U.S. patent application number 17/682633 was filed with the patent office on 2022-06-16 for cell surface coupling of nanoparticles.
The applicant listed for this patent is MASSACHUSETTS INSTITUTE OF TECHNOLOGY (MITN1). Invention is credited to Darrell J. IRVINE, Li TANG, Yiran ZHENG.
Application Number | 20220185860 17/682633 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220185860 |
Kind Code |
A1 |
IRVINE; Darrell J. ; et
al. |
June 16, 2022 |
CELL SURFACE COUPLING OF NANOPARTICLES
Abstract
The present disclosure is directed, in some embodiments, to
methods and compositions of comprising a cell having a
non-internalizing receptor, and a nanoparticle surface-modified
with a ligand that binds to the non-internalizing receptor.
Inventors: |
IRVINE; Darrell J.;
(Arlington, MA) ; ZHENG; Yiran; (Cambridge,
MA) ; TANG; Li; (Quincy, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MASSACHUSETTS INSTITUTE OF TECHNOLOGY (MITN1) |
Cambridge |
MA |
US |
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Appl. No.: |
17/682633 |
Filed: |
February 28, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17318590 |
May 12, 2021 |
11261226 |
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17682633 |
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15236186 |
Aug 12, 2016 |
11034752 |
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17318590 |
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62204337 |
Aug 12, 2015 |
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International
Class: |
C07K 14/55 20060101
C07K014/55; C07K 14/705 20060101 C07K014/705; C07K 14/54 20060101
C07K014/54; A61K 9/127 20060101 A61K009/127; C07K 16/28 20060101
C07K016/28; C07K 14/715 20060101 C07K014/715; A61K 47/68 20060101
A61K047/68; A61K 47/69 20060101 A61K047/69; A61K 47/60 20060101
A61K047/60; A61K 39/00 20060101 A61K039/00; C07K 14/725 20060101
C07K014/725; C07K 16/30 20060101 C07K016/30; C07K 19/00 20060101
C07K019/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under Grant
No. R01 CA172164 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of treating cancer in a subject comprising
administering a composition to the subject, wherein the composition
comprises: a carrier cell that includes a cytokine receptor on its
surface; and a protein nanogel that includes cytokines that
interact with the cytokine receptor and are linked to each other
via a linker.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/318,590, filed May 12, 2021, now pending,
which application is a divisional of U.S. patent application Ser.
No. 15/236,186, filed on Aug. 12, 2016, now U.S. Pat. No.
11,034,752, which claims priority to and the benefit of U.S.
Provisional Patent Application No. 62/204,337, filed Aug. 12, 2015,
the entire contents of which are herein incorporated by
reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format via EFS-Web and
is hereby incorporated by reference in its entirety. Said ASCII
copy, created Feb. 28, 2022, is named
"MITN1-004DVCN_Sequence-Listing.txt" and is 14050 bytes in
size.
BACKGROUND OF THE INVENTION
[0004] Combining nanostructure (e.g.: nanoparticle-and liposome)
based drug delivery strategies with cell-based therapies has
created a lot of opportunities in developing novel therapeutic
modalities with improved efficacy and reduced toxicity for the
treatment of various diseases. For example, nanoparticles
containing supporting drugs (e.g., cytokines) can be coupled with
therapeutic cells (e.g., tumor reactive T cells) to boost the
cell-based therapy (Cancer Res. 2007, 67, 300-308; Nat. Med. 2010,
16, 1035-1041). Alternatively, certain types of cells (e.g.,
antigen-specific T cells, mesenchymal stem cells) can be used as
carriers to specifically target drug-contained nanoparticles to
disease site to improve therapeutic efficacy (Nat. Med. 2005, 11,
1073-1081; ACS Nano 2011, 5, 7462-7470).
SUMMARY OF THE INVENTION
[0005] This present disclosure provides, in some aspects, a method
for efficiently and stably coupling nanostructures to a carrier
cell surface (e.g., T cell surface) with minimum cellular
internalization, permitting extracellular drug (e.g., cytokine and
small molecule) delivery in vivo for various biological
applications, such as targeted immunotherapy. Typically, coupling
of a particle (or nanostructure) to the surface of a T cells
triggers internalization of the particle (or nanostructure)
through, for example, T cell receptor-mediated endocytosis or
membrane permeation. Such internalization mechanisms prevent T
cells, which naturally home in vivo to particular tissues, from
being utilized as carrier cells for the delivery of, for example,
therapeutic or diagnostic agents. Surprisingly, experimental
results provided herein show that nanostructures surface-modified
with (e.g., conjugated to), for example, a ligand that binds to
CD45 (e.g., an anti-CD45 antibody) are maintained at the surface of
T cells expressing CD45. Thus, the present disclosure unexpectedly
shows that not all T cell receptors promote receptor-mediated
endocytosis. Some molecules, such as CD45, are capable of
maintaining particles at the surface of the carrier cell. Also
surprising are results showing that CD45, in particular, even when
expressed in combination with internalizing T cell receptors,
minimizes particles (e.g.: nanostructures) coupled to the cell
surface from being internalized.
[0006] Further, experimental results show, unexpectedly, that a
coupling efficiency of, for example, 89.6% can be achieved when
adding a polycation (e.g., poly-L-lysine) to the surface of a
nanostructure prior to performing a coupling reaction with a
carrier cell (e.g., T cell). As an example, human IL-15Sa protein
nanogels were coupled to activated T cells using a method of the
present disclosure for adoptive cell therapy, and highly efficient
expansion of the transferred T cells was achieved with
substantially reduced toxicity in mice compared with soluble human
IL-15Sa at an equivalent dose. Such results were unexpected given
the cellular toxicity known to result from delivery of DNA coated
and condensed using a polycationic coatings.
[0007] The compositions provided herein are useful to improve the
efficiency and stability of coupling of nanostructures to a carrier
cell surface (e.g., T cell surface) for a variety of biomedical and
pharmaceutical applications such as, for example, therapeutic and
prophylactic (e.g., drug delivery) as well as diagnostic (e.g.,
imaging and tracking) applications.
[0008] Thus, some aspects of the present disclosure provide
compositions comprising a carrier cell having a cell surface
coupling receptor, and a nanostructure surface-modified with a
ligand that binds to the cell surface coupling receptor. In certain
embodiments, the carrier cell is a nucleated carrier cell. In
certain embodiments the nucleated carrier cell homes to a tumor. In
certain embodiments, the composition comprises a nucleated carrier
cell having a cell surface coupling receptor coupled to a
nanostructure surface-modified with a ligand that binds to the cell
surface coupling receptor. In some embodiments, the ligand is
selected from the group consisting of antibodies, soluble protein
receptors, cytokines, peptides, small molecules, co-factors,
hormones and neurotransmitters.
[0009] In some embodiments, the cell surface coupling receptor is
CD45.
[0010] In some embodiments, the ligand is an anti-CD45 antibody. In
some embodiments, the anti-CD45 antibody is a human anti-CD45
antibody or a humanized anti-CD45 antibody. In some embodiments,
the anti-CD45 antibody is an anti-CD45 monoclonal antibody. In some
embodiments, the anti-CD45 monoclonal antibody is selected from the
group consisting of BC8, 4B2, 9.4 and GAP8.3.
[0011] In some embodiments, the ligand is bound to the cell surface
coupling receptor, thereby linking, or coupling, the nanostructure
to the nucleated carrier cell.
[0012] In some embodiments, the ligand binds to the cell surface
coupling receptor.
[0013] In some embodiments, the ligand binds to the cell surface
coupling receptor and at least 50% (e.g., at least 60%, at least
70%, at least 80%, at least 90%, or at least 95%) of the ligand
(e.g., anti-CD45 antibody) remains on the surface of the cell for
at least 24 hours (e.g., at least 36 hours, or at least 48
hours).
[0014] In some embodiments, the carrier cell is a T cell, a B cell,
a Natural Killer (NK) cell or hematopoietic progenitor cell. In
some embodiments, the carrier cell is a T cell. For example, the T
cell may be a CD8.sup.+ T cell or a CD4.sup.+ T cell. In some
embodiments, the T cell is an adoptively transferred T cell. In
some embodiments, the T cell is a chimeric antigen receptor (CAR) T
cell.
[0015] In some embodiments, the nanostructure is a nanoparticle or
a liposome.
[0016] In some embodiments, the nanoparticle is selected from the
group consisting of: protein nanogels, nucleic acid nanogels and
solidified polymers.
[0017] In some embodiments, the nanostructure is a liposome. In
some embodiments, the liposome is an interbilayer-crosslinked
multilamellar vesicle (ICMV) or a unilamellar vesicle.
[0018] In some embodiments, the nanoparticle is a protein nanogel.
In some embodiments, the protein nanogel is a carrier-free protein
nanogel.
[0019] In some embodiments, the nanostructure has a diameter of 1
to 1000 nanometers (nm). For example, the nanostructure may have a
diameter of 50 to 500 nm.
[0020] In some embodiments, the ligand is covalently conjugated to
the nanostructure. In some embodiments, the ligand is covalently
conjugated to the nanostructure via a maleimide-thiol reaction.
[0021] In some embodiments, the nanostructure comprises an agent.
In some embodiments the nanostructure is a protein nanogel or a
liposome comprising an agent. In some embodiments, the agent is
selected from the group consisting of therapeutic agents,
prophylactic agents, diagnostic agents and imaging agents. In some
embodiments, the agent is selected from the group consisting of
proteins, nucleic acids and small molecule drugs. In some
embodiments, the agent is a biologically active protein. For
example, the biologically active protein is a cytokine, such as
IL-2, IL-15 or IL-15-superagonist (IL-15-superagonist is also
referred to as IL-15Sa or IL-15SA).
[0022] In some embodiments, the nanostructure (e.g.: protein
nanogel) comprises on its surface a polycation. In some
embodiments, the polycation is polylysine. In some embodiments, the
polycation is polyethylene glycol-b-polylysine or polyethylene
glycol-g-polylysine.
[0023] In some embodiments, the compositions comprise a nucleated
carrier cell covalently conjugated to a nanostructure comprising a
surface polycation.
[0024] Some aspects of the present disclosure provide compositions
comprising a T cell having a CD45 receptor, coupled to a protein
nanogel that comprises a polycation and a ligand (e.g., anti-CD45
antibody) that binds the CD45 receptor.
[0025] Some aspects of the present disclosure provide compositions
comprising a T cell having a CD45 receptor, coupled to a protein
nanogel that comprises an agent, a polycation and a ligand (e.g.,
anti-CD45 antibody) that binds the CD45 receptor.
[0026] Some aspects of the present disclosure provide compositions
comprising a T cell having a CD45 receptor, coupled to a liposome
comprising a ligand (e.g., anti-CD45 antibody) that binds the CD45
receptor.
[0027] Some aspects of the present disclosure provide compositions
comprising a T cell having a CD45 receptor, coupled to a liposome
comprising an agent and a ligand (e.g., anti-CD45 antibody) that
binds the CD45 receptor.
[0028] Some embodiments of the present disclosure provide
compositions comprising a nucleated carrier cell that homes to a
tumor and is coupled to a nanostructure comprising a biologically
active protein, wherein the carrier cell comprises a CD45 receptor
and is coupled to the nanostructure with a ligand that binds the
CD45 receptor or the carrier cell comprises a negatively charged
cell membrane and the nanostructure comprises a polycation surface
which interacts electrostatically with the cell membrane, and
wherein the nanostructure is a protein nanogel or a liposome.
[0029] In in certain aspects the carrier cell comprises a CD45
receptor and is coupled to the nanostructure with a ligand that
binds the CD45 receptor.
[0030] In certain aspects the ligand is an anti-CD45 monoclonal
antibody.
