U.S. patent application number 15/735949 was filed with the patent office on 2018-05-24 for methods and polypeptides for modulation of immunoresponse.
This patent application is currently assigned to Mackay Medical Foundation The Presbyterian Church In Taiwan Mackay Memorial Hospital. The applicant listed for this patent is Yen-Ta LU, Mackay Medical Foundation The Presbyterian Church In Taiwan Mackay Memorial Hospital. Invention is credited to Chia-Ming CHANG, Yen-Ta LU, Tsai-Yin WEI.
Application Number | 20180141991 15/735949 |
Document ID | / |
Family ID | 57502886 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180141991 |
Kind Code |
A1 |
LU; Yen-Ta ; et al. |
May 24, 2018 |
METHODS AND POLYPEPTIDES FOR MODULATION OF IMMUNORESPONSE
Abstract
A method for modulating an immunoresponse includes binding a
TREM-like transcript 1 (TLT-1) polypeptide to an immune cell,
wherein the binding of the TLT-1 polypeptide to the immune cell
suppresses immunoresponse. A method for treating and/or preventing
a disease associated with immune hyper-reactivity includes
administering the TLT-1 polypeptide to a subject in need
thereof.
Inventors: |
LU; Yen-Ta; (Taipei, TW)
; CHANG; Chia-Ming; (Taipei, TW) ; WEI;
Tsai-Yin; (Changhua County, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LU; Yen-Ta
Mackay Medical Foundation The Presbyterian Church In Taiwan Mackay
Memorial Hospital |
Taipei City, Taiwan
Taipei |
|
CN
TW |
|
|
Assignee: |
Mackay Medical Foundation The
Presbyterian Church In Taiwan Mackay Memorial Hospital
Taipei
TW
|
Family ID: |
57502886 |
Appl. No.: |
15/735949 |
Filed: |
June 12, 2016 |
PCT Filed: |
June 12, 2016 |
PCT NO: |
PCT/CN2016/085452 |
371 Date: |
December 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62174681 |
Jun 12, 2015 |
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62174673 |
Jun 12, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/59 20170801;
A61K 45/06 20130101; C07K 2317/24 20130101; A61K 39/3955 20130101;
C07K 2319/70 20130101; A61K 38/00 20130101; C07K 16/2845 20130101;
C07K 2317/21 20130101; A61P 37/06 20180101; C07K 14/705 20130101;
A61K 38/1774 20130101; A61K 47/643 20170801; C07K 14/70503
20130101; C07K 2317/76 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61K 38/17 20060101 A61K038/17; A61P 37/06 20060101
A61P037/06; A61K 47/59 20060101 A61K047/59; A61K 47/64 20060101
A61K047/64; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for modulating an immunoresponse, comprising binding a
TREM-like transcript 1 (TLT-1) polypeptide to an immune cell,
wherein the binding of the TLT-1 polypeptide to the immune cell
suppresses immunoresponse.
2. The method of claim 1, wherein the binding of TLT-1 polypeptide
to the immune cell treats and/or prevents a disease associated with
immune hyper-reactivity.
3. The method of claim 2, wherein the disease associated with
immune hyper-reactivity is an autoimmune disease, hypersensitivity
reaction, transplantation rejection or graft versus host
disorder.
4. The method of claim 1, wherein the said immune cell is monocyte
or neutrophil.
5. The method of claim 1, wherein the TLT-1 polypeptide is a
polypeptide comprising an amino acid sequence comprising at least 5
amino acids of SEQ ID NO: 1 or at least about 80% amino acid
sequence identity to SEQ ID NO: 1, or a biologically active
fragment or a variant thereof.
6. The method of claim 5, wherein the polypeptide comprises an
amino acid sequence comprising at least 5 amino acids of SEQ ID NO:
2 or at least about 80% amino acid sequence identity to the amino
acid sequence set forth in SEQ ID NO: 2, or a biologically active
fragment or a variant thereof.
7. The method of claim 5, wherein the polypeptide comprises an
amino acid sequence comprising at least 5 amino acids of SEQ ID NO:
3 or at least about 80% amino acid sequence identity to the amino
acid sequence set forth in SEQ ID NO: 3, or a biologically active
fragment or a variant thereof.
8. The method of claim 5, wherein the polypeptide comprises the
amino acid sequence set forth in SEQ ID NO: 1, 2 or 3.
9. A polypeptide, comprising an amino acid sequence comprising at
least 5 amino acids of SEQ ID NO: 1 or at least about 80% amino
acid sequence identity to SEQ ID NO: 1, or a biologically active
fragment or a variant thereof, or at least 5 amino acids of SEQ ID
NO: 2 or at least about 80% amino acid sequence identity to SEQ ID
NO: 2, or a biologically active fragment or a variant thereof, or
at least 5 amino acids of SEQ ID NO: 3 or at least about 70% amino
acid sequence identity to SEQ ID NO: 3, or a biologically active
fragment or a variant thereof.
10. The polypeptide of claim 9, wherein the polypeptide comprises
the amino acid sequence set forth in SEQ ID NO: 1 or 2 or 3.
11.-14. (canceled)
15. The polypeptide of claim 9, wherein said polypeptide is
pegylated or conjugated with a tissue target molecule, an albumin,
or a serum albumin binding peptide.
16. The polypeptide of claim 9, wherein said polypeptide is fused
with a tissue target molecule, an albumin, or a serum albumin
binding peptide.
17. The polypeptide of claim 9, wherein the polypeptide is
formulated as a composition comprising a pharmaceutically
acceptable carrier.
18. The polypeptide of claim 17, which the composition further
comprises an additional immunosuppressor.
19. The polypeptide of claim 18, wherein the additional
immunosuppressor is glucocorticoid, cytostatic, alkylating agent,
antimetabolite, methotrexate, azathioprine, mercaptopurine,
dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin,
anti-CD20 antibody, muromonab-CD3, basiliximab, daclizumab,
ciclosporin, tacrolimus, sirolimus, interferon, opioid, TNF binding
protein, mycophenolate, fingolimod, or myriocin.
