accomplished by any method known
in the art. Non-limiting examples of useful routes of delivery
include oral, rectal, fecal (by enema), and via naso/oro-gastric
gavage, as well as parenteral, intraperitoneal, intravenous,
intradermal, transdermal, intrathecal, nasal, and intracheal
administration. The active agent may be systemic after
administration or may be localized by the use of regional
administration, intramural administration, or use of an implant
that acts to retain the active dose at the site of implantation.
The formulation can include added ingredients to improve
palatability, improve shelf-life, improve absorption, impart
nutritional benefits, and the like.
[0162] The useful dosages of the compounds and formulations of the
disclosure can vary widely, depending upon the nature of the
disease, the patient's medical history, the frequency of
administration, the manner of administration, the clearance of the
agent from the host, and the like. The initial dose may be larger,
followed by smaller maintenance doses. The dose may be administered
as infrequently as weekly or biweekly, or fractionated into smaller
doses and administered daily, semi-weekly, etc., to maintain an
effective dosage level. It is contemplated that a variety of doses
can be effective to achieve a therapeutic effect. While it is
possible to use a compound of the present disclosure for therapy as
is, it may be preferable to administer it in a pharmaceutical
formulation, e.g., in admixture with a suitable pharmaceutical
excipient, diluent or carrier selected with regard to the intended
route of administration and standard pharmaceutical practice. The
excipient, diluent and/or carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not deleterious to the recipient thereof. Acceptable
excipients, diluents, and carriers for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington: The Science and Practice of Pharmacy. Lippincott
Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of
pharmaceutical excipient, diluent, and carrier can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. Although there are no physical limitations
to delivery of the formulations of the present disclosure, oral
delivery is preferred for delivery to the digestive tract because
of its ease and convenience, and because oral formulations readily
accommodate additional mixtures, such as milk, yogurt, and infant
formula.
[0163] Oral delivery may also include the use of nanoparticles that
can be targeted, e.g., to the GI tract of the subject, such as
those described in Yun et al., Adv Drug Deliv Rev. 2013,
65(6):822-832 (e.g., mucoadhesive nanoparticles, negatively charged
carboxyl ate- or sulfate-modified particles, etc.). Non-limiting
examples of other methods of targeting delivery of compositions to
the GI tract are discussed in U.S. Pat. Appl. Pub. No. 2013/0149339
and references cited therein (e.g., pH sensitive compositions [such
as, e.g., enteric polymers which release their contents when the pH
becomes alkaline after the enteric polymers pass through the
stomach], compositions for delaying the release [e.g., compositions
which use hydrogel as a shell or a material which coats the active
substance with, e.g., in vivo degradable polymers, gradually
hydrolyzable polymers, gradually water-soluble polymers, and/or
enzyme degradable polymers], bioadhesive compositions which
specifically adhere to the colonic mucosal membrane, compositions
into which a protease inhibitor is incorporated, a carrier system
being specifically decomposed by an enzyme present in the colon).
Oral pharmaceutical dosage forms include, by way of example and
without limitation, solid, gel and liquid. Solid dosage forms
include tablets, capsules, granules, and bulk powders. Oral tablets
include compressed, chewable lozenges and tablets which may be
enteric-coated, sugar-coated or film-coated. Capsules may be hard
or soft gelatin capsules, while granules and powders may be
provided in non-effervescent or effervescent form with the
combination of other ingredients known to those skilled in the
art.
[0164] For oral administration, the active ingredient(s) can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions. The active component(s) can be encapsulated in gelatin
capsules together with inactive ingredients and powdered carriers,
such as glucose, lactose, sucrose, mannitol, starch, cellulose or
cellulose derivatives, magnesium stearate, stearic acid, sodium
saccharin, talcum, magnesium carbonate. Examples of additional
inactive ingredients that may be added to provide desirable color,
taste, stability, buffering capacity, dispersion or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, and edible white ink. Similar diluents
can be used to make compressed tablets. Both tablets and capsules
can be manufactured as sustained release products to provide for
continuous release of medication over a period of hours. Compressed
tablets can be sugar coated or film coated to mask any unpleasant
taste and protect the tablet from the atmosphere, or enteric-coated
for selective disintegration in the gastrointestinal tract. Liquid
dosage forms for oral administration can contain coloring and
flavoring to increase patient acceptance. In certain embodiments,
the formulations are solid dosage forms, such as capsules or
tablets. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or agents of a similar
nature: a binder; a diluent; a disintegrating agent; a lubricant; a
glidant; a sweetening agent; and a flavoring agent. If oral
administration is desired, the agent could be provided in a
composition that protects it from the acidic environment of the
stomach. For example, the composition can be formulated in an
enteric coating that maintains its integrity in the stomach and
releases the active agent in the intestine. The composition may
also be formulated in combination with an antacid or other such
ingredient.
[0165] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The agents can
also be administered as a component of an elixir, suspension,
syrup, wafer, sprinkle, chewing gum or the like. A syrup may
contain, in addition to the active agents, sucrose as a sweetening
agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials
which do not impair the desired action, or with materials that
supplement the desired action, such as antacids, H2 blockers, and
diuretics.
[0166] Pharmaceutically acceptable carriers included in tablets are
binders, lubricants, diluents, disintegrating agents, coloring
agents, flavoring agents, and wetting agents. Enteric-coated
tablets, because of the enteric-coating, resist the action of
stomach acid and dissolve or disintegrate in the neutral or
alkaline intestines. Sugar-coated tablets are compressed tablets to
which different layers of pharmaceutically acceptable substances
are applied. Film-coated tablets are compressed tablets which have
been coated with a polymer or other suitable coating. Multiple
compressed tablets are compressed tablets made by more than one
compression cycle utilizing the pharmaceutically acceptable
substances previously mentioned. Coloring agents may also be used
in the above dosage forms. Flavoring and sweetening agents are used
in compressed tablets, sugar-coated, multiple compressed and
chewable tablets. Flavoring and sweetening agents are useful in the
formation of chewable tablets and lozenges.
[0167] Liquid oral dosage forms include aqueous solutions,
emulsions, suspensions, solutions and/or suspensions reconstituted
from non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Aqueous solutions
include, for example, elixirs and syrups. Emulsions are either
oil-in-water or water-in-oil.
[0168] Formulations suitable for parenteral administration include
aqueous and nonaqueous, isotonic sterile injection solutions, which
can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and nonaqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives.
[0169] Solutions or suspensions can include any of the following
components, in any combination: a sterile diluent, including by way
of example without limitation, water for injection, saline
solution, fixed oil, polyethylene glycol, glycerine, propylene
glycol or other synthetic solvent; antimicrobial agents, such as
benzyl alcohol and methyl parabens; antioxidants, such as ascorbic
acid and sodium bisulfite; chelating agents, such as
ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,
citrates and phosphates; and agents for the adjustment of tonicity,
such as sodium chloride or dextrose. In instances in which the
agents exhibit insufficient solubility, methods for solubilizing
agents may be used. Such methods are known to those of skill in
this art, and include, but are not limited to, using co-solvents,
such as, e.g., dimethylsulfoxide (DMSO), using surfactants, such as
TWEEN.RTM. 80, or dissolution in aqueous sodium bicarbonate.
Pharmaceutically acceptable derivatives of the agents may also be
used in formulating effective pharmaceutical compositions.
[0170] The composition can contain along with the active agent, for
example and without limitation: a diluent such as lactose, sucrose,
dicalcium phosphate, or carboxymethylcellulose; a lubricant, such
as magnesium stearate, calcium stearate and talc; and a binder such
as starch, natural gums, such as gum acacia gelatin, glucose,
molasses, polyvinylpyrrolidone, celluloses and derivatives thereof,
povidone, crospovidones and other such binders known to those of
skill in the art. Liquid pharmaceutically administrable
compositions can, for example, be prepared by dissolving,
dispersing, or otherwise mixing an active agent as defined above
and optional pharmaceutical adjuvants in a carrier, such as, by way
of example and without limitation, water, saline, aqueous dextrose,
glycerol, glycols, ethanol, and the like, to thereby form a
solution or suspension. If desired, the pharmaceutical composition
to be administered may also contain minor amounts of nontoxic
auxiliary substances such as wetting agents, emulsifying agents, or
solubilizing agents, pH buffering agents and the like, such as, by
way of example and without limitation, acetate, sodium citrate,
cyclodextrin derivatives, sorbitan monolaurate, triethanolamine
sodium acetate, triethanolamine oleate, and other such agents.
Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art (e.g., Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th
Edition, 1975). The composition or formulation to be administered
may, in any event, contain a quantity of the active agent in an
amount sufficient to alleviate the symptoms of the treated
subject.
[0171] The active agents or pharmaceutically acceptable derivatives
may be prepared with carriers that protect the agent against rapid
elimination from the body, such as time release formulations or
coatings. The compositions may include other active agents to
obtain desired combinations of properties.
[0172] Parenteral administration, generally characterized by
injection, either subcutaneously, intramuscularly or intravenously,
is also contemplated herein. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients include, by way of
example and without limitation, water, saline, dextrose, glycerol
or ethanol. In addition, if desired, the pharmaceutical
compositions to be administered may also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, pH buffering agents, stabilizers, solubility enhancers, and
other such agents, such as, for example, sodium acetate, sorbitan
monolaurate, triethanolamine oleate and cyclodextrins.
