U.S. patent application number 16/342146 was filed with the patent office on 2019-08-22 for nanoparticles having molecules that bind or block pd-l1 and uses in treating cancer.
The applicant listed for this patent is Emory University. Invention is credited to Erica Bozeman, Lily Yang.
Application Number | 20190256573 16/342146 |
Document ID | / |
Family ID | 61906049 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190256573 |
Kind Code |
A1 |
Yang; Lily ; et al. |
August 22, 2019 |
Nanoparticles Having Molecules That Bind or Block PD-L1 and Uses In
Treating Cancer
Abstract
This disclosure relates to nanoparticles comprising a surface
molecule that binds or blocks PD-L1. In certain embodiments, the
disclosure relates to methods of using peptides or nanoparticles
disclosed herein for the treatment of cancer. In certain
embodiments, the disclosure relates to methods of using
nanoparticles disclosed herein for therapeutic and diagnostic
applications.
Inventors: |
Yang; Lily; (Atlanta,
HA) ; Bozeman; Erica; (North Wales, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emory University |
Atlanta |
GA |
US |
|
|
Family ID: |
61906049 |
Appl. No.: |
16/342146 |
Filed: |
October 16, 2017 |
PCT Filed: |
October 16, 2017 |
PCT NO: |
PCT/US2017/056810 |
371 Date: |
April 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62408141 |
Oct 14, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0093 20130101;
A61K 38/4886 20130101; A61K 47/6939 20170801; C07K 14/70521
20130101; A61K 45/06 20130101; C12Y 304/21073 20130101; A61K 31/405
20130101; A61K 31/405 20130101; A61K 51/08 20130101; A61K 2300/00
20130101; A61K 31/4245 20130101; A61K 49/0002 20130101; A61P 35/02
20180101; C07K 14/70596 20130101; A61K 47/62 20170801; A61K 38/177
20130101; A61K 49/1866 20130101; B82Y 5/00 20130101; G01N 33/574
20130101; A61K 38/482 20130101; A61K 31/7088 20130101; C12Y
304/2408 20130101; A61K 47/6929 20170801; C07K 2319/21 20130101;
A61K 31/4245 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61K 51/08 20060101 A61K051/08; A61K 47/62 20060101
A61K047/62; A61K 47/69 20060101 A61K047/69; G01N 33/574 20060101
G01N033/574; A61P 35/02 20060101 A61P035/02; A61K 49/00 20060101
A61K049/00; A61K 49/18 20060101 A61K049/18 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under
R01CA202846-01, R01CA154129A-01, U01CA151810-02, and F32CA189633-01
all awarded by the NIH. The government has certain rights in the
invention.
Claims
1. A peptide comprising NWNRLSPSNQTEKQAAP (SEQ ID NO: 8) or
variants thereof and CGAISLHPKAKIEE (SEQ ID NO: 9) or variants
thereof.
2. The peptide of claim 1, wherein the variant of SEQ ID NO: 8 has
at least 70 or 80 percent sequence identity.
3. The peptide of claim 1, wherein the variant of SEQ ID NO: 8 has
up to 3 amino acid substitutions, deletions, and/or additions.
4. The peptide of claim 1, wherein the variant of SEQ ID NO: 9 has
at least 80 percent sequence identity.
5. The peptide of claim 1, wherein the variant of SEQ ID NO: 9 has
up to 2 amino acid substitutions, deletions, and/or additions.
6. The peptide of claim 1, wherein peptide comprises SEQ ID NO:
2.
7. A nanoparticle comprising the peptide of claim 1.
8. The nanoparticle of claim 7, further comprising conjugated ATF
or ATF-MMP14 and/or a chemotherapy agent.
9. The nanoparticle of claim 7, further comprising a
Indoleamine-2,3-dioxygenase (IDO) inhibitor such as indoximod or
epacadostat.
10. A pharmaceutical composition comprising a nanoparticle of claim
7 and a pharmaceutically acceptable excipient.
11. A method of treating cancer comprising administering an
effective amount of a nanoparticle of claim 7 to a subject in need
thereof.
12. The method of claim 11, wherein the cancer is carcinoma,
lymphoma, blastoma, sarcoma, and leukemia, non-small cell lung,
squamous cell, small-cell lung, peritoneum, hepatocellular,
gastrointestinal, pancreatic, glioma, cervical, ovarian, liver,
bladder, hepatoma, breast, colon, colorectal, endometrial or
uterine, salivary gland, kidney, liver, prostate, vulval, thyroid,
hepatic, leukemia and other lymphoproliferative disorders, and
various types of head and neck.
13. A nucleic acid sequence comprising a sequence encoding the
peptide (SEQ ID NO: 8 or 9) from claim 1.
14. A vector comprising a nucleic acid sequence of claim 13.
15. A cell comprising a vector of claim 14.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/408,141 filed Oct. 14, 2016. The entirety of
this application is hereby incorporated by reference for all
purposes.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA
THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)
[0003] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is 16049PCT_ST25.txt. The
text file is 15 KB, was created on Oct. 16, 2017, and is being
submitted electronically via EFS-Web.
BACKGROUND
[0004] Cancer treatment is typically approached by a combination of
surgery along with chemotherapy and/or radiation. These approaches
often neglect to eliminate completely small metastatic tumors.
PD-L1 is expressed in tumor cells and tumor associated stromal
cells, such as fibroblasts and macrophages. Preclinical and
clinical studies have investigated the efficacy of monoclonal
antibody therapies that act as immune checkpoint blockades in
multiple cancer types. FDA approval of immune checkpoint blocking
related therapeutic antibodies include ipilimumab (anti-CTLA-4) for
melanoma, nivolumab (anti-PD-1) for melanoma, NSCLC and RCC, and
pembrolizumab (anti-PD-1) for melanoma and NSCLC. Several cancers
including pancreatic cancer are generally non-responsive to these
therapies. Thus, there remains a need to develop improved
therapeutic approaches.
[0005] Bozeman et al. report targeted chemotherapy delivery using
theranostic nanoparticles and PD-L1 blockade in an orthotopic mouse
pancreatic cancer model. In: Proceedings of the AACR Special
Conference: Tumor Immunology and Immunotherapy: A New Chapter;
December 1-4, 2014; Orlando, Fla. Philadelphia (Pa.): AACR; Cancer
Immunol Res 2015; 3(10 Suppl): Abstract nr A60. See also Zhou et
al. IGF1 Receptor Targeted Theranostic Nanoparticles for Targeted
and Image-Guided Therapy of Pancreatic Cancer. ACS Nano, 2015,
9(8):7976-91. Bombelli et al. Nanoparticle therapies for future
metastatic melanoma treatment. Lancet Oncol. 2014, 15(1):e22-32.
Yang E, et al. Theranostic Nanoparticles Carrying Doxorubicin
Attenuate Targeting Ligand Specific Antibody Responses Following
Systemic Delivery. Theranostics, 2015, 5(1):43-61. Huang J, et al.
Casein-coated Iron Oxide Nanoparticles for High MRI Contrast
Enhancement and Efficient Cell Targeting. ACS applied materials
& interfaces, 2013, 5(11):4632-4639. Lee G Y, et al.
Theranostic Nanoparticles with Controlled Release of Gemcitabine
for Targeted Therapy and MRI of Pancreatic Cancer. ACS nano, 2013,
7(3):2078-2089. See WO 2012/031205, WO 2013/0343996,
CN103304638.
[0006] References cited herein are not an admission of prior
art.
SUMMARY
[0007] This disclosure relates to nanoparticles comprising a
surface molecule that binds or blocks PD-L1. In certain
embodiments, the disclosure relates to methods of using peptides or
nanoparticles disclosed herein for the treatment of cancer. In
certain embodiments, the disclosure relates to methods of using
nanoparticles disclosed herein for therapeutic and diagnostic
applications.
[0008] In certain embodiments, the molecule that binds or blocks of
PD-L1 is a peptide comprising or consisting of NWNRLSPSNQTEKQAAP
(SEQ ID NO: 8) or variants thereof and/or CGAISLHPKAKIEE (SEQ ID
NO: 9) or variants thereof. In certain embodiments, the molecule
that binds or blocks of PD-L1 is a peptide comprising or consisting
of NWYRMSPSNQTDKLAA (SEQ ID NO: 12) or variants thereof and/or
CGAISLAPKAQIKE (SEQ ID NO: 13) or variants thereof.
[0009] In certain embodiments, the variant of SEQ ID NO: 8 has at
least 70, 80, 85, 90, or 95% percent sequence identity. In certain
embodiments, the variant of SEQ ID NO: 8 has up to 1 or 2 or 3
amino acid substitutions, deletions, and/or additions. In certain
embodiments, the variant of SEQ ID NO: 9 has at least 80, 85, 90,
or 95% percent sequence identity. In certain embodiments, the
variant of SEQ ID NO: 9 has up to 1 or 2 amino acid substitutions,
deletions, and/or additions.
[0010] In certain embodiments, the variant of SEQ ID NO: 12 has at
least 70, 80, 85, 90, or 95% percent sequence identity. In certain
embodiments, the variant of SEQ ID NO: 12 has up to 1 or 2 or 3
amino acid substitutions, deletions, and/or additions. In certain
embodiments, the variant of SEQ ID NO: 13 has at least 80, 85, 90,
or 95% percent sequence identity. In certain embodiments, the
variant of SEQ ID NO: 13 has up to 1 or 2 amino acid substitutions,
deletions, and/or additions.
[0011] In certain embodiments, the molecule that binds or blocks of
PD-L1 is a peptide comprising or consisting of SEQ ID NO: 1, 2, 3
or variants thereof.
[0012] In certain embodiments, the disclosure relates to targeted
delivery of nanoparticles into tumors mediated by PD-L1 blocking
peptides. In certain embodiments, the disclosure contemplates
nanoparticles comprising PD-1 peptides or fragments or PD-1 like
peptides with dual binding domains that are fusions with a
poly-histidine tag or other heterologous peptide. The peptides may
be conjugated to the nanoparticles through affinity interaction
with NTA-Cu that is covalently linked to an outer polymer,
configured to form ligand-metal complexes with the poly-histidine
tag. The peptides may be conjugated to the nanoparticles through an
outer polymer covalently linked to NTA and a metal configured to
form ligand-metal complexes with the poly-histidine tag.
[0013] In certain embodiments, the disclosure contemplates targeted
delivery of PD-L1 blocking peptides, such as those comprising of
consisting of SEQ ID NO: 8 or variants and/or SEQ ID NO: 9 or
variants, such as SEQ ID NO: 1, 2, 3, or variants, into tumors to
reduce potential systemic effects of blocking the PD-1 and PD-L1
interaction on the regulation of normal immune responses.
[0014] In certain embodiments, the disclosure contemplates methods
of treating cancer comprising administering and effective amount of
a peptide disclosed herein or a nanoparticle comprising a surface
peptide disclosed herein such as those comprising of consisting of
SEQ ID NO: 8 or variants and/or SEQ ID NO: 9 or variants, such as
SEQ ID NO: 1, 2, 3, or variants in combination with a nanoparticle
comprising an amino-terminal fragment (ATF) of uPA wherein the
nanoparticle optionally further comprise a chemotherapy agent to a
subject in need thereof. In certain embodiments, the components are
on a single particle or are contained on two particles, e.g., a
first nanoparticle comprising a surface peptide such as those
comprising or consisting SEQ ID NO: 8 or variants and/or SEQ ID NO:
9 or variants, such as SEQ ID NO: 1, 2, 3, or variants and a second
nanoparticle comprising an amino-terminal fragment (ATF) of uPA or
ATF-MMP14 catalytic domain fusion peptide further comprising a
chemotherapy agent. ATF-Matrix MetalloProtease 14 (MMP14) can be
used to break tumor stromal barrier so that the PD1-like peptide
conjugated nanoparticles can be delivered into tumor center and
bind to PDL-1 expressing tumor and stromal cells. In certain
embodiments, nanoparticles disclosed herein comprising or
consisting of SEQ ID NO: 8 or variants and/or SEQ ID NO: 9 or
variants, such as SEQ ID NO: 1, 2, 3, or variants and further
comprises the amino-terminal fragment (ATF) of uPA or ATF-MMP14 on
the outer surface of the particle.
[0015] In certain embodiments, the components are on a single
particle or are contained on two particles, e.g., a first
nanoparticle comprising a surface peptide comprising or consisting
of SEQ ID NO: 8 or variants and/or SEQ ID NO: 9 or variants, such
as SEQ ID NO: 1, 2, 3, or variants and a second nanoparticle
comprising an amino-terminal fragment (ATF) of uPA further
comprising a chemotherapy agent. In certain embodiments,
nanoparticles disclosed herein comprise a peptide such as those
comprising or consisting of SEQ ID NO: 8 or variants and/or SEQ ID
NO: 9 or variants, such as SEQ ID NO: 1, 2, 3, or variants and
further comprises the amino-terminal fragment (ATF) of uPA on the
outer surface of the particle.
[0016] In certain embodiments, variants of NWNRLSPSNQTEKQAAP (SEQ
ID NO: 8) are selected from NWNRMSPSNQTEKQAAP (SEQ ID NO: 14),
NWNRMSPSNQTDKQAAP (SEQ ID NO: 15), NWNRMSPSNQTDKLAAP (SEQ ID NO:
16), NWNRLSPSNQTEKLAAP (SEQ ID NO: 17), NWNRLSPSNQTDKLAAP (SEQ ID
NO: 18), NWYRMSPSNQTEKQAAP (SEQ ID NO: 19), NWYRMSPSNQTDKQAAP (SEQ
ID NO: 20), NWYRMSPSNQTDKLAAP (SEQ ID NO: 21), NWYRLSPSNQTEKLAAP
(SEQ ID NO: 22), and NWYRLSPSNQTDKLAAP (SEQ ID NO: 23).
[0017] In certain embodiments, variants of CGAISLHPKAKIEE (SEQ ID
NO: 9) are selected from CGAISLAPKAKIEE (SEQ ID NO: 24),
CGAISLAPKAQIEE (SEQ ID NO: 25), CGAISLAPKAQIKE (SEQ ID NO: 26),
CGAISLHPKAKIKE (SEQ ID NO: 27), and CGAISLHPKAQIKE (SEQ ID NO:
28).
[0018] In certain embodiments, the disclosure contemplates a
peptide comprising NWYRMSPSNQTDKLAAPXXXXCGAISLAPKAQIKE (SEQ ID NO:
29), NWYRMSPSNQTDKLAAPXXXCGAISLAPKAQIKE (SEQ ID NO: 30),
NWYRMSPSNQTDKLAAPXXXXXCGAISLAPKAQIKE (SEQ ID NO: 31) or variants
wherein each X is individually and independently at each occurrence
any amino acid, histidine, or glycine.
[0019] In certain embodiments, this disclosure relates to a nucleic
acid sequence that encodes a peptide disclosed herein. In further
embodiments, this disclosure relates to a vector comprising a
nucleic acid sequence that encodes a peptide disclosed herein. In
certain embodiments, this disclosure relates to a cell comprising a
vector comprising a nucleic acid sequence that encodes a peptide
disclosed herein. In certain embodiments, this disclosure relates
to an expression system comprising a vector comprising a nucleic
acid sequence that encodes a peptide disclosed herein.
[0020] In certain embodiments, this disclosure relates to the
isolated peptide comprising or consisting of any of the sequences
disclosed herein such as SEQ ID NO: 1, 2, or 3 or variant, wherein
the variant has at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
92, 95, 98 percent sequence identity or similarity. In certain
embodiments, the variant has one or more or up to 1, 2, 3, 4, 5, 6,
or 7 amino acid substitutions, deletions, and/or additions. In
certain embodiments, the substitution is a conserved substitution.
In certain embodiments, this disclosure relates to the peptide
variant of SEQ ID NO: 1 that is capable of binding or blocking
PD-L1.