[0031] In certain aspects the carrier cell comprises a negatively
charged cell membrane and the nanostructure comprises a polycation
surface which interacts electrostatically with the cell membrane.
In certain aspects, the polycation is polylysine. In certain
aspects, the polycation is polyethylene glycol-b-polylysine
(PEG-PLL).
[0032] In some embodiments, the composition comprises a
nanostructure wherein the nanostructure is a protein nanogel and
the protein nanogel comprises a plurality of biologically active
proteins reversibly and covalently crosslinked to each other
through a degradable linker. In some embodiments, the degradable
linker is a redox responsive linker that comprises a disulfide
bond.
[0033] In some embodiments, the composition comprises a
nanostructure wherein the nanostructure is a liposome and the
liposome comprises a plurality of biologically active proteins. In
certain aspects the liposome is unilamellar or an
interbilayer-crosslinked multilamellar vesicle.
[0034] In some embodiments, the composition comprises a carrier
cell wherein the carrier cell is a T cell, a B cell a Natural
Killer (NK) cell or a hematopoietic progenitor cells. In some
aspects, the carrier cell is a T cell. In some aspects, the T cell
is a CD8.sup.+ T cell or a CD4.sup.+ T cell. In some aspects, the T
cell is an adoptively transferred T cell. In some aspects, the T
cell is a chimeric antigen receptor (CAR) T cell.
[0035] In some embodiments, the composition comprises a
nanostructure comprising biologically active protein wherein the
biologically active protein is selected from the group consisting
of antibodies, antibody fragments, soluble protein receptors and
cytokines. In some aspects, the cytokine is IL-2, IL-7, IL-15 (or
superagonist/mutant forms of these cytokines e.g.: IL-15Sa), IL-12,
IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand. In some aspects, the
cytokine is IL-15-Sa. In some aspects, the IL-15Sa comprises a
complex comprising a dimeric IL-15R.alpha.Su/Fc and two IL-15N72D
molecules. In some aspects, the dimeric IL-15R.alpha.Su/Fc
comprises an amino acid sequence set forth in SEQ ID NO: 2 and the
IL-15N72D molecule comprises an amino acid sequence set forth in
SEQ ID NO: 1.
[0036] In some embodiments the composition comprises a
pharmaceutically acceptable carrier. In some aspects the
composition is useful as a medicament for delivering a biologically
active protein to a subject having a tumor.
[0037] Certain aspects of the disclosure provide methods of
treating cancer in a subject comprising administering to a subject
in need thereof a composition as described herein.
[0038] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) that homes to
a tumor coupled to a nanostructure comprising a biologically active
protein, wherein (a) the carrier cell comprises a CD45 receptor and
is coupled to the nanostructure with a ligand that binds the CD45
receptor; or (b) the carrier cell comprises a negatively charged
cell membrane and the nanostructure comprises a polycation surface
which interacts electrostatically with the cell membrane; or (c)
the carrier cell comprises a CD45 receptor and is coupled to the
nanostructure with a ligand that binds the CD45 receptor, and the
carrier cell comprises a negatively charged cell membrane and the
nanostructure comprises a polycation surface which interacts
electrostatically with the cell membrane.
[0039] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: T cell) comprising a
CD45 receptor and a nanostructure comprising a biologically active
protein, wherein the carrier cell is coupled to the nanostructure
with a ligand that binds the CD45 receptor.
[0040] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (T cell) that homes to a tumor
coupled to a nanostructure comprising a biologically active
protein, wherein the carrier cell comprises a negatively charged
cell membrane and the nanostructure comprises a polycation surface
which interacts electrostatically with the cell membrane.
[0041] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell comprising a CD45 receptor and
a protein nanogel, wherein the carrier cell is coupled to the
protein nanogel with a ligand that binds the CD45 receptor, and
wherein the protein nanogel comprises a plurality of biologically
active proteins reversibly and covalently crosslinked to each other
through a degradable linker.
[0042] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) that homes to
a tumor coupled to a protein nanogel, wherein the carrier cell
comprises a negatively charged cell membrane and the protein
nanogel comprises a polycation surface which interacts
electrostatically with the cell membrane, and wherein the protein
nanogel comprises a plurality of biologically active proteins
reversibly and covalently crosslinked to each other through a
degradable linker.
[0043] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) comprising a
CD45 receptor and a liposome comprising a plurality of biologically
active proteins, wherein the carrier cell is coupled to the
liposome with a ligand that binds the CD45 receptor.
[0044] Also provided herein are methods of producing a composition
comprising a carrier cell coupled to a nanostructure, the method
comprising modifying the surface of a nanostructure to contain a
ligand (e.g., anti-CD45 antibody) that binds to a cell surface
coupling receptor (e.g., CD45) located on the surface of a carrier
cell; and combining the nanostructure and the carrier cell
comprising the cell surface coupling receptor under conditions that
result in binding of the ligand to the cell surface coupling
receptor on the surface of the carrier cell, thereby producing a
cell coupled to a nanostructure.
[0045] Further provided herein are methods of producing a
composition comprising a carrier cell coupled to a nanostructure,
the method comprising combining (a) a nanostructure surface
modified to contain a ligand (e.g., anti-CD45 antibody) that binds
to a cell surface coupling receptor (e.g., CD45) located on the
surface of a carrier cell and (b) the carrier cell comprising the
cell surface coupling receptor, wherein the nanostructure and
carrier cell are combined under conditions that result in binding
of the ligand to the cell surface coupling receptor on the surface
of the carrier cell, thereby producing a carrier cell coupled to a
nanostructure.
[0046] Some aspects of the present disclosure provide methods of
producing a composition comprising a carrier cell coupled to a
nanostructure, the method comprising combining (a) a nanostructure
surface modified to contain a chemical linker with (b) a polycation
under conditions that result in a nanostructure with positive
charges on its surface which interacts electrostatically with the
cell membrane; and covalently conjugating the nanostructure to a
cell, thereby producing a carrier cell coupled to a
nanostructure.
[0047] Also provided herein are methods of producing a composition
comprising a carrier cell coupled to a nanostructure, the method
comprising combining (a) a nanostructure surface modified to
contain a chemical linker and ligand that binds to a cell surface
coupling receptor located on the surface of a carrier cell with (b)
a polycation under conditions that result in a nanostructure with
positive charges on its surface which interacts electrostatically
with the cell membrane; combining the nanoparticle with the carrier
cell comprising the cell surface coupling receptor under conditions
that result in binding of the ligand to the cell surface coupling
receptor; and covalently conjugating the nanostructure with
positive charges on its surface which interacts electrostatically
with the cell membrane to the carrier cell comprising the cell
surface coupling receptor, thereby producing a carrier cell coupled
to a nanostructure.
[0048] One aspect of the disclosure provides a method for
delivering an agent (e.g., a biologically active protein) by
administering to a subject a composition comprising a nucleated
carrier cell that homes to a tumor coupled to a nanostructure
(e.g., nanoparticle or liposome) comprising an agent, wherein the
carrier cell comprises a cell surface coupling receptor, and
wherein the nanostructure is coupled to the carrier cell with a
ligand that binds to the cell surface coupling receptor, such that
the agent is released from the nanostructure in vivo.
[0049] Other aspects of the disclosure relate to methods for
delivering an agent (e.g., a biologically active protein) by
administering to a subject a composition comprising a nucleated
carrier cell that homes to a tumor coupled to a nanostructure
(e.g., nanoparticle or liposome) comprising an agent, wherein the
nanostructure comprises a surface associated with a polycation
(e.g., polylysine), such that the agent is released from the
nanostructure in vivo.
[0050] Yet other aspects of the disclosure relate to methods for
delivering a biologically active protein (e.g., a cytokine) by
administering to a subject a composition comprising a nucleated
carrier cell that homes to a tumor coupled to a nanostructure
(e.g., nanoparticle or liposome) comprising a biologically active
protein, wherein the carrier cell comprises a CD45 receptor coupled
to the nanostructure with a ligand that binds the CD45 receptor or
the carrier cell comprises a negatively charged cell membrane and
the nanostructure comprises a polycation surface which interacts
electrostatically with the cell membrane, and wherein the
nanostructure is a protein nanogel or a liposome, such that the
biologically active protein is released from the nanostructure in
vivo.
[0051] In other aspects, the disclosure provides methods for
delivering an immunostimulatory cytokine by administering to a
subject a composition comprising a T cell that homes to a tumor
coupled to a nanostructure comprising an immunostimulatory
cytokine, wherein the T cell comprises a CD45 receptor coupled to
the nanostructure with a ligand that binds the CD45 receptor or the
T cell comprises a negatively charged cell membrane and the
nanostructure comprises a polycation surface which interacts
electrostatically with the cell membrane, or both, and wherein the
nanostructure is a protein nanogel or a liposome, such that the
immunostimulatory cytokine is released from the nanostructure in
vivo.
[0052] In another aspect, the disclosure provides a method for
delivering an immunostimulatory cytokine to a tumor comprising
administering to a subject having a tumor a tumor-reactive T cell
coupled to a nanogel comprising an immunostimulatory cytokine,
wherein the T cell comprises a CD45 receptor coupled to the nanogel
with a ligand that binds the CD45 receptor and wherein the T cell
comprises a negatively charged cell membrane and the nanogel
comprises a polycation surface which interacts electrostatically
with the cell membrane, such that the immunostimulatory cytokine is
released from the nanostructure in vivo.
[0053] In another aspect, the disclosure provides a method for
delivering an immunostimulatory cytokine to a tumor comprising
administering to a subject having a tumor a chimeric antigen
receptor (CAR) T cell coupled to a nanostructure (e.g., nanogel or
liposome) comprising an immunostimulatory cytokine, wherein the CAR
T cell comprises a CD45 receptor coupled to the nanostructure with
a ligand that binds the CD45 receptor or wherein the CAR T cell
comprises a negatively charged cell membrane and the nanostructure
comprises a polycation surface which interacts electrostatically
with the cell membrane, or both, such that the immunostimulatory
cytokine is released from the nanostructure in vivo.
[0054] Other aspects of the disclosure provide methods for
maintaining, stimulating or enhancing activity of a T cell in a
subject comprising administering to a subject a composition
comprising a T cell coupled to a nanostructure (e.g., nanogel or
liposome) comprising an immunostimulatory cytokine, wherein the T
cell comprises a CD45 receptor coupled to the nanostructure with a
ligand that binds the CD45 receptor or wherein the T cell comprises
a negatively charged cell membrane and the nanostructure comprises
a polycation surface which interacts electrostatically with the
cell membrane, or both, such that the immunostimulatory cytokine is
released from the nanostructure in vivo and activity of the T cell
is maintained, stimulated or enhanced.
[0055] Yet other aspects of the disclosure relate to methods for
maintaining, stimulating or enhancing activity of a T cell located
in the environment of a tumor, comprising administering to a
subject having a tumor a carrier T cell that homes to the tumor
coupled to a nanostructure (e.g., nanogel or liposome) comprising
an immunostimulatory cytokine, wherein the carrier T cell comprises
a CD45 receptor coupled to the nanostructure with a ligand that
binds the CD45 receptor or wherein the carrier T cell comprises a
negatively charged cell membrane and the nanostructure comprises a
polycation surface which interacts electrostatically with the cell
membrane, or both, and wherein release of the immunostimulatory
cytokine from the nanogel or liposome maintains, stimulates or
enhances activity of a T cell located in the environment of the
tumor.
[0056] In some embodiments, the subject has a tumor. In related
embodiments, the cell is a tumor-reactive T cell. In other related
embodiments, the carrier cell homes to the tumor or to the tissue
in which the tumor exists (e.g., lymphoid tissue). In some
embodiments, the tumor is a lymphoma and the agent is an antibody,
such as an anti-CD20 antibody, or a chemotherapy, such as
fludaribine. Other agents having therapeutic effect on lymphoma may
be used in place of or in addition to anti-CD20 antibody or
fludaribine.