[0173] Implantation of a slow-release or sustained-release system,
such that a constant level of dosage is maintained (e.g., U.S. Pat.
No. 3,710,795) is also contemplated herein. Briefly, an inhibitor
of Nt5e or A1R is dispersed in a solid inner matrix (e.g.,
polymethylmethacrylate, polybutylmethacrylate, plasticized or
unplasticized polyvinylchloride, plasticized nylon, plasticized
polyethyleneterephthalate, natural rubber, polyisoprene,
polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate
copolymers, silicone rubbers, polydimethylsiloxanes, silicone
carbonate copolymers, hydrophilic polymers such as hydrogels of
esters of acrylic and methacrylic acid, collagen, cross-linked
polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl
acetate) that is surrounded by an outer polymeric membrane (e.g.,
polyethylene, polypropylene, ethylene/propylene copolymers,
ethylene/ethyl acrylate copolymers, ethylene/vinylacetate
copolymers, silicone rubbers, polydimethyl siloxanes, neoprene
rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride
copolymers with vinyl acetate, vinylidene chloride, ethylene and
propylene, ionomer polyethylene terephthalate, butyl rubber
epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,
ethylene/vinyl acetate/vinyl alcohol terpolymer, and
ethylene/vinyloxyethanol copolymer) that is insoluble in body
fluids. The agent diffuses through the outer polymeric membrane in
a release rate controlling step. The percentage of active agent
contained in such parenteral compositions is highly dependent on
the specific nature thereof, as well as the activity of the agent
and the needs of the subject.
[0174] Lyophilized powders can be reconstituted for administration
as solutions, emulsions, and other mixtures or formulated as solids
or gels. The sterile, lyophilized powder is prepared by dissolving
an agent provided herein, or a pharmaceutically acceptable
derivative thereof, in a suitable solvent. The solvent may contain
an excipient which improves the stability or other pharmacological
component of the powder or reconstituted solution, prepared from
the powder. Excipients that may be used include, but are not
limited to, dextrose, sorbital, fructose, corn syrup, xylitol,
glycerin, glucose, sucrose or other suitable agent. The solvent may
also contain a buffer, such as citrate, sodium or potassium
phosphate or other such buffer known to those of skill in the art
at, typically, about neutral pH. Subsequent sterile filtration of
the solution followed by lyophilization under standard conditions
known to those of skill in the art provides the desired
formulation. Generally, the resulting solution can be apportioned
into vials for lyophilization. Each vial can contain, by way of
example and without limitation, a single dosage (10-1000 mg, such
as 100-500 mg) or multiple dosages of the agent. The lyophilized
powder can be stored under appropriate conditions, such as at about
4.degree. C. to room temperature. Reconstitution of this
lyophilized powder with water for injection provides a formulation
for use in parenteral administration. For reconstitution, about
1-50 mg, such as about 5-35 mg, for example, about 9-30 mg of
lyophilized powder, is added per mL of sterile water or other
suitable carrier. The precise amount depends upon the selected
agent. Such amount can be empirically determined.
[0175] The inventive composition or pharmaceutically acceptable
derivatives thereof may be formulated as aerosols for application,
e.g., by inhalation or intranasally (e.g., as described in U.S.
Pat. Nos. 4,044,126, 4,414,209, and 4,364,923). These formulations
can be in the form of an aerosol or solution for a nebulizer, or as
a microtine powder for insufflation, alone or in combination with
an inert carrier such as lactose. In such a case, the particles of
the formulation can, by way of example and without limitation, have
diameters of less than about 50 microns, such as less than about 10
microns.
[0176] The agents may be also formulated for local or topical
application, such as for application to the skin and mucous
membranes (e.g., intranasally), in the form of nasal solutions,
gels, creams, and lotions.
[0177] Other routes of administration, such as transdermal patches
are also contemplated herein. Transdermal patches, including
iontophoretic and electrophoretic devices, are well known to those
of skill in the art. For example, such patches are disclosed in
U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301,
6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and
5,860,957.
Kits of the Invention
[0178] The present invention also provides kits useful in the
practice of the methods of the invention. In some embodiments,
these kits comprise detection reagents that specifically bind the
nanosensors of the present invention, such as for example and not
limitation, at least one of the detectable peptide sequences. The
kits typically include a probe that comprises an antibody or
nucleic acid sequence that specifically binds to polypeptides or
polynucleotides of the invention, such as for example and not
limitation, at least one of the detectable peptide sequences
comprising the amino acid sequence of SEQ ID NOs 2-136, as well as
genes encoding these sequences, and a label for detecting the
presence of the probe. The kits may include several antibodies
specific for, or polynucleotide sequences encoding, the
polypeptides of the invention. The kits may further comprise
control probes for detection of a control nucleic acid or a control
protein in order to provide a control level of the nucleic acid or
protein, and/or other standards or controls. The probe is
optionally detectably labeled.
[0179] The kit may contain in separate containers a nucleic acid or
antibody (either already bound to a solid matrix or packaged
separately with reagents for binding them to the matrix), control
formulations (positive and/or negative), and/or a detectable label
such as fluorescein, green fluorescent protein, rhodamine, cyanine
dyes, Alexa dyes, luciferase, radiolabels, among others.
Instructions for carrying out the assay may also be included in the
kit. The assay may, for example and not limitation, be in the form
of a Northern hybridization, sandwich ELISA or protein antibody
array.
[0180] Reagents for detecting biomarkers of the present invention
can be immobilized on a solid matrix such as a porous strip to form
at least one detectable peptide sequence detection site. The
measurement or detection region of the porous strip may include a
plurality of sites containing an antibody or nucleic acid. A test
strip may also contain sites for negative and/or positive controls.
Alternatively, control sites can be located on a separate strip
from the test strip. Optionally, the different detection sites may
contain different amounts of immobilized antibodies or nucleic
acids, e.g., a higher amount in the first detection site and lesser
amounts in subsequent sites. Upon the addition of test sample, the
number of sites displaying a detectable signal provides a
quantitative indication of the amount of detectable peptide
sequence present in the sample. The detection sites may be
configured in any suitably detectable shape and are typically in
the shape of a bar or dot spanning the width of a test strip.
[0181] Alternatively, the kit contains a nucleic acid substrate
array comprising one or more nucleic acid sequences. The nucleic
acids on the array specifically identify one or more nucleic acid
sequences adapted to bind a nucleic acid sequence encoding a
detectable peptide sequence comprising at least one of SEQ ID NOs
2-136. The substrate array can be on, e.g., a solid substrate or
"chip". Alternatively, the substrate array can be a solution
array.
[0182] A kit of the invention can also provide reagents for primer
extension and amplification reactions. For example, in some
embodiments, the kit may further include one or more of the
following components: a reverse transcriptase enzyme, a DNA
polymerase enzyme (such as, e.g., a thermostable DNA polymerase), a
polymerase chain reaction buffer, a reverse transcription buffer,
and deoxynucleoside triphosphates (dNTPs).
[0183] Alternatively (or in addition), a kit can include reagents
for performing a hybridization assay for nucleic acid(s) and/or
proteins. The detecting agents can include nucleotide analogs
and/or a labeling moiety, e.g., directly detectable moiety such as
a fluorophore (fluorochrome) or a radioactive isotope, or
indirectly detectable moiety, such as a member of a binding pair,
such as biotin, or an enzyme capable of catalyzing a non-soluble
colorimetric or luminometric reaction. In addition, the kit may
further include at least one container containing reagents for
detection of electrophoresed nucleic acids. Such reagents include
those which directly detect nucleic acids, such as fluorescent
intercalating agent or silver staining reagents, or those reagents
directed at detecting labeled nucleic acids, such as, but not
limited to, ECL reagents. A kit can further include DNA or RNA
isolation or purification means as well as positive and negative
controls. Alternatively, the kit may include at least one container
containing reagents for detection of electrophoresed proteins. Such
reagents include those which directly detect proteins, such as
Coomassie blue or other staining reagents including fluorescent
staining agents, or those reagents directed at detecting labeled
proteins. A kit can further include protein isolation or
purification means as well as positive and negative controls. A kit
can also include a notice associated therewith in a form prescribed
by a governmental agency regulating the manufacture, use or sale of
diagnostic kits. Detailed instructions for use, storage and
trouble-shooting may also be provided with the kit. A kit can also
be optionally provided in a suitable housing that is preferably
useful for robotic handling in a high throughput setting.
[0184] The components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container. The container will generally include
at least one vial, test tube, flask, bottle, syringe, and/or other
container means, into which the solvent is placed, optionally
aliquoted. The kits may also comprise a second container means for
containing a sterile, pharmaceutically acceptable buffer and/or
other solvent.
[0185] Where there is more than one component in the kit, the kit
also will generally contain a second, third, or other additional
container into which the additional components may be separately
placed. However, various combinations of components may be
comprised in a container.
[0186] Such kits may also include components that preserve or
maintain DNA or RNA, such as reagents that protect against nucleic
acid degradation. Such components may be nuclease or RNase-free or
protect against RNases, for example. Such kits may also include
components that preserve or maintain proteins, such as reagents
that protect against protein degradation. Any of the compositions
or reagents described herein may be components in a kit.