[0021] In certain embodiments, the disclosure contemplates that the
peptide disclosed herein comprise or consist of amino acid
sequences, or N-terminal or C-terminal amino acid sequences,
disclosed herein such that the amino acids in the sequence are less
than 150, 100, or 50 amino acids. In certain embodiments, the
disclosure contemplates that the peptide disclosed herein comprise
or consist of amino acid sequences, or N-terminal or C-terminal
amino acid sequences, disclosed herein such that the amino acids in
the sequence are less than 45 or 40 or 35 amino acids.
[0022] In certain embodiments, this disclosure relates to a
nanoparticle comprising a peptide disclosed herein wherein the
nanoparticle comprises 20 to 30 or 10 to 40 or 10 to 60 or 10 to
100 of the peptide moieties bound to the exterior of the particle.
In certain embodiments, the nanoparticle comprises
copper-nitrilotriacetate complexes (NTA-Cu) and the peptide
comprises a poly-histidine sequence wherein the peptide is bound to
the particle by a complex of the poly histidine and copper complex.
In certain embodiments, this disclosure relates to a nanoparticle
comprising a core comprising metallic nanoparticles, such as iron
oxide, gold, or silver, or polymeric nanoparticles. In certain
embodiments, the core has an average diameter of 3 to 200 nm. In
certain embodiments, the core has an average diameter of 4 and 10
or 3 to 20 or 3 to 50 nm.
[0023] In certain embodiments, nanoparticles disclosed herein
further comprise conjugated ATF or ATF-MMP14 and/or a chemotherapy
agent. In certain embodiments, nanoparticles disclosed herein
further comprise a indoleamine-2,3-dioxygenase (IDO) inhibitor such
as indoximod or epacadostat.
[0024] In certain embodiments, this disclosure relates to a
pharmaceutical composition comprising a peptide disclosed herein or
a nanoparticle disclosed herein and a pharmaceutically acceptable
excipient. In further embodiments, this disclosure relates to a
pharmaceutical composition, in an aqueous phosphate buffer
solution. In certain embodiments, this disclosure relates to a
pharmaceutical composition in the form of a pill, capsule, tablet,
cream, or aerosol.
[0025] In certain embodiments, this disclosure relates to a method
of treating cancer comprising administering an effective amount of
a peptide disclosed herein or a nanoparticle comprising a peptide
disclosed herein or a peptide disclosed herein, e.g., a peptide
such as those comprising SEQ ID NO: 8 or variants and/or SEQ ID NO:
9 or variants, such as SEQ ID NO: 1, 2, 3, or variants, to a
subject in need thereof.
[0026] In certain embodiments, this disclosure relates to a method
of treating cancer comprising administering an effective amount of
a peptide disclosed herein or a nanoparticle comprising a peptide
disclosed herein or a peptide disclosed herein, e.g., a peptide
such as those comprising SEQ ID NO: 12 or variants and/or SEQ ID
NO: 13 or variants, such as SEQ ID NO: 1, 2, 3, or variants, to a
subject in need thereof.
[0027] In certain embodiments, the cancer is mediated by PD-L1. In
certain embodiments, the cancer is selected from carcinoma,
lymphoma, blastoma, sarcoma, and leukemia, non-small cell lung,
squamous cell, small-cell lung, peritoneum, hepatocellular,
gastrointestinal, pancreatic, glioma, cervical, ovarian, liver,
bladder, hepatoma, breast, colon, colorectal, endometrial or
uterine, salivary gland, kidney, liver, prostate, vulval, thyroid,
hepatic, leukemia and other lymphoproliferative disorders, and
various types of head and neck. In certain embodiments, the cancer
can be primary or metastatic tumors.
[0028] In further embodiments, this disclosure relates to methods
of treating cancer further comprising administering a second
nanoparticle and/or chemotherapy agent, additional
immunomodulators, or indoleamine 2,3-dioxygenase (IDO) inhibitors,
to the subject.
[0029] In certain embodiments, this disclosure relates to a method
for cancer diagnosis comprising administering an effective amount
of a peptide disclosed herein or nanoparticle disclosed herein to a
subject in need thereof and detecting the particle about the area
of a cancerous cell or tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1A illustrates PD-1 protein partial sequences (SEQ ID
NO: 10 and SEQ ID NO: 11) and two domains in the PD-1 like peptide
having SEQ ID NO: 8 and SEQ ID NO: 9.
[0031] FIG. 1B illustrated two PD-1 like peptides. Peptide PD-1 (Y)
has SEQ ID NO: 2. Peptide PD-1 (Lin) has SEQ ID NO: 3.
[0032] FIG. 2 illustration the PD-1 like peptides conjugated to
polymeric coated nanoparticle (NP).
[0033] FIG. 3A shows data from a competition-binding assay. 2 .mu.g
of FITC labeled PD-1 peptides were mixed with 0.5. 20 .mu.g of
anti-mouse PD-L1 antibody and then incubated with the KC cells for
2 hours. Unbound peptides and antibodies were washed off. Relative
fluorescent intensity of KC cells was measured for each group.
[0034] FIG. 3B shows data from a PD-1 peptide pull-down assay. PD-1
Ling or PD-1 Y peptides were conjugate to NTA-Ni beads and added to
tumor cell lysates for 2 to 3 hrs. The beads were spin-down
recovered. Protein fraction from the beads was examined by Western
blot to identify PD-L1 protein that was pulled down by PD-1
peptides.
[0035] FIG. 4A illustrates a treatment protocol. Mouse Panc02
pancreatic cancer model was used.
[0036] FIG. 4B shows Prussian blue staining of frozen tumor tissue
sections. Dots show IONP positive cells.
[0037] FIG. 5 shows Prussian blue staining showed PD-1Y conjugated
IONPs bound to primary cultures of human pancreatic cancer cells
derived from a human pancreatic cancer patient derived tumor
xenograft.
[0038] FIG. 6 data from flow cytometry analysis of CD4 and CD8 TIL
cells isolated from s.c. pancreatic tumors following systemic
deliveries of PD-1 Lin-IONP or PD-1 Y-IONP.
[0039] FIG. 7A illustrates a treatment protocol. Three i.v.
injections of 100 .mu.g/injection of anti-PD-L1 antibody or
equivalent amount of PD-1Y-IONP.
[0040] FIG. 7B shows data on tumor growth curves during
treatment.
[0041] FIG. 7C shows data on mean tumor weight of each treatment
group.
[0042] FIG. 8 shows data on the evaluation of pancreatic cancer
specific T cell cyto-toxicity in the KC mouse pancreatic cancer
model. Mice bearing orthotropic KC tumors received three i.p.
deliveries of PD-1 Lin-IONP or PD-1 Y-IONP once every 3 days. CD3+
T cells were isolated from splenocytes and co-cultured with the KC
cells for 72 hours. AlarmaBlue.TM. cell proliferation assay was
used to determine the percentage of viable cells in each group.
O.D. value from the KC cell culture without the addition of CD3+ T
cells is used as 100%.
[0043] FIG. 9A illustration of uPAR targeted ATF-IONP-Cisplatin and
treatment protocol. The combination of uPAR-targeted delivery of
theranostic IONPs with immune checkpoint blocking using an
anti-PD-L antibody enhanced therapeutic response in the Panc02
mouse pancreatic tumor model.
[0044] FIG. 9B shows data on mean tumor weight of each treatment
group following four treatments.
[0045] FIG. 9C shows data on mean volume of ascites in tumor
bearing mice following different treatments.
[0046] FIG. 10 shows data on targeted delivery of theranostic IONPs
into tumors promoted infiltration of CD8+ cytotoxic T cells into
pancreatic tumor tissues. Pdx1-Cre;LSL-K-rasG12D (KC) mouse
pancreatic cancer cell line derived orthotopic tumor.
Immunofluorescence labeling of T cells. Red fluorescence labeling
are CD3 and CD8+ T cells. Pdx1-Cre;LSL-K-rasG12D (KC) mouse
pancreatic cancer cell line derived orthotopic tumor. Tumor bearing
mice received 4.times. i.v. deliveries of the nanoparticles
PD-1Y-IONP and/or AFTmmp14-IONP-Dox. Bar figure shows the
quantification result of CD3 and CD8 cells in tumor tissue
sections. For to six tissue sections were examined for each mouse
group.
[0047] FIG. 11A shows data indicating an effect of targeted therapy
of pancreatic cancer by systemic delivery of stroma breaking
ATFmmp14-HANP/SN38 in a human pancreatic cancer PDX model. The
percentage of tumor growth inhibition following five weekly i.v.
injections of 5 mg/Kg SN38 equivalent dose of free SN38 or
HANP/SN38. PDX tumor weight of the no treatment control group was
used as a reference. Significant tumor growth inhibition was found
in the mouse group treated with ATFmmp14-HANP/SN38 compared to free
SN38 and HANP/SN38 treated mouse groups.
[0048] FIG. 11B shows data on targeted therapy of drug resistant
breast cancer following systemic delivery of ATFmmp14 conjugated
HANP carrying SN38. Human breast cancer patient tissue derived
xenograft models were used for the study, including ER+ and triple
negative breast cancer. The figure shows the tumor growth
inhibition in the ER+ breast cancer PDX model.
[0049] FIG. 12 shows analysis of binding affinity of two PD1
peptide mimetics and specific interaction site on PD-L1.
DETAILED DESCRIPTION
[0050] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, and as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
disclosure will be limited only by the appended claims.
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0052] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0053] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0054] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of medicine, organic chemistry,
biochemistry, molecular biology, pharmacology, and the like, which
are within the skill of the art. Such techniques are explained
fully in the literature.
[0055] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
[0056] The term "comprising" in reference to a peptide having an
amino acid sequence refers a peptide that may contain additional
N-terminal (amine end) or C-terminal (carboxylic acid end) amino
acids, i.e., the term is intended to include the amino acid
sequence within a larger peptide. The term "consisting of" in
reference to a peptide having an amino acid sequence refers a
peptide having the exact number of amino acids in the sequence and
not more or having not more than a rage of amino acids specified in
the claim. In certain embodiments, the disclosure contemplates that
the "N-terminus of a peptide may consist of an amino acid
sequence," which refers to the N-terminus of the peptide having the
exact number of amino acids in the sequence and not more or having
not more than a rage of amino acids specified in the claim however
the C-terminus may be connected to additional amino acids, e.g., as
part of a larger peptide. Similarly, the disclosure contemplates
that the "C-terminus of a peptide may consist of an amino acid
sequence," which refers to the C-terminus of the peptide having the
exact number of amino acids in the sequence and not more or having
not more than a rage of amino acids specified in the claim however
the N-terminus may be connected to additional amino acids, e.g., as
part of a larger peptide.
[0057] The term "nanoparticle" refers to a molecular conglomerate
of about between 1 and 1000 nm in diameter. One more molecules or
biomolecules linked to the nanoparticle typically refers to
covalently attaching the molecules or biomolecules to a polymer
based exterior or coating.
[0058] Within certain embodiment, the compositions and methods
disclosed herein may be utilized with a variety of polymer-coated
particle such as, e.g., quantum dots (QDs), metal particles, gold,
silver, iron, and iron-oxide nanoparticles (IONPs).
[0059] "PD-L1" refers to programmed death-ligand 1, also known as
CD274 and B7H1. The amino acid sequence of full-length PD-L1 is
provided in GenBank as accession number NP_054862.1. PD-L1 is a 290
amino acid protein with extracellular IgV-like and IgC-like domains
(amino acids 19-239 of full length PD-L1), a transmembrane domain
and an intracellular domain of approximately 30 amino acids. PD-L1
is constitutively expressed on many cells such as antigen
presenting cells (e.g., dendritic cells, macrophages, and B-cells)
and on hematopoietic and non-hematopoietic cells (e.g., vascular
endothelial cells, pancreatic islets, and sites of immune
privilege). PD-L1 is also expressed on a wide variety of tumors,
and virally-infected cells and is a component of the
immunosuppressive milieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1
binds to one of two T-cell co-inhibitors PD-1 and B7-1.
[0060] "PD-1" refers to the programmed death-1 protein, a T-cell
co-inhibitor, also known as CD279. The amino acid sequence of
full-length human PD-1 is provided in GenBank as accession number
NP_005009.2 (SEQ ID NO: 1)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN
TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN
DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG
LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP
EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL. PD-1 is a
member of the CD28/CTLA-4/ICOS family of T-cell co-inhibitors. PD-1
is a 288-amino acid protein with an extracellular N-terminal
domain, which is IgV-like, a transmembrane domain and an
intracellular domain containing an immunoreceptor tyrosine-based
inhibitory (ITIM) motif and an immunoreceptor tyrosine-based switch
(ITSM) motif (Chattopadhyay et al 2009, Immunol. Rev.). The PD-1
receptor has two ligands, PD-L1 and PD-L2.
[0061] An "isolated" peptide refers one that its sequence was
synthesized chemically or by recombinant techniques and
purified/isolated after synthesis. The peptide sequence is not
purified from naturally occurring environment but may be derived
from genetically modified cells or plants, or by chemical
synthesis.
[0062] A "specific binding" refers to binding by molecules, such as
polynucleotides, antibodies, and other ligands, that are able to
bind to or recognize a binding partner (or a limited number of
binding partners) to a substantially higher degree than to other,
similar biological entities.
[0063] A "subject" is defined to include any living animal or
human. The term "non-human animal" includes all vertebrates, e.g.,
mammals and non-mammals, such as non-human primates, sheep, dog,
cow, chickens, amphibians, reptiles, etc. A subject or non-human
animal is "treated" if one or more beneficial or desired results,
including desirably clinical results, are obtained. For purposes of
this disclosure, beneficial or desired clinical results include,
but are not limited to, one or more of the following: decreasing
one or more symptoms resulting from the disease, increasing the
quality of life of those suffering from the disease, decreasing the
dose of other medications required to treat the disease, delaying
the progression of the disease, and/or prolonging survival of
individuals.
[0064] A "nucleic acid," or "oligonucleotide," is defined as a
polymer of nucleotides. As used herein, a "nucleotide" is given its
ordinary meaning as used in the art, i.e., a molecule comprising a
sugar moiety, a phosphate group, and a base (usually nitrogenous).
Typically, the nucleotide comprises one or more bases connected to
a sugar-phosphate backbone (a base connected only to a sugar
moiety, without the phosphate group, is a "nucleoside"). The sugars
within the nucleotide can be, for example, ribose sugars (a
"ribonucleic acid," or "RNA"), or deoxyribose sugars (a
"deoxyribonucleic acid," or "DNA"). In some cases, the polymer can
comprise both ribose and deoxyribose sugars. Examples of bases
include, but not limited to, the naturally occurring bases (e.g.,
adenosine or "A," thymidine or "T," guanosine or "G," cytidine or
"C," or uridine or "U"). In some cases, the polymer can also
comprise nucleoside analogs (e.g., aracytidine, inosine,
isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine,
2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine,
2'-deoxyuridine, 3-nitorpyrrole, 4-methylindole, 4-thiouridine,
4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine,
5-bromocytidine, 5-iodouridine, inosine, 6-azauridine,
6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine,
8-azidoadenosine, benzimidazole, N6-methyladenosine,
pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine,
5-propynylcytidine, 5-propynyluridine, 5-bromouridine,
5-fluorouridine, 5-methylcytidine, 7-deazaadenosine,
7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, 2-thiocytidine, etc.), chemically or
biologically modified bases (e.g., methylated bases), intercalated
bases, modified sugars (e.g., 2'-fluororibose, 2'-aminoribose,
2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides
arabinose, hexose, etc.), modified phosphate moieties (e.g.,
phosphorothioates or 5'-N-phosphoramidite linkages), and/or other
naturally and non-naturally occurring bases substitutable into the
polymer, including substituted and unsubstituted aromatic moieties.
In some cases, the polynucleotide can include DNA, RNA, modified
DNA, modified RNA, antisense oligonucleotides, expression plasmid
systems, nucleotides, modified nucleotides, nucleosides, modified
nucleosides, intact genes, or combinations thereof. Other examples
of polynucleotides include interfering RNA, natural or unnatural
siRNAs, shRNAs, microRNAs, ribozymes, DNA plasmids, antisense
oligonucleotides, randomized oligonucleotides, or ribozymes. A
nucleic acid sequence may be composed of DNA nucleotides, RNA
nucleotides or a combination of both types and may include natural
nucleotides, chemically modified nucleotides and synthetic
nucleotides.
