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PEPTIDES AND COMPOSITIONS FOR TARGETED TREATMENT AND IMAGING
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No.
62/717,929,
filed August 13, 2018, U.S. Provisional Patent Application No. 62/751,303,
filed October 26,
2018, U.S. Provisional Patent Application No. 62/836823, filed April 22, 2019,
U.S. Provisional
Patent Application No. 62/843,835, filed May 06, 2019, and to U.S. Provisional
Patent
Application No. 62/875287 filed July 17, 2019, each of which are incorporated
herein by
reference in their entireties and for all purposes.
FIELD OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating
cancer and
other diseases related to activated immune cells using modulators of the TREM-
1/DAP-12
signaling pathway. The compositions, including peptides and peptide variants,
modulate TREM-
1-mediated immunological response as standalone and combination-therapy
treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory
therapies in
patients. In one embodiment, the present invention relates to targeted
treatment, prevention
and/or detection of cancer including but not limited to lung cancer including
non-small cell lung
cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial
giant cell tumor,
.. pigmented villonodular synovitis, cancer cachexia, etc., and other cancers
associated with
myeloid cell activation and recruitment. Additionally, the present invention
relates to the targeted
treatment, prevention and/or detection of scleroderma including but not
limited to calcinosis,
Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia
syndrome
(CREST). The invention further relates to personalized medical treatments.
BACKGROUND OF THE INVENTION
Administration of therapeutic peptides often causes activation of nontarget
cells and
leads to undesired side effects and increases risk of undesired immunogenic
effects. Limitations
generally attributed to therapeutic peptides are: a short half-life in the
circulation because of their
rapid degradation by proteolytic enzymes of the digestive system and blood
plasma; rapid
removal from the circulation by the liver (hepatic clearance) and kidneys
(renal clearance); poor
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ability to cross physiological barriers, such as the blood-brain barrier.
Because of therapeutic
peptides having general hydrophilicity; high conformational flexibility, and
use resulting
sometimes in a lack of selectivity involving interactions with different
receptors/targets (poor
specific biodistribution), described in part in Vlieghe, et al. Drug Discov
Today 2010, 15:40-56.
Consequently, there is need for more effective formulations of therapeutic
peptides to
improve their targeted delivery, prolonged circulatory half-life,
biocompatibility and therapeutic
efficiency.
SUMMARY OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating
cancer and
other diseases related to activated immune cells using modulators of the TREM-
1/DAP-12
signaling pathway. The compositions, including peptides and peptide variants,
modulate TREM-
1-mediated immunological response as standalone and combination-therapy
treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory
therapies in
patients. In one embodiment, the present invention relates to targeted
treatment, prevention
and/or detection of cancer including but not limited to lung cancer including
non-small cell lung
cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial
giant cell tumor,
pigmented villonodular synovitis, cancer cachexia, etc., and other cancers
associated with
myeloid cell activation and recruitment. Additionally, the present invention
relates to the targeted
treatment, prevention and/or detection of scleroderma including but not
limited to calcinosis,
Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia
syndrome
(CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and
therapeutic
compositions comprising such trifunctional peptides for use in treating
diseases related to
activated immune cells. In some embodiments, each trifunctional peptide is
capable of at least
three functions: 1) mediating formation of naturally long half-life
lipopeptide/lipoprotein
particles upon interaction with lipoproteins, 2) facilitation of the targeted
delivery to cells of
interest and/or sites of disease, and 3) treatment, prevention, and/or
detection of a disease or
condition. In some embodiments, each trifunctional peptide is capable of at
least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide
particles upon binding to
lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of
interest and/or sites of
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disease, and 3) treatment, prevention, and/or detection of a disease or
condition. In certain
embodiments, the present invention relates to amphipathic trifunctional
peptides consisting of
two amino acid domains, wherein upon interaction with plasma lipoproteins, one
amino acid
domain mediates formation of naturally long half-life lipopeptide/lipoprotein
particles and
.. targets these particles to macrophages, whereas the other amino acid domain
inhibits the TREM-
1/DAP-12 receptor signaling complex expressed on macrophages. The invention
further relates to
personalized medical treatments for cancer that involve targeting specific
cancers by their tumor
environment. The invention further relates to personalized medical treatments
for scleroderma
(systemic sclerosis, SSc). More specifically, the invention provides for
treatment of scleroderma
or a related autoimmune or a fibrotic condition by using modulators of the
TREM-1/DAP-12
pathway standalone or together with other antifibrotic therapies and the use
of such combinations
in the treatment of scleroderma.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof, said method comprising administering to said patient a
therapeutically effective
.. amount of at least one modulator that is effective for modulating the TREM-
1/DAP-12 signaling
pathway together with a therapeutically amount of an anticancer vaccine, an
anticancer
immunotherapy agent, anti-cancer immunomodulatory agent, an additional
anticancer
therapeutic, radiation therapy, surgery or a combination thereof. In one
embodiment, said method
further comprises administering said modulator together with a
pharmaceutically acceptable
.. excipient, carrier, diluents, or a combination thereof. In some
embodiments, said carrier is
selected from the group consisting of lipids, proteins or polypeptides, and
mixtures thereof. In
one embodiment, said method further comprises prior to administering the first
dose of said
modulator, the subject received a prior therapy selected from the group
consising of an
anticancer vaccine, an anticancer immunotherapy agent, anti-cancer
immunomodulatory agent,
an additional anticancer therapeutic, radiation therapy, surgery or a
combination thereof.
However it is not meant to limit such prior therapies. In some embodiments,
said cancer recurred
or progressed after the prior therapy. In some embodiments, said
administration of said
modulator to said patient is continued as a long-term maintenance treatment
for duration between
about two weeks to about five years, preferably said administration is
continued for duration of
.. up to one year. In some embodiments, said anticancer vaccine is selected
from the group
consisting of Gardasil, Cervarix, Sipuleucel-T/Provenge, and the like. In some
embodiments,
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said anticancer immunotherapy agent is selected from the group consisting of
Alemtuzumab,
Ipilimumab, Ofatumumab, Nivolumab, Pembrolizumab, Rituximab, Blinatumomab,
Daratumumab, Trastuzumab, Cetuximab, Elotuzumab, adoptive T-cell therapy, T-
Vec,
Interferon, Interleukin, and a combination thereof In some embodiments, said
anticancer
immunomodulatory agent is selected from the group consisting of thalidomide,
lenolidomide,
pomalidomide, and a combination thereof In some embodiments, said additional
anticancer
therapeutic is selected from the group consisting of an alkylating agent, a
tubulin inhibitor, a
topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2
inhibitor, a PARP
inhibitor, a tyrosine kinase inhibitor, CSF-1/CSF-1R inhibitor, doxorubicin,
gemcitabine,
entrectinib, epirubicin, vinblastine, etoposide, topotecan, bleomycin,
mytomycin c, and the like.
In some embodiments, said alkylating agent is selected from the group
consisting of
Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan,
Streptozocin,
Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide,
Chlorambucil,
Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin,
Nedaplatin, Cisplatin,
Carboplatin, Oxaliplatin, and the like. In some embodiments, said tubulin
inhibitor is selected
from the group consisting of Taxol, Docetaxel, Abraxane, Vinblastin,
Epothilone, Colchicine,
Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin
52, IDN-5109,
and the like. In some embodiments, said topoisomerase inhibitor is a
topoisomerase I inhibitor
selected from the group consisting of Irinotecan, Topotecan, Camptothecins
(CPT), and the like.
In some embodiments, said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from
the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins,
ellipticine, and
the like. In some embodiments, said proteasome inhibitor is selected from the
group consisting of
Velcade (bortezomib), and Kyprolis (carfilzomib), and the like. In some
embodiments, said
CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736,
AZ07762, A-
69002, and A-641397, and the like. In some embodiments, said PARP inhibitor is
selected from
the group consisting of Olaparib, Talazoparib, ABT-888, (veliparib), KU-59436,
AZD-2281,
AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231, and the like. In some
embodiments, said
tyrosine kinase inhibitor is selected from the group consisting of
pexidartinib, entrectinib,
matinib mesylate (STI571; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774;
Tarceva), lapatinib
(GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib
(PTK787/ZK222584),
sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101), and the
like. In some
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embodiments, said CSF-1/CSF-1R inhibitor is selected from the group consisting
of CSF-1R
kinase inhibitor, an antibody that binds CSF-1R and is capable of blocking
binding of CSF-1
and/or IL-34 to CSF-1R, and the like. In some embodiments, said CSF-1R kinase
inhibitor is
imatinib, nilotinib or PLX3397. In some embodiments, said radiation therapy is
selected from the
group consisting of X-rays, ion beams, electron beams, gamma-rays, UV-rays,
and decay of a
radioactive isotope, or a combination thereof. In some embodiments, said
surgery is surgical
tumor resection. In some embodiments, said cancer is lung cancer including non-
small cell lung
cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma,
melanoma, leukemia,
central nervous system cancer, stomach cancer, prostate, colon cancer,
colorectal cancer, brain
cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer,
skin cancer,
osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, prostate
cancer, thyroid
cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma
multiforme, stomach
cancer, bladder cancer, head and neck cancer, cervical cancer, giant cell
tumor of the tendon
sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis and
other cancers in
which myeloid cells are involved or recruited and cancer cachexia. In some
embodiments, said at
least one said modulator comprises a variant peptide sequence that is capable
of binding TREM-
1/DAP-12 and reducing or blocking TREM-1/DAP-12 activity (signaling and/or
activation). In
some embodiments, said variant peptide sequence comprises at least one D-amino
acid. In some
embodiments, said variant peptide sequence is a cyclic peptide. In some
embodiments, said
variant peptide sequence is derived from transmembrane domain sequences of
human or animal
TREM-1 and/or its signaling subunit, DAP-12, or a combination thereof. In some
embodiments,
said variant peptide sequence comprises LR12 and/or LP17 peptide variants and
the like or a
combination thereof In some embodiments, said modulator comprises at least one
isolated
antibody or fragment thereof, that is capable of specifically binding TREM-
1/DAP-12 and which
is capable of reducing or blocking TREM-1/DAP-12 activity (signaling and/or
activation). In one
embodiment, said method further comprises a diagnostic method. In one
embodiment, said
diagnostic method is performed prior to administering the first dose of said
modulator to predict
response of said patient to a therapy of the method of claim 1. In some
embodiments, said
diagnostic method comprises isolating a biological sample from said patient
and determining in
.. said sample the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of
CD68-positive
cells or a combination thereof, wherein the higher is the expression level of
CSF-1, CSF-1R, IL-
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6, TREM-1 or the higher is number of CD68-positive cells or a combination
thereof, the better
the patient is predicted to respond to a therapy of the method of claim 1. In
some embodiments,
said method comprises: (a) administering to said patient an amount of at least
one said modulator
of the method of claim 1 that is capable of binding TREM-1 and is conjugated
to at least one
imaging probe, or a combination thereof, in a detectably effective amount; (b)
imaging at least a
portion of the patient; (c) detecting the labeled probe, wherein the location
and amount of the
labeled probe corresponds to at least one symptom of the myeloid cell-related
cancer condition
and correlates with the TREM-1 expression levels and the higher the levels
are, the better the
patient is predicted to respond to a therapy of the method of claim 1. In some
embodiments, said
an imaging probe is selected from the group comprising Gd(III), Mn(II),
Mn(III), Cr(II), Cr(III),
Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III),
Eu(II), Eu(III), and
Er(III), T1201, K42, In", Fe.59, Tc99m, cr51
, Ga67, Ga68, cu64, Rb82,m099, Dy165,
Fluorescein,
Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, C11, N13, 015,
Br76, Kr81, Diatrizoate,
Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a
combination thereof.
The present invention encompasses the discovery that it is possible to combine
multiple
functions in one amphipathic polypeptide amino acid sequence to confer a
variety of properties
on the resulting peptide and provides novel peptides and compounds, which are
capable of
executing at least, three functions: 1) mediation of formation of naturally
long half-life
lipopeptide/lipoprotein particles (LP) upon interaction with native
lipoproteins, 2) facilitation of
the targeted delivery to cells of interest and/or sites of disease, and 3)
treatment, prevention,
and/or detection of a disease or condition. In one embodiment, said peptides
and compounds of
the present invention are used in combinations thereof The peptides and
compounds of the
present invention and combinations thereof have a wide variety of uses,
particularly in the areas
of oncology, transplantology, dermatology, hepatology, ophthalmology,
cardiovascular diseases,
sepsis, autoimmune diseases, neurodegenerative diseases and other diseases and
conditions.
They also are useful in the production of medical devices (for example,
medical implants and
implantable devices).
In some embodiments, the invention provides a synthetic trifunctional peptide
comprising: (a) a first amino acid domain that does not interact with native
lipoproteins in
isolated form, wherein said first amino acid domain is at least 3 amino acids
in length and is
capable of treating, preventing and/or detecting an immune-related disease or
condition; and (b)
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a second amino acid domain that mediates formation of lipopeptide/lipoprotein
particles upon
interaction of the peptide with native lipoproteins and targets these
particles to cells of interest
and/or sites of disease or condition, which second amino acid domain is at
least 6 amino acids in
length and has an amphipathic alpha helical amino acid sequence. In some
embodiments, said
first amino acid domain comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-
Leu-Val-Phe,
wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine,
Lys is lysine, and Val
is valine. In some embodiments, said first amino acid domain comprises amino
acid sequence
Met-Trp-Lys-Thr-Pro-Thr-Leu-Lys-Tyr-Phe, wherein Met is methionine, Trp is
tryptophan, Lys
is lysine, Thr is threonine, Pro is proline, Leu is leucine, Tyr is tyrosine,
and Phe is
phenylalanine. In some embodiments, said second amino acid domain comprises
amino acid
sequence Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val, wherein Tyr is
tyrosine, Leu is
leucine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic
acid, Met is
methionine, Arg is arginine, and Val is valine. In some embodiments, said
second amino acid
domain comprises amino acid sequence Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-
His-Val,
wherein Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is
arginine, Asp,
asparagine, Ala is alanine, His is histidine, and Val is valine. In some
embodiments, said first
amino acid domain and/or said second amino acid domain are conjugated to at
least one imaging
probe.
In some embodiments, the invention provides a method of imaging an immune-
related
disease or condition, comprising a) providing; i) a patient having at least
one symptom of a
disease or condition in which immune cells are involved or recruited, and ii)
a compound of
claim 8, wherein the composition has an affinity for immune receptors; b)
administering said
composition to said patient in a detectably effective amount, c) imaging at
least a portion of the
patient; and d) detecting the labeled probe, wherein the location of the
labeled probe corresponds
to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-
related
disease or condition, comprising: a) providing; i) a patient having at least
one symptom of a
disease or condition in which immune cells are involved or recruited, and ii)
the composition of
claim 1 capable of modulating immune receptors; b) administering said
composition to said
patient under conditions such that said at least one symptom is reduced. In
some embodiments,
said immune-related disease or condition is selected from the group comprising
cancer including
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but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain
and skin cancers,
cancer cachexia, heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke,
bacterial infectious diseases, acquired immune deficiency syndrome (AIDS),
allergic diseases,
acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic
shock, multiple
sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic
arthritis, Sjogrens,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
sepsis, inflammatory lung diseases (e.g., interstitial pneumonitis and
asthma), retinopathy (e.g.,
retinopathy of prematurity and diabetic retinopathy), neurodegenrative
diseases (e.g.,
Alzheimer's, Parkinson's and Huntington's diseases), gastroenterological
diseases and conditions
(e.g. inflammatory bowel disease, Crohn's disease, celiac disease), Guillain-
Barre syndrome,
Hashimoto's disease, pernicious anemia, primary biliary cirrhosis, chronic
active hepatitis,
alcohol-induced liver disease, nonalcoholic fatty liver disease and non-
alcoholic steatohepatitis,
skin problems (e.g. atopic dermatitis, psoriasis, pemphigus vulgaris),
cardiovascular problems
(e.g. autoimmune pericarditis), allergic diathesis (e.g. delayed type
hypersensitivity), contact
dermatitis, herpes simplex/zoster, respiratory conditions (e.g. allergic
alveolitis), inflammatory
conditions (e.g. myositis), ankylosing spondylitis, tissue/organ transplant
(e.g., heart/lung
transplants) rejection reactions, brain and spinal cord injuries, and other
diseases and conditions
where immune cells are involved or recruited.
In some embodiments, the invention provides a synthetic trifunctional peptide
comprising: (a) a first amino acid domain that does not interact with native
lipoproteins in
isolated form, which first amino acid domain is at least 3 amino acids in
length and is capable of
treating, preventing and/or detecting an immune-related disease or condition;
and (b) a second
amino acid domain that mediates formation of lipopeptide/lipoprotein particles
upon interaction
of the peptide with native lipoproteins and targets these particles to cells
of interest and/or sites
of disease or condition, which second amino acid domain is at least 6 amino
acids in length and
has an amphipathic alpha helical amino acid sequence. In some embodiments,
said first amino
acid domain comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe,
wherein
Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is
lysine, and Val is valine.
In some embodiments, said first amino acid domain comprises amino acid
sequence Met-Trp-
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Lys-Thr-Pro-Thr-Leu-Lys-Tyr-Phe, wherein Met is methionine, Trp is tryptophan,
Lys is lysine,
Thr is threonine, Pro is proline, Leu is leucine, Tyr is tyrosine, and Phe is
phenylalanine. In some
embodiments, said second amino acid domain comprises amino acid sequence Trp-
Gln-Glu-Glu-
Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val, wherein Tyr is tyrosine, Leu is leucine, Gin
is glutamine,
Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg
is arginine, and Val
is valine. In some embodiments, said the second amino acid domain comprises
amino acid
sequence Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val, wherein Gly is
glycine, Glu is
glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is
alanine, His is
histidine, and Val is valine. In some embodiments, said the first amino acid
domain and/or the
second amino acid domain are conjugated to at least one imaging probe.
In some embodiments, the invention provides a method of imaging an immune-
related
disease or condition, comprising a) providing; i) a patient having at least
one symptom of a
disease or condition in which immune cells are involved or recruited, and ii)
a compound of
claim 8, wherein the composition has an affinity for immune receptors; b)
administering said
composition to said patient in a detectably effective amount c) imaging at
least a portion of the
patient; and d) detecting the labeled probe, wherein the location of the
labeled probe corresponds
to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-
related
disease or condition, comprising: a) providing; i) a patient having at least
one symptom of a
disease or condition in which immune cells are involved or recruited, and ii)
the composition of
claim 1 capable of modulating immune receptors; b) administering said
composition to said
patient under conditions such that said at least one symptom is reduced. In
some embodiments,
said immune-related disease or condition is selected from the group comprising
cancer including
but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain
and skin cancers,
cancer cachexia, heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke,
bacterial infectious diseases, acquired immune deficiency syndrome (AIDS),
allergic diseases,
acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic
shock, multiple
sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic
arthritis, Sjogrens,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
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sepsis, inflammatory lung diseases (e.g., interstitial pneumonitis and
asthma), retinopathy (e.g.,
retinopathy of prematurity and diabetic retinopathy), neurodegenrative
diseases (e.g.,
Alzheimer's, Parkinson's and Huntington's diseases), gastroenterological
diseases and conditions
(e.g. inflammatory bowel disease, Crohn's disease, celiac disease), Guillain-
Barre syndrome,
Hashimoto's disease, pernicious anemia, primary biliary cirrhosis, chronic
active hepatitis,
alcohol-induced liver disease, nonalcoholic fatty liver disease and non-
alcoholic steatohepatitis,
skin problems (e.g. atopic dermatitis, psoriasis, pemphigus vulgaris),
cardiovascular problems
(e.g. autoimmune pericarditis), allergic diathesis (e.g. delayed type
hypersensitivity), contact
dermatitis, herpes simplex/zoster, respiratory conditions (e.g. allergic
alveolitis), inflammatory
conditions (e.g. myositis), ankylosing spondylitis, tissue/organ transplant
(e.g., heart/lung
transplants) rejection reactions, and other diseases and conditions where
immune cells are
involved or recruited.
The present disclosure provides novel peptides and compounds, which are
capable of
executing three functions: 1) assistance in the self-assembly of naturally
long half-life
lipopeptide particles upon interaction with lipoproteins, 2) facilitation of
the targeted delivery to
cells of interest and/or sites of disease, and 3) treatment, prevention,
and/or detection of a disease
or condition. In one embodiment, said peptides and compounds of the present
invention form
synthetic lipopeptide particles upon binding to lipid or lipid mixtures.
In some embodiments, the invention provides a synthetic trifunctional
polypeptide
comprising at least one peptide domain of 3 to 35 amino acids in length having
a C-terminal
amino acid and at least one amphipathic domain of 6 to 45 to amino acids in
length comprising
an amphipathic lipopeptide having an N-terminal amino acid, wherein said first
domain's C-
terminal amino acid is attached to said second domain's N-terminal amino acid.
In one
embodiment, said synthetic trifunctional polypeptide further comprises an
imaging agent. In one
embodiment, said synthetic trifunctional polypeptide further comprises a
therapeutic agent. In
one embodiment, said synthetic trifunctional polypeptide further comprises a
targeting agent. In
one embodiment, said synthetic trifunctional polypeptide further comprises a
lipopeptide
nanoparticle.
In some embodiments, the invention provides a population of spherical
lipopeptide
nanoparticles or discoidal lipopeptide nanoparticles comprising a plurality of
synthetic
trifunctional polypeptides, wherein said synthetic trifunctional polypeptide
comprising at least
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one peptide domain of 3 to 35 amino acids in length having a C-terminal amino
acid and at least
one amphipathic domain of 6 to 45 to amino acids in length comprising an
amphipathic
lipopeptide having an N-terminal amino acid, wherein said first domain's C-
terminal amino acid
is attached to said second domain's N-terminal amino acid.
In some embodiments, the invention provides a method of treating an immune-
related
disease or condition, comprising: a) providing; i) a patient having at least
one symptom of a
disease or condition in which immune cells are involved or recruited, and ii)
a synthetic
trifunctional polypeptide comprising at least one peptide domain of 3 to 35
amino acids in length
having a C-terminal amino acid and at least one amphipathic domain of 6 to 45
to amino acids in
length comprising an amphipathic lipopeptide having an N-terminal amino acid,
wherein said
first domain's C-terminal amino acid is attached to said second domain's N-
terminal amino acid,
wherein said trifunctional polypeptide is capable of modulating immune
receptors; b)
administering said synthetic trifunctional polypeptide to said patient under
conditions such that
said at least one symptom is reduced.
The invention relates to personalized medical treatments for cancer that
involve targeting
specific cancers by their tumor environment. More specifically, the invention
provides for
treatment of various cancers by using inhibitors of the TREM-1/DAP-12 pathway.
These
inhibitors include peptide variants and compositions that modulate the TREM-1-
mediated
immunological responses beneficial for the treatment of cancer. In addition,
the invention
provides for predicting the efficacy of TREM-1-targeted therapies in various
cancers by
analyzing biological samples for the presence of myeloid cells and for the
TREM-1 expression
levels. In one embodiment, the peptides and compositions of the present
invention modulate
TREM-1/DAP-12 receptor complex expressed on macrophages. In one embodiment,
the peptides
and compositions of the invention are conjugated to an imaging probe. In one
embodiment, the
invention provides for detecting the TREM-1-expressing cells and tissues in an
individual with
cancer using imaging techniques and the peptides and compositions of the
invention containing
an imaging probe. In one embodiment, the peptides and compositions of the
invention are used
in combinations thereof. In one embodiment, the peptides and compositions of
the invention are
used in combinations with other anticancer therapeutic agents. In one
embodiment, the present
invention relates to the targeted treatment, prevention and/or detection of
cancer including but
not limited to pancreatic cancer, breast cancer, liver cancer, multiple
myeloma, leukemia,
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bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain
cancer, ovarian
cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer
cachexia.
The invention provides for a method of treating cancer in an individual in
need thereof by
administering to the individual an effective amount of an inhibitor of the
TREM-1/DAP-12
pathway. In one aspect, the inhibitors are selected from peptide variants and
compositions that
suppress tumor growth by modulating the TREM-1/DAP-12 signaling pathway. In
one
embodiment, any or both the domains comprise minimal biologically active amino
acid
sequence. In one embodiment, the peptide variant comprises a cyclic peptide
sequence. In one
embodiment, the peptide variant comprises a disulfide-linked dimer. In one
embodiment, the
peptide variant includes amino acids selected from the group of natural and
unnatural amino
acids including, but not limited to, L-amino acids, or D-amino acids. In one
embodiment, an
imaging probe and/or an additional therapeutic agent is conjugated to the
peptide variants and
compositions of the invention. In one embodiment, the imaging agent is a Gd-
based contrast
agent (GBCA) for magnetic resonance imaging (MRI). In one embodiment, the
imaging agent is
.. a [64Cu]-containing imaging probe for imaging systems such as a positron
emission tomography
(PET) imaging systems (and combined PET/computer tomography (CT) and PET/MM
systems).
In one embodiment, the peptides and compositions of the invention are used in
combinations
thereof. In one embodiment, the peptides and compositions of the invention are
used in
combinations with other anticancer therapeutic agents. In certain embodiments,
the peptide
.. variants and compositions of the present invention are incorporated into
long half-life synthetic
lipopeptide particles (SLP). In certain embodiments, the peptide variants and
compositions of the
invention may incorporate into lipopeptide particles (LP) in vivo upon
administration to the
individual. In certain embodiments, the peptides and compositions of the
invention can cross the
blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier
(BTB). Thus, in
one aspect, the invention provides for a method for suppressing tumor growth
in an individual in
need thereof by administering to the individual an amount of a TREM-1
inhibitor that is effective
for suppressing tumor growth.
In some embodiments, methods of treating a proliferative disorder involving a
synovial
joint and/or tendon sheath in a subject are provided, comprising administering
to the subject an
effective amount of a compound or composition that modulates TREM-1/DAP-12
activity. In
some embodiments, the proliferative disorder is selected from pigmented
villonodular synovitis
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(PVNS), giant cell tumor of the tendon sheath (GCTTS), and tenosynovial giant
cell tumor
(TGCT) such as diffuse type tenosynovial gian cell tumor (dtTGCT). In some
embodiments, the
disorder is pigmented villonodular synovitis/diffuse type tenosynovial gian
cell tumor
(PVNS/dtTGCT).
In some embodiments, the PVNS tumor volume is reduced by at least 30% or at
least
40% or at least 50% or at least 60% or at least 70% after administration of at
least two, at least
three, at least four, at least five, at least six, at least seven, at least
eight, at least nine, or at least
ten doses of the compound or composition that modulates TREM-1/DAP-12
activity. In some
embodiments, the tumor volume is tumor volume in a single joint. In some
embodiments, the
single join is selected from a hip joint and a knee joint. In some
embodiments, the tumor volume
is total tumor volume in all joints affected by PVNS. In some embodiments, the
subject
experiences one or more than one of the following improvements in symptoms:
(a) a reduction in
joint pain, (b) an increase range of motion in a joint, and (c) an increase in
functional capacity of
a joint, following at least one dose of the compound or composition.
In some embodiments, the compounds or compositions of the present invention
are
selected peptide variants and compositions (see, e.g., US 9,981,004; US
8,513,185; US
9,815,883; US 9,273,111; US 8,013,116) that modulate the TREM-1/DAP-12
signaling pathway.
In certain embodiments, the present invention relates to amphipathic
trifunctional peptides
consisting of two amino acid domains, wherein upon interaction with plasma
lipoproteins, one
amino acid domain mediates formation of naturally long half-life
lipopeptide/lipoprotein
complexes and targets these complexes to macrophages, whereas the other amino
acid domain
inhibits the TREM-1/DAP-12 receptor signaling complex expressed on
macrophages. In one
embodiment, the peptide variant comprises a cyclic peptide sequence. In one
embodiment, the
peptide variant comprises a disulfide-linked dimer. In one embodiment, the
peptide variant
includes amino acids selected from the group of natural and unnatural amino
acids including, but
not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging
probe and/or an
additional therapeutic agent is conjugated to the compounds and compositions
of the invention.
In certain embodiments, the compounds and compositions of the present
invention are
incorporated into long half-life synthetic lipopeptide complexes (LPC). In
certain embodiments,
the compounds and compositions of the invention may incorporate into natural
lipoprotein
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particles (LP) in vivo upon administration to the individual. See, e.g., US
20110256224 and
(Sigalov 2014, Shen and Sigalov 2017, Shen et al. 2017, Rojas et al. 2018,
Tornai et al. 2019).
In certain embodiments, the preferred TREM-1 modulatory compounds and
compositions
are TREM-1 inhibitory peptide sequences such e.g., as GF9 described in
(described in (Sigalov
2014, Rojas et al. 2017, Shen and Sigalov 2017, Shen and Sigalov 2017) and
disclosed in (US
8,513,185 and US 9,981,004) or LR12 and LP17 (described in Gibot, et al.
Infect Immun 2006,
74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J
Immunol 2007, 37:456-
466; Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J
Clin Pharmacol
2018, in press; Zhou, et al. Int Immunopharmacol 2013, 17:155-161; and
disclosed in Faure, et
.. al., US 8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US
9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136). In certain embodiments, the
preferred
TREM-1 modulatory compounds and compositions are antibodies that bind and
block TREM-1
such e.g., as those disclosed in US 10,189,902. In some embodiments,
combinations of different
TREM-1 modulatory compounds and compositions of the invention is used.
In another aspect, the invention provides for a method of predicting the
efficacy of
TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a
biological sample from the individual; (b) determining the number of myeloid
cells in the
biological sample; (c) determining the expression levels of TREM-1 in the
cells contained within
the biological sample; (d) measuring the level of soluble form of the human
TREM-1 receptor in
the biological sample. See, e.g., US 8,021,836.
In some embodiments, prior to administering the first dose of the compound or
composition that modulates the TREM-1/DAP-12 receptor complex signaling, the
subject
receives a first therapy selected from surgical synovectomy, radiation beam
therapy, radio
isotope synovectomy, and joint replacement. In some embodiments, the PVNS
recurred or
progressed after the first therapy. In some embodiments, the compound or
composition of the
present invention is administered prior to a therapy selected from surgical
synovectomy,
radiation beam therapy, radio isotope synovectomy, and joint replacement. In
some
embodiments, the tumor is unresectable. In some embodiments, the subject has
not received prior
therapy with imatinib, nilotinib or a CSF1/CSF1R inhibitor, while in other
embodiments the
subject has received prior treatment with imatinib, nilotinib or a CSF1/CSF1R
inhibitor. In some
embodiments, the subject has not received prior treatment with a CSF1/CSF1R
inhibitor, while
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in other embodiments the subject has received prior treatment with a
CSF1/CSF1R inhibitor. In
some embodiments, the compound or composition that modulates the TREM-1/DAP-12
receptor
complex signaling is administered with imatinib, nilotinib, a CSF1/CSF1R
inhibitor, anti-
programmed cell death protein 1 (anti-PD1) or anti-programmed cell death
ligand 1 (PDL1)
antibodies.
In one embodiment the compound or composition of the present invention is
provided as
a pharmaceutical composition for intravenous administration. In one
embodiment, the compound
or composition of the present invention is provided as a pharmaceutical
composition for oral
administration. In one embodiment, the compound is administered once a day. In
one
embodiment, the compound is administered twice a day. In one embodiment, the
method
includes administering to the patient one or more additional therapeutic
compounds. In one
embodiment, the one or more additional therapeutic compound is selected from
one or more of a
Btk tyrosine kinase inhibitor, an Erbb2 tyrosine kinase receptor inhibitor; an
Erbb4 tyrosine
kinase receptor inhibitor, an mTOR inhibitor, a thymidylate synthase
inhibitor, an EGFR
tyrosine kinase receptor inhibitor, an epidermal growth factor antagonist, a
Fyn tyrosine kinase
inhibitor, a kit tyrosine kinase inhibitor, a Lyn tyrosine kinase inhibitor, a
NK cell receptor
modulator, a PDGF receptor antagonist, a PARP inhibitor, a poly ADP ribose
polymerase
inhibitor, a poly ADP ribose polymerase 1 inhibitor, a poly ADP ribose
polymerase 2 inhibitor, a
poly ADP ribose polymerase 3 inhibitor, a galactosyltransferase modulator, a
dihydropyrimidine
dehydrogenase inhibitor, an orotate phosphoribosyltransferase inhibitor, a
telomerase modulator,
a mucin 1 inhibitor, a mucin inhibitor, a secretin agonist, a TNF related
apoptosis inducing
ligand modulator, an IL-17 gene stimulator, an interleukin-17E ligand, a
neurokinin receptor
agonist, a cyclin G1 inhibitor, a checkpoint inhibitor, a PD-1 inhibitor, a PD-
Li inhibitor, a
CTLA4 inhibitor, a topoisomerase I inhibitor, an Alk-5 protein kinase
inhibitor, a connective
tissue growth factor ligand inhibitor, a notch-2 receptor antagonist, a notch-
3 receptor antagonist,
a hyaluronidase stimulator, a MEK-1 protein kinase inhibitor; MEK-2 protein
kinase inhibitor, a
GM-C SF receptor modulator; TNF alpha ligand modulator, a mesothelin
modulator, an
asparaginase stimulator, a caspase-3 stimulator; caspase-9 stimulator, a PKN3
gene inhibitor, a
hedgehog protein inhibitor; smoothened receptor antagonist, an AKT1 gene
inhibitor, a DHFR
inhibitor, a thymidine kinase stimulator, a CD29 modulator, a fibronectin
modulator, an
interleukin-2 ligand, a serine protease inhibitor, a D4OLG gene stimulator;
TNFSF9 gene
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stimulator, a 2-oxoglutarate dehydrogenase inhibitor, a TGF-beta type II
receptor antagonist, an
Erbb3 tyrosine kinase receptor inhibitor, a cholecystokinin CCK2 receptor
antagonist, a Wilms
tumor protein modulator, a Ras GTPase modulator, an histone deacetylase
inhibitor, a cyclin-
dependent kinase 4 inhibitor A modulator, an estrogen receptor beta modulator,
a 4-1BB
inhibitor, a 4-1BBL inhibitor, a PD-L2 inhibitor, a B7-H3 inhibitor, a B7-H4
inhibitor, a BTLA
inhibitor, a HVEM inhibitor, aTIM3 inhibitor, a GAL9 inhibitor, a LAG3
inhibitor, a VISTA
inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor and a CD66e
modulator. In one
embodiment, the one or more additional therapeutic compounds is selected from
one or more of
bavituximab, IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483,
TG-01,
.. mocetinostat, LOAd-703, CPI-613, upamostat, CRS-207, NovaCaps, trametinib,
Atu-027,
sonidegib, GRASPA, trabedersen, nastorazepide, Vaccell, oregovomab,
istiratumab, refametinib,
regorafenib, lapatinib, selumetinib, rucaparib, pelareorep, tarextumab,
PEGylated hyaluronidase,
varlitinib, aglatimagene besadenovec, GB S-01, GI-4000, WF-10, galunisertib,
afatinib, RX-
0201, FG-3019, pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizin,
yttrium (90Y)
clivatuzumab tetraxetan, brivudine, nimotuzumab, algenpantucel-L,
tegafur+gimeracil+oteracil
potassium+calcium folinate, olaparib, ibrutinib, pirarubicin, Rh-Apo2L,
tertomotide,
tegafur+gimeracil+oteracil potassium, tegafur +gimeracil +oteracil potassium,
masitinib, Rexin-
G, mitomycin, erlotinib, adriamycin, dexamethasone, vincristine,
cyclophosphamide,
fluorouracil, topotecan, taxol, interferons, platinum derivatives, taxane,
paclitaxel, vinca
alkaloids, vinblastine, anthracyclines, doxorubicin, epipodophyllotoxins,
etoposide, cisplatin,
rapamycin, methotrexate, actinomycin D, dolastatin 10, colchicine, emetine,
trimetrexate,
metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating
agents,
chlorambucil, 5-fluorouracil, campthothecin, metronidazole, Gleevec, Avastin,
Vectibix,
abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine,
amifostine, anastrozole,
arsenic trioxide, asparaginase, azacitidine, AZD9291, BCG Live, bevacuzimab,
fluorouracil,
bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine,
camptothecin,
carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cladribine,
clofarabine,
cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa, daunorubicin,
denileukin,
dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride,
dromostanolone
propionate, epirubicin, epoetin alfa, estramustine, etoposide phosphate,
etoposide, exemestane,
filgrastim, floxuridine fludarabine, fulvestrant, gefitinib, gemcitabine,
gemtuzumab, goserelin
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acetate, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide,
imatinib mesylate,
interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole,
leucovorin, leuprolide
acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine,
6-MP, mesna,
methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone,
nelarabine,
nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate,
pegademase,
pegaspargase, pegfilgrastim, pemetrexed di sodium, pentostatin, pipobroman,
plicamycin,
porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab,
rociletinib, sargramostim,
sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide,
teniposide, VM-26,
testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab,
trastuzumab,
tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine, zoledronate,
zoledronic acid, pembrolizumab, nivolumab, D3I-308, mDX-400, BGB-108, MEDI-
0680, SHR-
1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab, atezolizumab,
avelumab, BMS-
936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX and KY-1003. In
one
embodiment, the one or more additional therapeutic compound is FOLFIRINOX. In
one
embodiment, the one or more additional therapeutic compounds are gemcitabine
and paclitaxel.
In one embodiment, the one or more additional therapeutic compounds are
gemcitabine and nab-
paclitaxel.
In some embodiments, the invention provides diagnostic markers to prognose the
response to TREM-1 therapy. In some embodiments, the invention provides
prognostic markers
to prognose the response to TREM-1 therapy. It is not meant to limit the
markers to those
described herein.
Accordingly, the invention provides for a method of treating cancer in an
individual in
need thereof by administering to the individual a therapeutically effective
amount of at least one
modulator which affects myeloid cells by action on the TREM-1/DAP-12 signaling
pathway
together with a therapeutically effective amount of an anticancer vaccine, an
anticancer
immunotherapy agent, anti-cancer immunomodulatory agent, an additional
anticancer
therapeutic, radiation therapy, surgery or a combination thereof. The subject
of the present
invention includes any human subject who has been diagnosed with, has symptoms
of, or is at
risk of developing a cancer or a pre- or post-cancerous condition.
The invention relates to personalized combination-therapy treatments for
cancer that
involve targeting specific cancers by their tumor environment. More
specifically, the invention
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provides a method for treating various cancers by using modulators of the TREM-
1/DAP-12
pathway together with other cancer therapies and the use of such combinations
in the treatment
of cancer. In certain embodiments, these modulators may possess the antitumor
activity. In some
embodiments, these modulators may not possess the antitumor activity. In one
embodiment,
these modulators include peptide variants and compositions that are capable of
binding TREM-1
and reducing or blocking TREM-1 activity (signaling and/or activation). In one
embodiment
these peptide variants and compositions modulate the TREM-1-mediated
immunological
responses beneficial for the treatment of cancer. In one embodiment, the
peptides and
compositions of the present invention modulate TREM-1/DAP-12 receptor complex
expressed on
monocytes, macrophages and neutrophils. In one embodiment, the peptides and
compositions of
the present invention modulate TREM-1/DAP-12 receptor complex expressed on
tumor-
associated macrophages. In one embodiment, the invention provides a method for
predicting the
efficacy of standalone or combination-therapy treatment that involve TREM-1-
targeting
therapies in various cancers by analyzing biological samples from cancer
patients for the
presence of myeloid cells and for the expression levels of TREM-1, CSF-1, CSF-
1R, IL-6 and
other markers. In one embodiment, the peptides and compositions of the
invention are
conjugated to an imaging probe. In one embodiment, the invention provides for
detecting the
TREM-1-expressing cells and tissues in an individual with cancer using imaging
techniques and
the peptides and compositions of the invention containing an imaging probe. In
one embodiment,
the peptides and compositions of the invention are used in combinations
thereof In one
embodiment, the peptides and compositions of the invention are used in
combinations with other
anticancer therapeutic agents. In one embodiment, the present invention
relates to the targeted
treatment, prevention and/or detection of cancer including but not limited to
lung cancer
including non-small cell lung cancer (NSCLC), pancreatic cancer, breast
cancer, liver cancer,
multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system
(CNS) cancer,
stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain
cancer, gastrointestinal
cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer,
osteosarcoma, endometrial
cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma,
neurofibroma, glioma,
glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer,
pigmented
villonodular synovitis (PVNS) and other cancers in which myeloid cells are
involved or recruited
and cancer cachexia.
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In some embodiments, cancer is selected from the list including but not
limited to lung
cancer including NSCLC, pancreatic cancer, breast cancer, liver cancer,
multiple myeloma,
melanoma, leukemia, bladder cancer, central nervous system (CNS) cancer,
stomach cancer,
prostate cancer, colorectal cancer, colon cancer, brain cancer,
gastrointestinal cancer, gastric
cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial
cancer, esophageal
cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma,
glioblastoma,
glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell
tumor of the tendon
sheath (GCTTS), tenosynovial giant cell tumor (TGCT; also referred to in the
art as TSGCT),
PVNS and other cancers in which myeloid cells are involved or recruited and
cancer cachexia.
In some embodiments, the modulators of the TREM-1/DAP-12 signaling pathway are
capable of suppressing tumor growth in the subject. In another aspect, the
modulators are capable
of delaying the development of cancer in the subject. In another aspect, the
modulators are
capable of reducing tumor size in the subject. In another aspect, the
modulators are capable of
treating cancer in the subject. In another aspect, the modulators are capable
of treating cancer in
the subject. In another aspect, the modulators are capable of increasing
survival of the subject.
In some embodiments, the modulators are capable of binding TREM-1 and reducing
or
blocking TREM-1 activity (signaling and/or activation). In some embodiments,
the modulators
comprise peptide variants and compositions that are capable of binding TREM-1
and reducing or
blocking TREM-1 activity (signaling and/or activation) together with a
pharmaceutically
acceptable excipient, carrier, diluent, salt or a combination thereof In some
embodiments, the
modulators comprise antibodies or fragments thereof that are capable of
binding TREM-1 and
reducing or blocking TREM-1 activity (signaling and/or activation) together
with a
pharmaceutically acceptable excipient, carrier, diluent, salt or a combination
thereof.
The methods of combination therapy featured in the present invention may
result in a
synergistic effect, wherein the effect of a combination of compounds or other
therapeutic agents
is greater than the sum of the effects resulting from administration of any of
the compounds or
other therapeutic agents as single agents. A synergistic effect may also be an
effect that cannot
be achieved by administration of any of the compounds or other therapeutic
agents as single
agents. The synergistic effect may include, but is not limited to, an effect
of treating cancer by
reducing tumor size, inhibiting tumor growth, or increasing survival of the
subject. The
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synergistic effect may also include reducing cancer cell viability, inducing
cancer cell death, and
inhibiting or delaying cancer cell growth.
In another aspect, the invention provides for a method of predicting the
efficacy of
TREM-1 targeted therapies in an individual with cancer by: (a) obtaining a
biological sample
from the individual; (b) determining the number of myeloid cells in the
biological sample; (c)
determining the expression levels of TREM-1 in the cells contained within the
biological sample.
In another aspect, the invention provides for a method of detecting TREM-1
expression
levels in an individual with cancer by: (a) administering to the individual
the peptide variants and
composition of the present invention having an affinity for TREM-1 and an
imaging probe in a
detectably effective amount; (b) imaging at least a portion of the patient;
(c) detecting the labeled
probe, wherein the location of the labeled probe corresponds to at least one
symptom of the
myeloid cell-related condition.
In certain embodiments, the invention provides for a diagnostic method of
detecting
TREM-1 expression levels in an individual with cancer by: (a) administering to
the individual
the modulators of TREM-1 transmembrane signaling having an affinity for TREM-1
and an
imaging probe in a detectably effective amount; (b) imaging at least a portion
of the patient; (c)
detecting the labeled probe, wherein the location of the labeled probe
corresponds to at least one
symptom of the myeloid cell-related cancer condition and correlates with the
TREM-1
expression levels and the higher the levels are, the better the patient is
predicted to respond to a
TREM-1 inhibitory therapy using the modulators of the TREM-1/DAP-12 signaling
pathway as
standalone therapy or in combinations with other anticancer treatments.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof by modulating immune system activity, said method comprising
administering to
said patient an amount of a TREM-1 inhibitor that is effective for inhibiting
the TREM-1/DAP-
12 signaling pathway and suppressing tumor growth, or a combination thereof In
one
embodiment, said method further comprises administering the amount of the TREM-
1 inhibitor
together with a pharmaceutically acceptable excipient, carrier, diluents, or a
combination thereof.
In one embodiment, said method further comprises administering to said patient
an anticancer
vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory
agent, an
additional anticancer therapeutic, radiation therapy or a combination thereof.
In some
embodiments, said anticancer vaccine is selected from the group consisting of
Gardasil,
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Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer
immunotherapy
agent is selected from the group consisting of Alemtuzumab, Ipilimumab,
Nivolumab,
Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof.
In some
embodiments, said anti-cancer immunomodulatory agent is selected from the
group consisting of
thalidomide, lenolidomide, pomalidomide, and a combination thereof.. In some
embodiments,
said additional anti-cancer therapeutic is selected from the group consisting
of an alkylating
agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a
CHK1 inhibitor, a
CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine,
etoposide, topotecan,
bleomycin, and mytomycin c.. In some embodiments, said alkylating agent is
selected from the
group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine,
Uramustine, BuSulfan,
Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide,
Cyclophosphamide,
Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate,
Satraplatin, Nedaplatin,
Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin
inhibitor is selected
from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone,
Colchicine,
Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin
52, and IDN-
5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected
from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some
embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor
selected from the
group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and
ellipticine. In
some embodiments, said proteasome inhibitor is selected from the group
consisting of Velcade
(bortezomib), and Kyprolis (carfilzomib).. In some embodiments, said CHK1
inhibitor is
selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002,
and A-64
1397. In some embodiments, said PARP inhibitor is selected from the group
consisting of
Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-
15, 'NO-
.. 1001, ONO-2231 and the like. In some embodiments, said radiation therapy is
administered to
said patient.. In some embodiments, said at least one said TREM-1 inhibitor
comprises a variant
TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences
of human
or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some
embodiments, said at
least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants
and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-
12
expression levels in a patient with cancer in need thereof, said method
comprising administering
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to said patient an amount of a TREM-1 inhibitor that is conjugated to at least
one imaging probe,
or a combination thereof. In some embodiments, said imaging probe is selected
from the group
comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),
Pr(III), Nd(III) Sm(III),
Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42,
In", Fe.59, Tc99111, Cr51,
Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein,
F18, Xe133, 1125,
1131, 1123, p32, C11, N13, 015, Br76, Kr81,
Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol,
Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1
inhibitor comprises a
variant TREM-1 inhibitory peptide sequence derived from transmembrane domain
sequences of
human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some
embodiments,
said at least one said TREM-1 inhibitor comprises LR1 2 and/or LP1 7 peptide
variants and the
like.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof by modulating immune system activity, said method comprising
administering to
said patient an amount of a TREM-1 inhibitor that is effective for inhibiting
the TREM-1/DAP-
1 5 12 signaling pathway and suppressing tumor growth, or a combination
thereof In some
embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory
peptide sequence
derived from transmembrane domain sequences of human or animal TREM-1 and/or
its
signaling subunit, DAP-12, thereof In some embodiments, said a variant TREM-1
inhibitory
peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-
Phe,
wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine,
Lys is lysine, and Val
is valine. In some embodiments, said variant TREM-1 inhibitory peptide
sequence is conjugated
to at least one unmodified or modified amphipathic peptide sequence. In some
embodiments,
said an unmodified or modified amphipathic peptide sequence is derived from
amino acid
sequences of apolipoproteins selected from the group consisting of A-I, A-II,
A-IV, B, C-I, C-II,
.. C-III, and E, and any combination thereof. In some embodiments, said a
modified amphipathic
peptide sequence derived from amino acid sequences of apolipoproteins selected
from the group
consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination
thereof contains at
least one amino acid residue which is chemically or enzymatically modified. In
some
embodiments, said a chemically or enzymatically modified amino acid residue is
oxidized,
halogenated or nitrated. In some embodiments, said an oxidized amino acid
residue is the
methionine residue. In some embodiments, said an unmodified amphipathic
peptide sequence is
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derived from an apolipoprotein A-I amino acid sequence and comprises amino
acid sequence
Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-
Lys-Val-
Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine,
Phe is
phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is
glutamic acid, Met is
methionine, Arg is arginine, and Val is valine. In some embodiments, said an
unmodified
amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid
sequence and
comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-
His-Val-
Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly
is glycine, Glu is
glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is
alanine, His is
histidine, Val is valine, and Thr is threonine. In some embodiments, said a
modified amphipathic
peptide sequence is derived from an apolipoprotein A-I amino acid sequence and
comprises
amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-
Glu-
Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is
leucine, Asp,
asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is
tryptophan, Glu is
glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is
valine. In some
embodiments, said a modified amphipathic peptide sequence is derived from an
apolipoprotein
A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-
Met(0)-
Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro
is proline,
Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine
sulfoxide, Arg is arginine,
Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is
threonine. In some
embodiments, said B is conjugated to an additional peptide sequence to enhance
the targeting
efficacy. In some embodiments, said an additional peptide sequence comprises
amino acid
sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp
is asparagine. In
some embodiments, said A is conjugated to at least one additional therapeutic
agent to enhance
the therapeutic efficacy. In some embodiments, said an additional therapeutic
agent is selected
from the group of anticancer, antibacterial, antiviral, autoimmune, anti-
inflammatory and
cardiovascular agents, antioxidants, therapeutic peptides, and any combination
thereof In some
embodiments, said anticancer therapeutic agent is selected from the group
comprising paclitaxel,
valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination
thereof In some
embodiments, said A and/or B are conjugated to at least one imaging probe. In
some
embodiments, said an imaging probe is selected from the group comprising
Gd(III), Mn(II),
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Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III),
Yb(III) Dy(III), Ho(III),
Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, Cr51, Ga67, Ga68,
Cu64, Rb82,Mo99,
Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123,
P32, 11, N13, 015, Br76,
Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol,
Iodixanol.
In some embodiments, the invention provides a method of making a synthetic
lipopeptide
nanoparticle, said method comprising: a) co-dissolving a predetermined amount
of a mixture of
neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a
predetermined amount of triglycerides and/or cholesteryl ester; b) drying the
mixture of step (a)
under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a
predetermined amount of
sodium cholate; and ii. a predetermined amount of the compound of claim 1; for
a time period
sufficient to allow the components to self-assemble into synthetic lipopeptide
particles; d)
removing sodium cholate from the mixture of step (c); and e) isolating
particles that have a size
of between about 5 to about 200 nm diameter. In some embodiments, said lipid
is conjugated to
at least one imaging probe. In some embodiments, said an imaging probe is
selected from the
group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),
Pr(III), Nd(III)
Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III),
T1201, K42 In", Fe=59/
TC99111, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein,
Carboxyfluorescein, Calcein, F18,
xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81,
Diatrizoate, Metrizoate, Isopaque, Ioxaglate,
Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected
from the group
comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a
sphingolipid, a
cationic lipid, a diacylglycerol, and a triacylglycerol. In some embodiments,
said phospholipid is
selected from the group comprising phosphatidylcholine (PC),
phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG),
cardiolipin (CL),
sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In
some
embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) a labeled
probe, wherein the
labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an
affinity for
TREM-1 and an imaging probe; b) administering said composition to said patient
in a detectably
effective amount c) imaging at least a portion of the patient; and d)
detecting the labeled probe,
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wherein the location of the labeled probe corresponds to at least one symptom
of the myeloid
cell-related condition. In some embodiments, said a myeloid cell-related
condition is selected
from the group comprising cancer including but not limited to lung,
pancreatic, breast, stomach,
prostate, colon, brain and skin cancers, cancer cachexia, heart disease,
atherosclerosis, peripheral
artery disease, restenosis, stroke, bacterial infectious diseases, acquired
immune deficiency
syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory
bowel disease,
empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis,
autoimmune
diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus
erythematosus, non-
specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus
vulgaris),
granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid
granulomatosis,
Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g.,
sepsis,
inflammatory lung diseases such as interstitial pneumonitis and asthma,
inflammatory bowel
disease such as Crohn's disease, inflammatory arthritis retinopathy such as
retinopathy of
prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and
Huntington's diseases),
transplant (e.g., heart/lung transplants) rejection reactions, and other
diseases and conditions
where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid
cell-related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) the
compositions of claims 1,
3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition
to said patient
under conditions such that said at least one symptom is reduced. In some
embodiments, said a
myeloid cell-related condition is selected from the group comprising cancer
including but not
limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin
cancers, cancer
cachexia, heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke, bacterial
infectious diseases, acquired immune deficiency syndrome (AIDS), allergic
diseases, acute
radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric
ischemia,
hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid
arthritis, Sjogrens,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as
interstitial pneumonitis
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and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory
arthritis
retinopathy such as retinopathy of prematurity and diabetic retinopathy,
Alzheimer's, Parkinson's
and Huntington's diseases), transplant (e.g., heart/lung transplants)
rejection reactions, and other
diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) a labeled probe,
wherein the labeled
probe includes the compositions of claims 1, 5, 6, and 21-25 having an
affinity for TCR and an
imaging probe; b) administering said composition to said patient in a
detectably effective amount
c) imaging at least a portion of the patient; and d) detecting the labeled
probe, wherein the
location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group
including but not
limited to include, but are not limited to, systemic lupus erythematosus,
rheumatoid arthritis,
multiple sclerosis, scleroderma, type I diabetes, gastroenterological
conditions e.g. inflammatory
bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome,
Hashimoto's disease,
pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin
problems e.g. atopic
dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis,
allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis,
AIDS virus, herpes
simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory
conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases
and conditions where
T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-
related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) the compositions
of claims 1, 5, 6,
and 23 capable of inhibiting TCR; b) administering said composition to said
patient under
conditions such that said at least one symptom is reduced. In some
embodiments, said a T cell-
related condition is selected from the group including but not limited to
include, but are not
limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, scleroderma,
type I diabetes, gastroenterological conditions e.g. inflammatory bowel
disease, Crohn's disease,
celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious
anaemia, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis,
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pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis,
allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes
simplex/zoster, respiratory
conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis,
ankylosing spondylitis,
tissue/organ rejection, and other diseases and conditions where T cells are
involved or recruited.
In some embodiments, the invention provides a method of reducing pain in a
subject with
pigmented villonodular synovitis (PVNS) tumor, comprising administering to the
subject an
amount of a TREM-1 modulator that is effective for inhibiting the TREM-1/DAP-
12 signaling
pathway and capable of reducing pain in PVNS subjects independently of tumor
response. In
some embodiments, said PVNS tumor has a tumor volume. In some embodiments,
said
inhibition reduces said PVNS tumor volume by at least 30% after administration
of at least two,
at least three, at least four, at least five, at least six, at least seven, at
least eight, at least nine, or
at least ten doses of the modulator that inhibits the TREM-1/DAP-12 signaling
pathway. In some
embodiments, said tumor volume is tumor volume in a single joint.In some
embodiments, said
single joint is selected from a hip joint and a knee joint.In some
embodiments, said tumor
volume is total tumor volume in all joints affected by PVNS. In some
embodiments, said
modulator is an antibody. In some embodiments, prior to administering the
first dose of said
antibody, the subject received a prior therapy selected from surgical
synovectomy, radiation
beam therapy, radio isotope synovectomy, joint replacement and CSF1/CSF1R
inhibitor. In some
embodiments, said PVNS recurred or progressed after the prior therapy. In some
embodiments,
said antibody is administered prior to a therapy selected from surgical
synovectomy, radiation
beam therapy, radio isotope synovectomy, and joint replacement, or wherein the
subject has a
tumor that is unresectable. In some embodiments, said subject has not received
prior treatment
with a CSF1R inhibitor. In one embodiment, said method further comprises
administering the
amount of the TREM-1 modulator together with a pharmaceutically acceptable
excipient, carrier,
diluents, or a combination thereof.In one embodiment, said method further
comprises
administering the amount of the TREM-1 modulator together with an amount of an
anticancer
vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory
agent, an
additional anticancer therapeutic, radiation therapy, or a combination
thereof. In some
embodiments, said anticancer vaccine is selected from the group consisting of
Gardasil,
Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer
immunotherapy
agent is selected from the group consisting of Alemtuzumab, Ipilimumab,
Nivolumab,
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Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof
In some
embodiments, said anti-cancer immunomodulatory agent is selected from the
group consisting of
thalidomide, lenolidomide, pomalidomide, and a combination thereof In some
embodiments,
said additional anti-cancer therapeutic is selected from the group consisting
of an alkylating
.. agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome
inhibitor, a CHK1 inhibitor, a
CHK2 inhibitor, PARP inhibitor, CSF1/CSF1R inhibitor, doxorubicin, epirubicin,
vinblastine,
etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said
alkylating agent
is selected from the group consisting of Dacarbazine, Procarbazine,
Carmustine, Lomustine,
Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine,
Cyclophasphamide,
Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin
tetranitrate,
Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some
embodiments, said
tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel,
Vinblastin,
Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin,
Dolastin 10,
Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase
inhibitor is a
topoisomerase I inhibitor selected from the group consisting of Irinotecan,
Topotecan, and
Camptothecins (CPT). In some embodiments, said topoisomerase inhibitor is a
topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide,
Teniposide,
Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome
inhibitor is selected
from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).
In some
.. embodiments, said CHK1 inhibitor is selected from the group consisting of
TCS2312, PF-
0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP
inhibitor is
selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-
59436, AZD-2281,
AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like. In some
embodiments, said
CSF1/CSF1R inhibitor is selected from the group consisting of CSF1R kinase
inhibitor, an
antibody that binds CSF1R and the like. In some embodiments, said CSF1R kinase
inhibitor is
imatinib or nilotinib. In some embodiments, said CSF1R kinase inhibitor is
PLX3397. In some
embodiments, said anti-CSF1R antibody blocks binding of CSF1 and/or IL-34 to
CSF1R. In
some embodiments, said anti-CSF1R antibody inhibits ligand-induced CSF1R
phosphorylation
in vitro. In some embodiments, said antibody is a humanized antibody. In some
embodiments, a
radiation therapy is administered to said patient. In some embodiments, said
at least one said
TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence
derived from
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transmembrane domain sequences of human or animal TREM-1 and/or its signaling
subunit,
DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor
comprises
LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-
12
expression levels in a patient with cancer in need thereof, said method
comprising administering
to said patient an amount of a TREM-1 inhibitor that is conjugated to at least
one imaging probe,
or a combination thereof.
In some embodiments, the invention provides a method of predicting the
efficacy of
TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a
.. biological sample from the individual; (b) determining the number of
myeloid cells in the
biological sample; (c) determining the expression levels of TREM-1 in the
cells contained within
the biological sample; (d) measuring the level of soluble form of the human
TREM-1 receptor in
the biological sample.