[0187] In some embodiments, the kit further comprises an apparatus
for collecting a bodily fluid sample, e.g., a urine, blood,
lymphatic fluid, saliva and/or plasma sample, from a subject. In
other embodiments, the kit further comprises instructions for using
the collection apparatus and/or the reagents comprising the
kit.
Computer-Implemented Aspects
[0188] As understood by those of ordinary skill in the art, the
methods and information described herein may be implemented, in all
or in part, as computer executable instructions on known computer
readable media. For example, the methods described herein may be
implemented in hardware. Alternatively, the method may be
implemented in software stored in, for example, one or more
memories or other computer readable medium and implemented on one
or more processors. As is known, the processors may be associated
with one or more Controllers, calculation units and/or other units
of a computer system, or implanted in firmware as desired. If
implemented in software, the routines may be stored in any computer
readable memory such as in RAM, ROM, flash memory, a magnetic disk,
a laser disk, or other storage medium, as is also known. Likewise,
this software may be delivered to a computing device via any known
delivery method including, for example, over a communication
channel such as a telephone line, the Internet, a wireless
connection, etc., or via a transportable medium, such as a computer
readable disk, flash drive, and the like.
[0189] More generally, and as understood by those of ordinary skill
in the art, the various steps described above may be implemented as
various blocks, operations, tools, modules and techniques which, in
turn, may be implemented in hardware, firmware, software, or any
combination of hardware, firmware, and/or software. When
implemented in hardware, some or all of the blocks, operations,
techniques, etc. may be implemented in, for example, a custom
integrated circuit (IC), an application specific integrated circuit
(ASIC), a field programmable logic array (FPGA), a programmable
logic array (PLA), etc.
[0190] When implemented in software, the software may be stored in
any known computer readable medium such as on a magnetic disk, an
optical disk, or other storage medium, in a RAM or ROM or flash
memory of a computer, processor, hard disk drive, optical disk
drive, tape drive, etc. Likewise, the software may be delivered to
a user or a computing system via any known delivery method
including, for example, on a computer readable disk or other
transportable computer storage mechanism.
[0191] Thus, another aspect of the disclosure is a system that is
capable of carrying out a part or all of a method of the
disclosure, or carrying out a variation of a method of the
disclosure as described herein in greater detail. Exemplary systems
include, as one or more components, computing systems,
environments, and/or configurations that may be suitable for use
with the methods and include, but are not limited to, personal
computers, server computers, hand-held or laptop devices,
multiprocessor systems, microprocessor-based systems, set top
boxes, programmable consumer electronics, network PCs,
minicomputers, mainframe computers, distributed computing
environments that include any of the above systems or devices, and
the like. In some variations, a system of the disclosure includes
one or more machines used for analysis of biological material
(e.g., genetic material), as described herein. In some variations,
this analysis of the biological material involves a chemical
analysis and/or a nucleic acid amplification.
[0192] The computer may operate in a networked environment using
logical connections to one or more remote computers, such as a
remote computer via a network interface controller (NIC). The
remote computer may be a personal computer, a server, a router, a
network PC, a peer device or other common network node, and
typically includes many or all of the elements described above
relative to the computer. The logical connection between the NIC
and the remote computer may include a local area network (LAN), a
wide area network (WAN), or both, but may also include other
networks. Such networking environments are commonplace in offices,
enterprise-wide computer networks, intranets, and the Internet. The
remote computer may also represent a web server supporting
interactive sessions with the computer; or in the specific case of
location-based applications may be a location server or an
application server.
[0193] In some embodiments, the network interface may use a modem
when a broadband connection is not available or is not used. It
will be appreciated that the network connection shown is exemplary
and other means of establishing a communications link between the
computers may be used.
EXAMPLES
[0194] The present disclosure is also described and demonstrated by
way of the following examples. However, the use of these and other
examples anywhere in the specification is illustrative only and in
no way limits the scope and meaning of the disclosure or of any
exemplified term. Likewise, the disclosure is not limited to any
particular preferred embodiments described here. Indeed, many
modifications and variations of the disclosure may be apparent to
those skilled in the art upon reading this specification, and such
variations can be made without departing from the disclosure in
spirit or in scope. The disclosure is therefore to be limited only
by the terms of the appended claims along with the full scope of
equivalents to which those claims are entitled.
Example 1
Development of the Disclosed Compositions and Methods of Use
Engineering Synthetic Biomarkers to Sense GzmB Activity
[0195] To sense cytotoxic activity of CD8 T cells, the synthetic
biomarkers consisted of a nanoparticle core conjugated with peptide
substrates specific for Granzyme B (GzmB) (FIG. 2A). The use of a
nanoparticle chaperone achieved two goals: it extended the
half-life of surface-conjugated peptides by preventing renal
clearance, and through passive targeting, accumulated in tissues
with fenestrated endothelium including secondary lymphoid organs
(e.g., spleen, lymph nodes) as well as sites of inflammation as
occurs during graft rejection (22, 23). The inventors chose iron
oxide nanoparticles (IONP) because they are FDA-approved for
clinical use including as anemia therapies, contrast agents for
imaging, and thermal ablation (24). The second component of the
synthetic biomarkers was a peptide substrate designed to sense a
target protease of interest, which in this case is GzmB, the key
cytotoxic effector protease secreted by CD8 T cells (25). These
substrates were further modified with a reporter which allows
detection of cleaved peptide fragments in urine. While the present
compositions used a fluorescent reporter to analyze urine samples
with a simple fluorescent assay, peptide substrates can be labelled
with different barcoded technologies such as isobaric mass tags for
multiplexing analysis, ligand encoded reporters for point of care
paper-based tests (17-20). To ensure biocompatibility and improved
circulation half-life of synthetic biomarkers, the inventors
decorated nanoparticle surface with polyethylene glycol (PEG),
which reduces nanoparticle uptake by the reticuloendothelial system
(RES) (26). Hydrodynamic size profiling by dynamic light scattering
(DLS) showed that PEGylated nanoparticles were stable in both PBS
and mouse plasma, with the Z-average in plasma was 47 nm (FIG. 7A).
The circulation half-life of synthetic biomarkers was .about.3
hours in mice, which was consistent with previously reported values
for clinically approved IONPs (FIG. 7B) (27).
[0196] The inventors first set out to identify peptide substrates
that are sensitive to cleavage by recombinant GzmB. From published
literature (28-31), 13 candidate substrates (FIG. 6) were pooled,
each spanning 6-8 amino acids in length and containing conserved
residues isoleucine at position P4 and aspartic acid at P1--the
position immediately N-terminal of the cleavage site. The inventors
varied the amino acids at other locations, length of the substrate,
and flexible spacers on both sides of the designed substrate. From
a library, the inventors identified the sequence AIEFD|SGc (small
case letters are amino acids synthesized as d-stereoisomers) as the
substrate that produced the highest rate of cleavage by recombinant
GzmB (FIG. 2A, FIG. 6). Michaelis-Menten kinetics analyses were
performed to assess the efficiency of GzmB cleavage of substrate on
nanoparticle surface. Fitted k.sub.cat/K.sub.M was
1.09.times.10.sup.4 M.sup.-1s.sup.-1, similar to reported values of
GzmB cleaving free substrates of similar sequences (FIG. 2B) (28,
31), which shows that presentation of substrates on nanoparticle
surface does not affect GzmB cleavage. In vivo, the coagulation and
complement cascades contain ubiquitous circulating proteases that
can degrade the synthetic biomarkers. Therefore, to test
specificity of the substrate for GzmB, the inventors studied
synthetic biomarker cleavage by key recombinant proteases as well
as activated coagulation and complement cascades in blood samples.
Increases in fluorescent intensity were observed only in samples
containing recombinant GzmB (FIGS. 2C, 2D, and 2E). Furthermore,
the formation of membrane attack complex (MAC) was not detected
when synthetic biomarkers were incubated with serum samples,
indicating that the synthetic biomarkers did not activate the
complement cascade via interaction with foreign surface (32) (FIG.
2F). Overall, these data showed that synthetic biomarkers are
specific for GzmB and are not activated by proteases in the
coagulation and complement cascades.
Synthetic Biomarkers Amplify Cytotoxic Signals from Alloreactive T
Cells
[0197] Next, the inventors sought to investigate the use of
synthetic biomarkers to sense GzmB during cytotoxic activity by
recipient T cells against donor target cells expressing
alloantigens. GzmB is the central effector protease that CD8 T
cells utilize to kill target cells. In activated T cells, GzmB is
upregulated and contained in cytolytic granules, and upon TCR-pMHC
engagement, these granules are lysed to release GzmB that enters
target cells through perforin-mediated pore formation (25). Inside
target cells, GzmB serves as potent initiator of the apoptosis
cascade by either direct cleavage to activate proapoptotic protease
Caspase 3 or induction of mitochondrial disruption (25, 33).
Previously, elevated levels of GzmB in blood has been used to
monitor the efficacy of CAR T cell therapy against B cell leukemia
(34). In solid tumors, GzmB expression in longitudinal tumor
biopsies could be used to differentiate non-responders from
responders to immune blockade therapies (35). In transplant
rejection, presence of GzmB mRNA transcript in patient urine
samples has been shown to correlate to episodes of acute rejection
(13, 14).