[0065] "Amino acid sequence" is defined as a sequence composed of
any one of the 20 naturally appearing amino acids, amino acids,
which have been chemically modified, or composed of synthetic amino
acids. The terms "protein" and "peptide" refer to compounds
comprising amino acids joined via peptide bonds and are used
interchangeably. As used herein, where "amino acid sequence" is
recited herein to refer to an amino acid sequence of a protein
molecule. An "amino acid sequence" can be deduced from the nucleic
acid sequence encoding the protein.
[0066] Sequence "identity" refers to the number of matching
residues (expressed as a percentage) in a sequence alignment
between two sequences of the alignment. As used herein, percentage
identity of an alignment is calculated using the number of
identical positions divided by the greater of the shortest sequence
or the number of equivalent positions excluding overhangs wherein
internal gaps are counted as an equivalent position. For example,
the polypeptides GGGGGG and GGGGT have a sequence identity of 4 out
of 5 or 80%. For example, the polypeptides GGGPPP and GGGAPPP have
a sequence identity of 6 out of 7 or 85%.
[0067] Percent "similarity" is used to quantify the similarity
between two sequences of the alignment. This method is identical to
determining the identity except that certain amino acids do not
have to be identical to have a match. Amino acids are classified as
matches if they are among a group with similar properties according
to the following amino acid groups: Aromatic--F Y W; hydrophobic--A
V I L; Charged positive: R K H; Charged negative--D E; Polar--S T N
Q.
[0068] The terms "variant" when used in reference to a polypeptide
refer to an amino acid sequence that differs by one or more amino
acids from another, usually related polypeptide. The variant may
have "conservative" changes, wherein a substituted amino acid has
similar structural or chemical properties. One type of conservative
amino acid substitutions refers to the interchangeability of
residues having similar side chains. For example, a group of amino
acids having aliphatic side chains is glycine, alanine, valine,
leucine, and isoleucine; a group of amino acids having
aliphatic-hydroxyl side chains is serine and threonine; a group of
amino acids having amide-containing side chains is asparagine and
glutamine; a group of amino acids having aromatic side chains is
phenylalanine, tyrosine, and tryptophan; a group of amino acids
having basic side chains is lysine, arginine, and histidine; and a
group of amino acids having sulfur-containing side chains is
cysteine and methionine. Preferred conservative amino acids
substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. More rarely, a variant may have
"non-conservative" changes (e.g., replacement of a glycine with a
tryptophan). Similar minor variations may also include amino acid
deletions or insertions (in other words, additions), or both.
Guidance in determining which and how many amino acid residues may
be substituted, inserted or deleted without abolishing biological
activity may be found using computer programs well known in the
art, for example, DNAStar software. Variants can be tested in
functional assays. Certain variants have less than 10%, and
preferably less than 5%, and still more preferably less than 2%
changes (whether substitutions, deletions, and so on).
[0069] The term "recombinant" when made in reference to a nucleic
acid molecule refers to a nucleic acid molecule which is comprised
of segments of nucleic acid joined together by means of molecular
biological techniques. The term "recombinant" when made in
reference to a protein or a polypeptide refers to a protein
molecule which is expressed using a recombinant nucleic acid
molecule.
[0070] The terms "vector" or "expression vector" refer to a
recombinant nucleic acid containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism
or expression system, e.g., cellular or cell-free. Nucleic acid
sequences necessary for expression in prokaryotes usually include a
promoter, an operator (optional), and a ribosome binding site,
often along with other sequences. Eukaryotic cells are known to
utilize promoters, enhancers, and termination and polyadenylation
signals.
[0071] Protein "expression systems" refer to in vivo and in vitro
(cell free) systems. Systems for recombinant protein expression
typically utilize cells transfecting with a DNA expression vector
that contains the template. The cells are cultured under conditions
such that they translate the desired protein. Expressed proteins
are extracted for subsequent purification. In vivo protein
expression systems using prokaryotic and eukaryotic cells are well
known. Some proteins are recovered using denaturants and
protein-refolding procedures. Common expression systems, for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell, include E. coli host cells
and plasmid vectors, insect host cells and baculovirus vectors, and
mammalian host cells and vectors. Other examples of host cells
include, without limitation, prokaryotic cells (such as bacteria)
and eukaryotic cells (such as yeast cells, mammalian cells, insect
cells, plant cells, etc.). Specific examples include E. coli,
Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g.,
Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as
primary or established mammalian cell cultures (e.g., produced from
lymphoblasts, fibroblasts, embryonic cells, epithelial cells,
nervous cells, adipocytes, etc.). Examples also include mouse
SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC
CRL1580), CHO cell in which a dihydrofolate reductase gene
(hereinafter referred to as "DHFR gene") is defective, rat
YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to
as "YB2/0 cell"), and the like.
[0072] In vitro (cell-free) protein expression systems typically
use translation-compatible extracts of whole cells or compositions
that contain components sufficient for transcription, translation
and optionally post-translational modifications such as RNA
polymerase, regulatory protein factors, transcription factors,
ribosomes, tRNA cofactors, amino acids and nucleotides. In the
presence of an expression vectors, these extracts and components
can synthesize proteins of interest. Cell-free systems typically do
not contain proteases and enable labeling of the protein with
modified amino acids. Some cell free systems incorporated encoded
components for translation into the expression vector. See, e.g.,
Shimizu et al., Cell-free translation reconstituted with purified
components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara &
Chong, Nucleic Acids Research, 2010, 38(13): e141, both hereby
incorporated by reference in their entirety.
[0073] In certain embodiments, the disclosure relates to
recombinant peptides comprising sequences disclosed herein or
variants or fusions thereof wherein the amino terminal end or the
carbon terminal end of the amino acid sequence are optionally
attached to a heterologous amino acid sequence, label, or reporter
molecule. In certain embodiments, the disclosure relates to
recombinant peptides comprising sequences disclosed herein or
variants or fusions thereof wherein the selected amino acid
sequence that are critical for the high affinity binding to the
target molecule are optionally attached to a heterologous amino
acid sequence, label, or reporter molecule.
[0074] The term "fusion" when used in reference to a polypeptide
refers to a chimeric protein containing a protein of interest
joined to an exogenous protein fragment (the fusion partner). The
fusion partner may serve various functions, including enhancement
of solubility of the polypeptide of interest, and provide new
function of the peptide, as well as providing an "affinity tag" to
allow purification of the recombinant fusion polypeptide from a
host cell or from a supernatant or from both. If desired, the
fusion partner may be removed from the protein of interest after or
during purification.
[0075] A "label" refers to a detectable compound or composition
that is conjugated directly or indirectly to another molecule, such
as an antibody or a protein, to facilitate detection of that
molecule. Specific, non-limiting examples of labels include
fluorescent tags, enzymatic linkages, and radioactive isotopes. In
one example, a "label receptor" refers to incorporation of a
heterologous polypeptide in the receptor. A label includes the
incorporation of a radiolabeled amino acid, a fluorescent dye, or
the covalent attachment of biotinyl moieties to a polypeptide that
can be detected by marked avidin (for example, streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or colorimetric methods). Various methods of
labeling polypeptides and glycoproteins are known in the art and
may be used. Examples of labels for polypeptides include, but are
not limited to, the following: radioisotopes or radionucleotides
(such as .sup.35S or .sup.131I) fluorescent labels (such as
fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors,
or near infrared dyes), enzymatic labels (such as horseradish
peroxidase, beta-galactosidase, luciferase, alkaline phosphatase),
chemiluminescent markers, biotinyl groups, predetermined
polypeptide epitopes recognized by a secondary reporter (such as a
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags), or magnetic
agents, such as gadolinium chelates. In some embodiments, labels
are attached by spacer arms of various lengths to reduce potential
steric hindrance.
[0076] In certain embodiments, the disclosure relates to the
recombinant vectors comprising a nucleic acid encoding a peptide
disclosed herein or fusion protein thereof and optionally a
selectable marker. A "selectable marker" is a nucleic acid
introduced into a recombinant vector that encodes a polypeptide
that confers a trait suitable for artificial selection or
identification (report gene), e.g., beta-lactamase confers
antibiotic resistance, which allows an organism expressing
beta-lactamase to survive in the presence antibiotic in a growth
medium. Another example is thymidine kinase, which makes the host
sensitive to ganciclovir selection. It may be a screenable marker
that allows one to distinguish between wanted and unwanted cells
based on the presence or absence of an expected color. For example,
the lac-z-gene produces a beta-galactosidase enzyme, which confers
a blue color in the presence of X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside). If recombinant
insertion inactivates the lac-z-gene, then the resulting colonies
are colorless. There may be one or more selectable markers, e.g.,
an enzyme that can complement to the inability of an expression
organism to synthesize a particular compound required for its
growth (auxotrophic) and one able to convert a compound to another
that is toxic for growth. URA3, an orotidine-5' phosphate
decarboxylase, is necessary for uracil biosynthesis and can
complement ura3 mutants that are auxotrophic for uracil. URA3 also
converts 5-fluoroorotic acid into the toxic compound
5-fluorouracil. Additional contemplated selectable markers include
any genes that impart antibacterial resistance or express a
fluorescent protein.
[0077] Examples include, but are not limited to, the following
genes: ampr, cam.sup.r, tet.sup.r, blasticidin.sup.r, neo.sup.r,
hyg.sup.r, abx.sup.r, neomycin phosphotransferase type II gene
(nptII), p-glucuronidase (gus), green fluorescent protein (gfp),
egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15,
chloramphenicol acetyltransferase (cat), alkaline phosphatase
(phoA), bacterial luciferase (luxAB), bialaphos resistance gene
(bar), phosphomannose isomerase (pmi), xylose isomerase (xylA),
arabitol dehydrogenase (atlD), UDP-glucose:galactose-1-phosphate
uridyltransferasel (galT), feedback-insensitive .alpha. subunit of
anthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR),
benzyladenine-N-3-glucuronide, E. coli threonine deaminase,
glutamate 1-semialdehyde aminotransferase (GSA-AT), D-amino
acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like
protein (pflp), trehalose-6-P synthase gene (AtTPS1), lysine
racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan
synthase beta 1 (AtTSB1), dehalogenase (dhlA), mannose-6-phosphate
reductase gene (M6PR), hygromycin phosphotransferase (HPT), and
D-serine ammonialyase (dsdA).
[0078] In certain embodiments, the recombinant vector optionally
comprises a mammalian, human, insect, viral, bacterial, bacterial
plasmid, yeast associated origin of replication or gene such as a
gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS,
and INS nucleotide segment or gene selected from tat, rev, nef,
vif, vpr, vpu, and vpx or structural genes selected from gag, pol,
and env.
[0079] In certain embodiments, the recombinant vector optionally
comprises a gene vector element (nucleic acid) such as a selectable
marker region, lac operon, a CMV promoter, a hybrid chicken
B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA
polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter,
internal ribosome entry site (IRES) sequence, cis-acting woodchuck
post regulatory element (WPRE), scaffold-attachment region (SAR),
inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag
coding region, metal affinity tag coding region, streptavidin
binding peptide tag coding region, polyHis tag coding region, HA
tag coding region, MBP tag coding region, GST tag coding region,
polyadenylation coding region, SV40 polyadenylation signal, SV40
origin of replication, Col E1 origin of replication, f1 origin,
pBR322 origin, or pUC origin, TEV protease recognition site, loxP
site, Cre recombinase coding region, or a multiple cloning site
such as having 5, 6, or 7 or more restriction sites within a
continuous segment of less than 50 or 60 nucleotides or having 3 or
4 or more restriction sites with a continuous segment of less than
20 or 30 nucleotides.
[0080] "Radiation therapy" is defined as a cancer treatment that
uses high-energy x-rays or other types of radiation to kill cancer
cells or keep them from growing. There are two types of radiation
therapy. External radiation therapy, which uses a machine outside
the body to send radiation to the cancer. Internal radiation
therapy uses a radioactive substance sealed in needles, seeds,
wires, or catheters that are placed directly into or near the
cancer. The way the radiation therapy is administer is directly
dependent on the type and stage of the cancer.
[0081] "Chemoradiation therapy" is defined as a therapy that
combines chemotherapy and radiation therapy to increase the effects
of both.
[0082] "Cancer" refers any of various cellular diseases with
malignant neoplasms characterized by the proliferation of cells. It
is not intended that the diseased cells must actually invade
surrounding tissue and metastasize to new body sites. Cancer can
involve any tissue of the body and have many different forms in
each body area. Within the context of certain embodiments, whether
"cancer is reduced" may be identified by a variety of diagnostic
manners known to one skill in the art including, but not limited
to, observation the reduction in size or number of tumor masses or
if an increase of apoptosis of cancer cells observed, e.g., if more
than a 5% increase in apoptosis of cancer cells is observed for a
sample compound compared to a control without the compound. It may
also be identified by a change in relevant biomarker or gene
expression profile, such as PSA for prostate cancer, HER2 for
breast cancer, or others. The cancer to be treated in the context
of the present disclosure may be any type of cancer or tumor.
Targeted Delivery of Theranostic Nanoparticles Carrying Immune
Modulators for Activation of Tumor-Specific Immune Responses
[0083] Due to their small size (3-10 nm), iron oxide nanoparticles
(IONPs) are able to utilize the enhanced permeability and retention
(EPR) effect to extravasate through the abnormal, "leaky" tumor
vasculature and actively target PD-L1 over-expressing cells in
tumor cells and tumor microenvironment in comparison to
conventional antibody-based therapies that are delivered all over
the body in normal organs and tissues. One of the major challenges
in immunotherapy is that tumor antigens are poorly immunogenic and
many immunogenic mutant proteins localize inside tumor cells. The
combination of treatment using tumor cell-targeted nanoparticle
drug carriers with PD-L1 targeted nanoparticles has the potential
to destroy tumor cells to expose intracellular, immunogenic tumor
antigens and promote macrophages taking up and processing the
antigens for presentation to T and B cells. Meanwhile, inhibition
of PD-L1 mediated immune suppression function by the PD-L1 blocking
peptide conjugated nanoparticles should enhance overall
tumor-specific T cell responses. Conventional anti-PD1 or PDL1
antibody-mediated immunotherapies lack the function of enhancing
presentation of immunogenic intracellular mutant proteins. The
short PD-L1 blocking peptide reported herein have dual functions of
tumor targeting and blocking PD-L1 that is highly expressed by many
tumor cells as well as tumor stromal fibroblasts and macrophages.
Furthermore, about 20 to 30 peptides could be conjugated on the
surface of one nanoparticle, which should enhance the efficiency of
PD-1/PD-L1 inhibition, whereas an anti-PD-L1 antibody can only
block one PD-L1 molecule. An advantage of delivering PD-L1 blocking
agents, such as antibodies or peptides, using nanoparticles is the
limited bio-distribution in vivo that should only include the tumor
microenvironment and macrophages or the RES system in the liver and
spleen. However, conventional PD-1 or PD-L1 antibody therapies
often have systemic effects and leads to dysregulation of the
immune system and possible autoimmune diseases.
[0084] One of the major challenges in immunotherapy of human
cancers enriched in tumor stroma is that the stroma barrier
prevents efficient intratumoral delivery and distribution of PD-L1
or PD1 antibodies. For example, results of recent clinical trials
using antibody therapy to block PD-L1 and PD1 did not show
significant therapeutic response in pancreatic cancer patients. The
major issues for a poor response in pancreatic cancer are thought
to be due to extensive tumor stroma that consists of 50% to 80% of
the tumor mass in pancreatic cancer tissue creates a physical
barrier to block antibody delivery and infiltration of T cells into
tumor tissues. Receptor targeted nanoparticle drug carriers
disclosed herein have the potential to improve the therapeutic
response of immunotherapy through the following mechanisms: 1)
targeted delivery of a high level of nanoparticles into tumors that
promotes massive infiltration of immune cells, including T cells
and antigen presenting cells into tumor center areas to create
pro-immune environment to facilitate the activation of immune
responses; 2) destroying tumor cells to release tumor specific
antigens; 3) breaking tumor stromal barriers by theranostic
nanoparticles that increase T cell infiltration into deep tumor
tissues; and 4) targeted delivery of PD1 like peptide conjugated
nanoparticles into tumor that blocks PD-L1 on tumor cells and
stromal fibroblasts and macrophages to activate tumor specific T
cell responses and to enhance the effect of cytotoxic T cells.