[0057] In some embodiments, the subject has an autoimmune disease.
In some embodiments, the subject has an infection. In some
embodiments, the subject is in need of hematopoietic reconstitution
as a result of, for example, myeloablative chemotherapy and/or
radiation.
[0058] In some embodiments, the cell is a gut-specific T cell. In
some embodiments, the cell is a skin-specific T cell.
[0059] In some embodiments, the cell is autologous to the subject.
In some embodiments, the cell is activated prior to administration
to the subject. In some embodiments, the cell is genetically
engineered, such as, for example a chimeric antigen receptor (CAR)
T cell.
[0060] In some embodiments, the agent is an imaging agent. In some
embodiments, the agent is an immunostimulatory protein, such as a
cytokine. In some embodiments, the cytokine is IL15SA. In some
embodiments, the agent is an antigen. In some embodiments, the
agent is an adjuvant. In some embodiments, the adjuvant is a TLR
ligand. The TLR ligand may function to stimulate antigen-specific
immune responses (typically in the presence of exogenous or
endogenous antigens) and/or antigen-non-specific immune responses.
Thus, the TLR ligand may be used in the presence or absence of an
antigen. In some embodiments, the agent is an antibody or an
antibody fragment. In some embodiments, the agent is a drug. In
some embodiments, the agent is a chemical compound. In some
embodiments, the agent is a nucleic acid. In some embodiments, the
nucleic acid is an siRNA.
[0061] In some embodiments, the agents are anti-cancer agents
including anti-cancer antibodies, cancer antigens, anti-cancer
chemotherapeutic agents, and the like.
[0062] In various embodiments, the agents may be used at doses that
are below doses required to achieve the same effects in vivo
following systemic administration. In some instances, the doses are
at least 2 times less, at least 5 times less, at least 10 times
less, at least 20 times less, at least 50 times less, or at least
100 times less than the required systemic dose.
[0063] In some embodiments, the cell is covalently bound to a
plurality of nanostructures. In some embodiments, the plurality of
nanostructures comprises the same or different agents.
[0064] In some embodiments, the agent acts in an autocrine manner
(i.e., it acts upon the carrier cell itself). In some embodiments,
the agent acts in a paracrine manner (i.e., it acts upon cells
other than the cell carrier, such as cells at the site of a tumor
in vivo). In still other embodiments, the agent acts in both an
autocrine and a paracrine manner
[0065] These and other aspects and embodiments will be described in
greater detail herein. This disclosure is not limited in its
application to the details of construction and/or the arrangement
of components set forth in the following description or illustrated
in the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] The accompanying drawings are not intended to be drawn to
scale. For purposes of clarity, not every component may be labeled
in every drawing.
[0067] FIGS. 1A-1C show internalization kinetics of liposomes with
surface antibodies, .alpha.-CD2, .alpha.-CD8, .alpha.-CD11,
.alpha.-CD45, and/or .alpha.-Thy1.1.
[0068] FIG. 2 shows a schematic of an example of preparation and
surface modification of cytokine nanogels for cell surface
coupling.
[0069] FIG. 3A shows cell surface reduction rates of resting and
primed CD8.sup.+ T cells.
[0070] FIG. 3B shows anti-CD45 mediated anchoring of nanogels to
the T cell surface enables TCR-responsive protein cargo
release.
[0071] FIG. 4 shows IL-15Sa nanogels promote T cell expansion in
vitro.
[0072] FIGS. 5A-5E shows IL-15Sa nanogels promote specific
expansion of adoptively transferred T cells in tumors.
Specifically, counts of adoptively transferred (ACT)
Thy1.1.sup.+CD8.sup.+ T-cells (squares) and endogenous
Thy1.1.sup.-CD8.sup.+ T-cells (triangles) in blood (FIG. 5A,
normalized by volume) and tumours (FIG. 5B, normalized by weight).
FIGS. 5C-5E show ratios of counts of ACT CD8.sup.+ T-cells in the
group of T+aCD45/IL-15Sa-NG to that of T+free IL-15Sa in different
tissues (FIG. 5C), counts of Ki67.sup.+ ACT CD8.sup.+ T-cells in
tumours analysed by intracellular staining and flow cytometry (FIG.
5D), and counts of polyfunctional ACT CD8.sup.+ T-cells in tumours
by intracellular cytokine staining (FIG. 5E).
[0073] FIGS. 6A-6E show IL-15Sa nanogels increase the therapeutic
window for adjuvant cytokine delivery during ACT, for example, with
increase tolerability up to 80 .mu.g as measured by body weight
normalized to day 7 (FIG. 6A), counts of cytokine.sup.+ endogenous
CD8.sup.+ T-cells (FIG. 6B) and ACT CD8.sup.+ T-cells (FIG. 6C) in
blood and serum cytokine levels (TNF-.alpha., FIG. 6D; IL-6, FIG.
6E).
[0074] FIGS. 7A-7C show TCR signaling-responsive protein nanogels
improve the efficacy of murine T-cell and human CAR T cell
therapies.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Provided herein, in some aspects, are methods and
compositions for efficiently and stably coupling a nanostructure to
a carrier cell surface with minimum cellular internalization,
permitting, for example, carrier cell drug delivery in vivo for
targeted immunotherapy. Typically, linking or coupling a
nanostructure (e.g., a synthetic nanoparticle or liposome) to the
surface of a T cell, for example, triggers receptor-mediated
endocytosis. To minimize or prevent nanostructures from being
endocytosed by carrier cells, the nanostructures can be coupled to
the surface of the carrier cell though covalent modifications (see,
e.g., US20150110740 A1, incorporated herein by reference). The
present disclosure is based, at least in part, on surprising
results showing that the coupling of a nanostructure to a surface
of a cell is facilitated by expressing a receptor with prolonged
residence time at the surface of the cell and attaching a cognate
ligand to the nanostructure. Surprisingly, certain cell surface
receptors (e.g., CD45) are capable of maintaining nanostructures at
the surface of a cell, rather than triggering the classical
endocytotic internalization pathway. These cell surface coupling
receptors increase loading and/or coupling efficiency of the
nanostructure to the cell surface. Such cell surface receptors are
referred to herein as "cell surface coupling receptors". These cell
surface coupling receptors have a prolonged cell surface residence
time and/or are maintained on the cell surface with minimal
internalization.
[0076] Accordingly, in some aspects, the present disclosure
provides compositions comprising nucleated carrier cells that
express at least one cell surface coupling receptor coupled to one
or more nanostructures modified to provide a ligand that binds to
the cell surface coupling receptor. One such example is a T cell
that expresses CD45 coupled to a nanostructure via an anti-CD45
antibody provided by the nanostructure.
[0077] Also provided herein are compositions for improving the
coupling efficiency and increase loading of nanostructures (e.g.:
protein nanogels) to carrier cells through the use of a polycation,
which, when added to the surface of a nanostructure prior to
performing a coupling reaction, functions to neutralize
negatively-charged T cells. In effect, the polycation acts as a
"magnet" to bring the nanostructure (e.g.: nanogel) in close
proximity to the cell during a coupling reaction, thereby
increasing the efficiency of the coupling reaction.
[0078] Nanostructures for Coupling to Carrier Cells
[0079] Nanostructures of the present disclosure generally are
microscopic particles having at least one dimension less than 1000
nm (e.g., less than 500, less than 250, less than 100 nm). The term
"nanostructure" encompasses liposomes and nanoparticles as
described herein. In some embodiments, nanostructures are
synthetic. That is, the nanostructures do not occur naturally.
[0080] In some embodiments, nanostructures are synthesized to
comprise one or more reactive groups on their exterior surface for
reaction with reactive groups on cell carriers. These nanostructure
reactive groups include, without limitation, thiol-reactive
maleimide head groups, haloacetyl (e.g., iodoacetyl) groups,
imidoester groups, N-hydroxysuccinimide esters, and pyridyl
disulfide groups. These reactive groups react with groups on the
carrier cell surface and, thus, provide coupling of the
nanostructures to the cell surface. It should be understood that
when surface modified in this manner, the nanostructures are
intended for use with specific carrier cells having "complementary"
reactive groups (i.e., reactive groups that react with those of the
nanostructures). In some embodiments, the nanostructures do not
integrate into the lipid bilayer that comprises the cell
surface.
[0081] Nanostructures may be covalently conjugated or
non-covalently conjugated to the carrier cells. The process of
conjugating a nanostructure to a cell is referred to herein as a
"coupling reaction." Covalent conjugation typically provides a more
stable (and thus longer) association between the nanostructures and
the carrier cells. Covalent conjugation in some embodiments also
can provide stability and, thus, more sustained localized delivery
of agents in vivo. Non-covalent conjugation includes without
limitation absorption onto the cell surface and/or lipid bilayer of
the cell membrane.
[0082] In some embodiments, covalent attachment is achieved through
a maleimide-thiol reaction, wherein there is a two-step process in
which carrier cells are first incubated with maleimide-bearing
nanostructures to allow conjugation to the cell surface, followed
by in situ PEGylation with thiol-terminated polyethylene glycol
(PEG) to cap remaining maleimide groups of the particles and avoid
particle-mediated crosslinking of cells. With this approach,
substantial numbers of nanostructures with diameters in the 100-300
nm range have been conjugated to cell types used commonly in cell
therapy. This strategy allows particles ranging from simple
liposomes (e.g., with an aqueous drug-loaded core) to more complex
lipid-coated polymer or DNA-based nanostructures to be stably
attached to carrier cells. Importantly, the linkage chemistry is
benign and non-toxic as evidenced in part by the conjugation of up
to 139 (.+-.29) .about.200 nm-diameter lipid-coated nanoparticles
to the surface of cells without any deleterious effect (see U.S.
Patent Application Publication No. 2011/0293705).
[0083] In some embodiments, a nanostructure is a polymersome.
Polymersomes are a class of artificial vesicles that may be used
for drug delivery or as artificial organelles. The vesicle
membranes are made of block copolymers. Polymersomes have an
ability to encapsulate and transport molecules in the central water
pool or sequestered in their hydrophobic bilayer membrane.
Polymersomes differ from liposomes in that they are generally more
highly stable and the bilayer membranes of polymersomes are often
relatively impermeable, thereby hindering the release of
encapsulated molecules. Polymersomes are useful for encapsulating
and protecting sensitive molecules, such as drugs, enzymes, other
proteins and peptides, and DNA and RNA fragments. Polymersomes have
the ability to load both hydrophilic and hydrophobic molecules,
making them excellent candidates to use as drug delivery
systems.
[0084] In some embodiments, nanostructures comprise a lipid bilayer
on the outermost surface of the nanostructure. This bilayer may be
comprised of one or more lipids of the same or different type.
Examples include, without limitation, phospholipids such as
phosphocholines and phosphoinositols. Specific examples include,
without limitation, DMPC, DOPC, DSPC, and various other lipids such
as those recited below. The size of a protein nanostructure may be
determined at least two ways: based on its "dry size" and based on
its "hydrodynamic size." In certain embodiments, the "dry size" of
a nanostructure (e.g.: nanogel) refers to the diameter of the
nanostructure (e.g.: nanogel) as a dry solid. The dry size of a
nanostructure (e.g.: nanogel) may be determined, for example, by
transmission electron microscopy.