[0198] To test synthetic biomarkers in the context of T cell
mediated cytotoxicity, the inventors used transgenic OT-1 T cells,
which recognize the peptide epitope SIINFEKL from chicken ovalbumin
(OVA), in a T cell killing assay (36). To verify intracellular
expression of GzmB in plate-activated OT-1 T cells, the inventors
performed flow analysis and confirmed that GzmB levels were
elevated in OT-1 T cells after engagement with EG7-OVA target cells
compared to EL4 controls (FIGS. 8A-8B). To quantify the cleavage
activity of GzmB, a fluorogenic assay in which a substrate can
fluoresce upon cleavage by active GzmB was used to measure GzmB
activity inside target cells (FIG. 3A). It was observed that GzmB
activity inside EG7-OVA cells was 5 to 7-fold higher than EL-4
cells when coincubated with OT1 T cells at T cell to target cell
ratios from 1:1 to 10:1 (***P, n=3, FIGS. 3B, 3C). Because
synthetic biomarkers are designed to monitor extracellular protease
activity, the amount of secretory GzmB was measured and
significantly elevated level of GzmB in coculture supernatant of
OT1 T cells and EG7-OVA cells versus EL4 controls was detected,
with a 10-fold improvement observed at T cell to target cell ratio
of 10:1 (****P, n=3, FIG. 3D). The synthetic biomarkers amplified
cytotoxic signal from transgenic OT1 T cells killing EG7-OVA target
cells (FIGS. 3E, 3F). Furthermore, the initial velocity of probe
activation was strongly correlated to the amount of secretory GzmB
in coculture supernatant (FIG. 3D, 3G). After showing that the
synthetic biomarkers could sense transgenic T cell killing with
only one unique TCR-pMHC interaction, the inventors investigated
their ability to sense alloreactive T cell killing, which is a
polyclonal T cell response against alloantigens. For this
experiment, splenocytes from donor BALB/c mice were co-incubated
with splenocytes and lymphocytes from recipient C57BL/6 mice
bearing skin grafts before adding synthetic biomarkers to monitor
cytotoxic activity (FIG. 3H). Increased activation kinetics of
synthetic biomarkers in cocultures containing T cells from mice
bearing allografts was detected (FIG. 3I). Thus, the engineered
GzmB-sensing synthetic biomarkers are useful for detection of
alloreactive T cell killing.
GzmB is Upregulated at the Onset of Acute Cellular Rejection
[0199] During ACR episodes, host CD8 T cells activate through
direct or indirect pathways by interaction with donor- or
host-derived antigen presenting cells (APC) respectively, expand
rapidly in secondary lymphoid organs (e.g., spleen and lymph
nodes), and secrete cytotoxic granules containing GzmB to mediate
allograft rejection (3, 4). Currently, the core biopsy is used to
examine for histological hallmarks of ACR--which directly result
from T cell activity--including T cell infiltration, inflammatory
cytokines, morphological changes, tissue remodeling (6). In
contrast to these morphological biomarkers, which are downstream
and lack predictive value, the ability to detect proteases that
drive disease pathology can allow anticipation of patient
trajectory, such as monitoring MMP activity in liver fibrosis
progression and regression (17-21).
[0200] First, the inventors sought to validate the expression of
GzmB at the onset of acute rejection using the well-established
BALB/c to B6 mouse model of skin transplantation (FIG. 4A). To
quantify skin graft health, the inventors assigned a score of 4 for
healthy grafts, a score of 0 for rejected grafts, and intermediate
scores based on features such as the ratio of viable to necrotic
skin and the presence of ulcerations or scabs. Nine days after
transplant, allograft scores began to decrease significantly
compared to isograft controls (2.6 vs. 3.9, P<0.0001; FIGS. 4B,
4C and FIG. 9) and reached an endpoint when grafts were completely
rejected two weeks post-transplant. To investigate localized
expression of GzmB during rejection, the inventors stained skin
grafts and tissue sections and found significant upregulation of
CD8 and GzmB in skin allografts (FIG. 4D, FIGS. 10A, 10B). By
contrast, GzmB was not detected in plasma samples throughout the
course of rejection (FIG. 4E). To investigate the kinetics of GzmB
expression relative to the rate of graft rejection, the inventors
analyzed CD8 T cells in splenocytes and lymphocytes from mice
bearing skin grafts before and during the peak of rejection (day 5
to day 9). It was found that GzmB levels in CD8 T cells expressing
the activation marker CD44 were first elevated on day 7 when
allograft and isograft scores were indistinguishable (FIG. 4C), and
continued increasing throughout the course of allograft rejection
(FIG. 4F). Taken together, these data have shown that GzmB
expression is localized to skin allografts and secondary lymphoid
organs and upregulated before the onset of acute rejection.
Urine Analyses Predict Early Acute Rejection of Skin Allografts
[0201] During the onset of acute rejection, damaged associated
molecular patterns (DAMPS) trigger the release of proinflammatory
cytokines (e.g., TNF-alpha, IL-6) by innate immune cells that
increase vessel permeability to enhance local blood flow and immune
cell infiltration (4, 22, 23). Previously, this localized
vasodilation was exploited to deliver nanomedicines to inflammatory
tissues including atherosclerotic plaques and tumors (37-39). Thus,
the inventors sought to quantify the extent by which synthetic
biomarkers accumulate in allografts during inflammation and
rejection relative to healthy tissues. The inventors intravenously
administered surface-labelled nanoparticles to C57BL/6 mice
transplanted with both skin allografts and isografts on the same
recipient at the onset of rejection (day 7) to allow quantification
by full-body fluorescent imaging (FIG. 5A). Whereas both skin graft
scores were statistically identical (FIG. 4C), the inventors found
a 6-fold higher accumulation of nanoparticles in allografts
compared to isografts or healthy skin (***P, n=3) (FIGS. 5B, 5C,
and FIG. 11). This result was further supported by biodistribution
studies, where in addition to nanoparticle localization in skin
allografts, the inventors also observed passive targeting to organs
with fenestrated endothelium such as liver and secondary lymphoid
organs (spleen, draining lymph nodes) over other major organs
(brain, heart, kidney, lung) (FIG. 5D and FIG. 12). This transport
of nanoparticles across porous vasculature is consistent with
well-established studies in nanomedicine (37, 39). To confirm size
dependent filtration into urine, either labelled free peptides or
labelled nanoparticles were administered to skin graft mice. It was
observed that only free peptides cleared into urine (FIGS.
13A-13D).
[0202] Last, the inventors investigated the ability of synthetic
biomarkers to predict the onset of ACR by shedding a fluorescent
reporter into urine (FIG. 5E). To track in vivo GzmB cleavage
activity, on day 7 synthetic biomarkers were administered to mice
bearing skin grafts and several organs of interest were collected
for near infra-red (NIR) imaging. GzmB activity in allograft mice,
with respect to isograft mice, was 3-fold higher in the kidney due
to rapid clearance of the small fluorophores after cleavage (*P,
n=3-4, FIG. 5F). Using the same probe, the inventors performed full
body imaging and only detected fluorescent signals from the
bladders of allograft mice (FIG. 5G). Next, synthetic biomarkers
were administered to mice bearing skin grafts before (day -4) and
after (day 7) transplant surgeries were performed to establish a
diagnostic metric via urine fluorescence. Pre-graft urine signals
served as an internal baseline for each mouse to control for urine
clearance rate and confounding variables associated with transplant
surgery. The inventors did not detect significant elevation in
post-graft urine signals, with respect to pre-graft signals, from
naive mice, isograft mice, and allograft mice depleted of CD8 T
cells. In contrast, post-graft urine signals from allograft mice
significantly elevated by more than 2-fold when compared to
pre-graft signals (***P, n=7, FIG. 5H). Meanwhile, graft scores of
allografts and isografts were still undifferentiated (FIG. 4C).
Receiver-operating-characteristic (ROC) analysis showed that the
synthetic biomarkers could distinguish between accepting isografts
and rejecting allografts with an area under the curve (AUC) of
0.969 (95% CI was 0.892 to 1.045) (FIG. 5I). This AUC value
demonstrated greatly improved diagnostic power over other promising
noninvasive diagnostic platforms, where AUC values fall between 0.7
and 0.9. Overall, noninvasive administration of GzmB sensing
synthetic biomarkers allowed for prediction of ACR in a skin graft
mouse model of transplant rejection and provided a promising
diagnostic platform to replace tissue biopsy.
Discussion
[0203] In transplantation medicine, the core biopsy is considered
the "gold" standard for diagnosing anti-graft activity. However,
the biopsy is associated with significant patient morbidity and
sampling error. Thus, there is a clinical need to develop
noninvasive, sensitive, and specific diagnostics that accurately
predict acute rejection episodes. Herein are described
activity-based nanosensors consisting of a nanoparticle core
decorated with peptide substrates to sense the proteolytic activity
of GzmB during antigen-specific CD8 T cell killing. In skin graft
mouse models, synthetic biomarkers passively accumulate in
allograft tissue during T-cell mediated rejection where they are
cleaved by GzmB secreted by alloreactive T cells. These cleavage
events trigger a pharmacokinetic switch where the cleaved peptide
fragments, due to their small size, are filtered into urine to
produce a noninvasive signal that predicts the onset of ACR.