[0085] In order to minimize adverse side effects as well as to
enhance intratumoral delivery and tumor-specific immune responses,
IONPs were developed carrying peptide-based antagonists to PD-L1 to
demonstrate tumor targeting efficiency. It is contemplated that
these nanoparticles can be used as monotherapy and as a combination
therapy with targeted theranostic nanoparticles carrying
chemotherapeutic drugs.
[0086] Preliminary studies used i.p. delivery of unconjugated
anti-mouse PD1 antibody in combination with uPAR targeted
nanoparticles carrying a chemotherapy drug, cisplatin, demonstrated
the feasibility of blocking PD-1/PD-L1 interaction enhanced
anti-tumor growth effects of a receptor targeted nanoparticle
carrying a chemotherapy drug, Cisplatin (Cys), in a mouse
pancreatic cancer model. However, an antibody has a relatively
large size of 2.times.8 nm and is too large for conjugation to the
magnetic iron oxide nanoparticle with a core size of 5 nm that is
optimized to intratumoral delivery. Therefore, two PD1-like
peptides were designed by selecting the key PD-L1 binding domains
of PD1 amino sequences and fusing into a short peptide ligand with
35 (PD1Y) or 37 (PD1Linear) amino acids. These PD1-like peptides
have several necessary amino acid modifications to retain domain
structures, a short his tag (4) for conjugation and a cysteine
(Cys), for labeling fluorescence dye molecules. In one, a short his
tag (4.times.) was strategically placed in the middle of the PD-1
like peptide (PD1Y) to create a 3-D structure for PD-L1 binding. In
another, designated PD-1(Lin), the peptide is linear, PD-L1 binding
domains are fused together with his tag at the carboxyl-terminal.
Each peptide contains a poly-histidine tag for conjugation to
nitrilotriacetic acid-copper (NTA-Cu) functionalized IONPs to
ensure the correct orientation of the PD1 bind domains.
Nanoparticles
[0087] This disclosure relates to nanoparticles comprising
peptide-based antagonist of PD-L1 as a targeting moiety. In certain
embodiments, the targeting moiety is a peptide comprising SEQ ID
NO: 1, 2, or 3 or variants thereof. When reference is made to a
particle or nanoparticle comprising a peptide, it is understood
that the peptide is bound to the particle through a polymer
coating, either through covalent bonds or other binding
interactions, e.g., hydrophobic or hydrophilic binding or chelating
interactions. In certain embodiments, a particle or nanoparticle
comprising a peptide, and the peptide is bound to the particle
mediated by interaction of the short his-tag with NTA-Cu that is
conjugated to polymer coating of the nanoparticle.
[0088] Within certain embodiment, the compositions and methods
disclosed herein may be utilized with a variety of polymer-coated
particle such as, e.g., quantum dots (QDs), metal particles, gold,
silver, iron, and iron-oxide nanoparticles (IONPs). IONPs are
typically prepared with a mean particle diameter of 3-200 nm. IONPs
may be prepared by aging a stoichiometric mixture of ferrous and
ferric salts in aqueous media under basic conditions. Control over
particle size (3-20 nm) and shape is provided by adjusting the pH,
ionic strength and the concentration of the growth solution. The
nanoparticles can be functionalized in situ using additives such as
organic compounds (e.g. sodium citric) or polymers (e.g. dextran,
polyvinyl alcohol). Other metals such as gold, cobalt, nickel, and
manganese may be incorporated into the material.
[0089] High-temperature decomposition of Fe(CO).sub.5 in organic
solvents is another way to prepare IONPs. Size (3-19 nm) can be
varied using alternative temperatures. Flame spray pyrolysis yields
a range of magnetite, maghemite and wustite (FeO) particles IONPs.
Iron precursor such as Fe(CO).sub.5 and Fe(NO.sub.3).sub.3 may be
used. Flame spray pyrolysis can be used to produce different
nanoparticles (TiO.sub.2, ZrO.sub.2, silica, etc.) as well as
hybrid particles (e.g. silica-IONPs).
[0090] Hydroxyl groups on the IONP provide a place for synthetic
attachment of different functional groups. A range of chemistries
can be used to stabilize metal nanoparticles, exploiting
electrostatic, hydrophobic, chelating and covalent interactions.
Carboxylic acid groups can interact with the surface of IONPs by
coordination processes. IONP synthesis in organic solvents is
typically conducted in oleic acid. A polymer coating on the IONPs
is preferred. Polymer attachment to the IONP surface by an
initiator fixed to the surface of the IONPs and the polymer is
grown from the surface. Alternatively, a functional, pre-formed
polymer is grafted onto IONPs in situ. Copolymers with hydrophobic
groups, carboxylic acid groups, polyethylene glycols, or amine
groups are contemplated. Polymers with a hydrophilic block and a
hydrophobic block are contemplated. See Yang et al., Clin Cancer
Res, 2009 15:4722; Lin et al., Small, 2008, 4(3):334-341; Yu et
al., Nanotechnology, 2006, 17:4483-4487; Park et al., J. Mater.
Chem., 2009, 19, 6412-6417; Boyer et al. NPG Asia Mater., 2010,
2(1):23-30, Kim et al., Nanotechnology, 2011, 22, 155101; all
hereby incorporated by reference in their entirety.
[0091] Conjugating molecules or polypeptides to the polymers can be
accomplished using a variety of methods. Typically, primary amine
containing compounds and proteins may be conjugated to the
carboxylic acid groups on the polymer mediated by a coupling
reagent such as EDAC. See Yang et al., Small, 2009, 5(2):235-43,
hereby incorporated by reference in its entirety. Other coupling
methods are contemplated, e.g., poly-histidine sequence may be
incorporated by recombinant methods into a polypeptide sequence of
the targeting moiety. A poly-histidine chelating agent may be
coupled to the polymer surface, e.g., NTA-Ni or NTA-Cu. Mixing the
histidine tagged polypeptide sequence attaches it to the polymer
surface linked through the chelating agent NTA. The
avidin/streptavidin-biotin interactions may be used, e.g., biotin
may be coupled to the polymer surface and streptavidin may be
expressed as a chimera with the targeting moiety.
[0092] In addition to the peptides disclosed herein, the particles
may comprise a second targeting moiety. In certain embodiments the
targeting moiety is an amino-terminal fragment (ATF) of uPA, e.g.,
amino terminal fragment (ATF, 135 aa) of human uPA (17 kDa) (SEQ ID
NO: 5) SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFY
RGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCY
VQVGLKPLVQECMVHDCADGK or (ATF, 68 aa) of human uPA (SEQ ID NO: 6)
SNELHQVP SNCDCLNGGTCVSNKYF SNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFY
RGKASTDTMG, or human ATF-MMP14 (SEQ ID NO: 7)
MSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHF
YRGKASTDGAPIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRVWESATPLRFRE
VPYAYIREGHEKQADIMIFFAEGFHGDSTPFDGEGGFLAHAYFPGPNIGGDTHFD SAEPW
TVRNEDLNGNDIFLVAVHELGHALGLEHSSDPSAIMAPFYQWMDTENFVLPDDDRRGI
QQLYGGESGFPTKMPPQPRTTSRPSVPDKPKNPTYGPNIHHHHHH).
[0093] ATF or ATF-MMP14 may be produced from E. coli BL21 bacterial
expression system using a pET20a plasmid (Invitrogen, Grand Island,
N.Y.) containing the ATF or ATF-MMP14 cDNA sequence. Urokinase
plasminogen activator (uPA) is a serine protease that regulates
multiple pathways involved in matrix degradation, cell motility,
metastasis and angiogenesis. Interaction of the N-terminal growth
factor domain of uPA with its cellular receptor (uPAR) results in
the conversion of the plasminogen to a serine protease, which is a
central regulator of the activation of other proteases including
the matrix metalloproteinases (MMPs). Studies have shown that the
uPA/uPAR complex controls the motility of both tumor and
endothelial cells. In addition to its role in activation of the
process for degradation of extracellular matrix, uPAR also
activates .alpha.5.beta.1 integrin and ERK signaling through
interaction with EGFR and induces cell proliferation. Additionally,
the uPA/uPAR complex can bind to the matrix protein, vitronectin,
in association with transmembrane integrins, and activate
intracellular signaling molecules such as the protein kinases,
promoting cell adhesion, proliferation, and migration.
[0094] The uPAR-binding domain of uPA is located to the
amino-terminal fragment (ATF) of uPA. Studies have shown that ATF
is a potent uPA binding antagonist to its high affinity receptor
(uPAR) at the surface of both tumor and endothelial cells. Systemic
or local delivery of a non-catalytic amino-terminal fragment (ATF)
of uPA (residues 1-135) using an adenoviral vector or conjugated
peptides prevents the formation of the uPA/uPAR complex, thus
inhibiting tumor growth and angiogenesis. Yang et al., Clin Cancer
Res., 2009, 15(14):4722-32, hereby incorporated by reference in its
entirety, discuss the preparation of targeted iron oxide
nanoparticle using a recombinant peptide containing the
amino-terminal fragment of urokinase-type plasminogen activator
(uPA) conjugated to magnetic iron oxide nanoparticles
amino-terminal fragment conjugated-iron oxide nanoparticle
(ATF-IONP). This nanoparticle targets uPA receptor, which is
overexpressed in breast cancer tissues.
[0095] In certain embodiments, the second targeting moiety is a
moiety that binds EGFR or HER-2. The human epidermal growth factor
receptor (EGFR) family includes EGFR (HER-1), EGFR-2 (HER-2),
EGFR-3 (Her-3) and EGFR 4 (HER-4). The ligands that bind to EGFRs
are divided into EGFR-like ligands such as EGF and TGF-.alpha., and
the heregulins. These ligands bind to EGFR monomers to promoter
receptor dimerization and oligomerization that ultimately results
in the activation of the EGFR signaling pathway. This EGFR
signaling pathway plays a role in the regulation of cell
proliferation, survival and differentiation.
[0096] Human breast carcinomas with a triple negative subtype
express high levels of the EGF receptors. Her-2 positive subtype
breast cancer expresses a high level of Her-2 receptor.
Overexpression of those receptors have been associated with highly
aggressive breast cancer types and a poor response to therapeutic
agents. Prior preclinical and clinical studies have shown that
blocking the EGFR or HER-2 via monoclonal antibodies or inhibition
of EGFR tyrosine kinase with small molecule inhibitors inhibits the
growth of breast cancers and sensitize chemotherapy responses.
Single-chain antibodies to EGFR that contain the specific EGFR
binding region but lack the Fc region have been isolated from human
scFv phage display libraries. Yang et al., Small, 2009,
5(2):235-43, hereby incorporated by reference in its entirety,
discuss the preparation of EGFR targeted nanoparticles conjugating
a single-chain anti-EGFR antibody (ScFvEGFR). A high affinity Her-2
binding affibody conjugated magnetic iron oxide nanoparticle has
been shown to target to Her-2 expressing human ovarian tumors,
allowing non-invasive tumor imaging in an orthotopic human ovarian
cancer model in nude mice. (Satpathy M, & Yang L et al. Small,
2014; 10(3):544-55).
[0097] Iron oxide nanoparticles conjugated to a purified antibody
that selectively binds to the epidermal growth factor receptor
(EGFR) deletion mutant (EGFRvIII) present on human glioblastoma
multiforme (GBM) cells were used for therapeutic targeting and MRI
contrast enhancement of experimental glioblastoma, both in vitro
and in vivo, after convection-enhanced delivery (CED). See
Hadjipanayis et al., Cancer Res, 2010, 70:6303, hereby incorporated
by reference in its entirety. In certain embodiments, the
disclosure relates to targeting moiety that is an antibody or
antibody mimetic to EGFR or EGFRvIII for use in treating
glioblastoma multiforme.
[0098] In certain embodiments, the second targeting moiety is a
monoclonal antibody-610 that targets a surface antigen for use in
treating colon carcinoma. See Cerdan et al., Magn Reson Med, 1989,
12:151-63 1989, hereby incorporated by reference in its
entirety.
[0099] In certain embodiments, the second targeting moiety is an
antibody to carcinoembryonic antigen (CEA) that targets CEA for use
in treating colon tumors. See Tiefenauer et al., Magn Reson
Imaging, 1996, 14:391-402, hereby incorporated by reference in its
entirety.
[0100] In certain embodiments, the second targeting moiety is a
monoclonal antibody L6 that targets a surface antigen for use in
treating intracranial tumor. See Remsen et al., Am J Neuroradiol,
1996, 17:411-18, hereby incorporated by reference in its
entirety.
[0101] In certain embodiments, the second targeting moiety is
transferrin that targets transferrin receptor for use in treating
carcinoma. See Kresse et al., Magn Reson Med, 1998, 40:236-42,
hereby incorporated by reference in its entirety.
[0102] In certain embodiments, the second targeting moiety is a
monoclonal antibody to Her-2, e.g., Herceptin, which targets Her-2
receptors for use in treating breast cancer. See Lee et al., Nat
Med, 2007, 13:95-9; Artemov et al., Magn Reson Med, 2003, 49:403-8;
and Huh et al., J Am Chem Soc, 2005, 127:12387-91, all hereby
incorporated by reference in their entirety.
[0103] In certain embodiments, the second targeting moiety is the
EPPT peptide that targets underglycosylated mucin-1 antigen
(uMUC-1) for use in treating breast, colon, pancreas, and lung
cancer. See Moore et al., Cancer Res, 2004, 64:1821-7, hereby
incorporated by reference in its entirety.
[0104] In certain embodiments, the second targeting moiety is folic
acid that targets folate receptor for use in treating mouth
carcinoma and cervical cancer. See Chen et al., PDA J Pharm Sci
Technol, 2007, 61:303-13; Sun et al., Small, 2006, 4:372-9; and
Sonvico et al., Bioconjug Chem, 2005, 16:1181-8, all hereby
incorporated by reference in their entirety.
[0105] In certain embodiments, the second targeting moiety is
methotrexate that targets folate receptor for use in treating
cervical cancer. See Kohler et al., Langmuir, 2005, 21:8858-64,
hereby incorporated by reference in its entirety.
[0106] In certain embodiments, the second targeting moiety is a
monoclonal antibody A7 that targets colorectal tumor antigen for
use in treating colorectal carcinoma. See Toma et al., Br J Cancer,
2005, 93:131-6, hereby incorporated by reference in its
entirety.
[0107] In certain embodiments, the second targeting moiety is
chlorotoxin peptide that targets membrane-bound
matrixmetalloproteinase-2 (MMP-2) for use in treating glioma. See
Veiseh et al., Nano Lett, 2005, 5:1003-8, hereby incorporated by
reference in its entirety.
[0108] In certain embodiments, the second targeting moiety is F3
peptide that targets surface-localized tumor vasculature for use in
treating glioma. See Reddy et al., Clin Cancer Res, 2006,
12:6677-86, hereby incorporated by reference in its entirety.
[0109] In certain embodiments, the second targeting moiety is iRGD
or RGD4C that targets integrins for use in treating melanoma and
epidermoid carcinoma. See Zhang et al., Cancer Res, 2007,
67:1555-62 and Uchida et al., J Am Chem Soc, 2006, 128:16626-33,
both hereby incorporated by reference in their entirety.
[0110] In certain embodiments, the second targeting moiety is
luteinizing hormone releasing hormone (LHRH) that targets LHRH
receptor for use in treating breast cancer. See Leuschner et al.,
Breast Cancer Res Treat, 2006, 99:163-76, hereby incorporated by
reference in its entirety.