20. A method for treating and/or preventing a disease associated
with immune hyper-reactivity, comprising administering the
polypeptide of claim 9 to a subject in need thereof.
21. The method of claim 20, wherein the disease is an autoimmune
disease, a hypersensitivity reaction, a transplantation rejection
or a graft versus host disorder.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to modulation of an
immunoresponse. Particularly, the present invention relates to
methods and polypeptides for modulation of an immunoresponse by
binding TLT-1 directly to immune cells and triggering cells to
express immunosuppressive phenotypes.
BACKGROUND OF THE INVENTION
[0002] Platelets are crucial mediators of hemostasis. Recent
advances suggest that platelets can influence both innate and
adaptive immune responses. Activated platelets can release soluble
mediators, such as soluble CD40L, that interact with leukocytes to
modulate inflammatory processes and may contribute to immune
dysregulation in patients with sepsis. TREM-like transcript-1
(TLT-1) was observed exclusively in the alpha-granule of resting
platelet megakaryocytes and on the surface of activated platelets.
TLT-1 is a member of the TREM family consisting of a single V-set
immunoglobulin (Ig) domain, a short cytoplasmic tail, and a charged
residue in the transmembrane domain. A recent study using
antibodies against TLT-1 revealed the role of proteins in
thrombin-induced platelet aggregation (Giomarelli B, Washington V
A, Chisholm M M, Quigley L, McMahon J B, et al. (2007) Inhibition
of thrombin-induced platelet aggregation using human single-chain
Fv antibodies specific for TREM-like transcript-1. Thromb Haemost
97: 955-963). U.S. Pat. No. 7,553,936 B2 relates to methods and
compositions for modulating platelet activity, and methods and
compositions for treating a disease or disorder associated with
platelet activity in a subject, comprising administering a single
chain anti-TREM-like transcript-1 (TLT-1) antibody or a functional
fragment or variant thereof in an amount effective to modulate
platelet activity.
[0003] Small soluble fragments of the TLT-1 extracellular domain
(12 and 14 kDa) were observed in normal human serum but not in
plasma. Studies have shown that TLT-1 may serve as a regulator for
hemostasis by linking with fibrinogen to facilitate platelet
aggression, and have also demonstrated significant correlations
between high levels of soluble TLT-1 (sTLT-1) and disseminated
intravascular coagulation scores. US 20040180409 A1 provides TLT-1
nucleic acid and protein molecules useful as modulating agents in
regulating a variety of cellular processes, e.g., blood clotting
and immune response. Prolonged sTLT-1 expression in the plasma has
been associated with reduced survival in patients with septic shock
(Washington A V, Gibot S, Acevedo I, Gattis J, Quigley L, et al.
(2009) TREM-like transcript-1 protects against
inflammation-associated hemorrhage by facilitating platelet
aggregation in mice and humans. J Clin Invest 119: 1489-1501).
US20130029921 A1 discloses that TLT-1 and TLT-1 derived peptides
exhibit anti-inflammatory properties by specifically inhibiting
TREM-1 activity. Recently, Derive et al. demonstrated that sTLT-1
may bind to the soluble TREM-1 ligand, thus interfering with
leukocyte activation (Derive M Bouazza Y, Sennoun N, Marchionni S,
Quigley L, et al. (2012) Soluble TREM-like transcript-1 regulates
leukocyte activation and controls microbial sepsis. J Immunol 188:
5585-5592). Collectively, these clinical and in vitro studies of
sTLT-1 suggest that it may have dual effects, playing a role in
platelet aggression and mediating leukocyte function during
sepsis.
[0004] Thus, there is still a need to develop an agent or therapy
for modulation of immunoresponse.
SUMMARY OF THE INVENTION
[0005] The invention provides a method for modulating an
immunoresponse, comprising binding a TREM-like transcript 1 (TLT-1)
polypeptide to an immune cell, wherein the binding of the TLT-1
polypeptide to the immune cell suppresses immunoresponse. In one
embodiment, the binding of TLT-1 polypeptide the immune cell treats
and/or prevents a disease associated with immune hyper-reactivity;
preferably, the disease is an autoimmune disease, hypersensitivity
reaction, transplantation rejection or graft versus host disorder.
In some embodiments, the TLT-1 polypeptides are those described
herein below.
[0006] The invention also provides a polypeptide, comprising an
amino acid sequence comprising at least 5 amino acids of SEQ ID NO:
1, 2 or 42 or at least about 50% amino acid sequence identity to
SEQ ID NO: 1, 2 or 42, or a biologically active fragment or a
variant thereof. In some embodiments, the polypeptide comprises an
amino acid sequence having at least about 80% amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 1, 2 or
42; preferably, the polypeptide comprises an amino acid sequence
set forth in SEQ ID NO: 1, 2 or 42. In one embodiment, the said
polypeptide is recombinant polypeptide or synthetic
polypeptide.
[0007] In some embodiments, the polypeptide is pegylated or
conjugated with a tissue target molecule, an albumin or a serum
albumin binding peptide. In some embodiments, the polypeptide is
fused with a tissue target molecule, an albumin or a serum albumin
binding peptide.
[0008] The invention further provides a composition comprising a
TLT-1 polypeptide of the invention and a pharmaceutically
acceptable carrier. Also provided is a method for treating and/or
preventing a disease associated with immune hyper-reactivity,
comprising administering the TLT-1 polypeptide of the invention or
a composition comprising the TLT-1 polypeptide to a subject.
[0009] The invention further provides a TLT-1 fusion protein,
comprising the TLT-1 polypeptide of the invention fused with one or
more heterogeneous polypeptide or other TLT-1 polypeptide of the
invention.
[0010] The invention further provides a pegylated TLT-1
polypeptide, comprising one or more polyethylene glycol (PEG)
molecules operably linked to at least one amino acid residue in the
N-terminal of the TLT-1 polypeptide of the invention.
[0011] The invention further provides a TLT-1 polypeptide
conjugate, comprising a TLT-1 polypeptide of the invention
conjugated to an immunosuppressive agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows interaction between TLT-1 and leukocytes. PBMCs
were incubated with rsTLT-1 for 1 hour and stained with biotin-goat
anti-TLT-1 antibodies APC-avidin to detect the surface binding of
TLT-1. (A) depicts representative FACS histogram of various
leukocytes in the presence or absence of rsTLT-1 treatment.