[0204] The use of a nanoparticle chaperon increases circulating
half-life of substrate peptides which otherwise are cleared from
the body by renal filtration within minutes after IV
administration. This enhanced pharmacokinetics enables passive
delivery of peptides to inflamed allograft tissues, which are
active areas of T-cell mediated acute rejection. Though passive
accumulation of synthetic biomarkers is significant in skin
allograft, which is a thin and small piece of tissue, significantly
higher accumulation would occur in allografts of larger and more
solid organs. Furthermore, it is possible to functionalize the
nanoparticle scaffolds with organ-specific ligands to further
enhance delivery and detection signals. The inventors used IONPs as
the carrier of substrate peptides because they are FDA-approved and
have great translation potential. These synthetic biomarkers are
well tolerated upon repeated administration and have relatively
short circulation half-life (3 hours), which allows multiple time
point sampling to more accurately monitor progression of graft
heath. Besides using IONPs, substrate peptides can be coated on
other nanoscale scaffolds (e.g., PEG, protein-based carriers, PLGA)
to tune their pharmacokinetics and presentation to optimize urine
signals. While upregulation of GzmB has been detected in patients
with acute rejection episodes, this expression is mostly localized
to disease tissues, making in vitro diagnosis with a simple blood
draw especially challenging. The invented synthetic biomarkers
monitor in vivo GzmB activity at sites of active rejection and
offer two potential methods of amplifying detection signals: (1)
enzymatic turnovers allows one endogenous copy of GzmB to cleave
thousands of synthetic substrate peptides while (2) renal clearance
allows concentration of this disease signal in a small urine
volume.
[0205] The inventors have developed a sensitive probe to detect the
activity of GzmB, which is the key effector protease during T cell
cytotoxicity. Previous studies have found a correlation between
elevations of GzmB RNA transcripts in patient urine samples to
acute allograft rejection episodes (13, 14). Instead of looking at
GzmB abundance that does not differentiate between Serpin-bound
(inactive) from active form of GzmB (15, 40), these synthetic
biomarkers sense proteolytic activity of GzmB as a predictor for
acute cellular rejection. A single biomarker reflective of a
fundamental cellular process or mechanism can be broadly applied to
detect or monitor a range of disease conditions. For example,
fluorodeoxyglucose positron-emission tomography (FDG-PET) imaging,
which only monitors glucose metabolism, are valuable in diagnosis,
staging, and monitoring treatments of cancers and neurodegenerative
disorders (41-43), Likewise, synthetic biomarkers that monitor GzmB
activity can be used in the diagnosis of immune conditions related
to T cell cytotoxicity which include graft versus host disease
(GvHD), autoimmune diseases, and immuno-oncology. Moreover, the
sensitivity of a single biomarker is dependent on the setting in
which it is recommended for clinical use. For example, monitoring
the levels of a single biomarker over time in high-risk patients
can significantly increase detection sensitivity. For example,
prostate-specific antigen (PSA) lacks specificity and sensitivity
for general population screening; however, it is an excellent
biomarker when used in high-risk patients such as monitoring
recurrent after radiation therapies (44).
[0206] Moving forward, this platform can be readily expanded into a
multiplex probe set using mass barcodes (17) to obtain a protease
signature that reflects a more complete picture of patient
pathology. Multiplexing analysis improves diagnostic specificity by
simultaneous monitoring the activities of disease-associated
proteases on the background of T cell mediated cytotoxicity. This
capability can be valuable in differentiating acute organ rejection
from conditions such bacterial infections, viral infections, and
cancers, which all involve T cell cytotoxicity but are also driven
by unique protease subsets (bacterial proteases, viral proteases,
and tumor-derived proteases) (45-47). Furthermore, it is possible
to distinguish acute rejection, which is mediated by T cell
effector proteases (GzmB), from chronic rejection, which is driven
by complement (Cls, Clr) and fibrotic (ADAMTS1) proteases (5, 10,
48, 49). The ability to profile protease signatures at various
stages of rejection might allow researchers to learn more about
unique disease mechanisms to develop better therapies that prolong
graft survival and improve patient outcomes.
Materials and Methods
NP Synthesis and Characterization
[0207] Aminated IONPs were synthesized in house per published
protocol. GzmB-sensing peptides were synthesized by Tufts
University Core Facility peptide synthesis service. Aminated IONPs
were first reacted to the biofunctional crosslinker Succinimidyl
Iodoacetate (SIA; Thermo) for 2 hours at room temperature (RT) and
excess SIA were removed by buffer exchange using Amicon spin filter
(30 kDa, Millipore). Sulfhydryl-terminated peptides and
Polyethylene Glycol (PEG; LaysanBio, M-SH-20K) were mixed with
NP-SIA (90:20:1 molar ratio) and reacted overnight at RT in the
dark to obtain fully conjugated synthetic biomarkers. Synthetic
biomarkers were purified on a Superdex 200 Increase 10-300 GL
column using AKTA Pure FPLC System (GE Health Care). Ratios of FITC
per IONP were determined using absorbance of FITC (488 nm,
.epsilon.=78,000 cm-1M-1) and IONP (400 nm,
.epsilon.=2.07.times.10.sup.6 cm.sup.-1M.sup.-1) measured with
Cytation 5 Plate Reader (Biotek). DLS measurements of synthetic
biomarkers were done in PBS or mouse plasma at RT using Zetasizer
Nano ZS (Malvern). For half-life characterization, the inventors
administered via IV 20 ug of labelled IONPs to CFW Mice (Charles
River). At several time points following NP administration, blood
was collected into heparin-coated Capillary Tubes (VWR) via
retro-orbital collection and imaged using Odyssey CLx Imaging
System (LI-COR).
In Vitro Protease Cleavage Assays
[0208] Synthetic biomarkers (6 nM by NP, 300 nM by peptide) were
incubated in PBS+1% bovine serum albumin (BSA; Sigma) at 37.degree.
C. with murine Granzyme B (0.2 .mu.M, Peprotech), human thrombin
(HaemTech), mouse thrombin (HaemTech), mouse plasmin (HaemTech),
Clr (Sigma), Cls (Sigma), Factor D (Sigma), Factor I (Sigma), MASP2
(Biomatik). Sample fluorescence were measured for 60 minutes using
Cytation 5 plate reader (Biotek). To optimize GzmB substrate, a
library of potential substrates was synthesized by Tufts University
Core Facility peptide synthesis service and conjugated to IONPs.
Cleavage assays of nanoparticles decorated with these substrates
with recombinant GzmB were performed, and data was fitted to
compare initial cleavage velocities. To determine Michaelis-Menten
constants, cleavage assays with GzmB were performed at different
substrate concentrations. To initiate coagulation cascade, citrated
plasma was mixed with synthetic biomarkers before addition of
calcium chloride (15 mM, Sigma). To initiate complement activation,
Control Human Serum (Sigma) was mixed with synthetic biomarkers
before addition of Heat Aggregated Gamma Globulin (HAGG; Quidel)
per the manufacturer's protocol. After measuring fluorescence for 1
hour, supernatants were collected and measured for formation of MAC
complex using MicroVue CH50 Eq EIA Kit (Quidel).
GzmB Characterization in Transgenic T Cell Cocultures
[0209] EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640
supplemented with 10% FBS and 25 mM HEPES (Gibco). EG7-OVA cultures
were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells
were isolated from OT1 (Jackson Labs) splenocytes by MACS using
CD8a Microbeads (Miltenyi). Cells were activated by seeding in
96-well plates coated with anti-mouse CD3e (Clone: 145-2C11, BD)
and anti-mouse CD28 (Clone: 37.51, BD) at 2.times.10.sup.6 cells/ml
in RPMI 1640 supplemented with 10% FBS, 100 U/ml
penicillin-streptomycin, 1.times. non-essential amino acids
(Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 30
U/ml hIL-2 (Roche). After 2 days, cells were transferred to
uncoated plates. On day 5, 1.times.10.sup.6 activated OT1 T cells
were coincubated with 1.times.10.sup.6 EL4 or EG7-OVA cells for 2
hours at 37.degree. C. and stained for GzmB using anti-mouse GzmB
(Clone: NGZB, eBioScience) and Intracellular Fixation &
Permeabilization Buffer Set (eBioScience, 88-8824-00). To measure
GzmB activity inside target cells, the inventors coincubated
activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at
various T cell to target cell ratios and stained using GranToxiLux
Kit (OncoImmunin, GTL702-8). To measure secretory GzmB, the
inventors collected coculture supernatant of OT1 with target cells
and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience,
BMS6029).
NP Assays Sensing T Cell Killing
[0210] To sense transgenic T cell killing, CD8+ OT1 T cells were
isolated and activated per above protocol. On day 5 post
activation, 1.times.10.sup.6 OT1 T cells were coincubated with
1.times.106 EL4 or EG7-OVA target cells for 2 hours at 37.degree.
C. Coculture supernatants were mixed with synthetic biomarkers (2
nM by NP, 100 nM by peptides) and fluorescence were monitored for 1
hour at 37.degree. C. To sense alloreactive T cell killing, on day
7 post-transplant, CD8 T cells were isolated from splenocytes and
lymphocytes of skin graft mice. 5.times.10.sup.5 CD8 T cells from
skin graft mice were restimulated with 5.times.10.sup.5 splenocytes
from BALB/c Mice (Charles River) for 6 hours at 37.degree. C.