[0085] In other embodiments, the "hydrodynamic size" of a
nanostructure (e.g.: liposome) refers to the diameter of the
nanostructure (e.g.: liposome) as a hydrated gel (e.g., a liposome
in an aqueous buffer). The hydrodynamic size of the nanostructure
(e.g.: liposomes) may be determined, for example, by dynamic light
scattering. Methods for measuring nanostructure (e.g.: liposomes)
diameter by dynamic light scattering are known in the art (see, for
example, Malvern Instruments, Ltd. on the world wide web at:
malvern.co.uk)
[0086] In some embodiments, the dry size of a nanostructure is less
than 100 nm. In some embodiments, the dry size of a nanostructure
is less than 95 nm, less than 90 nm, less than 85 nm, less than 80
nm, less than 75 nm, less than 70 nm, less than 65 nm, or less than
60 nm. In some embodiments, the dry size of a nanostructure is 40
to 90 nm, 40 to 80 nm, 40 to 70 nm, 40 to 60 nm, 50 to 90 nm, 60 to
80 nm, 50 to 70 nm, or 50 to 60 nm. In some embodiments, the dry
size of a nanostructure is 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65
nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm or 95 nm.
[0087] In some embodiments, the average dry size of a nanostructure
within a plurality of nanostructures is less than 100 nm. In some
embodiments, the average dry size of a nanostructure within such a
plurality varies by no more than 5% or 10%. In some embodiments,
the average dry size of a nanostructure (e.g., nanogel) within a
plurality of nanostructures is less than 95 nm, less than 90 nm,
less than 85 nm, less than 80 nm, less than 75 nm, less than 70 nm,
less than 65 nm, or less than 60 nm. In some embodiments, the
average dry size of a nanostructure (e.g., nanogel) within a
plurality of nanostructures is 40 to 90 nm, 40 to 80 nm, 40 to 70
nm, 40 to 60 nm, 50 to 90 nm, 60 to 80 nm, 50 to 70 nm, or 50 to 60
nm. In some embodiments, the dry size of a nanostructure is 40 nm,
45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90
nm or 95 nm.
[0088] In some embodiments, the hydrodynamic size of a
nanostructure is less than 100 nm. In some embodiments, the dry
size of a nanostructure is less than 95 nm, less than 90 nm, less
than 80 nm, less than 85 nm, or less than 75 nm. In some
embodiments, the hydrodynamic size of a nanostructure is 70 to 90
nm, 70 to 85 nm, 70 to 80 nm, 75 to 90 nm, 75 to 85 nm, 75 to 80
nm, 80 to 90 nm, 80 to 85 nm or 85 to 90 nm. In some embodiments,
the hydrodynamic size of a nanostructure is 70 nm, 75 nm, 80 nm, 85
nm, 90 nm, or 95 nm. In some embodiments, the hydrodynamic size of
a nanostructure is 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm,
87 nm, 88 nm, 89 nm or 90 nm.
[0089] In some embodiments, the average hydrodynamic size of a
nanostructure within a plurality of nanostructures is less than 100
nm. In some embodiments, the average hydrodynamic size of a
nanostructure within such a plurality varies by no more than 5% or
10%. In some embodiments, the average hydrodynamic size of a
nanostructure within a plurality of nanostructures is less than 95
nm, less than 90 nm, less than 80 nm, less than 85 nm, or less than
75 nm. In some embodiments, the average hydrodynamic size of a
within a plurality of nanostructures is 70 to 90 nm, 70 to 85 nm,
70 to 80 nm, 75 to 90 nm, 75 to 85 nm, 75 to 80 nm, 80 to 90 nm, 80
to 85 nm or 85 to 90 nm. In some embodiments, the average
hydrodynamic size of a nanostructure within a plurality of
nanostructures is 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, or 95 nm. In
some embodiments, the average hydrodynamic size of a nanostructure
within a plurality of nanostructures is 80 nm, 81 nm, 82 nm, 83 nm,
84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm or 90 nm.
[0090] In some embodiments, nanostructures (e.g.: protein
nanogels)) are provided in a dry, solid form, such as a lyophilized
form. In other embodiments, nanostructures (e.g.: liposomes) are
provided in a hydrated form, such as in aqueous or otherwise liquid
solution.
[0091] Nanoparticles
[0092] The term "nanoparticle" includes nanogels (e.g: protein
nanogels and nucleic acid nanogels), solid colloidal nanoparticles,
magnetic nanoparticles, nobel metal nanoparticles, semiconductor
nanoparticles, multimodal nanoparticles, composite nanoparticles,
and other nanoparticles typically used for biomedical applications
(see, e.g., Blanco-Andujar et al. Annu. Rep. Prog. Chem., Sect. A,
2010, 106, 553-568, incorporated by reference herein). It should be
understood, however, that the term "nanoparticles" does not
encompass viruses or viral particles or liposomes, although, in
some embodiments, non-infectious virus-like particles (VLPs), which
do not contain viral genetic material, are contemplated herein.
Thus, in some embodiments, a composition comprises a VLP coupled to
the surface of a cell. Nanoparticles are distinguished from films
or other structurally layered polymers matrices.
[0093] In some embodiments, nanoparticles are synthetic. That is,
the nanoparticles no not occur naturally. In some embodiments,
nanoparticles are biodegradable and, thus, are not magnetic.
Biodegradable nanoparticles may be synthesized using methods known
in the art, including, without limitation, solvent evaporation, hot
melt microencapsulation, solvent removal and spray drying.
Exemplary methods for synthesizing nanoparticles are described by
Bershteyn et al., Soft Matter 4:1787-1787, 2008 and in U.S. Patent
Application Publication No. 2008/0014144 A1, the specific teachings
of which relating to nanoparticle synthesis are incorporated herein
by reference.
[0094] In some embodiments, nanoparticles release their agent
"payload" over a number of days as a function of their degradation
profile in vivo. Nanoparticles which are biodegradable in nature
gradually degrade in an aqueous environment such as occurs in vivo.
If the agents are dispersed throughout the nanoparticles, then
their release occurs as the outermost layers of the nanoparticle
degrade or as the pores within the nanoparticle enlarge. Release
kinetic studies show that, in some embodiments, protein and
small-molecule drugs can be released from biodegradable
nanoparticles over time-courses ranging from 1 day to 2 weeks.
Thus, in some embodiments biodegradable nanoparticles function to
gradually release their payload into the environment of the target
site(s).
[0095] In some embodiments, the diameter of a nanoparticle is, for
example, 1-1000 nanometers (nm). In some embodiments, the diameter
is 20-750 nm, 20-500 nm, or 20-250 nm. In some embodiments, the
diameter is 50-750 nm, 50-500 nm, 50-250 nm, or 100-300 nm. In some
embodiments, the diameter is 100 nm, 150 nm, 200 nm, 250 nm or 300
nm.
[0096] In some embodiments, a nanoparticle is comprised of one or
more solidified polymers, arranged in a random manner Exemplary
synthetic polymers which can be used to form biodegradable
nanoparticles include, without limitation, aliphatic polyesters,
poly (lactic acid) (PLA), poly (glycolic acid) (PGA), co-polymers
of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL),
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric
acid), poly(valeric acid), and poly(lactide-co-caprolactone), and
natural polymers such as alginate and other polysaccharides
including dextran and cellulose, collagen, chemical derivatives
thereof, including substitutions, additions of chemical groups such
as for example alkyl, alkylene, hydroxylations, oxidations, and
other modifications routinely made by those skilled in the art),
albumin and other hydrophilic proteins, zein and other prolamines
and hydrophobic proteins, copolymers and mixtures thereof. In
general, these materials degrade either by enzymatic hydrolysis or
exposure to an aqueous environment in vivo, by surface or bulk
erosion.
[0097] A polymer is a natural or synthetic large molecule, or
macromolecule, composed of many repeated, polymerized monomer
subunits. Polymers can be formed from up to thousands of only one
monomer or of a combination of monomers (copolymers). Synthetic
polymers include, without limitation, plastics such as polystyrene
and polyethylene. Natural polymers include, without limitation,
biopolymers such as DNA and proteins. The large molecular mass of
polymers relative to small molecule compounds produces unique
physical properties, including toughness, viscoelasticity, and a
tendency to form glasses and semicrystalline structures rather than
crystals.
[0098] In some embodiments, protein nanoparticles (e.g., protein
nanogels, including protein-polymer nanogels) of the present
disclosure do not contain carrier proteins or other carrier
molecules. For example, in some embodiments, protein nanoparticles
do not contain albumin (e.g., bovine serum albumin (BSA)). Carrier
proteins typically facilitate the diffusion and/or transport of
different molecules. It should be understood that the term "carrier
protein," as used herein, refers to a protein that does not
adversely affect a biologically-active protein of a protein
nanoparticle. In some embodiments, a carrier protein is an inert
protein. Thus, in some embodiments, carrier proteins are not
biologically active. Nanoparticles of the present disclosure, in
some embodiments, do not require carrier proteins or other carrier
molecules to facilitate their transport to and into cells and
tissue in vivo.
[0099] Nanogels
[0100] In some embodiments, a nanoparticle is a nanogel, such as a
protein nanogel or a nucleic acid nanogel. Nanogels may be composed
of, for example, a hydrogel comprising particles formed by
chemically or physically cross-linked polymer networks of nanoscale
size. In some embodiments, nanogels may be composed of swollen
nanosized networks composed of polymer chains that are hydrophilic
or amphiphilic. In some embodiments, nanogels may be composed of
swollen chemically cross-linked networks of cationic and neutral
polymers, e.g., branched polyethylenimine (PEI) and polyethylene
glycol (PEG) (PEG-cl-PEI). In some embodiments, nanogels may be
composed of physically cross-linked cholesterol-modified
polysaccharides (e.g., pullulan, mannan, amilopectin and dextran).
Nanogels may be utilized as carriers for the transport of drugs,
and can be designed to incorporate biologically active molecules
through the formation of salt bonds, hydrogen bonds, or hydrophobic
interactions.
[0101] In some embodiments, a nanoparticle is nucleic acid nanogel,
wherein the nanoparticles are comprised of a nucleic acid internal
core. Such "DNA nanoparticles" (or DNA-gel nanoparticles) are
described in greater detail in published U.S. Patent Application
Publication No. 20070148246. It is to be understood that the
nucleic acid core of such nanoparticles may act as a scaffold for
the agents being delivered in vivo and/or it may act as the agent
itself. An exemplary protocol for synthesizing DNA nanoparticles is
provided in U.S. Patent Application Publication No. 2011/0293705,
incorporated herein by reference.
[0102] In some embodiments, the nanostructure (or nanoaparticle) is
a protein nanogel. A protein nanogel refers to a plurality of
proteins crosslinked (e.g., reversibly and covalently crosslinked)
to each other through a degradable linker (see, e.g., US20150110740
A1, incorporated herein by reference). The proteins of a nanogel
are reversibly crosslinked via the degradable linker (e.g., a
disulfide linker), such that under physiological conditions, the
linker degrades and releases the intact, biologically-active
protein. In other embodiments, proteins of nanogels are reversibly
linked to functional groups through a degradable linker such that
under physiological conditions, the linker degrades and releases
the intact, biologically-active protein. In each instance, the
proteins are considered to be reversibly modified, as described
below.
[0103] A protein that is "reversibly linked to another protein"
herein refers to a protein that is attached (e.g., covalently
attached) to another protein through a degradable linker. Such
proteins are considered to be linked (e.g., crosslinked) to each
other through the degradable linker. In some embodiments, nanogels
contain a single (e.g., single type of) biologically-active protein
(e.g., IL-2, IL-15, IL-15Sa, IL-2-Fc, IL-15-Fc, or IL-15Sa-Fc),
while in other embodiments, nanostructures contain more than one
(e.g., 2, 3, 4, 5 or more) of biologically-active protein (e.g., a
combination of different proteins such as IL-2 and IL-15 (or
IL-15SA)). For example, a protein nanogel may contain a combination
of Protein A and Protein B, wherein Protein A is linked to Protein
A, Protein A is linked to Protein B and/or Protein B is linked to
Protein B.