In some embodiments, the invention provides a method of diagnosing disease of
the
proliferative disorder in a subject, wherein said disease is PVNS or TGCT,
which method
comprises the steps of (a) measuring a level of the soluble form of the human
TREM-1 receptor
in a biological sample obtained from said subject; (b) comparing the measured
level of the
soluble form of the human TREM-1 receptor in the sample with a mean level in a
control
population of individuals not PVNS or TGCT; (c) correlating elevated levels of
the soluble form
of the human TREM-1 receptor with the presence or extent of said proliferative
disease. In some
embodiments, said step of measuring the level of the soluble form of the human
TREM-1
receptor comprises the steps of: (a) contacting said biological sample with a
compound capable
of binding the soluble form of the human TREM-1 receptor; (b) detecting the
level of the soluble
form of the human TREM-1 receptor present in the sample by observing the level
of binding
between said compound and the soluble form of the human TREM-1 receptor.In one
embodiment, said method further comprises comprising the steps of measuring
the level of the
soluble form of the human TREM-1 receptor in a second or further sample from
said subject, the
first and second or further samples being obtained at different times; and
comparing the levels in
the samples to indicate the progression or remission of the proliferative
disease. In some
embodiments, said sample is selected from the group consisting of whole blood,
blood serum,
blood, plasma, urine, bronchoalveolar lavage fluid and synovial liquid. In
some embodiments,
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said sample is from synovial fluid. In some embodiments, said sample is from
blood serum or
blood plasma. In some embodiments, said sample is a human sample. In some
embodiments, said
compound specifically binds the soluble form of the human TREM-1 receptor. In
some
embodiments, said compound capable of binding the soluble form of the human
TREM-1
receptor is an antibody raised against all or part of the TREM-1 receptor. In
some embodiments,
said level of soluble form of the human TREM-1 receptor is measured by an
immunochemical
technique. In one embodiment, said method further comprisesan additional step
of measuring the
level of TREM-1-Ligand in one or more biological samples obtained from said
subject. In some
embodiments, said imaging probe is selected from the group comprising Gd(III),
Mn(II),
Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III),
Yb(III) Dy(III), Ho(III),
Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67,
Ga68, Cu64,
Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125,
1131, 1123,
P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate,
Iopamidol,
Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1
inhibitor comprises a
variant TREM-1 inhibitory peptide sequence derived from transmembrane domain
sequences of
human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some
embodiments,
said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide
variants and the
like.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof, said method comprising administering to said patient an amount
of a TREM-1
inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway
and suppressing
tumor growth and an amount of an anticancer vaccine, an anticancer
immunotherapy agent, anti-
cancer immunomodulatory agent, an additional anticancer therapeutic, radiation
therapy, or a
combination thereof. In one embodiment, said method further comprises
administering the
amount of the TREM-1 inhibitor together with a pharmaceutically acceptable
excipient, carrier,
diluents, or a combination thereof In some embodiments, said anticancer
vaccine is selected
from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In
some
embodiments, said anticancer immunotherapy agent is selected from the group
consisting of
Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon,
Interleukin, and
a combination thereof In some embodiments, said anti-cancer immunomodulatory
agent is
selected from the group consisting of thalidomide, lenolidomide, pomalidomide,
and a
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combination thereof In some embodiments, said additional anti-cancer
therapeutic is selected
from the group consisting of an alkylating agent, a tubulin inhibitor, a
topoisomerase inhibitor,
proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor,
doxorubicin,
epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In
some
embodiments, said alkylating agent is selected from the group consisting of
Dacarbazine,
Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin,
Altreamine,
Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil,
Fluorouracil
(5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin,
Carboplatin, and
Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the
group consisting of
Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS
347550, Rhizoxin,
Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some
embodiments, said
topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group
consisting of
Irinotecan, Topotecan, and Camptothecins (CPT). In some embodiments, said
topoisomerase
inhibitor is a topoisomerase II inhibitor selected from the group consisting
of Amsacrine,
.. Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some
embodiments, said
proteasome inhibitor is selected from the group consisting of Velcade
(bortezomib), and
Kyprolis (carfilzomib). In some embodiments, said CHK1 inhibitor is selected
from the group
consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some
embodiments, said PARP inhibitor is selected from the group consisting of
Olaparib, ABT-888,
(veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-
2231 and
the like. In some embodiments, a radiation therapy is administered to said
patient. In some
embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-
1 inhibitory
peptide sequence derived from transmembrane domain sequences of human or
animal TREM-1
and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at
least one said
TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-
12
expression levels in a patient with cancer in need thereof, said method
comprising administering
to said patient an amount of a TREM-1 inhibitor that is conjugated to at least
one imaging probe,
or a combination thereof. In some embodiments, said an imaging probe is
selected from the
.. group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe
(III), Pr(III), Nd(III)
Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III),
T1201, K42, ml ii, Fe.59,
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Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein,
Carboxyfluorescein,
Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81,
Diatrizoate,
Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some
embodiments, said at
least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide
sequence
derived from transmembrane domain sequences of human or animal TREM-1 and/or
its
signaling subunit, DAP-12, thereof. In some embodiments, said at least one
said TREM-1
inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof, said method comprising administering to said patient an amount
of a TREM-1
inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway
and suppressing
tumor growth and an amount of an anticancer vaccine, an anticancer
immunotherapy agent, anti-
cancer immunomodulatory agent, an additional anticancer therapeutic, radiation
therapy, or a
combination thereof. In one embodiment, said TREM-1 inhibitor comprises a
variant TREM-1
inhibitory peptide sequence derived from transmembrane domain sequences of
human or animal
TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments,
said a variant
TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-
Ser-Lys-Ser-
Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser
is serine, Lys is
lysine, and Val is valine. In some embodiments, said variant TREM-1 inhibitory
peptide
sequence is conjugated to at least one unmodified or modified amphipathic
peptide sequence. In
some embodiments, said an unmodified or modified amphipathic peptide sequence
is derived
from amino acid sequences of apolipoproteins selected from the group
consisting of A-I, A-II, A-
IV, B, C-I, C-II, C-III, and E, and any combination thereof. In some
embodiments, said a
modified amphipathic peptide sequence derived from amino acid sequences of
apolipoproteins
selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III,
and E, and any
combination thereof contains at least one amino acid residue which is
chemically or
enzymatically modified. In some embodiments, said a chemically or
enzymatically modified
amino acid residue is oxidized, halogenated or nitrated. In some embodiments,
said an oxidized
amino acid residue is the methionine residue. In some embodiments, said an
unmodified
amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid
sequence and
comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-
Glu-Met-
Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu
is leucine, Asp,
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asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is
tryptophan, Glu is
glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some
embodiments, said
an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-
I amino acid
sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-
Ala-Arg-
Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is
leucine, Gly is
glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp,
asparagine, Ala is alanine,
His is histidine, Val is valine, and Thr is threonine. In some embodiments,
said a modified
amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid
sequence and
comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-
Glu-
Met(0)-Glu-Leu-Tyr-Arg-Gin-Lys-Val-Glu, wherein Pro is proline, Tyr is
tyrosine, Leu is
leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is
lysine, Trp is tryptophan,
Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val
is valine. In some
embodiments, said a modified amphipathic peptide sequence is derived from an
apolipoprotein
A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-
Met(0)-
Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro
is proline,
Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine
sulfoxide, Arg is arginine,
Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is
threonine. In some
embodiments, said B is conjugated to an additional peptide sequence to enhance
the targeting
efficacy. In some embodiments, said an additional peptide sequence comprises
amino acid
sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp
is asparagine. In
some embodiments, said A is conjugated to at least one additional therapeutic
agent to enhance
the therapeutic efficacy. In some embodiments, said an additional therapeutic
agent is selected
from the group of anticancer, antibacterial, antiviral, autoimmune, anti-
inflammatory and
cardiovascular agents, antioxidants, therapeutic peptides, and any combination
thereof In some
embodiments, said anticancer therapeutic agent is selected from the group
comprising paclitaxel,
valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination
thereof In some
embodiments, said A and/or B are conjugated to at least one imaging probe. In
some
embodiments, said an imaging probe is selected from the group comprising
Gd(III), Mn(II),
Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III),
Yb(III) Dy(III), Ho(III),
Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67,
Ga68, Cu64,
Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125,
1131, 1123,
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P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate,
Iopamidol,
Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic
lipopeptide
nanoparticle, said method comprising: a) co-dissolving a predetermined amount
of a mixture of
neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a
predetermined amount of triglycerides and/or cholesteryl ester; b) drying the
mixture of step (a)
under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a
predetermined amount of
sodium cholate; and ii. a predetermined amount of the compound of claim 1; for
a time period
sufficient to allow the components to self-assemble into synthetic lipopeptide
particles; d)
removing sodium cholate from the mixture of step (c); and e) isolating
particles that have a size
of between about 5 to about 200 nm diameter. In some embodiments, said lipid
is conjugated to
at least one imaging probe. In some embodiments, said an imaging probe is
selected from the
group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),
Pr(III), Nd(III)
Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III),
T1201, K42, ml ii, Fe.59,
Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein,
Carboxyfluorescein,
Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81,
Diatrizoate,
Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some
embodiments, said lipid
is selected from the group comprising cholesterol, a cholesteryl ester, a
phospholipid, a
glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a
triacylglycerol. In some
.. embodiments, said phospholipid is selected from the group comprising
phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),
phosphatidylinositol (PI),
phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic
acid (PA), and
any combination thereof In some embodiments, said lipid is polyethylene
glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) a labeled
probe, wherein the
labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an
affinity for
TREM-1 and an imaging probe; b) administering said composition to said patient
in a detectably
effective amount c) imaging at least a portion of the patient; and d)
detecting the labeled probe,
wherein the location of the labeled probe corresponds to at least one symptom
of the myeloid
cell-related condition. In some embodiments, said a myeloid cell-related
condition is selected
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from the group comprising cancer including but not limited to lung,
pancreatic, breast, stomach,
prostate, colon, brain and skin cancers, cancer cachexia, heart disease,
atherosclerosis, peripheral
artery disease, restenosis, stroke, bacterial infectious diseases, acquired
immune deficiency
syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory
bowel disease,
empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis,
autoimmune
diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus
erythematosus, non-
specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus
vulgaris),
granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid
granulomatosis,
Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g.,
sepsis,
inflammatory lung diseases such as interstitial pneumonitis and asthma,
inflammatory bowel
disease such as Crohn's disease, inflammatory arthritis retinopathy such as
retinopathy of
prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and
Huntington's diseases),
transplant (e.g., heart/lung transplants) rejection reactions, and other
diseases and conditions
where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid
cell-related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) the
compositions of claims 1,
3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition
to said patient
under conditions such that said at least one symptom is reduced. In some
embodiments, said a
myeloid cell-related condition is selected from the group comprising cancer
including but not
limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin
cancers, cancer
cachexia, heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke, bacterial
infectious diseases, acquired immune deficiency syndrome (AIDS), allergic
diseases, acute
radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric
ischemia,
hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid
arthritis, Sjogrens,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as
interstitial pneumonitis
and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory
arthritis
retinopathy such as retinopathy of prematurity and diabetic retinopathy,
Alzheimer's, Parkinson's
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and Huntington's diseases), transplant (e.g., heart/lung transplants)
rejection reactions, and other
diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) a labeled probe,
wherein the labeled
probe includes the compositions of claims 1, 5, 6, and 21-25 having an
affinity for TCR and an
imaging probe; b) administering said composition to said patient in a
detectably effective amount
c) imaging at least a portion of the patient; and d) detecting the labeled
probe, wherein the
location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, the invention provides a method of treating a T cell-
related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) the compositions
of claims 1, 5, 6,
and 23 capable of inhibiting TCR; b) administering said composition to said
patient under
conditions such that said at least one symptom is reduced. In some
embodiments, said a T cell-
related condition is selected from the group including but not limited to
include, but are not
limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, scleroderma,
type I diabetes, gastroenterological conditions e.g. inflammatory bowel
disease, Crohn's disease,
celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious
anaemia, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis,
allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes
simplex/zoster, respiratory
conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis,
ankylosing spondylitis,
tissue/organ rejection, and other diseases and conditions where T cells are
involved or recruited.
In another aspect, the invention provides for a method of predicting response
of the
subject to the treatment by using the modulators of TREM-1/DAP-12 signaling
pathway in
standalone or combination-therapy regimen by: (a) obtaining a biological
sample from the
subject; (b) determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or
number of
CD68-positive TAMs or a combination thereof, wherein the higher is the
expression of CSF-1,
CSF-1R, IL-6, TREM-1 or the higher is number of CD68-positive TAMs or a
combination
thereof, the better the patient is predicted to respond to a therapy that
involves the modulators.
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In some embodiments, the invention provides for a method of diagnosing cancer
in which
myeloid cells are involved or recruited in the subject and/or predicting
response of the subject to
the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in
standalone or
combination-therapy regimen by: (a) administering to said patient an amount of
at least one
modulator capable of binding TREM-1 that is conjugated to at least one imaging
probe, or a
combination thereof, in a detectably effective amount; (b) imaging at least a
portion of the
patient; (c) detecting the labeled probe, wherein the location and amount of
the labeled probe
corresponds to at least one symptom of the myeloid cell-related cancer
condition and the TREM-
1 expression levels and the higher the expression level is, the better the
patient is predicted to
respond to a therapy that involves the modulators.
The invention relates to personalized medical treatments for scleroderma
(systemic
sclerosis, SSc). More specifically, the invention provides for treatment of
scleroderma or a
related autoimmune or a fibrotic condition by using modulators of the TREM-
1/DAP-12
pathway standalone or together with other antifibrotic therapies and the use
of such combinations
in the treatment of scleroderma. In certain embodiments, these modulators may
possess the
antifibrotic activity. In some embodiments, these modulators may not possess
the antifibrotic
activity. In certain embodiments, these modulators may possess the anti-
inflammatory activity.
In one embodiment, these modulators include peptide variants and compositions
that are capable
of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or
activation). In
one embodiment these peptide variants and compositions modulate the TREM-1-
mediated
immunological responses beneficial for the treatment of scleroderma or a
related autoimmune or
a fibrotic condition. In one embodiment, the peptides and compositions of the
present invention
modulate TREM-1/DAP-12 receptor complex expressed on monocytes, macrophages
and
neutrophils. In one embodiment, the peptides and compositions of the present
invention
modulate TREM-1/DAP-12 receptor complex expressed on SSc-associated
macrophages. In one
embodiment, the invention provides a method for predicting the efficacy of
standalone or
combination-therapy treatment that involve TREM-1-targeting therapies in
scleroderma by
analyzing biological samples from cancer patients for the presence of myeloid
cells and for the
expression levels of TREM-1, CSF-1, CSF-1R, IL-6 and other markers. In one
embodiment, the
peptides and compositions of the invention are conjugated to an imaging probe.
In one
embodiment, the invention provides for detecting the TREM-1-expressing cells
and tissues in an
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individual with scleroderma using imaging techniques and the peptides and
compositions of the
invention containing an imaging probe. In one embodiment, the peptides and
compositions of the
invention are used in combinations thereof In one embodiment, the peptides and
compositions
of the invention are used in combinations with other antifibrotic therapeutic
agents. In one
embodiment, the present invention relates to the targeted treatment,
prevention and/or detection
of scleroderma including but not limited to calcinosis, Raynaud's phenomenon,
esophageal
dysmotility, scleroderma, or telangiectasia syndrome (CREST).
The invention provides for a method of treating scleroderma (S Sc) or a
related
autoimmune or a fibrotic condition in an individual in need thereof by
administering to the
individual an effective amount of an inhibitor of the TREM-1/DAP-12 pathway.
In one aspect,
the inhibitors are selected from peptide variants and compositions that
suppress tumor growth by
modulating the TREM-1/DAP-12 signaling pathway. In one embodiment, any or both
the
domains comprise minimal biologically active amino acid sequence. In one
embodiment, the
peptide variant comprises a cyclic peptide sequence. In one embodiment, the
peptide variant
comprises a disulfide-linked dimer. In one embodiment, the peptide variant
includes amino acids
selected from the group of natural and unnatural amino acids including, but
not limited to, L-
amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an
additional
therapeutic agent is conjugated to the peptide variants and compositions of
the invention. In one
embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic
resonance
imaging (MM). In one embodiment, the imaging agent is a [64Cu]-containing
imaging probe for
imaging systems such as a positron emission tomography (PET) imaging systems
(and combined
PET/computer tomography (CT) and PET/MRI systems). In one embodiment, the
peptides and
compositions of the invention are used in combinations thereof. In one
embodiment, the peptides
and compositions of the invention are used in combinations with other
antifibrotic therapeutic
agents. In certain embodiments, the peptide variants and compositions of the
present invention
are incorporated into long half-life synthetic lipopeptide particles (SLP). In
certain embodiments,
the peptide variants and compositions of the invention may incorporate into
lipopeptide particles
(LP) in vivo upon administration to the individual. In certain embodiments,
the peptides and
compositions of the invention can cross the blood-brain barrier (BBB), blood-
retinal barrier
(BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention
provides for a method
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for suppressing tumor growth in an individual in need thereof by administering
to the individual
an amount of a TREM-1 inhibitor that is effective for suppressing inflammation
and fibrosis.
Some aspects of the invention provide methods for treating scleroderma or
related
autoimmune or a fibrotic condition in a subject by administering a
therapeutically effective
amount of a TREM-1 inhibitor to the subject in need of such a treatment. In
some embodiments,
scleroderma is a systemic sclerosis, which is a systemic autoimmune disease or
systemic
connective tissue disease. SSc is often characterized by deposition of
collagen in the skin. In
some cases, SSc involves deposition of collagen in organs, such as the
kidneys, heart, lungs
and/or stomach.
In other embodiments, scleroderma is a diffuse scleroderma. Diffuse
scleroderma
typically affects the skin and organs such as the heart, lungs,
gastrointestinal tract, and kidneys.
Still in other embodiments, scleroderma is a limited scleroderma that affects
primarily the skin
including, but not limited to, that of the face, neck and distal elbows and
knees. Still in other
embodiments, scleroderma is a limited scleroderma. In some instances, the
limited scleroderma
includes clinical conditions that affect the hands, arms, and face. In other
instances, clinical
conditions associated with the limited scleroderma include, calcinosis,
Raynaud's phenomenon,
esophageal dysfunction, sclerodactyl), telangiectasias and pulmonary arterial
hypertension. Yet
in other instances, scleroderma is a localized scleroderma.
In another aspect, the invention provides for a method of predicting the
efficacy of
TREM-1 targeted therapies in an individual with scleroderma by: (a) obtaining
a biological
sample from the individual; (b) determining the number of myeloid cells in the
biological
sample; (c) determining the expression levels of TREM-1 in the cells contained
within the
biological sample.
In another aspect, the invention provides for a method of detecting TREM-1
expression
levels in an individual with scleroderma by: (a) administering to the
individual the peptide
variants and composition of the present invention having an affinity for TREM-
1 and an imaging
probe in a detectably effective amount; (b) imaging at least a portion of the
patient; (c) detecting
the labeled probe, wherein the location of the labeled probe corresponds to at
least one symptom
of the myeloid cell-related condition.
The present invention provides the compounds and compositions for TREM-1-
targeted
treatment of SSc and the methods for predicting the efficacy of these
compositions. The
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invention further provides a method of using these compounds and compositions.
These and
other objects and advantages of the invention, as well as additional inventive
features, will be
apparent from the description of the invention provided herein.
In some embodiments, the invention provides a method for treating cancer in a
patient in
need thereof by modulating immune system activity, said method comprising
administering to
said patient an amount of a TREM-1 inhibitor that is effective for inhibiting
the TREM-1/DAP-
12 signaling pathway and suppressing tumor growth, or a combination thereof In
one
embodiment, said method further comprises administering the amount of the TREM-
1 inhibitor
together with a pharmaceutically acceptable excipient, carrier, diluents, or a
combination
thereof.In one embodiment, said method further comprises administering to said
patient an
anticancer vaccine, an anticancer immunotherapy agent, anti-cancer
immunomodulatory agent,
an additional anticancer therapeutic, radiation therapy or a combination
thereof In some
embodiments, said anticancer vaccine is selected from the group consisting of
Gardasil,
Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer
immunotherapy
agent is selected from the group consisting of Alemtuzumab, Ipilimumab,
Nivolumab,
Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof
In some
embodiments, said anti-cancer immunomodulatory agent is selected from the
group consisting of
thalidomide, lenolidomide, pomalidomide, and a combination thereof In some
embodiments,
said additional anti-cancer therapeutic is selected from the group consisting
of an alkylating
agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a
CHK1 inhibitor, a
CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine,
etoposide, topotecan,
bleomycin, and mytomycin c. In some embodiments, said alkylating agent is
selected from the
group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine,
Uramustine, BuSulfan,
Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide,
Cyclophosphamide,
Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate,
Satraplatin, Nedaplatin,
Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin
inhibitor is selected
from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone,
Colchicine,
Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin
52, and IDN-
5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected
from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some
embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor
selected from the
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group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and
ellipticine. In
some embodiments, said proteasome inhibitor is selected from the group
consisting of Velcade
(bortezomib), and Kyprolis (carfilzomib). In some embodiments, said CHK1
inhibitor is selected
from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64
1397. In
some embodiments, said PARP inhibitor is selected from the group consisting of
Olaparib, ABT-
888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001,
ONO-2231
and the like.In one embodiment, said method further comprises a radiation
therapy administered
to said patient. In some embodiments, said at least one said TREM-1 inhibitor
comprises a
variant TREM-1 inhibitory peptide sequence derived from transmembrane domain
sequences of
human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some
embodiments,
said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide
variants and the
like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-
12
expression levels in a patient with cancer in need thereof, said method
comprising administering
to said patient an amount of a TREM-1 inhibitor that is conjugated to at least
one imaging probe,
or a combination thereof. In some embodiments, said an imaging probe is
selected from the
group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),
Pr(III), Nd(III)
Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III),
T1201, K42, In", Fe.59,
Tc99m, Cr51, Ga67, Ga68, CU64, Rb82,M099, DY165, Fluorescein,
Carboxyfluorescein, Calcein, F18,
Xel", 1125, 1131, 1123, p32, C11, N13, 015, Br76, Kr81,
Diatrizoate, Metrizoate, Isopaque, Ioxaglate,
Iopamidol, Iohexol, Iodixanol. In some embodiments, said TREM-1 inhibitor
comprises a
variant TREM-1 inhibitory peptide sequence derived from transmembrane domain
sequences of
human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some
embodiments,
said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide
variants and the
like.
In some embodiments, the invention provides a method for treating cancer in a
patient in need
thereof by modulating immune system activity, said method comprising
administering to said
patient an amount of a TREM-1 inhibitor that is effective for inhibiting the
TREM-1/DAP-12
signaling pathway and suppressing tumor growth, or a combination thereof. In
some
embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory
peptide sequence
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derived from transmembrane domain sequences of human or animal TREM-1 and/or
its
signaling subunit, DAP-12, thereof In some embodiments, said variant TREM-1
inhibitory
peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-
Phe,
wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine,
Lys is lysine, and Val
.. is valine. In some embodiments, said variant TREM-1 inhibitory peptide
sequence is conjugated
to at least one unmodified or modified amphipathic peptide sequence. In some
embodiments,
said unmodified or modified amphipathic peptide sequence is derived from amino
acid
sequences of apolipoproteins selected from the group consisting of A-I, A-II,
A-IV, B, C-I, C-II,
C-III, and E, and any combination thereof. In some embodiments, said modified
amphipathic
peptide sequence derived from amino acid sequences of apolipoproteins selected
from the group
consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination
thereof contains at
least one amino acid residue which is chemically or enzymatically modified. In
some
embodiments, said chemically or enzymatically modified amino acid residue is
oxidized,
halogenated or nitrated. In some embodiments, said oxidized amino acid residue
is the
methionine residue. In some embodiments, said unmodified amphipathic peptide
sequence is
derived from an apolipoprotein A-I amino acid sequence and comprises amino
acid sequence
Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-
Lys-Val-
Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine,
Phe is
phenylalanine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is
glutamic acid, Met is
.. methionine, Arg is arginine, and Val is valine. In some embodiments, said
unmodified
amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid
sequence and
comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-
His-Val-
Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly
is glycine, Glu is
glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is
alanine, His is
histidine, Val is valine, and Thr is threonine. In some embodiments, said
modified amphipathic
peptide sequence is derived from an apolipoprotein A-I amino acid sequence and
comprises
amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-
Glu-
Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is
leucine, Asp,
asparagine, Phe is phenylalanine, Gln is glutamine, Lys is lysine, Trp is
tryptophan, Glu is
glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is
valine. In some
embodiments, said modified amphipathic peptide sequence is derived from an
apolipoprotein A-I
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amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-
Met(0)-Arg-
Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is
proline, Leu
is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide,
Arg is arginine,
Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is
threonine. In some
embodiments, said B is conjugated to an additional peptide sequence to enhance
the targeting
efficacy. In some embodiments, said an additional peptide sequence comprises
amino acid
sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp
is asparagine. In
some embodiments, said A is conjugated to at least one additional therapeutic
agent to enhance
the therapeutic efficacy. In some embodiments, said an additional therapeutic
agent is selected
from the group of anticancer, antibacterial, antiviral, autoimmune, anti-
inflammatory and
cardiovascular agents, antioxidants, therapeutic peptides, and any combination
thereof In some
embodiments, said anticancer therapeutic agent is selected from the group
comprising paclitaxel,
valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination
thereof In some
embodiments, said A and/or B are conjugated to at least one imaging probe. In
some
embodiments, said an imaging probe is selected from the group comprising
Gd(III), Mn(II),
Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III),
Yb(III) Dy(III), Ho(III),
Eu(II), Eu(III), and Er(III), T1201, K42, In Fe.59, TC99M, cr51
, Ga67, Ga68, cu64
, Rb82,m099,
DY165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123,
P32, Cll, N13, 015, Br76,
Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol,
Iodixanol.
In some embodiments, the invention provides a method of making a synthetic
lipopeptide
nanoparticle, said method comprising: a) co-dissolving a predetermined amount
of a mixture of
neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a
predetermined amount of triglycerides and/or cholesteryl ester; b) drying the
mixture of step (a)
under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a
predetermined amount of
sodium cholate; and ii. a predetermined amount of the compound of claim 1; for
a time period
sufficient to allow the components to self-assemble into synthetic lipopeptide
particles; d)
removing sodium cholate from the mixture of step (c); and e) isolating
particles that have a size
of between about 5 to about 200 nm diameter. In some embodiments, said lipid
is conjugated to
at least one imaging probe. In some embodiments, said imaging probe is
selected from the group
comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),
Pr(III), Nd(III) Sm(III),
Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42,
In Fe.59, TC99M, Cr51,
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Ga67, Ga68, CU64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein,
F18, Xe133, 1125,
1131, 1123, P32, C11, N13, 015, Br76, Kr81,
Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol,
Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group
comprising
cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a
sphingolipid, a cationic lipid, a
diacylglycerol, and a triacylglycerol. In some embodiments, said phospholipid
is selected from
the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG),
cardiolipin (CL),
sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In
some
embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) a labeled
probe, wherein the
labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an
affinity for
TREM-1 and an imaging probe; b) administering said composition to said patient
in a detectably
effective amount c) imaging at least a portion of the patient; and d)
detecting the labeled probe,
wherein the location of the labeled probe corresponds to at least one symptom
of the myeloid
cell-related condition. In some embodiments, said myeloid cell-related
condition is selected from
the group comprising cancer including but not limited to lung, pancreatic,
breast, stomach,
prostate, colon, brain and skin cancers, cancer cachexia, heart disease,
atherosclerosis, peripheral
artery disease, restenosis, stroke, bacterial infectious diseases, acquired
immune deficiency
syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory
bowel disease,
empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis,
autoimmune
diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus
erythematosus, non-
specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus
vulgaris),
granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid
granulomatosis,
Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g.,
sepsis,
inflammatory lung diseases such as interstitial pneumonitis and asthma,
inflammatory bowel
disease such as Crohn's disease, inflammatory arthritis retinopathy such as
retinopathy of
prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and
Huntington's diseases),
transplant (e.g., heart/lung transplants) rejection reactions, and other
diseases and conditions
where myeloid cells are involved or recruited.
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In some embodiments, the invention provides a method of treating a myeloid
cell-related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which myeloid cells are involved or recruited, and ii) the
compositions of claims 1,
3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition
to said patient
under conditions such that said at least one symptom is reduced. In some
embodiments, said
myeloid cell-related condition is selected from the group comprising cancer
including but not
limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin
cancers, cancer
cachexia, heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke, bacterial
infectious diseases, acquired immune deficiency syndrome (AIDS), allergic
diseases, acute
radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric
ischemia,
hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid
arthritis, Sjogrens,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as
interstitial pneumonitis
and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory
arthritis
retinopathy such as retinopathy of prematurity and diabetic retinopathy,
Alzheimer's, Parkinson's
and Huntington's diseases), transplant (e.g., heart/lung transplants)
rejection reactions, and other
diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-
related
condition, comprising a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) a labeled probe,
wherein the labeled
probe includes the compositions of claims 1, 5, 6, and 21-25 having an
affinity for TCR and an
imaging probe; b) administering said composition to said patient in a
detectably effective amount
c) imaging at least a portion of the patient; and d) detecting the labeled
probe, wherein the
location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group
including but not
limited to include, but are not limited to, systemic lupus erythematosus,
rheumatoid arthritis,
multiple sclerosis, scleroderma, type I diabetes, gastroenterological
conditions e.g. inflammatory
bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome,
Hashimoto's disease,
pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin
problems e.g. atopic
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dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis,
allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis,
AIDS virus, herpes
simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory
conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases
and conditions where
T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-
related
condition, comprising: a) providing; i) a patient having at least one symptom
of a disease or
condition in which T cells are involved or recruited, and ii) the compositions
of claims 1, 5, 6,
and 23 capable of inhibiting TCR; b) administering said composition to said
patient under
conditions such that said at least one symptom is reduced. In some
embodiments, said T cell-
related condition is selected from the group including but not limited to
include, but are not
limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, scleroderma,
type I diabetes, gastroenterological conditions e.g. inflammatory bowel
disease, Crohn's disease,
celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious
anaemia, primary
biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis,
allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes
simplex/zoster, respiratory
conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis,
ankylosing spondylitis,
tissue/organ rejection, and other diseases and conditions where T cells are
involved or recruited.
The details of one or more embodiments of the invention are set forth in the
accompanying Figures (Drawings) and Detailed Description of The Invention, as
described
herein and below. Other features, objects, and advantages of the invention
will be apparent from
the summary, description, figures and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following figures form part of the present specification and are included
to further
illustrate embodiments of the present invention. The invention may be better
understood by
reference to the figures in combination with the detailed description of the
specific embodiments
presented herein.
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The patent or application file contains at least one drawing executed in
color. Copies of
this patent or patent application publication with color drawing(s) will be
provided by the Office
upon request and payment of the necessary fee.
FIG. 1 presents an exemplary schematic representation of one embodiment of a
trifunctional
peptide of the present invention comprising amino acid domains A and B where
amino acid
domain A represents a therapeutic peptide sequence with or without an attached
drug compound
and/or imaging probe that functions to treat, prevent and/or detect a disease
or condition,
whereas amino acid domain B represents an amphipathic alpha helical peptide
sequence, with or
without an additional targeting peptide sequence, and functions to 1) assist
in the self-assembly
of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with
lipids or lipid
mixtures in vitro, for use in transporting these trifunctional peptides as
lipoprotein nanoparticles
to sites of interest in vitro or in vivo and/or 2) form long half-life
lipopeptide/lipoprotein particles
upon interaction with endogenous lipoproteins for transporting these
trifunctional peptides to the
sites of interest. Endogenous lipoproteins may be lipoproteins added to or
found in cell cultures,
or lipoproteins in a mammalian body.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related
trifunctional
peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE,
M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino
acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and
functions to
treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22
amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a
sulfoxidized
methionine residue and functions to assist in the self-assembly of synthetic
lipopeptide particles
(SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
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FIG. 3 presents a schematic representation of one embodiment of a TREM-1-
related
trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising
amino acid
domains A and B. Depending on lipid mixture compositions added to the
peptides, sub 50 nm-
sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-
1/TRIOPEP-
sSLP) morphology are self-assembled upon binding of the trifunctional peptide
to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
FIG. 4A illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic
peptide
sequence and functions to treat and/or prevent a TREM-1-related disease or
condition (shown for
atherosclerosis), whereas domain B represents a 22 amino acids-long
apolipoprotein A-I helix 4
or 6 peptide sequence with a sulfoxidized methionine residue and functions to
assist in the self-
assembly of synthetic lipopeptide particles (SLP) and to target the particles
to TREM-1-
expressing macrophages as applied to the treatment and/or prevention of
atherosclerosis. While
not being bound to any particular theory, it is believed that chemical and/or
enzymatic
modification of protein sequence in domain B leads to the recognition of SLP
of the present
invention by the macrophage scavenger receptors and results in an irreversible
binding to and
consequent uptake by macrophages of such particles. It is further believed
that accumulation of
these particles in intraplaque macrophages is accompanied by accumulation of
TRIOPEP in
these cells. In contrast, native HDL particles that contain only unmodified
apolipoprotein
molecules are not recognized by intraplaque macrophages and return to the
circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide
sequence and
functions to treat and/or prevent a TREM-1-related disease or condition (shown
for cancer),
whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6
peptide sequence
with a sulfoxidized methionine residue and functions to assist in the self-
assembly of synthetic
lipopeptide particles (SLP) and to target the particles to TREM-1-expressing
macrophages as
applied to the treatment and/or prevention of cancer. While not being bound to
any particular
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theory, it is believed that chemical and/or enzymatic modification of protein
sequence in domain
B leads to the recognition of SLP of the present invention by the macrophage
scavenger
receptors and results in an irreversible binding to and consequent uptake by
macrophages of such
particles. It is further believed that accumulation of these particles in
tumor-associated
macrophages is accompanied by accumulation of TRIOPEP in these cells. In
contrast, native
HDL particles that contain only unmodified apolipoprotein molecules are not
recognized by
tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane
interactions
between TREM-1 and DAP-12 by the trifunctional peptide of the present
invention comprising
two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1
inhibitory
therapeutic peptide sequence, whereas domain B is a 22 amino acids-long
apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While
not being bound to
any particular theory, it is believed that this disruption results in "pre-
dissociation" of a receptor
complex and upon ligand stimulation, leads to inhibition of TREM-1 and
silencing the TREM-1
signaling pathway.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine
residue-
containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with
TREM-1 in
the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and
a merged
image (FIG. 6C).
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic
lipopeptide
particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that
contain an equimolar
mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31
and GE 31
(TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The
post 4 h
incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-
1/TRIOPEP-
sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine
residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified
methionine
residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and
TREM-
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1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4
(white
bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001
to 0.001 as
compared with 4 h incubation time.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-
stimulated
macrophages incubated for 24 hour (hr) at 37 C with an equimolar mixture of
the sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles
(SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged
macrophages.
FIG. 9A-C presents the exemplary data showing that scavenger receptors SR-A
and SR-B1
mediate the macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-
1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and
the SCHOOL
mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-
1/DAP12
receptor complex expressed on macrophages leads to phosphorylation of the
DAP12 cytoplasmic
signaling domain and subsequent downstream inflammatory cytokine response
(left panel). SR-
mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by
macrophages
results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-
penetrate into the
cell membrane and block intramembrane interactions between TREM-1 and DAP12,
thereby
preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1,
right panel).
(FIG. 9A2, left panel) Activation of the TREM-1/DAP12 receptor complex
expressed on
Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling
domain, subsequent
SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2,
right panel)
SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells
results in the
release of GF9 (GA31 or GE31) into the cytoplasm; GF9 self-penetrates the cell
membrane and
blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing
DAP12
phosphorylation and the downstream signaling cascade.
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FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-
1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is
largely driven by
SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774
macrophages were
cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-
HDL, cells
were treated for 1 hour at 37 C with 40 i.tM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 i.tM BLT-1, as indicated. Cells were then incubated
for either 4 hours
or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars)
or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence
intensities of lysates were measured and normalized to the protein content.
Results are expressed
as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of
GF9-HDL and
GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation
protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL,
signaling chain
homo-oligomerization.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post
lipopolysaccharide
(LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline
(PBS),
dexamethasone (DEX), control peptide and with an equimolar mixture of the
sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
.. 31 and GE 31 in free form or incorporated into synthetic lipopeptide
particles (SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
Control peptide represents an equimolar mixture of two peptides, each of them
comprising two
amino acid domains A and B where domain A represents a non-functional 9 amino
acids-long
sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5
is substituted
with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22
amino acids-
long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as
compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as
compared with PBS-treated animals.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice
treated with an
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equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX,
paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into
synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared
with
vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549
xenograft mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form
or
incorporated into synthetic lipopeptide particles (SLP) particles of discoidal
(TREM-
1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX,
paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth
(FIG. 14A) and
TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration
(FIG. 14B,
FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an
equimolar
mixture of the sulfoxidized methionine residue-containing TREM-1-related
trifunctional peptides
(TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic
lipopeptide
particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical
(TREM-
1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the
mean SEM (n
= 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus
vehicle). (FIG. 14C)
Representative F4/80 images from BxPC-3-bearing mice treated using different
free and sSLP-
bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale
bar = 200 p.m.
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FIG. 15A-B presents the exemplary data showing improved survival of
lipopolysaccharide
(LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized
methionine
residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide
particles (SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6
mice treated with
increasing concentrations of an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in
free form.
FIG. 17A-B presents the exemplary data showing average clinical arthritis
score (FIG. 17A) and
mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the
difference
.. between beginning (day 24) and final (day 38) BWs of the collagen-induced
arthritis (CIA) mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated
into synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p
<0.001 as compared with vehicle-treated or naive animals.
FIG. 18A-D presents the exemplary data showing reduction of pathological
retinal
neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-
1 (FIG. 18C)
and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-
induced
.. retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP)
particles of spherical
morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-
treated
animals.
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FIG. 19 presents exemplary data showing penetration of the blood-brain barrier
(BBB) and
blood-retinal barrier (BRB) by systemically (intraperitoneally) administered
rhodamine B-
labeled spherical self-assembled particles (sSLP) that contain Gd-containing
contrast agent (Gd-
sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-
sSLP) or
-- an equimolar mixture of the sulfoxidized methionine residue-containing TREM-
1-related
trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the
expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and
FIG. 20B a-
-- Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates
of mice with
(alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF)
group; # indicates
significance level compared to the non-treated alcohol-fed group. o indicates
significance level
compared to the vehicle-treated alcohol-fed group. The significant levels are
as follows: *, 0.05
> P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses
the
production of alanine aminotransferase (ALT) in mice with alcoholic liver
disease (ALD), as
measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in
addition to
improving indicators of liver disease and inflammation. * indicates
significance level compared
to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide
particles of spherical
morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no
TREM-1
inhibitory peptide GF9. # indicates significance level compared to the non-
treated alcohol-fed
group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1
pathway inhibition
in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-
1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A)
Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-
1/TRIOPEP-
sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over
TREM-1 peptide
alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
-- staining, and the lipid content was analyzed by ImageJ (FIG. 21B). *
indicates significance level
compared to the nontreated PF group; * indicates significance level compared
to the nontreated
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alcohol-fed group; indicates significance level compared to the vehicle-
treated alcohol-fed
group. The numbers of the symbols sign the significant levels as the
following: **OP < 0.05;
WooP < 0.01;*"/###P <0001; ****P < 0 .0001. *** , 0.001 > P> 0.0001; ##, 0.01
> P> 0.001.
FIG. 22 presents an exemplary schematic representation of one embodiment of a
TREM-1-
related trifunctional peptide (TREM-1/TRIOPEP) G-HV21 of the present invention
comprising
amino acid domains A and B where domain A represents a 9 amino acids-long
human TREM-1
inhibitory therapeutic peptide sequence GF9 and functions to treat and/or
prevent a TREM-1-
related disease or condition, whereas domain B represents a 12 amino acids-
long amino acid
sequence GV12 that contains a sulfoxidized methionine residue and is derived
from human
apolipoprotein A-I amino acid sequence. While not being bound to any
particular theory, it is
believed that a resulting amphipathic alpha helical peptide G-HV21 upon
interaction with native
lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles
and targets these
particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1,
triggering receptor
expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 23 presents an exemplary schematic representation of one embodiment of a
TREM-1-
related trifunctional peptide (TREM-1/TRIOPEP) G-KV21 of the present invention
comprising
amino acid domains A and B where domain A represents a 9 amino acids-long
human TREM-1
inhibitory therapeutic peptide sequence GF9 and functions to treat and/or
prevent a TREM-1-
related disease or condition, whereas domain B represents a 12 amino acids-
long amino acid
sequence WV12 that contains a sulfoxidized methionine residue and is derived
from human
apolipoprotein A-I amino acid sequence. While not being bound to any
particular theory, it is
believed that a resulting amphipathic alpha helical peptide G-KV21 upon
interaction with native
lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles
and targets these
particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1,
triggering receptor
expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 24 presents an exemplary schematic representation of one embodiment of a
TREM-1-
related control peptide G-TE21 of the present invention comprising amino acid
domains A and B
where domain A represents a 9 amino acids-long human TREM-1 inhibitory
therapeutic peptide
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sequence GF9, whereas domain B represents a 12 amino acids-long amino acid
sequence TE12
that contains a sulfoxidized methionine residue and is derived from bovine
serum albumin amino
acid sequence. While not being bound to any particular theory, it is believed
that a resulting non-
amphipathic peptide G-TE21 does not interact with native lipoproteins and
therefore does not
form naturally long half-life lipopeptide/lipoprotein particles.
Abbreviations: TREM-1,
triggering receptor expressed on myeloid cells-1.
FIG. 25 presents an exemplary schematic representation of one embodiment of a
TCR-related
trifunctional peptide (TCR/TRIOPEP) M-VE32 of the present invention comprising
amino acid
domains A and B where domain A represents a 10 amino acids-long human TCR
inhibitory
therapeutic peptide sequence MF10 and functions to treat and/or prevent a TCR-
related disease
or condition, whereas domain B represents a 22 amino acids-long amino acid
sequence PE22 that
is derived from human apolipoprotein A-I amino acid sequence. While not being
bound to any
particular theory, it is believed that a resulting amphipathic alpha helical
peptide M-VE32 upon
-- interaction with native lipoproteins, forms naturally long half-life
lipopeptide/lipoprotein
particles. Abbreviations: TCR, T cell receptor; TRIOPEP, trifunctional
peptide.
FIG. 26 presents a schematic representation of one embodiment of a TCR-related
control peptide
M-TK32 of the present invention comprising amino acid domains A and B where
domain A
represents a 10 amino acids-long human TCR inhibitory therapeutic peptide
sequence MF10,
whereas domain B represents a random 22 amino acids-long amino acid sequence
LK22. While
not being bound to any particular theory, it is believed that a resulting non-
amphipathic peptide
M-TK32 does not interact with native lipoproteins and therefore does not form
naturally long
half-life lipopeptide/lipoprotein particles. Abbreviations: TCR, T cell
receptor.
FIG. 27 presents an exemplary schematic representation and the exemplary data
showing that
ultracentrifugation of whole mouse serum with added rho B-labeled TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) G-HV21 and G-KV21 results in
floatation of these
peptides with mouse lipoproteins. In contrast, when added to whole mouse
serum, rho B-labeled
TREM inhibitory peptide GF9 or rho B-labeled TREM-1-related control peptide G-
TE21
sedimentate with serum proteins upon ultracentrifugation. When added to
delipoproteinized
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mouse serum that does not contain lipoproteins, rho B-labeled TREM-1/TRIOPEP G-
HV21 and
G-KV21 sedimentate with serum proteins upon ultracentrifugation. While not
being bound to
any particular theory, it is believed that TREM-1/TRIOPEP G-HV21 and G-KV21
interact with
native lipoproteins of a whole mouse serum and/or their lipid components and
form
lipopeptide/lipoprotein particles that mimic serum lipoproteins and float
under the same
ultracentrifugation conditions. Abbreviations: TREM-1, triggering receptor
expressed on
myeloid cells-1; rho B, rhodamine B.
FIG. 28 presents exemplary data showing the endocytosis of rho B-labeled GF9,
G-TE21, G-
HV-21 and G-KV21 by macrophages in the absence (white bars) or presence (black
bars) of
HDL. In contrast to GF9 and TREM-1-related control peptide G-TE21, the in
vitro macrophage
uptake of TREM-1/TRIOPEP G-HV21 and G-KV21 significantly increases in the
presence of
HDL. ***,p < 0.001 (presence vs. absence of HDL). Abbreviations: HDL, high
density
lipoproteins; rho B, rhodamine B; n.s., not significant.
FIG. 29A-C shows exemplary images depicting colocalization of the sulfoxidized
methionine
residue-containing TREM-1/TRIOPEP G-KV21 (pre-incubated with HDL) with TREM-1
in the
J774 cell membrane FIG. 29A. FIG. 29B TREM-1 immunostaining. FIG. 29C merged
image.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL,
high density
-- lipoproteins.
FIG. 30A illustrates a hypothesized molecular mechanism of action of TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for
atherosclerosis).
While not being bound to any particular theory, it is believed that upon
interaction with native
lipoproteins including HDL, the modified methionine residue in the TREM-
1/TRIOPEP domain
B mediates the recognition of the formed lipopeptide/lipoprotein particles by
macrophage
scavenger receptors and results in an irreversible binding to and consequent
uptake by
macrophages of such particles. It is further believed that accumulation of
these particles in
intraplaque macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released within
these cells. In contrast, native HDL particles are not recognized by
intraplaque macrophages and
return to the circulation.
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FIG. 30B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-
1; HDL,
high-density lipoproteins.
FIG. 31A illustrates a hypothesized molecular mechanism of action of TREM-1-
related
-- trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for
cancer). While
not being bound to any particular theory, it is believed that upon intreaction
with native
lipoproteins including HDL, the modified methionine residue in the TREM-
1/TRIOPEP domain
B mediates the recognition of the formed lipopeptide/lipoprotein particles by
macrophage
scavenger receptors and results in an irreversible binding to and consequent
uptake by
-- macrophages of such particles. It is further believed that accumulation of
these particles in
tumor-associated macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released
within these cells. In contrast, native HDL particles are not recognized by
intraplaque
macrophages and return to the circulation.
FIG. 31B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-
1; HDL,
-- high-density lipoproteins.
FIG. 32 illustrates one embodiment of a specific disruption of intramembrane
interactions
between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide (TREM-
1/TRIOPEP)
of the present invention delivered to and released within TREM-1-expressing
cells by the
-- lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native
lipoproteins. While not being bound to any particular theory, it is believed
that this disruption
results in "pre-dissociation" of a receptor complex and upon ligand
stimulation, leads to
inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
Abbreviations: TREM-1,
triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-activation
protein 12; M-
-- CSF/CSF-1, macrophage colony stimulating factor-1; MCP-1/CCL2, monocyte
chemoattractant
protein-1; IL, interleukin; TNF, tumor necrosis factor.
FIG. 33 presents exemplary data showing cytokine production by LPS-stimulated
macrophages
incubated for 24 h at 37 C with GF9, G-TE21, G-HV21 and G-KV21 in the presence
of HDL. In
-- contrast to GF9 and TREM-1-related control peptide G-TE21, TREM-1/TRIOPEP G-
HV21 and
G-KV21 significantly inhibit the cytokine release in the presence of HDL. In
the absence of
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HDL, G-HV21 does not affect the cytokine production. ***,p <0.001 (vs. medium
+ HDL).
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS,
lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; HDL, high
density lipoproteins.
FIG. 34A-C presents exemplary LPS-challenged J774 macrophages: Cytokine
release data
showing that scavenger receptors SR-A and SR-B1 mediate the macrophage
endocytosis of
TREM-1/TRIOPEP G-HV21 and G-KV21 in the presence of HDL. (FIG. 34A) Schematic
representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1
blockade.
Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads
to
phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent
downstream
inflammatory cytokine response (left panel). SR-mediated macrophage
endocytosis of the
lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native
lipoproteins (shown for HDL) results in the release of TREM-1/TRIOPEP into the
cytoplasm.
Then, the released TREM-1/TRIOPEP self-penetrate into the cell membrane and
block
intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12
phosphorylation and downstream signaling cascade (FIG. 34A, right panel).
Macrophage
endocytosis of G-HV21 and G-KV21 in the presence of HDL in vitro is SR-
mediated in a time-
dependent manner and is largely driven by SR-A (B, C). J774 macrophages were
cultured at
37 C overnight with medium. Before adding G-HV21 and G-KV21, cells were
treated for 1 h at
37 C with 40 [tM cytochalasin D, 400 [tg/mL fucoidan (FIG. 34B) or 10 [tM BLT-
1 (FIG. 34C)
as indicated. Cells were then incubated for either 4 h or 22 h with medium
containing HDL and 2
[tM rho B-labeled G-KV21 (gray bars) or G-HV21 (black bars), respectively.
Cells were lysed
and rho B fluorescence intensities of lysates were measured and normalized to
the protein
content. Results are expressed as the mean SEM (n = 3). *,p < 0.05; **,p <
0.01; ****, p <
0.0001 versus uptake of G-HV21 and G-KV21 in the absence of inhibitor.
Abbreviations:
TREM-1, triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-
activation protein
12; M-CSF/CSF-1, macrophage colony stimulating factor; MCP-1/CCL2, monocyte
chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor; HDL,
high density
lipoproteins; BLT-1, blocker of lipid transport-1; rho B, rhodamine B; SR,
scavenger receptor.
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FIG. 35 presents exemplary data showing serum cytokine production at 90 min
post LPS
challenge in mice treated at 1 h before LPS challenge with PBS, DEX, GF9, TREM-
1-related
control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21
and G-KV21. In
contrast to GF9 and G-TE21, G-HV21 and G-KV21 significantly inhibit the LPS-
induced
cytokine release. ***,p < 0.001 as compared with PBS-treated animals.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS,
lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; DEX,
dexamethasone; PBS,
phosphate-buffer saline.
FIG. 36A-B presents the exemplary data showing survival of LPS-challenged mice
treated with
PBS (vehicle), TREM-1-related control peptide G-TE21, TREM-1-related
trifunctional peptides
G-HV21 and G-KV21 (FIG. 36A) or with TREM-1 inhibitory peptide GF9 at
different doses
(FIG. 36B). In contrast to G-TE21, G-HV21 and G-KV21 significantly improve
survival of
septic mice (FIG. 36A). When administered at a dose of 5 mg/kg, GF9 does not
affect survival of
septic mice, while at 25 mg/g, GF9 improves survival. In contrast, high dose
of GF9, 150 mg/kg,
contributes to earlier death as compared with control animals treated with
vehicle only (FIG.
36B). **,p < 0.01 as compared with vehicle-treated animals. Abbreviations:
TREM-1, triggering
receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; PBS, phosphate-
buffer saline.
FIG. 37A-B presents the exemplary data showing tumor growth in the human non-
small cell
lung cancer H292 mouse xenograft (FIG. 37A) and A549 mouse xenograft (FIG.
37B) xenograft
mice treated with PBS (vehicle), PTX, TREM-1-related control peptide G-TE21 or
with TREM-
1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-
HV21 and G-
KV21 significantly inhibit the tumor growth. ****,p < 0.0001 as compared with
vehicle-treated
animals. Abbreviations: PTX, paclitaxel; PBS, phosphate-buffer saline.
FIG. 38 presents exemplary A549 mouse xenograft data showing average tumor
weights in the
A549 xenograft mice treated with PBS (vehicle), PTX, TREM-1-related control
peptide G-TE21
or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast
to G-TE21, G-
HV21 and G-KV21 significantly decrease the tumor weight. **,p < 0.01 as
compared with
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vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed
on myeloid cells-
1; PTX, paclitaxel; PBS, phosphate-buffer saline; n.s., not significant.
FIG. 39A-B presents exemplary data showing tumor growth (A) and,infiltration
of macrophages
into the tumor as evaluated by F4/80 staining (B) in the human pancreatic
cancer BxPC-3
xenograft mice treated with PBS (vehicle), TREM-1-related control peptide G-
TE21 or with
TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-
TE21, G-HV21
and G-KV21 in a BxPC-3 mouse xenograft significantly inhibits the tumor growth
(FIG. 389)
and reduce macrophage infiltration into the tumor (FIG. 39B). **,p < 0.01,
****,p < 0.0001
(versus vehicle). Abbreviations: TREM-1, triggering receptor expressed on
myeloid cells-1;
PBS, phosphate-buffer saline; n.s., not significant.
FIG. 40A-B presents exemplary data showing PANC-1 mouse xenograft tumor growth
(FIG.
40A) and survival (FIG. 40B) in the human pancreatic cancer PANC-1 xenograft
mice treated
with PBS (vehicle) and TREM-1-related trifunctional peptide G-KV21 with or
without
chemotherapy treatment (GEM+ABX). G-KV21 sensitizes the tumor to chemotherapy
(FIG.
40A) and significantly improves survival (FIG. 40B). The median survival times
(FIG. 40B) are
indicated in parentheses. Abbreviations: TREM-1, triggering receptor expressed
on myeloid
cells-1; PBS, phosphate-buffer saline; GEM, gemcitabine; ABX, Abraxane
(nanoparticle
albumin-bound paclitaxel).
FIG. 41 presents the exemplary data showing average weights of Healthy C57BL/6
mice treated
with TREM-1-related control peptide G-TE21 or with TREM-1-related
trifunctional peptides G-
HV21 and G-KV21. No toxicity was observed for all three peptides.
Abbreviations: TREM-1,
triggering receptor expressed on myeloid cells-1.
FIG. 42A-B presents exemplary data showing average clinical arthritis score
(Collagen-induced
arthritis: Score FIG. 42A) and Collagen-induced arthritis: Body weight change
mean BW
changes (FIG. 42B) calculated as a percentage of the difference between
beginning (day 24) and
final (day 38) BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-
related control
peptide G-TE21, TCR-related control peptide M-TK32, TCR-related trifunctional
peptide M-
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VE32 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In
contrast to the
relevant control peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the
disease (FIG. 42A)
and are well-tolerated by arthritic mice (FIG. 42B). *,p < 0.05, **,p < 0.01;
***,p < 0.001 as
compared with vehicle-treated animals. Abbreviations: TREM-1, triggering
receptor expressed
on myeloid cells-1; CIA, collagen-induced arthritis; PBS, phosphate-buffer
saline; DEX,
dexamethasone; TCR, T cell receptor; BW, body weight.
FIG. 43A-D Oxygen-induced retinopathy presents exemplary data showing
pathological RNV
(FIG. 43A) and avascular (FIG. 43B) areas as well as expression of TREM-1
(FIG. 43C) and M-
CSF (FIG. 43D) in the retina of the mice with OIR treated with PBS (vehicle),
TREM-1-related
control peptide G-TE21 or TREM-1-related trifunctional peptide G-KV21. In
contrast to G-
TE21, G-KV21 significantly suppresses pathological RNV and inhibits tissue
expression of
TREM-1 and M-CSF. *,p < 0.05, **,p < 0.01; ***,p < 0.001 as compared with
vehicle-treated
animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-
1; OIR,
oxygen-induced retinopathy; PBS, phosphate-buffer saline; M-CSF, macrophage
colony
stimulating factor; RNV, retinal neovascularization.
FIG. 44 presents exemplary data showing penetration of the BBB and BRB by
systemically
(mice ¨ intraperitoneally; rats and rabbits ¨ intravenously) administered
rhodamine B-labeled
TREM-1-related trifunctional peptide G-KV21. Abbreviations: TREM-1, triggering
receptor
expressed on myeloid cells-1; BBB, blood-brain barrier; BRB, blood-retinal
barrier.
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the
expression of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1,
(FIG. 45C)
TNF-a, (FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (FIG. 45F) RANTES at
the mRNA
level as measured in whole-liver lysates by real-time quantitative PCR. *
indicates significance
level compared to nontreated PF group; # indicates significance level compared
to nontreated
alcohol-fed group; o indicates significance level compared to vehicle-treated
alcohol-fed group.
Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P < 0.01; ***
/000 P < 0.001;
****P < 0.0001. Abbreviation: CCL, chemokine (C-C motif) ligand.
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FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade
reduces
inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver
lysates as
measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was
analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as
a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance
level compared to the nontreated PF group; # indicates significance level
compared to the
nontreated alcohol-fed group; o indicates significance level compared to the
vehicle-treated
alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** /<figref></figref> P < 0.0001.
FIG. 47A-H Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1
inhibition
suppresses the mRNA expression of macrophage cell markers in the liver as
measured by real-
time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated
F4/80 as
shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of
neutrophil cell markers in the liver as measured by real-time quantitative
PCR. (FIG. 47G, H)
Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by
IHC. * indicates
significance level compared to the nontreated PF group; # indicates
significance level compared
to the nontreated alcohol-fed group; o indicates significance level compared
to the vehicle-
treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
.. ### P < 0.001; **** /<figref></figref> P < 0.0001.
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved
in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid
accumulation marker
(ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in
whole
liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level
compared to the nontreated alcohol-fed group; o indicates significance level
compared to the
vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o
P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
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FIG. 49A presents a schematic representation of one embodiment of the proposed
role of
inhibition of TREM-1 expressed on tumor-associated macrophages (TAMs) in
pancreatic cancer.
Pancreatic ductal adenocarcinoma cells, cancer-associated fibroblasts (CAFs)
and TAMs play a
role in generating a tumor favorable microenvironment, in part by producing
such cytokines and
growth factors as interleukin (IL)-1a, IL-6 and macrophage colony-stimulating
factor (M-CSF).
FIG. 49B presents a schematic representation of one embodiment of suppressing
tumor
favorable microenvironment by inhibition of TREM-1 expressed on tumor-
associated
macrophages (TAMs) and reduction of cytokines and growth factors including but
not limited to
interleukin (IL)-6, IL-1, monocyte chemoattractant protein-1 (MCP-1; also
referred to in the art
as CCL2) and macrophage colony-stimulating factor 1 (CSF-1; also referred to
in the art as M-
CSF). These prognostic factors are involved in tumorigenesis, cancer
progression, metastasis,
and even in the response to cancer treatment. The figure further presents a
schematic
representation of one embodiment of modulating the TREM-1/DAP-12 signaling
pathway by
type I TREM-1 inhibitors that bind either TREM-1 (type Ia inhibitors; e.g.,
anti-TREM-1
blocking antibodiesõ etc.) or its ligand (type Ib inhibitors; e.g., inhibitory
peptides LP17 and
LR12 that act as a decoy TREM-1 receptor), thereby blocking binding between
TREM-1 and its
yet uncertain ligand(s).
FIG. 50 presents a schematic representation of one embodiment of TREM-1
modulatory peptide
variants and compositions of the present invention that are rationally
designed using the
Signaling Chain HOmoOLigomerization (SCHOOL approach) to inhibit TREM-1 in a
ligand-
independent manner by blocking intramembrane interactions between TREM-1 and
its signaling
partner DAP-12 (type II inhibitors). These SCHOOL peptides can be employed in
either free
form or incorporated into macrophage-targeted (macrophage-specific) synthetic
lipopeptide
particles (SLP), which allows them to reach their site of action from either
outside (Route 1) or
inside the cell (Route 2).
FIG. 51A-F shows images of one embodiment depicting colocalization of the TREM-
1
modulatory peptide GF9 (GFLSKSLVF) with trifunctional TREM-1 in the cell
membrane. Fig.
51A shows exemplary peptide GF9. Fig. 51B and 51E shows exemplary TREM-1. Fig.
51C and
F shows exemplary merged Images. Fig. 51A shows exemplary inhibitory peptide
GE31
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((GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue with TREM-1 in the cell membrane.