Monocytes, PMNs, and lymphocytes are gated based on their FSC/SSC
properties (heavy-line histogram, rsTLT-1 treated cells;
dashed-line histogram, untreated cells; thin dashed-line histogram,
appropriate isotype control). TLT-1 bound to monocytes and PMNs,
but not to lymphocytes. (B) depicts binding of various
concentrations of rsTLT-1 to monocytes and PMNs. PMNs and monocytes
were able to bind with rsTLT-1 in a dose dependent manner. Values
are presented as mean.+-.SEM from 3 independent experiments.
[0013] FIG. 2 shows the effect of sTLT-1 on the surface expression
of HLA-DR and PD-L1 molecules in monocytes. (A) Monocytes were
incubated with rsTLT-1 (10 .mu.g/mL) for 3 days. At the indicated
time point, the cells were harvested and the HLA-DR and PD-L1
molecules were analyzed using flow cytometry. Treatment with
rsTLT-1 leads to a trend with down-regulation of HLA-DR expression.
By contrast, rsTLT-1 priming up-regulated the levels of PD-L1
expression on monocytes. (B) Monocytes were cultured with different
concentrations of rsTLT-1 for 2 days. The cells were then harvested
and the HLA-DR and PD-L1 molecules were analyzed using flow
cytometry. Surface molecule expression is presented as the mean
fluorescence intensity (MFI) relative to each day that the cells
were treated with the control medium (dashed line). Values are
presented as mean.+-.SEM from 3 independent experiments.
[0014] FIG. 3 shows the effect of TLT-1 polypeptides on the surface
expression of PD-L1 molecules in monocytes. (A) depicts structural
characteristics of extracellular domain of TLT-1 and designed TLT-1
polypeptides. (B) shows that monocytes were cultured with different
lengths of TLT-1 polypeptide (10 .mu.g/ml) and 10 .mu.g/ml rsTLT-1
for 1 day. The cells were then harvested and the PD-L1 molecules
were analyzed using flow cytometry. Surface molecule expression is
presented as the MFI relative to each day that the cells were
treated with the medium control (dashed line).
[0015] FIG. 4 shows the effect of TLT-1 15-63 polypeptides on the
surface expression of HLA-DR molecules in monocytes. Monocytes were
cultured with 10 .mu.g/ml TLT-1 15-63 polypeptide and 10 .mu.g/ml
rsTLT-1 for 2 days. The cells were then harvested and the HLA-DR
molecules were analyzed using flow cytometry. Surface molecule
expression is presented as the MFI relative to each day that the
cells were treated with the medium control (dashed line).
DETAILED DESCRIPTION OF THE INVENTION
[0016] Before the present composition, methods, and isolation
methodologies are described, it is to be understood that this
invention is not limited thereto, as such compositions, methods,
and conditions may vary. It is also to be understood that the
terminology used herein is for purposes of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only in the appended
claims.
[0017] The present invention unexpectedly found that TLT-1 directly
binds to immune cells and trigger cells to express
immunosuppressive phenotypes; i.e., down-regulation of HLA-DR and
up-regulation of PD-L1 by binding. The induction of PD-L1 and
reduction of HLA-DR by TLT-1 or TLT-1 polypeptides can therefore be
useful in treating diseases associated with immune
hyper-reactivity, such as autoimmune diseases, hypersensitivity
reactions, transplantation rejections and graft versus host
disorders.
Definitions
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, as
it will be understood that modifications and variations are
encompassed within the spirit and scope of the instant
disclosure.
[0019] Unless otherwise specified, "a" or "an" means one or
more.
[0020] As used herein, the amino acid residues are abbreviated as
follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp;
D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E),
glutamine (Gln; Q), glycine (Gly; G), histidine (His; H),
isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine
(Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;
S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and
valine (Val; V).
[0021] As used herein, the term "PD-L1" refers to programmed
death-ligand 1 (PD-L1), cluster of differentiation 274 (CD274) or
B7 homolog 1 (B7-H1). PD-L1 is a 40 kDa type 1 transmembrane
protein that plays a major role in suppressing the immune system
during particular events such as pregnancy, autoimmune disease,
cancer, sepsis, and other infectious diseases such as mycobacterium
tuberculosis, cytomegalovirus, and hepatitis.
[0022] As used herein, the term "monocyte," also called mononuclear
white cell, belongs to a type of white blood cell involved in
first-line defensive mechanism and is recognized as able to
differentiate into a dendritic cell or macrophage precursor.
Monocytes normally move in the blood system. In response to
external stimulating signals, monocytes secrete many
immuno-regulatory cytokines, move to the site of infection in the
tissues and differentiate into macrophages.
[0023] As used herein, the term "modulating" includes "increasing"
or "stimulating," as well as "decreasing" or "reducing," typically
in a statistically significant or a physiologically significant
amount as compared to a control.
[0024] As used herein, "identity" refers to a relationship between
two or more polypeptide or protein sequences, as determined by
comparing the sequences. In the art, "identity" also refers to the
degree of sequence relatedness between polypeptides or proteins, as
determined by the match between strings of such sequences.
"Identity" can be readily calculated by known bioinformational
methods. The "percent identity" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3) using its default parameters.
[0025] As used herein, the term "biologically active fragment"
means an amino acid fragment of a polypeptide encompassed by the
invention, said fragment also having the efficacy of the
polypeptide described herein.
[0026] As used herein, the term "variant" refers to an amino acid
sequence which is of the wild type or which has been altered by
substitution, insertion, cross-over, deletion, and/or other genetic
operation. For purposes of the present disclosure, a variant is not
limited to a particular method by which it is generated. In some
embodiments, a variant sequence can have increased, decreased, or
substantially similar activities or properties in comparison to the
parental sequence. In some embodiments, the polypeptide may contain
one or more amino acid residues that have been mutated as compared
to the amino acid sequence of the wild type polypeptide. In some
embodiments, one or more amino acid residues of the polypeptide can
be held constant, invariant, or not substituted as compared to a
parent polypeptide in the variant polypeptides making up the
plurality. In some embodiments, the parent polypeptide is used as
the basis for generating variants with improved robustness or other
properties. Variants can also differ in at least one of secondary
structure, tertiary structure, and degree of foldedness.