[0104] A protein that is "reversibly linked to a functional group,"
or a protein that is "reversibly modified," herein refers to a
protein that is attached (e.g., covalently attached) to a
functional group through a degradable linker. Such a protein may be
referred to herein as a "protein conjugate" or a "reversibly
modified protein conjugate"--the terms may be used interchangeably
herein. It should be understood that proteins and polymers each
contain functional groups to which a protein can be linked via a
reversible linker (e.g., degradable linker such as a disulfide
linker). Examples of protein conjugates and reversibly modified
proteins, as provided herein, include without limitation, a protein
reversibly linked (e.g., via a degradable linker) to another
protein, a protein reversibly linked to a polymer, and a protein
reversibly linked to another functional group. It should be
understood that the term "protein" also includes fusion
proteins.
[0105] The degradable linkers provided herein, in some embodiments,
comprise an N-hydroxysuccinimide ester, which is capable of
reacting with proteins at neutral pH (e.g., about 6 to about 8, or
about 7) without denaturing the protein. In some embodiments, the
degradable linkers are "redox responsive" linkers, meaning that
they degrade in the presence of a reducing agent (e.g.,
glutathione, GSH) under physiological conditions (e.g.,
20-40.degree. C. and/or pH 6-8), thereby releasing intact protein
from the compound to which it is reversibly linked. An example of a
degradable linker for use in accordance with the present disclosure
is the following:
##STR00001##
The linker of Formula I contains a disulfide, which is cleaved in
the presence of a reducing agent. For example, under physiological
conditions, the disulfide bond of the linker of Formula I is
cleaved by glutathione.
[0106] Proteins may be linked (e.g., covalently linked) to a
degradable linker through any terminal or internal --NH.sub.2
functional group (e.g., side chain of a lysine). Thus, an
intermediate species formed during the reversible modification of a
protein with a degradable linker of Formula I is the following:
##STR00002##
[0107] Reversibly modified proteins provided herein can, in some
embodiments, be formed or self-assemble into various nanogels
including, without limitation, protein-hydrophilic polymer
conjugates (e.g., reversibly modified with polyethylene glycol
(PEG)), protein-hydrophobic polymer conjugates (e.g., reversibly
modified PLA or PLGA), bulk crosslinked protein hydrogels,
crosslinked protein nanogel particles, protein nanocapsules with
different shell materials (e.g., silica), protein-conjugated
nanostructures (e.g., liposome, micelle, polymeric nanoparticles,
inorganic nanoparticles). Likewise, proteins crosslinked to each
other, as provided herein, in some embodiments, can be formed or
can self-assemble into protein nanostructures.
[0108] It is contemplated, in some embodiments that the proteins of
a nanogel are irreversibly and covalently crosslinked via the
non-degradable linker N-hydroxysulfosuccunimide linker.
[0109] The polymer, in some embodiments, may be crosslinked to the
surface of the nanogel (e.g., to proteins exposed at the surface of
the nanogel). In some embodiments, a protein nanogel is wrapped in
a polymer-based, or silica, nanoshell. A nanoshell may be formed,
in some embodiments, by polymerizing functional groups (e.g.,
silanes) of a protein conjugate with a crosslinker (e.g.,
silane-PEG-silane) in the presence of a catalyst (e.g., NaF).
[0110] It is contemplated that in some embodiments the proteins of
the nanogel may comprise protein agents as described supra. In some
embodiments, the protein nanogel may comprise, without limitation,
protein(s) selected from the group consisting of therapeutic
proteins, prophylactic proteins, diagnostic proteins, and imaging
proteins. Examples of proteins for use in accordance with the
present disclosure include, without limitation, antibodies, single
chain antibodies, antibody fragments, enzymes, fusion proteins,
co-factors, receptors, ligands, transcription factors and other
regulatory factors, some antigens (as discussed below), cytokines,
chemokines, and the like. The proteins may or may not be naturally
occurring. Other proteins are contemplated and may be used in
accordance with the disclosure. Such nanogels typically do not
contain inert carrier proteins, such as albumin.
[0111] In some embodiments, proteins of the nanogel are
biologically active proteins, such as protein agents described
supra. In certain embodiments the protein agents are
immunomodulatory proteins (e.g., immunostimulatory or
immunoinhibitory proteins) as described herein. In certain aspects
it is contemplated that the immunomodulatory protein is programmed
death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4
(CTLA-4), interleukin 10 (IL-10) or transforming growth factor beta
(TGF-beta).
[0112] In some embodiments, the proteins of the nanogel are fusion
proteins of a biologically active protein fused to an
immunoglobulin Fc domain, referred to as an Fc fusion protein
(e.g., human IgG1 Fc fusion protein). In some embodiments, the
biologically active protein of the nanogel is a cytokine,
including, without limitation, IL-12, IL-15, IL-15Sa, IL-18, IL-2,
and CCL5. In some embodiments the proteins of the nanogel are
cytokines fused to Fc domains (e.g., human IgG1 Fc domains).
[0113] Proteins of the nanogel may be modified in a binary solvent
that is compatible with proteins. For example, in some embodiments,
a binary solvent includes aqueous buffer and a water-miscible
organic solvent, such as phosphate buffered saline (PBS) and
dimethyl sulfoxide (DMSO), and is used for reversibly modifying a
protein with a degradable linker. The ratio of the aqueous buffer
(e.g., PBS) to organic phase (e.g., DMSO) may be within a range of
about 50:1 to about 20:1. In some embodiments, the ratio of
inorganic phase to organic phase is about 30:1 to about 20:1, or
about 25:1 (e.g., 500 .mu.L:20 .mu.L). In some embodiments, the
organic solvent is less than 5% of the total volume of the binary
buffer or the reaction containing the binary buffer.
[0114] Methods of producing protein nanogels are described in
US20150110740 A1, incorporated herein by reference.
[0115] Liposomes
[0116] In some embodiments, a nanostructure is a liposome.
Liposomes are closed vesicles comprising at least one lipid bilayer
and an internal aqueous compartment. Liposomes may be anionic,
neutral or cationic. They may be unilamellar or multilamellar.
Liposomes may comprise, without limitation, unilamellar vesicle
lipids, multilamellar vesicle lipids and extruded lipids including
DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to
yield DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and
cholesterol, and DDAB and cholesterol. Methods for preparation of
multilamellar vesicle lipids are known in the art (see, e.g., U.S.
Pat. No. 6,693,086, the teachings of which relating to
multilamellar vesicle lipid preparation are incorporated herein by
reference). Extruded lipids are prepared in a similar manner but
are then extruded through filters of decreasing size, as described
in Templeton et al., Nature Biotech, 15:647-652, 1997, the
teachings of which relating to extruded lipid preparation are
incorporated herein by reference.
[0117] Liposomes may be surface modified during or after synthesis
to include reactive groups complementary to the reactive groups on
the carrier cells. Such reactive groups include without limitation
maleimide groups. As an example, liposomes may be synthesized to
include maleimide conjugated phospholipids such as without
limitation DSPE-MaL-PEG2000.
[0118] In some embodiments, a nanostructure is an
interbilayer-crosslinked multilamellar vesicles (ICMVs). ICMVs are
a form of multilamellar lipid vesicle (MLV). MLVs are nano- or
microspheres having a shell that is comprised of two or more
concentrically arranged lipid bilayers. As used herein, adjacent or
apposed lipid bilayers (or lipid bilayer surfaces) intend bilayers
or surfaces that are in close proximity to each other but that are
otherwise distinct and typically physically separate. This term
does not typically mean the relationship between the two monolayers
of a single bilayer.
[0119] Like the stabilized MLV described above, ICMV are nano- or
microspheres having a shell that is comprised of two or more
concentrically arranged lipid bilayers that are conjugated to each
other as described herein. The number of lipid bilayers in the
stabilized multilamellar vesicles, including the ICMV, may vary
from about 2-30, but is more commonly in the range of 2-15. The
bilayers are typically comprised of lipids having hydrophilic heads
and hydrophobic tails that are arranged in a manner similar to a
cell membrane (i.e., with the hydrophilic heads exposed to
typically an aqueous environment and the hydrophobic tails buried
in the bilayer).
[0120] ICMVs are stabilized via crosslinks (e.g., covalent
crosslinks) between their lipid bilayers, and they are therefore
referred to as "interbilayer crosslinked" MLV. As used herein, this
means that at least two lipid bilayers in the shell of the vesicle
are crosslinked to each other. The crosslinked bilayers are
typically those that are apposed or adjacent to each other. Most or
all of the lipid bilayers in the shell may be crosslinked to their
apposing lipid bilayer in the shell. There may be one or more
crosslinks between lipid bilayers. Typically, there may be numerous
crosslinks between lipid bilayers. The arrangement and positioning
of such crosslinks may be random or non-random. The degree of
crosslinks (and thus the resultant stability of the vesicles) may
depend upon the proportion of functionalized lipids (or other lipid
bilayer components) used to make the vesicles and the crosslinking
conditions (including, for example, time of incubation of the
vesicles with a crosslinker). It should be understood that the
higher the proportion of functionalized lipids (or other lipid
bilayer components) in the vesicles, the more crosslinks that are
formed, all other factors and parameters being equal. Similarly,
the more favorable the conditions towards crosslinking, the greater
degree of crosslinking that is achieved.
[0121] Methods of Producing Nanostructures
[0122] Provided herein are methods of producing nanostructures. An
example of a nanostructure is a protein nanogel, such as a protein
nanogel that contains intact, biologically-active proteins but does
not contain a carrier (e.g., albumin, BSA). In some embodiments, a
method of producing a carrier-free, biologically-active protein
nanogel comprises contacting a protein with a degradable linker
under conditions that permit reversible covalent crosslinking of
proteins to each other through the degradable linker, thereby
producing a carrier-free, biologically-active protein nanogel. In
some embodiments, a method further comprises contacting the protein
nanogel with a polymer under conditions that permit crosslinking of
the polymer to proteins of the protein nanogel, thereby producing a
carrier-free, biologically-active protein-polymer nanogel. In some
embodiments, a plurality of protein nanogels or a plurality of
protein-polymer nanogels is produced.
[0123] Typically, conditions that permit reversible covalent
crosslinking of proteins to each other through a degradable linker
include contacting the proteins with degradable linkers at a
temperature of 4.degree. C. to 25.degree. C. (e.g., 4.degree. C.,
5.degree. C., 6.degree. C., 7.degree. C., 8.degree. C., 9.degree.
C., 10.degree. C., 11.degree. C., 12.degree. C., 13.degree. C.,
14.degree. C., 15.degree. C., 16.degree. C., 17.degree. C.,
18.degree. C., 19 .degree. C., 20.degree. C., 21.degree. C.,
22.degree. C., 23.degree. C., 24.degree. C. or 25.degree. C.). In
some embodiments, proteins are incubated with the degradable
linkers in an aqueous buffer (e.g., PBS) at a temperature of
4.degree. C. to 25.degree. C. (e.g., room temperature). In some
embodiments, proteins are incubated with the degradable linkers in
an aqueous buffer (e.g., PBS) at a temperature of no greater than
30.degree. C. In some embodiments, conditions that permit
reversible covalent crosslinking of proteins to each other through
a degradable linker include contacting proteins with degradable
linkers for 30 minutes to two hours, or 30 minutes to one hour
(e.g., 30, 35, 40, 45, 50, 55 or 60 minutes). In some embodiments,
proteins are incubated with the degradable linkers in an aqueous
buffer (e.g., PBS) for 30 minutes to two hours, or 30 minutes one
hour.