Coculture supernatants were mixed with synthetic biomarkers (2 nM
by NP, 100 nM by peptides) and fluorescence were monitored for 2
hours at 37.degree. C.
Skin Graft Scoring
[0211] Skin grafts were qualitatively scored on a scale ranging
from 0-4 per established protocol by the Emory Transplant Center.
Scoring involved direct observation and palpation of the graft and
surrounding tissue. A score of 0 was characterized by complete
tissue necrosis, stiffness, and severe discoloration. A score of 4
was characterized by incorporation with surrounding tissue,
pliability, and healthy pigmentation.
GzmB Characterization in Skin Graft Mouse Model
[0212] For histological analysis, tissues were collected from skin
graft mice at day 7 post-transplant. All tissues were fixed in 4%
paraformaldehyde (EMS) overnight at 4.degree. C., washed with PBS
and stored in 70% ethanol (VWR) until paraffin-embedding,
sectioning, and staining for GzmB and CD8 (Winship Pathology Core).
To analyze blood level of GzmB, citrated plasma samples were
collected from skin graft mice during rejection and diluted to 10%
before performing ELISA with Granzyme B Mouse ELISA Kit
(eBioScience, BMS6029). For flow cytometry analysis,
1.times.10.sup.6 splenocytes or lymphocytes from skin graft mice
were restimulated with 1.times.10.sup.6 BALB/c splenocytes for 6
hours at 37.degree. C. before staining for GzmB using anti-mouse
GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation &
Permeabilization Buffer Set (eBioScience).
NP Pharmacokinetics
[0213] On day 7 post-transplant, skin graft mice were administered
with either NPs (20 ug) or peptides (10 nmol) labelled with VivoTag
S-750 (VT750; PerkinElmer). For organ biodistribution, whole mice
were imaged with IVIS Spectrum CT Imaging System (PerkinElmer)
while excised organs were imaged with Odyssey CLx Imaging System
(LI-COR) after 24 hours. For urine pharmacokinetics, whole mice
were imaged with IVIS Spectrum CT Imaging System (PerkinElmer)
after 30-90 minutes.
Urinary Prediction of Acute Cellular Rejection
[0214] To track cleaved fragments after in vivo GzmB cleavage, on
day 7 post-transplant, VT750-labelled synthetic biomarkers (10 nmol
by peptides) were administered to skin graft mice. Major organs
were excised and imaged with Odyssey CLx Imaging System (LI-COR)
while mouse bladders were imaged with IVIS Spectrum CT Imaging
System (PerkinElmer) after 90 minutes. All urinalysis experiments
were done in paired setup. 4 days before and 7 days after
surgeries, skin graft mice were administered with FITC-labelled
Synthetic biomarkers (10 nmol by peptides). Urine were collected
after 90 minutes by placing mice over 96-well plates. FITC in urine
was purified by a magnetic separation assay using Dynabeads
(Thermo, 65501) coated with anti-FITC (GeneTex, GTX10257).
Fluorescent signals were measured with Cytation 5 Plate Reader
(Biotek). Concentrations of FITC were calculated using a free FITC
ladder and normalized with urine volume. For CD8 depletion study,
mice were given anti-mouse CD8 (clone: 53-6.7, BioXCell) for 3
consecutive days following with booster shots every 3 days after.
Flow cytometry analysis of splenocytes and lymphocytes were
performed with anti-mouse CD3 (clone: 17A2, Biolegend), anti-mouse
CD4 (clone: RM4-5, Biolegend), anti-mouse CD8 (clone: KT15,
Serotec) to confirm success of depletion.
Software and Statistical Analysis
[0215] Graphs were plotted and appropriate statistical analyses
were conducted using GraphPad Prism (*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001; all error bars depict s.e.m.).
Quantification of histological images was performed on ImageJ
Whole-mouse fluorescent data were analyzed using Living Image
(PerkinElmer). Whole-organ fluorescent data were analyzed using
Image Studio (LI-COR). Flow cytometry data were analyzed using
FlowJo X (FlowJo, LLC).
[0216] While several possible embodiments are disclosed above,
embodiments of the present disclosure are not so limited. These
exemplary embodiments are not intended to be exhaustive or to
unnecessarily limit the scope of the disclosure, but instead were
chosen and described in order to explain the principles of the
present disclosure so that others skilled in the art may practice
the disclosure. Indeed, various modifications of the disclosure in
addition to those described herein will become apparent to those
skilled in the art from the foregoing description. Such
modifications are intended to fall within the scope of the appended
claims. The scope of the disclosure is therefore indicated by the
following claims, rather than the foregoing description and
above-discussed embodiments, and all changes that come within the
meaning and range of equivalents thereof are intended to be
embraced therein.
[0217] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference in their entirety as if physically
present in this specification.
TABLE-US-00001 TABLE OF SEQUENCES SEQ ID NO. Type Source Sequence*
1 Protein Synthetic IIGGHEVKPHSRPYMALLSIKDQQPEAICGFLIREDFVLTAAHCEG
SIINVTLGAHNIKEQEKTQQVIPMVKCIPHPDYNPKTFSNDIMLLKLK
SKAKRTRAVRPLNLPRRNVNVKPGDVCYVAGWGRMAPMGKYSNT
LQEVELTVQKDRECESYFKNRYNKTNQICAGDPKTKRASFRGDSGG
PLVCKKVAAGIVSYGYKDGSPPRAFTKVSSFLSWIKKTMKSS 2 Protein Synthetic
AIEPDGSC 3 Protein Synthetic ASGIEPDSGGSC 4 Protein Synthetic
AKSKIEFDFGVKKC 5 Protein Synthetic AIEPDSGC 6 Protein Synthetic
AIEPDGSSKC 7 Protein Synthetic AIEPDSGSKC 8 Protein Synthetic
AKSIEPDGSSKC 9 Protein Synthetic AKSIEPDSGSKC 10 Protein Synthetic
AIEFDGSC 11 Protein Synthetic AIEFDSGC 12 Protein Synthetic
AIEFDSGSKC 13 Protein Synthetic AKSIEFDSGSKC 14 Protein Synthetic
AIEFDSGVSKC 15 Protein Synthetic AIEPDGSc 16 Protein Synthetic
AsGIEPDSGGsc 17 Protein Synthetic AksKIEFDEGVKkc 18 Protein
Synthetic AIEPDSGc 19 Protein Synthetic AIEPDGSskc 20 Protein
Synthetic AIEPDSGskc 21 Protein Synthetic AksIEPDGSskc 22 Protein
Synthetic AksIEPDSGskc 23 Protein Synthetic AIEFDGSc 24 Protein
Synthetic AIEFDSGc 25 Protein Synthetic AIEFDSGskc 26 Protein
Synthetic AksIEFDSGskc 27 Protein Synthetic AIEFDSGVskc 28 Protein
Synthetic EGVNDNEEGFFSAR 29 Protein Synthetic eGyndneeGffsar 30
Protein Synthetic AIEPDGSC 31 Protein Synthetic ASGIEPDSGGSC 32
Protein Synthetic AKSKIEFDFGVKKC 33 Protein Synthetic AIEPDSGC 34
Protein Synthetic AIEPDGSSKC 35 Protein Synthetic AIEPDSGSKC 36
Protein Synthetic AKSIEPDGSSKC 37 Protein Synthetic AKSIEPDSGSKC 38
Protein Synthetic AIEFDGSC 39 Protein Synthetic AIEFDSGC 40 Protein
Synthetic AIEFDSGSKC 41 Protein Synthetic AKSIEFDSGSKC 42 Protein
Synthetic AIEFDSGVSKC 43 Protein Synthetic aIEPDGSc 44 Protein
Synthetic asGIEPDSGGsc 45 Protein Synthetic aksKIEFDFGVKkc 46
Protein Synthetic aIEPDSGc 47 Protein Synthetic aIEPDGSskc 48
Protein Synthetic aIEPDSGskc 49 Protein Synthetic aksIEPDGSskc 50
Protein Synthetic aksIEPDSGskc 51 Protein Synthetic aIEFDGSc 52
Protein Synthetic aIEFDSGc 53 Protein Synthetic aIEFDSGskc 54
Protein Synthetic aksIEFDSGskc 55 Protein Synthetic aIEFDSGVskc 56
Protein Synthetic EGVNDNEEGFFSARKAIEPDGSC 57 Protein Synthetic
EGVNDNEEGFFSARKASGIEPDSGGSC 58 Protein Synthetic
EGVNDNEEGFFSARKAKSKIEFDFGVKKC 59 Protein Synthetic
EGVNDNEEGFFSARKAIEPDSGC 60 Protein Synthetic
EGVNDNEEGFFSARKAIEPDGSSKC 61 Protein Synthetic
EGVNDNEEGFFSARKAIEPDSGSKC 62 Protein Synthetic
EGVNDNEEGFFSARKAKSIEPDGSSKC 63 Protein Synthetic
EGVNDNEEGFFSARKAKSIEPDSGSKC 64 Protein Synthetic
EGVNDNEEGFFSARKAIEFDGSC 65 Protein Synthetic
EGVNDNEEGFFSARKAIEFDSGC 66 Protein Synthetic
EGVNDNEEGFFSARKAIEFDSGSKC 67 Protein Synthetic
EGVNDNEEGFFSARKAKSIEFDSGSKC 68 Protein Synthetic
EGVNDNEEGFFSARKAIEFDSGVSKC 69 Protein Synthetic
eGyndneeGffsarKaIEPDGSc 70 Protein Synthetic
eGyndneeGffsarKasGIEPDSGGsc 71 Protein Synthetic
eGyndneeGffsarKaksKIEFDFGVKkc 72 Protein Synthetic
eGyndneeGffsarKaIEPDSGc 73 Protein Synthetic
eGyndneeGffsarKaIEPDGSskc 74 Protein Synthetic
eGyndneeGffsarKaIEPDSGskc 75 Protein Synthetic
eGyndneeGffsarKaksIEPDGSskc 76 Protein Synthetic
eGyndneeGffsarKaksIEPDSGskc 77 Protein Synthetic
eGyndneeGffsarKaIEFDGSc 78 Protein Synthetic
eGyndneeGffsarKaIEFDSGc 79 Protein Synthetic
eGyndneeGffsarKaIEFDSGskc 80 Protein Synthetic
eGyndneeGffsarKaksIEFDSGskc 81 Protein Synthetic
eGyndneeGffsarKaIEFDSGVskc 82 Protein Synthetic AAPVRSL 83 Protein
Synthetic ALDPRSF 84 Protein Synthetic ATQNKAS 85 Protein Synthetic