[0027] As used herein, the terms "peptide," "polypeptide" and
"protein" each refer to a molecule comprising two or more amino
acid residues joined to each other by peptide bonds. These terms
encompass, e.g., native and artificial proteins, protein fragments
and polypeptide analogs (such as variants, and fusion proteins) of
a protein sequence as well as post-translationally, or otherwise
covalently or non-covalently, modified proteins. A peptide,
polypeptide, or protein may be monomeric or polymeric.
[0028] As used herein, the term "polypeptide fragment" refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion as compared to a corresponding full-length protein.
Fragments can also result from proteolytic (or other) processing,
which, for example, results in variation in the amino and/or
carboxy terminus of from one to five amino acids from that
predicted. A fragment can further comprise, at either or both of
its ends, one or more additional amino acids, for example, a
sequence of amino acids from a different naturally-occurring
protein or an artificial amino acid sequence (e.g., an artificial
linker sequence or a tag protein).
[0029] As used herein, the term "pharmaceutically acceptable
carrier" refers to an ingredient in a pharmaceutical formulation,
other than an active ingredient, which is nontoxic to a subject. A
pharmaceutically acceptable carrier includes, but is not limited
to, a buffer, excipient, stabilizer, or preservative.
[0030] As used herein, the term "subject" refers to a vertebrate,
preferably a mammal, more preferably a human. Mammals include, but
are not limited to, humans, farm animals, sport animals, and
pets.
[0031] As used herein, the term "effective amount" refers to an
amount sufficient to effect beneficial or desired clinical results.
An effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount is an amount that is sufficient to diagnose, palliate,
ameliorate, stabilize, reverse, slow or delay the progression of
the disease state.
[0032] As used herein, the terms "treatment," "treating," "treat"
and the like generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease or symptom from occurring in a
subject which may be predisposed to the disease or symptom but has
not yet been diagnosed as having it; (b) inhibiting the disease
symptom, i.e., arresting its development; or (c) relieving the
disease symptom, i.e., causing regression of the disease or
symptom.
[0033] The term "preventing" as used herein refers to a
preventative or prophylactic measure that stops a disease state or
condition from occurring in a patient or subject. Prevention can
also include reducing the likelihood of a disease state or
condition from occurring in a patient or subject and impeding or
arresting the onset of said disease state or condition.
[0034] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
Modulation of Immunoresponse
[0035] The present invention unexpectedly found that the direct
binding of TLT-1 to immune cells and triggers cells to express
immunosuppressive phenotypes through down-regulation of HLA-DR and
up-regulation of PD-L1.
[0036] In one aspect, the present invention provides a method for
modulating an immunoresponse, comprising binding a TREM-like
transcript 1 (TLT-1) polypeptide to an immune cell, wherein the
binding of the TLT-1 polypeptide to the immune cell suppresses
immunoresponse. In one embodiment, the TLT-1 polypeptides are those
described herein below.
[0037] In one embodiment, the present invention provides a method
of treating and/or preventing a disease associated with immune
hyper-reactivity, comprising administering an effective amount of a
TLT-1 polypeptide to a subject. In some embodiments, the diseases
include but are not limited to, autoimmune diseases,
hypersensitivity reactions, transplantation rejections and graft
versus host disorder.
[0038] The expression of PD-L1 can be induced and HLA-DR can be
reduced by binding a TLT-1 polypeptide to monocytes, whereby
treating diseases associated with immune hyper-reactivity. Any
TLT-1 polypeptide capable of binding to immune cells can achieve
the above-mentioned modulation on PD-L1 and HLA-DR, including
full-length TLT-1 and polypeptide fragments thereof.
TLT-1 Polypeptides
[0039] The invention surprisingly found that a sequence of the
TLT-1 extracellular domain is responsible for binding to immune
cells; preferably, a sequence comprising at least 30 amino acids
(TLT-1 34-63), a sequence comprising at least 49 amino acids (TLT-1
15-63) or a sequence comprising at least 147 amino acids (TLT-1
16-162). The expression of PD-L1 can be induced and HLA-DR can be
reduced by the engagement of TLT-1 polypeptides on immune cells,
suggesting that the induction of PD-L1 and reduction of HLA-DR by
TLT-1 or TLT-1 polypeptides can therefore be useful in treating
diseases associated with immune hyper-reactivity such as autoimmune
diseases, hypersensitivity reactions, transplantation rejections
and graft versus host disorders.
[0040] In another aspect, the invention provides a polypeptide,
comprising an amino acid sequence comprising at least 5 amino acids
of SEQ ID NO: 1 or at least about 80% amino acid sequence identity
to SEQ ID NO: 1, or a biologically active fragment or a variant
thereof
TABLE-US-00001 (SEQ ID NO: 1) ILVQCHYRLQDVKAQKVWCRFLPEGCQPLV
[0041] In some embodiments, the polypeptide has at least 80%, e.g.,
at least 80%, 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100%, amino acid sequence identity to
SEQ ID NO: 1.
[0042] In one embodiment, the polypeptide comprises an amino acid
sequence comprising 5 amino acids of SEQ ID NO: 2 or at least about
80% amino acid sequence identity to the amino acid sequence set
forth in SEQ ID NO: 2, or a biologically active fragment or a
variant thereof
TABLE-US-00002 (SEQ ID NO: 2)
GQGIVGSLPEVLQAPVGSSILVQCHYRLQDVKAQKVWCRFLPEGCQPLV
[0043] In some embodiments, the polypeptide has at least 80%, e.g.,
at least 80%, 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100%, amino acid sequence identity to
SEQ ID NO: 2.
[0044] In one embodiment, the polypeptide comprises an amino acid
sequence comprising 5 amino acids of SEQ ID NO: 3 or at least about
70% amino acid sequence identity to the amino acid sequence set
forth in SEQ ID NO: 42, or a biologically active fragment or a
variant thereof.