FIG. 51B shows images of one embodiment depicting colocalization of the TREM-1
modulatory
peptide GF9 (GFLSKSLVF) and trifunctional TREM-1
FIG. 52 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a
carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP)
morphology
suppresses tumor growth in experimental pancreatic cancer. As described
herein, after tumors in
AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice
were
randomized into groups and intraperitoneally (i.p.) administered once daily 5
times per week
(5qw) with either vehicle (black diamonds), GF9 (dark gray squares), GF9-
loaded discoidal SLP
(GF9-dSLP, light gray circles) or GF9-loaded spherical SLP (GF9-sSLP, white
circles) at
indicated doses. Treatment persisted for 31, 29 and 29 days for mice
containing AsPC-1, BxPC-3
and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated
and plotted. All
results are expressed as the mean SEM (n = 6 mice per group). On the final
day of treatment,
tumor volumes were compared between the drug-treated and control groups. **, p
< 0.01; ***, p
<0.001; ****, p < 0.0001 (versus vehicle).
FIG. 53 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a
carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP)
morphology (GF9-
dSLP and GF9-sSLP, respectively) suppresses tumor growth in experimental
pancreatic cancer
without affecting body weight (well-tolerable in long term-treated mice). As
described herein,
after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of
150-200 mm3,
mice were randomized into groups and intraperitoneally (i.p.) administered
once daily 5 times
per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares),
GF9-dSLP (light
gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment
persisted for 31, 29 and
29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts,
respectively. Body
weighs are plotted. All results are expressed as the mean SEM (n = 6 mice
per group).
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FIG. 54 presents the exemplary data of one embodiment showing that treatment
with synthetic
lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology
loaded with an
equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide
residue and the 31 amino acids-long TREM-1 modulatory peptide GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in
experimental pancreatic cancer. As described herein, after tumors in AsPC-1-,
BxPC-3- or
Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized
into groups
and intraperitoneally (i.p.) administered once daily 5 times per week (5qw)
with either vehicle
(black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white
triangles) at
indicated doses. Treatment persisted for 31, 29 and 29 days for mice
containing AsPC-1, BxPC-3
and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated
and plotted. All
results are expressed as the mean SEM (n = 6 mice per group). On the final
day of treatment,
tumor volumes were compared between the drug-treated and control groups. **,p
< 0.01; ***,p
<0.001; ****, p < 0.0001 (versus vehicle).
FIG. 55 presents the exemplary data of one embodiment showing that treatment
with synthetic
lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology
loaded with an
equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide
residue and the 31 amino acids-long TREM-1 modulatory peptide GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in
experimental pancreatic cancer without affecting body weight (i.e. well
tolerable by long term-
treated mice). As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-
l-bearing mice
reached a volume of 150-200 mm3, mice were randomized into groups and
intraperitoneally (i.p.)
administered once daily 5 times per week (5qw) with either vehicle (black
diamonds), GA/E31-
dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated
doses. Treatment
persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-
1 tumor
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xenografts, respectively. Body weighs are plotted. All results are expressed
as the mean SEM
(n = 6 mice per group).
FIG. 56 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into synthetic
lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology
(GF9-dSLP and
GF9-sSLP, respectively) prolongs survival in experimental pancreatic cancer.
As described
herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a
volume of 150-200
3
MM , mice were randomized into groups and intraperitoneally (i.p.)
administered once daily 5
times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray
circles), GF9-dSLP
(light gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment
persisted for 31, 29
and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts,
respectively.
Kaplan-Meier survival curves are shown for AsPC-1-, BxPC-3- or Capan-l-bearing
mice (n = 6
mice per group). **, p < 0.01; ***,p < 0.001 by log-rank test (versus
vehicle).
FIG. 57 presents the exemplary data of one embodiment showing that treatment
with synthetic
lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology
loaded with an
equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide
residue and the 31 amino acids-long TREM-1 modulatory peptide GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue (GA/E31-dSLP and GA/E31-sSLP, respectively) prolongs survival in
experimental
pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or
Capan-l-bearing
mice reached a volume of 150-200 mm3, mice were randomized into groups and
intraperitoneally
(i.p.) administered once daily 5 times per week (5qw) with either vehicle
(black diamonds),
GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at
indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3
and Capan-1
tumor xenografts, respectively. Kaplan-Meier survival curves are shown for
AsPC-1-, BxPC-3-
or Capan-l-bearing mice (n = 6 mice per group). **, p < 0.01; ***, p < 0.001
by log-rank test
(versus vehicle).
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FIG. 58 presents the exemplary data of one embodiment showing that the
antitumor efficacy of
TREM-1 blockade correlates with the intratumoral macrophage content in
experimental
pancreatic cancer. Antitumor efficacy is expressed as percent
treatment/control (% T/C) values
calculated using the following formula: % T/C = 100 x AT/AC where T and C are
the mean
tumor volumes of the drug-treated and control groups, respectively, on the
final day of the
treatment; AT is the mean tumor volume of the drug-treated group on the final
day of the
treatment minus mean tumor volume of the drug-treated group on initial day of
dosing; and AC is
the mean tumor volume of the control group on the final day of the treatment
minus mean tumor
volume of the control group on initial day of dosing. Intratumoral macrophage
content was
quantified by F4/80 staining using F4/80 antibodies. Data are shown for the
groups of AsPC-1-,
BxPC-3- and Capan-1- bearing mice treated with free and SLP-bound TREM-1
modulatory
peptides GF9 (GFLSKSLVF), GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and
GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide
residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 59 presents the exemplary data of one embodiment showing that TREM-1
blockade
suppresses intratumoral macrophage infiltration in experimental pancreatic
cancer. Intratumoral
macrophage content was quantified by F4/80 staining using F4/80 antibodies.
Data are shown for
the groups of BxPC-3-bearing mice treated with either vehicle (black bars),
free GF9
(GFLSKSLVF, dark grey bars), GF9 incorporated into a carrier, e.gs. synthetic
lipopeptide
particle of spherical morphology (GF9-sSLP, light grey bars) and sSLP that
contain an equimolar
mixture of TREM-1 modulatory peptides
GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA where M(0) is a methionine sulfoxide
residue) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a
methionine sulfoxide residue) (GA/E31-sSLP, white bars) (n = 4 mice per
group).
FIG. 60 presents the exemplary data of one embodiment showing the
representative F4/80
images demonstrating that TREM-1 blockade suppresses intratumoral macrophage
infiltration in
experimental pancreatic cancer. Intratumoral macrophage content was quantified
by F4/80
staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-
bearing mice treated
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with either vehicle, free GF9 (GFLSKSLVF), GF9 incorporated into synthetic
lipopeptide
particle of spherical morphology (GF9-sSLP) and sSLP that contain an equimolar
mixture of
TREM-1 modulatory peptides GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and
GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide
residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 61 presents the exemplary data of one embodiment showing that TREM-1
blockade
suppresses serum proinflammatory cytokines in xenograft mouse models of
pancreatic cancer.
Serum interleukin- 1 a (IL-1a), IL-6 and macrophage colony-stimulating factor
(M-CSF/CSF-1)
levels were analyzed on study days 1 and 8 in AsPC-1-, BxPC-3- and Capan-l-
bearing mice
treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9
(dark gray
squares) or GF9-loaded spherical synthetic lipopeptide particles (GF9-sSLP,
white circles) at
indicated doses. Results are expressed as the mean SEM (n = 5 mice per
group). *,p < 0.05;
**,p < 0.01; ***,p <0.001; ****,p < 0.0001 (versus vehicle).
FIG. 62 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex
(GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor
1 (CSF1) and
interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the
AsPC-1 xenograft
mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were
analyzed on study
days 1 and 8 in AsPC-1-bearing mice treated daily 5 times per week (5qw) with
either vehicle
(black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated
doses. Results are
expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 63 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex
(GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor
1 (CSF1) and
interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the
BxPC-3 xenograft
mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were
analyzed on study
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days 1 and 8 in BxPC-3-bearing mice treated daily 5 times per week (5qw) with
either vehicle
(black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated
doses. Results are
expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 64 presents the exemplary data of one embodiment showing that treatment
with free
TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex
(GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor
1 (CSF1) and
interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the
CAPAN-1
xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels
were analyzed
on study days 1 and 8 in CAPAN-1-bearing mice treated daily 5 times per week
(5qw) with
either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black
circles) at indicated
doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <
0.05; **, p <
0.01; ***, p <0.001; ****, p <0.0001 (versus vehicle).
FIG. 65 presents the exemplary data of one embodiment showing that combining
of
Gemcitabine and Abraxane chemotherapy with TREM-1 modulatory peptide GF9
(GFLSKSLVF) incorporated into synthetic lipopeptide particle (SLP) of
spherical (sSLP)
morphology (GF9-sSLP) has a synergistic effect in experimental pancreatic
cancer. As described
herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3,
mice were
randomized into groups and intraperitoneally (i.p.) administered with either
vehicle (black
diamonds; once daily 5 times per week, 5qw), GF9-sSLP (black squares; once
daily 5 times per
week, 5qw), Gemcitabine and Abraxane (black circles; days 1, 4, 8, 11, 15) or
GF9-sSLP (once
daily 5 times per week, 5qw) in combination with Gemcitabine and Abraxane
(days 1, 4, 8, 11,
15) (Black triangles). Treatment with GF9-sSLP persisted for 28 days. Mean
tumor volumes are
calculated and plotted. All results are expressed as the mean SEM (n = 9
mice per group). On
the final day of treatment, tumor volumes were compared between the
Gemcitabine+Abraxane-
treated and GF9-sSLP+Gemcitabine+Abraxane-treated groups. **, p < 0.01 (versus
chemotherapy alone treated group).
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FIG. 66 presents the exemplary data showing penetration of the blood-brain
barrier (BBB) and
blood-retinal barrier (BRB) by systemically (intraperitoneally) administered
rhodamine B-
labeled spherical synthetic lipopeptide particles (sSLP) that contain Gd-
containing contrast agent
(Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 modulatory peptide GF9
(GF9-
sSLP) or an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1
modulatory peptides, i.e. 31 amino acids-long TREM-1 modulatory peptide GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide
residue and the 31 amino acids-long TREM-1 modulatory peptide GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue GA 31 and GE 31 (GA/E31-sSLP).
Fig. 67 presents the exemplary data of one embodiment showing that TREM-1
blockade with
GF9, GF9 incorporated into the carrier - spherical synthetic lipopeptide
particles (GF9-sSLP) or
sSLP that carried an equimolar mixture of the 31 amino acids-long TREM-1
modulatory peptide
GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine
sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide
residue (GA/E31-sSLP) significantly reduces tissue expression of colony-
stimulating factor 1
(C SF-1) and TREM-1 in the retina of mice with oxygen-induced retinopathy
(OIR) at postnatal
day 17 (P17). Representative Western blots of retinal lysates from OIR mice
are shown. The
membrane was probed for TREM-1, reprobed for CSF-1 and then for 13-actin.
Values in the bar
graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs. vehicle-
treated mice.
Fig. 68 presents the exemplary data of one embodiment showing that combining
gemcitabine
(GEM) and abraxane (ABX) chemotherapy with TREM-1 modulatory peptide GF9
(GFLSKSLVF) incorporated into a carrier, e.g. synthetic lipopeptide particle
(SLP) of spherical
(sSLP) morphology (GF9-sSLP) has a synergistic therapeutic effect in
experimental pancreatic
cancer. As described herein, after tumors in PANC-1-bearing mice reached a
volume of 150-200
mm3, mice were randomized into groups and intraperitoneally (i.p.)
administered at indicated
doses with either vehicle (black diamonds; once daily 5 times per week, 5qw),
GF9-LPC (black
circles-black squares; once daily 5 times per week, 5qw), GEM and ABX (black
squares-(black
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circles; days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw)
in combination with
GEM and ABX (days 1, 4, 8, 11, 15) (half black half white hexagons-Black
triangles). Treatment
with GF9-LPC persisted for 28 days. Mean tumor volumes are calculated and
plotted. All results
are expressed as the mean SEM (n = 9 mice per group). On the day 88, tumor
volumes were
compared between the GEM+ABX-treated and GF9-sSLP+GEM+ABX-treated groups. *, p
<
0.05 (versus GEM+ABX-treated group), second set of symbols are used in the
longer term
studies.
Fig. 69 presents the exemplary data of one embodiment showing that TREM-1
blockade
treatment with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into a ,
e.g.
synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP)
alone,
lipopeptide complex (GF9-LPC) alone or in combination with gemcitabine (GEM)
and abraxane
(ABX) chemotherapy is well tolerable in mice with human PANC-1 pancreatic
cancer
xenografts. As described herein, after tumors in PANC-1-bearing mice reached a
volume of 150-
200 mm3, mice were randomized into groups and intraperitoneally (i.p.)
administered at
indicated doses with either vehicle (black diamonds; once daily 5 times per
week, 5qw), GF9-
LPC (black circles; once daily 5 times per week, 5qw), GEM and ABX (black
squares; days 1, 4,
8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with
GEM and ABX
(days 1, 4, 8, 11, 15) (half black half white hexagons). Treatment with GF9-
LPC (GF9-sSLP)
persisted for 28 days. Body weighs are plotted. All results are expressed as
the mean SEM (n =
6 mice per group).
Fig. 70 presents the exemplary data of one embodiment showing that treatment
with TREM-1
modulatory peptide GF9 incorporated into a carrie, e.g. synthetic lipopeptide
complex (GF9-
LPC) and particle (SLP) of spherical (sSLP) morphology (GF9-sSLP),
synergistically prolongs
survival rate in experimental pancreatic cancer (e.g. PANC-1) when combined
with gemcitabine
(GEM) and abraxane (ABX) chemotherapy. As described herein, after tumors in
PANC-1-
bearing mice reached a volume of 150-200 mm3, mice were randomized into groups
and
intraperitoneally (i.p.) administered at indicated doses with either vehicle
(once daily 5 times per
week, 5qw), GF9-LPC (once daily 5 times per week, 5qw), GEM and ABX (days 1,
4, 8, 11, 15)
or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX
(days 1, 4,
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8, 11, 15). Treatment with GF9-LPC persisted for 28 days. Kaplan-Meier
survival curves are
shown for PANC-1-bearing mice (n = 6 mice per group). *, p < 0.05 by log-rank
test (versus
GEM+AB X).
Fig. 71 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) is well tolerable in mice up to at least
300 mg/kg. As
described herein, healthy C57BL/6 mice were intraperitoneally (i.p.)
administered daily for 7
consecutive days with GF9 at indicated doses Mouse body weight (BW) was
measured daily.
Results are expressed as the mean SEM (n = 4 mice per group).
Fig.72 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) suppresses tumor growth in experimental
pancreatic cancer. As
described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice
reached a volume
of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.)
administered
.. once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9
(white circles),
GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at
indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3
and Capan-1
tumor xenografts, respectively. Mean tumor volumes are calculated and plotted.
All results are
expressed as the mean SEM (n = 6 mice per group).
Fig.73 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
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sulfoxide residue (GA/E31-LPC) is well tolerable in mice with human pancreatic
cancer
xenografts. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-
bearing mice
reached a volume of 150-200 mm3, mice were randomized into groups and
intraperitoneally
(i.p.) administered once daily 5 times per week (5qw) with either vehicle
(black diamonds), GF9
(white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white
circles) at
indicated doses. Treatment persisted for 31, 29 and 29 days for mice
containing AsPC-1, BxPC-3
and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All
results are expressed
as the mean SEM (n = 6 mice per group).
Fig.74 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) suppresses tumor growth as effectively as 20
mg/kg paclitaxel
and is well tolerable in mice with human non-small cell lung cancer
xenografts. As described
herein, after tumors in A549-bearing mice reached a volume of 150-200 mm3,
mice were
randomized into groups and intraperitoneally (i.p.) administered once daily 5
times per week
(5qw) with either vehicle (black diamonds), paclitaxel (black squares), GF9
(white circles), GF9-
LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated
doses. Treatment
persisted for 21 days. Mean tumor volumes are calculated and plotted. Body
weighs are plotted.
All results are expressed as the mean SEM (n = 6 mice per group).
Fig.75 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) suppresses intratumoral macrophage infiltration
in
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experimental pancreatic cancer. Intratumoral macrophage content was quantified
by F4/80
staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-
bearing mice treated
with either vehicle, GF9, GF9-LPC or GA/E31-LPC at indicated doses. Treatment
persisted for
21 days. All results are expressed as the mean SEM (n = 4 mice per group).
Scale bar = 200
Elm.
Fig.76 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) ameliorates arthritis in mice with collagen-
induced arthritis
(CIA). As described herein, starting on day 24 after immunization, mice with
CIA were
intraperitoneally (i.p.) administered daily for 14 consecutive days with
vehicle (black diamonds),
dexamethasone (black squares), GF9 (white circles), GF9-LPC (black circles)
and GA/E31-LPC
(half black half white circles) at indicated doses. Daily clinical scores were
given on a scale of 0-
5 for each of the paws on days 24-38. On day 38, mice were killed and the
histopathological
examination of mouse joints was performed. Histopathological scores of
inflammation (I),
pannus (P), cartilage damage (CD), bone resorption (BR) and periosteal new
bone formation
(PBF) are shown. Summed histopathology scores were calculated as the sum of
all five
histopathological parameters. All results are expressed as the mean SEM (n =
10 mice per
group).
Fig.77 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue is well tolerable in mice with collagen-induced arthritis
(CIA). Mouse body
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weight (BW) was measured every other day from day 24 to day 38. Mean BW
changes were
calculated as a percentage of the difference between beginning (day 24) and
final (day 38) BWs
of the CIA mice intraperitoneally (i.p.) treated daily for 14 consecutive days
with vehicle,
dexamethasone, GF9, GF9-LPC and GA/E31-LPC at indicated doses. All results are
expressed
as the mean SEM (n = 10 mice per group).
Fig.78 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) prevents pathological appearances from collagen-
induced
arthritis (CIA) in mice. As described herein, toluidine blue staining of the
joints from mice with
CIA treated with TREM-1 inhibitory GF9 sequences or control peptide GF9-G
(GFLSGSLVF)
was performed. Photomicrographs of fore paws, hind paws, knees and ankles from
representative
mice are shown for each treatment group. For paws (original magnification 16x)
and ankles
(original magnification 40x), arrows identify affected joints. For knees
(original magnification
100x), large arrow identifies cartilage damage, small arrow identifies pannus
and arrowhead
identifies bone resorption. W, wrist; S, synovium.
Fig.79 presents the exemplary data of one embodiment showing that treatment
with free TREM-
1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) reduces plasma cytokines in mice with collagen-
induced
arthritis (CIA). Plasma was collected on days 24, 30 and 38 from arthritic
mice treated with
vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) and
GA/E31-LPC (half
black half white circles). Plasma samples were analyzed for concentrations of
interleukin-lb (IL-
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lb), IL-6, and colony-stimulating factor 1 (CSF1). Results are expressed as
the mean SEM (n =
mice per group).
Fig. 80 presents the exemplary data of one embodiment showing that treatment
with free TREM-
5 1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide
complex (GF9-
LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-
long TREM-1
modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0)
is a methionine sulfoxide residue and the 31 amino acids-long TREM-1
modulatory peptide
GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine
sulfoxide residue (GA/E31-LPC) significantly reduces tissue expression of
colony-stimulating
factor 1 (CSF1) and TREM-1 in the retina of mice with oxygen-induced
retinopathy (OIR) at
postnatal day 17 (P17). Representative Western blots of retinal lysates from
OIR mice are
shown. The membrane was probed for TREM-1, reprobed for CSF1 and then for 13-
actin. Values
in the bar graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs.
vehicle-treated
mice.
Fig. 81 shows exemplary illustrations of peptide GF9 blocking TREM-1 signaling
by disruption
of intramembrane interactions with its signaling partner, DAP-12. One example
of a comparision
of current approaches (upper) with a SCHOOL approach (lower), e.g. Route 1.
Fig. 82 shows exemplary illustrations of LPC delivering of peptide GF9 to
macrophages, as two
exemplary embodiments, e.g. each as Route 2.
Fig. 83 shows exemplary results using Pancreas Cancer: PANC-1 Xenografts
demonstrating GF9
treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX,
ABX) and Adding GF9 treatment sensitizes the tumor to chemotherapy and at
least triples
survival rate.
Fig. 84 shows exemplary results using Pancreas Cancer: AsPC-1 Xenografts
demonstrating GF9
treatment alone does not inhibit tumor growth.Adding of the GF9 treatment
sensitizes the tumor
to chemotherapy. NOTE: Most tumors ¨ abscessed.
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Fig. 85 shows exemplary results using Pancreas Cancer: MiaPaca-2 Xenografts
demonstrating
GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine,
GEM + nab-
PTX, ABX) and Adding of the GF9 treatment to chemo does not affect.
Fig. 86 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts
demonstrating GF9
treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX,
ABX) and Adding of the GF9 treatment to chemo does not significantly affect
survival rate.
Fig. 87 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts
demonstrating GF9
treatment reduces macrophage content in the tumor, Vehicle, 2.5 mg/kg GF9-LPC
(5 qw, 4 wk).
Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 88 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts
demonstrating GF9
treatment reduces serum cytokine levels, Vehicle, 2.5 mg/kg GF9-LPC. Shen and
Sigalov, Mol
Pharm 2017,14:4572, 2017.
Fig. 89 shows exemplary results using Pancreas Cancer: Xenografts
demonstrating GF9
Treatment is Non-Toxic. Free GF9 tolerability (upper). GF9-LPC* tolerability
(lower). * Shown
for PANC-1 xenograft
Fig. 90 shows exemplary results demonstrating that GF9 peptide is well-
tolerable by healthy
mice up to at least, 300 mg/kg.
Fig. 91 shows exemplary results demonstrating that in mice with collagen-
induced arthritis
(CIA), GF9 suppresses arthritis as effectively as dexamethasone (DEX). Study
Day (Treatment:
Days 24-38). I, inflammation; P, pannus; CD, cartilage damage; BR, bone
resorption; PBF,
periosteal new bone formation. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
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Fig. 92 shows exemplary results demonstrating that in mice with collagen-
induced arthritis
(CIA), GF9 treatment reduces serum IL-lb\ TNFal, IL-6 and CSF-1. Shen and
Sigalov, Mol Pharm
2017,14:4572, 2017.
Fig. 93 shows exemplary results demonstrating that in mice with collagen-
induced arthritis
(CIA), GF9 treatment is well-tolerable: no body weight changes or other
clinical symptoms are
observed. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 94 shows exemplary results demonstrating that in NSCLC: A549 Xenografts,
GF9 inhibits
tumor growth as effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014,
21:208.
Fig. 95 shows exemplary results demonstrating that in Capan-1 xenografts, GF9
inhibits tumor
growth and reduces serum cytokines, including CSF-1 (but not VEGF). Shen and
Sigalov, Mol
Pharm 2017,14:4572, 2017.
Fig. 96 shows exemplary results demonstrating that GF9 is well-tolerated by
long term treated
cancer mice inCapan-1 Xenografts and A549 Xenografts, GF9 inhibits tumor
growth as
effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014, 21:208.
Fig. 97A-C shows exemplary current approaches for blocking TREM-1 binding to
its uncertain
ligand (Bouchon et al. 2001, Schenk et al. 2007, Gibot et al. 2008, Gibot et
al. 2009, Murakami
et al. 2009, Luo et al. 2010, Derive et al. 2013, Derive et al. 2014) Fig. 97A
In contrast, GF9
self-penetrates into the membrane and disrupts TREM-1 / DAP12 interactions
Fig. 97B when
colocalizes with TREM-1 Fig. 97C. Fig. 97A. CURRENT. Fig. 97B. SCHOOL Fig.
97C.
CONFOCAL.
Fig. 98A-B shows exemplary results demonstrating that GF9 is non-toxic in
healthy mice Fig.
98A and reduces TREM-1 and M-CSF overexpression in the retina of mice with
oxygen-induced
retinopathy Fig. 98B. Fig. 98A Graph. Fig. 98B Blot.
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Fig. 99A-C shows exemplary results demonstrating that Oxidized apo A-I
peptides in LPC
increase J774 intracellular uptake of GF9-LPC in vitro Fig. 99A, 99B and
enable in vivo delivery
to macrophages Fig. 99C (as shown using magnetic resonance imaging (Mill) and
confocal
microscopy (Sigalov 2014, Sigalov 2014, Shen and Sigalov 2017)). Fig. 99A. IN
VITRO. Fig.
.. 99B. CONFOCAL red: Rho B-PE; green: 488-GF9; blue: 405-apo A-I PE22. Fig.
99C. MOUSE
AORTA.
Fig. 100A-D shows exemplary results demonstrating that GF9-dLPC (disks) and
GF9-sLPC
(spheres) reduce LPS-induced cytokine release in vitro Fig. 100A and in vivo
Fig. 100B and
prolong survival Fig. 100C (Sigalov 2014). In cancer mice, GF9 and GF9-LPC
treatments inhibit
production of CSF-1/M-CSF but not VEGF Fig. 100D (Shen and Sigalov 2017). Fig.
100A.
CYTOKINES IN VITRO. Fig. 100B. CYTOKINES IN VIVO. Fig. 100C. SURVIVAL IN LPS-
INDUCED SEPTIC MICE. Fig. 100D. M-CSF / VEGF RELEASE IN CANCER MICE.
Fig. 101 shows exemplary results demonstrating that Different rate and
efficiency of GF9-dLPC
and GF9-sLPC in vitro uptake by J774 macrophages (Sigalov 2014).
Fig. 102 shows exemplary results demonstrating that Stability of GF9-LPC. GF9-
LPC AT 4 C.
Fig. 103A-D shows exemplary results demonstrating that GF9-LPC daily i.p.
administered at 2.5
mg/kg suppress the expression of TREM-1, MCP-1/CCL2 and early fibrosis marker
molecules in
mice with ALD. Indicates significance level compared to nontreated pair-fed
group; # indicates
significance level compared to nontreated alcohol-fed group. Significance
levels are as follows:
<0.05; **/#4, p < 0.01; ***,p <0.001; ****/ p <0.0001. Fig. 102A. TREM-1.
Fig.
102B. MCP-1/CCL2. Fig. 102C. Pro-Colllalpha. Fig. 102D. alpha-SMA.
Fig. 104A-D shows exemplary results demonstrating that GF9 and GF9-LPC daily
i.p.
administered are well-tolerated Fig. 104A, suppress macrophage infiltration
into the tumor Fig.
104B, 104C and inhibit release of CSF-1/M-CSF but not VEGF Fig. 104D. Scale
bar = 200 pm.
*,p< 0.05; **,p < 0.01; ***,p < 0.001; ****,p < 0.0001 (vs vehicle). Fig.
104A. BODY
WEIGHT. Fig. 104B. INTRATITMORAL MACROPHAGE INFILTRATION ¨ INHIBITION
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BY GF9 AND GF9-LPC. Fig. 104C. MACROPHAGE INFILTRATION. Fig. 104D. M-CSF /
VEGF RELEASE IN CANCER MICE.
Fig. 105A- C shows exemplary results demonstrating that in mice with
autoimmune arthritis,
GF9, discoidal GF9-LPC (GF9-dHDL) and spherical GF9-LPC (GF9-sHDL) i.p.
administered
daily are well-tolerated Fig. A, ameliorate the disease Fig. 105B and inhibit
production of
cytokines and M-CSF Fig. C (Shen and Sigalov 2017). Fig. 105A. BODY WEIGHT
CHANGES.
Fig. 105B. ARTHRITIS AMELIORATION. Fig. 105C. CYTOKINE RELEASE IN
ARTHRITIC MICE.
DEFINITIONS
The term, "composition", as used herein, refers to any mixture of substances
comprising
a peptide and/or compound contemplated by the present invention. Such a
composition may
include the substances individually or in any combination.
As used herein the term "lipoprotein" such as VLDL (very low density
lipoproteins),
LDL (low density lipoproteins) and HDL (high density lipoproteins), refers to
lipoproteins found
in the serum, plasma and lymph, in vivo, related to lipid transport. The
chemical composition of
each lipoprotein differs, for examples, HDL has a higher proportion of protein
versus lipid,
whereas the VLDL has a lower proportion of protein versus lipid. When
referring to lipoproteins,
the term "native" refers to naturally-occurring (e.g., a "wild-type")
lipoproteins.
The terms "AP0A1 HUMAN", "Apolipoprotein A-I", "Apolipoprotein A-1", "AP0A1",
"ApoA-I", "Apo-AI", "ApoA-1", "apo-Al", "apoA-1" and "Apo-Al" refer to the
naturally
occurring human protein listed in the UniProt Knowledgebase (UniProtKB,
www.uniprot.org)
under the name "AP0A1 HUMAN". The protein amino acid sequence can be found
under the
entry UniProt KB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647). The terms
"AP0A2 HUMAN", "Apolipoprotein A-II", Apolipoprotein A-2", "AP0A2", "ApoA-II",
"Apo-All", "ApoA-2", "apo-A2", "apoA-2" and "Apo-A2" refer to the naturally
occurring
human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org)
under the
name "AP0A2 HUMAN". The protein amino acid sequence can be found under the
entry
.. UniProt KB/Swiss-Prot P02652 (http://www.uniprot.org/uniprot/P02652).
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The term "TREM receptor", as used herein, refers to a member of TREM receptor
family
including: TREM-1, TREM-2, TREM-3 and TREM-4. The terms "TREM1 HUMAN", "TREM-
1 receptor", "TREM-1 receptor subunit", "TREM-1 subunit", and "TREM-1
recognition
subunit" refer to the naturally occurring human protein listed in the UniProt
Knowledgebase
(UniProtKB, www.uniprot.org) under the name "TREM1 HUMAN". The protein amino
acid
sequence can be found under the entry UniProt KB/Swiss-Prot Q9NP99.
The term "TREM receptor", as used herein, refers to a member of TREM receptor
family: TREM-1, TREM-2, TREM-3 and TREM-4.
As used herein, the term "T cell receptor" or "TCR" refers to a complex of
membrane
proteins that participate in the activation of T cells in response to the
presentation of antigen. The
TCR is responsible for recognizing antigens bound to major histocompatibility
complex
molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0)
chain, although in
some cells the TCR consists of gamma and delta (y/6) chains. TCRs may exist in
alpha/beta and
gamma/delta forms, which are structurally similar but have distinct anatomical
locations and
functions. Each chain is composed of two extracellular domains, a variable and
constant domain,
in some embodiments, the TCR may be modified on any ceil comprising a TCR,
including, for
example, a helper T cell, a cytotoxic T cell, a memory T ceil, regulatory T
cell, natural killer T
cell, and gamma delta T cell.
As employed herein and understood by the ordinary skill in the art, "amino
acid domain"
is a contiguous polymer of at least 2 amino acids joined by peptide bond(s).
The domain may be
joined to another amino acid or amino acid domain by one or more peptide
bonds. An amino acid
domain can constitute at least two amino acids at the N-terminus or C-terminus
of a peptide or
can constitute at least two amino acids in the middle of a peptide.
The term "antibody" herein refers to a protein, derived from a germline
immunoglobulin
sequence, which is capable of specifically binding to an antigen (TREM-1) or a
portion thereof.
The term includes full length antibodies of any class or isotype (that is,
IgA, IgE, IgG, IgM
and/or IgY) and any single chain or fragment thereof. An antibody that
specifically binds to an
antigen, or portion thereof, may bind exclusively to that antigen, or portion
thereof, or it may
bind to a limited number of homologous antigens, or portions thereof.
As used herein, a "peptide" and "polypeptide" comprises a string of at least
two amino
acids linked together by peptide bonds. A peptide generally represents a
string of between
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approximately 2 and 200 amino acids, more typically between approximately 6
and 64 amino
acids. Peptide may refer to an individual peptide or a collection of peptides.
Inventive peptides
typically contain natural amino acids, although non-natural amino acids (i.e.,
compounds that do
not occur in nature but that can be incorporated into a polypeptide chain and/
or amino acid
analogs as are known in the art may alternatively be employed. In particular,
D-amino acids may
be used.
As employed herein and understood by the ordinary skill in the art, "peptide
sequence",
or "amino acid sequence", is the order in which amino acid residues, connected
by peptide
bonds, lie in the chain in peptides. The sequence is generally reported from
the N-terminal end
containing free amino group to the C-terminal end containing free carboxyl
group. "Peptide
sequence" is often called "protein sequence" if it represents the primary
structure of a protein
(http://en.wikipedia.org/wiki/Peptide sequence).
Peptides and compositions of the present invention made synthetically may
include
substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally
occurring or
.. unnatural amino acid). Examples of non-naturally occurring amino acids
include D-amino acids,
an amino acid having an acetylaminomethyl group attached to a sulfur atom of a
cysteine, a
pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6,
neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl
glycine, N-methyl
isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and
.. methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and
ornithine is basic. Proline
may be substituted with hydroxyproline and retain the conformation conferring
properties.
Naturally occurring residues are divided into groups based on common side
chain
properties: as described herein. Analogues may be generated by substitutional
mutagenesis and
retain the biological activity of the original trifunctional peptides.
Examples of substitutions
identified as "conservative substitutions" are shown in TABLE 1. If such
substitutions result in a
change not desired, then other type of substitutions, denominated "exemplary
substitutions" in
TABLE 1, or as further described herein in reference to amino acid classes,
are introduced and
the products screened for their capability of executing three functions.
The term "amphipathic" is used herein to describe a molecule that has both
polar and
.. non-polar parts and as such, has two different affinities, as a polar end
that is attracted to water
and a nonpolar end that is repelled by it. An amphipathic helix is defined as
an alpha helix with
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opposing polar and nonpolar faces oriented along the long axis of the helix.
As well known in the
art, amino acid sequences can be screened for amphipathic helixes and an
amphipathicity score
can be calculated using a variety of computer programs available online (see,
for example,
http ://www.tcdb . org/progs/?tool=pepwheel, http ://lbqp.unb . br/NetWhe el
s/, https ://np s a-
prabi.ibcp.fr/cgi-bin/npsa automat.pl?page=/NPSA/npsa amphipaseek.html,
http ://rzlab .ucr. e du/scripts/wheel/whe el .cgi, http ://heli quest. ipm c.
cnrs.fr/cgi-
bin/ComputParams.py) or other techniques including but not limiting to those
described in Jones,
et al. J Lipid Res 1992, 33:287-296.
As used herein, the term "aptamer" or "specifically binding oligonucleotide"
refers to an
oligonucleotide that is capable of forming a complex with an intended target
substance.
In the present disclosure, the term "modified peptide" is used to describe
chemically or
enzymatically, or chemically and enzymatically modified oligopeptides,
oligopseudopeptides,
polypeptides, and pseudopolypeptides (synthetic or otherwise derived),
regardless of the nature
of the chemical and/or enzymatic modification. The term "pseudopeptide" refers
to a peptide
where one or more peptide bonds are replaced by non-amido bonds such as ester
or one or more
amino acids are replaced by amino acid analogs. The term "peptides" refers not
only to those
comprised of all natural amino acids, but also to those which contain
unnatural amino acids or
other non-coded structural units. The terms "peptides", when used alone,
include pseudopeptides.
It is worth mentioning that "modified peptides" have utility in many
biomedical applications
because of their increased stability against in vivo degradation, superior
pharmacokinetics, and
altered immunogenicity compared to their native counterparts.
The term "modified peptides," as employed herein, also includes oxidized
peptides.
The term "oxidized peptide" refers to a peptide in which at least one amino
acid residue
is oxidized.
The term "analog", as used herein, includes any peptide having an amino acid
sequence
substantially identical to one of the sequences specifically shown herein in
which one or more
residues have been conservatively substituted with a functionally similar
residue and which
displays the abilities as described herein. Examples of conservative
substitutions include the
substitution of one non-polar (hydrophobic) residue such as isoleucine,
valine, leucine or
methionine for another, the substitution of one polar (hydrophilic) residue
for another such as
between arginine and lysine, between glutamine and asparagine, between glycine
and serine, the
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substitution of one basic residue such as lysine, arginine or histidine for
another, or the
substitution of one acidic residue, such as aspartic acid or glutamic acid for
another.
The term "conservative substitution", as used herein, also includes the use of
a
chemically derivatized residue in place of a non-derivatized residue provided
that such peptide
displays the requisite inhibitory function on myeloid cells as specified
herein. The term
derivative includes any chemical derivative of the peptide of the invention
having one or more
residues chemically derivatized by reaction of side chains or functional
groups.
The term "homolog" or "homologous" when used in reference to a polypeptide
refers to a
high degree of sequence identity between two polypeptides, or to a high degree
of similarity
between the three-dimensional structures or to a high degree of similarity
between the active site
and the mechanism of action. In a preferred embodiment, a homolog has a
greater than 60%
sequence identity, and more preferably greater than 75% sequence identity, and
still more
preferably greater than 90% sequence identity, with a reference sequence.
As applied to polypeptides, the term "substantial identity" means that two
peptide
sequences, when optimally aligned, such as, for example, by the programs
KALIGN, DOTLET,
LALIGN and DIALIGN (https://www.expasy.org/tools) using default gap weights,
share at least
80 percent sequence identity, preferably at least 90 percent sequence
identity, more preferably at
least 95 percent sequence identity or more (e.g., 99 percent sequence
identity). Preferably,
residue positions which are not identical differ by conservative amino acid
substitutions.
The term "modified peptides," as employed herein, also includes oxidized
peptides. The
term "oxidized peptide" refers to a peptide in which at least one amino acid
residue is oxidized.
The term "oxidation status" refers to a metric of the extent to which specific
amino acid residues
are replaced by corresponding oxidized amino acid residues in a peptide. The
term "extent of
oxidation" refers to the degree to which potentially oxidizable amino acids in
a peptide have
undergone oxidation. For example, if the peptide contains a single tyrosine
residue which is
potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34
Dalton (i.e., the
approximate difference in mass between chlorine and hydrogen) indicates
oxidation of tyrosine
to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine
residue which is
potentially oxidized to methionine sulfoxide, then an increase in mass of 16
Dalton (i.e., the
difference in mass between methionine and methionine containing one extra
oxygen) indicates
oxidation of methionine to methionine sulfoxides.
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The term "oxidation status" refers to a metric of the extent to which specific
amino acid
residues are replaced by corresponding oxidized amino acid residues in a
peptide. The term
"extent of oxidation" refers to the degree to which potentially oxidizable
amino acids in a peptide
have undergone oxidation. For example, if the peptide contains a single
tyrosine residue which is
potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34
Dalton (i.e., the
approximate difference in mass between chlorine and hydrogen) indicates
oxidation of tyrosine
to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine
residue which is
potentially oxidized to methionine sulfoxide, then an increase in mass of 16
Dalton (i.e., the
difference in mass between methionine and methionine containing one extra
oxygen) indicates
oxidation of methionine to methionine sulfoxides.
The oxidation status can be measured by metrics known to the arts of protein
and peptide
chemistry (as disclosed in Caulfield, US 8,114,613 and Hazen, et al., US
8,338,110, herein
incorportaed by reference) including, without limitation, assay of the number
of oxidized
residues, mass spectral peak intensity, mass spectral integrated area, and the
like. In some
embodiments, oxidation status is reported as a percentage, wherein 0% refers
to no oxidation and
100% refers to complete oxidation of potentially oxidizable amino acid
residues within apo A-I
or apo A-II peptide fragments.
The term "potentially subject to oxidation," "potentially oxidizable amino
acid residues",
and the like refer to an amino acid which can undergo oxidation, for example
by nitration or
chlorination.
A "biologically active peptide motif' is a peptide that induces a phenotypic
response or
change in an appropriate cell type when the cell is contacted with the
peptide. The peptide may
be present either in isolated form or as part of a larger polypeptide or other
molecule. The ability
of the peptide to elicit the response may be determined, for example, by
comparing the relevant
parameter in the absence of the peptide (e.g., by mutating or removing the
peptide when
normally present within a larger polypeptide). Phenotypic responses or changes
include, but are
not limited to, enhancement of cell spreading, attachment, adhesion,
proliferation, secretion of an
extracellular matrix (ECM) molecule, or expression of a phenotype
characteristic of a particular
differentiated cell type.
As used herein, a "minimal biologically active sequence" refers to the minimum
length of
a sequence of a peptide that has a specific biological function. In a first
example,
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-IVILLAGGFLSKSLVFSVLFA- (e.g., Domain A, SEQ ID NO. 47) is a biologically
active
TREM-1 inhibitory sequence corresponding to the human TREM-1 transmembrane
domain,
wherein -GFLSKSLVF- (e.g. Domain A, SEQ ID NO. 1) has the sole function of
TREM-1
inhibition. Thus, in this case, -GFLSKSLVF- (Domain A, SEQ ID NO. 1) is a
"minimal
biologically active sequence." In a second example, the sequence ¨
PLGEEMRDRARAHVDALRTHLARGD, and an internal sequence -GEEMRDRARAHVRGD-
(Domain B, SEQ ID NO. 5) contains the sequence -RGD-; -RGD- has a cell
attachment function.
However, -PLGEEMRDRARAHVDALRTHLARGD and -GEEMRDRARAHVRGD- (Domain
B, SEQ ID NO. 5) also functions to assist in the formation of naturally long
half-life
lipopeptide/lipoprotein particles upon interaction with native lipoproteins
and to promote binding
of these particles with scavenger receptor type I (SR-B1). Thus, in this case,
both -
PLGEEMRDRARAHVDALRTHLARGD- andGEEMRDRARAHVRGD- (Domain B, SEQ ID
NO. 5) in addition to -RGD- are considered a "minimal biologically active
sequence." In another
example, the sequence -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO.
...) contains the sequence -RGD-; -RGD-has a cell attachment function.
However, -
GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) also has the functions
of inhibition of TREM-1, assistance in the self-assembly of naturally long
half-life lipopeptide
particles upon binding to lipid or lipid mixtures particle and of interaction
with scavenger
receptor type I (SRBI). Thus, in this case, both -
GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) and -RGD- are
considered a "minimal biologically active sequence." As is understood from the
present
invention, the first and second amino acid domains of a resulting peptide
contain at least one
minimal biologically active sequence. This minimal biologically active
sequence is any length of
sequence from an original peptide sequence. Moreover, with the exception of
the amino acids of
the minimal biologically active sequence, the amino acids of any or both amino
acid domain can
be exchanged, added or removed according to the design of the molecule to
adjust its overall
hydrophilicity and/or net charge. In certain embodiments, the minimal
biologically active
sequence refers to any one of the sequences provided in TABLE 2.
The term "imaging agent" or "imaging probe" as used herein refers to contrast
agents
used in imaging techniques such as computed tomography (CT), gamma-
scintigraphy, positron
emission tomography (PET), single photon emission computed tomography (SPECT),
magnetic
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resonance imaging (MM), and combined imaging techniques in order to improve
diagnostic
performance of medical imaging.
The term "labeling substance or label or labeled probe" refers to a substance
that can
image whether there is a binding between the modulator and the cellular
component (e.g.,
TREM-1/DAP-12 receptor complex), and can visualize the binding by a pattern.
It may include
radioactive materials, fluorescent or emitting materials.
The term "carrier" as used herein, refers to a biocompatible nanoparticle that
facilitates
administration of a pharmaceutical agent to an individual.
The term "encapsulation" as used herein refers to the enclosure of a molecule,
such as
trifunctional peptides and compounds of the present invention, inside the
nanoparticle. The term
"incorporation" as used herein refers to imbibing or adsorbing the
trifunctional peptides and
compounds onto the nanoparticle. The terms "reconstituted" and "recombinant"
as used herein
both refer to synthetic lipopeptide particles that represent both discoidal
and spherical
nanoparticles and mimic native HDL particles.
As used herein, "naturally occurring" means found in nature. A naturally
occurring
biomolecule is, in general, synthesized by an organism that is found in nature
and is unmodified
by the hand of man, or is a degradation product of such a molecule. A molecule
that is
synthesized by a process that involves the hand of man (e.g., through chemical
synthesis not
involving a living organism or through a process that involves a living
organism that has been
manipulated by the hand of man or is descended from such an organism) but that
is identical to a
molecule that is synthesized by an organism that is found in nature and is
unmodified by the
hand of man is also considered a naturally occurring molecule.
A "site of interest" on a target as used herein is a site to which modified
peptides and
compounds of the present invention bind.
The term "target site", as used herein, refers to sites/tissue areas of
interest.
As used in this invention, the terms "target cells" or "target tissues" refer
to those cells or
tissues, respectively that are intended to be targeted using the compositions
of the present
invention delivered in accord with the invention. Target cells or target
tissues take up or link
with the modified peptides and compounds of the invention. As used in this
invention, the terms
"target cells" or "target tissues" refer to those cells or tissues,
respectively that are intended to be
treated and/or visualized in imaging techniques such as CT, gamma-
scintigraphy, PET, SPECT,
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Mill, and combined imaging techniques, using the compositions of the present
invention
delivered in accord with the invention. Target cells are cells in target
tissue, and the target tissue
includes, but is not limited to, atherosclerotic plaques, vascular endothelial
tissue, abnormal
vascular walls of tumors, solid tumors, tumor-associated macrophages, and
other tissues or cells
related to cancer, cardiovascular, inflammatory, autoimmune diseases, and the
like. Further,
target cells include virus-containing cells, and parasite-containing cells.
Also included among
target cells are cells undergoing substantially more rapid division as
compared to non-target
cells.
The term "target cells" also includes, but is not limited to, microorganisms
such as
bacteria, viruses, fungi, parasites, and infectious agents. Thus, the term
"target cell" is not limited
to living cells but also includes infectious organic particles such as
viruses. "Target
compositions" or "target biological components" include, but are not be
limited to: toxins,
peptides, polymers, and other compounds that may be selectively and
specifically identified as
an organic target that is intended to be visualized in imaging techniques
using the compositions
of the present invention.
The term "therapeutic agent" or "drug" as used herein refers to any compound
or
composition having preventive, therapeutic or diagnostic activity, primarily
but not exclusively
in the treatment of patients with macrophage (myeloid cell)-related diseases.
The term "myeloid
cells" include monocytes, macrophages, neutrophils, basophils, eosinophils,
erythrocytes, and
megakaryocytes to platelets.
The terms "macrophage-associated", "macrophage-mediated", and "macrophage-
related
diseases" include diseases associated with macrophages as disclosed in Low and
Turk, US
8,916,167, herein incorportaed by reference in its entirety.
The term "plaque" includes, for example, an atherosclerotic plaque.
The term "myeloid cell-mediated pathology" (or "myeloid cell-related
pathologies", or
"myeloid cell-mediated disorder, or "myeloid cell-related disease"), as used
herein, refers to any
condition in which an inappropriate myeloid cell response is a component of
the pathology. The
term is intended to include both diseases directly mediated by myeloid cells,
and also diseases in
which an inappropriate myeloid cell response contributes to the production of
abnormal
antibodies, antibodies, as well as graft rejection.
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The term "ligand-induced myeloid cell activation", as used herein, refers to
myeloid cell
activation in response to the stimulation by the specific ligand.
The term "stimulation", as used herein, refers to a primary response induced
by ligation
of a cell surface moiety. For example, in the context of receptors, such
stimulation entails the
ligation of a receptor and a subsequent signal transduction event. With
respect to stimulation of a
myeloid cell, such stimulation refers to the ligation of a myeloid cell
surface moiety that in one
embodiment subsequently induces a signal transduction event, such as binding
the TREM-
1/DAP- 12 complex. Further, the stimulation event may activate a cell and up-
regulate or down-
regulate expression or secretion of a molecule.
The term "ligand", or "antigen", as used herein, refers to a stimulating
molecule that
binds to a defined population of cells. The ligand may bind any cell surface
moiety, such as a
receptor, an antigenic determinant, or other binding site present on the
target cell population. The
ligand may be a protein, peptide, antibody and antibody fragments thereof,
fusion proteins,
synthetic molecule, an organic molecule (e.g., a small molecule), or the like.
Within the
specification and in the context of myeloid cell stimulation, the ligand (or
antigen) binds the
TREM receptor and this binding activates the myeloid cell.
The term "activation", as used herein, refers to the state of a cell following
sufficient cell
surface moiety ligation to induce a noticeable biochemical or morphological
change. Within the
context of myeloid cells, such activation, refers to the state of a myeloid
cell that has been
sufficiently stimulated to induce production of interleukin (IL) 1, 6 and/or 8
(IL-1, IL-6 and/or
IL-8, respectively) and tumor necrosis factor alpha (TNF-alpha),
differentiation of primary
monocytes into immature dendritic cells, and enhancement of inflammatory
responses to
microbial products. Within the context of other cells, this term infers either
up or down
regulation of a particular physico-chemical process.
The term "inhibiting myeloid cell activation" (or "TREM-mediated cell
activation"), as
used herein, refers to the slowing of myeloid cell activation, as well as
completely eliminating
and/or preventing myeloid cell activation.
The term, "treating a disease or condition", as used herein, refers to
modulating myeloid
cell activation including, but not limited to, decreasing cytokine production
and differentiation of
primary monocytes into immature dendritic cells and/or slowing myeloid cell
activation, as well
as completely eliminating and/or preventing myeloid cell activation. Myeloid
cell-related
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diseases and/ or conditions treatable by modulating myeloid cell activation
include, but are not
limited to, cancer including but not limited to lung cancer, pancreatic
cancer, multiple myeloma,
melanoma, leukemia, prostate cancer, breast cancer, liver cancer, bladder
cancer, stomach
cancer, prostate cancer, colon cancer, colorectal cancer, CNS cancer,
melanoma, ovarian cancer,
gastrointestinal cancer, renal cancer, or osteosarcoma and other cancers,
brain and skin cancers,
endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer,
neuroblastoma,
neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck
cancer, cervical
cancer, giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell
tumor (TGCT;
also referred to in the art as TSGCT), PVNS and other cancers in which myeloid
cells are
involved or recruited, cancer cachexia, in addition to ALD, atherosclerosis,
allergic diseases,
acute radiation syndrome, inflammatory bowel disease, empyema, alcohol-induced
liver disease,
nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, acute
mesenteric ischemia,
hemorrhagic shock, multiple sclerosis, autoimmune diseases, including but not
limited to, atopic
dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and
other rheumatic
diseases, sepsis, diabetic retinopathy and retinopathy of prematurity,
Alzheimer's, Parkinson's
and Huntington's diseases, and other myeloid cell-related inflammatory
conditions eg myositis,
tissue/organ rejection, brain and spinal cord injuries. Other exemplary
cancers include, but are
not limited to, adrenocortical carcinoma, acquired immune deficiency syndrome
(AIDS)-related
cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the
anal canal,
appendix cancer, childhood cerebellar astrocytoma, childhood cerebral
astrocytoma, basal cell
carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct
cancer,
intrahepatic bile duct cancer, bladder cancer, uringary bladder cancer, bone
and joint cancer,
osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor,
brain stem glioma,
cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,
medulloblastoma,
supratentorial primitive neuroectodeimal tumors, visual pathway and
hypothalamic glioma,
bronchial adenomas/carcinoids, carcinoid tumor, nervous system cancer, nervous
system
lymphoma, central nervous system lymphoma, cervical cancer, childhood cancers,
chronic
lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative
disorders,
cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary
Syndrome,
esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor,
extrahepatic bile
duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder
cancer,
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gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ
cell tumor, ovarian
germ cell tumor, gestational trophoblastic tumor glioma, Hodgkin lymphoma,
hypopharyngeal
cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine
pancreas), Kaposi
Sarcoma, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute
myeloid leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell
leukemia, lip and oral
cavity cancer, lung cancer, small cell lung cancer, AIDS-related lymphoma, non-
Hodgkin
lymphoma, primary central nervous system lymphoma, Waldenstram
macroglobulinemia,
medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma,
mesothelioma
malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer
of the tongue,
multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic
syndromes,
myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia,
acute myeloid
leukemia, multiple myeloma, chronic myeloproliferative disorders,
nasopharyngeal cancer,
neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian
epithelial cancer,
ovarian low malignant potential tumor, islet cell pancreatic cancer, paranasal
sinus and nasal
.. cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma,
pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary
tumor, plasma cell
neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal
cancer, renal
pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland
cancer, ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma,
uterine cancer,
.. uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel
cell skin
carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell
carcinoma, supratentorial
primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma,
thymoma and
thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis
and ureter and other
urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial
uterine cancer,
.. uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and
Wilm's Tumor.
The term "detectable" refers to the ability to detect a signal over the
background signal.
In accordance with the present disclosure, "a detectably effective amount" of
the labeled
probe of the present disclosure is defined as an amount sufficient to yield an
acceptable image
using equipment that is available for clinical use. A detectably effective
amount of the labeled
probe of the present disclosure may be administered in more than one
injection. The detectably
effective amount of the labeled probe of the present disclosure can vary
according to factors such
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as the degree of susceptibility of the individual, the age, sex, and weight of
the individual,
idiosyncratic responses of the individual, and the like.
Detectably effective amounts of the probe of the present disclosure can also
vary
according to instrument and film-related factors. Optimization of such factors
is well within the
level of skill in the art.
The term "in vivo imaging" as used herein refers to methods or processes in
which the
structural, functional, molecular, or physiological state of a living being is
examinable without
the need for a life-ending sacrifice.
The term "inhibiting T cell activation", as used herein, refers to the slowing
of T cell
activation, as well as completely eliminating and/or preventing T cell
activation.
The term "T cell-mediated pathology" (or "T cell-related pathologies", or "T
cell-
mediated disorder, or "T cell-related disease"), as used herein, refers to any
condition in which
an inappropriate T cell response is a component of the pathology. The term is
intended to include
both diseases directly mediated by T cells, and also diseases in which an
inappropriate T cell
response contributes to the production of abnormal antibodies, as well as
graft rejection.
The term "treating a T cell-mediated disease or condition", as used herein,
refers to
modulating T cell activation including, but not limited to, decreasing
cellular proliferation,
cytokine production and performance of regulatory or cytolytic effector
functions and/or slowing
T cell activation, as well as completely eliminating and/or preventing T cell
activation. T cell-
related diseases and/or conditions treatable by modulating T cell activation
include, but are not
limited to, systemic lupus erythematosus, rheumatoid arthritis, psoriatic
arthritis, multiple
sclerosis, type I diabetes, gastroenterological conditions e.g. inflammatory
bowel disease,
Crohn's disease, celiac, Guillain-Barre syndrome, Hashimotos disease,
pernicious anaemia,
primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic
dermatitis, psoriasis,
pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis,
allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes
simplex/zoster, respiratory
conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis,
ankylosing spondylitis,
tissue/organ rejection.
The term, "subject" or "patient", as used herein, refers to any individual
organism. For
example, the organism may be a mammal, such as a primate (i.e., for example, a
human) or a
laboratory animal. Further, the organism may be a domesticated animal (i.e.,
for example, cats,
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dogs, etc.), livestock (i.e., for example, cattle, horses, pigs, sheep, goats,
etc.), or a laboratory
animal (i.e., for example, mouse, rabbit, rat, guinea pig, etc.).
The term, "therapeutically effective amount", "therapeutically effective dose"
or
"effective amount", as used herein, refers to an amount needed to achieve a
desired clinical result
.. or results (e.g. inhibiting receptor-mediated cell activation) based upon
trained medical
observation and/or quantitative test results. The potency of any administered
peptide or
compound determines the "effective amount" which can vary for the various
compounds that
inhibit myeloid cell activation (i.e., for example, compounds inhibiting TREM
ligand-induced
myeloid cell activation and/or TCR-mediated T cell activation). Additionally,
the "effective
amount" of a compound may vary depending on the desired result, for example,
the level of
myeloid cell activation inhibition desired. The "therapeutically effective
amount" necessary for
inhibiting differentiation of primary monocytes into immature dendritic cells
may differ from the
"therapeutically effective amount" necessary for preventing or inhibiting
cytokine production.
The term, "agent", as used herein, refers to any natural or synthetic compound
(i.e., for
example, a peptide, a peptide variant, or a small molecule).
The term, "intrinsic helicity", as used herein, refers to the helicity which
is adopted by a
peptide in an aqueous solution. The term, "induced helicity", as used herein,
refers to the helicity
which is adopted by a peptide when in the presence of a helicity inducer,
including, but not
limited to, trifluoroethanol (TFE), detergents (e.g., sodium dodecyl sulfate,
SDS) or lipids.
The term "therapeutic drug", as used herein, refers to any pharmacologically
active
substance capable of being administered which achieves a desired effect. Drugs
or compounds
can be synthetic or naturally occurring, non-peptide, proteins or peptides,
oligonucleotides or
nucleotides, polysaccharides or sugars. Drugs or compounds may have any of a
variety of
activities, which may be stimulatory or inhibitory, such as antibiotic
activity, antiviral activity,
antifungal activity, steroidal activity, cytotoxic, cytostatic, anti-
proliferative, anti-inflammatory,
analgesic or anesthetic activity, or can be useful as contrast or other
diagnostic agents.
The term "effective dose" as used herein refers to the concentration of any
compound or
drug contemplated herein that results in a favorable clinical response. In
solution, an effective
dose may range between approximately 1 ng/ml and 100 mg/ml, preferably between
100 ng/ml
and 10 mg/ml, but more preferably between 500 ng/ml and 1 mg/ml.
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The term "effective amount" or "therapeutically effective amount" refers to an
amount of
a drug effective to treat a disease or disorder in a subject. In certain
embodiments, an effective
amount refers to an amount effective, at dosages and for periods of time
necessary, to achieve the
desired therapeutic or prophylactic result. A therapeutically effective amount
of the compound or
composition of the invention that modulate TREM-1/DAP-12 receptor complex
signaling may
vary according to factors such as the disease state, age, sex, and weight of
the individual, and the
ability of the compound or composition to elicit a desired response in the
individual. A
therapeutically effective amount encompasses an amount in which any toxic or
detrimental
effects of the compound or composition are outweighed by the therapeutically
beneficial effects.
As one example, in some embodiments, the expression "effective amount" refers
to an amount of
the compound or composition that is effective for treating cancer.
A "prophylactically effective amount" refers to an amount effective, at
dosages and for
periods of time necessary, to achieve the desired prophylactic result.
Typically, but not
necessarily, since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease,
the prophylactically effective amount would be less than the therapeutically
effective amount.
A "induction therapy" refers to the first treatment given for a disease. It is
often part of a
standard set of treatments, such as surgery followed by chemotherapy and
radiation. When used
by itself, induction therapy is the one accepted as the best treatment. If it
doesn't cure the disease
or it causes severe side effects, other treatment may be added or used
instead. Also called first-
line therapy, primary therapy, and primary treatment.
A "maintenance therapy" refers to a medical therapy that is designed to help a
primary
treatment succeed. For example, maintenance chemotherapy may be given to
people who have a
cancer in remission in an attempt to prevent a relapse. In other words,
treatment that is given to
help keep cancer from coming back after it has disappeared following the
initial therapy. It may
include treatment with drugs, vaccines, or antibodies that kill cancer cells
or keep tumor
unfavorable microenvironment, and it may be given for a long time. This form
of treatment is
also a common approach for the management of many incurable, chronic diseases
such as
periodontal disease, Crohn's disease or ulcerative colitis.
Administration "in combination with" one or more further therapeutic agents
includes
.. simultaneous (concurrent) and consecutive (sequential) administration in
any order.
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A "pharmaceutically acceptable carrier" refers to a non-toxic solid,
semisolid, or liquid
filler, diluent, encapsulating material, formulation auxiliary, or carrier
conventional in the art for
use with a therapeutic agent that together comprise a "pharmaceutical
composition" for
administration to a subject. A pharmaceutically acceptable carrier is non-
toxic to recipients at the
dosages and concentrations employed and is compatible with other ingredients
of the
formulation. The pharmaceutically acceptable carrier is appropriate for the
formulation
employed. For example, if the therapeutic agent is to be administered orally,
the carrier may be a
gel capsule. If the therapeutic agent is to be administered subcutaneously,
the carrier ideally is
not irritable to the skin and does not cause injection site reaction.