ACYLD 86 Protein Synthetic AIETDGS 87 Protein Synthetic ALEVDCY 88
Protein Synthetic AEPLFAERK 89 Protein Synthetic AMNPKFA 90 Protein
Synthetic ALQRIYKC 91 Protein Synthetic AKSVARTLLVKC 92 Protein
Synthetic AEEKQRIIGC 93 Protein Synthetic AQRQRIIGGC 94 Protein
Synthetic ALGRGGSC 95 Protein Synthetic AKYLGRSYKVC 96 Protein
Synthetic ARALERGLQDC 97 Protein Synthetic ASLGRKIQIC 98 Protein
Synthetic AGLQRALEIC 99 Protein Synthetic AKVFMGRVYDPC 100 Protein
Synthetic ASSTGRNGFKC 101 Protein Synthetic AKTTGGRIYGGC 102
Protein Synthetic ADPRGGSC 103 Protein Synthetic AVPRGGSC 104
Protein Synthetic ALPSRSSKIC 105 Protein Synthetic AHRGRTLEIC 106
Protein Synthetic ASTGRNGFKC 107 Protein Synthetic AQQKRKIVLC 108
Protein Synthetic AQARKIVLC 109 Protein Synthetic AQARGGSC 110
Protein Synthetic AIPENFF 111 Protein Synthetic AKEEEGL 112 Protein
Synthetic ANLVYMV 113 Protein Synthetic ARLAAIT 114 Protein
Synthetic ASLSRLT 115 Protein Synthetic APLGVRGK 116 Protein
Synthetic ADEVDNK 117 Protein Synthetic ADEVDGV 118 Protein
Synthetic ADEVDRD 119 Protein Synthetic ADEVDGV 120 Protein
Synthetic ALEVDCY
121 Protein Synthetic AAAPVc 122 Protein Synthetic AAAPAc 123
Protein Synthetic AAAPLc 124 Protein Synthetic AAAPMc 125 Protein
Synthetic AAAPFc 126 Protein Synthetic GGFPRSGGGc 127 Protein
Synthetic AGFPRSGGGc 128 Protein Synthetic AIKFFSAc 129 Protein
Synthetic ALRQRESc 130 Protein Synthetic AAEFRHDc 131 Protein
Synthetic AAAPFc 132 Protein Synthetic AQFVLTEc 133 Protein
Synthetic ARETYGEc 134 Protein Synthetic AATVYVDc 135 Protein
Synthetic APLDKKRc 136 Protein Synthetic ADKVKAQc *Lowercase
letters indicate the D form of the amino acid.
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Sequence CWU 1
1
1151226PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Ile Ile Gly Gly His Glu Val Lys Pro His Ser
Arg Pro Tyr Met Ala1 5 10 15Leu Leu Ser Ile Lys Asp Gln Gln Pro Glu
Ala Ile Cys Gly Phe Leu 20 25 30Ile Arg Glu Asp Phe Val Leu Thr Ala
Ala His Cys Glu Gly Ser Ile 35 40 45Ile Asn Val Thr Leu Gly Ala His
Asn Ile Lys Glu Gln Glu Lys Thr 50 55 60Gln Gln Val Ile Pro Met Val
Lys Cys Ile Pro His Pro Asp Tyr Asn65 70 75 80Pro Lys Thr Phe Ser
Asn Asp Ile Met Leu Leu Lys Leu Lys Ser Lys 85 90 95Ala Lys Arg Thr
Arg Ala Val Arg Pro Leu Asn Leu Pro Arg Arg Asn 100 105 110Val Asn
Val Lys Pro Gly Asp Val Cys Tyr Val Ala Gly Trp Gly Arg 115 120
125Met Ala Pro Met Gly Lys Tyr Ser Asn Thr Leu Gln Glu Val Glu Leu
130 135 140Thr Val Gln Lys Asp Arg Glu Cys Glu Ser Tyr Phe Lys Asn
Arg Tyr145 150 155 160Asn Lys Thr Asn Gln Ile Cys Ala Gly Asp Pro
Lys Thr Lys Arg Ala 165 170 175Ser Phe Arg Gly Asp Ser Gly Gly Pro
Leu Val Cys Lys Lys Val Ala 180 185 190Ala Gly Ile Val Ser Tyr Gly
Tyr Lys Asp Gly Ser Pro Pro Arg Ala 195 200 205Phe Thr Lys Val Ser
Ser Phe Leu Ser Trp Ile Lys Lys Thr Met Lys 210 215 220Ser
Ser22528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Ala Ile Glu Pro Asp Gly Ser Cys1
5312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ala Ser Gly Ile Glu Pro Asp Ser Gly Gly Ser Cys1
5 10414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ala Lys Ser Lys Ile Glu Phe Asp Phe Gly Val Lys
Lys Cys1 5 1058PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Ala Ile Glu Pro Asp Ser Gly Cys1
5610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ala Ile Glu Pro Asp Gly Ser Ser Lys Cys1 5
10710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Ala Ile Glu Pro Asp Ser Gly Ser Lys Cys1 5
10812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Ala Lys Ser Ile Glu Pro Asp Gly Ser Ser Lys Cys1
5 10912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ala Lys Ser Ile Glu Pro Asp Ser Gly Ser Lys Cys1
5 10108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Ala Ile Glu Phe Asp Gly Ser Cys1
5118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Ala Ile Glu Phe Asp Ser Gly Cys1
51210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Ala Ile Glu Phe Asp Ser Gly Ser Lys Cys1 5
101312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Ala Lys Ser Ile Glu Phe Asp Ser Gly Ser Lys
Cys1 5 101411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Ala Ile Glu Phe Asp Ser Gly Val Ser
Lys Cys1 5 10158PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(8)..(8)D-amino acid 15Ala Ile Glu
Pro Asp Gly Ser Cys1 51612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(2)D-amino
acidMOD_RES(11)..(12)D-amino acid 16Ala Ser Gly Ile Glu Pro Asp Ser
Gly Gly Ser Cys1 5 101714PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(3)D-amino
acidMOD_RES(13)..(14)D-amino acid 17Ala Lys Ser Lys Ile Glu Phe Asp
Phe Gly Val Lys Lys Cys1 5 10188PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(8)..(8)D-amino acid
18Ala Ile Glu Pro Asp Ser Gly Cys1 51910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(8)..(10)D-amino acid 19Ala Ile Glu Pro Asp Gly Ser
Ser Lys Cys1 5 102010PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(8)..(10)D-amino acid
20Ala Ile Glu Pro Asp Ser Gly Ser Lys Cys1 5 102112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(2)..(3)D-amino acidMOD_RES(10)..(12)D-amino acid
21Ala Lys Ser Ile Glu Pro Asp Gly Ser Ser Lys Cys1 5
102212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(3)D-amino
acidMOD_RES(10)..(12)D-amino acid 22Ala Lys Ser Ile Glu Pro Asp Ser
Gly Ser Lys Cys1 5 10238PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(8)..(8)D-amino acid
23Ala Ile Glu Phe Asp Gly Ser Cys1 5248PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(8)..(8)D-amino acid 24Ala Ile Glu Phe Asp Ser Gly
Cys1 52510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(10)D-amino acid 25Ala Ile Glu Phe Asp
Ser Gly Ser Lys Cys1 5 102612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(2)..(3)D-amino
acidMOD_RES(10)..(12)D-amino acid 26Ala Lys Ser Ile Glu Phe Asp Ser
Gly Ser Lys Cys1 5 102711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(9)..(11)D-amino acid
27Ala Ile Glu Phe Asp Ser Gly Val Ser Lys Cys1 5
102814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg1 5 102914PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(3)..(8)D-amino acidMOD_RES(10)..(14)D-amino acid 29Glu
Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg1 5
10308PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Ala Ile Glu Pro Asp Gly Ser Cys1
53112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Ala Ser Gly Ile Glu Pro Asp Ser Gly Gly Ser
Cys1 5 103214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 32Ala Lys Ser Lys Ile Glu Phe Asp Phe
Gly Val Lys Lys Cys1 5 10338PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Ala Ile Glu Pro Asp Ser Gly
Cys1 53410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ala Ile Glu Pro Asp Gly Ser Ser Lys Cys1 5
103511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Lys Ala Ile Glu Pro Asp Ser Gly Ser Lys Cys1 5
103612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Ala Lys Ser Ile Glu Pro Asp Gly Ser Ser Lys
Cys1 5 103712PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Ala Lys Ser Ile Glu Pro Asp Ser Gly
Ser Lys Cys1 5 10388PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 38Ala Ile Glu Phe Asp Gly Ser Cys1
5398PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Ala Ile Glu Phe Asp Ser Gly Cys1
54010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Ala Ile Glu Phe Asp Ser Gly Ser Lys Cys1 5
104112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Ala Lys Ser Ile Glu Phe Asp Ser Gly Ser Lys
Cys1 5 104212PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 42Lys Ala Ile Glu Phe Asp Ser Gly Val
Ser Lys Cys1 5 10438PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(8)..(8)D-amino acid 43Ala Ile Glu Pro Asp Gly Ser Cys1
54412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(2)D-amino
acidMOD_RES(11)..(12)D-amino acid 44Ala Ser Gly Ile Glu Pro Asp Ser
Gly Gly Ser Cys1 5 104514PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(3)D-amino
acidMOD_RES(14)..(15)D-amino acid 45Ala Lys Ser Lys Ile Glu Phe Asp
Phe Gly Val Lys Lys Cys1 5 10468PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(8)..(8)D-amino acid 46Ala Ile Glu Pro Asp Ser Gly Cys1
54710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(8)..(10)D-amino
acid 47Ala Ile Glu Pro Asp Gly Ser Ser Lys Cys1 5
104810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(8)..(10)D-amino
acid 48Ala Ile Glu Pro Asp Ser Gly Ser Lys Cys1 5