TABLE-US-00003 (SEQ ID NO: 3)
QGIVGSLPEVLQAPVGSSILVQCHYRLQDVKAQKVWCRFLPEGCQPLVSS
AVDRRAPAGRRTFLTDLGGGLLQVEMVTLQEEDAGEYGCMVDGARGPQIL
HRVSLNILPPEEEEETHKIGSLAENAFSDPAGSANPLEPSQDEKSIP
In some embodiments, the polypeptide has at least 70%, e.g., at
least 70%, 80%, 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100%, amino acid sequence identity to
SEQ ID NO: 3.
[0045] In some embodiments, the invention provides a polypeptide
comprising one or more insertion, substitution and/or deletion in
any of TLT-1 amino acid sequences described herein. Preferably, the
TLT-1 amino acid sequence is SEQ ID NO:1, 2 or 3.
[0046] Polypeptides can be produced through recombinant methods and
chemical synthesis. In addition, functionally equivalent
polypeptides may find use, where the equivalent polypeptide may
contain deletions, additions or substitutions of amino acid
residues that result in a silent change, thus producing a
functionally equivalent differentially expressed on pathway gene
product. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. "Functionally equivalent," as used herein, refers to a
protein capable of exhibiting a substantially similar in vivo
activity.
[0047] The polypeptides may be produced by recombinant DNA
technology or synthetic technology using techniques well known in
the art. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing coding
sequences and appropriate transcriptional/translational control
signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic techniques and in vivo
recombination/genetic recombination. Alternatively, RNA capable of
encoding the polypeptides of interest may be chemically
synthesized.
[0048] Typically, the coding sequence is placed under the control
of a promoter that is functional in the desired host cell to
produce relatively large quantities of the gene product. An
extremely wide variety of promoters is well known and can be used
in the expression vectors of the invention depending on the
particular application. Ordinarily, the promoter selected depends
upon the cell in which the promoter is to be active. Other
expression control sequences such as ribosome binding sites,
transcription termination sites and the like are also optionally
included. Constructs that include one or more of these control
sequences are termed "expression cassettes." Expression can be
achieved in prokaryotic and eukaryotic cells utilizing promoters
and other regulatory agents appropriate for the particular host
cell. Exemplary host cells include, but are not limited to, E.
coli, other bacterial hosts, yeast, and various higher eukaryotic
cells such as the COS, CHO and HeLa cells lines and myeloma cell
lines.
[0049] Once expressed, the recombinant polypeptides can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, ion exchange and/or size
exclusivity chromatography, gel electrophoresis and the like (see,
generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.
(1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification, Academic Press, Inc. N.Y. (1990)).
[0050] As an alternative to recombinant methods, polypeptides can
be chemically synthesized. Such methods typically include
solid-state approaches, but can also utilize solution based
chemistries and combinations or combinations of solid-state and
solution approaches. Examples of solid-state methodologies for
synthesizing proteins are described by Merrifield (1964) J. Am.
Chem. Soc. 85:2149; and Houghton (1985) Proc. Natl. Acad. Sci.,
82:5132. Fragments of polypeptides of the invention protein can be
synthesized and then joined together. Methods for conducting such
reactions are described by Grant (1992) Synthetic Peptides: A User
Guide, W.H. Freeman and Co., N.Y.; and in "Principles of Peptide
Synthesis," (Bodansky and Trost, ed.), Springer-Verlag, Inc. N.Y.,
(1993).
[0051] In another aspect, the invention provides a TLT-1 fusion
protein, comprising the TLT-1 polypeptide of the invention fused
with one or more heterogeneous polypeptide or other TLT-1
polypeptide of the invention. The TLT-1 polypeptide covalently
links to one or more heterogeneous polypeptide or another TLT-1
polypeptide of the invention, either directly or via an amino acid
linker. The polypeptides forming the fusion protein are typically
linked C-terminus to N-terminus, although they can also be linked
C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus
to C-terminus. The polypeptides of the fusion protein can be in any
order and may include more than one of either or both of the
constituent polypeptides. Examples of the heterogeneous polypeptide
include, but are not limited to, an albumin, a serum albumin
binding peptide, a cell penetrating peptide (CPP), a tissue
targeting molecule and an immunosuppressive peptide. Preferably,
the heterogeneous polypeptide is an albumin or a serum albumin
binding peptide.
[0052] The invention also provided a pegylated TLT-1 polypeptide,
comprising one or more polyethylene glycol (PEG) molecules operably
linked to at least one amino acid residue in the N-terminal of the
TLT-1 polypeptide of the invention. PEGylation of the molecules can
be carried out, e.g., according to the methods described in
Youngster et al., Curr Pharm Des (2002), 8:2139. Any kind of
polyethylene glycol is suitable for the present invention provided
that the PEG-polypeptide is still functionally active which can be
assayed according to methods known in the art. Preferably, the
polyethylene glycol of the present invention is PEG 1000, 2000,
3000, 5000, 10000, 15000, 20000, 30000, 40000 or 50000. In one
example, the pegylated TLT-1 polypeptide comprises a monomeric
TLT-1 polypeptide. In another example, the pegylated molecule
comprises is an oligomeric TLT-1 polypeptide. In yet another
example, the pegylated TLT-1 polypeptide comprises a multiarm PEG,
wherein one or more monomeric TLT-1 polypeptide are operably linked
to the multiarm PEG.
[0053] The invention also comprises a TLT-1 polypeptide conjugate,
comprising a TLT-1 polypeptide of the invention conjugated to an
immunosuppressive agent, a tissue target molecule, an albumin or a
serum albumin binding peptide.
Compositions of Polypeptide of the Invention
[0054] In another aspect, the present invention provides
compositions comprising a TLT-1 polypeptide of the invention. In
some embodiments, such compositions may be administered to
subjects. In some embodiments, the TLT-1 polypeptide of the
invention may be provided in a composition that comprises one or
more other components, including, but not limited to,
pharmaceutically acceptable carriers, adjuvants, wetting or
emulsifying agents, pH buffering agents, preservatives, and/or any
other components suitable for the intended use of the compositions.