The term "administered" or "administering" a drug or compound, as used herein,
refers to
any method of providing a drug or compound to a patient such that the drug or
compound has its
intended effect on the patient. For example, one method of administering is by
an indirect
mechanism using a medical device such as, but not limited to a catheter,
syringe etc. A second
exemplary method of administering is by a direct mechanism such as, local
tissue administration
(i.e., for example, extravascular placement), oral ingestion, transdermal
patch, topical, inhalation,
suppository etc.)
The term, "agent", as used herein, refers to any natural or synthetic compound
(i.e., for
example, a peptide, a peptide variant, or a small molecule).
The term, "composition", as used herein, refers to any mixture of substances
comprising
a peptide and/or compound contemplated by the present invention. Such a
composition may
include the substances individually or in any combination.
The term "modulator" used in this invention refers to a substance and/or
compositions
contemplated by the present invention or a combination thereof with capacity
to inhibit (e.g.,
"antagonist" activity) a functional property of biological activity or process
(e.g., reducing or
blocking TREM-1/DAP-12 activity ¨ signaling and/or activation); such
inhibition can be
contingent on the occurrence of a specific event, such as reduction or
blockade of a signal
transduction pathway, and/or can be manifest only in particular cell types.
For instance, small
molecules such as drugs, proteins such as antibodies, hormones or growth
factors, protein
domains, protein motifs, and peptides or a combination thereof can act as a
modulator.
The term "tissue sample" refers to a collection of similar cells obtained from
a tissue of a
subject. The source of the tissue sample may be solid tissue as from a fresh,
frozen and/or
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preserved organ or tissue sample or biopsy or aspirate; blood or any blood
constituents; bodily
fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,
synovial fluid, or interstitial
fluid; cells from any time in gestation or development of the subject. In some
embodiments, a
tissue sample is a synovial biopsy tissue sample and/or a synovial fluid
sample. In some
.. embodiments, a tissue sample is a synovial fluid sample. The tissue sample
may also be primary
or cultured cells or cell lines. Optionally, the tissue sample is obtained
from a disease
tissue/organ. The tissue sample may contain compounds that are not naturally
intermixed with
the tissue in nature such as preservatives, anticoagulants, buffers,
fixatives, nutrients, antibiotics,
or the like. A "control sample" or "control tissue", as used herein, refers to
a sample, cell, or
tissue obtained from a source known, or believed, not to be afflicted with the
disease for which
the subject is being treated.
For the purposes herein a "section" of a tissue sample means a part or piece
of a tissue
sample, such as a thin slice of tissue or cells cut from a solid tissue
sample.
The term "anti-inflammatory drug" means any compound, composition, or drug
useful
for preventing or treating inflammatory disease.
The term "medical device", as used herein, refers broadly to any apparatus
used in
relation to a medical procedure. Specifically, any apparatus that contacts a
patient during a
medical procedure or therapy is contemplated herein as a medical device.
Similarly, any
apparatus that administers a drug or compound to a patient during a medical
procedure or
therapy is contemplated herein as a medical device. "Direct medical implants"
include, but are
not limited to, urinary and intravascular catheters, dialysis catheters, wound
drain tubes, skin
sutures, vascular grafts and implantable meshes, intraocular devices,
implantable drug delivery
systems and heart valves, and the like. "Wound care devices" include, but are
not limited to,
general wound dressings, non-adherent dressings, burn dressings, biological
graft materials, tape
closures and dressings, surgical drapes, sponges and absorbable hemostats.
"Surgical devices"
include, but are not limited to, surgical instruments, endoscope systems
(i.e., catheters, vascular
catheters, surgical tools such as scalpels, retractors, and the like) and
temporary drug delivery
devices such as drug ports, injection needles etc. to administer the medium. A
medical device is
"coated" when a medium comprising an anti-inflammatory drug (i.e., for
example, the peptides,
compositions, and compounds of the present invention) becomes attached to the
surface of the
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medical device. This attachment may be permanent or temporary. When temporary,
the
attachment may result in a controlled release of an inflammatory drug.
DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating
cancer and
other diseases related to activated immune cells using modulators of the TREM-
1/DAP-12
signaling pathway. The compositions, including peptides and peptide variants,
modulate TREM-
1-mediated immunological response as standalone and combination-therapy
treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory
therapies in
patients. In one embodiment, the present invention relates to targeted
treatment, prevention
and/or detection of cancer including but not limited to lung cancer including
non-small cell lung
cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial
giant cell tumor,
pigmented villonodular synovitis, cancer cachexia, etc., and other cancers
associated with
myeloid cell activation and recruitment. Additionally, the present invention
relates to the targeted
treatment, prevention and/or detection of scleroderma including but not
limited to calcinosis,
Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia
syndrome
(CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and
therapeutic
compositions comprising such trifunctional peptides for use in treating
diseases related to
activated macrophages. In some embodiments, each trifunctional peptide is
capable of at least
three functions: 1) mediating formation of naturally long half-life
lipopeptide/lipoprotein
particles upon interaction with lipoproteins, 2) facilitation of the targeted
delivery to cells of
interest and/or sites of disease, and 3) treatment, prevention, and/or
detection of a disease or
condition. In certain embodiments, the present invention relates to
amphipathic trifunctional
peptides consisting of two amino acid domains, wherein upon interaction with
plasma
lipoproteins, one amino acid domain mediates formation of naturally long half-
life
lipopeptide/lipoprotein particles and targets these particles to macrophages,
whereas the other
amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex
expressed on
macrophages.
As described herein, surprisingly it was found that potentially therapeutic
trifunctional
peptides of the present invention are capable of executing at least, three
functions (trifunctional
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peptides): 1) assistance in the self-assembly of naturally long half-life
lipopeptide particles upon
binding to lipid or lipid mixtures in vitro, i.e. incorporation of the
trifunctional peptides as part of
the lipid portion of synthetic/recombinant HDLs, then after administration; 2)
facilitation of the
targeted delivery to cells of interest and/or sites of disease, and 3)
treatment, prevention, and/or
detection of a disease or condition. Thus in some embodiments, trifunctional
peptides, after
mixing with lipids in vitro, may assist in the self-assembly of synthetic
lipopeptide particles
(SLP) upon binding to a lipid or to lipids in mixtures. In the methods of the
present invention, the
SLP of interest are synthetic nanoparticles that mimic human lipoproteins as
recombinant
(r)HDLs. While not being bound to any particular theory, it is believed that
this interaction and
ability to form lipopeptide/lipoprotein particles is mediated by the
amphipathic alpha helical
sequences of the trifunctional peptides described herein.
Another surprising discovery was that administration of potentially
therapeutic
trifunctional peptides of the present invention, that were not in rHDL
formulations, showed: 1)
mediation of formation of naturally long half-life lipopeptide/lipoprotein
particles (LP) upon
interaction with native lipoproteins in vivo, 2) facilitation of the targeted
delivery to cells of
interest and/or sites of disease, and 3) treatment, prevention, and/or
detection of a disease or
condition. Thus in some embodiments, free trifunctional peptides (i.e. not in
rHDL formulations)
as part of compounds of the present invention, after administration to
populations of cells or
administration to a mammal, may interact with native lipoproteins and form
trifunctional peptide
.. containing lipopeptide/lipoprotein particles in vivo.
Thus, potentially therapeutic trifunctional peptides of the present invention
were
synthesized and used for targeted treatment and imaging in vivo, as either
formulations with
HDLs or without, i.e. trifunctional peptides in a pharmaceutical formulation
without HDLs.
Advantageous of using the trifunctional peptides described herein in order to
solve
numerous problems administering therapeutic or diagnostic compounds include
avoiding high
dosages of other TAs (therapeutic agents) and imaging probes required; and the
lack of control
and reproducibility of formulations, especially in large-scale production. In
other words, using
trifunctional peptides described herein, including trifunctional peptide
formulations including
therapeutic drug compounds, would potentially lower the amount of drug needed
to reduce
symptoms of a disease.
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Another advantage is economic. Therapeutic peptides have relatively high
synthetic and
production costs, For example, the production cost of a 5000 Da molecular mass
peptide exceeds
the production cost of a 500 Da molecular mass small molecule, which in turn
exceeds the
production cost of a 500 Da molecular mass small molecule by more than 10-fold
up to less than
100-fold for each increase in magnitude of size. By combining three functions
in one peptide
significantly simplifies the manufacture of these trifunctional peptides as
targeted drugs, and as
delivery agents for drug compounds and imaging probes.
I. Trifunctional Peptides.
The present invention encompasses the discovery that it is possible to combine
multiple
functions in one polypeptide amino acid sequence, i.e. a trifunctional
peptide, in order to confer a
variety of properties on the resulting amphipathic multipeptide.
The present disclosure describes novel amphipathic trifunctional peptides and
therapeutic
compositions comprising such trifunctional peptides for use in treating
diseases related to
activated immune cells. In some embodiments, each trifunctional peptide is
capable of at least
three functions: 1) mediating formation of naturally long half-life
lipopeptide/lipoprotein
particles upon interaction with lipoproteins, 2) facilitation of the targeted
delivery to cells of
interest and/or sites of disease, and 3) treatment, prevention, and/or
detection of a disease or
condition. In some embodiments, each trifunctional peptide is capable of at
least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide
particles upon binding to
lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of
interest and/or sites of
disease, and 3) treatment, prevention, and/or detection of a disease or
condition. In certain
embodiments, the present invention relates to amphipathic trifunctional
peptides consisting of
two amino acid domains, wherein upon interaction with plasma lipoproteins, one
amino acid
domain mediates formation of naturally long half-life lipopeptide/lipoprotein
particles and
targets these particles to macrophages, whereas the other amino acid domain
inhibits the TREM-
1/DAP-12 receptor signaling complex expressed on macrophages. The invention
further relates to
personalized medical treatments for cancer that involve targeting specific
cancers by their tumor
environment.
In preferred embodiments, trifunctional peptides of the present invention
comprise two
amino acid domains (See FIG. 1): domain A that confers therapeutic and/or
diagnostic benefits
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in the context of the treatment, prevention, and/or detection of a disease or
condition; and
domain B that confers multiple benefits in the context of: 1A) formation of
long half-life
lipopeptide particles upon binding to lipid or lipid mixtures in vitro 1B)
formation of long half-
life LP upon interaction with lipoproteins in vivo, and 2) the targeted
delivery of the particles
formed to cells of interest and/or sites of disease or condition.
In one embodiment, the present invention includes a resulting trifunctional
peptide
comprising: (a) one amino acid domain that confers therapeutic and/or
diagnostic benefits in the
context of the treatment, prevention, and/or detection of a disease or
condition; and (b) another
amino acid domain that confers multiple benefits in the context of the self-
assembly of naturally
long half-life SLP and LP upon binding to lipid or lipid mixtures and targeted
delivery of the
particles formed to cells of interest and/or sites of disease or condition. In
one embodiment, any
or both the domains comprise minimal biologically active amino acid sequence.
In one
embodiment, the first amino acid domain comprises a cyclic peptide sequence.
In one
embodiment, the first amino acid domain comprises a disulfide-linked dimer. In
one
embodiment, any or both of the amino acid domains include amino acids selected
from the group
of natural and unnatural amino acids including, but not limited to, L-amino
acids, or D-amino
acids.
In one embodiment, one or both amino acid domains of the peptides and
compounds of
the present invention are conjugated to a drug compound (therapeutic agent:
TA). In one
.. embodiment, a therapeutic agent is selected from the group including, but
not limited to,
anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and
cardiovascular agents,
antioxidants, and therapeutic peptides. In one embodiment, the therapeutic
agent is a
hydrophobic therapeutic agent. The therapeutic agent may also be selected from
the group
comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin,
etoposide, and any
combination thereof.
In one embodiment, one or both amino acid domains of the peptides and
compounds of
the present invention are conjugated to an imaging probe. In one embodiment,
the imaging agent
is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MM). In
one
embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging
systems such
as a positron emission tomography (PET) imaging systems (and combined
PET/computer
tomography (CT) and PET/MM systems). In one embodiment, an imaging probe
and/or an
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additional therapeutic agent is conjugated to any or both of the domains. In
one embodiment, the
peptides and compounds of the present invention are used in combinations
thereof
Although many examples describe or show results of using trifunctional
peptides in
formulations with rHDLs, it is not meant to limit the use of these
trifunctional peptide sequences
in HDL formulations. Conversely, examples describing or showing results of
using trifunctional
peptides alone, or in formulations without rHDLs is not meant to limit the use
of such
trifunctional peptides without rHDLs. Thus, in certain embodiments, the
trifunctional peptides of
the present invention may be administered within rHDLs, or administered in
pharmaceutical
formulations as part of rHDLs. In other embodiments, the trifunctional
peptides of the present
invention may be administered without rHDLs, or administered in pharmaceutical
formulations
without rHDLs.
In one embodiment, the peptides of the present invention form lipopeptide
particles in
vitro. In one embodiment, the peptides of the present invention form
lipopeptide particles in vivo.
In certain embodiments, the present invention relates to peptides consisting
of two amino acid
domains, wherein upon binding to lipid or lipid mixtures, one amino acid
domain assists in the
self-assembly of naturally long half-life lipopeptide particles and targets
these particles to
macrophages, whereas another amino acid domain inhibits TREM-1/DAP-12 receptor
complex
expressed on macrophages.
In certain embodiments, the present invention relates to peptides comprising
at least two
amino acid domains, wherein upon binding to lipid or lipid mixtures, the first
amino acid domain
assists in the self-assembly of naturally long half-life lipopeptide particles
and targets these
particles to macrophages, whereas the second amino acid domain inhibits TREM-
1/DAP-12
receptor complex expressed on macrophages.
In certain embodiments, the peptides of the present invention self-assemble
upon binding
to lipid or lipid mixtures in vitro to form synthetic lipopeptide particles
(SLP) that mimic human
lipoproteins and have a long half-life in a bloodstream. In one embodiment,
the peptides and
compounds of the present invention interact with endogenous lipoproteins in
vivo and form long
half-life LP. In one embodiment, the peptides and compounds of the present
invention are used
in combinations thereof.
The peptides and compounds of the present invention and combinations thereof
alone as
well as the SLP formed upon their binding to lipid or lipid mixtures have a
wide variety of uses,
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particularly in the areas of oncology, transplantology, dermatology,
hepatology, ophthalmology,
cardiovascular diseases, sepsis, autoimmune diseases, neurodegenerative
diseases and other
diseases and conditions. They also are useful in the production of medical
devices (for example,
medical implants and implantable devices).
The invention disclosed herein provides for methods of treating cancer using
inhibitors of
the TREM-1 pathway. These inhibitors include peptide variants and compositions
that modulate
the TREM-1-mediated immunological responses beneficial for the treatment of
cancer. The
invention also provides for predicting the efficacy of TREM-1-targeted
therapies in various
cancers by analyzing biological samples for the presence of myeloid cells and
for the TREM-1
expression levels. In one embodiment, the present invention relates to the
targeted treatment,
prevention and/or detection of cancer including but not limited to pancreatic
cancer, breast
cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer,
stomach cancer,
prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin
cancer, osteosarcoma
and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using
inhibitors of
the TREM-1 pathway. These inhibitors include peptide variants and compositions
that modulate
the TREM-1-mediated immunological responses beneficial for the treatment of
cancer. The
invention also provides for predicting the efficacy of TREM-1-targeted
therapies in various
cancers by analyzing biological samples for the presence of myeloid cells and
for the TREM-1
expression levels. In one embodiment, the present invention relates to the
targeted treatment,
prevention and/or detection of cancer including but not limited to pancreatic
cancer, breast
cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer,
stomach cancer,
prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin
cancer, osteosarcoma
and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using
modulators
of the TREM-1/DAP-12 signaling pathway. These compounds and compositions
modulate the
TREM-1-mediated immunological responses beneficial for the treatment of cancer
in standalone
and combination-therapy treatment regimen. The invention also provides for
predicting the
efficacy of TREM-1 modulatory therapies in patients with various cancers. In
one embodiment,
the present invention relates to the targeted treatment, prevention and/or
detection of cancer
including but not limited to lung cancer including non-small cell lung cancer,
pancreatic cancer,
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breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder
cancer, central
nervous system cancer, stomach cancer, prostate cancer, colorectal cancer,
colon cancer, brain
cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer,
skin cancer,
osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid
cancer,
.. neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme,
head and neck
cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial
giant cell tumor,
pigmented villonodular synovitis, and other cancers in which myeloid cells are
involved or
recruited and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using
inhibitors of
the TREM-1 pathway. These inhibitors include peptide variants and compositions
that modulate
the TREM-1-mediated immunological responses beneficial for the treatment of
cancer. The
invention also provides for predicting the efficacy of TREM-1-targeted
therapies in various
cancers by analyzing biological samples for the presence of myeloid cells and
for the TREM-1
expression levels. In one embodiment, the present invention relates to the
targeted treatment,
prevention and/or detection of cancer including but not limited to pancreatic
cancer, breast
cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer,
stomach cancer,
prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin
cancer, osteosarcoma
and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating scleroderma
using
modulators of the TREM-1/DAP-12 signaling pathway. These compounds and
compositions
modulate the TREM-1-m edi ated immunological responses beneficial for the
treatment of
scleroderma or a related autoimmune or a fibrotic condition in standalone and
combination-
therapy treatment regimen. The invention also provides for predicting the
efficacy of TREM-1
modulatory therapies in patients with scleroderma. In one embodiment, the
present invention
relates to the targeted treatment, prevention and/or detection of scleroderma
including but not
limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility,
scleroderma, or
tel angi ectasi a syndrome (CREST).
In one embodiment, the present invention relates to the targeted treatment,
prevention
and/or detection of cancer including but not limited to lung, pancreatic,
breast, stomach, prostate,
colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic
diseases, acute radiation
syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia,
hemorrhagic
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shock, multiple sclerosis, autoimmune diseases, including but not limited to,
atopic dermatitis,
lupus, scleroderma, rheumatoid arthritis and other rheumatic diseases, sepsis
and other
inflammatory diseases or other condition involving myeloid cell activation
and, more
particularly, TREM receptor-mediated cell activation, including but not
limited to diabetic
retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and
Huntington's diseases.
The disclosure also provides for a method of treating, preventing and/or
detecting an
immune-related condition. The method comprises providing a composition
comprising peptides
and compounds of the present disclosure and/or a synthetic nanoparticle formed
upon their
binding to lipid or lipid mixtures, a patient having at least one symptom of a
disease or condition
in which the immune system is involved, and administering the composition to
the patient under
conditions such that said one symptom is reduced. The immune-related condition
of the method
may include a heart disease, atherosclerosis, peripheral artery disease,
restenosis, stroke, multiple
sclerosis, the cancers (e.g., sarcoma, lymphoma, leukemia, carcinoma and
melanoma), bacterial
infectious diseases, acquired immune deficiency syndrome (AIDS), allergic
diseases,
autoimmune diseases (e.g., atopic dermatitis, psoriasis, rheumatoid arthritis,
Sjogren's syndrome,
scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's
disease,
psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,
tuberculosis,
sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's
disease,
inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as
chronic obstructive
.. pulmonary disease ( COPD ), interstitial pneumonitis and asthma,
retinopathy such as diabetic
retinopathy and retinopathy of prematurity, inflammatory bowel disease such as
Crohn's disease,
and inflammatory arthritis), liver diseases (e.g., alcoholic liver disease and
nonalcoholic fatty
liver disease), neurodegenerative diseases such as Alzheimer's, Parkinson's
and Huntington's
diseases, and transplant (e.g., heart/lung transplants) rejection reactions.
The invention relates to personalized medical treatments for scleroderma. More
specifically, the invention provides for treatment of scleroderma or a related
autoimmune or a
fibrotic condition by using inhibitors of the TREM-1/DAP-12 pathway. These
inhibitors include
peptide variants and compositions that modulate the TREM-1-mediated
immunological
responses beneficial for the treatment of scleroderma. In addition, the
invention provides for
predicting the efficacy of TREM-1-targeted therapies in scleroderma by
analyzing biological
samples for the presence of myeloid cells and for the TREM-1 expression
levels. In one
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embodiment, the peptides and compositions of the present invention modulate
TREM-1/DAP-12
receptor complex expressed on macrophages. In one embodiment, the peptides and
compositions
of the invention are conjugated to an imaging probe. In one embodiment, the
invention provides
for detecting the TREM-1-expressing cells and tissues in an individual with
scleroderma using
imaging techniques and the peptides and compositions of the invention
containing an imaging
probe. In one embodiment, the peptides and compositions of the invention are
used in
combinations thereof In one embodiment, the peptides and compositions of the
invention are
used in combinations with other antifibrotic therapeutic agents. In one
embodiment, the present
invention relates to the targeted treatment, prevention and/or detection of
scleroderma including
but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility,
scleroderma, or
telangiectasia syndrome (CREST).
Trifunctional Peptides In rHDL (SLP) Formulations.
In one embodiment, the SLP self-assembled upon binding of the peptides and
compounds
.. of the present invention and combinations thereof to lipid or lipid
mixtures are discoidal or
spherical in shape. While the size of the particles is preferably between 5 nm
and 50 nm, the
diameter may be up to 200 nm. In one embodiment, the lipid of the particles
may include
cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a
sphingolipid, a cationic lipid, a
diacylglycerol, or a triacylglycerol. And further, the phospholipid may
include
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine
(PS),
phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL),
sphingomyelin (SM), or
phosphatidic acid (PA). And even further, the cationic lipid can be 1,2-
dioleoy1-3-
trimethylammonium-propane (DOTAP). The lipid of the synthetic nanoparticle may
be
polyethylene glycol(PEG)ylated. In certain embodiments, the peptides and
compounds of the
present invention and/or the SLP and LP formed by these peptides and compounds
may pass the
blood-brain barrier (BBB). In one embodiment, the peptides and compounds of
the present
invention and/or the SLP and LP formed by these peptides and compounds may
pass the blood-
retinal barrier (BRB). In one embodiment, the peptides and compounds of the
present invention
and/or the SLP and LP formed by these peptides and compounds may pass the
blood-tumor
barrier (BTB).
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In certain embodiments, the peptides and compounds of the present invention
include an
amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or
E. In one
embodiment, the peptides and compounds of the present invention include an
amino acid
sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E and
Arginine-glycine-
aspartic acid (RGD)-peptide sequence. In certain embodiments, the peptides and
compounds of
the present invention include an amino acid sequence derived from
transmembrane domain
sequences of human or animal cell-surface receptors and of signaling subunits
thereof In certain
embodiments, the peptides and compounds of the present invention include an
amino acid
sequence derived from virus membrane fusion and structural proteins. In one
embodiment, the
peptides and compounds of the present invention include an amino acid sequence
derived from
apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E conjugated to a targeting
moiety to enhance the
targeting efficacy of the therapeutic agent. The targeting moiety may include
a polypeptide, an
antibody, a receptor, a ligand, a peptidomimetic agent, an aptamer or a
product of phage display.
In one embodiment, the amino acid domains of the peptides and compounds of the
present invention comprise unmodified or modified peptide sequences. The
modified peptide
sequence may contain at least one amino acid residue which is chemically or
enzymatically
modified. The modified amino acid residue may be an oxidized amino acid
residue. The oxidized
amino acid residue may be a methionine residue. The modified peptide sequence
may contain at
least one amino acid residue, which is oxidized, halogenated, or nitrated. The
modified peptide
sequence may include an amphipathic amino acid sequence.
In certain embodiments, the present invention relates to the targeted
treatment or
prevention of inflammatory or other condition involving myeloid cell
activation and, more
particularly, TREM receptor-mediated cell activation, such as cancer including
but not limited
to, lung, pancreatic, breast, stomach, prostate, colon, brain and skin
cancers, cancer cachexia,
atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory
bowel disease,
empyema, alcohol-induced liver disease, nonalcoholic fatty liver disease and
non-alcoholic
steatohepatitis, acute mesenteric ischemia, hemorrhagic shock, multiple
sclerosis, sepsis, diabetic
retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and
Huntington's diseases,
autoimmune diseases, including but not limited to, atopic dermatitis, lupus,
scleroderma,
rheumatoid arthritis and other rheumatic diseases.
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In one embodiment, the present invention provides a pharmaceutical composition
comprising the peptides and compounds and combinations thereof alone or the
SLP
nanoparticles self-assembled upon binding of these peptides and compounds to
lipid or lipid
mixtures.
A. TREM-1-related Trifunctional peptides.
TREM-1 is expressed on the majority of innate immune cells and to a lesser
extent on
parenchymal cells. Upon activation, TREM-1 can directly amplify an
inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly
associated with
infectious diseases, recent evidences demonstrate that TREM-1 receptor and its
signaling
pathways contribute to the pathology of non-infectious acute and chronic
inflammatory diseases,
including but not limiting to, rheumatoid arthritis, atherosclerosis, ischemia
reperfusion-induced
tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases,
retinopathies, and
cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res
2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-
2768, Tornai, et al.
Hepatology Communications 2018, in press, and Kuai, et al. US 2008/0247955).
In certain embodiments, a resulting trifunctional peptide of the present
invention
comprises two amino acid domains, wherein one domain comprises a variant TREM-
1 inhibitory
amino acid sequence and functions to inhibit TREM-1/DAP-12 receptor complex
expressed on
myeloid cells (e.g. macrophages), whereas another amino acid domain comprises
the chemically
and/or enzymatically modified amino acid sequence derived from apolipoprotein
amino acid
sequences and functions to assist in the self-assembly of SLP upon binding to
lipid or lipid
mixtures in vitro and/or to form LP in vivo, respectively, and to target these
particles to myeloid
cells (e.g. macrophages). In one embodiment, the TREM-1 inhibitory amino acid
domain is the
.. N-terminal domain of a resulting peptide. In one embodiment, the TREM-1
inhibitory amino
acid domain is the C-terminal domain of a resulting peptide. In one
embodiment, the TREM-1
inhibitory amino acid domain comprises a cyclic peptide sequence. In one
embodiment, the
TREM-1 inhibitory amino acid domain comprises a disulfide-linked dimer. In one
embodiment,
the TREM-1 inhibitory amino acid domain includes the group of natural and
unnatural amino
acids including, but not limited to, L-amino acids, or D-amino acids. In one
embodiment, an
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imaging agent is conjugated to the TREM-1 inhibitory amino acid domain or to
the
apolipoprotein amino acid sequence-derived domain or to both.
In some preferred embodiments, TREM-1-related peptides and associated
compositions
of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of
cell
receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of
modulating
TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself
2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006,
27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct
Biol 2018,
111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are
herein
incorporated by reference in their entirety.
In some preferred embodiments, peptides and compositions of this class further
comprise
the domain B comprising at least one modified or unmodified amphipathic alpha
helical peptide
fragment, such as a apo A-I and/or A-II peptide fragment, to form upon
interaction with lipid
and/or lipid mixtures. In certain embodiments, exemplary trifunctional
peptides comprise the
domain B comprises with the amino acid sequence selected from the amino acid
sequences of the
major HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22
amino acid residue-long peptide sequence of the apo A-I helix 4. In one
embodiment, this
sequence contains a modified amino acid residue. In one embodiment, this
modified amino acid
residue is methionine sulfoxide. In one embodiment, the domain B of the
peptides and
compositions of the invention comprises 22 amino acid residue-long peptide
sequence of the apo
A-I helix 6. In one embodiment, this sequence contains a modified amino acid
residue. In one
embodiment, this modified amino acid residue is methionine sulfoxide.
FIG. 1 presents an exemplary schematic representation of one embodiment of a
trifunctional
peptide of the present invention comprising amino acid domains A and B where
amino acid
domain A represents a therapeutic peptide sequence with or without an attached
drug compound
and/or imaging probe that functions to treat, prevent and/or detect a disease
or condition,
whereas amino acid domain B represents an amphipathic alpha helical peptide
sequence, with or
without an additional targeting peptide sequence, and functions to 1) assist
in the self-assembly
of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with
lipids or lipid
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mixtures in vitro, for use in transporting these trifunctional peptides as
lipoprotien nanoparticles
to sites of interest in vitro or in vivo and/or 2) form long half-life
lipopeptide/lipoprotein particles
upon interaction with endogenous lipoproteins for transporting these
trifunctional peptides to the
sites of interest. Endogenous lipoproteins may be lipoproteins added to or
found in cell cultures,
or lipoprotiens in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-
related
trifunctional peptides, TREM-1/TRIOPEP GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA,
M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine
residues of the
peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA)
(SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human
TREM-1
(UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a
(UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)). Peptide
GFLSKSLVF is also described without an attached apo I peptide domain, in, for
examples, WO
2011/047097 "Inhibition of trem receptor signaling with peptide variants."
Publication Date:
21.04.2011, U59981004B2 "Inhibition of TREM receptor signaling with peptide
variants."
Published June 5, 2014, each of which is herein incorporated by reference in
its entirety.
Sequence information was downloaded 10-25-, 10-26- or 10-27-2019.
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1,
Homo
sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo
sapiens
isoform CRA a:
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MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVF SVLF AVTLR SF VP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid
cells 1,
Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTL SF SWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid
cells 1,
Homo sapiens:
MRKTRLW GLLWMLF V SELRAATKLTEEKYELKEGQ TLDVKCDYTLEKF A S S QKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KST.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related
trifunctional
peptide (TREM-1/TRIOPEP). GE31 (GFL SKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE,
M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino
acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and
functions to
treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22
amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a
sulfoxidized
methionine residue and functions to assist in the self-assembly of synthetic
lipopeptide particles
(SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
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Imaging of TREM-1 expression.
Another way to evaluate the TREM-1 expression level is to use imaging
(visualization)
techniques and procedures. In one embodiment, FIG. 50 shows that the
fluorescently labeled
TREM-1/TRIOPEP peptide GE31 delivered to macrophages by the SLP particles
colocalizes
with TREM-1 expressed on these cells. See also (Rojas et al. 2018). As
described herein and in
(Rojas et al. 2018), TREM-1 inhibitory therapy using the modulators of the
TREM-1/DAP-12
signaling pathway results in reduction of tissue TREM-1 expression as measured
by Western
Blot (See Fig. 13).
In certain embodiments, the capability of the modulators of the TREM-1/DAP-12
signaling pathway described herein, including but not limited to, anti-TREM-1
blocking
antibodies and fragments thereof, TREM-1 inhibitory SCHOOL peptides (e.g.,
GF9) and
trifunctional TREM-1 inhibitory peptides including but not limited to, GA31
and GE31, to
colocalize with TREM-1 can be used to visualize (image) this receptor and
evaluate its
expression/level in the areas of interest. In one embodiment, for this
purpose, an imaging probe
(e.g. [64cu-.j,
see TABLE 3) can be conjugated to the peptide sequences, GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 27) and/or GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0),
methionine sulfoxide) (SEQ ID NO. 26). In one embodiment, methionine residues
of the
peptides GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. In
one embodiment, imaging (visualization) of TREM-1 levels using the labeled
modulators
described herein and the PET and/or other imaging techniques can be used to
diagnose GBM
and/or to select and monitor novel GBM therapies as disclosed in WO
2017083682A1 and
described in (Johnson et al. 2017, Liu et al. 2019). In certain embodiments,
imaging
(visualization) of TREM-1 levels can be used to diagnose other TREM-1-related
diseases and
conditions as well as to monitor novel therapies for these diseases and
conditions.
GF9 immunotherapy targets pathways restricted to pathological conditions and
is highly
competitive. In some embodiments, safe and effective GF9 therapies are
contemplated for use on
pancreatic cancer (PC) to be used in combination with standard first-line
treatments:
FOLFIRINOX (5-FU, leucovorin, irinotecan and oxaliplatin) or Gemzar +
ABRAXANE .
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In some embodiments, advantages for using free GF9 peptide for treating PVNS
include but are
not limited to: Low toxicity; Proven efficacy in vivo, including joints; easy
formulation
development; easy scale-up process; Easy and fast GMP production; Low cost of
production;
and Stable and easy to store.
Therapy*
* Shown for Cancer Acute toxicity Risk of side effects Administration Cost
Indications
Systemic /
GF9 immunotherapy Intranasal /
LOW LOW LOW
(as described herein) Pulmonary /
Oral
Cytotoxic drugs
(Gemzar, Abraxane, HIGH HIGH Systemic / Oral
HIGH
Temozol omi de)
Biologies LOW HIGH
Systemic HIGH
(Bevacizumab,
Canakinumab)
In certain embodiments, other preferred TREM-1-related trifunctional peptides
and
compositions of this class comprise the domain A comprising the TREM-1
inhibitory peptide
sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-
2830; Gibot,
et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am
Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in
press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et
al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and
Derive, US 9,255,136, each of which is herein incorporated by reference in its
entirety) while the
domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures,
SLP structures that
can be spherical or discoidal. In some embodiments, resulting trifunctional
peptide sequences
may be radiolabeled and/or contain unmodified or modified methionine residues
(TABLE 2)
including but not limiting to, the following
sequences:
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LQEEDAGEYGCNIPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide
(SEQ ID NO 7), LQEEDAGEYGCNIPYLDDFQKKWQEEM(0)ELYRQKVE (M(0),
methionine sulfoxide (SEQ ID NO
8),
LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide
(SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0),
methionine sulfoxide (SEQ ID NO 10).
SLP (rHDL) structures that can be spherical or discoidal (described herein and
in e.g.,
Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014,
21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in its entirety). The inclusion of an amphipathic
apo A-I sequences
aids the assistance in the self-assembly of SLP and the structural stability
of the particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31
(GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In
one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-
assembly of
nanosized SLP of discoidal or spherical morphology (dSLP and sSLP,
respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-
related
trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising
amino acid
domains A and B. Depending on lipid mixture compositions added to the
peptides, sub 50 nm-
sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-
1/TRIOPEP-
sSLP) morphology are self-assembled upon binding of the trifunctional peptide
to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
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In one embodiment, this provides targeted delivery of the SLP constituents
including
TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one
embodiment, this
provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to
tumor-
associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic
peptide
sequence and functions to treat and/or prevent a TREM-1-related disease or
condition (example,
for atherosclerosis), whereas domain B represents a 22 amino acids-long
apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and
functions to assist in
the self-assembly of synthetic lipopeptide particles (SLP) and to target the
particles to TREM-1-
expressing macrophages as applied to the treatment and/or prevention of
atherosclerosis. While
not being bound to any particular theory, it is believed that chemical and/or
enzymatic
modification of protein sequence in domain B leads to the recognition of SLP
of the present
invention by the macrophage scavenger receptors and results in an irreversible
binding to and
consequent uptake by macrophages of such particles. It is further believed
that accumulation of
these particles in intraplaque macrophages is accompanied by accumulation of
TRIOPEP in
these cells. In contrast, native HDL particles that contain only unmodified
apolipoprotein
molecules are not recognized by intraplaque macrophages and return to the
circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide
sequence and
functions to treat and/or prevent a TREM-1-related disease or condition
(example, for cancer),
whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6
peptide sequence
with a sulfoxidized methionine residue and functions to assist in the self-
assembly of synthetic
lipopeptide particles (SLP) and to target the particles to TREM-1-expressing
macrophages as
applied to the treatment and/or prevention of cancer. While not being bound to
any particular
theory, it is believed that chemical and/or enzymatic modification of protein
sequence in domain
B leads to the recognition of SLP of the present invention by the macrophage
scavenger
receptors and results in an irreversible binding to and consequent uptake by
macrophages of such
particles. It is further believed that accumulation of these particles in
tumor-associated
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macrophages is accompanied by accumulation of TRIOPEP in these cells. In
contrast, native
HDL particles that contain only unmodified apolipoprotein molecules are not
recognized by
tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one
embodiment,
this colocalization is accompanied by a specific disruption of intramembrane
interactions
between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the
present
invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1
upon ligand
binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and
Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein
incorporated by reference
in its entirety.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane
interactions
between TREM-1 and DAP-12 by the trifunctional peptide of the present
invention comprising
two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1
inhibitory
therapeutic peptide sequence, whereas domain B is a 22 amino acids-long
apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While
not being bound to
any particular theory, it is believed that this disruption results in "pre-
dissociation" of a receptor
complex and upon ligand stimulation, leads to inhibition of TREM-1 and
silencing the TREM-1
signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present
invention colocalizes
with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys
Acta 2018,
1864:2761-2768, each of which is herein incorporated by reference in its
entirety). In certain
embodiments, the capability of the TREM-1-related trifunctional peptides and
compounds of the
present invention including but not limiting to, TREM-1/TRIOPEP GA31 and TREM-
1/TRIOPEP GE31, to colocalize with TREM-1 can be used to visualize (image)
this receptor and
evaluate its expression in the areas of interest. In one embodiment, for this
purpose, an imaging
probe (e.g. [64Cu], see TABLE 2) can be conjugated to the TREM-1/TRIOPEP
sequences, GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine
sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-
1 levels using
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PET and/or other imaging techniques can be used to diagnose glioblastoma
multiforme (GBM)
and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017,
19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments,
imaging
(visualization) of TREM-1 levels can be used to diagnose other TREM-1-related
diseases and
conditions as well as to monitor novel therapies for these diseases and
conditions.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine
residue-
containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with
TREM-1 in
the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and
a merged
image (FIG. 6C).
As described herein (see FIG. 7), sulfoxidation of methionine residues in the
TREM-
1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis
of the SLP
containing an equimolar mixture of these peptides (designated as TREM-
1/TRIOPEP), TREM-
1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is
herein
incorporated by reference in its entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic
lipopeptide
particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that
contain an equimolar
mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31
and GE 31
(TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The
post 4 h
incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-
1/TRIOPEP-
sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine
residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified
methionine
residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and
TREM-
1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4
(white
bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001
to 0.001 as
compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free
and
SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-
mediated
release of pro-inflammatory cytokines, both in vitro (FIG. 8) and in vivo (in
serum) (FIG. 10).
.. While not being bound to any particular theory, it is believed that this
indicates that similarly to
TREM-1-inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014,
21:208-219;
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Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell
Mol Med 2017,
21:2524-2534, each of which is herein incorporated by reference in its
entirety), TREM-1-related
trifunctional peptides can reach their site of action from both outside (free
TREM-1/TRIOPEP)
and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon
administration,
free TREM-1/TRIOPEP may form LP in vivo and/or interact with native
lipoproteins, resulting
in formation of HDL-mimicking LP. In one embodiment, these LP may further
target the cells of
interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-
stimulated
macrophages incubated for 24 h at 37 C with an equimolar mixture of the
sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles
(SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged
macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post
lipopolysaccharide
(LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline
(PBS),
dexamethasone (DEX), control peptide and with an equimolar mixture of the
sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles
(SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
Control peptide represents an equimolar mixture of two peptides, each of them
comprising two
amino acid domains A and B where domain A represents a non-functional 9 amino
acids-long
sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5
is substituted
with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22
amino acids-
long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as
compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as
compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased
uptake
described herein, is mediated by macrophage scavenger receptors (SR)
including, but not
limiting to, SR-A and SR-B1 (see FIG. 9A1,A2-C). While not being bound to any
particular
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theory, it is believed that in one embodiment, this colocalization is
accompanied by a specific
disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-
1-related
trifunctional peptide of the present invention (see FIG. 9A), resulting in
ligand-independent
inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol
Pharm 2017,
14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of
which is
herein incorporated by reference in its entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-
1/DAP12 receptor
complex expressed on macrophages and presents the exemplary data showing that
scavenger
receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP (GF9-
HDL) and
GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1
signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel)
Activation
of the TREM-1/DAP12 receptor complex expressed on macrophages leads to
phosphorylation of
the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory
cytokine
response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and
GE31 peptide
inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into
the cytoplasm,
which self-penetrate into the cell membrane and block intramembrane
interactions between
TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream
signaling
cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-
1/DAP12
receptor complex expressed on Kupffer cells leads to phosphorylation of the
DAP12 cytoplasmic
signaling domain, subsequent SYK recruitment, and the downstream inflammatory
cytokine
response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9
peptide
inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into
the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions
between TREM-1
and DAP12, thereby preventing DAP12 phosphorylation and the downstream
signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-
1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is
largely driven by
SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774
macrophages were
cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-
HDL, cells
were treated for 1 hour at 37 C with 40 [tM cytochalasin D and either (FIG.
9B) 400 g/mL
fucoidan or (FIG. 9C) 10 M BLT-1, as indicated. Cells were then incubated for
either 4 hours
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or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars)
or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence
intensities of lysates were measured and normalized to the protein content.
Results are expressed
as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of
GF9-HDL and
GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation
protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL,
signaling chain
homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14 demonstrate that TREM-1/TRIOPEP in
free
.. and SLP-bound forms inhibits tumor growth, reduces infiltration of
macrophages into the tumor
in mouse models of NSCLC and PC and is well-tolerated by cancer mice during
the treatment
period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582, each of which
is herein
incorporated by reference in its entirety).
IG. 11A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX,
paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into
synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****, p < 0.0001 as
compared with
vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549
xenograft mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form
or
incorporated into synthetic lipopeptide particles (SLP) particles of discoidal
(TREM-
1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX,
paclitaxel.
<0.0001 as compared with vehicle-treated animals.
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FIG. 14A-C presents the exemplary data showing inhibition of tumor growth
(FIG. 14A) and
TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration
(FIG. 14B,
FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an
equimolar
mixture of the sulfoxidized methionine residue-containing TREM-1-related
trifunctional peptides
(TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic
lipopeptide
particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical
(TREM-
1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the
mean SEM (n
= 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus
vehicle). (FIG. 14C)
Representative F4/80 images from BxPC-3-bearing mice treated using different
free and sSLP-
bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale
bar = 200 m.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound
forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-
induced septic
shock.
FIG. 15A-B presents the exemplary data showing improved survival of
lipopolysaccharide
(LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized
methionine
residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide
particles (SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy
mice
at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6
mice treated with
increasing concentrations of an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in
free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-
bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA)
and is well-
tolerated by arthritic mice during the treatment period of 2 weeks (see Shen
and Sigalov. J Cell
Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference
in its entirety).
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FIG. 17A-B presents the exemplary data showing average clinical arthritis
score (FIG. 17A) and
mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the
difference
between beginning (day 24) and final (day 38) BWs of the collagen-induced
arthritis (CIA) mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated
into synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p
<0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents
pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated
by these mice
during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018,
1864:2761-2768, each
of which is herein incorporated by reference in its entirety).
FIG. 18A-D presents the exemplary data showing reduction of pathological
retinal
neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-
1 (FIG. 18C)
and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-
induced
retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP)
particles of spherical
morphology (TREM-1/TRIOPEP-sSLP). * * *, p < 0.001 as compared with vehicle-
treated
animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, herein
incorporated by
reference in its entirety, systemically administered human apo A-I accumulates
in murine brain.
It is also known that transcytosis of HDL in brain microvascular endothelial
cells is mediated by
SRBI (see Fung, et al. Front Physiol 2017, 8:841, herein incorporated by
reference in its
entirety). However, until tested as described herein, it was not known that a
self-assembled SLP
of the present invention comprising a trifunctional peptide was capable of
crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the
present
invention may cross the BBB, BRB and BTB, thus delivering their constituents
including but not
limiting to, TREM-1/TRIOPEP, GF9, GA31 and GE31, to the areas of interest in
the brain,
retina and tumor. In certain embodiments, FIG. 63 demonstrates that the
fluorescently labeled
sSLP described herein may cross the BBB, BRB and BTB, thus delivering their
constituents
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including but not limiting to, GBCA imaging probe to the areas of interest in
the brain, retina and
tumor.
While not being bound to any particular theory, it is believed that the brain-
, retina-, and
tumor-penetrating capabilities of these SLP can be mediated by interaction of
SRBI with the
domain B amino acid sequences that correspond to the sequences of human apo A-
I helices 4
and/or 6 (see e.g. Liu, et al. J Biol Chem 2002, 277:21576-21584, herein
incorporated by
reference in its entirety).
In certain embodiments, these capabilities of the peptides and compositions of
the present
invention can be used to diagnose, treat and/or prevent cancers (including
brain cancer), diabetic
retinopathy and retinopathy of prematurity, neurodegenerative diseases
including Alzheimer's,
Parkinson's and Huntington's diseases and other diseases and conditions where
delivery of the
peptides and compositions of the invention to the brain, retina and/or tumor
is needed.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier
(BBB) and
blood-retinal barrier (BRB) by systemically (intraperitoneally) administered
rhodamine B-
labeled spherical self-assembled particles (sSLP) that contain Gd-containing
contrast agent (Gd-
sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-
sSLP) or
an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
A mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels
of early fibrosis markers Pro-Colla and a-SMA were significantly increased in
alcohol-fed mice
compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of
these makers
was remarkably attenuated in the vehicle-treated group and, importantly,
further decreased by the
TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the
expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and
FIG. 20B cc-
Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of
mice with
(alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF)
group; # indicates
significance level compared to the non-treated alcohol-fed group. o indicates
significance level
compared to the vehicle-treated alcohol-fed group. The significant levels are
as follows: *, 0.05
> P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
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TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in
liver.
Serum ALT levels obtained during week 5 of the alcohol feeding showed
significant increases in
alcohol-fed mice compared to PF controls. This ALT increase was attenuated in
both TREM-1
inhibitor-treated groups, indicating attenuation of liver injury (Fig. 21A).
Surprisingly, vehicle
.. treatment (HDL) also showed a similar protective effect (Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0
staining in livers
of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig. 21B-D)
and H& (Fig.
21D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1
inhibitor-treated mice
compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses
the
production of alanine aminotransferase (ALT) in mice with alcoholic liver
disease (ALD), as
measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in
addition to
improving indicators of liver disease and inflammation. * indicates
significance level compared
to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide
particles of spherical
morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no
TREM-1
inhibitory peptide GF9. # indicates significance level compared to the non-
treated alcohol-fed
group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1
pathway inhibition
in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-
1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A)
Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-
1/TRIOPEP-
sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over
TREM-1 peptide
alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates
significance level
compared to the nontreated PF group; * indicates significance level compared
to the nontreated
alcohol-fed group; indicates significance level compared to the vehicle-
treated alcohol-fed
group. The numbers of the symbols sign the significant levels as the
following: **OP < 0.05;
WooP < 0.01;*"/#"P <0.001; ****P < 0 .0001. *** , 0.001 > P > 0.0001; ##, 0.01
> P > 0.001.
B. TCR-Related Trifunctional Peptides
The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation,
in
.. protecting the organism from infectious agents, and in the function of T-
cells. The TCR is a
complex of a heterodimer of TCRa and TCRb chains, which are responsible for
antigen
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recognition and interaction with the major histocompatibility complex (MHC)
molecules of
antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are
responsible for
transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr
2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios,
et al. US
20120077732, each of which is herein incorporated by reference in its
entirety).
The preferred TCR-related peptides and compositions of this class comprise the
domain
A comprising the TCR modulatory peptide sequences designed using a well-known
in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948,
each of which is herein incorporated by reference in its entirety). The
preferred peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic alpha helical peptide fragment. As described above, the
inclusion of an
amphipathic amino acid sequences aids the assistance in the ability to
interact with native
lipoproteins in a bloodstream in vivo and to form naturally long half-life
lipopeptide/lipoprotein
particles LP. It further aids the ability to provide targeted delivery to the
sites of interest. It
further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the
domain
A comprising the TCR modulatory peptide sequences designed using a well-known
in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948,
each of which is herein incorporated by reference in its entirety). The
preferred peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
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and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). As described
above, the
inclusion of an amphipathic apo A-I sequences aids the assistance in the self-
assembly of SLP
and the structural stability of the particle formed, particularly when the
particle has a discoidal
shape. It further aids the ability to provide targeted delivery to the cells
of interest. It further aids
the ability to interact with lipids and/or lipoproteins in a bloodstream in
vivo and form LP that
mimic native lipoproteins.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides:
TCR/TRIOPEP MA32
(MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12),
TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE)
(SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA)
(SEQ ID NO 15), and TCR/TRIOPEP
GE32
(GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of TCRa
chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific
ligand-
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independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self
Nonself 2010,
1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem
Struct Biol
2018, 111:61-99, each of which is herein incorporated by reference in its
entirety). In one
embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP
LA32
(LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not
being bound to any particular theory, it is believed that in one embodiment,
these peptides
colocalize with TCR in the cell membrane and selectively disrupt intramembrane
interactions of
TCRb chain with the CD3eg heterodimer, resulting to specific ligand-
independent inhibition of
TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99,
each of which is
herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP
YA32
(YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3zz
homodimer with TCRa chain, resulting to specific ligand-independent inhibition
of TCR upon
antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32
(IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3ed
heterodimer with TCRa chain, resulting to specific ligand-independent
inhibition of TCR upon
antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
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1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of
which is herein
incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP
FA32
(FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3eg
heterodimer with TCRb chain, resulting to specific ligand-independent
inhibition of TCR upon
antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of
which is herein
incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP
IA32e
(IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3eg and
CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to
specific ligand-
independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self
Nonself 2010,
1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem
Struct Biol 2018,
111:61-99, each of which is herein incorporated by reference in its entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the TCR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
inhibit TCR can be used
to treat and/or prevent TCR-related diseases and conditions including but not
limiting to, allergic
diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune
disease e.g. systemic
lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes,
Guillain-Barre syndrome,
Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel
disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
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allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other
disorder where T
cells are involved/recruited
In certain embodiments, the capability of the TCR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
colocalize with TCR can
be used to visualize (image) this receptor and evaluate its expression in the
areas of interest. In
64cut
one embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be
conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging
(visualization) of TCR
levels using PET and/or other imaging techniques can be used to diagnose TCR-
related diseases
and conditions as well as to monitor novel therapies for these diseases and
conditions.
C. NKG2D-Related Trifunctional Peptides
NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The
NKG2D is a complex of an NKG2D chain, which is responsible for ligand
recognition, and
DAP10 homodimer, which is responsible for transmembrane signal transduction
(see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004,
25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224,
each of which is
herein incorporated by reference in its entirety). NKG2D ligands show a
restricted expression in
normal tissues, but they are frequently overexpressed in cancer and infected
cells. The binding of
NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of
the cells
expressing these molecules. The mechanisms involved in the expression of the
ligands of
NKG2D play a role in the recognition of stressed cells by the immune system
and represent a
promising therapeutic target for improving the immune response against cancer
or autoimmune
disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et
al. Am J Physiol
Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl
Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-
Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is
herein
incorporated by reference in its entirety).
The preferred NKG2D-related peptides and compositions of this class comprise
the
domain A comprising the NKG2D modulatory peptide sequences designed using a
well-known
in the art novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization
model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-
39; Sigalov.
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Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018,
111:61-9, each
of which is herein incorporated by reference in its entirety). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in its entirety). As described above, the inclusion
of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36
(IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and
NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that
in one embodiment,
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these peptides colocalize with NKG2D in the cell membrane and selectively
disrupt
intramembrane interactions of NKG2D chain with the DNAX-activation protein 10
(DAP-10)
signaling homodimer, resulting to specific ligand-independent inhibition of
NKG2D upon ligand
stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self
Nonself 2010, 1:192-224;
.. and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is
herein
incorporated by reference in its entirety). In one embodiment, methionine
residues of
NKG2D/TRIOPEP peptides are modified.
In certain embodiments, the capability of the NKG2D-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit NKG2D can
be used to treat and/or prevent NKG2D-related diseases and conditions
including but not limiting
to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and
any other disorder
where NKG2D cells are involved/recruited. In one embodiment, the present
invention provides
methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
NKG2D can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu],
see TABLE 2) can
be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging
(visualization)
of NKG2D levels using PET and/or other imaging techniques can be used to
diagnose NKG2D-
related diseases and conditions as well as to monitor novel therapies for
these diseases and
conditions.
D. GPVI-Related Trifunctional Peptides.
In recent years, the central activating platelet collagen receptor,
glycoprotein (GP) VI, has
emerged as a promising antithrombotic target because its blockade or antibody-
mediated
depletion in circulating platelets was shown to effectively inhibit
experimental thrombosis and
thromboinflammatory disease states, such as stroke, without affecting
hemostatic plug formation.
GPVI is a complex of an GPVI chain, which is responsible for ligand
recognition, and FcRg
homodimer, which is responsible for transmembrane signal transduction (see
e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-
589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost
2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends
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Pharmacol Sci 2012, 33:583-590; Ungerer, etal. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, each of which is herein incorporated by
reference in its
entirety). The binding of GPVI to collagen or other antagonists ligands
induces platelet adhesion,
activation and aggregation. Platelet activation is a step in the pathogenesis
of ischemic cardio-
.. and cerebrovascular diseases, which represent the leading causes of death
and severe disability
worldwide. Although existing antiplatelet drugs have proved beneficial in the
clinic, their use is
limited by their inherent effect on primary hemostasis, making the
identification of novel
pharmacological targets for platelet inhibition a goal of cardiovascular
research.
The preferred GPVI-related peptides and compositions of this class comprise
the domain
.. A comprising the GPVI modulatory peptide sequences designed using a well-
known in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb
Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692,
each of
which is herein incorporated by reference in its entirety). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in its entirety). As described above, the inclusion
of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
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In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative GPVI-related trifunctional peptides:
GPVI/TRIOPEP GA32
(GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not
being bound to any particular theory, it is believed that in one embodiment,
these peptides
colocalize with GPVI in the cell membrane and selectively disrupt
intramembrane interactions of
GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-
independent
inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself
2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol
2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets
2008,
12:677-692, each of which is herein incorporated by reference in its
entirety). In one
embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit GPVI can be
used to treat and/or prevent GPVI-related diseases and conditions including
but not limiting to,
ischemic and thromboinflammatory diseases, and any other disorder where
platelets are
involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
GPVI can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu],
see TABLE 2) can
be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging
(visualization) of
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GPVI levels using PET and/or other imaging techniques can be used to diagnose
GPVI-related
diseases and conditions as well as to monitor novel therapies for these
diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides
The DAP10 and DAP12 signaling subunits are highly conserved in evolution and
associate with a large family of receptors in hematopoietic cells, including
dendritic cells,
plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells,
monocytes,
macrophages, natural killer cells, and some B and T cells. Some receptors are
able to associate
with either DAP10 or DAP12, which contribute unique intracellular signaling
functions. DAP-
10- and DAP-12-associated receptors have been shown to recognize both host-
encoded ligands
and ligands encoded by microbial pathogens, indicating that they play a role
in innate immune
responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends
Pharmacol Sci
2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self
Nonself 2010, 1:4-
39; Sigalov. Self Nonself 2010, 1:192-224, each of which is herein
incorporated by reference in
its entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this
class
comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide
sequences,
respectively, designed using a well-known in the art novel model of cell
receptor signaling, the
Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and
DAP-12-
associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends
Immunol 2004,
25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of
which is herein
incorporated by reference in its entirety). The preferred peptides and
compositions of this class
further comprise the domain B comprising at least one modified or unmodified
amphipathic apo
A-I and/or A-II peptide fragment to form upon interaction with lipid and/or
lipid mixtures, SLP
.. structures that can be spherical or discoidal (described herein and in
e.g., Sigalov. Contrast
Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-
219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015,
10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell
Mol Med
2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et
al. Biochim
.. Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by
reference in its
entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the
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assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32
(LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-
10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one
embodiment, these
peptides colocalize with DAP-10-associated cell receptors in the cell membrane
and selectively
disrupt intramembrane interactions of the receptor with the DAP-10 signaling
homodimer,
resulting to specific ligand-independent inhibition of the receptor upon
ligand stimulation (see
e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-
224; Sigalov. Adv
Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated
by reference in
its entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are
modified.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32
(VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-
12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one
embodiment, these
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peptides colocalize with DAP-12-associated cell receptors in the cell membrane
and selectively
disrupt intramembrane interactions of the receptor with the DAP-12 signaling
homodimer,
resulting to specific ligand-independent inhibition of the receptor upon
ligand stimulation (see
e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-
224; Sigalov. Adv
Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated
by reference in
its entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are
modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used
to treat and/or
prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can
be used to
visualize (image) these receptors and evaluate their expression in the areas
of interest. In one
64cut
embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to
the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging
(visualization) of levels of the DAP-10- and DAP-12-associated receptors using
PET and/or
other imaging techniques can be used to diagnose any diseases and conditions
where these
receptors are involved as well as to monitor novel therapies for these
diseases and conditions.
F. EGFR-Related Trifunctional Peptides.
The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is
the best
studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is
overexpressed
on the surface of 25-30% of breast cancer cells, and it has been associated
with a high risk of
relapse and death. EGFR amplification and mutations have been associated with
many
carcinomas. In particular, the EGFR pathway appears to play a role in
pancreatic carcinoma. See
e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol
Cell 2004, 15:3464-
3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol
2004, 25:583-
589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-
224. Short
hydrophobic peptides corresponding to the transmembrane domains of EGFR, ErB2
and insulin
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receptors inhibit specifically the autophosphorylation and signaling pathway
of their cognate
receptor (see Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474).
The preferred EGFR-related peptides and compositions of this class comprise
the domain
A comprising the EGFR modulatory peptide sequences, designed using a well-
known in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each
of which is
herein incorporated by reference in its entirety). The preferred peptides and
compositions of this
class further comprise the domain B comprising at least one modified or
unmodified amphipathic
apo A-I and/or A-II peptide fragment to form upon interaction with lipid
and/or lipid mixtures,
SLP structures that can be spherical or discoidal (described herein and in
e.g., Sigalov. Contrast
Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-
219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015,
10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell
Mol Med
2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et
al. Biochim
Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by
reference in its
entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the
assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
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embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47
(SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47
(SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in
one embodiment, these
peptides colocalize with EGFR in the cell membrane and selectively disrupts
intramembrane
interactions between the receptors, resulting to specific ligand-independent
inhibition of the
receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself
2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein
incorporated by
reference in its entirety). In one embodiment, methionine residues of
EGFR/TRIOPEP peptides
are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides
and
compounds of the present invention including but not limiting to those
described above, to
inhibit the receptors of the EGFR and/or ErB receptor families, respectively,
can be used to treat
and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
colocalize with the receptors of the EGFR and/or ErB receptor families can be
used to visualize
(image) these receptors and evaluate their expression in the areas of
interest. In one embodiment,
64co
for this purpose, an imaging probe (e.g. [ can be conjugated to the
EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the
receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be
used to diagnose
any diseases and conditions where these receptors are involved as well as to
monitor novel
therapies for these diseases and conditions.
G. Additional Trifunctional Peptides
Additional therapeutic peptide sequences and/or other therapeutic agents can
comprise
the domain A of the peptides and compositions of the present invention.
Additional examples are
provided in, for e.gs., Vlieghe, et al. Drug Discov Today 2010, 15:40-56;
Tsung, et al. Shock
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2007, 27:364-369; Chang, et al. PLoS One 2009, 4:e4171; Tjin Tham Sjin, et al.
Cancer Res
2005, 65:3656-3663; Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336;
Khan, et al. Hum
Immunol 2002, 63:1-7; Banga. Therapeutic peptides and proteins: formulation,
processing, and
delivery systems. 2nd ed. Boca Raton, FL: Taylor & Francis Group; 2006;
Stevenson. Curr
Pharm Biotechnol 2009, 10:122-137; Wu and Chi, US 9,387,257; Wu, et al., US
8,415,453;
Faure, et al., US 8,013,116; Faure, et al., US 9,273,111; Eggink and Hoober,
US 7,811,995;
Eggink and Hoober, US 8,496,942; Morgan and Pandha. US 2012/0177672 Al;
Broersma, et al.,
US 5,681,925), each of which is herein incorporated by reference in its
entirety.