104912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(3)D-amino
acidMOD_RES(10)..(12)D-amino acid 49Ala Lys Ser Ile Glu Pro Asp Gly
Ser Ser Lys Cys1 5 105012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(3)D-amino
acidMOD_RES(10)..(12)D-amino acid 50Ala Lys Ser Ile Glu Pro Asp Ser
Gly Ser Lys Cys1 5 10518PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(8)..(8)D-amino acid 51Ala Ile Glu Phe Asp Gly Ser Cys1
5528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(9)..(9)D-amino
acid 52Ala Ile Glu Phe Asp Ser Gly Cys1 55310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)D-amino acidMOD_RES(8)..(10)D-amino acid
53Ala Ile Glu Phe Asp Ser Gly Ser Lys Cys1 5 105412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(3)D-amino acidMOD_RES(10)..(12)D-amino acid
54Ala Lys Ser Ile Glu Phe Asp Ser Gly Ser Lys Cys1 5
105511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(9)..(11)D-amino
acid 55Ala Ile Glu Phe Asp Ser Gly Val Ser Lys Cys1 5
105623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Gly Ser Cys
205727PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ser Gly Ile Glu Pro Asp Ser Gly Gly Ser Cys
20 255829PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Lys Ser Lys Ile Glu Phe Asp Phe Gly Val Lys
Lys Cys 20 255923PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Glu Gly Val Asn Asp Asn Glu Glu Gly
Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Ser Gly Cys
206025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Gly Ser Ser Lys Cys 20
256125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Ser Gly Ser Lys Cys 20
256227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Lys Ser Ile Glu Pro Asp Gly Ser Ser Lys Cys
20 256327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Lys Ser Ile Glu Pro Asp Ser Gly Ser Lys Cys
20 256423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Gly Ser Cys
206523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Cys
206625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Ser Lys Cys 20
256727PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Lys Ser Ile Glu Phe Asp Ser Gly Ser Lys Cys
20 256826PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser
Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Val Ser Lys Cys 20
256923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(23)D-amino acid 69Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Gly Ser Cys
207027PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(17)D-amino
acidMOD_RES(26)..(27)D-amino acid 70Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ser Gly Ile Glu Pro Asp Ser
Gly Gly Ser Cys 20 257129PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(3)..(8)D-amino acidMOD_RES(10)..(14)D-amino
acidMOD_RES(16)..(18)D-amino acidMOD_RES(28)..(29)D-amino acid
71Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Lys Ala1
5 10 15Lys Ser Lys Ile Glu Phe Asp Phe Gly Val Lys Lys Cys 20
257223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(23)D-amino acid 72Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Ser Gly Cys
207325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(25)D-amino acid 73Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Pro Asp Gly Ser Ser
Lys Cys 20 257425PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(3)..(8)D-amino acidMOD_RES(10)..(14)D-amino
acidMOD_RES(16)..(16)D-amino acidMOD_RES(23)..(25)D-amino acid
74Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Lys Ala1
5 10
15Ile Glu Pro Asp Ser Gly Ser Lys Cys 20 257527PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(18)D-amino
acidMOD_RES(25)..(27)D-amino acid 75Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Lys Ser Ile Glu Pro Asp Gly
Ser Ser Lys Cys 20 257627PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(3)..(8)D-amino acidMOD_RES(10)..(14)D-amino
acidMOD_RES(16)..(18)D-amino acidMOD_RES(25)..(27)D-amino acid
76Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Lys Ala1
5 10 15Lys Ser Ile Glu Pro Asp Ser Gly Ser Lys Cys 20
257723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(23)D-amino acid 77Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Gly Ser Cys
207823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(23)D-amino acid 78Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Cys
207925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(23)..(25)D-amino acid 79Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Ser
Lys Cys 20 258027PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)D-amino
acidMOD_RES(3)..(8)D-amino acidMOD_RES(10)..(14)D-amino
acidMOD_RES(16)..(18)D-amino acidMOD_RES(25)..(27)D-amino acid
80Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Lys Ala1
5 10 15Lys Ser Ile Glu Phe Asp Ser Gly Ser Lys Cys 20
258126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)D-amino acidMOD_RES(3)..(8)D-amino
acidMOD_RES(10)..(14)D-amino acidMOD_RES(16)..(16)D-amino
acidMOD_RES(24)..(26)D-amino acid 81Glu Gly Val Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala Arg Lys Ala1 5 10 15Ile Glu Phe Asp Ser Gly Val
Ser Lys Cys 20 25827PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 82Ala Ala Pro Val Arg Ser Leu1
5837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Ala Leu Asp Pro Arg Ser Phe1 5847PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Ala
Thr Gln Asn Lys Ala Ser1 5855PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 85Ala Cys Tyr Leu Asp1
5867PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 86Ala Ile Glu Thr Asp Gly Ser1 5877PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 87Ala
Leu Glu Val Asp Cys Tyr1 5889PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 88Ala Glu Pro Leu Phe Ala Glu
Arg Lys1 5897PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 89Ala Met Asn Pro Lys Phe Ala1
5908PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Ala Leu Gln Arg Ile Tyr Lys Cys1
59112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Ala Lys Ser Val Ala Arg Thr Leu Leu Val Lys
Cys1 5 109210PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 92Ala Glu Glu Lys Gln Arg Ile Ile Gly
Cys1 5 109310PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 93Ala Gln Arg Gln Arg Ile Ile Gly Gly
Cys1 5 10948PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 94Ala Leu Gly Arg Gly Gly Ser Cys1
59511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Ala Lys Tyr Leu Gly Arg Ser Tyr Lys Val Cys1 5
109611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Ala Arg Ala Leu Glu Arg Gly Leu Gln Asp Cys1 5
109710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Ala Ser Leu Gly Arg Lys Ile Gln Ile Cys1 5
109810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 98Ala Gly Leu Gln Arg Ala Leu Glu Ile Cys1 5
109912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Ala Lys Val Phe Met Gly Arg Val Tyr Asp Pro
Cys1 5 1010011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 100Ala Ser Ser Thr Gly Arg Asn Gly Phe
Lys Cys1 5 1010112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 101Ala Lys Thr Thr Gly Gly Arg Ile Tyr
Gly Gly Cys1 5 101028PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 102Ala Asp Pro Arg Gly Gly
Ser Cys1 51038PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 103Ala Val Pro Arg Gly Gly Ser Cys1
510410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 104Ala Leu Pro Ser Arg Ser Ser Lys Ile Cys1 5
1010510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Ala His Arg Gly Arg Thr Leu Glu Ile Cys1 5
1010610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 106Ala Ser Thr Gly Arg Asn Gly Phe Lys Cys1 5
1010710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Ala Gln Gln Lys Arg Lys Ile Val Leu Cys1 5
101089PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Ala Gln Ala Arg Lys Ile Val Leu Cys1
51098PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Ala Gln Ala Arg Gly Gly Ser Cys1
51107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Ala Ile Pro Glu Asn Phe Phe1
51117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Ala Lys Glu Glu Glu Gly Leu1
51127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Ala Asn Leu Val Tyr Met Val1
51137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Ala Arg Leu Ala Ala Ile Thr1
51147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Ala Ser Leu Ser Arg Leu Thr1
51158PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Ala Pro Leu Gly Val Arg Gly Lys1 5
* * * * *