Such compositions can take the form of solutions, suspensions,
emulsions and the like. The term "pharmaceutically acceptable
carrier" includes various diluents, excipients and/or vehicles in
which, or with which, the TLT-1 polypeptides, proteins, and/or
protein complexes of the invention can be provided. The
pharmaceutically acceptable carrier includes, but is not limited
to, carriers known to be safe for delivery to human and/or other
animal subjects, and/or approved by a regulatory agency of the
federal or a state government, and/or listed in the U.S.
Pharmacopeia, and/or other generally recognized pharmacopeia,
and/or receiving specific or individual approval from one or more
generally recognized regulatory agencies for use in humans and/or
other animals. Such pharmaceutically acceptable carriers, include,
but are not limited to, water, aqueous solutions (such as saline
solutions, buffers, and the like), organic solvents (such as
certain alcohols and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil) and the like.
[0055] In one embodiment, the TLT-1 polypeptide of the invention
may be provided in a composition that comprises one or more
additional active components, such as one or more additional
immunosuppressors. The immunosuppressors include but are not
limited to glucocorticoid, cytostatic, alkylating agent,
antimetabolite, methotrexate, azathioprine, mercaptopurine,
dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin,
anti-CD20 antibody, muromonab-CD3, basiliximab, daclizumab,
cyclosporin, tacrolimus, sirolimus, interferon, opioid, TNF binding
protein, mycophenolate, fingolimod and myriocin.
[0056] In some embodiments, the compositions of the invention
comprise an "effective amount" of a TLT-1 polypeptide of the
invention. An "effective amount" is an amount required to achieve a
desired end result. The amount of a TLT-1 polypeptide of the
invention that is effective to achieve the desired end result will
depend on a variety of factors including, but not limited to, the
species of the intended subject (e.g. whether human or some other
animal species), the age and/or sex of the intended subject, the
planned route of administration, the planned dosing regimen, the
seriousness of any ongoing diseases or conditions, and the like.
The effective amount--which may be a range of effective
amounts--can be determined by standard techniques without any undue
experimentation, for example using in vitro assays and/or in vivo
assays in the intended subject species or any suitable animal model
species. Suitable assays include, but are not limited to, those
that involve extrapolation from dose-response curves and/or other
data derived from in vitro and/or in vivo model systems. In some
embodiments the effective amount may be determined according to the
judgment of a medical or veterinary practitioner based on the
specific circumstances.
[0057] In one embodiment, an effective amount of the TLT-1
polypeptide ranges from about 0.0002 mg/kg to about 20 mg/kg body
weight per administration.
Administration Methods
[0058] In some embodiments, the present invention provides methods
that comprise administering the TLT-1 polypeptide of the invention
to a subject. Such methods may comprise methods for treating
subjects suffering from a disease associated with immune
hyper-reactivity, such as autoimmune diseases, hypersensitivity
reactions, transplantation rejections and graft versus host
disorders.
[0059] Subjects to which the TLT-1 polypeptides of the invention,
or compositions comprising such TLT-1 polypeptide, can be
administered (for example in the course of a method of treatment)
include any and all animal species. In some embodiments, the
subjects are mammalian species. Mammalian subjects include, but are
not limited to, humans, non-human primates, rodents, rabbits, and
ferrets.
[0060] Various delivery systems are known in the art and any
suitable delivery systems can be used to administer the
compositions of the present invention to subjects. Such delivery
systems include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral delivery systems. The compositions
of the present invention may be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local.
[0061] In some such embodiments, administration of a single dose is
preferred. However, in other embodiments, additional dosages can be
administered, by the same or different route, to achieve the
desired effect. In some embodiments, dosing regimens may comprise a
single administration. In other embodiments, dosing regimens may
comprise multiple administrations.
Examples
[0062] The materials and methods used in the following examples are
described below.
[0063] Human Cell Isolation and Cell Culture
[0064] White blood cell concentrates from healthy volunteers were
obtained from the Taiwan Blood Service Foundation (Taipei, Taiwan).
Written informed consent was obtained for participation in the
study, which was approved by the Institutional Review Board of the
MacKay Memorial Hospital. Human monocytes were isolated as
previously described. In brief, peripheral blood mononuclear cells
(PBMCs) were isolated using Ficoll-Paque Plus (GE Healthcare)
gradient centrifugation. The monocytes were further purified by
conducting CD14 selection using CD14 MACS microbeads (Miltenyi
Biotec). The purity of monocytes confirmed using flow cytometry
analysis was approximately 90%.
[0065] Generation of Recombinant Soluble TLT-1 and TLT-1
Polypeptide
[0066] For the generation of recombinant soluble TLT-1 (rsTLT-1),
pET28a(+)-rsTLT-1 encoding a human TLT-1 extracellular domain
(Glu16-Pro162) with a polyhistidine tag at the N-terminus was
expressed using E. coli and purified using Ni-NTA columns
(Novagen). The purity of the recombinant protein determined using
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and visualized using a coomassive blue stain was
>95%. The endotoxin contamination of the purified proteins was
examined using a LAL assay (QCL-1000). All proteins were sterile
and the endotoxin concentration was lower than the detectable limit
(<0.1 EU/.mu.g protein). The TLT-1 polypeptides were chemically
synthesized by Kelowna International Scientific Inc. (Taipei,
Taiwan).
[0067] Flow Cytometric Analysis
[0068] For the TLT-1 binding experiments, PBMCs were incubated
overnight in an AIM-V medium (Life Technologies) at 37.degree. C.
in a 5% CO.sub.2 humidified atmosphere to allow platelet shedding
from the cell surface. To screen leukocytes for the specific
binding of TLT-1, isolated PBMCs were incubated with various
concentrations of TLT-1 and competing peptide for 1 hour at
37.degree. C. The cells were then washed and stained with a
biotin-conjugated goat anti-TLT-1 antibody (R&D Systems).