In one embodiment, this domain comprises the Toll Like Receptor (TLR)
modulatory
sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in its entirety). As described above, the inclusion
of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
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embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32
(DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and
TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE)
(SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are
modified.
In certain embodiments, the capability of the TLR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
inhibit TLR can be used
to treat and/or prevent TLR-related diseases and conditions including but not
limiting to, sepsis
and other infectious diseases, and any other disorder where TLR receptors are
involved.
In certain embodiments, the capability of the TLR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
colocalize with TLR can
be used to visualize (image) this receptor and evaluate its expression in the
areas of interest. In
one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the
TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels
using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases
and conditions as
well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the
invention
comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory
sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in its entirety). As described above, the inclusion
of an amphipathic
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apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50
(SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50
(SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are
modified.
In certain embodiments, the capability of the ANPR-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit ANPRs can
be used to treat and/or prevent ANPR-related diseases and conditions including
but not limiting
to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are
involved.
In certain embodiments, the capability of the ANPR-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
ANPR can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated
to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of
ANPR levels
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using PET and/or other imaging techniques can be used to diagnose ANPR-related
diseases and
conditions as well as to monitor novel therapies for these diseases and
conditions.
In certain embodiments, other therapeutic agents including but not limiting
to, to those
described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R,
Wagman LD,
Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th
ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al.,
Therapy of
Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013,
49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene
Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating
Peptides. 2nd
edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman
and Altman,
US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne,
et al., US 9,173,891, each of which is herein incorporated by reference in its
entirety (see also
TABLE 2) can comprise the domain A of the peptides and compositions of the
present invention.
III. Lipoproteins And rHDLS.
Lipoproteins, inlcuding circulating lipoproteins in blood plasma, are natural
complexes
that contain both proteins (apolipoproteins, apo) and lipids bound to the
proteins, which allow
water-insoluble molecules such as fats to move through the water inside and
outside cells.
Lipoproteins serve to emulsify the lipid molecules. Examples include the
plasma lipoprotein
particles classified under high-density lipoproteins (HDL), which enable
cholesterol and other
hydrophobic lipid molecules to be carried in the bloodstream. In particular,
HDL transport
cholesterol and other water insoluble or poorly soluble lipids from the
peripheral tissues to the
liver.
The use of HDLs as delivery vehicles was proposed however in order to properly
function in vivo for delivery of drugs or imaging agents to sites of interest,
HDLs should mimic
native lipoproteins as close as possible. In a human body, HDL exists in two
forms: nascent or
discoidal HDL and spherical HDL. The use of isolated plasma lipoproteins,
including isolated
HDLs, as delivery vehicles is impractical.
However in vitro, long half-life lipoprotein particles that mimic native HDL
(as synthetic
sHDL or recombinant HDL, rHDL) can be readily reconstituted (synthesized) from
lipid
formulations and apolipoproteins (apo) resulting in, for example, sub 30 nm-
sized particles of
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discoidal or spherical morphology. Morphology of rHDLs is determined by the
composition of
lipid and apo mixtures and preparation procedures.
Many types of rHDLs were evaluated both clinically and experimentally as a
delivery
system for administering hydrophobic agents and for mitigating the toxic
effects associated with
administration of imaging probes such as Gd-containing contrast agents (GBCAs)
for magnetic
resonance imaging (MM).
As delivery vehicles, rHDL have several competitive advantages as compared
with other
delivery platforms: 1) apo A-I, a major HDL protein, is used for rHDL
preparation as it's
recombinant or synthesized peptide/protein represents an endogenous protein
that does not
trigger immunoreactions; 2) apo A-I's small size allows rHDL to pass through
blood vessel
walls, enter and then accumulate in the places of interest, including for
treatment and/or
detection, such as tumor sites, areas of disease, such as liver tissue, etc.,
or atherosclerotic
plaques; 3) rHDL's small particle size also allows for intravenous,
intramuscular and
subcutaneous applications; 4) rHDL's naturally long half-life extends the half-
life of incorporated
drugs and/or imaging agents in a bloodstream; and 5) a variety of drugs and
imaging agents can
be incorporated into this platform.
However, in order to properly function in vivo and as a result, to realize all
the
advantages mentioned above, rHDL should mimic native lipoproteins including
but not limited
to HDL as close as possible. This is a complicated task because two functions,
assistance in the
self-assembly of rHDL and therapeutic and/or imaging action in vivo, have to
be executed by at
least, two separate rHDL ingredients such as human apolipoprotein and
therapeutic agent and/or
imaging probe. In addition, in contrast to, for example, native HDL that are
normally target the
liver, rHDL have to be able to target other sites of interest such as, for
example, macrophages
which results in the need of targeting moieties thus adding the third function
of rHDL ingredients
¨ targeting. This hampers wider use of rHDL by difficulties in industrializing
the manufacture of
rHDL, along with rHDL' lack of stability and reproducibility. In addition, the
use of native or
recombinant human apolipoproteins significantly complicates development of the
commercial
product, drastically increases its cost and possesses potential clinical and
regulatory pitfalls.
An alternative, fully synthetic lipopeptide system for targeted treatment
and/or imaging
that closely mimics native lipoproteins and exhibits the advantageous
properties of rHDL as well
as superior stability, uniformity, ease of use, and reproducibility of
preparation is needed for
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administration and targeted delivery of therapeutic agents (e.g. anti-cancer
and anti-sepsis agents,
other anti-inflammatory drugs) and/or imaging probes. The invention provides
such a system and
a method of using the system (e.g., for delivery of anti-cancer, anti-
arthritic, anti-sepsis, anti-
angiogenic and other therapeutic agents and/or imaging probes to a subject).
These and other
objects and advantages of the invention, as well as additional inventive
features, will be apparent
from the description of the invention provided herein.
Additional contemplative advantages of a lipoprotein delivery platform
includes
increasing activity due to specific targeting, sequestration of the drug at
the target site, protection
of the drug from rapid metabolism, amplified therapeutic effect due to
packaging of numerous
drug molecules in each particle, and decreased toxicity due to altered
pharmacokinetics. Due to
the naturally long half-life of native discoidal and spherical HDL in normal
subjects being 12-20
hrs and 3-5 days, respectively, rHDL represent a promising versatile delivery
platform in
particular for therapeutic peptides that have a bloodstream half-life of
minutes.
For example, it would be desirable to combine in one molecule therapeutic
(and/or
diagnostic), particle forming and targeting functions. The invention addresses
these needs,
among others, and provides such a system/molecule and a method of using the
system (e.g., for
delivery of anti-cancer, anti-arthritic, anti-sepsis, anti-angiogenic, anti-
inflammatory and other
therapeutic agents and/or imaging probes to a subject). These and other
objects and advantages
of the invention, as well as additional inventive features, will be apparent
from the description of
the invention provided herein.
IV. Trifunctional Peptides In rHDL Formulations.
A. TREM-1-related Trifunctional peptides: TREM-1 Signaling
Pathway and Its
Blockade.
TREM-1 is expressed on the majority of innate immune cells and to a lesser
extent on
parenchymal cells. Upon activation, TREM-1 can directly amplify an
inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly
associated with
infectious diseases, recent evidences demonstrate that TREM-1 receptor and its
signaling
pathways contribute to the pathology of non-infectious acute and chronic
inflammatory diseases,
.. including but not limiting to, rheumatoid arthritis, atherosclerosis,
ischemia reperfusion-induced
tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases,
retinopathies, and
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cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res
2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-
2768, and Kuai, et
al. US 2008/0247955, each of which is herein incorporated by reference in its
entirety).
In some preferred embodiments, TREM-1-related peptides and associated
compositions
of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of
cell
receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of
modulating
TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself
2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006,
27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct
Biol 2018,
111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are
herein
incorporated by reference in their entirety. In some preferred embodiments,
peptides and
compositions of the present invention comprise the TREM-1 modulatory peptide
sequences
designed using a well-known in the art novel model of cell receptor signaling,
the Signaling
Chain HOmoOLigomerization model.
In some preferred embodiments, peptides and compositions of this class further
comprise
the domain B comprising at least one modified or unmodified amphipathic alpha
helical peptide
fragment, such as a apo A-I and/or A-II peptide fragment, to form upon
interaction with lipid
and/or lipid mixtures. In certain embodiments, exemplary trifunctional
peptides comprise the
domain B comprises with the amino acid sequence selected from the amino acid
sequences of the
major HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22
amino acid residue-long peptide sequence of the apo A-I helix 4. In one
embodiment, this
sequence contains a modified amino acid residue. In one embodiment, this
modified amino acid
residue is methionine sulfoxide. In one embodiment, the domain B of the
peptides and
compositions of the invention comprises 22 amino acid residue-long peptide
sequence of the apo
A-I helix 6. In one embodiment, this sequence contains a modified amino acid
residue. In one
embodiment, this modified amino acid residue is methionine sulfoxide.
In one embodiment, preferred peptides and compositions of the invention
further
comprise at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide fragment
capable upon interaction with lipid and/or lipid mixtures, to form synthetic
lipopeptide particles
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(SLP) structures that can be spherical or discoidal (described herein and in
e.g., Sigalov. Contrast
Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-
219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US 20130039948;
Shen, et al.
PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and
Sigalov. J
Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-
4582; Rojas, et
al. Biochim Biophys Acta 2018, 1864:2761-2768). The inclusion of an
amphipathic apo A-I
sequences in the peptides and compositions of the invention further aids the
ability to provide
targeted delivery to the cells of interest. It further aids the ability to
interact with lipids and/or
lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It further aids
the ability to cross the BBB, BRB and BTB.
FIG. 1 presents an exemplary schematic representation of one embodiment of a
trifunctional
peptide of the present invention comprising amino acid domains A and B where
amino acid
domain A represents a therapeutic peptide sequence with or without an attached
drug compound
and/or imaging probe that functions to treat, prevent and/or detect a disease
or condition,
whereas amino acid domain B represents an amphipathic alpha helical peptide
sequence, with or
without an additional targeting peptide sequence, and functions to 1) assist
in the self-assembly
of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with
lipids or lipid
mixtures in vitro, for use in transporting these trifunctional peptides as
lipoprotien nanoparticles
to sites of interest in vitro or in vivo and/or 2) form long half-life
lipopeptide/lipoprotein particles
upon interaction with endogenous lipoproteins for transporting these
trifunctional peptides to the
sites of interest. Endogenous lipoproteins may be lipoproteins added to or
found in cell cultures,
or lipoproteins in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-
related
trifunctional peptides, TREM-1/TRIOPEP GE31
(GFL5K5LVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFL5K5LVFPLGEEM(0)RDRARAHVDALRTHLA,
M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine
residues of the
peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA)
(SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human
TREM-1
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(UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a
(UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)).
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1,
Homo
sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo
sapiens
isoform CRA a:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid
cells 1,
Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid
cells 1,
Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KST.
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FIG. 2 presents schematic representations of embodiments of a TREM-1-related
trifunctional
peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE,
M(0), methionine sulfoxide) comprising amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino
acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and
functions to
treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22
amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a
sulfoxidized
methionine residue and functions to assist in the self-assembly of synthetic
lipopeptide particles
(SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
In certain embodiments, other preferred TREM-1-related trifunctional peptides
and
compositions of this class comprise the domain A comprising the TREM-1
inhibitory peptide
sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-
2830; Gibot,
et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am
Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in
press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et
al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and
Derive, US 9,255,136, each of which is herein incorporated by reference in
it's entirety) while
the domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures,
SLP structures that
can be spherical or discoidal. In some embodiments, resulting trifunctional
peptide sequences
may be radiolabeled and/or contain unmodified or modified methionine residues
(TABLE 2)
including but not limiting to, the following sequences:
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide
(SEQ ID NO 7), LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE (M(0),
methionine sulfoxide (SEQ ID NO 8),
LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide
(SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0),
methionine sulfoxide (SEQ ID NO 10).
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SLP (rHDL) structures that can be spherical or discoidal (described herein and
in e.g.,
Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014,
21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorpoated by referene in it's entirety). The inclusion of an amphipathic apo
A-I sequences aids
the assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31
(GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In
one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-
assembly of
nanosized SLP of discoidal or spherical morphology (dSLP and sSLP,
respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-
related
trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising
amino acid
domains A and B. Depending on lipid mixture compositions added to the
peptides, sub 50 nm-
sized SLP particles of discoidal (TREM-1/TRIOPEP-d5LP) or spherical (TREM-
1/TRIOPEP-
s5LP) morphology are self-assembled upon binding of the trifunctional peptide
to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain
homooligomerization; TREM-1,
triggering receptor expressed on myeloid cells-1.
In one embodiment, this provides targeted delivery of the SLP constituents
including
TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one
embodiment, this
provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to
tumor-
associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
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B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic
peptide
sequence and functions to treat and/or prevent a TREM-1-related disease or
condition (example,
for atherosclerosis), whereas domain B represents a 22 amino acids-long
apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and
functions to assist in
the self-assembly of synthetic lipopeptide particles (SLP) and to target the
particles to TREM-1-
expressing macrophages as applied to the treatment and/or prevention of
atherosclerosis. While
not being bound to any particular theory, it is believed that chemical and/or
enzymatic
modification of protein sequence in domain B leads to the recognition of SLP
of the present
invention by the macrophage scavenger receptors and results in an irreversible
binding to and
consequent uptake by macrophages of such particles. It is further believed
that accumulation of
these particles in intraplaque macrophages is accompanied by accumulation of
TRIOPEP in
these cells. In contrast, native HDL particles that contain only unmodified
apolipoprotein
molecules are not recognized by intraplaque macrophages and return to the
circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one
embodiment of a
trifunctional peptide (TRIOPEP) of the present invention comprising amino acid
domains A and
B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide
sequence and
functions to treat and/or prevent a TREM-1-related disease or condition
(example, for cancer),
whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6
peptide sequence
with a sulfoxidized methionine residue and functions to assist in the self-
assembly of synthetic
lipopeptide particles (SLP) and to target the particles to TREM-1-expressing
macrophages as
applied to the treatment and/or prevention of cancer. While not being bound to
any particular
theory, it is believed that chemical and/or enzymatic modification of protein
sequence in domain
B leads to the recognition of SLP of the present invention by the macrophage
scavenger
receptors and results in an irreversible binding to and consequent uptake by
macrophages of such
particles. It is further believed that accumulation of these particles in
tumor-associated
macrophages is accompanied by accumulation of TRIOPEP in these cells. In
contrast, native
HDL particles that contain only unmodified apolipoprotein molecules are not
recognized by
tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one
embodiment,
this colocalization is accompanied by a specific disruption of intramembrane
interactions
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between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the
present
invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1
upon ligand
binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and
Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein
incorporated by reference
in it's entirety
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane
interactions
between TREM-1 and DAP-12 by the trifunctional peptide of the present
invention comprising
two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1
inhibitory
therapeutic peptide sequence, whereas domain B is a 22 amino acids-long
apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While
not being bound to
any particular theory, it is believed that this disruption results in "pre-
dissociation" of a receptor
complex and upon ligand stimulation, leads to inhibition of TREM-1 and
silencing the TREM-1
signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present
invention colocalizes
with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys
Acta 2018,
1864:2761-2768). In certain embodiments, the capability of the TREM-1-related
trifunctional
peptides and compounds of the present invention including but not limiting to,
TREM-
1/TRIOPEP GA31 and TREM-1/TRIOPEP GE31, to colocalize with TREM-1 can be used
to
visualize (image) this receptor and evaluate its expression in the areas of
interest. In one
embodiment, for this purpose, an imaging probe (e.g. [64Cu], see TABLE 2) can
be conjugated to
the TREM-1/TRIOPEP sequences, GE31
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine
sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-
1 levels using
PET and/or other imaging techniques can be used to diagnose glioblastoma
multiforme (GBM)
and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017,
19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments,
imaging
(visualization) of TREM-1 levels can be used to diagnose other TREM-1-related
diseases and
conditions as well as to monitor novel therapies for these diseases and
conditions.
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FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine
residue-
containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with
TREM-1 in
the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and
a merged
image (FIG. 6C).
As described herein (see FIG. 7A-B), sulfoxidation of methionine residues in
the TREM-
1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis
of the SLP
containing an equimolar mixture of these peptides (designated as TREM-
1/TRIOPEP), TREM-
1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, all of which are
herein
incorporated in their entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic
lipopeptide
particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that
contain an equimolar
mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31
and GE 31
(TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The
post 4 h
incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-
1/TRIOPEP-
sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine
residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified
methionine
residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and
TREM-
1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4
(white
bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001
to 0.001 as
compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free
and
SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-
mediated
release of pro-inflammatory cytokines both in vitro (FIG. 8) and in vivo (FIG.
10). While not
being bound to any particular theory, it is believed that this indicates that
similarly to TREM-1-
inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014, 21:208-
219; Shen and
Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell Mol Med
2017, 21:2524-
2534, each of which is herein incorporated by reference in it's entirety),
TREM-1-related
trifunctional peptides can reach their site of action from both outside (free
TREM-1/TRIOPEP)
and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon
administration,
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free TREM-1/TRIOPEP may form LP in vivo and/or interact with native
lipoproteins, resulting
in formation of HDL-mimicking LP. In one embodiment, these LP may further
target the cells of
interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lb eta production by lipopoly sacchari de
(LPS)-stimulated
macrophages incubated for 24 h at 37 C with an equimolar mixture of the
sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles
(SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged
macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis
factor-alpha (TNF-
alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post
lipopolysaccharide
(LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline
(PBS),
dexamethasone (DEX), control peptide and with an equimolar mixture of the
sulfoxidized
methionine residue-containing TREM-1-related trifunctional peptides (TREM-
1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles
(SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
Control peptide represents an equimolar mixture of two peptides, each of them
comprising two
amino acid domains A and B where domain A represents a non-functional 9 amino
acids-long
sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5
is substituted
with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22
amino acids-
long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as
compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as
compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased
uptake
described herein, is mediated by macrophage scavenger receptors (SR)
including, but not
limiting to, SR-A and SR-B1 (see FIG. 9A-C). While not being bound to any
particular theory, it
is believed that in one embodiment, this colocalization is accompanied by a
specific disruption of
intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related
trifunctional
peptide of the present invention (see FIG. 9A), resulting in ligand-
independent inhibition of
TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017,
14:4572-4582
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and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is
herein incorporated
by reference in it's entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-
1/DAP12 receptor
complex expressed on macrophages and presents the exemplary data showing that
scavenger
.. receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP
(GF9-HDL) and
GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1
signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel)
Activation
of the TREM-1/DAP12 receptor complex expressed on macrophages leads to
phosphorylation of
the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory
cytokine
response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and
GE31 peptide
inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into
the cytoplasm,
which self-penetrate into the cell membrane and block intramembrane
interactions between
TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream
signaling
cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-
1/DAP12
receptor complex expressed on Kupffer cells leads to phosphorylation of the
DAP12 cytoplasmic
signaling domain, subsequent SYK recruitment, and the downstream inflammatory
cytokine
response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9
peptide
inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into
the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions
between TREM-1
and DAP12, thereby preventing DAP12 phosphorylation and the downstream
signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-
1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is
largely driven by
SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774
macrophages were
cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-
HDL, cells
were treated for 1 hour at 37 C with 401.IM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 1.1M BLT-1, as indicated. Cells were then incubated
for either 4 hours
or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars)
or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence
intensities of lysates were measured and normalized to the protein content.
Results are expressed
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as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of
GF9-HDL and
GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation
protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL,
signaling chain
homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14A-C demonstrate that TREM-1/TRIOPEP in
free and SLP-bound forms inhibits tumor growth, reduces infiltration of
macrophages into the
tumor in mouse models of NSCLC and PC and is well-tolerated by cancer mice
during the
treatment period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582).
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX,
paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into
synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared
with
vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549
xenograft mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form
or
incorporated into synthetic lipopeptide particles (SLP) particles of discoidal
(TREM-
1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX,
paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth
(FIG. 14A) and
TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration
(FIG. 14B,
FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an
equimolar
mixture of the sulfoxidized methionine residue-containing TREM-1-related
trifunctional peptides
(TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic
lipopeptide
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particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical
(TREM-
1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the
mean SEM (n
= 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus
vehicle). (FIG. 14C)
Representative F4/80 images from BxPC-3-bearing mice treated using different
free and sSLP-
-- bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale
bar = 200 pm.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound
forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-
induced septic
shock.
FIG. 15A-B presents the exemplary data showing improved survival of
lipopolysaccharide
(LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized
methionine
residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide
particles (SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
-- FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy
mice
at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6
mice treated with
increasing concentrations of an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in
free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-
bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA)
and is well-
tolerated by arthritic mice during the treatment period of 2 weeks (see Shen
and Sigalov. J Cell
-- Mol Med 2017, 21:2524-2534).
FIG. 17A-B presents the exemplary data showing average clinical arthritis
score (FIG. 17A) and
mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the
difference
between beginning (day 24) and final (day 38) BWs of the collagen-induced
arthritis (CIA) mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
-- related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31
incorporated into synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
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(TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p
<0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents
pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated
by these mice
during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018,
1864:2761-2768).
FIG. 18A-D presents the exemplary data showing reduction of pathological
retinal
neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-
1 (FIG. 18C)
and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-
induced
retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized
methionine residue-
containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP)
particles of spherical
morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-
treated
animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, systemically
administered human apo A-I accumulates in murine brain. It is also known that
transcytosis of
HDL in brain microvascular endothelial cells is mediated by SRBI (see Fung, et
al. Front Physiol
2017, 8:841). However, until tested, it was not known that a self-assembled
SLP of the present
invention comprising a trifunctional peptide was capable of crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the
present
invention may cross the BBB, BRB and BTB, thus delivering their constituents
including but not
limiting to, TREM-1/TRIOPEP to the areas of interest in the brain, retina and
tumor. While not
being bound to any particular theory, it is believed that the brain-, retina-,
and tumor-penetrating
capabilities of these SLP can be mediated by interaction of SRBI with the
domain B amino acid
sequences that correspond to the sequences of human apo A-I helices 4 and/or 6
(see e.g. Liu, et
al. J Biol Chem 2002, 277:21576-21584, each of which is herein incorporated by
reference in it's
entirety).
In certain embodiments, these capabilities of the peptides and compositions of
the present
invention can be used to diagnose, treat and/or prevent cancers (including
brain cancer), diabetic
retinopathy and retinopathy of prematurity, neurodegenerative diseases
including Alzheimer's,
Parkinson's and Huntington's diseases and other diseases and conditions where
delivery of the
peptides and compositions of the invention to the brain, retina and/or tumor
is needed.
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FIG. 19 presents exemplary data showing penetration of the blood-brain barrier
(BBB) and
blood-retinal barrier (BRB) by systemically (intraperitoneally) administered
rhodamine B-
labeled spherical self-assembled particles (sSLP) that contain Gd-containing
contrast agent (Gd-
sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-
sSLP) or
an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
Mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels of
early fibrosis markers Pro-Colla and a-SMA were significantly increased in
alcohol-fed mice
compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of
these makers
-- was remarkably attenuated in the vehicle-treated group and, importantly,
further decreased by the
TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the
expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and
FIG. 20B a-
Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of
mice with
(alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF)
group; # indicates
significance level compared to the non-treated alcohol-fed group. o indicates
significance level
compared to the vehicle-treated alcohol-fed group. The significant levels are
as follows: *, 0.05
> P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in
liver. Serum ALT levels obtained during week 5 of the alcohol feeding showed
significant
increases in alcohol-fed mice compared to PF controls. This ALT increase was
attenuated in both
TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig.
4A).
Surprisingly, vehicle treatment (HDL) also showed a similar protective effect
(Fig. 4A).
Consistent with steatosis, we found a significant increase in Oil Red 0
staining in livers
of alcohol-fed mice compared to PF controls (Fig. 4C). Oil Red 0 (Fig. 4B-D)
and H& (Fig.
4D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1
inhibitor-treated mice
compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
4B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses
the
production of alanine aminotransferase (ALT) in mice with alcoholic liver
disease (ALD), as
measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in
addition to
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improving indicators of liver disease and inflammation. * indicates
significance level compared
to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide
particles of spherical
morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no
TREM-1
inhibitory peptide GF9. # indicates significance level compared to the non-
treated alcohol-fed
group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1
pathway inhibition
in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-
1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A)
Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-
1/TRIOPEP-
sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over
TREM-1 peptide
alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates
significance level
compared to the nontreated PF group; * indicates significance level compared
to the nontreated
alcohol-fed group; indicates significance level compared to the vehicle-
treated alcohol-fed
group. The numbers of the symbols sign the significant levels as the
following: **OP < 0.05;
44/ P < 0.01;*/"413 <0001; ****P < 0.0001.***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
B. TCR-Related Trifunctional Peptides
The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation,
in
protecting the organism from infectious agents, and in the function of T-
cells. The TCR is a
complex of a heterodimer of TCRa and TCRb chains, which are responsible for
antigen
recognition and interaction with the major histocompatibility complex (MHC)
molecules of
antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are
responsible for
transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr
2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios,
et al. US
20120077732, each of which is herein incorporated by reference in it's
entirety).
The preferred TCR-related peptides and compositions of this class comprise the
domain
A comprising the TCR modulatory peptide sequences designed using a well-known
in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
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2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948,
each of which is herein incorporated by reference in it's entirety). The
preferred peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
-- unmodified amphipathic alpha helical peptide fragment. As described above,
the inclusion of an
amphipathic amino acid sequences aids the assistance in the ability to
interact with native
lipoproteins in a bloodstream in vivo and to form naturally long half-life
lipopeptide/lipoprotein
particles LP. It further aids the ability to provide targeted delivery to the
sites of interest. It
further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the
domain
A comprising the TCR modulatory peptide sequences designed using a well-known
in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948,
each of which is herein incorporated by reference in it's entirety). The
preferred peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in it's entirety). As described above, the inclusion
of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
-- and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins.
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In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP MA32
(MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12),
TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE)
(SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA)
(SEQ ID NO 15), and TCR/TRIOPEP GE32
(GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of TCRa
chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific
ligand-
independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self
Nonself 2010,
1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem
Struct Biol
2018, 111:61-99, each of which is herein incorporated by reference in it's
entirety). In one
embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP LA32
(LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not
being bound to any particular theory, it is believed that in one embodiment,
these peptides
colocalize with TCR in the cell membrane and selectively disrupt intramembrane
interactions of
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TCRb chain with the CD3eg heterodimer, resulting to specific ligand-
independent inhibition of
TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99,
each of which is
herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP YA32
(YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3zz
homodimer with TCRa chain, resulting to specific ligand-independent inhibition
of TCR upon
antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of
which is herein
incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32
(IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3ed
heterodimer with TCRa chain, resulting to specific ligand-independent
inhibition of TCR upon
antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of
which is herein
incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP FA32
(FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3eg
heterodimer with TCRb chain, resulting to specific ligand-independent
inhibition of TCR upon
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antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of
which is herein
incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32e
(IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being
bound to any particular theory, it is believed that in one embodiment, these
peptides colocalize
with TCR in the cell membrane and selectively disrupt intramembrane
interactions of CD3eg and
CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to
specific ligand-
independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self
Nonself 2010,
1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem
Struct Biol 2018,
111:61-99, all of which are herein incorporated by reference in their
entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified as
described herein.
In certain embodiments, the capability of the TCR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
inhibit TCR can be used
to treat and/or prevent TCR-related diseases and conditions including but not
limiting to, allergic
diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune
disease e.g. systemic
lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes,
Guillain-Barre syndrome,
Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel
disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other
disorder where T
cells are involved/recruited
In certain embodiments, the capability of the TCR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
colocalize with TCR can
be used to visualize (image) this receptor and evaluate its expression in the
areas of interest. In
one embodiment, for this purpose, an imaging probe (e.g. [64cu-.j,
see TABLE 2) can be
conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging
(visualization) of TCR
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levels using PET and/or other imaging techniques can be used to diagnose TCR-
related diseases
and conditions as well as to monitor novel therapies for these diseases and
conditions.
C. NKG2D-Related Trifunctional Peptides
NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The
NKG2D is a complex of an NKG2D chain, which is responsible for ligand
recognition, and
DAP10 homodimer, which is responsible for transmembrane signal transduction
(see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004,
25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224,
all of which are
herein incorporated by reference in their entirety). NKG2D ligands show a
restricted expression
in normal tissues, but they are frequently overexpressed in cancer and
infected cells. The binding
of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis
of the cells
expressing these molecules. The mechanisms involved in the expression of the
ligands of
NKG2D play a role in the recognition of stressed cells by the immune system
and represent a
promising therapeutic target for improving the immune response against cancer
or autoimmune
disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et
al. Am J Physiol
Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl
Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-
Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is
herein
incorporated by reference in it's entirety).
The preferred NKG2D-related peptides and compositions of this class comprise
the
domain A comprising the NKG2D modulatory peptide sequences designed using a
well-known
in the art novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization
model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-
39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018,
111:61-9, all
of which are herein incorporated by reference in their entirety). The
preferred peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
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Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the
assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36
(IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and
NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that
in one embodiment,
these peptides colocalize with NKG2D in the cell membrane and selectively
disrupt
intramembrane interactions of NKG2D chain with the DNAX-activation protein 10
(DAP-10)
signaling homodimer, resulting to specific ligand-independent inhibition of
NKG2D upon ligand
stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self
Nonself 2010, 1:192-224;
and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are
herein
incorporated by reference in their entirety). In one embodiment, methionine
residues of
NKG2D/TRIOPEP peptides are modified.
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In certain embodiments, the capability of the NKG2D-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit NKG2D can
be used to treat and/or prevent NKG2D-related diseases and conditions
including but not limiting
to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and
any other disorder
where NKG2D cells are involved/recruited. In one embodiment, the present
invention provides
methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
NKG2D can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu],
see TABLE 2) can
be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging
(visualization)
of NKG2D levels using PET and/or other imaging techniques can be used to
diagnose NKG2D-
related diseases and conditions as well as to monitor novel therapies for
these diseases and
conditions.
D. GPVI-Related Trifunctional Peptides
In recent years, the central activating platelet collagen receptor,
glycoprotein (GP) VI, has
emerged as a promising antithrombotic target because its blockade or antibody-
mediated
depletion in circulating platelets was shown to effectively inhibit
experimental thrombosis and
thromboinflammatory disease states, such as stroke, without affecting
hemostatic plug formation.
GPVI is a complex of a GPVI chain, which is responsible for ligand
recognition, and FcRg
homodimer, which is responsible for transmembrane signal transduction (see
e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-
589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost
2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends
Pharmacol Sci 2012, 33:583-590; Ungerer, et al. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, all of which are herein incorporated by
reference in their
entirety). The binding of GPVI to collagen or other antagonists ligands
induces platelet adhesion,
activation and aggregation. Platelet activation is a step in the pathogenesis
of ischemic cardio-
and cerebrovascular diseases, which represent the leading causes of death and
severe disability
worldwide. Although existing antiplatelet drugs have proved beneficial in the
clinic, their use is
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limited by their inherent effect on primary hemostasis, making the
identification of novel
pharmacological targets for platelet inhibition a goal of cardiovascular
research.
The preferred GPVI-related peptides and compositions of this class comprise
the domain
A comprising the GPVI modulatory peptide sequences designed using a well-known
in the art
novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb
Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692,
each of
which is herein incorporated by reference in it's entirety). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which
is herein
incorporated by reference in it's entirety). As described above, the inclusion
of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
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embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative GPVI-related trifunctional peptides: GPVI/TRIOPEP GA32
(GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not
being bound to any particular theory, it is believed that in one embodiment,
these peptides
colocalize with GPVI in the cell membrane and selectively disrupt
intramembrane interactions of
GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-
independent
inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself
2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol
2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets
2008,
12:677-692, each of which is herein incorporated by reference in it's
entirety). In one
embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit GPVI can be
used to treat and/or prevent GPVI-related diseases and conditions including
but not limiting to,
ischemic and thromboinflammatory diseases, and any other disorder where
platelets are
involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
GPVI can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu],
see TABLE 2) can
be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging
(visualization) of
GPVI levels using PET and/or other imaging techniques can be used to diagnose
GPVI-related
diseases and conditions as well as to monitor novel therapies for these
diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides
The DAP10 and DAP12 signaling subunits are highly conserved in evolution and
associate with a large family of receptors in hematopoietic cells, including
dendritic cells,
plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells,
monocytes,
macrophages, natural killer cells, and some B and T cells. Some receptors are
able to associate
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with either DAP10 or DAP12, which contribute unique intracellular signaling
functions. DAP-
10- and DAP-12-associated receptors have been shown to recognize both host-
encoded ligands
and ligands encoded by microbial pathogens, indicating that they play a role
in innate immune
responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends
Pharmacol Sci
2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self
Nonself 2010, 1:4-
39; Sigalov. Self Nonself 2010, 1:192-224, all of which are herein
incorporated by reference in
their entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this
class
comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide
sequences,
respectively, designed using a well-known in the art novel model of cell
receptor signaling, the
Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and
DAP-12-
associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010,
1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends
Immunol 2004,
25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of
which is herein
incorporated by reference in it's entirety). The preferred peptides and
compositions of this class
further comprise the domain B comprising at least one modified or unmodified
amphipathic apo
A-I and/or A-II peptide fragment to form upon interaction with lipid and/or
lipid mixtures, SLP
structures that can be spherical or discoidal (described herein and in e.g.,
Sigalov. Contrast
Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-
219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015,
10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell
Mol Med
2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et
al. Biochim
Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by
reference in it's
entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the
assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
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In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32
(LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-
10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one
embodiment, these
peptides colocalize with DAP-10-associated cell receptors in the cell membrane
and selectively
disrupt intramembrane interactions of the receptor with the DAP-10 signaling
homodimer,
resulting to specific ligand-independent inhibition of the receptor upon
ligand stimulation (see
e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-
224; Sigalov. Adv
Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated
by reference in
it's entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are
modified.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32
(VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-
12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one
embodiment, these
peptides colocalize with DAP-12-associated cell receptors in the cell membrane
and selectively
disrupt intramembrane interactions of the receptor with the DAP-12 signaling
homodimer,
resulting to specific ligand-independent inhibition of the receptor upon
ligand stimulation (see
e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-
224; Sigalov. Adv
Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated
by reference in
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it's entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are
modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used
to treat and/or
prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can
be used to
.. visualize (image) these receptors and evaluate their expression in the
areas of interest. In one
64cut
embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to
the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging
(visualization) of levels of the DAP-10- and DAP-12-associated receptors using
PET and/or
other imaging techniques can be used to diagnose any diseases and conditions
where these
receptors are involved as well as to monitor novel therapies for these
diseases and conditions.
6. EGFR-Related Trifunctional Peptides
The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is
the best
studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is
overexpressed
on the surface of 25-30% of breast cancer cells, and it has been associated
with a high risk of
relapse and death. EGFR amplification and mutations have been associated with
many
carcinomas. In particular, the EGFR pathway appears to play a role in
pancreatic carcinoma. See
e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol
Cell 2004, 15:3464-
3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol
2004, 25:583-
589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-
224, each of which is
herein incorporated by reference in it's entiretyy. Short hydrophobic peptides
corresponding to
the transmembrane domains of EGFR, ErB2 and insulin receptors inhibit
specifically the
autophosphorylation and signaling pathway of their cognate receptor (see
Bennasroune, et al.
Mol Biol Cell 2004, 15:3464-3474, all of which are herein incorporated by
reference in their
entirety).
The preferred EGFR-related peptides and compositions of this class comprise
the domain
A comprising the EGFR modulatory peptide sequences, designed using a well-
known in the art
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novel model of cell receptor signaling, the Signaling Chain
HOmoOLigomerization model,
capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself
2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol
2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, all of
which are
herein incorporated by reference in their entirety). The preferred peptides
and compositions of
this class further comprise the domain B comprising at least one modified or
unmodified
amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with
lipid and/or
lipid mixtures, SLP structures that can be spherical or discoidal (described
herein and in e.g.,
Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int
Immunopharmacol 2014,
21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al.
PLoS One 2015,
10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell
Mol Med
2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et
al. Biochim
Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by
reference in it's
entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the
assistance in the self-assembly of SLP and the structural stability of the
particle formed,
particularly when the particle has a discoidal shape. It further aids the
ability to provide targeted
delivery to the cells of interest. It further aids the ability to interact
with lipids and/or lipoproteins
in a bloodstream in vivo and form LP that mimic native lipoproteins. It
further aids the ability to
cross the BBB, BRB and BTB.
In one embodiment, TREM-1 inhibitory SCHOOL peptide GF9 described herein is
incorporated into SLP that contain apo A-I peptide fragments comprising 22
amino acid residue-
long peptide sequences of the apo A-I helix 4 and/or helix 6. In one
embodiment, the inclusion of
an amphipathic apo A-I sequences in the peptides and compositions of the
invention further aids
the ability to provide targeted delivery to the cells of interest. It further
aids the ability to interact
with lipids and/or lipoproteins in a bloodstream in vivo and form lipopeptide
particles (LP) that
mimic native lipoproteins. It further aids the ability to cross the blood-
brain barrier (BBB),
blood-retinal barrier (BRB) and blood-tumor barrier (BTB).
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
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acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47
(SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47
(SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in
one embodiment, these
peptides colocalize with EGFR in the cell membrane and selectively disrupts
intramembrane
interactions between the receptors, resulting to specific ligand-independent
inhibition of the
receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself
2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein
incorporated by
reference in their entirety). In one embodiment, methionine residues of
EGFR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides
and
compounds of the present invention including but not limiting to those
described above, to
inhibit the receptors of the EGFR and/or ErB receptor families, respectively,
can be used to treat
and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related
peptides and
compounds of the present invention including but not limiting to those
described above, to
colocalize with the receptors of the EGFR and/or ErB receptor families can be
used to visualize
(image) these receptors and evaluate their expression in the areas of
interest. In one embodiment,
64co
for this purpose, an imaging probe (e.g. [ can be conjugated to the
EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the
receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be
used to diagnose
any diseases and conditions where these receptors are involved as well as to
monitor novel
therapies for these diseases and conditions.
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F. Additional Trifunctional Peptides
Additional therapeutic peptide sequences (see e.g., Vlieghe, et al. Drug
Discov Today
2010, 15:40-56; Tsung, et al. Shock 2007, 27:364-369; Chang, et al. PLoS One
2009, 4:e4171;
Tjin Tham Sjin, et al. Cancer Res 2005, 65:3656-3663; Ladetzki-Baehs, et al.
Endocrinology
2007, 148:332-336; Khan, et al. Hum Immunol 2002, 63:1-7; Banga. Therapeutic
peptides and
proteins: formulation, processing, and delivery systems. 2nd ed. Boca Raton,
FL: Taylor &
Francis Group; 2006; Stevenson. Curr Pharm Biotechnol 2009, 10:122-137; Wu and
Chi, US
9,387,257; Wu, et al., US 8,415,453; Faure, et al., US 8,013,116; Faure, et
al., US 9,273,111;
Eggink and Hoober, US 7,811,995; Eggink and Hoober, US 8,496,942; Morgan and
Pandha. US
2012/0177672 Al; Broersma, et al., US 5,681,925, each of which is herein
incorporated by
reference in it's entirety) and/or other therapeutic agents can comprise the
domain A of the
peptides and compositions of the present invention.
In one embodiment, this domain comprises the Toll Like Receptor (TLR)
modulatory
sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369, herein incorpoated by
referene in it's
entirety). The preferred peptides and compositions of this class further
comprise the domain B
comprising at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide
fragment to form upon interaction with lipid and/or lipid mixtures, SLP
structures that can be
spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media
Mol Imaging 2014,
9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US
20110256224;
Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and
Sigalov. Sci
Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen
and Sigalov.
Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018,
1864:2761-2768,
each of which is herein incorporated by reference in it's entirety). As
described above, the
inclusion of an amphipathic apo A-I sequences aids the assistance in the self-
assembly of SLP
and the structural stability of the particle formed, particularly when the
particle has a discoidal
shape. It further aids the ability to provide targeted delivery to the cells
of interest. It further aids
the ability to interact with lipids and/or lipoproteins in a bloodstream in
vivo and form LP that
mimic native lipoproteins. It further aids the ability to cross the BBB, BRB
and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
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acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32
(DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and
TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE)
(SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are
modified.
In certain embodiments, the capability of the TLR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
inhibit TLR can be used
.. to treat and/or prevent TLR-related diseases and conditions including but
not limiting to, sepsis
and other infectious diseases, and any other disorder where TLR receptors are
involved.
In certain embodiments, the capability of the TLR-related peptides and
compounds of the
present invention including but not limiting to those described above, to
colocalize with TLR can
be used to visualize (image) this receptor and evaluate its expression in the
areas of interest. In
one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the
TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels
using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases
and conditions as
well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the
invention
comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory
sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred
peptides and
compositions of this class further comprise the domain B comprising at least
one modified or
unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon
interaction with
lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal
(described herein
.. and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov.
Int
Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
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Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016,
6:28672; Shen and
Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017,
14:4572-
4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, all of which
are herein
incorporated by reference in their entirety). As described above, the
inclusion of an amphipathic
apo A-I sequences aids the assistance in the self-assembly of SLP and the
structural stability of
the particle formed, particularly when the particle has a discoidal shape. It
further aids the ability
to provide targeted delivery to the cells of interest. It further aids the
ability to interact with lipids
and/or lipoproteins in a bloodstream in vivo and form LP that mimic native
lipoproteins. It
further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences
of the major
HDL protein constituent, apo A-I. In certain embodiments, this sequence
comprises 22 amino
acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment,
this sequence
contains a modified amino acid residue. In one embodiment, this modified amino
acid residue is
methionine sulfoxide. In one embodiment, the domain B of the peptides and
compositions of the
invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one
embodiment, this sequence contains a modified amino acid residue. In one
embodiment, this
modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of
representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50
(SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50
(SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are
modified.
In certain embodiments, the capability of the ANPR-related peptides and
compounds of
the present invention including but not limiting to those described above, to
inhibit ANPRs can
be used to treat and/or prevent ANPR-related diseases and conditions including
but not limiting
to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are
involved.
In certain embodiments, the capability of the ANPR-related peptides and
compounds of
the present invention including but not limiting to those described above, to
colocalize with
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ANPR can be used to visualize (image) this receptor and evaluate its
expression in the areas of
interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated
to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of
ANPR levels
using PET and/or other imaging techniques can be used to diagnose ANPR-related
diseases and
conditions as well as to monitor novel therapies for these diseases and
conditions.
In certain embodiments, other therapeutic agents including but not limiting
to, to those
described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R,
Wagman LD,
Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th
ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al.,
Therapy of
Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013,
49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene
Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating
Peptides. 2nd
edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman
and Altman,
US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne,
et al., US 9,173,891, all of which are herein incorporated by reference in
their entirety, (see also
TABLE 2) can comprise the domain A of the peptides and compositions of the
present invention.
V. Diseases Contemplated For Treatment Using Peptides and Compositions
Described
Herein.
A. Overview.
The present invention encompasses the recognition that it is possible to
produce
compositions that possess the advantages typically associated with a fully
synthetic material and
yet also possess certain desirable features of materials derived from natural
sources.
In some embodiments, peptides and compounds of the present invention, e.g.
trifunctional peptides, with rHDLs (including discoidal and/or spherical HDLs)
or without
rHDLs (such as in therapeitic compositions as free trifunctional peptides),
are contemplated for
use in preventative treatments for diseases associated with activated
macrophages and/or T-cells,
in particular for preventing one or more symptoms associated with the disease.
In some
embodiments, peptides and compounds of the present invention are contemplated
for use
preventative treatments for diseases associated with activated macrophages
and/or T-cells, in
particular for reducing one or more symptoms associated with the disease. In
some
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embodiments, peptides and compounds of the present invention are contemplated
for use
diagnostic applications for detecting/identifying; determining disease
progression; determining
results of disease treatment, for diseases associated with activated
macrophages and/or T-cells.
Such diseases associated with activated macrophages and/or T-cells include but
are not limited to
including but not limited to lung cancer, such as non small-cell lung cancer
(NSCLC); pancreatic
cancer (PC); glioblastoma multiforme (GBM, or brain cancer), with or without
radiation therapy;
breast cancer with or without radiation therapy; sepsis; retinopathy;
rheumatoid arthritis (RA);
sepsis; and alcoholic liver disease (ALD). Furthermore, diseases associated
with activated
macrophages and/or T-cells include but are not limited to 1) Alcohol-induced
neuroinflammation
.. and brain damage; 2) Radiation-induced multiple organ dysfunction syndrome;
3) Scleroderma;
4) Atopic dermatitis; 5) Atherosclerosis; 6) Alzheimer's, Parkinson's and/or
Huntington's
diseases. In one embodiment, the present invention relates to the targeted
treatment, prevention
and/or detection of cancer including but not limited to lung, pancreatic,
breast, stomach, prostate,
colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic
diseases, acute radiation
syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia,
hemorrhagic
shock, multiple sclerosis, liver diseases, autoimmune diseases, including but
not limited to,
atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic
arthritis and other rheumatic
diseases, sepsis and other inflammatory diseases or other condition involving
myeloid cell
activation and, more particularly, TREM receptor-mediated cell activation,
including but not
limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's,
Parkinson's and
Huntington's diseases.
Thus, in some embodiments, trifunctonal peptides as described herein are
contemplated
for administration to a subject for reducing a disease symptom. In some
embodiments,
trifunctonal peptides as described herein are contemplated for administration
to a subject for
delaying onset of a disease symptom. In some embodiments, trifunctonal
peptides as described
herein are contemplated for administration to a subject for preventing a
disease symptom. In
some embodiments, trifunctonal peptides as described herein are contemplated
for administration
to a subject receiving therapy for a disease. In some embodiments,
trifunctonal peptides as
described herein are contemplated for administration to a subject receiving
anti-cancer therapy.
In some embodiments, trifunctonal peptides as described herein, are
contemplated for
administration to a subject as anti-cancer therapy. In some embodiments,
trifunctonal peptides as
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described herein, further comprising a drug compound are contemplated for
administration to a
subject as anti-cancer therapy. In some embodiments, trifunctonal peptides as
described herein,
further comprising a Paclitaxel compound are contemplated for administration
to a subject as
anti-cancer therapy.
As disease progression of a liver in a subject proceeds from epatosteatosis,
steatohepatitis, and fibrosis to cirrhosis, it is contemplated that a
trifunctonal peptide as described
herein, is administered to said subject at any point along the disease
progression for reducing
disease progression, in part as described herein. Thus, in some embodiments,
trifunctonal
peptides as described herein are contemplated for administration to a subject
for reducing a liver
disease symptom, including but not limited to reducing one or more of ALT,
procollegen I-alpha
and alpha-SMA.
In some embodiments, trifunctonal peptides as described herein are
contemplated for
administration to a subject for reducing a liver disease symptom, in
combination with one or
more of steroid drugs, ursodiol, etc., in order to delay or prevent further
progression of liver
.. degeneration to cirrhosis. In some embodiments, trifunctonal peptides as
described herein are
contemplated for administration to a subject for reducing a liver disease
symptom in combination
with one or more of steroid drugs, ursodiol, etc., in order to improve
function of a diseased liver.
In some embodiments, trifunctonal peptides as described herein are
contemplated for
administration to a subject for treating severe hemorrhagic shock. In some
embodiments,
trifunctonal peptides as described herein are contemplated for administration
to a subject for
treating colitis and colitis-associated tumorigenesis.
In some embodiments, trifunctonal peptides as described herein are
contemplated for
administration to a subject for decreasing neovascularization.
In some embodiments, trifunctonal peptides as described herein are selected
from the
group consisting of G-KV21, G-HV21, G-TE21, M-VE32 and M-TK32, and mixtures
thereof. In
some embodiments, a trifunctonal peptide as described herein is GE31. In some
embodiments, a
trifunctonal peptide as described herein is GA31. In some embodiments, a
trifunctonal peptide as
described herein is a mixture of GE31 and GA31.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one
embodiment of
a trifunctional peptide (TRIOPEP) of the present invention comprising amino
acid domains A
and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic
peptide sequence
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and functions to treat and/or prevent a TREM-1-related disease or condition
(shown for cancer),
whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6
peptide sequence
with a sulfoxidized methionine residue and functions to assist in the self-
assembly of synthetic
lipopeptide particles (SLP) and to target the particles to TREM-1-expressing
macrophages as
applied to the treatment and/or prevention of cancer. While not being bound to
any particular
theory, it is believed that chemical and/or enzymatic modification of protein
sequence in domain
B leads to the recognition of SLP of the present invention by the macrophage
scavenger
receptors and results in an irreversible binding to and consequent uptake by
macrophages of such
particles. It is further believed that accumulation of these particles in
tumor-associated
macrophages is accompanied by accumulation of TRIOPEP in these cells. In
contrast, native
HDL particles that contain only unmodified apolipoprotein molecules are not
recognized by
tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
B. Cancer.
Approximately 8.8 million people are dying each year of cancer, amounting to
one out of six deaths globally, and cancer incidence is estimated to double by
2035 (Prager et al.
2018). Combination-therapy treatments for cancer have become more common, in
part due to the
perceived advantage of attacking the disease via multiple avenues. Although
many effective
combination-therapy treatments have been identified over the past few decades;
in view of the
continuing high number of deaths each year resulting from cancer, a continuing
need exists to
identify effective therapeutic regimens for use in anticancer treatment.
The present invention encompasses the recognition that it is possible to
prevent and treat
different types of cancer including but not limited to, pancreatic cancer,
multiple myeloma,
leukemia, prostate cancer, breast cancer, liver cancer, bladder cancer,
colorectal cancer, lung
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, or osteosarcoma
and other cancers,
and cancer cachexia by blocking the TREM-1 signaling pathway using the peptide
variants and
compositions that possess the advantages typically associated with a fully
synthetic material and
yet possess certain desirable features of materials derived from natural
sources. The invention
further encompasses the recognition that it is possible to use imaging
techniques and the peptide
variants and compositions of the invention conjugated to an imaging probe for
detecting the
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labeled probe in an individual with cancer, wherein the location of the
labeled probe corresponds
to at least one symptom of the myeloid cell-related condition. The invention
further encompasses
that it is possible to predict the efficacy of the peptides and compositions
of the invention by
determining the number of myeloid cells in the biological sample from the
individual with cancer
and determining the expression levels of TREM-1 in the cells contained within
the biological
sample.
Cancer continues to have a huge Social and economic impact. In 2011, 571,950
Americans died of cancer (-25% of all deaths), with US cancer-associated costs
of S263.8
billion: S102.8 billion for direct medical costs (total health expenditures);
$20.9 billion for
indirect morbidity costs (lost productivity); and S140.1 billion for indirect
mortality costs (lost
productivity from premature death).
Inflammatory responses play decisive roles at different stages of tumor
development,
including initiation, promotion, malignant conversion, invasion, and
metastasis (Grivennikov et
al. 2010). Inflammation also affects immune surveillance and responses to
therapy (Grivennikov
et al. 2010). Many solid tumors are characterized by a marked infiltration of
macrophages,
inflammatory cells, into the stromal compartment (Shih et al. 2006, Solinas et
al. 2009), a
process which is mediated by cancer-associated fibroblasts (CAFs) and plays a
key role in
disease progression and its response to therapy (see FIG. 49). These tumor-
associated
macrophages (TAMs) secrete a variety of growth factors, cytokines, chemokines,
and enzymes
.. that regulate tumor growth, angiogenesis, invasion, and metastasis (Shih et
al. 2006). See FIG.
49. High macrophage infiltration correlates with the promotion of tumor growth
and metastasis
development (Solinas et al. 2009, Grivennikov et al. 2010). In patients with
PC, macrophage
infiltration begins during the preinvasive stage of the disease and increases
progressively (Clark
et al. 2007). The number of TAMs is significantly higher in patients with
metastases (Gardian et
al. 2012). TREM-1 is upregulated in cancer and its overexpression correlates
with survival of
cancer patients. In NSCLC, TREM-1 expression in TAMs is associated with cancer
recurrence
and poor survival of patients with NSCLC: patients with low TREM-1 expression
have a 4-year
survival rate of over 60%, compared with less than 20% in patients with high
TREM-1
expression (Ho et al. 2008). Activation of the TREM-1/DAP-12 signaling pathway
results in
release of multiple cytokines, chemokines and growth factors most of which are
increased in
cancer patients and their upregulation correlates with poor prognosis (See
FIG. 1).
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The present invention encompasses the recognition that it is possible to
prevent and treat
different types of cancer in which myeloid cells are involved or recruited and
cancer cachexia by
combining cancer therapies with a therapeutically effective amount of at least
one compound
and/or composition ("modulator") which affects myeloid cells by action on the
TREM-1/DAP-12
signaling pathway.
The infiltrate of most solid tumors contains tumor-associated macrophages
(TAMs) that
are attracted by chemokines including CCL2 and represent attractive treatment
targets in
oncology (Shih et al. 2006, Mantovani et al. 2017). The increased TAM content
in NSCLC
(Yusen et al. 2018) is associated with poor prognosis in NSCLC (Welsh et al.
2005). TAM
recruitment, activation, growth and differentiation are regulated by CSF-1
(Elgert et al. 1998,
Laoui et al. 2014). Many tumor cells or tumor stromal cells have been found to
produce CSF-1,
which activates monocyte/macrophage cells through CSF-1 receptor (CSF-1R). The
level of
CSF-1 in tumors has been shown to correlate with the level of TAMs in the
tumor. Higher levels
of TAMs have been found to correlate with poorer patient prognoses in the
majority of cancers.
Increased pretreatment serum CSF-1 is a strong independent predictor of poor
survival in
NSCLC (Baghdadi et al. 2018). In addition, CSF-1 has been found to promote
tumor growth and
progression to metastasis in, for example, human breast cancer xenografts in
mice (Paulus et al.
2006). Further, CSF-1R plays a role in osteolytic bone destruction in bone
metastasis (Ohno et
al. 2006). TAMs promote tumor growth, in part, by suppressing anti-tumor T
cell effector
function through the release of immunosuppressive cytokines and the expression
of T cell
inhibitory surface proteins. Blockade of CSF-1 or CSF-1R not only suppresses
tumor
angiogenesis and lymphangiogenesis (Kubota et al. 2009) but also improves
response to T-cell
checkpoint immunotherapies that target programmed cell death protein 1 (PD-1),
programmed
death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu et
al. 2014).
Importantly, continuous CSF-1 inhibition affects pathological angiogenesis but
not healthy
vascular and lymphatic systems outside tumors (Kubota et al. 2009). In
contrast to blockade of
vascular endothelial growth factor (VEGF), interruption of CSF-1 inhibition
does not promote
rapid vascular regrowth (Kubota et al. 2009).
The present invention provides a method of treating these and other types of
cancers by using modulators of the TREM-1/DAP-12 signaling pathway that are
capable of
binding TREM-1 and modulating TREM-1/DAP-12 receptor complex activity in
combination-
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therapy treatments together with other cancer therapies. The invention further
provides the
methods for predicting response of a cancer patient to the treatment by using
these modulators in
combination-therapy regimen. These and other objects and advantages of the
invention, as well
as additional inventive features, will be apparent from the description of the
invention provided
herein.
The invention further encompasses the recognition that it is possible to
predict response
of the subject to the treatment by using the modulators of TREM-1/DAP-12
signaling pathway in
combination-therapy regimen by: (a) obtaining a biological sample from the
subject; (b)
determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of
CD68-positive
.. TAMs or a combination thereof, wherein the higher is the expression of CSF-
1, CSF-1R, IL-6,
TREM-1 or the higher is number of CD68-positive TAMs or a combination thereof,
the better
the patient is predicted to respond to a therapy that involves the modulators.
The invention further encompasses the recognition that it is possible to use
imaging
techniques and the modulators conjugated to an imaging probe for detecting the
labeled probe in
an individual with cancer in which myeloid cells are involved or recruited,
wherein the location
and the measured intensity of the labeled probe can diagnose cancer and/or
predict response of
the subject to the treatment by using the modulators of TREM-1/DAP-12
signaling pathway, the
higher the measured intensity of the labeled probe, the better the patient is
predicted to respond
to a therapy that involves the modulators.
1. Lung Cancer.
Lung cancer, including NSCLC, is the leading cause of cancer deaths worldwide
(Wong
et al. 2018) and has a poor prognosis. Despite advances made in chemotherapy,
NSCLC is
responsible for over 1.1 million deaths annually worldwide, and the 5-year
survival rate for
patients with NSCLC is reported to be only 15% or less than 18% (Zappa et al.
2016), showing
.. an urgent need for new therapies.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer (NSCLC) H292 (FIG. 11A) and A549 (FIG. 11B) xenograft
mice treated
with an equimolar mixture of the sulfoxidized methionine residue-containing
TREM-1-related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX,
paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
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FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into
synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared
with
vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in
the human non-
small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice
treated with an
equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-
related
trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into
synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared
with
vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549
xenograft mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form
or
incorporated into synthetic lipopeptide particles (SLP) particles of discoidal
(TREM-
1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX,
paclitaxel.
<0.0001 as compared with vehicle-treated animals.
2. Pancreatic cancer.
Pancreatic cancer (PC, 85% of which are pancreatic ductal adenocarcinomas,
PDAC) is
the fourth leading cause of cancer-related mortality across the world with
very poor clinical
outcome. (Ilic et al. 2016). Current treatments of PC marginally prolong
survival or relieve
symptoms in patients with PC (Ilic and Ilic 2016). There has been no
significant progress in the
field of targeted therapy for PC (Walker et al. 2014) and despite tremendous
efforts, the 5-year
survival rate remains less than 5% (Ilic and Ilic 2016).This highlights the
urgent need for novel
approaches to prevent and treat PC and other types of cancer. However, it
should be noted that
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the techniques and compositions listed and described herein are applicable to
a broad range of
other types of cancer and cancer cachexia. Other features and advantages of
the invention will
become apparent from the following detailed description. It should be
understood, however, that
the detailed description and the specific examples, while indicating preferred
embodiments of the
invention, are given by way of illustration, because various changes and
modifications within the
spirit and scope of the invention will become apparent to those skilled in the
art from this
detailed description.
Current treatments of PC marginally prolong survival or relieve symptoms in
patients
with PC (Schneider 2005). There has been no significant progress in the field
of targeted therapy
for PC (Walker and Ko 2014) and despite tremendous efforts, the 5-year
survival rate remains
less than 5% (2010).
3. Additional Neoplasms: Giant Cell Tumor and PVNS.
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the
inflammatory
response (Colonna et al. 2003) and is upregulated under inflammatory
conditions including
.. cancer (Wang et al. 2004). For downstream signal transduction, TREM-1 is
coupled to the
immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, DNAX
activation
protein of 12kDa (DAP12). TREM-1/DAP-12 receptor complex activation enhances
release of
multiple cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor
necrosis
factor-a (TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and colony-stimulating
factor 1 (referred
to herein as CSF1; also referred to in the art as M-CSF) (Schenk et al. 2007,
Lagler et al. 2009,
Sigalov 2014).