[0111] In certain embodiments, the second targeting moiety is CREKA
peptide that targets clotted plasma proteins for use in treating
breast cancer. See Simberg et al., Proc Natl Acad Sci USA, 2007,
104:932-6, hereby incorporated by reference in its entirety.
[0112] In certain embodiments, the second targeting moiety is an
antibody to prostate specific membrane antigen (PSMA) that targets
PSMA for use in treating prostate cancer. See Serda et al., Mol
Imaging, 2007, 6:277-88, hereby incorporated by reference in its
entirety.
[0113] In certain embodiments, the disclosure relates to
multifunctional nanoparticles comprising a targeting peptide
disclosed herein, the nanoparticle, and the cargo. The
nanoparticles can be either Quantum Dots (QDs) or gold
nanoparticles that can be imaged optically or iron oxide
nanoparticles (IONPs) that can be imaged via MRI. In certain
embodiments, the cargo is either a DNA cassette coding for a siRNA
against an oncogene or survival factor, a chemotherapy drug or
both.
[0114] Since siRNA is expressed from a RNA polymerase III (e.g., U6
or H1) promoter, a short hairpin siRNA (shRNA) gene may be cloned
into expression vectors containing a polymerase III promoter to
produce shRNAs from plasmid or viral vectors following transfecting
into cells. See Brummelkamp et al., Science, 2002, 296, 550-553;
Miyagishi & Taira, Nat. Biotechnol, 2002, 20, 497-500; McAnuff
et al, J. Pharm. Sci. 2007, 96, 2922-2930; Bot et al., Blood, 2005,
106, 1147-1153. The shRNAs are further processed into siRNAs by a
cellular endoribonuclease. DNA cassettes expressing shRNA
containing a U6 promoter and a shRNA gene can be synthesized by a
two-step PCR amplification protocol. See Castanotto et al., RNA,
2002, 8, 1454-1460 and Gou et al., FEBS Lett., 2003, 548,
113-118.
[0115] In certain embodiments provided herein is a particle that
contains a polymer-coated nanoparticle core, e.g., a fluorescent
quantum dot (QD) or MRI contrast enhancing magnetic iron oxide
nanoparticle (IONP), conjugated with about 10 to 20 DNA
nanocassettes that contain a U6 promoter and a shRNA gene for in
vivo siRNA gene expression following intracellular delivery. The
nanoparticle is conjugated to a targeting peptide disclosed herein
typically the amino terminal fragment (ATF) of the urokinase
plasminogen activator (uPA), which targets its cellular receptor,
uPAR. This receptor is highly expressed in tumors, angiogenic
endothelial, and stromal cells in many types of human cancers. See
Nielsen et al., Int. J. Cancer 2007, 120, 2086-2095; Blasi &
Carmeliet, Nat. Rev. Mol. Cell Biol. 2002, 3, 932-943; Pyke et al.,
Cancer Res, 1993, 53, 1911-1915.
[0116] In certain embodiments, the disclosure relates to particles
comprising a core coated with a polymer, wherein the polymer is
conjugated to a targeting moiety, a lysosomally degradable moiety,
and a therapeutic agent such as gemcitabine, doxorubicin, cytosine
arabinoside, mitomycin, or any therapeutic agent with that an amine
side group. In certain embodiments, the therapeutic agent is
cisplatin. In certain embodiments, the particle is a metal
nanoparticle or metal oxide nanoparticle, such as an iron oxide
nanoparticle or elemental iron core nanoparticle with an oxide
coat, or a quantum dot, e.g., those with a diameter of between
about 5 to 200 nm or 10 to 100 nm. In certain embodiments, the
lysosomally degradable moiety is the polypeptide GFLG (SEQ ID NO:
4) linked to the therapeutic agent. In certain embodiments, the
disclosure relates to compositions comprising a polymer conjugated
to a targeting moiety, lysosomally degradable moiety, and a
therapeutic agent which are described herein. In one example, the
lysosomally degradable moiety linked to the therapeutic agent is of
the formula:
##STR00001##
[0117] or salts or derivatives thereof optionally substituted with
one or more substituents. In certain embodiments, the polymer is an
amphiphilic polymer comprising a hydrophobic section further
comprising a hydrophobic chemotherapeutic agent.
[0118] In certain embodiments, the particle further comprises a
fluorescent dye, e.g., a
(3,3-dimethyl-indol-1-ium-1-yl)-N-alkylsulfonate dye or salt
thereof such as one of the formula:
##STR00002##
[0119] or salts or derivatives thereof optionally substituted with
one or more substituents wherein X is S or NH and n is 2 to 22 or n
is 4 to 22. In certain embodiments, the dye is conjugated to the
free thiol group on cysteine or free amino group of the peptides or
proteins.
METHODS OF USE
[0120] In certain embodiments, this disclosure relates to a method
of treating cancer comprising administering an effective amount of
a nanoparticle comprising a peptide disclosed herein or a peptide
disclosed herein, to a subject in need thereof.
[0121] In certain embodiments, this disclosure relates to a method
of treating cancer comprising administering an effective amount of
a nanoparticle comprising a peptide comprising SEQ ID NO: 1, 2, 3,
or variants to a subject in need thereof.
[0122] In certain embodiments, this disclosure relates to a method
of treating cancer comprising administering an effective amount of
a peptide comprising SEQ ID NO: 1, 2, 3, or variants to a subject
in need thereof. In certain embodiments, the peptide may be
administered in combination with a nanoparticle comprising an
amino-terminal fragment (ATF) of uPA, ATF-MMP14, or the other
peptide targeting ligands that may be incorporated into the
nanoparticle.
[0123] In certain embodiments, the disclosure contemplates a
combination chemotherapy comprising the administration of a first
agent in combination with a second agent, wherein the first agent
is a nanoparticle comprising a peptide having SEQ ID NO: 1, 2, 3,
or variant thereof, wherein the second agent is an anti-CTLA-4
antibody such as Ipilimumab, or anti-PD-1 antibody such as
nivolumab or pembrolizumab.
[0124] In certain embodiments, the disclosure contemplates a
combination chemotherapy comprising the administration of a first
agent in combination with a second agent, wherein the first agent
is a nanoparticle comprising a peptide having SEQ ID NO: 1, 2, 3,
or variant thereof, wherein the second agent is a nanoparticle
comprising a an amino-terminal fragment (ATF) of uPA or ATF-MMP14
and a chemotherapy agent attached to the nanoparticle or the
chemotherapy agent is encapsulated by a polymer around the core of
the particle.
[0125] In certain embodiments, the disclosure contemplates a
combination chemotherapy comprising the administration of a first
agent in combination with a second agent, wherein the first agent
is a nanoparticle comprising a peptide having SEQ ID NO: 1, 2, 3,
or variant thereof and an amino-terminal fragment (ATF) of uPA or
ATF-MMP14 and optionally a chemotherapy agent attached to the
nanoparticle or the chemotherapy agent is encapsulated by a polymer
around the core of the particle; and the second agent is an
anti-CTLA-4 antibody such as Ipilimumab, or anti-PD-1 antibody such
as nivolumab or pembrolizumab.
[0126] In certain embodiments, the cancer is mediated by PD-L1. In
certain embodiments, the cancer overexpresses a receptor of the
targeting molecule in tumor cells, tumor endothelial cells, or
tumor stromal fibroblasts compared to noncancerous tissue of an
organ containing the cancerous tumor. In certain embodiments, the
targeting molecule is an antibody or fragment, antibody mimetic,
inhibitor, or aptamer targeting a protein or glycoprotein expressed
on the surface of a cancerous cell. In certain embodiments, the
cancer over-expresses uPAR, EGFR, or HER-2. In certain embodiments,
the cancer is selected from pancreatic cancer, breast cancer,
prostate cancer, lung cancer, skin cancer, bladder cancer, brain
cancer, colon cancer, rectal cancer, kidney cancer, endometrial
cancer, and thyroid cancer.
[0127] In certain embodiments, the cancer is selected from
carcinoma, lymphoma, blastoma, sarcoma, and leukemia, non-small
cell lung, squamous cell, small-cell lung, peritoneum,
hepatocellular, gastrointestinal, pancreatic, glioma, cervical,
ovarian, liver, bladder, hepatoma, breast, colon, colorectal,
endometrial or uterine, salivary gland, kidney, liver, prostate,
vulval, thyroid, hepatic, leukemia and other lymphoproliferative
disorders, and various types of head and neck. In certain
embodiments, the cancer can be primary or metastatic tumors.
[0128] In further embodiments, this disclosure relates to methods
of treating cancer further comprising administering a particle or
peptide disclosed herein comprising a second chemotherapy agent or
administering a second chemotherapy to the subject separate from
any chemotherapy agent contained in or attached to the particle
and/or the surrounding polymer. In certain embodiments, particles
disclosed herein are administered an effective amount to treat a
subject diagnosed with cancer or a cancerous tumor. In certain
embodiments, the particles disclosed herein are administered in
combination with a second anti-cancer agent such as, but not
limited to, bevacizumab, gefitinib, erlotinib, temazolamide,
docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur,
raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea,
adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,
idarubicin, mitomycin-C, dactinomycin, mithramycin, vincristine,
vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide,
teniposide, amsacrine, topotecan, camptothecin, bortezomib,
anegrilide, tamoxifen, toremifene, raloxifene, droloxifene,
iodoxyfene fulvestrant, bicalutamide, flutamide, nilutamide,
cyproterone, goserelin, leuprorelin, buserelin, megestrol,
anastrozole, letrozole, vorazole, exemestane, finasteride,
marimastat, trastuzumab, cetuximab, dasatinib, imatinib,
combretastatin, thalidomide, and/or lenalidomide or combinations
thereof.
[0129] In certain embodiments, the methods disclosed herein may be
used in combination with radiation and chemoradiation therapy.
[0130] In certain embodiments, this disclosure relates to a method
for cancer diagnosis comprising administering an effective amount
of a peptide disclosed herein or nanoparticle disclosed herein to a
subject in need thereof and detecting the particle about the area
of a cancerous cell or tumor.
[0131] Also contemplated are malignancies located in the colon,
abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, hypophysis,
testicles, ovaries, thymus, thyroid), eye, head and neck, nervous
system (central and peripheral), lymphatic system, pelvis, skin,
soft tissue, spleen, thorax and genito-urinary apparatus and, more
particularly, childhood acute lymphoblastic leukemia, acute
lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid
leukemia, adrenocortical carcinoma, adult (primary) hepatocellular
cancer, adult (primary) liver cancer, adult acute lymphocytic
leukemia, adult acute myeloid leukemia, adult Hodgkin's disease,
adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult
non-Hodgkin's lymphoma, adult primary liver cancer, adult soft
tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant
tumors, anal cancer, astrocytoma, cancer of the biliary tract,
cancer of the bladder, bone cancer, brain stem glioma, brain
tumors, breast cancer, cancer of the renal pelvis and ureter,
primary central nervous system lymphoma, central nervous system
lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the
cervix, childhood (primary) hepatocellular cancer, childhood
(primary) liver cancer, childhood acute lymphoblastic leukemia,
childhood acute myeloid leukemia, childhood brain stem glioma,
childhood cerebellar astrocytoma, childhood brain astrocytoma,
childhood extracranial germ cell tumors, childhood Hodgkin's
disease, childhood Hodgkin's lymphoma, childhood visual pathway and
hypothalamic glioma, childhood lymphoblastic leukemia, childhood
medulloblastoma, childhood non-Hodgkin's lymphoma, childhood
supratentorial primitive neuroectodermal and pineal tumors,
childhood primary liver cancer, childhood rhabdomyosarcoma,
childhood soft tissue sarcoma, childhood visual pathway and
hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid
leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine
pancreatic islet cells carcinoma, endometrial cancer, ependymoma,
epithelial cancer, cancer of the oesophagus, Ewing's sarcoma and
related tumors, cancer of the exocrine pancreas, extracranial germ
cell tumor, extragonadal germ cell tumor, extrahepatic biliary
tract cancer, cancer of the eye, breast cancer in women, Gaucher's
disease, cancer of the gallbladder, gastric cancer,
gastrointestinal carcinoid tumor, gastrointestinal tumors, germ
cell tumors, gestational trophoblastic tumor, tricoleukemia, head
and neck cancer, hepatocellular cancer, Hodgkin's disease,
Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer,
intestinal cancers, intraocular melanoma, islet cell carcinoma,
islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney,
cancer of the larynx, cancer of the lip and mouth, cancer of the
liver, cancer of the lung, lymphoproliferative disorders,
macroglobulinemia, breast cancer in men, malignant mesothelioma,
malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult
primary metastatic squamous neck cancer, primary metastatic
squamous neck cancer, metastatic squamous neck cancer, multiple
myeloma, multiple myeloma/plasmatic cell neoplasia, myelodysplastic
syndrome, myelogenous leukemia, myeloid leukemia,
myeloproliferative disorders, paranasal sinus and nasal cavity
cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's
lymphoma during pregnancy, non-melanoma skin cancer, non-small cell
lung cancer, metastatic squamous neck cancer with occult primary,
buccopharyngeal cancer, malignant fibrous histiocytoma, malignant
fibrous osteosarcoma/histiocytoma of the bone, epithelial ovarian
cancer, ovarian germ cell tumor, ovarian low malignant potential
tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid
cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor,
neoplasia of plasmatic cells/multiple myeloma, primary central
nervous system lymphoma, primary liver cancer, prostate cancer,
rectal cancer, renal cell cancer, cancer of the renal pelvis and
ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary
glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer,
small intestine cancer, soft tissue sarcoma, squamous neck cancer,
stomach cancer, pineal and supratentorial primitive neuroectodermal
tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid
cancer, transitional cell cancer of the renal pelvis and ureter,
transitional renal pelvis and ureter cancer, trophoblastic tumors,
cell cancer of the renal pelvis and ureter, cancer of the urethra,
cancer of the uterus, uterine sarcoma, vaginal cancer, optic
pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's
macroglobulinemia, Wilms' tumor and any other hyperproliferative
disease, as well as neoplasia, located in the system of a
previously mentioned organ.
[0132] A "chemotherapy agent," "chemotherapeutic," "anti-cancer
agent" or the like, refer to molecules that are recognized to aid
in the treatment of a cancer. Contemplated examples include the
following molecules or derivatives such as temozolomide,
carmustine, bevacizumab, procarbazine, lomustine, vincristine,
gefitinib, erlotinib, cisplatin, carboplatin, oxaliplatin,
5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,
cytosine arabinoside, hydroxyurea, adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin, mithramycin, vinblastine, vindesine, vinorelbine,
paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine,
topotecan, camptothecin, bortezomib, anagrelide, tamoxifen,
toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant,
bicalutamide, flutamide, nilutamide, cyproterone, goserelin,
leuprorelin, buserelin, megestrol, anastrozole, letrozole,
vorozole, exemestane, finasteride, marimastat, trastuzumab,
cetuximab, dasatinib, imatinib, combretastatin, thalidomide,
azacitidine, azathioprine, capecitabine, chlorambucil,
cyclophosphamide, cytarabine, daunorubicin, doxifluridine,
epothilone, irinotecan, mechlorethamine, mercaptopurine,
mitoxantrone, pemetrexed, tioguanine, valrubicin and/or
lenalidomide or combinations thereof such as, cyclophosphamide,
methotrexate, 5-fluorouracil (CMF); doxorubicin, cyclophosphamide
(AC); mustine, vincristine, procarbazine, prednisolone (MOPP);
sdriamycin, bleomycin, vinblastine, dacarbazine (ABVD);
cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP);
bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin,
5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX);
methotrexate, vincristine, doxorubicin, cisplatin (MVAC).
[0133] In certain embodiments, the disclosure relates to methods of
optical and MRI imaging the nanoparticle in tumors. 3D-MRI enables
monitoring of intratumoral distribution of nanoparticles and tumor
responses to therapeutics contained on or in the nanoparticles.