[0124] In some embodiments, the concentration of the protein in the
aqueous buffer is 10 mg/mL to 50 mg/mL. For example, the
concentration of the protein in an aqueous buffer may be 10 mg/mL,
15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45
mg/mL or 50 mg/mL protein/aqueous buffer).
[0125] In some embodiments, the weight percentage of protein in a
carrier-free, biologically-active protein nanogel or
protein-polymer nanogel is at least 75% w/w. For example, the
weight percentage of protein in the carrier-free,
biologically-active protein-polymer nanogels is at least 80% w/w,
at least 85% w/w, at least 90% w/w, or at least 95% w/w. In some
embodiments, the weight percentage of protein in a carrier-free,
biologically-active protein nanogel or protein-polymer nanogel is
75% w/w to 90% w/w, 80% w/w to 90% w/w, or 85% w/w to 90% w/w.
[0126] Conditions that permit crosslinking of a polymer to proteins
of a protein nanogel include contacting the protein nanogel with a
polymer at a temperature of 4.degree. C. to 25.degree. C. (e.g.,
4.degree. C., 5.degree. C., 6.degree. C., 7.degree. C., 8.degree.
C., 9.degree. C., 10.degree. C., 11.degree. C., 12.degree. C.,
13.degree. C., 14.degree. C., 15.degree. C., 16.degree. C.,
17.degree. C., 18.degree. C., 19.degree. C., 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C. or
25.degree. C.). In some embodiments, protein nanogels are incubated
with the polymers in an aqueous buffer (e.g., PBS) at a temperature
of 4.degree. C. to 25.degree. C. (e.g., room temperature). In some
embodiments, protein nanogels are incubated with the polymers in an
aqueous buffer (e.g., PBS) at a temperature of no greater than
30.degree. C.
[0127] In some embodiments, conditions that permit crosslinking of
a polymer to proteins of a protein nanogel include contacting the
protein nanogel with a polymer for 30 minutes to two hours, or 30
minutes to one hour (e.g., 30, 35, 40, 45, 50, 55 or 60 minutes).
In some embodiments, protein nanogels are incubated with the
polymer in an aqueous buffer (e.g., PBS) for 30 minutes to two
hours, or 30 minutes one hour.
[0128] In some embodiments, methods of the present disclosure
specifically exclude contacting a protein with a degradable linker
in the presence of an organic solvent (e.g., an alcohol such as
ethanol or isopropanol). In some embodiments, methods of the
present disclosure specifically exclude contacting a protein
nanogel with a polymer in the presence of an organic solvent (e.g.,
an alcohol such as ethanol or isopropanol). Organic solvents may
adversely affect the biological activity of the proteins.
[0129] Other methods of producing nanostructures of the present
disclosure may comprise modifying a protein with a degradable
linker and polymerizable functional groups, and polymerizing the
polymerizable functional groups with a crosslinker and soluble
fluoride.
[0130] Proteins of the disclosure may be modified with, or
conjugated to, a degradable linker such as, for example, a redox
responsive linker. The modification may, in some embodiments, be a
covalent modification. Polymerizable functional groups may be
polymerized with a crosslinker in the presence of a soluble
fluoride catalyst. In some embodiments, the crosslinker is a
polymer (e.g., silane-PEG-silane). In some embodiments, the soluble
fluoride is sodium fluoride. In some embodiments, the soluble
fluoride is potassium fluoride.
[0131] Nanostructure Surface Coatings
[0132] Ligands for Binding Cell Surface Coupling Receptors
[0133] A ligand is any molecule that binds to another molecule,
such as a cell surface receptor (e.g., a cell surface coupling
receptor, such as CD45). Examples of ligands include antibodies
(also referred to as immunoglobulins), soluble proteins receptors
(e.g., CD22; Sgroi et al. Proc. Natl. Acad. Sci. USA, 92:4026-30,
1995), cytokines, peptides, small molecules, co-factors, hormones
and neurotransmitters. Other protein binding partners are
contemplated herein. Ligands can be incorporated in a nanoparticle
or liposome to facilitate coupling of the nanoparticle or liposome
to a carrier cell.
[0134] The term "antibody," as used herein, includes antibodies
typically comprising two large heavy chains and two small light
chains, antibody fragments (e.g., fragment antigen-binding (Fab)
and fragment crystallizable (Fc)) and recombinant proteins
containing antibody fragments (e.g., antigen-binding portions),
unless otherwise indicated. An antibody may be a monoclonal
antibody or a polyclonal antibody. In some embodiments, an antibody
is a human antibody or a humanized antibody.
[0135] In some embodiments, a ligand is an antibody, such as an
anti-CD45 antibody. In some embodiments, the anti-CD45 antibody is
a human monoclonal anti-CD45 antibody. In some embodiments, the
monoclonal anti-CD45 antibody is selected from the group consisting
of BC8 (ACCT.COPYRGT.: HB-10507.TM.), 4B2, 9.4 (ATTC.COPYRGT.:
HB-10508.TM.) and GAP8.3 (ATTC.COPYRGT.: HB-12.TM.). Thus, in some
embodiments, a nanostructure (e.g., protein nanogel or liposome) is
linked to an anti-CD45 antibody. In some embodiments, a protein
nanogel comprising a cytokine (e.g., IL-2, IL-15, IL-15-SA, or a
combination thereof) is linked to an anti-CD45 antibody, and then
coupled to a carrier cell (e.g., T cell) expressing CD45.
[0136] A nanostructure, in some embodiments, may comprise 10 to
10000, or more ligands, depending, in part, on the size of the
nanostructure. For example, a nanostructure may comprise 10 to
10000, 10 to 1000, 10 to 100 ligands, 100 to 10000, 100 to 1000, or
1000 to 10000 ligands.
[0137] In some embodiments, a ligand is linked to a nanostructure
through a crosslinking reaction. In some embodiments, for example,
where the nanostructure is a protein nanogel, the same crosslinker
used to produce the protein nanogel may be used to incorporate the
ligand.
[0138] In some embodiments, a ligand is linked to a nanostructure
(e.g., antibodies, such as anti-CD45) with a permanent (e.g.,
irreversible) linker. In some embodiments, the linker contains two
N-hydroxysulfosuccinimide (sulfo-NHS) groups. In some embodiments,
the linker contains two maleimide groups, or other reactive groups
known in the art for coupling molecules to antibodies. In some
embodiments the linker is a reversible linker (e.g.: a redox
responsive linker that degrades in the presence of a reducing agent
(e.g.: glutathione, GSH) under physiological conditions).
[0139] Nanostructures comprising ligands may be coupled to carrier
cells through a "coupling reaction," as described elsewhere herein,
which may be covalent or non-covalent. In some embodiments, a
nanostructure is coupled to a carrier cell through free surface
thiols, as described, for example, in U.S. Patent Application
Publication No. 2011/0293705.
[0140] Polycations
[0141] Some aspects of the present disclosure provide
nanostructures (e.g.: nanogels) comprising on their surface a
polycation. A polycation is a molecule or chemical complex having
positive charges at several sites. Generally, polycations have an
overall positive charge. Examples of polycations for use in
accordance with the present disclosure include, without limitation,
polylysine (poly-L-lysine and/or poly-D-lysine), poly(argininate
glyceryl succinate) (PAGS, an arginine-based polymer),
polyethyleneimine, polyhistidine, polyarginine, protamine sulfate,
polyethylene glycol-b-polylysine (PEG-PLL), or polyethylene
glycol-g-polylysine. Polycations may be used, as provided herein,
to improve adherence of a nanoparticle to a carrier cell (e.g.: T
cell) to increase efficiency of a coupling reaction. Specifically,
polycations, may be used to increase loading of a nanostructure
(e.g.: protein nanogel) to a carrier cell. Polycations may also be
used to increase the coupling or coupling efficiency of a
nanostructure to a carrier cell. It is thought that polycations
enhance electrostatic interactions between negatively-charged ions
of the cell membrane and positively-charged surface ions of the
nanogel.
[0142] In some embodiments, a polycation is added to the surface of
a nanogel prior to coupling the nanogel to the surface of a carrier
cell via a cell surface coupling receptor. In some embodiments, a
polycation (e.g., polyethylene glycol-b-polylysine or PEG-PLL) is
added to the surface of a nanogel prior to coupling the nanogel to
the surface of a cell without a cell surface coupling receptor. In
some embodiments the polycation is polyethylene
glycol-b-polylysine. In some embodiments the polycation is added to
a nanogel prior to coupling the nanogel to the surface of a carrier
cell with a cell surface coupling receptor. In some embodiments the
polycation is added to a nanogel with or without anti-CD45.
[0143] In some embodiments, adding a polycation to a nanostructure
increases the coupling efficiency (e.g., efficiency of coupling a
nanogel to a cell) to at least 50%. For example, adding a
polycation to a nanostructure may increase the coupling efficiency
that coupling efficiency to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90% or 95%. In some embodiments, adding a polycation to a
nanostructure increases the coupling efficiency to 60% to 90%, 70%
to 90%, or 80% to 90%.
[0144] Cell Surface Coupling Receptors for Carrier Cell
Coupling
[0145] Cell surface receptors (e.g., transmembrane receptors) are
proteins that mediate communication between a cell and its
extracellular environment. Extracellular ligands, including
cytokines, growth factors, hormones, neurotransmitters and cell
recognition molecules, bind to a cognate receptor, triggering
conformational changes that transduce an extracellular signal,
initiating intracellular signaling pathways. Cell surface receptors
regulate a number of biological pathways responsible for growth,
differentiation, proliferation and survival. Both T cells and B
cells contain cell surface receptors responsible for activating the
cells.
[0146] As indicated above, a "cell surface coupling receptor"
refers to a molecule (i.e.: cell surface receptor) located at the
surface of a cell (e.g.: a carrier cell) that binds to and
maintains cell surface localization of a cognate ligand (e.g., a
ligand linked to a nanoparticle). The cell surface coupling
receptor is a cell surface receptor that stably couples ligands to
the cell surface with little or no internalization over time. In
certain aspects, cell surface coupling receptors exhibit prolonged
cell surface retention and/or long cell surface half-life. In
certain aspects cell surface coupling receptors are
slowly-internalizing T-cell surface proteins. In certain aspects
the cell surface coupling receptor is a slowly-internalizing
receptor. Thus, aspects of the present disclosure provide
compositions comprising a nanostructure linked to the surface of a
carrier cell (e.g.: a nucleated carrier cell) via a binding
interaction between a cell surface coupling receptor on the carrier
cell and a cognate ligand located on the nanostructure.
[0147] In some embodiments, a cell surface coupling receptor (e.g.,
CD45), when bound to a cognate ligand (e.g., anti-CD45 antibody),
stably couples a nanostructure to a carrier cell. In some
embodiments, the nanostructure coupled to the carrier cell with a
cell surface coupling receptor exhibits prolonged surface
retention. In some embodiments, the nanostructure coupled to the
carrier cell with a cell surface coupling receptor exhibits a long
cell surface half-life.
[0148] In some embodiments, a cell surface coupling receptor (e.g.,
CD45), when bound to a cognate ligand (e.g., anti-CD45 antibody),
remains at the carrier cell surface (i.e., is not internalized) for
at least 24 hours. For example, a cell surface coupling receptor
bound to a cognate ligand may remain at the cell surface for at
least 30 hours, at least 36 hours, at least 42 hours, at least 48
hours, at least 54 hours, at least 60 hours, at least 66 hours, at
least 72 hours. In some embodiments, at least 50% (e.g., at least
60%, at least 70%, at least 80%, at least 90%, or at least 95%) of
the cell surface coupling receptor bound to a cognate ligand
remains at the cell surface for at least at least 24 hours (e.g.,
at least 30 hours, at least 36 hours, at least 42 hours, at least
48 hours, at least 54 hours, at least 60 hours, at least 66 hours,
at least 72 hours).