Binding of CSF1 or the interleukin 34 ligand (referred to herein as IL-34) to
CSF1
receptor (referred to herein as CSF1R) leads to receptor dimerization,
upregulation of CSF1R
protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues,
and downstream
signaling events. CSF1R activation by CSF1 or IL-34 leads to the trafficking,
survival,
proliferation, and differentiation of monocytes and macrophages, as well as
other monocytic cell
lineages such as osteoclasts, dendritic cells, and microglia.
Many tumor cells or tumor stromal cells have been found to produce CSF1, which
activates monocyte/macrophage cells through CSF1R. The level of CSF1 in tumors
has been
shown to correlate with the level of tumor-associated macrophages (TAMs) in
the tumor. Higher
levels of TAMs have been found to correlate with poorer patient prognoses in
the majority of
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cancers. In addition, CSF1 has been found to promote tumor growth and
progression to
metastasis in, for example, human breast cancer xenografts in mice (Paulus et
al. 2006). Further,
CSF1R plays a role in osteolytic bone destruction in bone metastasis (Ohno et
al. 2006). TAMs
promote tumor growth, in part, by suppressing anti-tumor T cell effector
function through the
release of immunosuppressive cytokines and the expression of T cell inhibitory
surface proteins.
Blockade of CSF1 or CSF1R not only suppresses tumor angiogenesis and
lymphangiogenesis
(Kubota et al. 2009) but also improves response to T-cell checkpoint
immunotherapies that target
programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen-4
(CTLA-4) (Zhu et
al. 2014). Importantly, continuous CSF1 inhibition affects pathological
angiogenesis but not
healthy vascular and lymphatic systems outside tumors (Kubota et al. 2009). In
contrast to
blockade of vascular endothelial growth factor (VEGF), interruption of CSF1
inhibition does not
promote rapid vascular regrowth (Kubota et al. 2009).
Giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor
(TGCT;
also referred to in the art as TSGCT), and pigmented villonodular synovitis
(PVNS) are the
common names for a group of rare proliferative disorders that involve synovial
joints and tendon
sheaths. PVNS is a solid tumor of the synovium with features of both reactive
inflammation and
clonal neoplastic proliferation in which CSF1 is over expressed. A common
translocation of the
CSF1 gene (1p13) to the COL6A3 promoter (2q35) is present in approximately 60%
of PVNS
patients. The translocation is accompanied by CSF1 overexpression in the
synovium. In addition,
approximately 40% of PVNS patients have CSF1 overexpression in the absence of
an identified
CSF1 translocation. The consistent presence of CSF1 overexpression in all
cases of PVNS and
reactive synovitis suggests both an important role for CSF1 in the spectrum of
synovial
pathologies and the utility of targeting the CSF1/CSF1R signaling pathway
therapeutically (West
et al. 2006). In PVNS, CSF1 overexpression is present in a minority of
synovial cells, whereas
the majority of the cellular infiltrate expresses CSF1R but not CSF1. This has
been characterized
as a tumor-landscaping effect with aberrant CSF1 expression in the tumor
cells, leading to the
abnormal accumulation of non-neoplastic cells that form a mass.
Surgery is the treatment of choice for patients with localized PVNS.
Recurrences occur in
8-20% of patients and are often managed by re-excision. Diffuse tenosynovial
giant cell tumor
(TGCT/PVNS or PVNS/dtTGCT) tends to recur more often (33-50%) and has a much
more
aggressive clinical course. Patients are often symptomatic and require
multiple surgical
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procedures during their lifetime and even amputation. For patients with
unresectable disease or
multiple recurrences, systemic therapy using CSF1R inhibitors may help delay
or avoid surgical
procedures and improve functional outcomes (Radi et al. 2011).
Imatinib, a non-specific inhibitor of CSF1R, has undergone evaluation in PVNS
patients
(Cassier et al. 2012). Twenty-nine patients from 12 institutions in Europe,
Australia, and the
United States were included. The median age was 41 years and the most common
site of disease
was the knee (n=17; 59%). Two patients had metastatic disease to the lung
and/or bone. Five of
27 evaluable patients had complete (n=1) or partial (n=4) responses per RECIST
for an overall
response rate of 19%. Twenty of 27 patients (74%) had stable disease.
Symptomatic
improvement was noted in 16 of 22 patients (73%) who were assessable for
symptoms. Despite a
high rate of symptomatic improvement and an overall favorable safety profile,
10 patients
discontinued treatment for toxicity or other reasons.
Pexidartinib (PLX3397), a potent, selective oral CSF1R inhibitor, that traps
the kinase in
the autoinhibited conformation, has undergone evaluation in TGCT patients (Tap
et al. 2015). A
total of 41 patients were enrolled in the dose-escalation study, and an
additional 23 patients were
enrolled in the extension study. In the extension study, 12 patients with
TGCTs had a partial
response and 7 patients had stable disease. The most common adverse events
included fatigue,
change in hair color, nausea, dysgeusia, and periorbital edema; adverse events
rarely led to
discontinuation of treatment. Despite treatment of TGCTs with PLX3397 resulted
in a prolonged
regression in tumor volume in most patients of this Phase 2 study, later the
Phase 3 study was
suspended after two reported cases of nonfatal, serious liver toxicity.
Anti-CSF1R antibodies alone or in combination with antibodies against PD1 or
against
PDL1, one of the ligands for PD1, were proposed as less toxic alternative
treatments for PVNS.
See, e.g., US Pat 10,040,858 B2 and US Pat 10,221,224. As with most
combination therapies,
the promise of increased clinical activity is accompanied by the risk of
additive toxicity and
therefore requires careful assessment.
Liver enzyme elevations can be considered a class effect of CSF1R-targeting
compounds
(Cannarile et al. 2017). In addition, the oversuppression of the CSF1/CSF1R
signaling pathway
may result in potential serious long term adverse effects (AEs). In animals,
CSF1 deficiency
results in a range of developmental abnormalities, including skeletal,
neurological, growth and
fertility defects (Michaelson et al. 1996, Hume et al. 2012, Jones et al.
2013).
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Thus, PVNS is a rare, locally aggressive neoplasm of the joint or tendon
sheath with
features of both reactive inflammation and clonal neoplastic proliferation in
which CSF-1 is over
expressed (Tap et al. 2015). Surgical resection is the primary treatment;
however, diffuse TGCT
is more difficult to resect and often involves total synovectomy, joint
replacement, or amputation
(Tap et al. 2015). There are no approved systemic therapies. Therefore,
alternative, less toxic and
more targeted treatments for PVNS are needed.
Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1
and is
a promising approach in a variety of inflammation-associated disorders
including cancer
(Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov
2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium
sulfate-induced
models of colitis, Treml-/- mice displayed significantly attenuated disease
that was associated
with reduced inflammatory infiltrates and diminished expression of pro-
inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased
lesion size upon
infection with Leishmania major (Weber et al. 2014). Furthermore, reduced
morbidity was
observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while
immune-associated pathologies were significantly reduced, Treml-/- mice were
equally capable
of controlling infections with L. major, influenza virus, but also Legionella
pneumophila as
Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have
problems
resolving infections with viruses or bacteria (Lanier 2009). Collectively,
these findings suggest
that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic
blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders
including CSF1-
dependend TGCTs holds considerable promise by blunting excessive inflammation
while
preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-
12
modulatory peptides and compositions for treatment of PVNS. These and other
objects and
advantages of the invention, as well as additional inventive features, will be
apparent from the
description of the invention provided herein.
Methods of treating tenosynovial giant cell tumor (TGCT) or pigmented
villonodular
synovitis (PVNS) with peptide variants and compositions that modulate activity
of the receptor
complex formed by triggering receptor expressed on myeloid cells 1 (TREM-1)
and DNAX
activation protein of 12kDa (DAP12) are provided.
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Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1
and is
a promising approach in a variety of inflammation-associated disorders
including cancer
(Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov
2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium
sulfate-induced
models of colitis, Treml-/- mice displayed significantly attenuated disease
that was associated
with reduced inflammatory infiltrates and diminished expression of pro-
inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased
lesion size upon
infection with Leishmania major (Weber et al. 2014). Furthermore, reduced
morbidity was
observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while
immune-associated pathologies were significantly reduced, Treml-/- mice were
equally capable
of controlling infections with L. major, influenza virus, but also Legionella
pneumophila as
Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have
problems
resolving infections with viruses or bacteria (Lanier 2009). Collectively,
these findings suggest
that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic
blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders
including CSF1-
dependend TGCTs holds considerable promise by blunting excessive inflammation
while
preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-
12
modulatory peptides and compositions for treatment of PVNS. These and other
objects and
advantages of the invention, as well as additional inventive features, will be
apparent from the
description of the invention provided herein.
4. Liver Cancer.
Globally, liver cancer is the fifth commonest cancer in 2012, accounting for
9.1% of all
cancer deaths worldwide with the overall 5-year relative survival rate for
patients with liver
cancer of 17%. Owing to its extremely aggressive nature and poor survival
rate, it remains an
important public health issue worldwide (Wong et al. 2017)
5. Breast Cancer.
Breast cancer is the most common malignancy in women around the world
(Ghoncheh et
al. 2016). Ii is the most common cancer in women, accounting for 25.1% of all
cancers. Breast
cancer incidence in developed countries is higher, while relative mortality is
greatest in less
developed countries (Ghoncheh et al. 2016). Despite significant improvements
in clinical
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outcomes within the field of breast cancer in the last 50 years, the triple-
negative breast cancer
(TNBC) subtype remains an area of huge unmet clinical need (Partridge et al.
2017).
6. Glioblastoma.
Glioblastoma Multiforme (GBM) is the most common and lethal type of brain
cancer
(Shergalis et al. 2018). For adults with GBM, treated with standard first-line
therapy ¨
concurrent radiation and temozolomide (TMZ) therapy followed by TMZ
monotherapy, the
median survival is about 14.6 months (Grossman et al. 2010, Shergalis et al.
2018). Little
progress has been made over the past several decades in the treatment of GBM,
highlighting an
urgent need for new therapies.
7. Colorectal Cancer.
Colorectal cancer (CRC) has a considerable impact on patients and healthcare
systems in
developed countries and around 25% of patients present with metastatic disease
that significantly
impacts on prognosis (Van Cutsem et al. 2013). For those with localized CRC of
stages I and II,
the 5-year survival rate is as high as 93%, declining to 60%, 42% and 25% for
patients with
stages IIIA, IIIB and IIIC, respectively. However, most patients with
metastatic CRC (stage IV)
are not curable, with the 5-year survival rate falling to less than 10%. While
early diagnosis of
CRC in recent years combined with advances in treatment has considerably
improved survival,
management of the disease remains challenging and further progress is needed
(Van Cutsem et
al. 2013).
Scleroderma, related autoimmune conditions and fibrotic conditions.
It is estimated that scleroderma or systemic sclerosis (SSc) affects 100,000-
300,000
Americans, predominantly young to middle aged women. Systemic sclerosis is a
progressive and
untreatable disease of unknown cause and high mortality. Fibrosis in SSc
resembles uncontrolled
.. wound healing, where healing occurs by intractable fibrosis rather than
normal tissue
regeneration.
It is believed that SSc is associated with the highest case-fatality rates
among the
rheumatic diseases or connective tissue diseases. Currently, there are no
validated biomarkers for
diagnosis. Furthermore, no effective disease-modifying therapies are currently
available. In fact,
.. while some treatment can alleviate the pain associated with SSc, to date no
therapy has been
shown to significantly alter survival. The pathogenesis of SSc is
characterized by early vascular
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injury, with inflammation followed by progressive tissue damage and fibrosis.
Excessive
production of collagen and ECM and accumulation of myofibroblasts in lesional
tissues are
believed to be responsible for progressive organ failure. Pathological
fibrosis resembles a normal
wound healing response that has become deregulated. It is estimated that
fibrosis accounts
for>25% of all deaths in the U.S. Thus, fibrosis represents one of the major
unmet medical
needs.
Accordingly, there is a need for an effective anti-fibrotic therapy.
Project Summary/Abstract
Scleroderma that includes localized scleroderma (LS) and systemic sclerosis
(SSc) is a
rare but devastating autoimmune disorder. Current therapies all have side
effects, are limited and
associate with 10 year survival of 55%, showing the need for novel approaches.
The long-term
goal of this project is to develop a new mechanism-based, efficient and well
tolerable
scleroderma therapy.
Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation
amplifier,
contributes to the development of fibrosis in SSc. In patients, number of
activated macrophages
in the fibrotic areas is increased and associates with fibrosis severity.
Activation of TREM-1
leads to overproduction of MCP-1/CCL2 and M-CSF/CSF-1, resulting in macrophage
recruitment to an injured area and the sclerotic lesion formation in rats with
scleroderma. In
animal models, TREM-1 blockade inhibits inflammation and ameliorates a variety
of
autoimmune diseases. The hypothesis of the "proof-of-concept" Phase I is that
TREM-1
blockade can prevent and treat scleroderma.
Current TREM-1 inhibitors all attempt to block binding of TREM-1 to its still
uncertain
ligand(s). To minimize risk of failure in clinical development, we developed a
first-in-class
ligand-independent TREM-1 inhibitory peptide GF9 that is well-tolerated and
can be formulated
into SignaBlok's long half-life macrophage-specific lipopeptide complexes
(LPC) to improve its
half-life and targeting to the inflammation areas. The major goal of the Phase
I study is to show
that TREM-1 blockade by GF9-LPC alleviates the disease in a bleomycin (BLM)-
induced mouse
model of scleroderma.
Phase I specific aims are to: 1) optimize TREM-1 inhibitory compositions for
their
functionality in vitro and pharmacokinetics in vivo and select the lead, 2)
test two doses of the
lead selected in a BLM-induced mouse model of scleroderma. We will generate,
optimize and
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select the lead based upon its functionality in vitro and its PK profile in
vivo. We will test two
doses of the lead for its ability to prevent and treat lung, heart, muscle and
skin fibrosis in a
mouse model of multiorgan fibrosis in vivo. Histology/IHC studies will be
performed. Serum
and tissue cytokines will be evlauated, nonlmiting examples including MCP-1,
CSF-1, VEGF,
TGF-beta, TNF-alpha, IL-6, and IL-1-beta, will be analyzed.
It is anticipated that the Phase I study will identify a novel, first-in-
class, well tolerable
agent as a powerful platform for development of an effective and well-
tolerable systemic
scleroderma therapy, thereby improving treatment and survival of patients. Its
anticipated safety
is supported by good tolerability of SignaBlok's GF9-based formulations by
long term-treated
mice. Prototypes of SignaBlok's LPC are well tolerated in humans. TREM-1
blockade by
SignaBlok competitor's inhibitory peptide LR12 (Inotrem, France) was safe in
healthy and septic
subjects. If successful, Phase I will be followed in Phase II by toxicology,
ADME, pharmacology
and CMC studies, filing an IND and subsequent evaluation in humans.
Project Narrative.
Scleroderma (also known as systemic sclerosis) is a rare autoimmune disorder
that affects
about 20 to 24 people per million population in the US each year, with the
majority being women
of childbearing age. There is no approved drug for scleroderma. Current
therapies all have side
effects, are limited and associated with 10 year survival of 55%, highlighting
the urgent need for
novel approaches The proposed research is anticipated to result in the
development of novel
mechanism-based first-in-class therapeutics that could substantially improve
treatment of
scleroderma and patient survival.
SPECIFIC AIMS.
The Product. The final product will represent a new mechanism-based,
efficient, stable, well
tolerable systemic immunomodulatory therapy for scleroderma in order to
significantly decrease
long-term disability, morbidity and mortality of the patients with scleroderma
and improve the
quality of their life.
Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes
et al. 2003, Helmick et al. 2008) with no approved drug available. Current
main treatments all
have side effects, are limited and associated with 10 year survival of 55%
(Badea et al. 2009,
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Kowal-Bielecka et al. 2009, Shah et al. 2013), highlighting an urgent need for
new therapies.
Macrophages are associated with fibrosis (Ishikawa et al. 1992, Kraling et al.
1995, Lech et al.
2013, Chia et al. 2015) and are recruited to inflammation sites by MCP-1 which
is significantly
elevated in patients with systemic sclerosis (SSc) (Hasegawa et al. 1999).
Activated
macrophages produce VEGF, IL-lbeta, TNFalpha, IL-6, TGFbeta and PDGF that play
a role in
scleroderma (Bonner et al. 1991, Clouthier et al. 1997, Yamamoto 2011,
Yamamoto et al. 2011,
Liu et al. 2013, Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R)
(Kitaba et al. 2012),
VEGF (Koca et al. 2016), TNFalpha (Koca et al. 2008) and TGFalpha (Varga et
al. 2009, Varga
et al. 2009) alleviate scleroderma but all may have serious side effects
including fatal infections
and sepsis (Varga 2004). CSF-1/M-CSF plays a role in pulmonary fibrosis that
occurs in 90% of
scleroderma patients (Baran et al. 2007). TREM-1 mediates release of MCP-
1/CCL2, TNFalpha,
IL-lbeta, IL-6 and CSF-1 (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov
2014, Shen et al. 2015). TREM-1 expression is increased in the lungs of mice
with BLM-induced
pulmonary fibrosis (Peng et al. 2016). Together, this implicates TREM-1 as a
new target to
develop a first-in-class therapy for scleroderma.
Innovation. At least two aspects: /. This is the first project to study TREM-1
blockade in an
animal model of scleroderma. 2. To block TREM-1, we use a proprietary peptide
GF9
formulated into macrophage-specific LipoPeptide Complexes (LPC) to extend its
half-life and
increase targeting (Sigalov 2014, Shen et al. 2017, Shen et al. 2017). Other
TREM-1 blockers
(e.g., LR12 peptide by Inotrem, France (Cuvier et al. 2018)) all attempt to
block binding of
currently uncertain ligands of TREM-1 and have a risk of failure in clinics,
while GF9 is an
advantageously ligand-independent.
Previously ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017,
Tornai et al.
2019), Preliminary Data), we found that TREM-1 blockade using GF9: ameliorates
disease in
mice with collagen-induced arthritis (CIA); reduces serum CSF-1, TNFalpha, IL-
lalpha, IL-6 in
mice with CIA, cancer, and liver disease; and inhibits expression of MCP-
1/CCL2, TNFalpha,
Pro-Colll-alpha and alpha -SMA in mice with liver disease.
The goal of this project is to develop TREM-1-targeting drug for the treatment
of
scleroderma.
Aim 1: Optimize TREM-1 inhibitory compositions for their functionality in
vitro and
pharmacokinetics in vivo and select the lead. GF9-LPC will be generated using
GF9, lipids and
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two modified peptides that mediate macrophage uptake of GF9-LPC and affect
their half-life in
vivo. We will vary lipid/peptide composition and peptide ratios to prepare
long half-life GF9-
LPC with fast and high uptake by J774 cells and high inhibitory effect on
cytokine release by
LPS-stimulated J774 cells. Three most promising GF9-LPC injectables selected
based on their
functionality in vitro will be tested in rats for their pharmacokinetic (PK)
profiles. To analyze
GF9 in animal serum, we will develop and validate an LC-MS assay with ZATA
Pharmaceuticals. Milestone 1 includes development of the long half-life lead,
which is efficient
in inhibiting cytokine release in vitro. Completion of the Aim 1 will answer
the question on the
possibility of generating of the lead optimized to provide fast, efficient and
long-lasting
therapeutic effect.
Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model
of
scleroderma. We have shown that chronic subcutaneous injection of BLM in mice
results in the
development of progressive multiple organ fibrosis with histological changes
in the skin, muscle
and lungs that resemble those seen in patients with SSc (Bhattacharyya et al.
2018,
Bhattacharyya et al. 2018). Two doses of the GF9-LPC lead generated in the Aim
1 will be
tested for its effect on lung, heart, muscle and skin fibrosis in this mouse
model. Studies will be
performed at Northwestern Scleroderma by lab of Dr. John Varga, a world-
renowned expert in
autoimmune diseases with special emphasis on scleroderma. Histology/IHC
studies will be
performed. Serum and tissue CCL2, CSF-1, VEGF, TGFbeta, TNFalpha, IL-6, and IL-
lbeta will
be analyzed. Milestone 2 includes in vivo testing of suitability of TREM-1
blockade to prevent
and treat scleroderma. Completion of the Aim 2 will answer a question about
feasibility of using
GF9-LPC as a first-in-class therapy for scleroderma.
The project is anticipated to identify the lead that will set the stage for
development of
first-in-class, safe and effective scleroderma therapies. If successful, Phase
I will be followed in
Phase II by toxicology, pharmacology ADME, PK/PD, and CMC studies, filing an
IND and
subsequent evaluation in humans.
Anticipated low toxicity of GF9 therapy is supported by the safety and well
tolerability of
300 mg/kg GF9 in healthy mice (Sigalov 2014) (while its therapeutic dose
varies from 2.5 mg/kg
for GF9-LPC to 25 mg/kg for free GF9 (Sigalov 2014, Shen and Sigalov 2015,
Rojas et al. 2017,
.. Shen and Sigalov 2017, Tornai et al. 2019)) and lack of body weight changes
in cancer and
arthritic mice long-term treated with GF9-LPC (Sigalov 2014, Shen and Sigalov
2017).
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Prototypes of SignaBlok's LPC are safe in humans (Newton et al. 2002, Kingwell
et al. 2013).
TREM-1 blockade using peptide LR12 developed by SignaBlok's top competitor
(Inotrem,
France) is safe in humans (Cuvier et al. 2018, Francois et al. 2018).
Successful completion of Phase I will provide the animal proof of concept that
might be
applicable not only to scleroderma but also to other rare musculoskeletal,
rheumatic or skin
diseases.
Research Strategy
Scleroderma: An unmet need for an effective and low toxic treatment options
Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes et al.
2003, Helmick et al. 2008) with no approved drug available. Current main
treatments all have
side effects, are limited and associated with 10 year survival of 55% (Badea
et al. 2009, Kowal-
Bielecka et al. 2009, Shah and Wigley 2013), highlighting an urgent need for
new therapies. The
long-term goal of the proposed project is to develop a novel, first-in-class,
efficient and well
tolerable systemic therapy for scleroderma.
Macrophages and scleroderma.
Macrophages are the predominant infiltrating cells in skin lesions of patients
with
scleroderma and are associated with fibrosis (Ishikawa and Ishikawa 1992,
Kraling et al. 1995,
Lech and Anders 2013, Chia and Lu 2015). MCP-1 recruits macrophages to
inflammation sites
and is significantly elevated in patients with systemic sclerosis (S Sc)
(Hasegawa et al. 1999).
Activated macrophages produce VEGF, IL-1-beta, TNFalpha, IL-6, TGF-beta and
PDGF,
which are of crucial importance in the profibrogenic role of fibroblasts in
scleroderma (Bonner et
al. 1991, Clouthier et al. 1997, Yamamoto 2011, Yamamoto and Katayama 2011,
Liu et al. 2013,
Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R) (Kitaba et al.
2012), VEGF (Koca
et al. 2016), TNF-alpha (Koca et al. 2008) and TGF-beta (Varga and Pasche
2009, Varga and
Whitfield 2009) alleviate scleroderma but all may have serious side effects
including fatal
infections and sepsis (Varga 2004). M-CSF plays a role in pulmonary fibrosis
that occurs in 90%
of scleroderma patients (Baran et al. 2007). In rats, elevated MCP-1 and M-CSF
lead to
macrophage recruitment in an injured area and to the lesion formation
(Juniantito et al. 2013).
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Inhibition of TRE11-1 signaling: A new approach to disorders associated with
systemic
inflammation
Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an inflammation
amplifier, plays a role in immune response (Bouchon et al. 2000, Bouchon et
al. 2001, Bleharski
et al. 2003, Colonna et al. 2003, Klesney-Tait et al. 2006, Tessarz et al.
2008) and is upregulated
upon inflammation (Wang et al. 2004, Gonzalez-Roldan et al. 2005, Koussoulas
et al. 2006,
Schenk et al. 2007). TREM-1 mediates release of multiple cytokines including
MCP-1, TNF 0,
IL-1 0, IL-6 and M-CSF (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov
2014, Shen and Sigalov 2015). TREM-1 blockade is a new approach to
inflammatory disorders
(Bouchon et al. 2001, Colonna and Facchetti 2003, Schenk et al. 2007, Gibot et
al. 2008, Ho et
al. 2008, Ford et al. 2009, Gibot et al. 2009, Murakami et al. 2009, Luo et
al. 2010, Pelham et al.
2014, Pelham et al. 2014, Bosco et al. 2016), In mice, TREM-1 blockade
inhibits M-CSF,
TNFalpha, IL-lbeta and IL-6, suppresses tumor growth and ameliorates
autoimmune arthritis
(Sigalov 2014, Shen and Sigalov 2017).
TREM-1 blockade blunts excessive inflammation but in contrast to single
cytokine
blockers, preserves the capacity for microbial control (Weber et al. 2014).
TREM-1 blockade
was suggested as a treatment of neonatal infection (Qian et al. 2014).
Endotoxic and septic mice
lacking DAP12, a signaling adapter of TREM-1, have improved survival (Turnbull
et al. 2005).
Humans lacking DAP12 do not have problems resolving infections (Lanier 2009).
Inhibition of TRE11-1 signaling: A new approach to preventing and treating
scleroderma
TREM-1 is overexpressed in the lungs of mice with BLM-induced pulmonary
fibrosis (Peng et
al. 2016). In experimental autoimmune arthritis, cancer and retinopathy, TREM-
1 blockade
reduces inflammation and inhibits the macrophage infiltration / activation
(Sigalov 2014, Shen
and Sigalov 2017, Shen and Sigalov 2017) (Section 3.3.3.1). In mice with
alcohol-induced liver
disease (ALD), TREM-1 blockade inhibits expression of TREM-1, MCP-1/CCL2, TNF
0, Pro-
Colll 0 and 0 -SMA (Tornai et al. 2019). Collectively, these findings
implicate TREM-1 as a
target for development of new therapy for scleroderma.
The main concepts of the proposed project: Silencing the scleroderma-related
TREM-1-
specific inflammatory response can be superior to anti-single cytokine
strategies in the treatment
of scleroderma in terms of safety and efficacy; Delivery of systemically
administered TREM-1
blockers to macrophages may have several advantages: (a) striking the target
cell population, (b)
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sparing other cells that have no (or marginal) effects on scleroderma, (c)
minimizing off-target
effects, and (d) reducing the therapeutic dose; and Rate and efficiency of
intracellular delivery of
TREM-1 blockers to macrophages may be important to provide a prompt and
effective
therapeutic response during scleroderma progression.
INNOVATION
TREM-1 Blockade
Major challenge. Current approaches (eg Inotrem's LR12) that all attempt to
block TREM-1
binding to its ligand(s) (Fig. 97A) have a risk of failure since exact nature
of TREM-1 ligand(s) is
still uncertain (Tammaro et al. 2017).
SignaBlok's solution. Using our new model of signaling, the Signaling Chain
HOmoOLigomerization (SCHOOL) model (Sigalov 2006, Sigalov 2010), we developed
a first-
in-class ligand-independent TREM-1 inhibitory peptide GF9 (US 8,513,185) that
disrupts
recognition and signaling functions of TREM-1 in the membrane (Fig. 97B)
(Sigalov 2010,
Sigalov 2013, Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017).
As other peptides (Graff et al. 2003, Lien et al. 2003, Gotthardt et al. 2004,
Ladner et al.
2004, Prive et al. 2006, Sato et al. 2006, Antosova et al. 2009, Koskimaki et
al. 2010), GF9 is
advantageous compared to large protein molecules. Mechanistically, GF9 self-
penetrates into the
cell membrane and can reach its site of action from both inside and outside
the cell (Fig. 97B
and Fig. 97C). GF9 is well-tolerated by healthy mice (up to 300 mg/kg; Fig.
98A). GF9 at 25
mg/kg in a free form or at 2.5 mg/kg when formulated into LipoPeptide
Complexes (LPC,
below), reduces tissue TREM-1 and M-CSF upon inflammation (shown on the
example of the
retina of mice with oxygen-induced retinopathy, OIR) (Fig. 98B), and
ameliorates diseases in
mouse models of cancer (Sigalov 2014, Shen and Sigalov 2017), retinopathy
(Rojas et al. 2018),
ALD (Tornai et al. 2019), sepsis and autoimmune arthritis (Sigalov 2014, Shen
and Sigalov
2017).
LPC mimic human High Density Lipoproteins (HDL) and consist of lipids and
peptides
of human apolipoprotein (apo) A-I, the major protein of HDL. In contrast to
native HDL, these
peptides contain naturally occurring modifications that target LPC to
macrophages. SignaBlok's
LPC can deliver GF9 to macrophages in vitro and in vivo (Fig. 99A-C) and
increase its
therapeutic efficacy (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov
2017). NOTE:
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GF9-LPC describes GF9 formulated into either discoidal (GF9-dLPC, short 612:
hrs) or spherical
(GF9-sLPC; long 612: days) LPC.
Epitope-based rational design of long half-life GF9-LPC fast and effective in
delivery of GF9.
GF9-LPC tested to date, all contained a fixed amount of GF9 and an equimolar
mixture
of oxidized (MetS0) 22-mer peptides with sequences from either helix 4 (PE22)
or 6 (PA22) of
human apo A-I. Although these modifications increase macrophage uptake of LPC
in vitro and
in vivo (Sigalov 2014, Sigalov 2014, Shen et al. 2015) (Fig. 99A), the uptake
can be optimized to
make it faster and more efficient. Oxidized PE22 and PA22 contain different
MetS0 epitopes for
binding to Scavenger Receptor (SR) SR-A (Apo A-I peptides contain putative
epitopes for
binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE. PA22: PLGEEM(0)RDRARAHVDALRTHLA)
(Neyen et al. 2009). In addition, PA22 contains an epitope for binding to
macrophage and
hepatocyte SR-BI (Liadaki et al. 2000, Cai et al. 2012). Its exposure affects
binding to SR-BI (de
.. Beer et al. 2001) and can determine the LPC half life.
APPROACH
Overall strategy, methodology, and analyses to be used to accomplish the
specific aims
Towards the overall goal of the proposed Phase I research, we will:
Aim 1. Optimize TREM-1 inhibitory compositions for their functionality in
vitro and
pharmacokinetics in vivo and select the lead.
1) generate and characterize GF9-LPC of different GF9/1pid/PE22/PA22
compositions; 2) use
J774 cells and the relevant antibodies to explore mechanisms of SR-mediated
uptake of GF9-
LPC; 3) use the mechanistic data to optimize GF9/1pid/PE22/PA22 compositions
and generate
.. long half-life GF9-LPC with high GF9 load and high rate and efficiency of
macrophage delivery
of GF9; 4) functionally test the generated GF9-LPC for inhibition of cytokine
release in LPS-
stimulated J774 cells; 5) develop an LC-MS assay for analysis of GF9 in rat
serum;
6) test three most promising formulations in PK studies in Sprague-Dawley (SD)
rats;
7) analyze the data obtained and select the lead GF9-LPC formulation for
further animal testing.
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Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model
of
scleroderma
1) test two doses of the GF9-LPC lead generated in the Aim 1 in preventative
and established
BLM-induced mouse models of scleroderma for its efficacy in preventing and
treating the
disease;
2) perform comprehensive histology / immunohistochemistry studies;
3) analyze serum and tissue GF9 and cytokines (LC-MS; ELISA).
Preliminary data Preliminary data, rationale, methodology, and analyses
to be used to
accomplish the Aim 1. Previously (Sigalov 2014), we showed that oxidation of
PE22 and PA22
results in increased in vitro J774 cell uptake of GF9-LPC (Fig. 99A-C) and
that GF9 (but not a
control peptide) either in a free form (not shown) or formulated into LPC of
discoidal (GF9-
dLPC) or spherical (GF9-sLPC) shape inhibits cytokine release both in vitro
and in vivo and
protects mice from LPS-induced sepsis-related death (ig. 100A-D). GF9-dLPC and
GF9-sLPC
both contained the same amount of GF9 and an equimolar mixture of oxidized
PE22 and PA22.
Rationale
GF9-dLPC and GF9-sLPC both inhibit LPS-stimulated cytokine release in vitro
and in vivo to
about the same degree (Fig. 100A, Fig. 100B) but their protective effect at
the dose of 5 mg/kg in
LPS-induced septic mice differs: GF9-sLPC provide less effective but longer-
lasting protection
as compared with GF9-dLPC (Fig. 100C). Further, despite the same GF9 load and
1:1
PE22:PA22 molar ratio, these GF9-LPC differ in rate and efficiency of the
macrophage uptake in
vitro (Fig. 101) (Sigalov 2014). Stronger protection by GF9-dLPC may result
from higher
efficiency and rate of their uptake (Fig. 101), while longer protection by GF9-
sLPC ¨ from their
longer half-life. Thus, uptake of GF9-LPC may depend on exposure of SR-binding
apo A-I
epitopes (Liu et al. 2002, Horiuchi et al. 2003) (Apo A-I peptides contain
putative epitopes for
binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE.
PA22: PLGEEM(0)RDRARAHVDALRTHLA) that affect both rate and efficiency of the
uptake.
In Phase I Aim 1, we will optimize exposure of SR-A- and SR-BI-binding
epitopes and GF9
content of long half-life GF9-LPC by varying of GF9/lipid/PE22/PA22 ratios to
increase GF9
load and rate and efficiency of its delivery in vivo and thus to provide
prompt, effective and long-
.. term therapeutic response.
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Methodology and analyses
Peptides. GF9 and two oxidized 22-mer peptides PE22 and PA22 will be ordered
from
Bachem, Inc. and characterized as described previously (Sigalov et al. 1998,
Sigalov et al. 2001,
Sigalov et al. 2002, Sigalov 2014, Shen et al. 2015, Shen and Sigalov 2017,
Shen and Sigalov
2017).
Long half-life GF9-LPC (spherical). Previously used non-optimized GF9-LPC be
synthesized
as described (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) and
used as a
reference in all in vitro studies. In some studies, GF9 and/or PE22 will be
Dylight (Dy) 488-
labeled. In some studies, GF9-LPC will be Rhodamine B (Rho B)-labeled.
Optimization. The following parameters will be varied: a) phospholipid chain
length; 2) lipid
composition; 3) lipid/PE22/PA22 composition/ ratio; and 4) GF9 content. The
obtained GF9-
LPC will be purified and their integrity, stability, and GF9 content will be
analyzed as reported
(Sigalov 2014, Shen and Sigalov 2017). As analyzed by Dynamic Light Scattering
(DLS), GF9-
LPC are stable at 4 C for at least, up to 6 months (Fig. 102).
In vitro macrophage uptake assay. GF9-LPC will be characterized by in vitro
macrophage
uptake assay as reported (Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai
et al. 2019). To
explore the mechanisms of GF9-LPC uptake, cells will be incubated with either
anti-SR-BI, anti-
SR-A, or isotype controls for 15 min on ice before adding Rho B-labeled GF9-
LPC with Dy 488-
labeled GF9 and/or PE22. After incubation, cells will be washed, lysed and
fluorescence and
protein concentrations in the lysates will be measured.
In vitro cytokine release. The assay will be performed in LPS-stimulated J774
macrophages
(Fig. 100B) as previously reported (Sigalov 2014).
Confocal analysis. J774A.1 cells will be grown at 37 C in 6 well tissue
culture plates
containing glass coverslips. After reaching target confluency of ¨ 50%, cells
will be incubated
for 6 h at 37 C with Rho B-GF9-LPC. In subsets of experiments, Rho B- GF9-LPC
that contain
Dylight 488-PE22 or Dylight 488-GF9 will be used. TREM-1 staining will be
performed as
described (Shen and Sigalov 2017). The slides will be imaged as reported (Shen
and Sigalov
2017).
Integrity and stability studies. RP-HPLC, SEC, and DLS will be used as
described (Sigalov
2014, Shen and Sigalov 2017, Shen and Sigalov 2017) to study structural
integrity and stability
of GF9-LPC.
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LC-MS for GF9 analysis in animal serum. LC-MS assay for analysis of GF9 in rat
serum in
PK studies in rats will be developed and validated (with ZATA). The assay will
include
ultracentrifugation step followed by LC-MS. The snap-frozen samples of rat
serum will be
ordered from BioreclamationIVT (Westbury, NY) and processed as reported
(Walther et al.
2011, Yanachkov et al. 2011, Yanachkova et al. 2015, Yanachkov et al. 2016).
GF9-LPC will be
added to serum and GF9 will be assayed by LC-MS. The assay will be validated
using the FDA
guidelines (https://www.fda.gov/downloads/drugs/guidances/ucm368107.pdf).
PK studies in SD rats. Animal studies will be provided by WBI. SignaBlok will
perform LC-
MS/histology/IHC. Sex as a biological variable. To exclude differences in PK
in male and
female rats (Shelnutt et al. 1999), we propose to use both sexes. 3 most
promising GF9-LPC
selected based on their TREM-1 inhibitory activity in vitro will be tested in
8 wk-old SD rats
(200-250 g) (3 groups; 3 males+3 females / group, 18 SD rats). Briefly, SD
rats will be IV
administered with 2.5 mg/kg GF9-sLPC. Serum samples will be collected at 8
post-injection
timepoints within 24 hrs, frozen and shipped to SignaBlok for LC-MS analysis
of GF9.
Ultracentrifugation of serum to float lipoproteins and GF9-LPC will be
performed as reported
(Sigalov et al. 1991, Sigalov 1993, Sigalov et al. 1997, Sigalov and Stern
1998, Sigalov and
Stern 2001). Briefly, 50 OL serum, 50 OL saline, 0.5 mM EDTA, and 130 OL KBr
(d = 1.37
g/mL) will be mixed (final d=1.21 g/mL) and centrifuged in a 42.2 TI rotor at
42,000 rpm for 12
h at 10 C. 50 OL will be taken from top, dialyzed for 4 h at 4 C and analyzed
for GF9 by LC-
MS.
Statistical analysis. GraphPad Prism will be used for statistical testing. In
in vitro uptake assay
and cytokine assay data, statistical significances will be determined by two-
tailed Student's t test
as described (Sigalov 2014). Results will be considered significant atp<0.05.
PK data will be
analyzed using PKSolver, a menu-driven add-in Microsoft Excel software (Zhang
et al. 2010).
Outcome measures
Stability of GF9-LPC will be tested by DLS (Fig. 102). GF9 in GF9-LPC will be
analyzed as
reported (Sigalov 2014, Shen and Sigalov 2017) and by LC-MS. In vitro J774
cell uptake will be
measured by Rho B fluorescence in cell lysates (Sigalov 2014). Activity of GF9-
LPC in
reduction of cytokine release by LPS-stimulated cells will be tested as
reported (Sigalov 2014).
In PK studies, half-life, Cmax, Tmax and the area under the AUC will be
analyzed.
Anticipated results and interpretations
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Native dHDL and sHDL have half-lives of 12-20 hrs and 3-5 days, respectively
(Scanu et al.
1962, Furman et al. 1964). We expect that formulation of GF9 into spherical
LPC will extend its
half-life closer to that for sHDL. Based on our preliminary data and (Sigalov
2014), we predict
that: 1) GF9-sLPC of different compositions will have different exposure of SR-
A and SR-BI
epitopes, and 2) use of SR inhibitors will allow to find the preferential
receptor involved in cell
uptake. We predict that PK profiles of GF9-LPC formulations with different
exposure of SR-A
and SR-BI epitopes will differ. Thus, we anticipate to optimize SR-A/SR-BI
epitope exposure
and prepare GF9-LPC with high in vitro efficacy and favorable PK in vivo.
Completion of Aim 1
will show the feasibility of using of epitope-based design to optimize GF9-LPC
for effective and
long-term inhibition of TREM-1 in vitro and in vivo. Milestone 1 includes
selection of the lead
based on its stability, in vitro activity and PK profile.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok, Drs. Tabatadze and
Yanachkov at
ZATA, and the WBI's staff have expertise in all methods (Yanachkov et al.
2011, Sigalov 2014,
Sigalov 2014, Shen et al. 2015, Yanachkova et al. 2015, Shen et al. 2016,
Yanachkov et al. 2016,
Shen and Sigalov 2017, Shen and Sigalov 2017, Yanachkov et al. 2017).
Preliminary data
Previously, using non-optimized GF9-LPC, we demonstrated that:
1) in mice with ALD, systemic 2.5 mg/kg GF9-LPC reduces TREM-1, MCP-1/CCL2,
early
fibrosis markers (alpha-smooth muscle actin [alpha-SMA] and procollagenl-alpha
[Pro-
Colll-alpha]) at the mRNA level (Tornai et al. 2019) Fig. 103A-D);
2) in cancer mice, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-
tolerated (Fig.
Fig. 104A), reduce macrophage infiltration into the tumor (Fig. 103B, Fig.
103C) and inhibit
CSF-1/M-CSF (Fig. 104D) (Shen and Sigalov 2017);
3) in mice with CIA, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-
tolerated
(Fig. 105AA), ameliorate arthritis (Fig. 104B) and inhibit IL-1-beta, IL-6,
TNF-alpha and
CSF-1/M-CSF (Fig. 105AC) (Shen and Sigalov 2017).
Rationale
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TREM-1 blockade by GF9-LPC suppress macrophage infiltration and activation,
reduce
cytokine, CSF-1/ M-CSF and early fibrosis markers and ameliorate disease in
ALD, cancer,
septic and arthritic mice ((Sigalov 2014, Shen and Sigalov 2017, Shen and
Sigalov 2017, Tornai
et al. 2019), Fig. 103A-D-Fig. 105A- C). This suggests that GF9-LPC will be
effective in the
treatment of scleroderma (Ishikawa and Ishikawa 1992, Kraling et al. 1995,
Lech and Anders
2013, Chia and Lu 2015). BLM mouse model is a valuable tool for drug
development for
scleroderma (Yamamoto et al. 1999, Yamamoto et al. 1999, Huber et al. 2007,
Beyer et al. 2010,
Kitaba et al. 2012, Avci et al. 2013, Artlett 2014, Toyama et al. 2016). We
have shown that
chronic subcutaneous (s.c.) injection of BLM in mice results in development of
progressive
multiple organ fibrosis (Bhattacharyya et al. 2018, Bhattacharyya et al.
2018). In Aim 2, we will
use this model to test GF9- LPC ability to prevent and treat organ fibrosis.
Serum and tissue
CCL2, CSF-1/M-CSF, VEGF, TGF 0, TNF 0, IL-6, and IL-10 will be analyzed.
Methodology and analyses
We will design perform and analyze animal studies with Dr. John Varga, M.D.
(Director,
Northwestern Scleroderma, Northwestern University Feinberg School of Medicine
(Chicago,
IL).
Sex (gender) as a biological variable. In a BLM-induced scleroderma mouse
model, while a
more pronounced fibrosis phenotype was reported for male compared with female
mice
(Ruzehaji et al. 2015), other data show no histologic differences between male
and female mice
(Yamamoto et al. 1999, Yamamoto et al. 1999) We suggest to use both sexes of
mice in this
project.
Mouse model of scleroderma. S.c. BLM delivery leads to slowly-progressive
fibrosis in
multiple organs with no mortality, and histological changes in the skin,
muscle and lungs that
resemble those seen in patients with SSc (Bhattacharyya et al. 2018,
Bhattacharyya et al. 2018).
8-12 wk-old C57BL6 mice (288 in total) will be randomized and divided into 3
arms by 12
groups of 8 mice per group (6 male and 6 female groups). In preventative model
(arms 1 and 2),
mice will receive s.c. injections of 10 mg/kg BLM or PBS daily for 10 days (5
days/week), along
with 2.5 or 5 mg/kg GF9-LPC by daily i.p. injections starting concurrently
with BLM, and will
be sacrificed on day 7 (arm 1) or 22 (arm 2). In established model (arm 3),
mice will receive 2.5
or 5 mg/kg GF9-LPC daily starting at day 15, and continue until sacrifice at
day 28. In all arms,
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control groups of mice will receive BLM or PBS alone daily for 10 (7, arm 1)
days or 2.5 or 5
mg/kg GF9-LPC alone daily until sacrifice at days 7, 22 or 28.
Statistical analysis. Statistical significance of differences in parameters of
fibrosis and
inflammation between control and treated mice will be determined by F-test.
Comparison among
three or more groups will be executed with one-way ANOVA followed by a post
hoc Tukey's
test. Based on our previous studies using this model (Bhattacharyya et al.
2018, Bhattacharyya et
al. 2018), a sample size of 8 mice in each group is chosen to give a power of
80% to detect 10%
difference in mean values between experimental and control groups, with a
significance level of
0.05.
Outcome measures
Experiments will test efficacy of treatment given as prevention, as well as
treatment, to
determine if TREM-1 inhibition can promote regression of established skin,
lung and heart
fibrosis and resolution of tissue damage. Clinical observations (daily) and
body weights (weekly)
will be made until termination. DRAIZE scoring will be recorded once weekly
for all groups.
Effect of TREM-1 blockade will be tested on early (day 3-7) inflammatory
changes and
monocyte/macrophage influx in the lungs and skin by IHC; subsequent
development of fibrotic
parenchymal changes (at day 10-20) by histology/IHC, biochemical and
functional assays.
Tissues will be collected, prepared, stained with H&E and Trichrome and
evaluated by board-
certified pathologist. Part of collected tissues will be homogenized and along
with blood and
FFPE tissue samples shipped to SignaBlok for GF9, cytokine and IHC analysis.
Tissue collagen
content will be determined by hydroxyproline assays (Bhattacharyya et al.
2016). Lung fibrosis
will be quantitated in histological lung sections using the modified Ashcroft
score determined
from 5 h.p.f. per mice (Hubner score) (Hubner et al. 2008). Skin hardness will
be measured using
a Vesmeter three times at the injection area. Dermal thickness will be
determined at three
randomly selected sites in each animal. a-SMA-positive cells will be counted.
Macrophage
infiltration will be evaluated by IHC. Serum and tissue CCL2, VEGF, CSF-1, TGF
0, TNF 0, IL-
6 and IL-1 0 will be analyzed by ELISA. Tissue TREM-1 expression will be
tested by Western
Blot.
Anticipated results and interpretations
These studies are expected to demonstrate if TREM-1 blockade using GF9-LPC
can, by attenuating
TLR4 activity in target organs, prevent, slow the progression, and promote the
recovery from, fibrotic
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injury in the skin, lungs, muscle and heart. Further, the results are expected
to indicate whether observed
beneficial effects are primarily due to attenuated early inflammation, reduced
fibrosis due to attenuated
activation of (myo)fibroblasts, or a combination of both of these mechanisms.
Based on our previous data
((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017); Fig. 100A-D,
Fig. 103A-D -Fig. 105A-
C), we predict that treatment with GF9-LPC will be well-tolerated and
associated with reductions in
levels of CCL2, CSF-1, TNF-beta, TGF-alpha, IL-6 and IL-lbeta. We expect that
GF9-LPC will be
effective in a dose-dependent manner and that LPC (no GF9) will be without
effect. Completion of Aim 2
will answer a question about the feasibility of using GF9-LPC as a first-in-
class therapy for scleroderma.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok and the Varga
laboratory's and animal facility'
staff have extensive expertise in all methods (Varga and Whitfield 2009,
Sigalov 2014, Shen et al.
2015, Shen and Sigalov 2016, Shen and Sigalov 2017, Shen and Sigalov 2017,
Bhattacharyya et al.
2018, Bhattacharyya et al. 2018, Yamashita et al. 2018, Lakota et al. 2019,
Tornai et al. 2019).
Final product. SignaBlok's GF9-LPC consist of phospholipids widely used in
pharmacology and
synthetic peptides, all derived from human sequences, suggesting the lack of
potential
immunogenicity. Lipoprotein- and peptide-based drug formulations are currently
on the
market (Chang et al. 2012, Adler-Moore et al. 2016) or in clinical trials
(Tricoci et al.
2015), which makes SignaBlok's efficient and well tolerable systemic therapy
for
scleroderma commercially viable.
Future directions. If successful, Phase I will be followed in Phase II where
to evaluate the
efficacy of TREM-1 blockade for mitigating organ fibrosis, we will use
complementary mouse
models of SSc, including the Tsk1/+ mouse, which (spontaneously) develop skin
fibrosis in the
absence of inflammation. Other administration schedules and regimen will be
tested. The lead
and its manufacturing technology will be further optimized and the more
detailed safety, TOX,
ADME, CMC and other IND-enabling studies will be performed. Upon completion,
an IND will
be filed for subsequent testing in humans.
Anticipated low toxicity of GF9-LPC is supported by the safety of 300 mg/kg
GF9 in healthy
mice (Sigalov 2014) (therapeutic doses are 25 mg/kg for GF9 or 2.5 mg/kg for
GF9-LPC), lack
of body weight changes in mice long-term treated with GF9-LPC (Sigalov 2014,
Shen et al.
2017, Tornai et al. 2019), and by the fact that prototypes of SignaBlok's LPC
were well tolerated
in humans (Newton and Krause 2002, Kingwell and Chapman 2013). TREM-1 blockade
using
inhibitory peptide LR12 which is in development by SignaBlok's top competitor
(Inotrem,
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France) was well tolerated in healthy and septic subjects (Cuvier et al. 2018,
Francois et al.
2018).
The decision to go to Phase II will be made if the significant (more than 50%)
decrease in
fibrosis is shown in treated mice as compared with those shown in control
mice.
Bibliography and References Cited.
1. Helmick CG, Felson DT, Lawrence RC, Gabriel S, Hirsch R, Kwoh CK, et
al. Estimates
of the prevalence of arthritis and other rheumatic conditions in the United
States. Part I. Arthritis
Rheum 2008; 58:15-25. PMID: 18163481.
2. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, et
al.
Estimates of the prevalence of arthritis and selected musculoskeletal
disorders in the United
States. Arthritis Rheum 1998; 41:778-99. PMID: 9588729.
3. Mayes MD, Lacey JV, Jr., Beebe-Dimmer J, Gillespie BW, Cooper B, Laing
TJ, et al.
Prevalence, incidence, survival, and disease characteristics of systemic
sclerosis in a large US
population. Arthritis Rheum 2003; 48:2246-55. PMID: 12905479.
4. Kowal-Bielecka 0, Landewe R, Avouac J, Chwiesko S, Miniati I, Czirjak L,
et al.
EULAR recommendations for the treatment of systemic sclerosis: a report from
the EULAR
Scleroderma Trials and Research group (EUSTAR). Ann Rheum Dis 2009; 68:620-8.
PMID:
19147617.
5. Badea I, Taylor M, Rosenberg A, Foldvari M. Pathogenesis and therapeutic
approaches
for improved topical treatment in localized scleroderma and systemic
sclerosis. Rheumatology
(Oxford) 2009; 48:213-21. PMID: 19022832.
6. Shah AA, Wigley FM. My approach to the treatment of scleroderma.
Mayo Clin Proc
2013; 88:377-93. PMID: 23541012.
7. Ishikawa 0, Ishikawa H. Macrophage infiltration in the skin of patients
with systemic
sclerosis. J Rheumatol 1992; 19:1202-6. PMID: 1404154.
8. Chia JJ, Lu TT. Update on macrophages and innate immunity in
scleroderma. Curr Opin
Rheumatol 2015; 27:530-6. PMID: 26352734.
9. Lech M, Anders HJ. Macrophages and fibrosis: How resident and
infiltrating
mononuclear phagocytes orchestrate all phases of tissue injury and repair.
Biochim Biophys Acta
2013; 1832:989-97. PMID: 23246690.
206
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
10. Kraling BM, Maul GG, Jimenez SA. Mononuclear cellular infiltrates in
clinically
involved skin from patients with systemic sclerosis of recent onset
predominantly consist of
monocytes/macrophages. Pathobiology 1995; 63:48-56. PMID: 7546275.
11. Hasegawa M, Sato S, Takehara K. Augmented production of chemokines
(monocyte
chemotactic protein-1 (MCP-1), macrophage inflammatory protein-lalpha (MIP-
lalpha) and
MIP-lbeta) in patients with systemic sclerosis: MCP-1 and MIP-lalpha may be
involved in the
development of pulmonary fibrosis. Clin Exp Immunol 1999; 117:159-65. PMID:
10403930.
12. Liu J, Copland DA, Hone S, Wu WK, Chen M, Xu Y, et al. Myeloid cells
expressing
VEGF and arginase-1 following uptake of damaged retinal pigment epithelium
suggests potential
mechanism that drives the onset of choroidal angiogenesis in mice. PLoS One
2013; 8:e72935.
PMID: 23977372.
13. Yamamoto T, Katayama I. Vascular changes in bleomycin-induced
scleroderma. Int J
Rheumatol 2011; 2011:270938. PMID: 22028717.
14. Yamamoto T. Autoimmune mechanisms of scleroderma and a role of
oxidative stress.
Self Nonself 2011; 2:4-10. PMID: 21776329.
15. Clouthier DE, Comerford SA, Hammer RE. Hepatic fibrosis,
glomerulosclerosis, and a
lipodystrophy-like syndrome in PEPCK-TGF-betal transgenic mice. J Clin Invest
1997;
100:2697-713. PMID: 9389733.
16. Bonner JC, Osornio-Vargas AR, Badgett A, Brody AR. Differential
proliferation of rat
lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -
BB isoforms
secreted by rat alveolar macrophages. Am J Respir Cell Mol Biol 1991; 5:539-
47. PMID:
1958381.
17. Manetti M. Deciphering the alternatively activated (M2) phenotype of
macrophages in
scleroderma. Exp Dermatol 2015; 24:576-8. PMID: 25869115.
18. Kitaba S, Murota H, Terao M, Azukizawa H, Terabe F, Shima Y, et al.
Blockade of
interleukin-6 receptor alleviates disease in mouse model of scleroderma. Am J
Pathol 2012;
180:165-76. PMID: 22062222.
19. Koca SS, Ozgen M, Dagli AF, Gozel N, Ozercan IH, Isik A. The
Protective Effects of
Bevacizumab in Bleomycin-Induced Experimental Scleroderma. Adv Clin Exp Med
2016;
25:249-53. PMID: 27627557.
207
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
20. Koca SS, Isik A, Ozercan IH, Ustundag B, Evren B, Metin K.
Effectiveness of etanercept
in bleomycin-induced experimental scleroderma. Rheumatology (Oxford) 2008;
47:172-5.
PMID: 18174229.
21. Varga J, Whitfield ML. Transforming growth factor-beta in systemic
sclerosis
.. (scleroderma). Front Biosci (Schol Ed) 2009; 1:226-35. PMID: 19482698.
22. Varga J, Pasche B. Transforming growth factor beta as a therapeutic
target in systemic
sclerosis. Nat Rev Rheumatol 2009; 5:200-6. PMID: 19337284.
23. Varga J. Antifibrotic therapy in scleroderma: extracellular or
intracellular targeting of
activated fibroblasts? Curr Rheumatol Rep 2004; 6:164-70. PMID: 15016348.
24. Baran CP, Opalek JM, McMaken S, Newland CA, O'Brien JIM, Jr., Hunter
MG, et al.
Important roles for macrophage colony-stimulating factor, CC chemokine ligand
2, and
mononuclear phagocytes in the pathogenesis of pulmonary fibrosis. Am J Respir
Crit Care Med
2007; 176:78-89. PMID: 17431224.
25. Lagler H, Sharif 0, Haslinger I, Matt U, Stich K, Furtner T, et al.
TREM-1 activation
.. alters the dynamics of pulmonary IRAK-M expression in vivo and improves
host defense during
pneumococcal pneumonia. J Immunol 2009; 183:2027-36. PMID: 19596984.
26. Dower K, Ellis DK, Saraf K, Jelinsky SA, Lin LL. Innate immune
responses to TREM-1
activation: overlap, divergence, and positive and negative cross-talk with
bacterial
lipopolysaccharide. J Immunol 2008; 180:3520-34. PMID: 18292579.
27. Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1--expressing
intestinal
macrophages crucially amplify chronic inflammation in experimental colitis and
inflammatory
bowel diseases. J Clin Invest 2007; 117:3097-106. PMID: 17853946.
28. Shen ZT, Sigalov AB. Novel Ligand-Independent Peptide Inhibitors of
Triggering
Receptor Expressed on Myeloid Cells 1 (TREM-1) and T Cell Receptor (TCR):
Efficacy in a
Collagen-Induced Arthritis Model Suggests New Targeted Treatment for
Rheumatoid Arthritis
[abstract]. Arthritis Rheumatol 2015; 67:1347-8. PMID.
29. Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1
suppresses tumor
growth in human lung cancer xenografts and prolongs survival of mice with
lipopolysaccharide-
induced septic shock. Int Immunopharmacol 2014; 21:208-19. PMID: 24836682.
208
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
30. Peng L, Zhou Y, Dong L, Chen RQ, Sun GY, Liu T, et al. TGF-betal
Upregulates the
Expression of Triggering Receptor Expressed on Myeloid Cells 1 in Murine
Lungs. Sci Rep
2016; 6:18946. PMID: 26738569.
31. Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide
inhibitors of
TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017; 21:2524-34.
PMID:
28382703.
32. Shen ZT, Sigalov AB. Novel TREM-1 Inhibitors Attenuate Tumor Growth and
Prolong
Survival in Experimental Pancreatic Cancer. Mol Pharm 2017; 14:4572-82. PMID:
29095622.
33. Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1
suppresses tumor
growth in human lung cancer xenografts and prolongs survival of mice with
lipopolysaccharide-
induced septic shock. Int Immunopharmacol 2014; 21:208-19. PMID: 24836682.
34. Cuvier V, Lorch U, Witte S, Olivier A, Gibot S, Delor I, et al. A first-
in-man safety and
pharmacokinetics study of nangibotide, a new modulator of innate immune
response through
TREM-1 receptor inhibition. Br J Clin Pharmacol 2018; 84:2270-9. PMID:
29885068.
35. Tornai D, Fun i I, Shen ZT, Sigalov AB, Coban S, Szabo G. Inhibition of
Triggering
Receptor Expressed on Myeloid Cells 1 Ameliorates Inflammation and Macrophage
and
Neutrophil Activation in Alcoholic Liver Disease in Mice. Hepatol Commun 2019;
3:99-115.
PMID: 30619998.
36. Bhattacharyya S, Wang W, Tamaki Z, Shi B, Yeldandi A, Tsukimi Y, et al.
Pharmacological Inhibition of Toll-Like Receptor-4 Signaling by TAK242
Prevents and Induces
Regression of Experimental Organ Fibrosis. Front Immunol 2018; 9:2434. PMID:
30405628.
37. Bhattacharyya S, Wang W, Qin W, Cheng K, Coulup S, Chavez S, et al.
TLR4-
dependent fibroblast activation drives persistent organ fibrosis in skin and
lung. JCI Insight
2018; 3. PMID: 29997297.
38. Rojas MA, Caldwell RB, Shen Z, Sigalov A. TREM-1 blockade prevents
vitreoretinal
neovascularization in a mouse model of retinopathy of prematurity [abstract].
Invest Ophthalmol
Vis Sci 2017; 58:3452. PMID.
39. Kingwell BA, Chapman MJ. Future of high-density lipoprotein infusion
therapies:
potential for clinical management of vascular disease. Circulation 2013;
128:1112-21. PMID:
24002713.
209
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
40. Newton RS, Krause BR. HDL therapy for the acute treatment of
atherosclerosis.
Atheroscler Suppl 2002; 3:31-8. PMID: 12573361.
41. Francois B, Wittebole X, Mira JP, Dugernier T, Gibot S, Derive M, et
al. P1 Safety and
pharmacodynamic activity of a novel TREM-1 pathway inhibitory peptide in
septic shock
patients: phase Ha clinical trial results. Intensive Care Med Exp 2018;
6(Suppl 1). PMID.