[0134] In certain embodiments, the disclosure relates to
nanoparticles coated with amphiphilic polymers conjugated with
molecules useful for targeting tumors, monitoring the location of
the nanoparticles administered to a subject by MRI, and viewing the
presence of the nanoparticles during optical image-guided
surgery.
[0135] In certain embodiments, the disclosure relates to uses of
particles disclosed herein as a theranostics. Theranostics are
therapeutics with physical properties that allows one to image
molecular accumulation of the vehicles in vivo. Yang et al.,
WO/2007/018647, disclose binding and internalization of tumor
targeted-iron oxide particles using MRI. See also Yang et al., J.
Biomed. Nanotechnol., 2008, 4, 439-449. Lammers et al.,
Biomaterials, 2009, 30(2):3466-3475, disclose the simultaneous
delivery of doxorubicin and gemcitabine to tumors in vivo using
polymeric drug carriers.
[0136] In certain embodiments, the disclosure relates to methods
comprising preoperatively administering a composition comprising
nanoparticles disclosed herein and monitoring the location of the
particles in the subject by detecting it by MRI (magnetic resonance
imaging) in an area of the subject. In certain embodiments, the
method further comprises the steps of operating on the subject in
the area of detected particles, imaging dye identified tumors
binding the targeting molecule, and surgically removing dye
identified tumors or tissue.
[0137] In certain embodiments, the disclosure relates to methods
comprising preoperatively administering cancer targeted
nanoparticles conjugated to dyes disclosed herein to a subject,
optically imaging a tumor that bind the nanoparticles
intra-operatively, and removing tumors targeted with the
nanoparticles.
[0138] In certain embodiments, the disclosure contemplates imaging
and effecting cancer cell lysis or other cell lysis with particles
using iron or iron oxide cores. See WO2009/120702.
[0139] In certain embodiments, the disclosure relates to targeting
of cancer by local hyperthermia using composition and methods
disclosed herein. Local hyperthermia can lead to induction of
apoptosis, heat-shock protein release, and chemotherapy agent
sensitivity of cancer cells by exposure of cancer cells containing
particles with an iron or iron oxide core to an alternating
magnetic fields (<1000 kHz) that are safe to normal cells.
[0140] In certain embodiments, the disclosure relates to methods
for lysis of a cancer cells comprising, administering to a subject
particles disclosed herein and adjusting magnetic fields proximate
the subject to cause cell lysis of cancer cell that absorb the
particles after administration. Typically, the magnetic field is an
oscillating magnetic field and the particles are heated to at least
37.degree. C. in vivo typically greater than 41.degree. C.
Pharmaceutical Compositions
[0141] In certain embodiments, the disclosure relates to
pharmaceutical compositions comprising particles disclosed herein
and a pharmaceutically acceptable excipient. In certain
embodiments, the composition is a pill or in a capsule or the
composition is an aqueous buffer, e.g., a pH between 6 and 8. In
certain embodiments, the pharmaceutically acceptable excipient is
selected from a filler, glidant, binder, disintegrant, lubricant,
and saccharide. Optionally, the pharmaceutical composition further
comprises a second anticancer agent.
[0142] Compositions suitable for parenteral injection may comprise
physiologically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers, diluents
solvents or vehicles include water, ethanol, polyols (propylene
glycol, polyethylene glycol, glycerol, and the like), suitable
mixtures thereof, vegetable (such as olive oil, sesame oil and
viscoleo) and injectable organic esters such as ethyl oleate.
[0143] Prevention of the action of microorganisms may be controlled
by addition of any of various antibacterial and antifungal agents,
example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. It may also be desirable to include isotonic agents, for
example sugars, sodium chloride, and the like. Prolonged absorption
of the injectable pharmaceutical form can be brought about by the
use of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0144] Solid dosage forms for oral administration include capsules,
tablets, pills, powders and granules. In such solid dosage forms,
the particles may be admixed with at least one inert customary
excipient (or carrier) such as sodium citrate or dicalcium
phosphate or: (a) fillers or extenders, as for example, starches,
lactose, sucrose, glucose, mannitol and silicic acid, (b) binders,
as for example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for
example, glycerol (d) disintegrating agents, as for example,
agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain complex silicates, and sodium carbonate, (e) solution
retarders, as for example paraffin, (f) absorption accelerators, as
for example, quaternary ammonium compounds, (g) wetting agents, as
for example cetyl alcohol, and glycerol monostearate, (h)
adsorbents, as for example, kaolin and bentonite, and (i)
lubricants, as for example, talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, or
mixtures thereof. In the case of capsules, tablets, and pills, the
dosage forms may also comprise buffering agents.
[0145] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar and as high molecular weight
polyethylene glycols, and the like.
[0146] Solid dosage forms such as tablets, capsules, pills, and
granules can be prepared with coatings and shells, such as enteric
coatings and others well known in the art. They may contain
opacifying agents, and can also be of such composition that they
release the particles in a certain part of the intestinal tract in
a delayed manner. Examples of embedding compositions which can be
used are polymeric substances and waxes.
[0147] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs. In addition to the particles, the liquid
dosage forms may contain inert diluents commonly used in the art,
such as water or other solvents, solubilizing agents and
emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in
particular, cottonseed oil, groundnut oil, corn germ oil, olive
oil, viscoleo, castor oil and sesame oil, glycerol,
tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid
esters of sorbitan or mixtures of these substances, and the
like.
[0148] Besides such inert diluents, the composition can also
include adjuvants, such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the particles, may contain suspending
agents, as for example, ethoxylated iso-stearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite agar-agar and
tragacanth, or mixtures of these substances, and the like.
[0149] Pharmaceutical compositions typically comprise an effective
amount of particles and a suitable pharmaceutical acceptable
carrier. The preparations can be prepared in a manner known per se,
which usually involves mixing the particles according to the
disclosure with the one or more pharmaceutically acceptable
carriers, and, if desired, in combination with other pharmaceutical
active compounds, when necessary under aseptic conditions.
Reference is made to U.S. Pat. Nos. 6,372,778, 6,369,086, 6,369,087
and 6,372,733 and the further references mentioned above, as well
as to the standard handbooks, such as the latest edition of
Remington's Pharmaceutical Sciences.
[0150] The pharmaceutical preparations of the disclosure are
preferably in a unit dosage form, and can be suitably packaged, for
example in a box, blister, vial, bottle, sachet, ampoule or in any
other suitable single-dose or multi-dose holder or container (which
can be properly labeled); optionally with one or more leaflets
containing product information and/or instructions for use.
Generally, such unit dosages will contain between 1 and 1000 mg,
and usually between 5 and 500 mg, of the particles of the
disclosure e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit
dosage.
[0151] The particles can be administered by a variety of routes
including the oral, ocular, rectal, transdermal, subcutaneous,
intravenous, intramuscular or intranasal routes, depending mainly
on the specific preparation used. The particles will generally be
administered in an "effective amount," by which it is meant any
amount of particles that, upon suitable administration, is
sufficient to achieve the desired therapeutic or prophylactic
effect in the subject to which it is administered. Usually,
depending on the condition to be prevented or treated and the route
of administration, such an effective amount will usually be between
0.01 to 1000 mg per kilogram body weight of the subject per day,
more often between 0.1 and 500 mg, such as between 1 and 250 mg,
for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per
kilogram body weight of the subject per day, which can be
administered as a single daily dose, divided over one or more daily
doses. The amount(s) to be administered, the route of
administration and the further treatment regimen can be determined
by the treating clinician, depending on factors such as the age,
gender and general condition of the subject and the nature and
severity of the disease/symptoms to be treated.
[0152] Formulations containing particles described herein can be
prepared using a pharmaceutically acceptable carrier composed of
materials that are considered safe and effective and can be
administered to an individual without causing undesirable
biological side effects or unwanted interactions. The carrier is
all components present in the pharmaceutical formulation other than
the active ingredient or ingredients. As generally used herein
"carrier" includes, but is not limited to, diluents, binders,
lubricants, disintegrators, fillers, pH modifying agents,
preservatives, antioxidants, solubility enhancers, and coating
compositions.
[0153] Carrier also includes all components of the coating
composition which can include plasticizers, pigments, colorants,
stabilizing agents, and glidants. Delayed release, extended
release, and/or pulsatile release dosage formulations can be
prepared as described in standard references such as
"Pharmaceutical dosage form tablets," eds. Liberman et. al. (New
York, Marcel Dekker, Inc., 1989), "Remington--The science and
practice of pharmacy," 20th ed., Lippincott Williams & Wilkins,
Baltimore, Md., 2000, and "Pharmaceutical dosage forms and drug
delivery systems," 6th Edition, Ansel et al., (Media, Pa.: Williams
and Wilkins, 1995). These references provide information on
carriers, materials, equipment and process for preparing tablets
and capsules and delayed release dosage forms of tablets, capsules,
and granules.
[0154] Examples of suitable coating materials include, but are not
limited to, cellulose polymers such as cellulose acetate phthalate,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate and hydroxypropyl
methylcellulose acetate succinate; polyvinyl acetate phthalate,
acrylic acid polymers and copolymers, and methacrylic resins that
are commercially available under the trade name EUDRAGIT.RTM. (Roth
Pharma, Westerstadt, Germany), zein, shellac, and
polysaccharides.
[0155] Disintegrants are used to facilitate dosage form
disintegration or "breakup" after administration, and generally
include, but are not limited to, starch, sodium starch glycolate,
sodium carboxymethyl starch, sodium carboxymethylcellulose,
hydroxypropyl cellulose, pregelatinized starch, clays, cellulose,
alginine, gums or cross linked polymers, such as cross-linked PVP
(Polyplasdone XL from GAF Chemical Corp).
[0156] Stabilizers are used to inhibit or retard decomposition
reactions which include, by way of example, oxidative
reactions.
[0157] A "pharmaceutical composition" or "pharmaceutically
acceptable" composition, is defined as a therapeutically effective
amount of one or more of the compositions described herein,
formulated together with one or more pharmaceutically acceptable
carriers (additives) and/or diluents. As described in detail, the
pharmaceutical compositions of the present disclosure can be
specially formulated for administration in solid or liquid form,
including those adapted for the following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection
as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as
a cream, ointment, or a controlled-release patch or spray applied
to the skin, lungs, or oral cavity; intravaginally or
intrarectally, for example, as a pessary, cream or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary and to
other mucosal surfaces.
[0158] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0159] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent material, involved in carrying or transporting the subject
compound from one organ, or portion of the body, to another organ,
or portion of the body. Each carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: sugars, such as lactose, glucose and sucrose; starches,
such as corn starch and potato starch; cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; pH buffered solutions;
polyesters, polycarbonates and/or polyanhydrides; and other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0160] Wetting agents, emulsifiers, and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0161] Examples of pharmaceutically-acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0162] The compositions of the present disclosure can be given in
dosages, generally, at the maximum amount while avoiding or
minimizing any potentially detrimental side effects. The
compositions can be administered in effective amounts, alone or in
a cocktail with other compounds, for example, other compounds that
can be used to treat a disease. An effective amount is generally an
amount sufficient to inhibit the disease within the subject.
[0163] One of skill in the art can determine what an effective
amount of the composition is by screening the composition using
known methods. The effective amounts may depend, of course, on
factors such as the severity of the condition being treated;
individual patient parameters including age, physical condition,
size, and weight; concurrent treatments; the frequency of
treatment; or the mode of administration. These factors are well
known to those of ordinary skill in the art and can be addressed
with no more than routine experimentation. In some cases, a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment.
[0164] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this disclosure can be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0165] The selected dosage level may depend upon a variety of
factors including the activity of the particular compound of the
present disclosure employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular compound being
employed, the duration of the treatment, other drugs, compounds
and/or materials used in combination with the particular compound
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0166] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the disclosure
employed in the pharmaceutical composition at levels lower than
that required to achieve the desired therapeutic effect and then
gradually increasing the dosage until the desired effect is
achieved.
[0167] In some embodiments, a compound or pharmaceutical
composition of the disclosure is provided to a subject chronically.
Chronic treatments include any form of repeated administration for
an extended period of time, such as repeated administrations for
one or more months, between a month and a year, one or more years,
or longer. In many embodiments, a chronic treatment involves
administering a compound or pharmaceutical composition of the
disclosure repeatedly over the life of the subject. For example,
chronic treatments can involve regular administrations, for example
one or more times a day, one or more times a week, or one or more
times a month. In general, a suitable dose such as a daily dose of
a compound of the disclosure will be that amount of the compound
that is the lowest dose effective to produce a therapeutic effect.
Such an effective dose will generally depend upon the factors
described above.
[0168] While it is possible for a composition of the present
disclosure to be administered alone, it can be administered as a
pharmaceutical formulation (composition) as described above.
Examples
Designed and Synthesized Two PD-L1 Blocking Peptides.
[0169] Although antibodies against PD-1 or PD-L1 have been used as
immune checkpoint blocker for immunotherapy, due to their bulky
size, conjugation of antibodies to the surface of the IONPs will
significantly increase the size of the IONPs thus potentially
inhibiting its efficient extravasation deep into tumor tissues.
Additionally, only 2 to 3 antibodies can be conjugated to a single
nanoparticle with a particle size of 20 nm.
[0170] To increase the efficiency of PD-L1 blocking in tumor
tissues, it is important to increase the numbers of PD-L1 blocking
ligands on each nanoparticles. It was discovered that 20 to 50
short peptides (35 AA) could be conjugated to a single nanoparticle
coated with polymer and NTA-cu. To select a peptide-blocking agent
with a high affinity in PD-1 binding, two PD-L1 blocking peptides
were designed and synthesized. Those peptides were derived from the
PD-L1 binding domains of PD-1, which would bind to the PD-L1
expressed by tumor cells and stromal fibroblasts and macrophages,
and inhibit the binding of PD-1+ cells, namely T cells, and
potentially rescue these cells from exhaustion and anergy, thus
enhancing localized, cellular immune responses.
[0171] Asterisk marked amino acids are identified as "hot spots" of
PD-1 for PD-L1 binding (FIG. 1A). PD-L1 blocking peptides were
prepared (FIG. 1B). PD-1(Y): two PD-L1 binding domains of PD-1
separated by his-tag (4.times.) for highly affinity binding and
PD-1(Lin): TwoPD-L1 binding domains fused as a single peptide with
his-tag at C-terminal.
[0172] Since the binding affinity of the native PD-1 to PD-L1 is
not very high (.about.8.5 .mu.M), amino acid adjustments were some
in the PD-1 peptide to improve the binding and secondary structure.
As shown in FIG. 1A, 5 of 17 amino acids in the first binding
domain, and 4 of 14 amino acids in the second binding domain have
been strategically replaced. For conjugation of such a short
peptide to nanoparticles, it is important to ensure the correct
orientation of the binding domains for high affinity binding. Four
histidine residues are added between two binding domains (PD-1 Y),
or by the end of the peptide (PD-1 (Lin)) for conjugating the
peptides to the NTA-Cu on the surface of nanoparticles. Structural
analysis of PD-1 Y and PD-1 (Lin) peptides revealed that the
presence of his-tag in the middle of PD-1 Y peptide created a "Y"
shaped peptide with two binding domain extended out for effective
binding to PD-L1. However, PD-1 (Lin) peptide has one binding
domain exposed but the second binding domain is covered by his-tag.
Thus, conjugation of PD-1(Lin) through His-tag-NTA-cu may interfere
with the binding of the second domain to PD-L1.