[0149] A non-limiting example of a cell surface coupling receptor
is CD45. CD45 is a 147 kDa single chain transmembrane member of the
protein tyrosine phosphatase (PTP) family, expressed in both T and
B cells, and is required for T and B cell activation. It contains a
highly glycosylated extracellular domain, a single transmembrane
segment, and two tandem intracellular catalytic domains. The two
catalytic domains are involved in src and JAK kinase signaling and
interact with antigen-receptor complexes. Thus, in some
embodiments, a nanostructure containing (e.g., at its surface) an
anti-CD45 antibody is linked, or coupled, to the surface of a
carrier cell expressing CD45. Internalization of such cell-surface
receptors is minimal. In some embodiments, 50% or more (or more
than 50%) of liposomes or nanoparticles (e.g.: nanogels) linked to
a carrier cell (e.g., T cell) expressing a cell surface coupling
receptor (e.g., CD45) are maintained at the surface of the cell
(i.e., not internalized) for at least 24 hours (e.g., 24, 30, 36,
42, 48, 54, 60, 66, 72 hours). For example, more than 55%, more
than 60%, more than 65%, more than 70%, more than 75%, more than
80%, more than 85%, or more than 90% of liposomes or nanoparticles
(e.g.: nanogels) linked to a cell expressing a cell surface
coupling receptor are maintained at the surface of the carrier cell
for at least 24 hours. In some embodiments, a cell surface coupling
receptor is a human (Homo sapiens) CD45 receptor. Examples of
nucleic acids encoding human CD45 receptors include, without
limitation, NCBI Reference Sequence Nos: NM_080921.3, NM_002838.4,
NR_052021.1 and NM_001267798.1.
[0150] In some embodiments, the cell surface coupling receptor is a
recombinant receptor (e.g., comprising nucleic acid obtained from
multiple sources).
[0151] In some embodiments, a carrier cell expresses at least 2
(e.g., at least 3, at least 4 or at least 5) cell surface coupling
receptors, including, for example, CD45.
[0152] In some embodiments, a carrier cell is engineered to express
a cell surface coupling receptor (e.g., CD45). A cell is engineered
if it contains, for example, an engineered nucleic acid. An
"engineered nucleic acid" is a nucleic acid that does not occur in
nature. It should be understood, however, that while an engineered
nucleic acid as a whole is not naturally-occurring, it may include
nucleotide sequences that occur in nature. In some embodiments, an
engineered nucleic acid comprises nucleotide sequences from
different organisms (e.g., from different species). Engineered
nucleic acids include recombinant nucleic acids and synthetic
nucleic acids. A "recombinant nucleic acid" is a molecule that is
constructed by joining nucleic acids (e.g., isolated nucleic acids,
synthetic nucleic acids or a combination thereof) and, in some
embodiments, can replicate in a living cell. A "synthetic nucleic
acid" is a molecule that is amplified or chemically, or by other
means, synthesized. A synthetic nucleic acid includes those that
are chemically modified, or otherwise modified, but can base pair
with naturally-occurring nucleic acid molecules. Recombinant and
synthetic nucleic acids also include those molecules that result
from the replication of either of the foregoing.
[0153] Thus, in some embodiments, a carrier cell (e.g., a T cell)
is engineered to express CD45. A carrier cell may contain, for
example, an engineered nucleic acid encoding CD45. Such nucleic
acids may be produced using standard molecular biology methods
(see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory
Manual, 2012, Cold Spring Harbor Press). An engineered nucleic acid
may be introduced into a cell by conventional methods, such as, for
example, electroporation (see, e.g., Heiser W. C. Transcription
Factor Protocols: Methods in Molecular Biology.TM. 2000; 130:
117-134), chemical (e.g., calcium phosphate or lipid) transfection
(see, e.g., Lewis W. H., et al., Somatic Cell Genet. 1980 May;
6(3): 333-47; Chen C., et al., Mol Cell Biol. 1987 August; 7(8):
2745-2752), transduction, conjugation, or microinjection of
purified nucleic acid (e.g., DNA) directly into the nucleus of the
cell (see, e.g., Capecchi M. R. Cell. 1980 November; 22(2 Pt 2):
479-88).
[0154] Carrier Cells for Coupling to Nanostructures
[0155] Aspects of the present disclosure provide compositions
comprising a nanostructure (e.g.: protein nanogel or liposome)
stably bound to a carrier cell (e.g., nucleated carrier cell) (or
more simply, "a cell"). Carrier cells are cells to which
nanoparticles are conjugated and which, when administered in vivo,
typically home to target site(s). Suitable target cells are chosen
based on their homing potential, their cell surface phenotype (for
conjugation to the nanoparticles). Cells, in some embodiments, may
be T cells (also referred to as T lymphocytes), B cells or natural
killer (NK) cells, and hematopoietic progenitor cells including,
without limitation, murine lineage-negative, Sca-1-positive and
c-kit-positive cells and their human counterparts. Substantial
levels of free thiol (--SH) groups exist on the surfaces of T
cells, B cells and hematopoietic progenitor cells, thereby
facilitating conjugation of nanostructures to such cells.
[0156] Carrier cells, in some embodiments, can extravasate from
blood vessels (particularly when administered by intravenous
injection) and thereby enter target tissues or organs. Red blood
cells typically are not able to exit the blood stream. Accordingly,
one important class of carrier cells includes nucleated carrier
cells. Thus, in some embodiments, carrier cells are not red blood
cells. In other embodiments, carrier cells are red blood cells.
[0157] Some embodiments of the present disclosure refer to isolated
carrier cells. Isolated carrier cells are cells that have been
separated from the environment in which they naturally occur (i.e.,
they are not present in vivo). T cells in vitro are an example of
an isolated carrier cell. It should be understood that carrier
cells may be isolated from their in vivo environment, conjugated to
nanostructures of the present disclosure, and then re-introduced in
vivo. Such carrier cells are still considered to be isolated
cells.
[0158] The carrier cells, in some embodiments, are autologous to a
subject being treated. In other embodiments, the carrier cells are
non-autologous (yet preferably major histocompatability complex
(MHC) matched cells).
[0159] The carrier cells typically have a half-life in vivo,
following administration (or re-infusion, in some instances) of at
least 48 hours, at least 3 days, at least 4 days, at least 5 days,
at least 6 days, at least 7 days, or more.
[0160] The carrier cells, in some embodiments, are genetically
engineered to express one or more factors including, without
limitation, co-stimulatory molecules or receptors including
chimeric receptors. In other embodiments, the carrier cells are not
genetically engineered. In some embodiments, the carrier cells are
isolated and naturally occurring (i.e., they have not been
genetically or otherwise engineered).
[0161] Depending on their nature and function, the carrier cells,
in some embodiments, are manipulated prior to conjugation with the
nanostructures. The carrier cells, however, need not be
surface-modified in order to facilitate conjugation of the
nanostructures. In some of embodiments, instead, reactive groups
that normally exist on the carrier cell surface are used without
having to incorporate reactive groups or other entities onto the
cell surface. As a result, such carrier cells do not require the
presence of exogenous entities such as antibodies or antibody
fragments, among others, on their surface in order to conjugate to
nanostructures.
[0162] Such manipulation may also involve activation of the carrier
cells, as is routinely performed for T cells. The carrier cells
may, in some embodiments, be expanded and/or activated (or
stimulated, as the terms are used interchangeably herein) in vitro
prior to mixing with nanostructures. Expansion and activation
protocols may vary depending on the carrier cell type but can
include incubation with one or more cytokines, incubation with one
or more cell types, and incubation with one or more antigens. If
the carrier cell is a T cell, then activation may be performed by
incubating the T cells with IL-2, IL-15, IL-15 superagonist
(IL-15SA or I1-15Sa), costimulatory molecules such as B7, B7.2,
CD40, antibodies to various T cell surface molecules including
antibodies to cell surface receptors, anti-CD3 antibodies,
anti-CD28 antibodies, anti-CTLA-4 antibodies, anti-CD40L
antibodies, and the like. In some embodiments, the carrier cells
and more particularly the T cells, are not coated with exogenous
antibodies on their cell surface (i.e., the cells have not been
contacted with antibodies or antibody fragments in vitro prior to
administration).
[0163] Expansion may be measured by proliferation assays involving
incorporation of radiolabeled nucleotides such as tritiated
thymidine. Activation may be measured by production of cytokines
such as IL-2, gamma-IFN, IL-1, IL-4, IL-6 and TNF, among others.
Other ways of measuring expansion and activation are known in the
art and may be used in accordance with the disclosure.
[0164] Carrier cells may be selected prior to administration to a
subject in order to enrich and thus administer higher numbers of
such cells in smaller volumes and/or to remove other, potentially
unwanted, cells from the administered composition. Selection may
involve positive or negative selection including, for example,
column or plate based enrichment protocols that are known in the
art.
[0165] The carrier cells may be eukaryotic cells, such as mammalian
cells (e.g., human cells). Alternatively, they may be non-mammalian
cells. In still other embodiments, the carrier cells may be
prokaryotic cells (e.g., bacterial cells). Several bacterial cell
types are of particular interest. For example, attenuated
salmonella typhimurium is under study as a candidate vector for
oral vaccine delivery (Xiang et al., Immunol Rev 222:117, 2008; and
Iweala et al., J Immunol 183(4):2252, 2009) and engineered E. coli
bacteria have been shown to be capable of specific homing to poorly
oxygenated tumors (Cheong et al., Science 314(5803):1308, 2006).
Bacteria offer new modes of administration and tissue site
targeting possibilities, such as oral administration and the
ability to target therapeutics to the gut and gut-associated
lymphoid tissues. Such microbial vectors may offer advantages
relative to autologous host cells in terms of creating
off-the-shelf ready-to-use cell-nanoparticles systems. Particles
conjugation to microbes can be achieved using the same suite of
chemical strategies described for mammalian cells. In some
instances, temporary removal of flagellar coats of microbes (e.g.,
via simple mechanical shearing as described by Rosu et al., J
Bacteriol 188(14):5196, 2006) can be used to achieve optimal
conjugation of particles to microbe cell bodies.
[0166] T Cells
[0167] In some embodiments, cells of the present disclosure are T
cells. T cells are lymphocytes of a type produced or processed by
the thymus gland and actively participating in the immune response.
T cells can be distinguished from other lymphocytes, such as B
cells and natural killer cells (NK cells), by the presence of a
T-cell receptor (TCR) on the cell surface. Examples of T cells for
use in accordance with the present disclosure include T helper
cells (T.sub.H cells, CD4.sup.+ T cells), cytotoxic T cells (Tc
cells, CTLs, CD8.sup.+ T cells), memory T cells, suppressor T cells
and natural killer T cells (NKT cells).
[0168] T helper cells aid other white blood cells in immunologic
processes, including the maturation of B cells into plasma cells
and memory B cells, and the activation of cytotoxic T cells and
macrophages. They are activated by the presentation of peptide
antigens by MHC class II molecules expressed on the surface of
antigen-presenting cells. Upon activation, T helper cells secrete
cytokines that further aid the immune response. T helper cells can
differentiate into subtypes, which secrete different cytokines,
causing different immune responses.
[0169] Cytotoxic T cells recognize their targets by binding to
antigens associated with MHC class I molecules, which are found on
the surfaces of all nucleated cells. They then destroy the
virus-infected or tumor cells. Cytotoxic T cells are also
implicated in transplant rejection. IL-10, adenosine, and other
molecules secreted by regulatory T cells maintain the cytotoxic T
cells in an inactive anergic state, preventing autoimmune
diseases.