42. Juniantito V, Izawa T, Yuasa T, Ichikawa C, Yano R, Kuwamura M, et al.
Immunophenotypical characterization of macrophages in rat bleomycin-induced
scleroderma.
Vet Pathol 2013; 50:76-85. PMID: 22700848.
43. Bouchon A, Dietrich J, Colonna M. Cutting edge: inflammatory responses
can be
triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.
J Immunol
2000; 164:4991-5. PMID: 10799849.
44. Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies
inflammation and
is a crucial mediator of septic shock. Nature 2001; 410:1103-7. PMID:
11323674.
45. Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, Colonna
M, et al. A role
for triggering receptor expressed on myeloid cells-1 in host defense during
the early-induced and
adaptive phases of the immune response. J Immunol 2003; 170:3812-8. PMID:
12646648.
46. Tessarz AS, Cerwenka A. The TREM-1/DAP12 pathway. Immunol Lett 2008;
116:111-
6. PMID: 18192027.
47. Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and
signal
integration. Nat Immunol 2006; 7:1266-73. PMID: 17110943.
48. Colonna M, Facchetti F. TREM-1 (triggering receptor expressed on
myeloid cells): a new
player in acute inflammatory responses. J Infect Dis 2003; 187 Suppl 2:S397-
401. PMID:
12792857.
49. Gonzalez-Roldan N, Ferat-Osorio E, Aduna-Vicente R, Wong-Baeza I,
Esquivel-Callejas
N, Astudillo-de la Vega H, et al. Expression of triggering receptor on myeloid
cell 1 and
histocompatibility complex molecules in sepsis and major abdominal surgery.
World J
Gastroenterol 2005; 11:7473-9. PMID: 16437719.
50. Koussoulas V, Vassiliou S, Demonakou M, Tassias G, Giamarellos-
Bourboulis EJ,
Mouktaroudi M, et al. Soluble triggering receptor expressed on myeloid cells
(5TREM-1): a new
mediator involved in the pathogenesis of peptic ulcer disease. Eur J
Gastroenterol Hepatol 2006;
18:375-9. PMID: 16538108.
210
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
51. Wang DY, Qin RY, Liu ZR, Gupta MK, Chang Q. Expression of TREM-1 mRNA
in
acute pancreatitis. World J Gastroenterol 2004; 10:2744-6. PMID: 15309732.
52. Gibot S, Massin F, Alauzet C, Montemont C, Lozniewski A, Bollaert PE,
et al. Effects of
the TREM-1 pathway modulation during mesenteric ischemia-reperfusion in rats.
Crit Care Med
2008; 36:504-10. PMID: 18091551.
53. Luo L, Zhou Q, Chen XJ, Qin SM, Ma WL, Shi HZ. Effects of the TREM-1
pathway
modulation during empyema in rats. Chin Med J (Engl) 2010; 123:1561-5. PMID:
20819512.
54. Murakami Y, Akahoshi T, Aoki N, Toyomoto M, Miyasaka N, Kohsaka H.
Intervention
of an inflammation amplifier, triggering receptor expressed on myeloid cells
1, for treatment of
autoimmune arthritis. Arthritis Rheum 2009; 60:1615-23. PMID: 19479878.
55. Gibot S, Massin F, Alauzet C, Derive M, Montemont C, Collin S, et al.
Effects of the
TREM 1 pathway modulation during hemorrhagic shock in rats. Shock 2009; 32:633-
7. PMID:
19333144.
56. Ho CC, Liao WY, Wang CY, Lu YH, Huang HY, Chen HY, et al. TREM-1
expression in
tumor-associated macrophages and clinical outcome in lung cancer. Am J Respir
Crit Care Med
2008; 177:763-70. PMID: 18096709.
57. Bosco MC, Raggi F, Varesio L. Therapeutic Potential of Targeting TREM-1
in
Inflammatory Diseases and Cancer. Curr Pharm Des 2016; 22:6209-33. PMID:
27568730.
58. Ford JW, McVicar DW. TREM and TREM-like receptors in inflammation and
disease.
Curr Opin Immunol 2009; 21:38-46. PMID: 19230638.
59. Pelham CJ, Agrawal DK. Emerging roles for triggering receptor expressed
on myeloid
cells receptor family signaling in inflammatory diseases. Expert Rev Clin
Immunol 2014;
10:243-56. PMID: 24325404.
60. Pelham CJ, Pandya AN, Agrawal DK. Triggering receptor expressed on
myeloid cells
receptor family modulators: a patent review. Expert Opin Ther Pat 2014;
24:1383-95. PMID:
25363248.
61. Weber B, Schuster S, Zysset D, Rihs S, Dickgreber N, Schurch C, et al.
TREM-1
Deficiency Can Attenuate Disease Severity without Affecting Pathogen
Clearance. PLoS Pathog
2014; 10:e1003900. PMID: 24453980.
62. Qian L, Weng W, Chen W, Sun CH, Wu J. TREM-1 as a potential therapeutic
target in
neonatal sepsis. Int J Clin Exp Med 2014; 7:1650-8. PMID: 25126161.
211
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
63. Turnbull IR, McDunn JE, Takai T, Townsend RR, Cobb JP, Colonna M. DAP12
(KARAP) amplifies inflammation and increases mortality from endotoxemia and
septic
peritonitis. J Exp Med 2005; 202:363-9. PMID: 16061725.
64. Lanier LL. DAP10- and DAP12-associated receptors in innate immunity.
Immunol Rev
.. 2009; 227:150-60. PMID: 19120482.
65. Derive M, Boufenzer A, Bouazza Y, Groubatch F, Alauzet C, Barraud D, et
al. Effects of
a TREM-like transcript 1-derived peptide during hypodynamic septic shock in
pigs. Shock 2013;
39:176-82. PMID: 23324887.
66. Derive M, Boufenzer A, Gibot S. Attenuation of responses to endotoxin
by the triggering
receptor expressed on myeloid cells-1 inhibitor LR12 in nonhuman primate.
Anesthesiology
2014; 120:935-42. PMID: 24270127.
67. Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC. TREM-
1 and its
potential ligands in non-infectious diseases: from biology to clinical
perspectives. Pharmacol
Ther 2017; 177:81-95. PMID: 28245991.
68. Sigalov AB. Immune cell signaling: a novel mechanistic model reveals
new therapeutic
targets. Trends Pharmacol Sci 2006; 27:518-24. PMID: 16908074.
69. Sigalov AB. The SCHOOL of nature. III. From mechanistic understanding
to novel
therapies. Self/Nonself - Immune Recognition and Signaling 2010; 1:192-224.
PMID.
70. Sigalov AB. New therapeutic strategies targeting transmembrane signal
transduction in
the immune system. Cell Adh Migr 2010; 4:255-67. PMID: 20519929.
71. Sigalov AB. Inhibition of TREM receptor signaling with peptide
variants. Pat. US
8,513,185. In: USPTO, ed. USA: SignaBlok, Inc., Shrewsbury, MA (US), 2013.
72. Ladner RC, Sato AK, Gorzelany J, de Souza M. Phage display-derived
peptides as
therapeutic alternatives to antibodies. Drug Discov Today 2004; 9:525-9. PMID:
15183160.
73. Antosova Z, Mackova M, Kral V, Macek T. Therapeutic application of
peptides and
proteins: parenteral forever? Trends Biotechnol 2009; 27:628-35. PMID:
19766335.
74. Gotthardt M, Boermann OC, Behr TM, Behe MP, Oyen WJ. Development and
clinical
application of peptide-based radiopharmaceuticals. Curr Pharm Des 2004;
10:2951-63. PMID:
15379661.
212
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
75. Koskimaki JE, Karagiannis ED, Tang BC, Hammers H, Watkins DN, Pili R,
et al.
Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth
in a small cell
lung cancer xenograft model. BMC Cancer 2010; 10:29. PMID: 20122172.
76. Lien S, Lowman HB. Therapeutic peptides. Trends Biotechnol 2003; 21:556-
62. PMID:
14624865.
77. Prive GG, Melnick A. Specific peptides for the therapeutic targeting of
oncogenes. Curr
Opin Genet Dev 2006; 16:71-7. PMID: 16377176.
78. Sato AK, Viswanathan M, Kent RB, Wood CR. Therapeutic peptides:
technological
advances driving peptides into development. Curr Opin Biotechnol 2006; 17:638-
42. PMID:
17049837.
79. Graff CP, Wittrup KD. Theoretical analysis of antibody targeting of
tumor spheroids:
importance of dosage for penetration, and affinity for retention. Cancer Res
2003; 63:1288-96.
PMID: 12649189.
80. Rojas MA, Shen ZT, Caldwell RB, Sigalov AB. Blockade of TREM-1 prevents
vitreoretinal neovascularization in mice with oxygen-induced retinopathy.
Biochim Biophys
Acta 2018; 1864:2761-8. PMID: 29730341.
81. Sigalov AB. Nature-inspired nanoformulations for contrast-enhanced in
vivo MR
imaging of macrophages. Contrast Media Mol Imaging 2014; 9:372-82. PMID:
24729189.
82. Sigalov AB. Methods and Compositions for Targeted Imaging. US
20130045161.
Publication Date: Feb 21, 2013.
83. Sigalov AB. Methods and Compositions for Targeted Delivery. US
20110256224.
Publication Date: Oct. 20, 2011. PMID.
84. Shen ZT, Zheng S, Gounis MJ, Sigalov AB. Diagnostic Magnetic Resonance
Imaging of
Atherosclerosis in Apolipoprotein E Knockout Mouse Model Using Macrophage-
Targeted
Gadolinium-Containing Synthetic Lipopeptide Nanoparticles. PLoS One 2015;
10:e0143453.
PMID: 26569115.
85. Neyen C, Pluddemann A, Roversi P, Thomas B, Cai L, van der Westhuyzen
DR, et al.
Macrophage scavenger receptor A mediates adhesion to apolipoproteins A-I and
E. Biochemistry
2009; 48:11858-71. PMID: 19911804.
86. Liadaki KN, Liu T, Xu S, Ishida BY, Duchateaux PN, Krieger JP, et al.
Binding of high
density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor
scavenger
213
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
receptor class B type I. Effect of lipid association and APOA-I mutations on
receptor binding. J
Biol Chem 2000; 275:21262-71. PMID: 10801839.
87. Cai L, Wang Z, Meyer JM, Ji A, van der Westhuyzen DR. Macrophage SR-BI
regulates
LPS-induced pro-inflammatory signaling in mice and isolated macrophages. J
Lipid Res 2012;
53:1472-81. PMID: 22589557.
88. de Beer MC, Durbin DM, Cai L, Jonas A, de Beer FC, van der Westhuyzen
DR.
Apolipoprotein A-I conformation markedly influences HDL interaction with
scavenger receptor
BI. J Lipid Res 2001; 42:309-13. PMID: 11181762.
89. Horiuchi S, Sakamoto Y, Sakai M. Scavenger receptors for oxidized and
glycated
proteins. Amino Acids 2003; 25:283-92. PMID: 14661091.
90. Liu T, Krieger M, Kan HY, Zannis VI. The effects of mutations in
helices 4 and 6 of
ApoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol
efflux suggest that
formation of a productive complex between reconstituted high density
lipoprotein and SR-BI is
required for efficient lipid transport. J Biol Chem 2002; 277:21576-84. PMID:
11882653.
91. Sigalov AB, Stern U. Enzymatic repair of oxidative damage to human
apolipoprotein A-
I. FEBS Lett 1998; 433:196-200. PMID: 9744793.
92. Sigalov AB, Stern U. Oxidation of methionine residues affects the
structure and stability
of apolipoprotein A-I in reconstituted high density lipoprotein particles.
Chem Phys Lipids 2001;
113:133-46. PMID: 11687233.
93. Sigalov AB, Stern U. Dihydrolipoic acid as an effective cofactor for
peptide methionine
sulfoxide reductase in enzymatic repair of oxidative damage to both lipid-free
and lipid-bound
apolipoprotein a-I. Antioxid Redox Signal 2002; 4:553-7. PMID: 12215223.
94. Yanachkov IB, Chang H, Yanachkova MI, Dix EJ, Berny-Lang MA, Gremmel T,
et al.
New highly active antiplatelet agents with dual specificity for platelet P2Y1
and P2Y12
adenosine diphosphate receptors. Eur J Med Chem 2016; 107:204-18. PMID:
26588064.
95. Yanachkov IB, Dix EJ, Yanachkova MI, Wright GE. P1,P2-diimidazoly1
derivatives of
pyrophosphate and bis-phosphonates--synthesis, properties, and use in
preparation of
dinucleoside tetraphosphates and analogs. Org Biomol Chem 2011; 9:730-8. PMID:
21082127.
96. Yanachkova M, Xu WC, Dvoskin S, Dix EJ, Yanachkov IB, Focher F, et al.
Prodrugs of
herpes simplex thymidine kinase inhibitors. Antivir Chem Chemother 2015; 24:47-
55. PMID:
26463822.
214
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
97. Walther DM, Mann M. Accurate quantification of more than 4000 mouse
tissue proteins
reveals minimal proteome changes during aging. Mol Cell Proteomics 2011;
10:M110 004523.
PMID: 21048193.
98. Shelnutt SR, Gunnell M, Owens SM. Sexual dimorphism in phencyclidine in
vitro
metabolism and pharmacokinetics in rats. J Pharmacol Exp Ther 1999; 290:1292-
8. PMID:
10454506.
99. Sigalov A, Alexandrovich 0, Strizevskaya E. Large-scale isolation and
purification of
human apolipoproteins A-I and A-II. J Chromatogr 1991; 537:464-8. PMID:
1904881.
100. Sigalov AB. Comparison of apolipoprotein A-I values assayed in
lyophilized and frozen
pooled human sera by a non-immunochemical electrophoretic method and by
immunoassay. Eur
J Clin Chem Clin Biochem 1993; 31:579-83. PMID: 8260529.
101. Sigalov AB, Petrichenko IE, Kolpakova GV. The ratio of non-
oxidized/oxidized forms of
apolipoprotein A-I can affect cholesterol efflux from human skin fibroblasts
mediated by high
density lipoprotein. Eur J Clin Chem Clin Biochem 1997; 35:395-6. PMID:
9189746.
.. 102. Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program for
pharmacokinetic and
pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs
Biomed 2010;
99:306-14. PMID: 20176408.
103. Scanu A, Hughes WL. Further characterization of the human serum D 1.063-
1.21, alpha-
lipoprotein. J Clin Invest 1962; 41:1681-9. PMID: 14497795.
104. Furman RH, Sanbar SS, Alaupovic P, Bradford RH, Howard RP. Studies of the
Metabolism of Radioiodinated Human Serum Alpha Lipoprotein in Normal and
Hyperlipidemic
Subjects. J Lab Clin Med 1964; 63:193-204. PMID: 14125106.
105. Shen ZT, Sigalov AB. SARS Coronavirus Fusion Peptide-Derived Sequence
Suppresses
Collagen-Induced Arthritis in DBA/1J Mice. Sci Rep 2016; 6:28672. PMID:
27349522.
.. 106. Yanachkov I, Zavizion B, Metelev V, Stevens LJ, Tabatadze Y,
Yanachkova M, et al.
Self-neutralizing oligonucleotides with enhanced cellular uptake. Org Biomol
Chem 2017;
15:1363-80. PMID: 28074950.
107. Huber LC, Distler JH, Moritz F, Hemmatazad H, Hauser T, Michel BA, et al.
Trichostatin A prevents the accumulation of extracellular matrix in a mouse
model of bleomycin-
induced skin fibrosis. Arthritis Rheum 2007; 56:2755-64. PMID: 17665426.
215
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
108. Toyama T, Asano Y, Akamata K, Noda S, Taniguchi T, Takahashi T, et al.
Tamibarotene
Ameliorates Bleomycin-Induced Dermal Fibrosis by Modulating Phenotypes of
Fibroblasts,
Endothelial Cells, and Immune Cells. J Invest Dermatol 2016; 136:387-98. PMID:
26967475.
109. Yamamoto T, Takagawa S, Katayama I, Yamazaki K, Hamazaki Y, Shinkai H, et
al.
Animal model of sclerotic skin. I: Local injections of bleomycin induce
sclerotic skin mimicking
scleroderma. J Invest Dermatol 1999; 112:456-62. PMID: 10201529.
110. Yamamoto T, Takahashi Y, Takagawa S, Katayama I, Nishioka K. Animal model
of
sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell
deficient WBB6F1-
W/W(V) mice. J Rheumatol 1999; 26:2628-34. PMID: 10606374.
111. Avci P, Sadasivam M, Gupta A, De Melo WC, Huang YY, Yin R, et al. Animal
models
of skin disease for drug discovery. Expert Opin Drug Discov 2013; 8:331-55.
PMID: 23293893.
112. Artlett CM. Animal models of systemic sclerosis: their utility and
limitations. Open
Access Rheumatol 2014; 6:65-81. PMID: 27790036.
113. Beyer C, Schett G, Distler 0, Distler JH. Animal models of systemic
sclerosis: prospects
and limitations. Arthritis Rheum 2010; 62:2831-44. PMID: 20617524.
114. Ruzehaji N, Avouac J, Elhai M, Frechet M, Frantz C, Ruiz B, et al.
Combined effect of
genetic background and gender in a mouse model of bleomycin-induced skin
fibrosis. Arthritis
Res Ther 2015; 17:145. PMID: 26025306.
115. Bhattacharyya S, Wang W, Morales-Nebreda L, Feng G, Wu M, Zhou X, et al.
Tenascin-
C drives persistence of organ fibrosis. Nat Commun 2016; 7:11703. PMID:
27256716.
116. Hubner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al.
Standardized
quantification of pulmonary fibrosis in histological samples. Biotechniques
2008; 44:507-11, 14-
7. PMID: 18476815.
117. Lakota K, Hanumanthu VS, Agrawal R, Cams M, Armanios M, Varga J. Short
lymphocyte, but not granulocyte, telomere length in a subset of patients with
systemic sclerosis.
Ann Rheum Dis 2019. PMID: 30679155.
118. Yamashita T, Lakota K, Taniguchi T, Yoshizaki A, Sato S, Hong W, et al.
An orally-
active adiponectin receptor agonist mitigates cutaneous fibrosis, inflammation
and microvascular
pathology in a murine model of systemic sclerosis. Sci Rep 2018; 8:11843.
PMID: 30087356.
.. 119. Chang HI, Yeh MK. Clinical development of liposome-based drugs:
formulation,
characterization, and therapeutic efficacy. Int J Nanomedicine 2012; 7:49-60.
PMID: 22275822.
216
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
120. Adler-Moore JP, Gangneux JP, Pappas PG. Comparison between liposomal
formulations
of amphotericin B. Med Mycol 2016; 54:223-31. PMID: 26768369.
121. Tricoci P, D'Andrea DM, Gurbel PA, Yao Z, Cuchel M, Winston B, et al.
Infusion of
Reconstituted High-Density Lipoprotein, CSL112, in Patients With
Atherosclerosis: Safety and
Pharmacokinetic Results From a Phase 2a Randomized Clinical Trial. J Am Heart
Assoc 2015;
4:e002171. PMID: 26307570.
122. Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide
inhibitors of
TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017. PMID:
28382703.
123. Deshmukh SV, Durston J, Shomer NH. Validation of the use of nonnaive
surgically
catheterized rats for pharmacokinetics studies. J Am Assoc Lab Anim Sci 2008;
47:41-5. PMID:
19049252.
124. Wang X, Song L, Li N, Qiu Z, Zhou S, Li C, et al. Pharmacokinetics and
biodistribution
study of paclitaxel liposome in Sprague-Dawley rats and Beagle dogs by liquid
chromatography-
tandem mass spectrometry. Drug Res (Stuttg) 2013; 63:603-6. PMID: 23842945.
125. Kuwahara Y, Shima Y, Shirayama D, Kawai M, Hagihara K, Hirano T, et al.
Quantification of hardness, elasticity and viscosity of the skin of patients
with systemic sclerosis
using a novel sensing device (Vesmeter): a proposal for a new outcome
measurement procedure.
Rheumatology (Oxford) 2008; 47:1018-24. PMID: 18440998.
Additional Advantages of Using Peptides and Compositions as described herein.
As well-known in the art and described in Irby, et al. Mol Pharm 2017, 14:1325-
1338,
most anticancer chemotherapeutic agents as well as many other therapeutic
agents (TA) are toxic
and hydrophobic and cannot be administered by themselves as pure chemicals but
have to be
included in biocompatible formulations to enhance solubility, increase
circulatory residence time
of the therapeutic agents, minimize the undesirable side effects and alleviate
drug resistance.
Numerous formulation approaches have been developed, including solid lipid
particles,
emulsions, liposomes, etc., however, the delivery of the poorly water soluble
(hydrophobic, or
lipophilic) pharmaceuticals remains especially problematic as most of the body
compartments,
including the blood circulation and intracellular fluids, represent an aqueous
environment. As a
result, the direct injection of hydrophobic TAs often results in harmful side
effects due to
hypersensitivity, hemolysis, cardiac and neurological symptoms.
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As described in Vlieghe, et al. Drug Discov Today 2010, 15:40-56, the main
limitations
generally attributed to therapeutic peptides are: a short half-life because of
their rapid
degradation by proteolytic enzymes of the digestive system and blood plasma;
rapid removal
from the circulation by the liver (hepatic clearance) and kidneys (renal
clearance); poor ability to
cross physiological barriers because of their general hydrophilicity; high
conformational
flexibility, resulting sometimes in a lack of selectivity involving
interactions with different
receptors/targets (poor specific biodistribution), causing activation of
several targets and leading
to side effects; eventual risk of immunogenic effects; and high synthetic and
production costs
(the production cost of a 5000 Da molecular mass peptide exceeds the
production cost of a 500
Da molecular mass small molecule by more than 10-fold but clearly not 100-
fold).
Consequently, there is need for more effective and low toxic therapies for PC
and other types of
cancer as well as new formulations of hydrophobic drugs and therapeutic
peptides to improve
their targeted delivery, prolonged half-life, biocompatibility and therapeutic
efficiency.
As described herein, it is surprisingly found that the peptides and
compositions of the
present invention capable of modulating the TREM-1 signaling pathway can be
synthesized and
used for targeted treatment of cancer and imaging. The advantageous
trifunctional peptides and
compositions are demonstrated by the present invention to solve numerous
problems which
otherwise are associated with high dosages of TAs and imaging probes required
and the lack of
control and reproducibility of formulations, especially in large-scale
production.
As many other solid tumors, PC is characterized by a marked infiltration of
macrophages
into the stromal compartment (Shih 2006, Solinas 2009), a process, which is
mediated by cancer-
associated fibroblasts (CAFs) (FIG. 49) and plays a role in disease
progression and its response
to therapy. These tumor-associated macrophages (TAMs) secrete a variety of
growth factors,
cytokines, chemokines, and enzymes that regulate tumor growth, angiogenesis,
invasion, and
metastasis (Feurino 2006, Lewis and Pollard 2006, Shih 2006). High macrophage
infiltration
correlates with the promotion of tumor growth and metastasis development (Lin
2006, Lin 2001,
Solinas 2009). In patients with PC, macrophage infiltration begins during the
pre-invasive stage
of the disease and increases progressively (Clark 2007). The number of TAMs is
significantly
higher in patients with metastases (Gardian 2012). Presence of TAMs in the PC
stroma correlates
with increased angiogenesis (Esposito 2004), a known predictor of poor
prognosis (Kuwahara
2003). TAM recruitment, activation, growth and differentiation are regulated
by macrophage
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colony-stimulating factor (M-CSF, also known as colony-stimulating factor 1,
CSF-1) (Elgert
1998, Varney 2005). High pretreatment serum M-CSF is a strong independent
predictor of poor
survival in PC patients (Groblewska 2007). In PC mouse models, blockade of M-
CSF or its
receptor not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota
2009) but also
improves response to T-cell checkpoint immunotherapies that target programmed
cell death
protein 1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu 2014).
Importantly,
continuous M-CSF inhibition affects pathological angiogenesis but not healthy
vascular and
lymphatic systems outside tumors (Kubota 2009). In contrast to blockade of
vascular endothelial
growth factor (VEGF), interruption of M-CSF inhibition does not promote rapid
vascular
regrowth (Kubota 2009). Collectively, these findings further suggest that
targeting TAMs is a
promising strategy for treating cancer (Bowman and Joyce 2014, Jinushi and
Komohara 2015,
Komohara 2016).
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the
inflammatory
response (Colonna and Facchetti 2003) and is upregulated under inflammatory
conditions
including including cancer (Ho et al. 2008, Yuan et al. 2014, Nguyen et al.
2015), brain and
spinal cord injuries (Li et al 2019) and acute pancreatitis (D. Y. Wang 2004).
For downstream
signal transduction, TREM-1 is coupled to the immunoreceptor tyrosine-based
activation motif
(ITAM)-containing adaptor, DNAX activation protein of 12 kDa (DAP-12).
Activation of
TREM-1/DAP-12 receptor complex enhances release of multiple cytokines
including monocyte
chemoattractant protein-1 (MCP-1; also referred to in the art as CCL2), tumor
necrosis factor-a
(TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and macrophage colony-stimulating
factor 1 (CSF-
1; also referred to in the art as M-CSF) (Schenk et al. 2007, Dower et al.
2008, Sigalov 2014,
Shen et al. 2017, Shen et al. 2017, Rojas et al. 2018, Tornai et al. 2019).
Most of these cytokines
are increased in cancer patients (Tjomsland et al. 2011, Rossi et al. 2015,
Yako et al. 2016,
Tsukamoto et al. 2018, Yoshimura 2018)and play a vital role in creating and
sustaining
inflammation in the tumor favorable microenvironment, thus affecting patient
survival.
TREM-1 activation enhances release of multiple cytokines including monocyte
chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interleukin-
la (IL-1a), IL-
113, IL-6 and M-CSF (Lagler 2009, Schenk 2007, Sigalov 2014). Most of these
cytokines are
increased in patients with PC (Tjomsland 2011, Yako 2016) and play a vital
role in creating and
sustaining inflammation in the tumor favorable microenvironment, thus
affecting patient
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survival. Inhibition of TREM-1 lowers levels of proinflammatory cytokines and
is a promising
approach in a variety of inflammation-associated disorders (Colonna and
Facchetti 2003, Pelham
and Agrawal 2014, Schenk 2007, Shen and Sigalov 2017, Sigalov 2014).
Importantly, in contrast
to cytokine blockers, blockade of TREM-1 can blunt excessive inflammation
while preserving
the capacity for microbial control (Weber 2014). In vitro silencing of TREM-1
suppresses cancer
cell invasion (Ho 2008). In patients with non-small cell lung cancer (NSCLC),
TREM-1
expression on TAMs is associated with cancer recurrence and poor survival:
patients with low
TREM-1 expression have a 4-year survival rate of over 60%, compared with less
than 20% in
patients with high TREM-1 expression (Ho 2008).
Inhibition of TREM-1 lowers levels of proinflammatory cytokines and chemokines
including CSF-1 (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017,
Rojas et al.
2018, Tornai et al. 2019) and as recently demonstrated in experimental cancer
including NSCLC,
pancreatic cancer and liver cancer, TREM-1 blockade inhibits tumor growth and
improves
survival (Wu et al. 2012, Sigalov 2014, Shen and Sigalov 2017, Wu et al.
2019). In vitro
silencing of TREM-1 suppresses cancer cell invasion (Ho et al. 2008). In
patients with NSCLC,
TREM-1 expression on TAMs is associated with cancer recurrence and poor
survival: patients
with low TREM-1 expression have a 4-year survival rate of over 60%, compared
with less than
20% in patients with high TREM-1 expression (Ho et al. 2008). Importantly, in
contrast to
cytokine blockers, blockade of TREM-1 can blunt excessive inflammation while
preserving the
capacity for microbial control (Weber et al. 2014). Septic mice lacking DAP-
12, a signaling
adapter of TREM-1, have improved survival (Turnbull et al. 2005). Humans
lacking DAP12 do
not have problems resolving infections (Lanier 2009). TREM-1 blockade is safe
in healthy and
septic subjects (Cuvier et al. 2018, Francois et al. 2018). Taken together,
these finding make
TREM-1 a promising therapeutic target in oncology.
The present invention provides the low toxic peptides and compositions for
TREM-1-
targeted treatment of cancer, e.g. PC, and other myeloid cell-related diseases
and conditions and
the methods for predicting the efficacy of these compositions. The invention
further provides a
method of using these peptides and compositions. These and other objects and
advantages of the
invention, as well as additional inventive features, will be apparent from the
description of the
invention provided herein.
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FIG. 14A-C presents the exemplary data showing inhibition of tumor growth
(FIG. 14A) and
TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration
(FIG. 14B,
FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an
equimolar
mixture of the sulfoxidized methionine residue-containing TREM-1-related
trifunctional peptides
(TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic
lipopeptide
particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical
(TREM-
1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the
mean SEM (n
= 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus
vehicle). (FIG. 14C)
Representative F4/80 images from BxPC-3-bearing mice treated using different
free and sSLP-
bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale
bar= 200 pm.
C. Sepsis; Severe Sepsis and Septic Shock.
Sepsis is another disorder with a high mortality rate. Currently, no approved
sepsis drugs
are available and over 30 drug candidates have failed late-stage clinical
trials. Sepsis refers to a
potentially life-threatening complication of an infection. Sepsis occurs when
endogenous
chemicals released into the bloodstream to fight the infection trigger
inflammatory responses
throughout the body. This inflammation can trigger a cascade of changes that
can damage
multiple organ systems, causing them to fail. If sepsis progresses to septic
shock, blood pressure
drops dramatically, which may lead to death.
Anyone can develop sepsis, but it's most common and most dangerous in older
adults or
those with weakened immune systems. Risk factors include but are not limited
to: young or
elderly; Have a compromised immune system; Are already very sick, often in a
hospital's
intensive care unit; Have wounds or injuries, such as burns; Have invasive
devices, such as
intravenous catheters or breathing tubes; etc.
Early treatment of sepsis, usually with antibiotics and large amounts of
intravenous
fluids, improves chances for survival. While any type of infection: ncluding
bacterial, viral or
fungal, can lead to sepsis, the most likely varieties include: Pneumonia;
Abdominal infection;
Kidney infection; Bloodstream infection (bacteremia); etc.
The incidence of sepsis appears to be increasing in the United States. The
causes of this
increase may include: Aging population. Americans are living longer, which is
swelling the
ranks of the highest risk age group ¨ people older than 65; Drug-resistant
bacteria. Many
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types of bacteria can resist the effects of antibiotics that once killed them.
These antibiotic-
resistant bacteria are often the root cause of the infections that trigger
sepsis.; Weakened
immune systems. More Americans are living with weakened immune systems, caused
by HIV,
cancer treatments or transplant drugs.; etc.
Sepsis ranges from less to more severe. As sepsis worsens, blood flow to vital
organs,
such as brain, heart and kidneys, becomes impaired. Sepsis can also cause
blood clots to form in
organs and in arms, legs, fingers and toes, leading to varying degrees of
organ failure and tissue
death (gangrene). Most people recover from mild sepsis, but the mortality rate
for septic shock is
nearly 50 percent. Also, an episode of severe sepsis may place you at higher
risk of future
infections.
Sepsis may present as a three-stage syndrome, starting with sepsis and
progressing
through severe sepsis to septic shock. The goal is to treat sepsis during its
early stage, before it
becomes more dangerous.the following symptoms, plus a probable or confirmed
infection: Body
temperature above 101 F (38.3 C) or below 96.8 F (36 C); Heart rate higher
than 90 beats a
minute; Respiratory rate higher than 20 breaths a minute, etc.
Severe sepsis refers to having at least one of the following signs and
symptoms, which
indicate an organ may be failing: Significantly decreased urine output; Abrupt
change in mental
status; Decrease in platelet count; Difficulty breathing; Abnormal heart
pumping function;
Abdominal pain; etc.
Septic shock refers to having at least one of the following signs and symptoms
of severe
sepsis, plus extremely low blood pressure that doesn't adequately respond to
simple fluid
replacement.
FIG. 15A-B presents the exemplary data showing improved survival of
lipopolysaccharide
(LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized
methionine
residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide
particles (SLP) particles of
discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP)
morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6
mice treated with
increasing concentrations of an equimolar mixture of the sulfoxidized
methionine residue-
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containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in
free form.
D. Rheumatoid arthritis (RA).
Rheumatoid arthritis (RA) refers to a chronic inflammatory disorder that can
affect more
than just your joints. In some people, the condition also can damage a wide
variety of body
systems, including the skin, eyes, lungs, heart and blood vessels.
RA affects as much as 1% of the worldwide population. There is no cure for RA
yet and
up to 80% or more of RA patients are disabled after 20 years of symptoms.
Unlike the wear-and-tear damage of osteoarthritis, rheumatoid arthritis
affects the lining
of your joints, causing a painful swelling that can eventually result in bone
erosion and joint
deformity.
The inflammation associated with rheumatoid arthritis is what can damage other
parts of
the body as well. While new types of medications have improved treatment
options dramatically,
severe rheumatoid arthritis can still cause physical disabilities.
Signs and symptoms of rheumatoid arthritis may include: Tender, warm, swollen
joints;
Joint stiffness that is usually worse in the mornings and after inactivity;
Fatigue, fever and
weight loss; etc.
Early rheumatoid arthritis tends to affect your smaller joints first,
particularly the joints
that attach your fingers to your hands and your toes to your feet.
As the disease progresses, symptoms often spread to the wrists, knees, ankles,
elbows,
hips and shoulders. In most cases, symptoms occur in the same joints on both
sides of your body.
About 40 percent of the people who have rheumatoid arthritis also experience
signs and
symptoms that don't involve the joints. Rheumatoid arthritis can affect many
nonjoint structures,
including: Skin; Eyes; Lungs; Heart; Kidneys; Salivary glands; Nerve tissue;
Bone marrow;
Blood vessels; etc.
Rheumatoid arthritis signs and symptoms may vary in severity and may even come
and
go. Periods of increased disease activity, called flares, alternate with
periods of relative remission
¨ when the swelling and pain fade or disappear. Over time, rheumatoid
arthritis can cause joints
to deform and shift out of place.
Rheumatoid arthritis increases your risk of developing: Osteoporosis.
Rheumatoid
arthritis itself, along with some medications used for treating rheumatoid
arthritis, can increase
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your risk of osteoporosis ¨ a condition that weakens your bones and makes them
more prone to
fracture. Rheumatoid nodules. These firm bumps of tissue most commonly form
around
pressure points, such as the elbows. However, these nodules can form anywhere
in the body,
including the lungs. Dry eyes and mouth. People who have rheumatoid arthritis
are much more
.. likely to experience Sjogren's syndrome, a disorder that decreases the
amount of moisture in your
eyes and mouth. Infections. The disease itself and many of the medications
used to combat
rheumatoid arthritis can impair the immune system, leading to increased
infections. Abnormal
body composition. The proportion of fat compared to lean mass is often higher
in people who
have rheumatoid arthritis, even in people who have a normal body mass index
(BMI). Carpal
.. tunnel syndrome. If rheumatoid arthritis affects your wrists, the
inflammation can compress the
nerve that serves most of your hand and fingers. Heart problems. Rheumatoid
arthritis can
increase your risk of hardened and blocked arteries, as well as inflammation
of the sac that
encloses your heart. Lung disease. People with rheumatoid arthritis have an
increased risk of
inflammation and scarring of the lung tissues, which can lead to progressive
shortness of breath.
Lymphoma. Rheumatoid arthritis increases the risk of lymphoma, a group of
blood cancers that
develop in the lymph system.
FIG. 17A-B presents the exemplary data showing average clinical arthritis
score (FIG. 17A) and
mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the
difference
between beginning (day 24) and final (day 38) BWs of the collagen-induced
arthritis (CIA) mice
treated with an equimolar mixture of the sulfoxidized methionine residue-
containing TREM-1-
related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated
into synthetic
lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and
spherical
(TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p
<0.001 as compared with vehicle-treated or naive animals.
FIG. 42A-B presents exemplary data showing average clinical arthritis score
(Collagen-induced
arthritis: Score 42A) and Collagen-induced arthritis: Body weight change mean
BW changes
(42B) calculated as a percentage of the difference between beginning (day 24)
and final (day 38)
BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-related control
peptide G-
TE21, TCR-related control peptide M-TK32, TCR-related trifunctional peptide M-
VE32 or with
TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to the
relevant control
peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the disease (A) and are
well-tolerated
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by arthritic mice (B). *, p <0.05, **,p <0.01; ***,p <0.001 as compared with
vehicle-treated
animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-
1; CIA,
collagen-induced arthritis; PBS, phosphate-buffer saline; DEX, dexamethasone;
TCR, T cell
receptor; BW, body weight.
E. Retinopathy.
Pathological retinal neovascularization (RNV) causes angiogenesis-related
vision
impairment in retinopathy of prematurity (ROP), diabetic retinopathy (DR), and
retinal vein
occlusion (RVO), which are the most common causes of vision loss and blindness
in each age
group. Conventional therapeutic options include laser ablation and the anti-
vascular endothelial
growth factor (VEGF) therapy, which both have their limitations and
complications. Laser
therapy is often accompanied by corneal edema, anterior chamber reaction,
intraocular
hemorrhage, cataract formation, and intraocular pressure changes, while the
VEGF-targeted
therapy can be complicated by damage of healthy vessels, potential side
effects on neurons, rapid
vascular regrowth upon interrupting the VEGF blockade, and limited
effectiveness in some
patients.
F. Cirrhosis Of The Liver And Alcoholic Liver Disease.
The human liver is located in the upper right side of the abdomen below the
ribs. It has
many essential body functions. These include: producing bile, which helps your
body absorb
dietary fats, cholesterol, and vitamins A, D, E, and K; storing sugar and
vitamins for later use by
the body; removing toxins such as alcohol and bacteria from your system:
creating blood clotting
proteins; etc.
Several of the most common causes of cirrhosis of the liver in the United
States are long-
term viral hepatitis C infection and chronic alcohol abuse. Obesity is also a
cause of cirrhosis,
although it is not as prevalent as alcoholism or hepatitis C. Obesity can be a
risk factor by itself,
or in combination with alcoholism and hepatitis C.
According to the The National Institute of Diabetes and Digestive and Kidney
Diseases
(NIDDK) and other components of the National Institutes of Health (NIH),
cirrhosis can develop
in women who drink more than two alcoholic drinks per day (including beer and
wine) for many
years. For men, drinking more than three drinks a day for years can put them
at risk for cirrhosis.
However, the amount is different for every person, and this doesn't mean that
everyone who has
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ever drunk more than a few drinks will develop cirrhosis. Cirrhosis caused by
alcohol is usually
the result of regularly drinking more than these amounts over the course of 10
or 12 years.
Cirrhosis causes the liver to shrink and harden. This makes it difficult for
nutrient-rich blood to
flow into the liver from the portal vein. The portal vein carries blood from
the digestive organs to
the liver. The pressure in the portal vein rises when blood can't pass into
the liver. The end result
is a serious condition called portal hypertension, in which the vein develops
high blood pressure.
The unfortunate consequence of portal hypertension is that this high-pressure
system causes a
backup, which leads to esophageal varices (like varicose veins), which can
then burst and bleed.
Cirrhosis of the liver refers to severe scarring of the liver and poor liver
function seen at the
terminal stages of chronic liver disease. The scarring is most often caused by
long-term exposure
to toxins such as alcohol or viral infections.
Alcoholic liver cirrhosis is directly related to alcohol intake and is the
final phase of
alcoholic liver disease. Symptoms including but not limited to: anemia (low
blood levels due to
too little iron); high blood ammonia level); high blood sugar levels;
leukocytosis (large amount
of white blood cells) ; unhealthy liver tissue when a sample is removed from a
biopsy and
studied in a laboratory; liver enzyme blood tests that show the level of
aspartate aminotransferase
(AST) is two times that of alanine aminotransferase (ALT); low blood magnesium
levels; low
blood potassium levels; low blood sodium levels; portal hypertension; etc.
Alcoholic liver cirrhosis can cause serious complications. This is known as
decompensated cirrhosis. Examples of these complications include: ascites, or
a buildup of fluid
in the stomach; encephalopathy, or mental confusion; internal bleeding, known
as bleeding
varices; jaundice, which makes the skin and eyes have a yellow tint; etc.
Those with this the more severe form of cirrhosis often require a liver
transplant to survive.; etc.
According to the Cleveland Clinic, patients with decompensated alcoholic liver
cirrhosis who
receive a liver transplant have a five-year survival rate of 70 percent.
Alcoholic liver disease (ALD) occurs after years of heavy drinking. The
chances of
getting liver disease go up the longer you have been drinking and more alcohol
you consume.
Typically, a person has drank heavily for at least eight years. The National
Institute on Alcohol
Abuse and Alcoholism defines heavy drinking as drinking five or more drinks in
one day on at
least five of the past 30 days.
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Symptoms of alcoholic liver cirrhosis typically develop when a person is
between the
ages of 30 and 40. A human body will be able to compensate for it's liver's
limited function in
the early stages of the disease. As the disease progresses, symptoms will
become more
noticeable. The disease is common in people between 40 and 50 years of age.
Men are more
likely to have this problem. However, Women are also more at-risk for
alcoholic liver disease.
Women don't have as many enzymes in their stomachs to break down alcohol
particles. Because
of this, more alcohol is able to reach the liver and make scar tissue.
Alcoholic liver disease can also have some genetic factors. For example, some
people are
born with a deficiency in enzymes that help to eliminate alcohol. Obesity, a
high-fat diet, and
having hepatitis C can also increase a person's likelihood they will have
alcoholic liver disease.
women may develop the disease after less exposure to alcohol than men. Some
people may have
an inherited risk for the disease. The disease is part of a progression. It
may start with fatty liver
disease, then progress to alcoholic hepatitis, and then to alcoholic
cirrhosis. However, it's
possible a person can develop alcoholic liver cirrhosis without ever having
alcoholic hepatitis.
When a person drinks alcohol heavily over the course of decades, the body
starts to
replace the liver's healthy tissue with scar tissue. Doctors call this
condition alcoholic liver
cirrhosis.
Alcoholic liver disease affects millions of people globally and often leads to
fibrosis and
cirrhosis. Liver cirrhosis is the 12th leading cause of death in the United
States and costs society
more than $15 billions annually. Despite this profound economic and health
impact, there are
currently no approved drugs to treat ALD. Current treatments including
corticosteroids,
immunosuppressants, and antioxidants have multiple shortcomings including a
high level of
serious side effects and insufficient efficacy.
slow the disease's progress and reduce your symptoms.
In some emboidments, either or both of the TREM-1 rHDLS and TREM-1
trifunctional
peptides may be used in combination with treatments including but not limited
to: Medications:
including but not limited to corticosteroids, calcium channel blockers,
insulin, antioxidant
supplements, and S-adenosyl-L-methionine (SAMe).; Nutritional Counseling:
Alcohol abuse can
lead to malnutrition.; Extra protein: Patients often require extra protein in
certain forms to help
reduce the likelihood for developing brain disease (encephalopathy).; Liver
Transplant.; etc.
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investigated the role of TREM-1 in ALD and the potential therapeutic effect of
the TREM-1
inhibitory GF9-HDL and GA/ E31-HDL formulations in the Lieber-DeCarli ALD
mouse model.
1. TREM-1 BLOCKADE AMELIORATES EXPRESSION OF EARLY
FIBROSIS MARKER GENES INDUCED BY CHRONIC ALCOHOL CONSUMPTION.
The clinical progression of ALD is associated with liver fibrosis.27 Our mouse
model of
ALD mimics the early phase of the human disease, yet mRNA levels of early
fibrosis markers
Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared
to PF controls
in the whole-liver samples (FIG. 20A-B). Induction of these makers was
remarkably attenuated
in the vehicle-treated group and further decreased by the TREM-1 inhibitory
formulations used
(FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the
expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and
FIG. 20B a-
Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of
mice with
(alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF)
group; # indicates
significance level compared to the non-treated alcohol-fed group. o indicates
significance level
compared to the vehicle-treated alcohol-fed group. The significant levels are
as follows: *, 0.05
> P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
2. TREM-1 INHIBITORY FORMULATIONS AND HDL
AMELIORATE CHRONIC ALCOHOL-INDUCED LIVER INJURY AND
STEATOSIS.
We evaluated the impact of the TREM-1 inhibitors on hepatocyte damage and
steatosis in liver. Serum ALT levels obtained during week 5 of the alcohol
feeding showed
significant increases in alcohol-fed mice compared to PF controls. This ALT
increase was
attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of
liver injury
(Fig. 21A). Interestingly, vehicle treatment (HDL) also showed a similar
protective effect
(Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0
staining in
livers of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig.
21B-D) and
H& (Fig. 21D) staining revealed attenuation of steatosis in the alcohol-fed
TREM-1
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inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated
alcohol-fed
groups (Fig. 21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses
the
production of alanine aminotransferase (ALT) in mice with alcoholic liver
disease (ALD), as
measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in
addition to
improving indicators of liver disease and inflammation. * indicates
significance level compared
to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide
particles of spherical
morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no
TREM-1
inhibitory peptide GF9. # indicates significance level compared to the non-
treated alcohol-fed
group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1
pathway inhibition
in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-
1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A)
Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-
1/TRIOPEP-
sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over
TREM-1 peptide
alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates
significance level
compared to the nontreated PF group; * indicates significance level compared
to the nontreated
alcohol-fed group; indicates significance level compared to the vehicle-
treated alcohol-fed
group. The numbers of the symbols sign the significant levels as the
following: **OP < 0.05;
WooP < 0.01;*"/###P <0.001; ****P < 0.0001. ***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
3. BLOCKADE OF TREM-1 SIGNALING REDUCES THE EXPRESSION
OF INFLAMMATION-ASSOCIATED GENES IN ALD IN MICE.
Previous reports showed that TREM-1 activation leads to the expression and
release of
proinflammatory cytokines and chemokines through nuclear factor kB activation,
which also
regulates the expression of TREM-1, providing a positive feedback loop on the
expression of the
receptor.4 Proinflammatory cytokine expression is increased in ALDA1-3123124,
therefore, we
hypothesized that TREM-1 signaling contributes to the amplification of
proinflammatory
pathways in ALD.
To evaluate this hypothesis, first we tested whole-liver mRNAs of Et0H-fed and
PF mice
with or without treatment with two different TREM-1 inhibitory formulations
and a vehicle
control in a 5-week alcohol administration model of ALD in mice.(25) We found
that mRNA
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levels of TREM-1 and MCP-1 were significantly increased in livers of alcohol-
fed mice
compared to PF controls (Fig. 1A,B).
In contrast, in mice treated with the TREM-1 inhibitors, both GF9-HDL and
GA/E31-
HDL inhibited alcohol-related changes in TREM-1; in addition, MCP-1 mRNA
levels
corresponded to those of the PF controls (Fig. 1A,B). Although induction of
TNF-a and IL-11s in
alcohol-fed mice did not reach statistical significance compared to PF
controls, TREM-1 block-
ade by GF9-HDL resulted in a significant inhibition of TNF-a mRNA in the
alcohol-fed mice
compared to vehicle treatment (Fig. 1C), while IL-lfi mRNA expression was also
significantly
attenuated by both the GF9-HDL and GA/E31-HDL formulations in the alcohol-fed
as well as in
the PF groups (Fig. ID). MIP-la mRNA levels were increased in alcohol-fed
mice, but TREM-1
blockade with GF9-HDL or GA/E31-HDL significantly attenuated this increase
compared to the
vehicle control (Fig. IE). Regulated on activation, normal T cell expressed,
and secreted
(RANTES) mRNA levels did not change regardless of alcohol feeding or TREM-1
treatment
(Fig. IF).
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the
expression
of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1, (FIG. 45C)
TNF-a,
(FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (F) RANTES at the mRNA level
as measured
in whole-liver lysates by real-time quantitative PCR. * indicates significance
level compared to
nontreated PF group; # indicates significance level compared to nontreated
alcohol-fed group; o
indicates significance level compared to vehicle-treated alcohol-fed group.
Significance levels
are as follows: * /#/o P <0.05; ** /##/oo P <0.01; *** /000 P <0.001; ****P
<0.0001.
Abbreviation: CCL, chemokine (C-C motif) ligand.
Next, we used specific ELISA kits to assess the protein levels of cytokines in
the serum
and in whole-liver lysates (Fig. 2). We found a significant increase in MCP-1
level in the serum
and liver and TNF-a in the liver of alcohol-fed mice compared to PF controls
(Fig. 2A-D). All
these alcohol-induced increases were prevented both in the serum and liver by
administration of
either TREM-1 inhibitor. Interestingly, we found attenuation of alcohol-
induced liver MCP-1
and TNF-a induction even in the vehicle-treated (HDL only) groups (Fig. 2A-C).
The increase in
total IL-lfs levels after alcohol feeding and its attenuation by TREM-1
inhibition did not reach
statistical significance (Fig. 2D).
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Because TREM-1 is a membrane-associated molecule that triggers SYK activation
as one
of its proximal signaling molecules and we previously found increased SYK
phosphorylation in
liver in ALD/24" we EBtested the levels of total and activated phospho-SYK
(p_syKY525/526) in
the livers. We found significantly increased total and p-SYKY525/s26 levels
after alcohol feeding
.. (Fig. 2E-G). Treatment with GA/E31-HDL significantly decreased the p-
SYKYS2S/526
levels in
alcohol-fed mice compared to the untreated and vehicle-treated alcohol-fed
groups, while GF9-
HDL decreased p-SYKYs25/526 levels compared to the vehicle-treated group.
(Fig. 2E,F).
FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade
reduces
inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver
lysates as
.. measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was
analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as
a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance
level compared to the nontreated PF group; # indicates significance level
compared to the
nontreated alcohol-fed group; o indicates significance level compared to the
vehicle-treated
alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** /<figref></figref> P < 0.0001.
5. BLOCKADE OF TREM-1 ACTIVATION REDUCES EXPRESSION OF
MACROPHAGE AND NEUTROPHIL MARKERS IN LIVER
In agreement with previous studies indicating that chronic alcohol use causes
hepatic
macrophage infiltration and activation/1'3'26^ we found increased expression
of the Kupffer
cell/macrophage markers F4/80 and CD68 at the mRNA level. Treatment with the
TREM-1
inhibitors significantly attenuated alcohol-induced expression of both F4/80
and CD68 in the
liver, indicating anti-significant decrease in F4/80 expression on paraffin-
embedded liver
sections by IHC in alcohol-fed mice treated with either GF9-HDL or GA/E31-HDL
compared to
the Et0H-fed vehicle-treated group (Fig. 3C,D).
Neutrophil infiltration of the liver is a characteristic of alcoholic
hepatitis; therefore, we
investigated markers associated with this cell population. Expression of the
neutrophil markers
Ly6G and MPO were significantly increased in livers of alcohol-fed mice
compared to PF
controls. This was fully prevented by TREM-1 blockade (Fig. 3E,F).
Interestingly, the HDL
vehicle alone also resulted in a decreasing trend of Ly6G and MPO expression
in alcohol-fed
mice; however, the GF9-HDL and GA/E31-HDL TREM-1 inhibitors significantly
attenuated
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Ly6G and MPO levels even when compared to the vehicle-treated alcohol-fed mice
(Fig. 3E,F).
MPO staining on IHC confirmed that both TREM-1 inhibitors significantly
reduced WO-
positive cell numbers compared to the untreated alcohol-fed control group
(Fig. 3G,H).
FIG. 47A-H. Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1
inhibition
suppresses the mRNA expression of macrophage cell markers in the liver as
measured by real-
time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated
F4/80 as
shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of
neutrophil cell markers in the liver as measured by real-time quantitative
PCR. (FIG. 47G, H)
Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by
IHC. * indicates
significance level compared to the nontreated PF group; # indicates
significance level compared
to the nontreated alcohol-fed group; o indicates significance level compared
to the vehicle-
treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
### P < 0.001; **** /<figref></figref> P < 0.0001.
6. TREM-1 INHIBITORY FORMULATIONS AND HDL AMELIORATE
CHRONIC ALCOHOL-INDUCED LIVER INJURY AND STEATOSIS
To further assess the effects of the TREM-1 inhibitors on mechanisms of lipid
metabolism, we tested genes involved in lipid synthesis (sterol regulatory
element binding
transcription factor 1 [SREBF1] and acetyl-coenzyme A carboxylase 1 [ACC1])
along with the
lipid accumulation marker perilipin-2 (ADRP) (FIG. 48A-C). Both TREM-1
inhibitors but not
vehicle treatment prevented alcohol-induced up-regulation of SREBF1, ACC1, and
ADRP at the
mRNA level (FIG. 48A-C). To assess lipid oxidation, we tested peroxisome
proliferator-
activated receptor a (PPARa), carnitine palmitoyl transferase 1A (CPT1A), and
medium-chain
acyl-coenzyme A dehydrogenase (MCAD) mRNA levels in whole-liver samples (FIG.
48D-F).
Alcohol feeding significantly reduced mRNA expression of PPARa and CPT1A,
while MCAD
had a decreasing trend. Both TREM-1 inhibitors as well as the vehicle
treatment significantly
increased PPARa and MCAD levels compared to the untreated alcohol-fed controls
(FIG. 48D-
F).
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved
in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid
accumulation marker
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(ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in
whole
liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level
compared to the nontreated alcohol-fed group; o indicates significance level
compared to the
vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o
P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
7. GF9HDL AND GA/E31HDL IS MAINLY MEDIATED BY S R A
We studied the uptake of GF9-HDL and GA/ E31-HDL in vitro in order to evaluate
potential mechanisms of targeted delivery of GF9 (GA/E31). Kupffer cells and
recruited hepatic
macrophages express high levels of SRs, including SR-A, that are involved in
phagocytosis and
removal of oxidatively damaged lipoproteins and cells from the blood
circulation.28'29 We
previously demonstrated intracellu-SR lar macrophage delivery of GF9, GA31,
and GE31 by
macrophage-targeted GF9-HDL and GA/E31-HDL, respectively, and hypothesized
that the
observed macrophage endocytosis of these complexes is SR mediated.16'17 See,
FIG 9A1 and
9A2. To further investigate the molecular mechanisms involved in this process,
we used J774
macrophages as a model for Kupffer cells and incubated them with rho B-labeled
GF9-HDL or
GA/ E31-HDL in the presence or absence of cytochalasin D, fucoidan, or BLT-1,
which are
known to inhibit all SRs,(30) SR-A,(31) or SR-BI,(32) respectively.
In the presence of cytochalasin D, which inhibits both SR-A and SR-BI, the
macrophage
uptake of both TREM-1 inhibitor complexes was significantly inhibited,
suggesting that this
uptake is SR mediated. Fucoidan, an SR-A inhibitor, substantially suppressed
endocytosis of
TREM-1 inhibitor complexes at 22 hours but not at 4 hours, indicating time-
dependent
mechanisms of SR-A-mediated endocytosis (Fig. 9B). In contrast, BLT-1, which
inhibits SR-BI,
similarly inhibited the uptake of the complexes at both time points but to a
lesser extent
compared with that of fucoidan (Fig. 9C), presumably because of lower
expression of SR-BI on
J774 macrophages(33'1) These findings suggest that SR-A is the main
contributor in SR-mediated
endocytosis of both GF9-HDL and GA/E31-HDL.
Interestingly, quantitatively determined macrophage uptake levels in the
presence or
absence of fucoidan or BLT-1 were similar for GF9-HDL and GA/E31-HDL (Fig.
7B). This
suggests that the combination of GF9 and apo AT peptide sequences in GA31 and
GE31
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sequences does not change the level and mechanisms of macrophage endocytosis
of GA/E31-
HDL compared with those of GF9-HDL.
8. Summary of TREM-1 in ALD.
Using a mouse model, significant up-regulation of TREM-1 was measured in
livers of
mice following chronic alcohol feeding. Treatment with novel ligand-
independent TREM-1
inhibitors reduced the expression of the TREM-1 molecule itself, attenuated or
fully prevented
alcohol-induced increases in proinflammatory cytokines at the mRNA level, and
inhibited SYK
activation. TREM-1 blockade provided by trifunctional peptides described
herein, results in
reduced macrophage and neutrophil infiltration and activation indicated by
reduced F4/80,
.. CD68, Ly6G, and MPO expression in the liver. These findings complement data
demonstrating
that TREM-1 blockade using GF9-HDL and GA/ E31-HDL suppresses macrophage
infiltration
of the tumor in cancer mice.(Reference 17) The TREM-1 inhibitors attenuated
alcohol-induced
liver steatosis. HDL and the TREM-1 inhibitors also attenuated liver injury
and markers of early
fibrosis in alcohol-fed mice. Interestingly, the HDL vehicle control showed
similar efficiency as
.. the inhibitory formulations at the protein level of the proinflammatory
cytokines. Thereforeitwas
also discovered that rHDL itself has some protective effects on ALD at the
level of ALT and lipid
oxidation.
While the ligand of TREM-1 is still unknown, it has been shown that TREM-1
activation
amplifies inflammation and synergizes with TLR signaling pathways.(34) It was
also observed
that bacterial infection and challenge with LPS or lipoteichoic acid increase
TREM-1
expression,(7) indicating a positive feedback loop among PAMP exposure, TREM-1
expression,
and inflammatory cytokine induction. Different DAMPs, such as 3-hydroxy-3-
methyl-glutaryl Bl
and heat shock protein 70, have been suggested to stimulate TREM-1,(35)' while
other studies
found cell (granulocyte and platelet)-surface-associated activators as
well.(35,36) Both PAMPs
and DAMPs are present in ALD, providing potential mechanisms for TREM-1 up-
regulation in
this disease. Alcohol induces changes in the gut microbiome and disrupts the
gut barrier
function, resulting in increased levels of endotoxin and microbial PAMPs in
circulation.(1,37)
Alcohol also causes hepatocyte damage that leads to the release of DAMP5,(23)
and these
processes contribute to TREM-1 activation.
TREM-1 signaling leads to phosphorylation and activation of SYK, which has
been
indicated as a major regulator in inflammatory processes in ALD.(38) TREM-1
also amplifies
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TLR4 signaling that involves activation of SYK, which has been indicated as a
downstream SYK
activation and phosphorylation.(38) Indeed, we found increased total and
phosphorylated SYK
levels in the livers of alcohol-fed mice that was attenuated by TREM-1
inhibitor administration.
A previous study showed that inhibition of SYK activation attenuates alcohol-
induced liver
inflammation, cell death, and steatosis, suggesting that the SYK pathway could
be a feasible
therapeutic target in ALD.(24) SYK is expressed in a wild spectrum of cells,
while TREM-1
inhibition may specifically modulate macrophages, neutrophils, and stellate
cells that each play a
role in ALD. Another advantage of TREM-1 inhibition is that it likely
attenuates signaling from
a broader spectrum ofTLRs, in addition toTLR4.
TREM-1 activation alone has been shown to increase the production of
proinflammatory
chemo-kines and cytokines.(39) Furthermore, simultaneous stimulation of TREM-1
and TLRs by
an agonistic anti-TREM-1 antibody and different TLR ligands synergized in the
induction of
these proinflammatory molecules. TREM-1 and TLR4 costimulated monocytes showed
increased production of MCP-1, IL-113, and IL-8. In contrast, the level of the
anti-inflammatory
cytokine IL-10 decreased when anti-TREM-1 antibody and the TLR3 ligand
poly(LC) or the
TLR4 ligand LPS simultaneously attached to their receptors.(40) Because self-
perpetuating
proinflammatory pathways are present in alcoholic hepatitis, interruption of
these pathways
using TREM-1 inhibition seems attractive.