TABLE-US-00001 (SEQ ID NO: 2, MW 4111.56) PD-1
(Y)-NWNRLSPSNQTEKQAAPHHHHCGAISLHPKAKIEE (SEQ ID NO: 3, MW: 3967.43)
PD-1(Lin)-NWNRLSPSNQTEKQAACGAISLHPKAKIEESPGHHHH
Conjugation of His Tagged PD-1(Y) or PD-1 (Lin) Peptides to 5 nm
Core Size and Polymer Coated IONPs that are Functionalized by
NTA-Cu on the Surface of IONPs
[0173] To determine the ability of PD-1 like peptides in binding to
and blocking PD-L1, both peptides were conjugated to the
nanoparticles mediated by a histidine (His) tag located either at
the C-terminus (Lin) or in the middle of the sequence (PD1 (Y)) to
NTA-Cu on the surface of IONPs (FIG. 2). PD-1 like peptides were
mixed with NTA-cu-IONP (core size 5 nm) at a molar ratio of
peptide. IONP at 20:1 for 4 hours at room temperature. Unbound
peptides were removed by 100K column spinning. If optical imaging
was performed, PD-1 like peptides were labeled with maleimide
NIR-830 dye. Ultra-small magnetic iron oxide nanoparticles (IONP)
with a core size of 3.5 having an anti-fouling polymer coating were
also produced.
Hyaluronic Acid Polymeric Nanoparticle (HANP) Using Cys-Thiol and
Maleimide Mediated Conjugation
[0174] Hyaluronic acid nanoparticles may be prepared by chemical
conjugation of hydrophobic molecules. The outer hydrophilic shell
prevents uptake by the reticuloendothelial system (RES). The
hydrophobic moieties may be attached through the carboxylic acid
group of HA using carbodiimide chemistry. Hyaluronic acid
nanoparticles (HANP) were prepared by covalently conjugating
hyaluronic acid (HA) with a hydrophobic moiety, 51-cholanic acid.
The resulting nanoparticle has hydrophilic surface and hydrophobic
inside, where hydrophobic chemotherapy drugs can be encapsulated.
After administrated in vivo, the enhanced permeability retention
(EPR) effect and inherent CD44 targetability make HANP a dual tumor
targeting drug carrier.
[0175] A urokinase-type plasminogen activator receptor and matrix
metalloprotease 14 fusion protein, ATFmmp14, were conjugated onto
surface of HANP/drug complex. HANP can be used to deliver
therapeutic agents, such as SN38, camptothecin, paclitaxel and many
other hydrophobic chemotherapy drugs as well as small molecule
drugs. These protease-linked HANPs have the ability to break tumor
stromal matrix and migrate inside tumor tissue to improve
intratumoral nanoparticle distribution and potentially increase
drug delivery into tumor cells.
[0176] The protease-linked and targeted HANP should have broad
applications in targeted cancer therapy for many types of human
cancers that have extensive tumor stromal components, such as
pancreatic, triple negative breast, skin, head and neck, liver,
sarcoma, and prostate cancers. The protease-linked and targeted
HANP is capable of carrying different hydrophobic chemotherapy
drugs for cancer-targeted therapy. Receptor targeted HANP for drug
delivery into uPAR, EGFR, IGF-1R, or Her-2, positive tumor cells. A
near infrared dye can be conjugated to the protease-linked
targeting ligands, providing optical imaging capability. This
stroma breaking polymeric drug delivery platform can be used for
the treatment of non-oncology diseases with severe fibrotic and
inflammatory tissues that prevent drug delivery into diseased cells
or targets, such as chronic viral hepatitis, liver cirrhosis, fat
liver, chronic tuberculosis, and atherosclerosis.
[0177] Hyaluronic acid polymeric nanoparticle (HANP) with a size of
about 150 nm were designed using a unique cysteine in the loop
region between two binding domains mediated by thiol side group
(See SEQ ID NO: 2 and 3) and maleimide. These methods conjugate
short peptides to the nanoparticle surface while retaining a good
binding affinity. Furthermore, about 20 to 30 peptides can be
conjugated to one nanoparticle. For near infrared optical imaging,
NIR 830 dye can also be conjugated to the peptides or
nanoparticles, resulting in two IONP based theranostic PDL1
blocking nanoparticles with MRI and optical imaging ability, and
one theranostic PDL1 polymeric HANP with NIR optical imaging
ability.
Specific Binding of PD-1 Like Peptides to PD-L1 Expressing Mouse
Pancreatic Tumor Cell Lines
[0178] Specific binding of PD-1 like peptides to PD-L1 has been
demonstrated in vitro using three different assays. First,
incubation of PD-1 Y or PD-1 Lin peptide conjugated IONPs with
mouse pancreatic cancer cell lines, including Kras transgenic mouse
pancreatic cancer derived KC and mouse pancreatic cancer Panc02
cells, led to the IONP binding tumors cells that were detected by
Prussian blue staining. Cells incubated with control non-targeted
poly-gly peptide conjugated IONPs had a very low level of
IONPs.
[0179] Next, a competition-binding assay was performed to determine
specificity of the PD-1 like peptides binding to PD-L1 on tumor
cell surface. Fluorescent dye (FITC) labeled PD-1 Y or PD-1 Lin
peptides (2 .mu.g/ml) were mixed with different concentrations of
an anti-mouse PD-L1 monoclonal antibody (BioX cell) that has been
shown to block PD-L1, and then added to tissue culture plate with
mouse pancreatic cancer KC cells. High levels of fluorescence
intensity on cells treated both PD-1 Y and PD-1 Lin peptides (FIG.
3A). However, the binding of PD-1 Y to cells could be reduced by
addition of a high concentration (20 .mu.g/ml) of the anti-PD-L1
antibody, suggesting that the PD-1 Y binding site on PD-L1 is the
same as the PD-L1 antibody (FIG. 12). On the other hand, PD-1 Lin
peptide bound to tumor cells at a higher level than PD-1 Y
peptides. Its binding could not be competed off by the high
concentration of anti-PD-L1 antibody (20 .mu.g/ml). It is possible
that in vitro, PD-1 Lin is able to bind PD-L1 with a high affinity
that anti-PD-L1 antibody failed to compete off. However, it is also
possible that PD-1 Lin peptide has a different binding site on
PD-L1 compared to the anti-PD-L1 antibody. However, based on the
computation analysis of peptide structures and interaction site as
shown in FIG. 1 to 2, it is likely that PD-1 Y peptide binds
specifically to the interaction sites of PD-1 to PD-L1.
[0180] A pull-down assay was conducted to confirm the binding of
PD-1 like peptides specifically to PD-L1 protein on tumor cells.
Cell lysates of mouse pancreatic cancer KPC cells derived from a
Kras/p53 transgenic mouse pancreatic tumor were incubated with
agarose beads coated with PD-1 like peptides through his-tag and
NTA-Ni. Both PD-1 like peptides pulled down a high level of PD-L1
proteins, indicating specific binding of the PD-1 like peptides to
PD-L1 (FIG. 3B).
PD-1 Like Peptides are Capable of Targeted Delivery of IONPs into
Orthotopic Pancreatic Tumors Following Intraperitoneal (i.p.) or
Systemic Delivery
[0181] PD-L1 is upregulated in many tumor cells and tumor stromal
cells. PD-1 like peptide-conjugated nanoparticles have the
potential to serve as tumor targeted nanoparticles to be delivered
into tumors for tumor imaging and immunotherapy. PD-1 like peptides
can be used for targeted delivery of nanoparticles into tumors
following systemic administration. Efficient delivery of PD-1 Y
conjugated IONPs into pancreatic tumors has been tested in three
mouse pancreatic tumor models and one human orthotopic pancreatic
cancer patient tissue derived xenograft (PDX) model.
[0182] An orthotopic mouse PANC02 pancreatic tumor model was
established by injecting 5.times.10.sup.4 of PANC02 cells directly
into the pancreas of the mice. Tumor bearing mice then received 5
treatments of i.p. delivery of NIR 830 dye labeled PD1-IONP
conjugates and a non-targeting control that was conjugated with a
poly-glycine peptide (Gly-IONPs) beginning on day 4 post tumor cell
implantation once every 3 days (FIG. 4A). Mice were sacrificed and
the abdominal cavity was opened to expose the location of the
orthotopic pancreatic tumors. NIR optical imaging was performed to
determine selective accumulation of the PD-1 like
peptide-conjugated IONPs into pancreatic tumors. High levels of
optical signal were detected in the orthotopic tumors, indicating
targeted delivery into the tumors. Histological analysis of tumor
tissues using Prussian blue staining revealed a high level of blue
iron containing cells in the tumor obtained from the mice that
received i.p. delivery of PD-1(Y)-IONPs (FIG. 4B). Low to
intermediate levels of IONP positive cells were seen in the tumors
of the mice treated with non-targeted IONPs or PD-1(Lin)-IONPs
(FIG. 4B). Taken together, PD-1 IONPs capable of binding to PD-L1
and expressing pancreatic tumor cells in vitro were developed. In
vivo targeting to orthotopic PANC02 tumors was demonstrated.
[0183] Effective targeted delivery of IONP by PD-1 like peptides
into the orthotopic pancreatic tumors following i.p. delivery was
also demonstrated in a mouse pancreatic KC tumor model that was
established by intra-pancreatic injection of the UN-KC-6141 tumor
cell line (KC) derived from the Pdx-1-Cre;LSL-K-rasG12D mice. One
week following tumor cell injection, the mice received i.p.
delivery of 800 pmol of NIR-830 dye labeled PD-1 Lin-IONP,
PD-1Y-IONP, or non-targeted Gly-IONP once every three days for
three injections. Non-invasive whole body optical imaging performed
48 hours following the last IONP injection showed strong optical
signals in the pancreatic tumor areas. A low level of the optical
signal was found in the tumor area of the mice injected with
non-targeted IONPs.
[0184] Following sacrificing mice, ex vivo optical imaging showed
the strongest optical signal in the tumor obtained from the mice
that received PD-1Y-IONP injection. NIR signal in the tumor
obtained from the mice that received PD-1Lin-IONP was lower than
that of the PD-1Y-IONP treated tumors, but was higher than that of
non-targeted Gly-IONP treated tumors. Histological analysis of
tumor tissues further confirmed the presence of a high level of
IONP positive cells (blue stained cells) in the pancreatic tumors
treated with PD-1Y-IONPs. A high level of NIR signal was also
co-localized with the IONP positive tumor cell areas. Therefore,
results from two different mouse pancreatic tumor models both
showed that PD-1Y-IONPs have a higher efficiency in targeted
delivery into pancreatic cancers compared to PD-1 Lin-IONPs.
[0185] Mouse PD-1 can bind to human PD-L1 and human PD-1 also binds
to mouse PD-L1. With a translational goal, a mouse PD-1 peptide
sequence was used to engineer the PD-1 like peptides so that the
same reagent can be used for future clinical applications.
PD-1Y-IONP was able to bind to primary culture of human pancreatic
cancer cells derived a human pancreatic cancer patient
tissue-derived xenograft (PDX) in mice (FIG. 5). I.p. delivery of
the NIR-830-dye-PD-1 Y-IONPs into the nude mice bearing the
orthotopic human pancreatic PDX tumor xenografts showed the
selective accumulation of the nanoparticles in the pancreatic
tumors. Therefore, PD-1 Y-IONP has the potential for further
development as a targeted immunotherapy agent for the applications
in human cancer patients.
[0186] The effect of systemic delivery of PD-1 peptide conjugated
IONPs on tumor growth and function of immune cells in tumors using
a subcutaneous (s.c) tumor model derived from KC tumor cell line
was examined. One of the studies used a mouse tumor model bearing
two s.c. tumors on the each side of the flank areas, derived from
the mouse pancreatic tumor KC cell line and the UN-KPC-691 (KPC)
cell line that was established from mouse pancreatic cancer from
Pdx1-Cre/LSL-KRAS/p53 R175H transgenic mice.
[0187] Following three systemic deliveries of PD-1 Lin-IONP or PD-1
Y-IONP for 48 hours, strong NIR optical signal was detected in both
KC and KPC tumors on the same mouse. Since KPC tumors grew slower
than KC tumor in the wild type Kras transgenic mice, the mean
signal intensity was lower than that of detected in KC tumor.
Consistent with results in orthotopic pancreatic tumor models using
i.p. delivery of the PD-1-IONPs, intravenous delivery of the
nanoparticles led to a higher level of the accumulation of PD-1
Y-IONP into s.c. KC or KPC tumors compared to that of PD-1 Lin-IONP
treated tumors. Results of ex vivo imaging of normal organs also
showed a very low signal in normal organs, including the liver,
spleen, lung, and heart. Low to intermediate levels of optical
signals were detected in some areas of intestine and kidney, which
may be due to elimination of breaking-down components of the
nanoparticles.
[0188] Forty-eight hours following the last of three systemic
deliveries of PD-1 Y-IONP or non-targeted Gly-IONP (800 pmol/each
dose) into the mice bearing s.c. KC tumors, T2-weighted MRI using
3T MRI scanner was performed on the mice. In comparison with the
MRI contrast in tumors before and after the nanoparticle
injections, we found marked MRI T2 signal decrease in the tumor
treated with PD-1 Y-IONPs, which provides another evidence of
targeted delivery into tumors. Results of this study also suggested
that possibility of using NIR-830-dye-PD-1Y-IONP as an imaging
probe for non-invasive detection of PD-1L expression in human
tumors as a therapeutic indication for PD-1/PD-L1 targeted
immunotherapy.
Targeted Delivery of PD-1 Lin and PD-1 Y-IONP into Pancreatic
Tumors Increase Both CD4 and CD8 TIL Cell Population
[0189] To evaluate the effect of targeted delivery of PD-1 peptide
conjugated IONPs on tumor growth and changes in immune cell
population and function, TIL cells were examined in the KC
pancreatic tumor model. Following three i.v. deliveries of
PD-1Lin-IONP or PD-1Y-IONP into mice bearing s.c. tumors, tumors
were collected and TIL cells were isolated. Cells were labeled with
CD4 and PD-1 antibodies or CD8 and PD-1 antibodies, and then
analyzed by flow cytometry. Targeted delivery of PD-1 Lin and PD-1
Y-IONP into pancreatic tumors increase both CD4 and CD8 TIL cell
population (FIG. 8). Nanoparticle-induced infiltration of TIL cells
into tumor tissues should create a pro-immune environment for
activation of tumor specific cytotoxic T cell responses. Although
the level of PD-1 Lin-IONP in tumors was found lower than
PD-1Y-IONP, similar levels of TIL cells were detected in both
groups. In another orthotopic KC tumor model, three i.p. deliveries
of PD-1Y-IONPs led to the increased level of CD8 TIL cells in both
tumor edge and tumor central areas.
In Vivo Delivery of PD-1 Peptide Conjugated IONPs Inhibited the
Growth of s.c. Pancreatic Tumors in the KC Cell Derived Transgenic
Pancreatic Tumor Model
[0190] To determine whether targeted delivery of PD-1 like peptide
conjugated IONPs into tumor tissues can block PD-L1 immune
checkpoint and produces an anti-tumor effect, the effect of
systemic delivery of the PD-1Y-IONPs and an anti-mouse PD-L1
antibody was compared on the growth of the KC tumors injected s,c.
into Kras wild type mice (FIG. 7A). Following four injections,
there was no apparent body weight change and other symptoms of
systemic toxicity. Significant inhibition of tumor growth was found
in mice treated with PD-1Y-IONP or anti-PD-L1 antibody compared to
non-targeted Gly-IONP control (p<0.05) (FIGS. 7B and C).
Pancreatic Cancer Specific T Cell Cyto-Toxicity in the KC Mouse
Pancreatic Cancer Model
[0191] To determine if tumor specific T cell response could be
activated by targeted delivery of PD-1-IONPs, splenocytes from the
orthotopic KC tumor bearing mice that received three i.p.
deliveries of PD-1 Lin-IONP or PD-1 Y-IONP were collected. A CD3
positive T cell population was isolated using magnetic beads and
then added to tissue culture plates with KC cells at a ratio of 1
tumor cell to 10 T cells. 72 hours following co-culture,
alarmaBlue.TM. cell proliferation was performed. There was a
significant decrease in cell viability in KC cells incubated with T
cells from the mice treated with PD-1Y-IONP, compared with no
treatment control or treated with PD-1 Lin-IONP (FIG. 8).