Monocytes, polymorphonuclear leukocytes (PMNs), and lymphocytes
were gated based on their FSC/SSC properties. To analyze the
surface phenotype of the TLT-1 or TLT-1 polypeptide primed
monocytes, the cells were incubated for 30 minutes on ice in the
dark with the following mAbs diluted in phosphate-buffered saline
(PBS) containing 1% BSA: HLA-DR-PE, PD-L1-FITC, CD14-PerCP (BD
Biosciences). The fluorescence was detected using FACS Calibur, and
data analysis was performed using FCS Express version 3 (De Novo
Software).
[0069] Statistical Analysis
[0070] Data were analyzed using Prism 6.0 (GraphPad) and expressed
as mean.+-.SEM. Comparisons between groups were performed using the
Student's t test. Correlations were determined using the Pearson's
correlation coefficient. P value <0.05 was considered
significant.
Example 1 Interaction Between TLT-1 and Leukocytes
[0071] To determine whether TLT-1 binds to leukocytes, we incubated
the leukocytes with 10 .mu.g/mL rsTLT-1 for 1 hour and the detected
cell surface binding of TLT-1 by using flow cytometry. As shown in
FIG. 1A, we detected the binding of rsTLT-1 with PMNs and
monocytes, but not with lymphocytes. In addition, we observed that
the binding of PMNs and monocytes bound rsTLT-1 was dose-dependent
(FIG. 1B). These results suggest that a receptor for TLT-1 is
present on human PMNs and monocytes.
Example 2 TLT-1 Alters the Surface Expression of HLA-DR and PD-L1
Molecules in Monocytes
[0072] To investigate whether the presence of sTLT-1 may modulate
the expression of HLA-DR and PD-L1 on monocytes, human monocytes
were first incubated with rsTLT-1 (10 .mu.g/mL) and the expression
of HLA-DR and PD-L1 were examined for 3 consecutive days. As shown
in FIG. 3A (left), HLA-DR was significantly up-regulated by rsTLT-1
within the first 24 hours followed by a rapid down-regulation of
its expression at later time points. PD-L1 was also significantly
increased in its expression within 24 hours but remained
up-regulated throughout the following 3-day experiment (FIG. 2A,
right). Secondly, we studied the dose-effect of sTLT-1 on the
expression of HLA-DR and PD-L1. FIG. 2B (left) showed that the
expression of HLA-DR on monocytes was decreased at 48 hours of
incubation with rsTLT-1, and this down-regulation was significantly
correlated with increasing concentrations of rsTLT-1. The
expression of PD-L1 was, by contrast, significantly up-regulated
with increasing concentrations of rsTLT-1 (FIG. 2B, right).
Collectively, the data suggest that monocytes stimulated by rsTLT-1
have immunosuppressive phenotypes.
Example 3 TLT-1 15-63 Polypeptides Alter the Surface Expression of
PD-L1 and HLA-DR Molecules in Monocytes
[0073] TLT-1 has a single extracellular Ig variable (Ig V) domain
which contains 9 beta strands forming 2 antiparallel beta sheets
locked together by a conserved disulfide bond between strands B and
strands F. In addition, TLT-1 has another disulfide bridge to
stabilize its C-C' beta hairpin. To screen the functional domain of
sTLT-1, we synthesized different lengths of polypeptides according
to its V-type Ig-structure (FIG. 3A). The polypeptide sequences are
shown in Table I. Human monocytes were incubated with different
polypeptides (10 .mu.g/mL) and the expression of PD-L1 was examined
for 24 hours. As shown in FIG. 3B, PD-L1 was significantly
increased in its expression within 24 hours in TLT-1 15-63
peptide-treated monocytes. In additions, treatment with TLT-1 34-63
peptide also induced PD-L1 expression. The expression of HLA-DR on
monocytes was also decreased at 48 hours of incubation with TLT-1
15-63 peptide (FIG. 4). Thus, TLT-1 15-63 can serve as a
therapeutic agent to treat hyper-immune related disease through
up-regulating PD-L1 expression and down-regulating HLA-DR
expression.
TABLE-US-00004 TABLE I TLT-1 polypeptides of the inventions were
designed mimicking different parts of its extracellular domain
Polypeptide name Sequence TLT-1 20-39 GSLPEVLQAPVGSSILVQCH (SEQ ID
NO: 4) TLT-1 15-46 GQGIVGSLPEVLQAPVGSSILVQCHYRLQ DVK (SEQ ID NO: 5)
TLT-1 15-53 GQGIVGSLPEVLQAPVGSSILVQCHYRLQ DVKAQKVWCR (SEQ ID NO: 6)
TLT-1 15-63 GQGIVGSLPEVLQAPVGSSILVQCHYRLQ DVKAQKVWCRFLPEGCQPLV (SEQ
ID NO: 7) TLT-1 40-63 YRLQDVKAQKVWCRFLPEGCQPLV (SEQ ID NO: 8) TLT-1
34-63 ILVQCHYRLQDVKAQKVWCRFLPEGCQP LV (SEQ ID NO: 9)
Sequence CWU 1
1
26130PRTartificialrecombinant polypeptide 1Ile Leu Val Gln Cys His
Tyr Arg Leu Gln Asp Val Lys Ala Gln Lys 1 5 10 15 Val Trp Cys Arg
Phe Leu Pro Glu Gly Cys Gln Pro Leu Val 20 25 30
249PRTartificialrecombinant polypeptide 2Gly Gln Gly Ile Val Gly
Ser Leu Pro Glu Val Leu Gln Ala Pro Val 1 5 10 15 Gly Ser Ser Ile
Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys 20 25 30 Ala Gln
Lys Val Trp Cys Arg Phe Leu Pro Glu Gly Cys Gln Pro Leu 35 40 45
Val 3147PRTartificialrecombinant polypeptide 3Gln Gly Ile Val Gly
Ser Leu Pro Glu Val Leu Gln Ala Pro Val Gly 1 5 10 15 Ser Ser Ile
Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys Ala 20 25 30 Gln
Lys Val Trp Cys Arg Phe Leu Pro Glu Gly Cys Gln Pro Leu Val 35 40
45 Ser Ser Ala Val Asp Arg Arg Ala Pro Ala Gly Arg Arg Thr Phe Leu
50 55 60 Thr Asp Leu Gly Gly Gly Leu Leu Gln Val Glu Met Val Thr
Leu Gln 65 70 75 80 Glu Glu Asp Ala Gly Glu Tyr Gly Cys Met Val Asp
Gly Ala Arg Gly 85 90 95 Pro Gln Ile Leu His Arg Val Ser Leu Asn
Ile Leu Pro Pro Glu Glu 100 105 110 Glu Glu Glu Thr His Lys Ile Gly
Ser Leu Ala Glu Asn Ala Phe Ser 115 120 125 Asp Pro Ala Gly Ser Ala
Asn Pro Leu Glu Pro Ser Gln Asp Glu Lys 130 135 140 Ser Ile Pro 145
420PRTartificialPolypeptide name TLT-1 20-39 4Gly Ser Leu Pro Glu
Val Leu Gln Ala Pro Val Gly Ser Ser Ile Leu 1 5 10 15 Val Gln Cys
His 20 532PRTartificialPolypeptide name TLT-1 15-46 5Gly Gln Gly
Ile Val Gly Ser Leu Pro Glu Val Leu Gln Ala Pro Val 1 5 10 15 Gly
Ser Ser Ile Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys 20 25
30 639PRTartificialPolypeptide name TLT-1 15-53 6Gly Gln Gly Ile
Val Gly Ser Leu Pro Glu Val Leu Gln Ala Pro Val 1 5 10 15 Gly Ser
Ser Ile Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys 20 25 30
Ala Gln Lys Val Trp Cys Arg 35 749PRTartificialPolypeptide name
TLT-1 15-63 7Gly Gln Gly Ile Val Gly Ser Leu Pro Glu Val Leu Gln
Ala Pro Val 1 5 10 15 Gly Ser Ser Ile Leu Val Gln Cys His Tyr Arg
Leu Gln Asp Val Lys 20 25 30 Ala Gln Lys Val Trp Cys Arg Phe Leu
Pro Glu Gly Cys Gln Pro Leu 35 40 45 Val
824PRTartificialPolypeptide name TLT-1 40-63 8Tyr Arg Leu Gln Asp
Val Lys Ala Gln Lys Val Trp Cys Arg Phe Leu 1 5 10 15 Pro Glu Gly
Cys Gln Pro Leu Val 20 930PRTartificialPolypeptide name TLT-1 34-63
9Ile Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys Ala Gln Lys 1
5 10 15 Val Trp Cys Arg Phe Leu Pro Glu Gly Cys Gln Pro Leu Val 20
25 30 1013PRTartificialPeptides identified by MS/MS tandem
analysis- Integrin beta2 (CD18) 10Arg Tyr Asn Gly Gln Val Cys Gly
Gly Pro Gly Arg Gly 1 5 10 1116PRTartificialPeptides identified by
MS/MS tandem analysis- Integrin beta2 (CD18) 11Lys Val Thr Tyr Asp
Ser Phe Cys Ser Asn Gly Val Thr His Arg Asn 1 5 10 15
1217PRTartificialPeptides identified by MS/MS tandem analysis-
Integrin beta2 (CD18) 12Lys Leu Ala Glu Asn Asn Ile Gln Pro Ile Phe
Ala Val Thr Ser Arg 1 5 10 15 Met 1317PRTartificialPeptides
identified by MS/MS tandem analysis- Integrin beta2 (CD18) 13Arg
Ser Asn Glu Phe Asp Tyr Pro Ser Val Gly Gln Leu Ala His Lys 1 5 10
15 Leu 1417PRTartificialPeptides identified by MS/MS tandem
analysis- Integrin beta2 (CD18) 14Lys Val Thr Ala Thr Glu Cys Ile
Gln Glu Gln Ser Phe Val Ile Arg 1 5 10 15 Ala
1519PRTartificialPeptides identified by MS/MS tandem analysis-
Integrin beta2 (CD18) 15Arg Gly Asp Cys Asp Gly Val Gln Ile Asn Val
Pro Ile Thr Phe Gln 1 5 10 15 Val Lys Val 1620PRTartificialPeptides
identified by MS/MS tandem analysis- Integrin beta2 (CD18) 16Lys
Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Val Thr His Arg Asn 1 5 10
15 Gln Pro Arg Gly 20 178PRTartificialPeptides identified by MS/MS
tandem analysis- Integrin-alphaM (CD11b) 17Arg Ser Gln Arg Ser Trp
Arg Leu 1 5 1812PRTartificialPeptides identified by MS/MS tandem
analysis- Integrin-alphaM (CD11b) 18Arg Tyr Val Ile Gly Val Gly Asp
Ala Phe Arg Ser 1 5 10 1915PRTartificialPeptides identified by
MS/MS tandem analysis- Integrin-alphaM (CD11b) 19Arg Ser Leu Pro
Ile Ser Leu Val Phe Leu Val Pro Val Arg Leu 1 5 10 15
2015PRTartificialPeptides identified by MS/MS tandem analysis- CD33
20Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 1 5
10 15 2114PRTartificialPeptides identified by MS/MS tandem
analysis- CD33 21Lys Leu Asp Gln Glu Val Gln Glu Glu Thr Gln Gly
Arg Phe 1 5 10 2218PRTartificialPeptides identified by MS/MS tandem
analysis- CD33 22Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu
Thr His Arg Pro 1 5 10 15 Lys Ile 2311PRTartificialPeptides
identified by MS/MS tandem analysis- HSP90 23Lys Tyr Ile Asp Gln
Glu Glu Leu Asn Lys Thr 1 5 10 2413PRTartificialPeptides identified
by MS/MS tandem analysis- HSP90 24Lys Glu Gln Val Ala Asn Ser Ala
Phe Val Glu Arg Val 1 5 10 2516PRTartificialPeptides identified by
MS/MS tandem analysis- HSP90 25Arg Gly Val Val Asp Ser Glu Asp Leu
Pro Leu Asn Ile Ser Arg Glu 1 5 10 15 2617PRTartificialPeptides
identified by MS/MS tandem analysis- HSP90 26Arg Asn Pro Asp Asp
Ile Thr Gln Glu Glu Tyr Gly Glu Phe Tyr Lys 1 5 10 15 Ser
* * * * *