By inducing TNF-a, IL-6, MCP-1, IL-8, and granulocyte-macrophage colony-
stimulating
factor and inhibiting IL-10 production, TREM-1 is involved in activation and
recruitment of
monocytes and modulation of inflammatory responses.(40) Furthermore, TREM-1
expression was
highly up-regulated on the surface of infiltrating monocytes and neutrophils
in human tissues
infected by bacteria, highlighting the importance of this receptor in these
processes.(7) In
alcoholic hepatitis, neutrophils infiltrate the liver, inducing oxidative
stress and cytotoxicity that
contributes to the high mortality of the disease.(2) We showed that these
processes can be
attenuated by TREM-1 inhibitors. Mechanistically, the GF9-HDL and GA/E31-HDL
formulations target the liver more efficiently than peptides alone and release
the TREM-1
inhibitory sequences inside the target cells where these peptides likely
inhibit TREM-1 signaling
by disrupting the intramembrane interactions of the TREM-1 receptor and its
signaling adaptor
molecule death-associated protein 12 (Fig. 27).(15-17)
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It was contemplated that observed preferential endocytosis of GF9-HDL and
GA/E31-
HDL by macrophages and hepatic clearance of these complexes is mediated by SR
recognition
of putative epitopes in the modified apo A-I peptide constituents of GF9-HDL
and GA/E31-
HDL.(16,17,19) Findings described herein indicate that GF9-HDL and GA/ E31-HDL
are
largely recognized by SR-A on macrophages (Fig. 9A-B). We also observed SR-BI-
mediated
uptake, which likely explains the previously observed hepatic clearance for
these complexes in
another animal model.(19) While these data confirm our hypothesis, future
studies are needed to
determine the clearance properties for GF9-HDL and GA/E31-HDL in ALD.
Further, our present study demonstrates that GF9-HDL and GA/E31-HDL exhibit
not
only similar macrophage uptake in vitro largely driven by SR-A (Fig. 9A1) but
also similar
therapeutic effect in a mouse model of ALD (Figs. 20-21). This is in line with
our previous
studies where GF9-HDL and GA/E31-HDL exhibited similar therapeutic activities
in cancer and
arthritic mice. (16,17) We suggest that SR-A epitopes are similarly exposed on
GA31 and GE31
in GA/ E31-HDL and on PA22 and PE22 in GF9-HDL, providing similar uptake of
these
complexes and as a result delivery of TREM-1 inhibitory GF9 peptide sequences
in vivo. The
use of GA/E31-HDL in the further development of effective and low-toxicity
therapy for ALD is
advantageous because it makes the entire manufacturing process easier and less
expensive. We
also suggest that the in vitro macrophage uptake assay can be potentially used
to predict the
outcomes for macrophage-targeted TREM-1 therapy in vivo.
In addition to attenuating inflammatory processes, the TREM-1 inhibitory
formulations
also ameliorated hepatocyte damage and steatosis. Serum ALT and liver
triglyceride levels were
both decreased in the GF9-HDL, GA/E31-HDL, and HDL-vehicle treated groups. The
vehicle
also had an inhibitory effect on TNF-a and MCP-1 protein levels as well as on
mRNA expression
of neutrophil and fibrosis markers, indicating that the HDL vehicle
formulation can attenuate
inflammation to a moderate extent. A previous study found evidence that HDL
can protect
hepatocytes from endoplasmic reticulum stress, (41) while other publications
reported a
scavenger function of HDL for LPS and lipoteichoic acid(42,43) that could
prevent immune cells
from being activated by those molecules.(42,43) Further, the observed moderate
beneficial effect
of HDL treatment alone on fatty acid oxidation markers in alcohol-exposed mice
(Fig. 47A-C) is
in line with data that demonstrate infusion of reconstituted HDL reduces fatty
acid oxidation in
patients with type 2 diabetes mellitus. (44) In human and rat plasma, apo A-I,
the major protein
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of HDL, has been shown to inhibit lipid peroxidation. (45) These data might
provide an
explanation for our findings of the hepatoprotective effects of HDL.
Our study shows that TREM-1 inhibitors with HDL formulation exerted
significant
inhibition on early signaling events of proinflammatory processes at ^the
level of cytokine
mRNA and the activated p-SYK protein levels compared to the HDL vehicle alone
in a mouse
model of ALD. This effect presumably would be even more obvious at the protein
level of
cytokines in a more severe liver injury. However, in mice, the most commonly
used 5-week
alcohol feeding that weused resulted in moderate liver damage and minimal(25)
inflammation,
which is a limitation of our study. As shown on the stained liver sections,
the GF9-HDL and
GA/E31-HDL formulations significantly inhibited immune cell infiltration and
steatosis
compared to the HDL vehicle only in mice with ALD. Thus, in some emboidments,
TREM-1
inhibitors, such as the trifuncitonal peptides described herein, are
contemplate for administration
to patients showing at least one sympton, or at risk of developing a sympton,
for ALD for
decreasing inflammation in liver tissue for reducing said symptom or
delaying/preventing said
sympton.
Materials and Methods, for example, in relation to experiments assocated with
treating
ALD.
REAGENTS AND CELLS
The murine macrophage J774A.1 cell line was purchased from ATCC (Manassas,
VA).
Cytochalasin D was purchased from MP Biomedicals (Solon, OH). Blocker of lipid
transport 1
(BLT-1) was purchased from Calbiochem (Torrey Pines, CA). Sodium cho-late,
cholesteryl
oleate, fucoidan, and other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO). 1-
Palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoy1-0T-
glycero-3-
phosphoeth-anolamine-N-(lissamine rhodamine B sulfonyl) (rho EEB-PE), and
cholesterol were
purchased from Avanti Polar Lipids (Alabaster, AL).
PEPTIDE SYNTHESIS
The following synthetic peptides were ordered from Bachem (Torrance, CA): one
9-mer
peptide, GFLSKSLVF (human TREM-1213- 221, GF9); two 22-mer methionine
sulfoxidized
peptides, PYLDDFQKKWQEEM(0)ELYRQKVE (H4) and PLG
EEM(0)RDRARAHVDALRTHLA (H6), which correspond to human apo A-I helices 4 (apo
A-
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1123.144) and 6 (apo A-I167-188), respectively; and two 31-mer methionine
sulfoxidized peptides,
GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE (GE31) and
GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (GA31).
LIPOPEPTIDE COMPLEXES
HDL-mimicking lipopeptide complexes of spherical morphology that contained
either
GF9 and an equimolar mixture of PE22 and PA22 (GF9-HDL) or an equimolar
mixture of GA31
and GE31 (GA/ E31-HDL) were synthesized using the sodium cholate dialysis
procedure,
purified, and characterized as described.(16-18, 22) For GF9-HDL, the initial
molar ratio was
125:6:2:3:1:210, corresponding to POPC:cholesterol:cholesteryl oleate:GF9:apo
A-I:sodium
cholate, respectively, where apo A-I was an equimolar mixture of PE22 and
PA22. For GA/E31-
HDL, the initial molar ratio was 125:6:2:1:210, corresponding to
POPC:cholesterol:cholesteryl
oleate:GA/ E31: sodium cholate, respectively, where GA/E31 was an equimolar
mixture of
GA31 and GE31.
IN VITRO MACROPHAGE UPTAKE OF GF9HDL AND GA/E31HDL
A quantitative in vitro macrophage assay of endo-cytosis of rho B-labeled HDL-
mimicking lipopeptide complexes by J774 macrophage was performed as
described.(18-20)
Briefly, BALB/c murine macrophage J774A.1 cells (ATCC) were cultured at 37 C
with 5% CO2
in Dulbecco's modified Eagle's medium (Cellgro Mediatech, Manassas, VA) with 2
mM
glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% heat-
inactivated fetal bovine
serum (Cellgro Mediatech) and grown to approximately 90% confluency in 12-well
tissue
culture plates (Corning Costar, Corning, NY). After reaching target
confluency, cells were
incubated for 1 hour in medium with or without fucoidan (400 ug/mL), BLT-1
((1011M), or
cytochalasin D (4011M). Cells were subsequently incubated for 4 hours and 22
hours at 37 C in
medium containing 2 [EM of rho B-labeled GF9-HDL or GA/E31-HDL (as calculated
for rho B).
Cells were washed twice using phosphate-buffered saline and lysed using
Passive Lysis
Buffer (Promega, Madison, WI). Rho B fluorescence was measured in the lysates
with 544-nm
excitation and 590-nm emission filters, using a Fluoroscan Ascent CF
fluorescence microplate
reader (Thermo Labsystems, Vantaa, Finland). Protein concentrations in the
lysates were
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measured using Bradford reagent (Sigma-Aldrich) and an MRX microplate reader
(Dynex
Technologies, Chantilly, VA) according to the manufacturer's recommended
protocol.
ANIMALS
C57BL/6 female mice (10- to 12-week-old) were purchased from the Jackson
Laboratory
(Bar Harbor, ME) and housed at the University of Massachusetts Medical School
(UMMS)
animal facility. Animals received humane care in accordance with protocols
approved by the
UMMS Institutional Animal Use and Care Committee. Mice (n = 6-9/group) were
acclimated to
a Lieber-DeCarli liquid diet of 5% eth-anol (Et0H) (volume [vol]/vol) over a
period of 1 week,
then maintained on the 5% diet for 4 weeks. Pair-fed (PF) control mice were
fed a calorie-
matched dextran-maltose diet. Animals had unrestricted access to water
throughout the entire
experimental period. In treated groups, mice were intraperitone-ally treated 5
days/week with
vehicle (empty HDL) or the TREM-1 inhibitory formulations GF9-HDL (2.5 mg of
GF9/kg) or
GA/E31-HDL (4 mg equivalent of GF9/kg) (SignaBlok, Shrewsbury, MA) from the
first day on
a 5% Et0H diet. At the end of all animal experiments, cheek blood samples were
collected in
serum collection tubes (BD Biosciences, San Jose, CA) and processed within an
hour. After
blood collections, mice were euthanized and liver samples were harvested and
stored at -80 C
until further analysis.
TOTAL PROTEIN ISOLATION FROM LIVER
Total protein was extracted from liver samples using radio immunoprecipitation
assay
buffer (BP-115; Boston BioProducts) supplemented with protease inhibitor
cocktail tablets
(11836153001; Roche) and Phospho Stop phosphatase inhibitor (04906837001;
Roche). Cell
debris was removed from cell lysates by 10 minutes centrifugation at 2,000
rpm.
BIOCHEMICAL ASSAYS AND CYTOKINES
Serum ALT levels were determined by the kinetic method using commercially
available
reagents from Teco Diagnostics (Anaheim, CA). Cytokine levels were measured in
serum
samples, and whole-liver lysates were diluted in assay diluent following the
manufacturer's
instructions. Specific anti-mouse enzyme-linked immunosorbent assay (ELISA)
kits were used
for the quantification of MCP-1, TNF-a (BioLegend Inc., San Diego, CA), and IL-
ip (R&D
Systems, Minneapolis, MN) levels. For normalization, the total protein
concentration of the
whole-liver lysate was determined using the Pierce bicinchoninic acid protein
assay.
WESTERN BLOT ANALYSIS
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Whole-liver proteins were boiled in Laemmli's buffer. Samples were resolved in
10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel under reducing
conditions, using
a Tris-glycine buffer system; resolved proteins were transferred onto a
nitrocellulose membrane.
SYK proteins were detected by specific primary antibodies (SYK, 2712 [Cell
Signaling];
phospho-SYKY525/526, ab58575 [Abeam]) followed by an appropriate secondary
horseradish
peroxidase-conjugated immunoglobulin G antibody from Santa Cruz Biotechnology,
p-actin,
detected by an ab49900 antibody (Abeam), was used as a loading control. The
specific
immunoreactive bands of interest were visualized by chemiluminescence (Bio-Rad
Laboratories)
using the Fujifilm LAS-4000 luminescent image analyzer.
RNA EXTRACTION AND QUANTITATIVE REALTIME POLYMERASE
CHAIN REACTION ANALYSIS
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen) according to the
manufacturer's instructions with on-column deoxyribonuclease treatment. RNA
was quantified
using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and
complementary DNA
synthesis was performed using the iScript Reverse Transcription Supermix (Bio-
Rad
Laboratories) and 1 ug total RNA. Real-time quantitative polymerase chain
reaction (PCR) was
performed using Bio-Rad iTaq Universal SYBR Green Supermix and a CFX96 real-
time
detection system (Bio-Rad Laboratories). Relative gene expression was
calculated by the
comparative AAACt method. The expression level of target genes was normalized
to the
housekeeping gene 18S ribosomal RNA in each sample, and the fold change in the
target gene
expression among experimental groups was expressed as a ratio. Primers were
synthesized by
IDT, Inc.; exemplary sequences are listed in Table 1.
LIVER HISTOPATHOLOGY
Sections of formalin-fixed paraffin-embedded liver specimens from mice were
stained
with hematoxylin and eosin (H&E) or F4/80 (MF48000; Thermo Fisher Scientific)
and MPO
(ab9535; Abeam) antibodies for immunohistochemistry (IHC). The fresh-frozen
samples were
stained with Oil Red 0 at the UMMS Diabetes and Endocrinology Research Center
histology
core facility.
STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism 7.02 (GraphPad
Software
Inc.). Significance levels were determined using one-way analysis of variance
followed by a
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post-hoc test for multiple comparisons. Data are shown as mean SEM, and
differences were
considered statistically significant when P < 0.05.
REFERENCES related to sections on ALD.
1) Szabo G, Bala S, Petrasek J, Gattu A. Gut-liver axis and sensing microbes.
Dig Dis
2010;28:737-744.
2) Bautista AP. Neutrophilic infiltration in alcoholic hepatitis. Alcohol
2002;27:17-21.
3) SzaboG, PetrasekJ, BalaS. Innate immunity and alcoholic liver disease. Dig
Dis
2012;30(Suppl 1.):55-60.
4) Tessarz AS, Cerwenka A. The TREM-1/DAP12 pathway. Immunol Lett 2008;116:111-
116.
5) Arts RJ, Joosten LA, Dinarello CA, Kullberg BJ, van der Meer JW, Netea MG.
TREM-1
interaction with the LPS/TLR4 receptor complex. Eur Cytokine Netw 2011;22:11-
14.
6) Campanholle G, Mittelsteadt K, Nakagawa S, Kobayashi A, Lin SL, Gharib SA,
et al. TLR-
2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells
during
sterile kidney injury. PLoS One 2013;8:e68640.
7) Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies
inflammation and is a
crucial mediator of septic shock. Nature 2001;410:1103-1107.
8) Zysset D, Weber B, Rihs S, Brasseit J, Freigang S, Riether C, et al. TREM-1
links
dyslipidemia to inflammation and lipid deposition in atherosclerosis. Nat
Commun
2016;7:13151.
9) Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1¨expressing intestinal
macrophages
crucially amplify chronic inflammation in experimental colitis and
inflammatory bowel
diseases. J Clin Invest 2007;117:3097-3106.
10) Liao R, Sun TW, Yi Y, Wu H, Li YW, Wang FX, et al. Expression of TREM-1 in
hepatic
stellate cells and prognostic value in hepatitis B-related hepatocellular
carcinoma. Cancer Sci
2012;103:984-992.
11) Wu J, Li J, Salcedo R, Mivechi NF, Trinchieri G, Horuzsko A. The
proinflammatory
myeloid cell receptor TREM-1 controls Kupffer cell activation and development
of
hepatocellular carcinoma. Cancer Res 2012;72:3977-3986.
12) ReadCB, KuijperJL,Hjorth SA, HeipelMD,TangX,Fleetwood AJ, et al. Cutting
edge:
identification of neutrophil PGLYRP1 as a ligand for TREM-1.J Immunol
2015;194:1417-
1421.
241
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
13) Sigalov AB. Multichain immune recognition receptor signaling: different
players, same
game? Trends Immunol 2004;25:583-589.
14) Sigalov AB. Immune cell signaling: a novel mechanistic model reveals new
therapeutic
targets. Trends Pharmacol Sci 2006;27:518-524.
15) Rojas MA, Shen ZT, Caldwell RB, Sigalov AB. Blockade of TREM-1 prevents
vitreoretinal
neovascularization in mice with oxygen-induced retinopathy. Biochim Biophys
Acta Mol
Basis Dis 2018;1864:2761-2768.
16) Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide
inhibitors of TREM-1
ameliorate collagen-induced arthritis. J Cell Mol Med 2017;21:2524-2534.
17) Shen ZT, Sigalov AB. Novel TREM-1 inhibitors attenuate tumor growth and
prolong
survival in experimental pancreatic cancer. Mol Pharm 2017;14:4572-4582.
18) Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1
suppresses tumor
growth in human lung cancer xenografts and prolongs survival of mice with
lipopolysaccharide-in-duced septic shock. Int Immunopharmacol 2014;21:208-219.
19) Shen ZT, Zheng S, Gounis MJ, Sigalov AB. Diagnostic magnetic resonance
imaging of
atherosclerosis in apolipoprotein E knockout mouse model using macrophage-
targeted
gadolinium-containing synthetic lipopeptide nanoparticles. PLoS One
2015;10:e0143453.
20) Sigalov AB. Nature-inspired nanoformulations for contrast-enhanced in vivo
MR imaging of
macrophages. Contrast Media Mol Imaging 2014;9:372-382.
21) Lieber CS, DeCarli LM. The feeding of alcohol in liquid diets: two decades
of applications
and 1982 update. Alcohol Clin Exp Res 1982;6:523-531.
22) Shen ZT, Sigalov AB. SARS coronavirus fusion peptide-derived sequence
suppresses
collagen-induced arthritis in DBA/If mice. Sci Rep 2016;6:28672.
23) Iracheta-Vellve A, Petrasek J, Satishchandran A, Gyongyosi B, Saha B,
Kodys K, et al.
Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome
activation and
protects from alcoholic steatohepatitis in mice. J Hepatol 2015;63:1147-1155.
24) Bukong TN, Iracheta-Vellve A, Saha B, Ambade A, Satishchandran A,
Gyongyosi B, et al.
Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell
death, and
steatosis in alcoholic liver disease. Hepatology 2016;64:1057-1071.
25) Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and
binge ethanol
feeding (the NIAAA model). Nat Protoc 2013;8:627-637.
242
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
26) Ju C, Mandrekar P. Macrophages and alcohol-related liver inflammation.
Alcohol Res
2015;37:251-262.
27) Teli MR, Day CP, Burt AD, Bennett MK, James OF. Determinants of
progression to
cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet 1995;346:987-990.
28) Terpstra V, van Berkel TJ. Scavenger receptors on liver Kupffer cells
mediate the in vivo
uptake of oxidatively damaged red blood cells in mice. Blood 2000;95:2157-
2163.
29) Zingg JIM, Ricciarelli R, Azzi A. Scavenger receptors and modified
lipoproteins: fatal
attractions? ITJBMB Life 2000;49:397-403.
30) Gu BJ, Saunders BM, Jursik C, Wiley JS. The P2X7-nonmuscle myosin membrane
complex
regulates phagocytosis of nonop-sonized particles and bacteria by a pathway
attenuated by
extracellular ATP. Blood 2010;115:1621-1631.
31) Sigola LB, Fuentes AL, Millis LM, Vapenik J, Murira A. Effects of Toll-
like receptor
ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis. Tissue
Cell
2016;48:389-396.
32) Yu M, Romer KA, Nieland TJ, Xu S, Saenz-Vash V, Penman M, et al.
Exoplasmic cysteine
Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-
molecule inhibitor
and normal lipid transport activity. Proc Natl Acad Sci USA 2011;108:12243-
12248.
33) Dong P, Xie T, Zhou X, Hu W, Chen Y, Duan Y, et al. Induction of
macrophage scavenger
receptor type BI expression by tamoxifen and 4-hydroxytamoxifen.
Atherosclerosis
2011;218:435-442.
34) Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC. TREM-1
and its
potential ligands in non-infectious diseases: from biology to clinical
perspectives. Pharmacol
Ther 2017;177:81-95.
35) El Mezayen R, El Gazzar M, Seeds MC, McCall CE, Dreskin SC, Nicolls MR.
Endogenous
signals released from necrotic cells augment inflammatory responses to
bacterial endotoxin.
Immunol Lett 2007;111:36-44.
36) Gibot S, Buonsanti C, Massin F, Romano M, Kolopp-Sarda MN, Benigni F, et
al.
Modulation of the triggering receptor expressed on the myeloid cell type 1
pathway in murine
septic shock. Infect Immun 2006;74:2823-2830.
37) Bala S, Marcos M, Gattu A, Catalano D, Szabo G. Acute binge drinking
increases serum
endotoxin and bacterial DNA levels in healthy individuals. PLoS One
2014;9:e96864.
243
CA 03109702 2021-02-12
WO 2020/036987
PCT/US2019/046392
38) Arts RJ, Joosten LA, van der Meer JW, Netea MG. TREM-1: intracellular
signaling
pathways and interaction with pattern recognition receptors. J Leukoc Biol
2013;93:209-215.
39) Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and
signal integration.
Nat Immunol 2006;7:1266-1273.
40) Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, Colonna M,
et al. A role for
triggering receptor expressed on myeloid cells-1 in host defense during the
early-induced and
adaptive phases of the immune response. J Immunol 2003;170:3812-3818.
41) Hong D, Li LF, Gao HC, Wang X, Li CC, Luo Y, et al. High-density
lipoprotein prevents
endoplasmic reticulum stress-induced downregulation of liver LOX-1 expression.
PLoS One
2015;10:e0124285.
42) Feingold KR, Grunfeld C. The role of HDL in innate immunity. J Lipid Res
2011;52:1-3.
43) Tobias PS, Ulevitch RJ. Control of lipopolysaccharide-high density
lipoprotein binding by
acute phase protein(s). J Immunol 1983;131:1913-1916.
44) Drew BG, Carey AL, Natoli AK, Formosa MF, Vizi D, Reddy-Luthmoodoo M, et
al.
Reconstituted high-density lipoprotein infusion modulates fatty acid
metabolism in patients
with type 2 diabetes mellitus. J Lipid Res 2011;52:572-581.
45) Mashima R, Yamamoto Y, Yoshimura S. Reduction of phosphatidylcholine
hydroperoxide
by apolipoprotein A-I: purification of the hydroperoxide-reducing proteins
from human blood
plasma. J Lipid Res 1998;39:1133-1140.
Taken together, this highlights the urgent need for novel approaches to
prevent, treat
and/or diagnose these diseases. However, it should be noted that the
techniques and
compositions listed and described herein are applicable to a broad range of
disease states
including, but not limiting to, cardiovascular disease, bacterial infectious
diseases, diabetes, and
autoimmune diseases. Other features and advantages of the invention will
become apparent from
the following detailed description. It should be understood, however, that the
detailed description
and the specific examples, while indicating preferred embodiments of the
invention, are given by
way of illustration, because various changes and modifications within the
spirit and scope of the
invention will become apparent to those skilled in the art from this detailed
description.
VI. Imaging probes.
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In one embodiment, one or both amino acid domains of the peptides and
compounds of
the present invention are conjugated to an imaging probe. In one embodiment,
the peptides and
compounds of the present invention are used in combinations thereof In one
embodiment, the
present invention relates to the targeted treatment, prevention and/or
detection of cancer
including but not limited to lung, pancreatic, breast, stomach, prostate,
colon, brain and skin
cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation
syndrome,
inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic
shock, multiple
sclerosis, liver diseases, autoimmune diseases, including but not limited to,
atopic dermatitis,
lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other
rheumatic diseases, sepsis
and other inflammatory diseases or other condition involving myeloid cell
activation and, more
particularly, TREM receptor-mediated cell activation, including but not
limited to diabetic
retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and
Huntington's diseases.
VII. Exempalry Methods Of Providing Synthetic (recombinant)
Lipopeptide
Particles (SLPs or rHDLs) And Synthetic Peptides.
In one embodiment, the invention provides methods for making SLPs. The method
comprises co-dissolving a predetermined amount of a mixture of neutral and/or
charged lipids.
The method further comprises drying the mixture under nitrogen. The method
even further
comprises co-dissolving the dried mixture with a predetermined amount of a
trifunctional peptide
or compound of the present invention or combinations thereof. The co-
dissolving is conducted
for a time period sufficient to allow the mixture to self-assemble into
structures whereby
particles are formed. The method further comprises isolating particles that
have a size of between
about 5 to about 200 nm diameter.
The lipid of the method may include PC, PE, PS, PI, PG, CL, SM, DOTAP or PA.
In
certain embodiments, the invention provides a method for making SLP comprising
co-dissolving
a predetermined amount of a mixture of neutral and/or charged lipids with a
predetermined
amount of cholesterol, a predetermined amount of triglycerides and/or
cholesteryl ester. The
method further comprises drying the mixture under nitrogen. The method even
further comprises
co-dissolving the dried mixture with a predetermined amount of sodium cholate
and a
predetermined amount of a trifunctional peptide or compound of the present
invention or
combinations thereof The co-dissolving is conducted for a time period
sufficient to allow the
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components to coalesce into particles. The method still further comprises
removing sodium
cholate from the mixture, and isolating particles that have a size of between
about 5 to about 200
nm diameter. The lipid of the method may include PC, PE, PS, PI, PG, CL, SM,
DOTAP, or PA.
In one embodiment, in the methods of the present disclosure, the peptides and
compounds of the invention are pre-formulated into synthetic lipopeptide
particles (SLP). In one
embodiment, SLPs are discoidal in shape. In one embodiment, SLPs are spherical
in shape.
While the size of the particles is preferably between 5 nm and 50 nm, the
diameter may be up to
200 nm. In one embodiment, the lipid of the particles may include cholesterol,
a cholesteryl
ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a
diacylglycerol, or a
triacylglycerol. And further, the phospholipid may include phosphatidylcholine
(PC),
phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol
(PI),
phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), or
phosphatidic acid (PA),
and any combinations thereof.
And even further, the cationic lipid can be 1,2-dioleoy1-3-trimethylammonium-
propane
(DOTAP). The lipid of the synthetic nanoparticle may be polyethylene
glycol(PEG)ylated. In
one embodiment, lipid is conjugated to at least one imaging probe.
In certain embodiments, an imaging probe is selected from the group comprising
Gd(III),
Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III),
Tb(III), Yb(III) Dy(III),
Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In",
Fe.59, TC99111, Cr51, Ga67, Ga68, Cu64,
Rb82,m099, Dy165,
Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32,
Cll,
N13, 015, Br76, Kr81,
Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol,
or a combination thereof.
In one embodiment, the imaging agent is a GBCA for MM. In one embodiment, the
imaging agent is a [64Cu]-containing imaging probe for imaging systems such as
PET imaging
systems (and combined PET/CT and PET/MRI systems). In one embodiment, the
peptides and
compositions of the invention are used in combinations thereof. In one
embodiment, the peptides
and compositions of the invention are used in combinations with other
anticancer therapeutic
agents. In certain embodiments, the modulators and compositions described
herein are
incorporated into long half-life SLP. In certain embodiments, the modulators
and compositions
described herein may incorporate into lipopeptide particles (LP) in vivo upon
administration to
the individual. In certain embodiments, the peptides and compositions of the
invention can cross
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the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor
barrier (BTB). Thus,
in one aspect, the invention provides for a method for suppressing tumor
growth in an individual
in need thereof by administering to the individual an amount of a TREM-1
inhibitor that is
effective for suppressing tumor growth.
A. Discoidal SLP (dSLP).
In one embodiment, the invention provides a method for making discoidal SLP
(dSLP).
The method comprises co-dissolving a predetermined amount of a mixture of
neutral and/or
charged lipids. The method further comprises drying the mixture under
nitrogen. The method
even further comprises co-dissolving the dried mixture with a predetermined
amount of a
trifunctional peptide or compound of the present invention or combinations
thereof The co-
dissolving is conducted for a time period sufficient to allow the mixture to
self-assemble into
structures whereby particles are formed. The method further comprises
isolating particles that
have a size of between about 5 to about 200 nm diameter.
B. Spherical SLP (sSLP).
In one embodiment, the invention provides a method for making spherical SLP
(sSLP)
comprising co-dissolving a predetermined amount of a mixture of neutral and/or
charged lipids
with a predetermined amount of cholesterol, a predetermined amount of
triglycerides and/or
cholesteryl ester. The method further comprises drying the mixture under
nitrogen. The method
even further comprises co-dissolving the dried mixture with a predetermined
amount of sodium
cholate and a predetermined amount of a trifunctional peptide or compound of
the present
invention or combinations thereof The co-dissolving is conducted for a time
period sufficient to
allow the components to coalesce into particles. The method still further
comprises removing
sodium cholate from the mixture, and isolating particles that have a size of
between about 5 to
about 200 nm diameter.
From second prov
C. Peptides.
Synthetic peptides, including trifunctional peptides of the present invention
may include
substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally
occurring or
unnatural amino acid). Examples of non-naturally occurring amino acids include
D-amino acids,
an amino acid having an acetylaminomethyl group attached to a sulfur atom of a
cysteine, a
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pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6,
neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl
glycine, N-methyl
isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and
methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and
ornithine is basic. Proline
may be substituted with hydroxyproline and retain the conformation conferring
properties.
Naturally occurring residues are divided into groups based on common side
chain
properties:
(1) hydrophobic: norleucine, methioninc (Met), Alanine (Ala), Valine (Val),
Leucine (Leu),
Isoleucine (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr),
Phenylalanine (Phe);
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr);
(3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu);
(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys),
Arginine (Arg);
(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);
(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine
(His);
(7) polar: Ser, Thr, Asn, Gln;
(8) basic positively charged: Arg, Lys, His; and;
(9) charged: Asp, Glu, Arg, Lys, His
Analogues may be generated by substitutional mutagenesis and retain the
biological
activity of the original trifunctional peptides. Examples of substitutions
identified as
"conservative substitutions" are shown in TABLE 1. If such substitutions
result in a change not
desired, then other type of substitutions, denominated "exemplary
substitutions" in Table 1, or as
further described herein in reference to amino acid classes, are introduced
and the products
screened for their capability of executing three functions.
TABLE 1. Amino acid substitutions.
Amino acid substitution
Original residue Exemplary substitution Conservative
substitution
Ala (A) Val, Leu, Ile Val
Arg (R) Lys, Gln, Asn Lys
Asn (N) Gln, His, Lys, Arg Gln
Asp (D) Glu Glu
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Cys (C) Ser Ser
Gin (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Asn, Gin, Lys, Arg Arg
Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu
Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile
Lys (K) Arg, Gin, Asn Arg
Met (M) Leu, Phe, Ile Leu
Phe (F) Leu, Val, Ile, Ala Leu
Pro (P) Gly Gly
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr Tyr
Tyr (Y) Trp, Phe, Thr, Ser Phe
Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu
TABLE 2A. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions
1 GFL SK SLVF GEEMRDRARAHV
2 GFL SKSLVFGEEM(0)RDRARAHV
3 GFL SKSLVFWQEEMELYRQKV
4 GFL SKSLVFWQEEM(0)ELYRQKV
GFL SR SLVF GEEMRDRARAHV
6 GFL SR SLVF GEEM(0)RDRARAHV
7 GFL SRSLVFWQEEMELYRQKV
8 GFL SRSLVFWQEEM(0)ELYRQKV
9 GLL SKSLVFGEEMRDRARAHV
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GLLSKSLVFGEEM(0)RDRARAHV
11 GLL SKSLVFWQEEMELYRQKV
12 GLL SKSLVFWQEEM(0)ELYRQKV
13 GFL SKSLVFGEEMRDRARAHVRGD
14 GFL SKSLVFWQEEMELYRQKVRGD
GFL SKSLVFPLGEEMRDRARAHVDALRTHLA
16 GFL SKSLVFPYLDDFQKKWQEEMELYRQKVE
17 GFL SKSLVFPLGEEM(0)RDRARAHVDALRTHLA
18 GFL SK SLVFPYLDDFQKKWQEEM(0)ELYRQKVE
19 GFL SKSLVFPLGEEMRDRARAHVDALRTHLARGD
GFL SKSLVFPYLDDFQKKWQEEMELYRQKVERGD
21 [64Cu]GFL SKSLVFGEEM(0)RDRARAHV
22 [64Cu]GFLSKSLVFWQEEM(0)ELYRQKV
23 [64Cu]GFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
24 [64Cu]GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
26 LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
27 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
28 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
29 LQEEDAGEYGCMGEEM(0)RDRARAHV
LQEEDAGEYGCMWQEEM(0)ELYRQKV
31 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
32 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
33 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
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34 [64Cu]LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
35 LQVTD SGLYRCVIYHPPGEEM(0)RDRARAHV
36 LQVTD SGLYRCVIYHPPWQEEM(0)ELYRQKV
37 MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
38 MWKTPTLKYFPYLDDF QKKWQEEMELYRQKVE
39 MWRTPTLRYFPLGEEMRDRARAHVDALRTHLA
40 MWRTPTLRYFPYLDDF QKKWQEEMELYRQKVE
41 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
42 [64Cu]MWKTPTLKYFPYLDDF QKKWQEEMELYRQKVE
43 GAR SMTLTVQARQLPLGEEMRDRARAHVDALRTHLA
44 GAR SMTLTVQARQLPYLDDF QKKWQEEMELYRQKVE
45 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
46 [64Cu]GARSMTLTVQARQLPYLDDF QKKWQEEMELYRQKVE
47 GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
48 GVLRLLLFKLPYLDDF QKKWQEEMELYRQKVE
49 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
50 [64Cu]GVLRLLLFKLPYLDDF QKKWQEEMELYRQKVE
51 LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
52 LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
53 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
54 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
5 YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
56 YLLDGILFIYPYLDDF QKKWQEEMELYRQKVE
57 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
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58 [64C u] YLLD GILF IYP YLDDF QKKWQEEMELYRQKVE
59 IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
60 IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
61 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
63 FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
64 FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
65 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
66 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
67 IVIVDIC IT GPLGEEMRDRARAHVDALRTHLA
68 IVIVDIC IT GP YLDDF QKKWQEEMELYRQKVE
69 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
70 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
71 IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA
72 IAVAMGIRFIIMVAPYLDDF QKKWQEEMELYRQKVE
73 GNLVRICLGAPLGEEMRDRARAHVDALRTHLA
74 GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE
75 [64C u] GNL VRICL GAPL GEEMRDRARAHVD ALRTHL A
76 [64C u] GNL VRICL GAP YLDDF QKKWQEEMELYRQKVE
77 VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA
78 VMGDLVLTVLPYLDDF QKKWQEEMELYRQKVE
79 [64Cu]VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA
80 [64C u] VIVIGDL VL T VLP YLDDF QKKWQEEMELYRQKVE
81 LVAADAVASLPLGEEMRDRARAHVDALRTHLA
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82 LVAADAVASLPYLDDF QKKWQEEMELYRQKVE
83 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
84 [64Cu]LVAADAVASLPYLDDF QKKWQEEMELYRQKVE
85 SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA
86 SIATGMVGALLLLLVVALGIGLFMRPYLDDF QKKWQEEMELYRQKVE
87 DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA
88 DIVKLTVYDCIRRRRRRRRRPYLDDF QKKWQEEMELYRQKVE
89 SLRRS SCF GGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA
90 SLRRS SCF GGRMDRIGAQ SGLGCNSFRYPYLDDF QKKWQEEMELYRQKVE
91 PtxGFL SK SLVFPLGEEMRDRARAHVDALRTHLA
92 PtxGFL SK SLVFPYLDDF QKKWQEEMELYRQKVE
93 PtxGFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
94 PtxGFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
95 IVILLAGGFL SK SLVF SVLF APL GEEMRDRARAHVD ALRTHL A
96 IVILLAGGFL SK SLVF S VLF APYLDDF QKKWQEEMELYRQKVE
TABLE 2B. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions
1 GFLSKSLVFPLGEEMRDRARAHVDALRTHLA
2 GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
3 GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
4 GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD
6 GFLSKSLVFPYLDDFQKKWQEEMELYRQKVERGD
7 [64C u] GF L SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
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8 [64Cu]GFL SKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
9 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
11 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
12 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
13 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
14 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
[64Cu]LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
16 [64Cu]LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
17 MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
18 MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE
19 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
[64Cu]MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE
21 GAR SMTLTVQARQLPLGEEMRDRARAHVDALRTHLA
22 GAR SMTLTVQARQLPYLDDF QKKWQEEMELYRQKVE
23 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
24 [64Cu]GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE
GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
26 GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE
27 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
28 [64Cu]GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE
29 LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE
31 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
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32 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
33 YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
34 YLLDGILFIYPYLDDF QKKWQEEMELYRQKVE
35 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
36 [64Cu]YLLDGILFIYPYLDDF QKKWQEEMELYRQKVE
37 IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
38 IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
39 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
40 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
41 FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
42 FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
43 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
44 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
45 IVIVDIC IT GPLGEEMRDRARAHVD ALRTHLA
46 IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
47 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
48 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
49 IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA
50 IAVAMGIRFIIMVAPYLDDF QKKWQEEMELYRQKVE
51 GNLVRICLGAPLGEEMRDRARAHVDALRTHLA
52 GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE
53 [64Cu]GNLVRICLGAPLGEEMRDRARAHVDALRTHLA
54 [64Cu]GNLVRICLGAPYLDDF QKKWQEEMELYRQKVE
55 VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA
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56 VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE
57 [64C1.1]VMGDLVLTVLPLGEEIVIRDRARAHVDALRTHLA
58 [64CU]VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE
59 LVAADAVASLPLGEEMRDRARAHVDALRTHLA
60 LVAADAVASLPYLDDFQKKWQEEMELYRQKVE
61 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]LVAADAVASLPYLDDFQKKWQEEMELYRQKVE
63 SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA
64 SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE
65 DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA
66 DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE
67 SLRRSSCFGGRMDRIGAQSGLGCNSFRYPLGEEMRDRARAHVDALRTHLA
68 SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE
69 Ptx-GFLSKSLVFPLGEEMRDRARAHVDALRTHLA
70 Ptx-GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
71 Ptx-GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
72 Ptx-GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
73 IVILLAGGFLSKSLVFSVLFAPLGEEMRDRARAHVDALRTHLA
74 IVILLAGGFL SKSLVF S VLF AP YLDDF QKKWQEEMEL YRQKVE
TREM-1 inhibitory trifunctional SCHOOL peptides
In certain embodiments, the present invention relates to amphipathic TREM-1
inhibitory
trifunctional peptides and therapeutic compositions comprising such
trifunctional peptides for
use in treating cancer in combination with other cancer therapies. In one
embodiment, these
peptides may possess the antitumor activity. In one embodiment, these peptides
may not possess
the antitumor activity.
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In some embodiments, each trifunctional peptide is capable of at least three
functions: 1)
mediating formation of naturally long half-life lipopeptide/lipoprotein
particles upon interaction
with lipoproteins, 2) facilitation of the targeted delivery to cells of
interest and/or sites of disease,
and 3) treatment, prevention, and/or detection of a disease or condition. In
some embodiments,
each trifunctional peptide is capable of at least three functions: 1)
mediating the self-assembly of
naturally long half-life lipopeptide particles upon binding to lipid or lipid
mixtures, 2) facilitation
of the targeted delivery to cells of interest and/or sites of disease, and 3)
treatment, prevention,
and/or detection of a disease or condition. In certain embodiments, the
present invention relates
to amphipathic trifunctional peptides consisting of two amino acid domains,
wherein upon
interaction with plasma lipoproteins, one amino acid domain mediates formation
of naturally
long half-life lipopeptide/lipoprotein particles and targets these particles
to macrophages,
whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor
signaling complex
expressed on myeloid cells including but not limited to, macrophages.
In one embodiment, the TREM-1 inhibitory trifunctional SCHOOL peptides
(TRIOPEPs)
of the present invention form self-assembling SLP in vitro. In one embodiment,
TRIOPEPs are
incorporated into self-assembled nanosized SLP of discoidal or spherical
morphology (dSLP and
sSLP, respectively) that contain apo A-I peptide fragments comprising 22 amino
acid residue-
long peptide sequences of the apo A-I helix 4 and/or helix 6. In one
embodiment, the TREM-1
inhibitory trifunctional SCHOOL peptides described herein form naturally long
half life
lipopeptide particles in vivo. In certain embodiments, the present invention
relates to peptides
consisting of two amino acid domains, wherein upon binding to lipid or lipid
mixtures, one
amino acid domain assists in the self-assembly of naturally long half-life
lipopeptide particles
and targets these particles to macrophages, whereas another amino acid domain
inhibits TREM-
1/DAP-12 receptor complex expressed on macrophages.
In some embodiments of the present inventions, TABLE 3 presents a list of the
peptides
and therapeutic compositions that includes, but is not limited to the
trifunctional SCHOOL
peptide-based TREM-1 inhibitors and therapeutic compositions that can be used
in order to treat
tumors in combinations with other cancer therapies or to predict response of
the subject to the
treatment by using the modulators of TREM-1/DAP-12 signaling pathway in
combination-
therapy regiment.
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Exemplary TREM-1 inhibitory trifunctional SCHOOL peptides include but are not
limited to, 31 amino acid-long peptide TREM-1 inhibitory peptides GA31
(GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID
NO. 26) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine
sulfoxide) (SEQ ID NO. 27). In one embodiment, methionine residues of the
peptides GE31
(GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and GA31
(GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. See
TABLE 3.
In one embodiment, any or both the domains comprise minimal biologically
active amino
acid sequence. In one embodiment, the peptide variant comprises a cyclic
peptide sequence. In
one embodiment, the peptide variant comprises a disulfide-linked dimer. In one
embodiment, the
peptide variant includes amino acids selected from the group of natural and
unnatural amino
acids including, but not limited to, L-amino acids, or D-amino acids.
In one embodiment, one or both amino acid domains of the peptides and
compounds of
the present invention are conjugated to a drug compound (TA). In one
embodiment, TA is
selected from the group including, but not limited to, anticancer,
antibacterial, antiviral,
autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, and
therapeutic
peptides. In one embodiment, the TA is a hydrophobic therapeutic agent. The TA
may also be
selected from the group comprising paclitaxel, valrubicin, doxorubicin,
taxotere, campotechin,
etoposide, and any combination thereof.
In one embodiment, one or both amino acid domains of the peptides and
compounds of
the present invention are conjugated to an imaging probe. In one embodiment,
the imaging agent
is GBCA for MM. In one embodiment, the imaging agent is a [64Cu]-containing
imaging probe
for imaging systems such as a PET imaging system and combined PET/CT and
PET/MRI
systems.
In one embodiment, an imaging probe and/or an additional TA is conjugated to
any or
both of the domains. In one embodiment, the peptides and compounds of the
present invention
are used in combinations thereof.
Embodiments Of TREM-1 inhibitory SCHOOL peptides.
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Normal transmembrane interactions between the TREM-1 and the DAP-12 dimer
forming a functional TREM-1/DAP-12 receptor complex comprise positively
charged lysine
amino acid within the TREM-1 transmembrane portion and negatively charged
aspartic acid
pairs in a DAP-12 dimer, thereby allowing subunit association (See FIG. 49).
In one embodiment, the simplest TREM-1 inhibitory SCHOOL agents would be
synthetic
peptides and their variants (SCHOOL peptides) that correspond to the TREM-1
and/or DAP-12
transmembrane domains or their functionally important minimal protein
sequences as disclosed
in US 8,513,185, US 9,981,004 and US 20190117725. Although it is not necessary
to understand
the mechanism of an invention, it is believed that interactions between a
lysine residue of
SCHOOL peptides that correspond to the TREM-1 transmembrane domain or its
functionally
important minimal protein sequence and an aspartic acid residue of a DAP-12
dimer disrupt the
interactions between TREM-1 and DAP-12 in the membrane, thereby
"disconnecting" TREM-1
and resulting in a non-functioning receptor. Accordingly, it is believed that
interactions between
an aspartic residue of SCHOOL peptides that correspond to the DAP-12
transmembrane domain
or its functionally important minimal protein sequence and lysine amino acid
residue of the
TREM-1 transmembrane domain disrupt the interactions between DAP-12 and TREM-1
in the
membrane, thereby "disconnecting" DAP-12 and resulting in a non-functioning
receptor. These
peptide variants and compositions possess the advantages typically associated
with a fully
synthetic material and yet possess certain desirable features of materials
derived from natural
sources.
In some embodiments of the present inventions, TABLE 3 presents a list of the
peptides
and therapeutic compositions that includes, but is not limited to the SCHOOL
peptide-based
TREM-1 inhibitors and their variants that can be designed as disclosed in US
8,513,185, US
9,981,004 and US 20190117725 and used in order to treat tumors in combinations
with other
cancer therapies or to predict response of the subject to the treatment by
using the modulators of
TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
In some embodiments, the SCHOOL peptides and their variants that inhibit TREM-
1
transmembrane signaling can be used in a free form. Exemplary TREM-1
inhibitory SCHOOL
peptides include but are not limited to, a 9 amino acid-long peptide TREM-1
inhibitory peptide
GF9 (GFLSKSLVF) disclosed in US 8,513,185, US 9,981,004 and US 20190117725 and
described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017,
Rojas et al. 2018).
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Although it is not necessary to understand the mechanism of an invention, it
is believed that free
SCHOOL peptide self-inserts into the cell membrane from outside the cell, co-
localizes with
TREM-1/DAP-12 receptor complex and disrupts the protein-protein interactions
between
TREM-1 and DAP-12, thereby resulting in a non-functional receptor complex that
does not
provide TREM-1 transmembrane signaling upon binding to a putative TREM-1
ligand(s) (See
FIG. 49, Route 1). In one embodiment, FIG. 50 demonstrates colocalization of
GF9 with the
TREM-1 in the cell membrane. These peptide variants and compositions possess
the advantages
typically associated with a fully synthetic material and yet possess certain
desirable features of
materials derived from natural sources.
As described in (Vlieghe et al. 2010, Lau et al. 2018), the main limitations
generally
attributed to therapeutic peptides are: a short half-life because of their
rapid degradation by
proteolytic enzymes of the digestive system and blood plasma; rapid removal
from the
circulation by the liver (hepatic clearance) and kidneys (renal clearance);
poor ability to cross
physiological barriers because of their general hydrophilicity; high
conformational flexibility,
resulting sometimes in a lack of selectivity involving interactions with
different receptors/targets
(poor specific biodistribution), causing activation of several targets and
leading to side effects;
eventual risk of immunogenic effects; and high synthetic and production costs
(the production
cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500
Da molecular
mass small molecule by more than 10-fold but clearly not 100-fold).
In some embodiments of the present invention, the SCHOOL peptides and their
variants
that inhibit TREM-1 transmembrane signaling can be formulated into self-
assembling SLP of
discoidal (sSLP) or spherical (sSLP) shape that mimic human naturally long
half-life high
density lipoproteins (HDL) and are disclosed in US 20130045161 and US
20110256224 and
described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017,
Rojas et al. 2018,
Tornai et al. 2019). Although it is not necessary to understand the mechanism
of an invention, it
is believed that these particles provide targeted delivery of the incorporated
SCHOOL peptides
to target cells and increase half life of these peptides in circulation. In
some embodiments, these
SLP contain the modified amphipathic apolipoprotein A-I peptide fragments that
not only assist
in the self-assembly of SLP but also provide targeted delivery of these
particles to target cells in
vitro and in vivo. In some embodiments, the modification represents a
sulfoxidation of
methionine amino acid residue in the apo A-I peptide sequence.
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In one embodiment, FIG. 49 presents a schematic representation of targeted
delivery of
the TREM-1 modulatory SCHOOL peptides by SLP to myeloid cells including but
not limited
to, macrophages including TAMs. Although it is not necessary to understand the
mechanism of
an invention, it is believed that SLP that contain TREM-1 modulatory SCHOOL
peptides
(exemplary shown for GF9) are endocytosed by macrophages through scavenger
receptor(s), and
then release the incorporated SCHOOL peptide, which self-inserts into the cell
membrane from
inside the cell, co-localizes with TREM-1/DAP-12 receptor complex and disrupts
the protein-
protein interactions between TREM-1 and DAP-12, thereby resulting in a non-
functional
receptor complex that does not induce TREM-1 transmembrane signaling upon
binding to a
putative TREM-1 ligand(s) (See FIG. 49, Route 2).
Modulators of TREM-1/DAP-12 signaling pathway.
Modulators (inhibitors) of TREM-1/DAP-12 signaling pathway can be
nonexclusively
divided into two major categories: those that inhibit TREM-1 transmembrane
signaling by
blocking binding of TREM-1 to its ligand(s) (type I inhibitors; See FIG. 49)
and those that
employ a ligand binding-independent mechanism of action and modulate (inhibit)
TREM-1-
mediated transmembrane signaling by disrupting protein-protein interactions
between TREM-1
and DAP-12 in the cell membrane (type II inhibitors; See FIG. 50). Type I
inhibitors can be, in
turn, subdivided into two subtypes: those that bind to TREM-1 (type Ia
inhibitors) and those that
bind to TREM-1 ligand(s) (type lb inhibitors).
Type I TREM-1 inhibitors.
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited
to,
antagonistic (blocking, inhibiting) anti-TREM-1 antibodies and/or their
fragments such as
antibodies that block and inhibit TREM-1 disclosed in US 9,000,127 and US
9,550,830 and
described in (Brynjolfsson et al. 2016). These TREM-1 inhibitors are believed
to block binding
of TREM-1 to its ligand(s) by binding to the extracellular domain of TREM-1
(type Ia inhibitors,
See FIG. 49).
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited
to,
synthetic peptides derived from a part of the extracellular domain of either
TREM-1 such as Pl,
P3 and LP17 peptides disclosed in US 20160193288, US 20150232531, US 8,013,836
and US
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9,273,111 and described in (Gibot et al. 2004, Gibot et al. 2006) or the TREM-
like transcript-1
(TLT-1) such as LR17 and LR12 peptides disclosed in US 20160193288, US
20160015773, US
20150232531, US 9,255,136; US 9,657,081 and US 9,815.883 and described in
(Derive et al.
2012). These TREM-1 inhibitors are believed to act as an endogenous decoy
receptor (type lb
inhibitors, See FIG. 1) by binding TREM-1 ligands and preventing their
engagement to
membrane-bound TREM-1 (Pelham et al. 2014).
In some embodiments of the present invention, the TREM-1 type I inhibitors can
be used
in order to treat tumors in combinations with other cancer therapies or to
predict response of the
subject to the treatment by using the modulators of TREM-1/DAP-12 signaling
pathway in
combination-therapy regiment.
In some embodiments of the present inventions, TABLE 3 presents a list of the
peptides
and peptide analogues that includes, but is not limited to the TREM-1 type lb
peptide inhibitors
and their variants that can be designed as disclosed in US 20160193288, US
20150232531, US
8,013,836, US 9,273,111, US 20160015773, US 9,255,136; US 9,657,081 and US
9,815.883 and
described in (Gibot et al. 2004, Gibot et al. 2006, Derive et al. 2012) and
used in order to treat
tumors in combinations with other cancer therapies.
Type II TREM-1 inhibitors..
Application of the Signaling Chain HOmoOLigomerization (SCHOOL) model of
receptor signaling described in (Sigalov et al. 2004, Sigalov 2004, Sigalov
2006, Sigalov 2018)
to the transmembrane signal transduction mediated by a TREM-1 receptor
suggested that an
inhibition of TREM-1/DAP-12 signaling may be achieved by using transmembrane-
targeted
agents (SCHOOL agents) which specifically disrupt interactions between TREM-1
and DAP-12
subunits in the cell membrane (See FIG. 2), thereby disconnecting TREM-1 and
DAP-12 and
.. resulting in a non-functioning TREM-1/DAP-12 receptor complex.
In some embodiments of the present invention, the TREM-1 type II inhibitors
can be
used in order to treat tumors in combinations with other cancer therapies or
to predict response of
the subject to the treatment by using the modulators of TREM-1/DAP-12
signaling pathway in
combination-therapy regiment.
As described in (Tammaro et al. 2017), although TREM-1 appears to be activated
by
damage associated molecular patterns (DAMPs) that are shared by other pattern
recognition
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receptors (PRRs), no TREM-1 specific (endogenous) ligand has been discovered
to date. It is
unknown why these ligands, specifically, share TREM-1 activation. Neither it
is known what
they have in common, but this information could certainly be of use in the
determination of new
specific ligands. This makes ligand binding-independent type II TREM-1
inhibitors
advantageous compared to type I inhibitors that attempt to block binding TREM-
1 to its yet
unknown ligand(s).
In some embodiments, type II TREM-1 inhibitors include but are not limited to,
the
TREM-1 inhibitory SCHOOL peptides. The preferred peptides and compositions of
the present
invention comprise the TREM-1 modulatory peptide sequences designed using the
SCHOOL
.. model of TREM-1 signaling and capable of modulating TREM-1 receptor
expressed on myeloid
cells as disclosed in US 8,513,185 and US 9,981,004 and described in (Sigalov
2010, Shen and
Sigalov 2017).
Listed below in TABLE 2 are reported transmembrane sequences of TREM-1 and DAP-
12 in a number of species. These regions are highly conserved and the
substitutions between
species are very conservative. This suggests a functional role for the
transmembrane regions of
both, TREM-1 and DAP-12, constituents of the complex. These regions strongly
interact
between themselves, thus maintaining the integrity of the TREM-1/DAP-12
receptor signaling
complex in resting cells. These transmembrane domains are short and should be
easily mimicked
by synthetic peptides and compounds. In some embodiments, synthetic peptides
and compounds
.. are contemplated that may provide successful treatment options in the
clinical setting.
TABLE 2C Sequence comparison of TREM-1 and DAP-12 transmembrane regions
(accession
codes are given in parenthesis).
SEQUENCE
SPECIES
TREM-1 DAP-12
HUMAN IVILLAGGFL SKSLVF S VLF A GVLAGIVMGDLVLTVLIALAV
(Q9NP99) (043914)
MOUSE VTI S VIC GLL SKSLVF IILF I GVLAGIVLGDLVLTLLIALAV
(Q9JKE2) (054885)
BOVIN IIIPAACGLL SKTLVFIGLF A GVLAGIVLGDLMLTLLIALAV
(Q6QUN5) (Q95J79)
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SHEEP not known GVLAGIVLGDLMLTLLIALAV
(Q95KS5)
RAT not known GVLAGIVLGDLVLTLLIALAV
(Q6X9T7)
PIG ILPAVCGLLSKSLVFIVLFVV GILAGIVLGDLVLTLLIALAV
(Q6TYI6) (Q9TU45)
CLUSTAL W 2.0 multiple sequence alignment:
HUMAN IVILLAGGFLSKSLVFSVLFA- 21 GVLAGIVMGDLVLTVLIALAV 21
MOUSE VTISVICGLLSKSLVFIILFI- 21 GVLAGIVLGDLVLTLLIALAV 21
BOVIN IIIPAACGLLSKTLVFIGLFA- 21 GVLAGIVLGDLMLTLLIALAV 21
SHEEP GVLAGIVLGDLMLTLLIALAV 21
RAT GVLAGIVLGDLVLTLLIALAV 21
PIG 21 GILAGIVLGDLVLTLLIALAV 21
ILPAVCGLLSKSLVFIVLFVV
*:***:*** ** *:*****:***:**:******
TABLE 3. Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and
Compositions.
##
Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions
1 IVILLAGGFLSKSLVFSVLFA
2 GFLSKSLVF
3 (GFLSKSLVF)2
4 GLLSKSLVF
GVLAGIVMGDLVLTVLIALAV
6 GIVMGDLVLT
7 IVMGDLVLT
8 LQEEDAGEYGCM
9 LQVTDSGLYRCVIYHPP
264
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GFL SK SLVF GEEMRDRARAHV
11 GFL SK SLVF GEEM(0)RDRARAHV
12 GFL SK SLVFWQEEMELYRQKV
13 GFL SK SLVFWQEEM(0)ELYRQKV
14 GFL SR SLVF GEEMRDRARAHV
GFL SR SL VF GEEM(0)RDRARAHV
16 GFL SRSLVFWQEEMELYRQKV
17 GFL SRSLVFWQEEM(0)ELYRQKV
18 GLL SK SLVF GEEMRDRARAHV
19 GLL SK SLVFGEEM(0)RDRARAHV
GLL SK SLVFWQEEMELYRQKV
21 GLL SK SLVFWQEEM(0)ELYRQKV
22 GFL SK SLVF GEEMRDRARAHVRGD
23 GFL SK SLVFWQEEMELYRQKVRGD
24 GFL SK SL VF PL GEEMRDRARAHVD ALRTHL A
GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
28 GFL SK SLVFPLGEEMRDRARAHVDALRTHLARGD
29 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVERGD
[64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
32 GFL SK SL VF PL GEEMRDRARAHVD ALRTHL A
33 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
265
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34 GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
36 GFL SK SLVFPLGEEMRDRARAHVDALRTHLARGD
37 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVERGD
38 [64Cu]GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
39 [64Cu]GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
40 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
41 LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
42 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
43 [64Cu]LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
44 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
45 LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
46 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
47 [64Cu]L QVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
48 LQEEDAGEYGCMGEEM(0)RDRARAHV
49 LQEEDAGEYGCMWQEEM(0)ELYRQKV
50 IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
51 IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
52 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
53 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
54 IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
5 IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
56 [64Cu]IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
57 [64Cu]IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
266
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58 PtxGFLSKSLVFPLGEEMRDRARAHVDALRTHLA
59 PtxGFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
60 PtxGFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
61 PtxGFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
62 IVILLAGGFLSKSLVFSVLFAPLGEEMRDRARAHVDALRTHLA
63 IVILLAGGFLSKSLVFSVLFAPYLDDFQKKWQEEMELYRQKVE
64 IVILLAGGFLSKSLVFSVLFAPLGEEM(0)RDRARAHVDALRTHLA
65 IVILLAGGFLSKSLVFSVLFAPYLDDFQKKWQEEM(0)ELYRQKVE
TABLE 3 - Continuted
## Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and
Compositions
1 IVILLAGGFLSKSLVFSVLFA
2 GFLSKSLVF
3 (GFLSKSLVF)2
4 GLLSKSLVF
GVLAGIVMGDLVLTVLIALAV
6 GIVMGDLVLT
7 IVMGDLVLT
8 LQEEDAGEYGCM
9 LQVTDSGLYRCVIYHPP
GFLSKSLVFGEEMRDRARAHV
11 GFLSKSLVFGEEM(0)RDRARAHV
12 GFLSKSLVFWQEEMELYRQKV
13 GFLSKSLVFWQEEM(0)ELYRQKV
14 GFLSRSLVFGEEMRDRARAHV
267
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15 GFL SR SL VF GEEM(0)RDRARAHV
16 GFL SRSLVFWQEEMELYRQKV
17 GFL SRSLVFWQEEM(0)ELYRQKV
18 GLL SK SLVF GEEMRDRARAHV
19 GLL SK SLVFGEEM(0)RDRARAHV
20 GLL SK SLVFWQEEMELYRQKV
21 GLL SK SLVFWQEEM(0)ELYRQKV
22 GFL SK SLVF GEEMRDRARAHVRGD
23 GFL SK SLVFWQEEMELYRQKVRGD
24 GFL SK SL VF PL GEEMRDRARAHVD ALRTHL A
25 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
28 GFL SK SLVFPLGEEMRDRARAHVDALRTHLARGD
29 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVERGD
30 [64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
32 GFL SK SL VF PL GEEMRDRARAHVD ALRTHL A
33 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
34 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
36 GFL SK SLVFPLGEEMRDRARAHVDALRTHLARGD
37 GFL SK SLVFPYLDDFQKKWQEEMELYRQKVERGD
38 [64C u] GF L SK SL VF PL GEEM (0)RDRARAHVD ALRTHL A
268
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