The Combination of uPAR Targeted Delivery of Theranostic IONPs with
Immune Check Point Blocking Using an Anti-PD-L Antibody
[0192] Current clinical trials using therapeutic antibodies to
block PD-1/PD-L1 failed to show a good therapeutic effect in
pancreatic cancer patients, which is thought due to a low
immunogenicity of pancreatic cancer tissues and presence of a dense
tumor stromal barrier that prevents efficient delivery of
antibodies into tumors as well as blocking infiltration of T cells
into the tumor tissues. Therefore, the combination therapy with
targeted theranostic nanoparticle carrying chemotherapy agents has
the potential to break tumor stromal barrier for improved delivery
of PD-1L blocking agents and to kill tumor cells for releasing
neo-tumor antigen. Delivery of nanoparticles into tumor tissue also
promotes infiltration of antigen presenting cells into tumor center
and increase TIL cell population.
[0193] The feasibility of the combination therapy was evaluated
using an anti-mouse PD-L1 monoclonal antibody (BioX cell) in
combination with uPAR-targeted ATF peptide conjugated IONP carrying
cisplatin in the orthotopic Panc02 pancreatic tumor model in C57/B6
mice. Following four i.p. deliveries of mouse (m)ATF-IONP-cisplatin
(5 mg/kg Cisplatin dose equivalent IONPs), control nanoparticles or
antibodies, significant tumor growth inhibition was found in the
tumor bearing mice received mATF-IONP-cisplatin alone or
mATF-IONP-cisplatin and anti-PD-L1 antibody. The combination of
mATF-IONP-cisplatin with anti-PD-L1 antibody showed enhanced
therapeutic effect in Panc02 tumors. There was also significant
reduction in the amount of ascites produced in the mice (FIG.
9A-C).
TABLE-US-00002 Avg Tumor Growth Avg Ascites Fluid Treatments
Inhibition (%) Inhibition (%) mATF-PEG-IONP-Cis 49.1 74.5
.alpha.PD-L1 29.9 55.6 mATF-PEG-IONP-Cis + 73.4 81.7
.alpha.PD-L1
[0194] Levels of CD4 or CD8+TIL cells were analyzed the in the
tumors after treatment. The combination therapy increased the
levels of CD4 and CD8 T cells in tumors. Therefore, results of this
study showed that the combination of targeted therapy using a
nanoparticle drug carrier with targeted delivery of PD-L1 blocking
nanoparticles is a promising approach for the development of
effective cancer therapeutic agents.
Targeted Delivery of IONPs and Theranostic IONPs into Tumors
Induces Immune Cell Infiltration for Enhanced Tumor Specific T Cell
Response
[0195] Immune therapy has not shown good responses in clinical
trials for the treatment of pancreatic cancer. One of the potential
mechanisms is thought to be the presence of tumor stromal barriers
that limit delivery of therapeutic antibodies into tumor cell nest.
Immunosuppressive cytokines also create a biological barrier for
immune cells and immune function. It has also been shown that human
pancreatic cancer tissues lack cytotoxic T cells. Additionally,
pancreatic cancer has a low immunogenicity compared to other cancer
types. It is important to combine cytotoxic agents to destroy tumor
cells and release potentially immunogenic mutant proteins.
[0196] Studies indicate that targeted delivery of theranostic IONPs
into pancreatic cancer tissue promoted infiltration of immune cells
into the tumor center. Four systemic deliveries of uPAR targeted
and stroma break PD1Y-IONP, ATFmmp14-IONP-Doxs, or the combination
of both nanoparticles resulted in a high level of nanoparticle
accumulation in pancreatic tumors in the Kras transgenic mouse
model. However, NIR signal was not detectable in the tumor tissue
section from NIR 830 dye labeled non-targeted IONP treated mouse.
Those frozen tissue sections were further labeled with antibodies
against CD3 and CD8. CD4 was not used since it also reacts with a
subpopulation of macrophages. Immunofluorescence labeling using
antibodies for total T cell (CD3) or cytotoxic T cell (CD8)
revealed that KPC tumor tissues have an intermediate level of CD3+
T cells but lack CD8+ cytotoxic T cells. These CD3+/CD8-T cells are
likely to be CD4 T cells with subpopulation of these cells being T
suppressor cells. However, ATPmmp14-IONP-Dox treated tumor tissues
had marked increases in CD8+ T cell infiltration and those T cells
infiltrated into and surrounded ductal tumor cells. A high
percentage of CD3+ T cells are also CD8+ cells, suggesting
significant increase in the cytotoxic T cells in the tumor tissue.
PD1Y-IONP treatment slightly increased CD3 and CD8+ T cells but
uPAR targeted ATPmmp14-IONP-Dox theranostic IONP or the combination
of both IONP treated tumors had marked increases in the CD8+ and
CD3+ T cells (FIG. 10). The observed effect of modulating T cell
populations in tumor tissues following targeted delivery of
theranostic IONPs is very significant for improving the therapeutic
response to anti-PD1/PDL-1 checkpoint medicated immunotherapy of
pancreatic cancer.
[0197] The effect of targeted delivery of nanoparticles on
promoting intratumoral infiltration of T cells has also been
demonstrated in the Pdx1-Cre;LSL-K-rasGl2D P53Trp53R (KPC)
transgenic mouse pancreatic cancer model using another theranostic
IONP. Systemic delivery of PD1Y, ATFmmp14 or dual ATFmmp14+PD1Y
conjugated ultrafine IONPs (3.5 nm core) carrying SN38 (derivative
of CPT-11) into the KPC mice led to targeted delivery into
orthotopic pancreatic tumors that is detectable by optical imaging.
Immunofluorescence labeling of tumor tissue sections revealed
increased numbers of CD3 and CD8 T cells in the tumors.
Determination of PDL1 Targeted Delivery of HANP Polymeric
Nanoparticles into Human Breast Cancer Tissue Derived Xenograft
(PDX) Model.
[0198] To determine if PD1Y peptide can be used for targeted
delivery of other types of nanoparticles and for immunotherapy of
different human tumors, PD1Y peptides are conjugated to HANP
polymeric nanoparticle (150 nm). Systemic delivery of PD1Y-HANP
into the nude mice bearing human breast cancer tissue derived
xenograft (PDX) model led to the accumulation of the NIR 830 dye
labeled HANP and allowed optical imaging of breast tumor in mice.
Ex vivo organ imaging showed an optical signal in the tumor. In
normal organs, signal was only found in the liver and kidney.
ATFmmp14-HANP/SN38 Treatment Improved Therapeutic Effects in Both
Pancreatic and Breast Cancer Using PDX Tumor Models
[0199] To increasing drug delivery into tumor after systemic
administration, Urokinase plasminogen activator receptor (uPAR)
fused matrix metalloproteinase 14 was conjugated onto HANP/SN38
complex. ATFmmp14-HANP/SN38 complex has great potential in
improving cancer therapeutic effects. Efficacy studies were carried
out using ATFmmp14-HANP/SN38 in a human pancreatic cancer patient
tissue derived xenograft (PDX) model in nude mice. SN38
(7-ethyl-10-hydroxy-camptothecin) is an active metabolite of
chemotherapy drug, irinotecan. We found that systemic delivery of
ATFmmp14-HANP/SN38 significantly inhibited the growth of orthotopic
tumors in a human pancreatic PDX tumor model (FIG. 11A). Induction
of a high level of apoptotic cell death and significant reduction
of the level of stromal fibroblasts were detected in tumor tissues
following the targeted therapy, compared with control groups. Our
results showed that HANP/SN38 complex more efficiently ablates
pancreatic cancer growth (88%) with ATFmmp14, while HANP/SN38
without ATFmmp14 only showed less than 50% tumor growth inhibition,
suggesting that breaking stroma barrier is helpful for improving
cancer therapy response. The effect of the targeted HANP was
further evaluated in human breast cancer PDX models. Significant
tumor growth inhibition was also found in those PDX models derived
from both ER+ and triple negative breast cancer patients (FIG.
11B).
Sequence CWU 1
1
311288PRTHomo sapiens 1Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val
Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp
Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu
Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe
Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser
Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp
Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu
Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110Ala Arg
Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120
125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro
Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val
Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser Leu Val Leu Leu Val
Trp Val Leu Ala Val Ile Cys 180 185 190Ser Arg Ala Ala Arg Gly Thr
Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205Leu Lys Glu Asp Pro
Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220Glu Leu Asp
Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro225 230 235
240Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly
Pro Arg 260 265 270Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys
Ser Trp Pro Leu 275 280 285235PRTArtificialSynthetic 2Asn Trp Asn
Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala1 5 10 15Pro His
His His His Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys 20 25 30Ile
Glu Glu 35337PRTArtificialSynthetic 3Asn Trp Asn Arg Leu Ser Pro
Ser Asn Gln Thr Glu Lys Gln Ala Ala1 5 10 15Cys Gly Ala Ile Ser Leu
His Pro Lys Ala Lys Ile Glu Glu Ser Pro 20 25 30Gly His His His His
3544PRTArtificialSynthetic 4Gly Phe Leu
Gly15135PRTArtificialSynthetic 5Ser Asn Glu Leu His Gln Val Pro Ser
Asn Cys Asp Cys Leu Asn Gly1 5 10 15Gly Thr Cys Val Ser Asn Lys Tyr
Phe Ser Asn Ile His Trp Cys Asn 20 25 30Cys Pro Lys Lys Phe Gly Gly
Gln His Cys Glu Ile Asp Lys Ser Lys 35 40 45Thr Cys Tyr Glu Gly Asn
Gly His Phe Tyr Arg Gly Lys Ala Ser Thr 50 55 60Asp Thr Met Gly Arg
Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu65 70 75 80Gln Gln Thr
Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu 85 90 95Gly Lys
His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp 100 105
110Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val
115 120 125His Asp Cys Ala Asp Gly Lys 130
135668PRTArtificialSynthetic 6Ser Asn Glu Leu His Gln Val Pro Ser
Asn Cys Asp Cys Leu Asn Gly1 5 10 15Gly Thr Cys Val Ser Asn Lys Tyr
Phe Ser Asn Ile His Trp Cys Asn 20 25 30Cys Pro Lys Lys Phe Gly Gly
Gln His Cys Glu Ile Asp Lys Ser Lys 35 40 45Thr Cys Tyr Glu Gly Asn
Gly His Phe Tyr Arg Gly Lys Ala Ser Thr 50 55 60Asp Thr Met
Gly657280PRTArtificialSynthetic 7Met Ser Asn Glu Leu His Gln Val
Pro Ser Asn Cys Asp Cys Leu Asn1 5 10 15Gly Gly Thr Cys Val Ser Asn
Lys Tyr Phe Ser Asn Ile His Trp Cys 20 25 30Asn Cys Pro Lys Lys Phe
Gly Gly Gln His Cys Glu Ile Asp Lys Ser 35 40 45Lys Thr Cys Tyr Glu
Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 50 55 60Thr Asp Gly Ala
Pro Ile Gln Gly Leu Lys Trp Gln His Asn Glu Ile65 70 75 80Thr Phe
Cys Ile Gln Asn Tyr Thr Pro Lys Val Gly Glu Tyr Ala Thr 85 90 95Tyr
Glu Ala Ile Arg Lys Ala Phe Arg Val Trp Glu Ser Ala Thr Pro 100 105
110Leu Arg Phe Arg Glu Val Pro Tyr Ala Tyr Ile Arg Glu Gly His Glu
115 120 125Lys Gln Ala Asp Ile Met Ile Phe Phe Ala Glu Gly Phe His
Gly Asp 130 135 140Ser Thr Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala
His Ala Tyr Phe145 150 155 160Pro Gly Pro Asn Ile Gly Gly Asp Thr
His Phe Asp Ser Ala Glu Pro 165 170 175Trp Thr Val Arg Asn Glu Asp
Leu Asn Gly Asn Asp Ile Phe Leu Val 180 185 190Ala Val His Glu Leu
Gly His Ala Leu Gly Leu Glu His Ser Ser Asp 195 200 205Pro Ser Ala
Ile Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Glu Asn 210 215 220Phe
Val Leu Pro Asp Asp Asp Arg Arg Gly Ile Gln Gln Leu Tyr Gly225 230
235 240Gly Glu Ser Gly Phe Pro Thr Lys Met Pro Pro Gln Pro Arg Thr
Thr 245 250 255Ser Arg Pro Ser Val Pro Asp Lys Pro Lys Asn Pro Thr
Tyr Gly Pro 260 265 270Asn Ile His His His His His His 275
280817PRTArtificialSynthetic 8Asn Trp Asn Arg Leu Ser Pro Ser Asn
Gln Thr Glu Lys Gln Ala Ala1 5 10 15Pro914PRTArtificialSynthetic
9Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu1 5
101030PRTArtificialSynthetic 10Glu Ser Phe Val Leu Asn Trp Tyr Arg
Met Ser Pro Ser Asn Gln Thr1 5 10 15Asp Lys Leu Ala Ala Phe Pro Glu
Asp Arg Ser Gln Pro Gly 20 25 301138PRTArtificialSynthetic 11Pro
Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn1 5 10
15Asp Ser Gly Thr Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile
20 25 30Lys Glu Ser Leu Arg Ala 351216PRTArtificialSynthetic 12Asn
Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala1 5 10
151314PRTArtificialSynthetic 13Cys Gly Ala Ile Ser Leu Ala Pro Lys
Ala Gln Ile Lys Glu1 5 101417PRTArtificialSynthetic 14Asn Trp Asn
Arg Met Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala1 5 10
15Pro1517PRTArtificialSynthetic 15Asn Trp Asn Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Gln Ala Ala1 5 10
15Pro1617PRTArtificialSynthetic 16Asn Trp Asn Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala Ala1 5 10
15Pro1717PRTArtificialSynthetic 17Asn Trp Asn Arg Leu Ser Pro Ser
Asn Gln Thr Glu Lys Leu Ala Ala1 5 10
15Pro1817PRTArtificialSynthetic 18Asn Trp Asn Arg Leu Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala Ala1 5 10
15Pro1917PRTArtificialSynthetic 19Asn Trp Tyr Arg Met Ser Pro Ser
Asn Gln Thr Glu Lys Gln Ala Ala1 5 10
15Pro2017PRTArtificialSynthetic 20Asn Trp Tyr Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Gln Ala Ala1 5 10
15Pro2117PRTArtificialSynthetic 21Asn Trp Tyr Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala Ala1 5 10
15Pro2217PRTArtificialSynthetic 22Asn Trp Tyr Arg Leu Ser Pro Ser
Asn Gln Thr Glu Lys Leu Ala Ala1 5 10
15Pro2317PRTArtificialSynthetic 23Asn Trp Tyr Arg Leu Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala Ala1 5 10
15Pro2414PRTArtificialSynthetic 24Cys Gly Ala Ile Ser Leu Ala Pro
Lys Ala Lys Ile Glu Glu1 5 102514PRTArtificialSynthetic 25Cys Gly
Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Glu Glu1 5
102614PRTArtificialSynthetic 26Cys Gly Ala Ile Ser Leu Ala Pro Lys
Ala Gln Ile Lys Glu1 5 102714PRTArtificialSynthetic 27Cys Gly Ala
Ile Ser Leu His Pro Lys Ala Lys Ile Lys Glu1 5
102814PRTArtificialSynthetic 28Cys Gly Ala Ile Ser Leu His Pro Lys
Ala Gln Ile Lys Glu1 5
102935PRTArtificialSyntheticX(1)..(35)Wherein X is any amino acid
29Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala1
5 10 15Pro Xaa Xaa Xaa Xaa Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala
Gln 20 25 30Ile Lys Glu
353034PRTArtificialSyntheticX(1)..(34)Wherein X is any amino acid
30Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala1
5 10 15Pro Xaa Xaa Xaa Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln
Ile 20 25 30Lys Glu3136PRTArtificialSyntheticX(1)..(36)Wherein X is
any amino acid 31Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp
Lys Leu Ala Ala1 5 10 15Pro Xaa Xaa Xaa Xaa Xaa Cys Gly Ala Ile Ser
Leu Ala Pro Lys Ala 20 25 30Gln Ile Lys Glu 35
* * * * *