[0170] Memory T cells, which may be either CD4.sup.+ or CD8.sup.+,
persist after an infection has been resolved. When re-exposed to
their cognate antigen, they expand to large numbers of effector T
cells to eliminate the pathogen more efficiently. Suppressor T
cells quell T cell-mediated immunity at the end of an immune
reaction and suppress autoreactive T cells that escaped negative
selection in the thymus. Together with helper T cells, they
constitute the regulatory T cells.
[0171] Natural killer T cells (not to be confused with natural
killer cells, discussed below) link the adaptive and innate immune
systems. Natural killer T cells recognize glycolipid antigen
presented by CD1d, instead of antigens presented by MHC molecules.
Upon activation, natural killer T cells are able to produce
cytokines and release cytolytic molecules.
[0172] B Cells
[0173] In some embodiments, cells of the present disclosure are B
cells. B cells are involved in humoral immunity in the adaptive
immune system. They are distinguishable by the B cell receptors
(BCRs) present on their outer surfaces. B cells make antibodies in
response to antigens, work as antigen-presenting cells, and develop
into memory B cells after activation stemming from antigen
interaction. In addition, B cells secrete cytokines which are used
in immune regulatory functions. B cells circulate in the blood and
lymphatic system, performing immune surveillance. They do not
produce antibodies until they have been fully activated. The B cell
receptor (BCR), a membrane-bound immunoglobulin present on the
surface of the B cell, binds to one specific antigen. B cells are
activated either in a T cell-dependent manner or a T
cell-independent manner Upon activation, a B cell becomes a plasma
B cell or a memory B cell. A B cell may also undergo an
intermediate differentiation step, whereby it hypermutates the
variable region of its immunoglobulin gene and may undergo class
switching. Regulatory B cells secrete IL-10 and TGF-.beta. and are
involved in immune regulation via various mechanisms.
[0174] T and B cells may be harvested from the peripheral blood of
a subject.
[0175] Natural Killer Cells
[0176] In some embodiments, cells of the present disclosure are
Natural killer (NK) cells. Natural killer cells are cytotoxic
lymphocytes of the innate immune response. They often lack
antigen-specific cell surface receptors, and are able to react
immediately without prior exposure to the pathogen. They contain
viral infections while the adaptive immune response generates
antigen-specific cytotoxic T cells to clear the infection
completely. Small granules in their cytoplasm include perforin and
granzymes, which are proteases. When these molecules are released
in close proximity to the target cell, the perforin creates pores
in the cell membrane of the target cell through which the granzymes
and other associated molecules can enter, resulting in apoptosis or
osmotic cell lysis. Natural killer cells also secrete
.alpha.-defensins, antimicrobial molecules that directly kill
bacteria by disrupting their cell walls, similar to the actions of
neutrophils. Cytokines, including IL-12, IL-15, IL-18, IL-2 and
CCL5, are released by cells in response to a viral infection and
signal the NK cell to the presence of viral pathogens in the
region. Natural killer cells secrete IFN.gamma. and TNF.alpha. in
response to viral infections. INF.gamma. activates macrophages for
phagocytosis and lysis, while TNF.alpha. promotes direct NK tumor
cell killing. Natural killer cells also have an immune surveillance
function, and are engaged in reciprocal interactions with dendritic
cells, macrophages, T cells and endothelial cells.
[0177] Hematopoietic Progenitor Cells
[0178] Hematopoietic progenitor cells may be obtained from a number
of sources including but not limited to cord blood, bone marrow,
mobilized peripheral blood and, in some instances, differentiated
embryonic stem cells.
[0179] Hematopoietic progenitor cells have been characterized in
the art. Such cells in the human generally have minimally a CD34+
phenotype, although they may also be CD59.sup.+, Thy1/CD90.sup.+,
CD38.sup.lo/neg, CD33.sup.-, and/or c-kit/CD117.sup.+. They also
are characterized as not expressing lineage specific markers. They
can be harvested from bone marrow, cord blood or peripheral blood
using affinity columns, magnetic beads, fluorescence activated cell
sorting (FACS), some combination thereof, and the like. These cells
have the ability to repopulate one or more hematopoietic lineages
upon transplantation. Preferably, these cells repopulate more than
one lineage, and even more preferably, all lineages. Repopulation
or population of lineages as used herein refers to the
differentiation of the stem cell into one or more lineages such
that progeny of the stem cell contribute to the make-up of that
lineage in the subject. It does not, however, require that the
entire lineage compartment derive from the transplanted cells,
however in some instances this may occur.
[0180] Isolated stem cells may be obtained by fractionating a
heterogeneous cell population according to one or more markers,
including by not limited to cell surface markers.
[0181] Chimeric Antigen Receptor (CAR) T Cells
[0182] T cells may be engineered to express chimeric antigen
receptors (CARs). In their simplest form, CARs contain an antigen
binding domain coupled with the transmembrane domain and the
signaling domain from the cytoplasmic tail of the CD3 .zeta. chain.
There is some evidence that the CD3 .zeta. chain is insufficient to
fully activate transduced T cells. Accordingly, CARs preferably
contain an antigen binding domain, a costimulatory domain, and a
CD3 .zeta. signaling domain. Using a costimulatory domain in
combination with the CD3 .zeta. signaling domain mimics the
two-signal model of T cell activation. For example, some
embodiments of the present disclosure relate to a chimeric antigen
receptor comprising an antigen binding domain, a costimulatory
domain, such as 4-1BB intracellular domain and a CD3 .zeta.
signaling domain. In some embodiments, the antigen binding domain
is fused to the costimulatory domain and a CD3 .zeta. signaling
domain via a linker, such as the CD8a hinge and transmembrance
domain.
[0183] In some embodiments, CAR T cells are engineered to express
antigen binding domains of a monoclonal antibody or antibody
fragment, such as, for example, a Fab or an scFv. In some
embodiments, a scFv is fused to the "BBz" chimeric antigen
receptor, such as for example, huEGFRscFv-BBz chimeric antigen
receptor (Johnson et. Al., 2015, Sci Transl Med., 7(275);
US2014/0271635A1, incorporated herein by reference). The
huEGFRscFv-BBz chimeric antigen receptor is a fusion protein
designed based on the heavy and light chains of the EGFR inhibitor,
cetuximab Specifically, the heavy and light chains of cetuximab
form a single-chain variable fragment that is fused to a portion of
the extracellular and transmembrane domains of human CD8.alpha.,
which links to the BBz signaling domain The BBz domain comprises
the intracellular domains of 4-1BB and CD3.zeta..
[0184] The CAR antigen binding domain can be an antibody or
antibody fragment, such as, for example, a Fab or an scFv.
Non-limiting examples of anti-cancer antibodies include the
following, without limitation:
[0185] trastuzumab (HERCEPTIN.TM. by Genentech, South San
Francisco, Calif.), which is used to treat HER-2/neu positive
breast cancer or metastatic breast cancer;
[0186] bevacizumab (AVASTIN.TM. by Genentech), which is used to
treat colorectal cancer, metastatic colorectal cancer, breast
cancer, metastatic breast cancer, non-small cell lung cancer, or
renal cell carcinoma;
[0187] rituximab (RITUXAN.TM. by Genentech), which is used to treat
non-Hodgkin's lymphoma or chronic lymphocytic leukemia;
[0188] pertuzumab (OMNITARG.TM. by Genentech), which is used to
treat breast cancer, prostate cancer, non-small cell lung cancer,
or ovarian cancer;
[0189] cetuximab (ERBITUX.TM. by ImClone Systems Incorporated, New
York, N.Y.), which can be used to treat colorectal cancer,
metastatic colorectal cancer, lung cancer, head and neck cancer,
colon cancer, breast cancer, prostate cancer, gastric cancer,
ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer,
renal cell cancer, prostate cancer, cervical cancer, or bladder
cancer;
[0190] IMC-1C11 (ImClone Systems Incorporated), which is used to
treat colorectal cancer, head and neck cancer, as well as other
potential cancer targets;
[0191] tositumomab and tositumomab and iodine I.sup.131 (BEXXAR.TM.
by Corixa Corporation, Seattle, Wash.), which is used to treat
non-Hodgkin's lymphoma, which can be CD20 positive, follicular,
non-Hodgkin's lymphoma, with and without transformation, whose
disease is refractory to Rituximab and has relapsed following
chemotherapy;
[0192] In.sup.111 ibirtumomab tiuxetan; Y.sup.90 ibirtumomab
tiuxetan; In.sup.111 ibirtumomab tiuxetan and Y.sup.90 ibirtumomab
tiuxetan (ZEVALIN.TM. by Biogen Idec, Cambridge, Mass.), which is
used to treat lymphoma or non-Hodgkin's lymphoma, which can include
relapsed follicular lymphoma; relapsed or refractory, low grade or
follicular non-Hodgkin's lymphoma; or transformed B-cell
non-Hodgkin's lymphoma;
[0193] EMD 7200 (EMD Pharmaceuticals, Durham, N.C.), which is used
for treating for treating non-small cell lung cancer or cervical
cancer;
[0194] SGN-30 (a genetically engineered monoclonal antibody
targeted to CD30 antigen by Seattle Genetics, Bothell, Wash.),
which is used for treating Hodgkin's lymphoma or non-Hodgkin's
lymphoma;
[0195] SGN-15 (a genetically engineered monoclonal antibody
targeted to a Lewis.gamma.-related antigen that is conjugated to
doxorubicin by Seattle Genetics), which is used for treating
non-small cell lung cancer;
[0196] SGN-33 (a humanized antibody targeted to CD33 antigen by
Seattle Genetics), which is used for treating acute myeloid
leukemia (AML) and myelodysplastic syndromes (MDS);
[0197] SGN-40 (a humanized monoclonal antibody targeted to CD40
antigen by Seattle Genetics), which is used for treating multiple
myeloma or non-Hodgkin's lymphoma;
[0198] SGN-35 (a genetically engineered monoclonal antibody
targeted to a CD30 antigen that is conjugated to auristatin E by
Seattle Genetics), which is used for treating non-Hodgkin's
lymphoma;
[0199] SGN-70 (a humanized antibody targeted to CD70 antigen by
Seattle Genetics), that is used for treating renal cancer and
nasopharyngeal carcinoma;
[0200] SGN-75 (a conjugate comprised of the SGN70 antibody and an
Auristatin derivative by Seattle Genetics); and
[0201] SGN-17/19 (a fusion protein containing antibody and enzyme
conjugated to melphalan prodrug by Seattle Genetics), which is used
for treating melanoma or metastatic melanoma.
[0202] It should be understood that the therapeutic antibodies to
be used in the methods of the present invention are not limited to
those described supra.
[0203] Agents For Use in Nanostructures
[0204] In some embodiments, the present disclosure provides methods
for delivering an agent comprising administering to a subject a
carrier cell covalently bound to a nanostructure that comprises an
agent, wherein the cell does not substantially internalize the
nanostructure and maintains the nanostructure on the cell surface,
and wherein the agent is released from the nanostructure in
vivo.
[0205] The present disclosure contemplates the delivery of agents
to particular cells, and thus potentially to localized regions or
tissues in vivo. As used herein, an "agent" is any atom or molecule
or compound that can be used to provide benefit to a subject
(including without limitation prophylactic or therapeutic benefit).
The agents of particular interest, in some embodiments, are those
that exert an effect on target cells, whether directly or
indirectly. Some agents may exert their effects on tumor cells,
pathogens, or pathogen-infected cells. The nature of the agent
depends on the particular application, as should be apparent.
[0206] Nanostructures (eg: protein nanogels) may carry the agent
internally including for example in pores or in a hollow core. The
nanostructures may carry the agent internally and on its
surface.
[0207] The present disclosure further contemplates that one or more
agents may be used alongside the nanostructures, although not
conjugated to or encapsulated within. For example, the
nanostructures may be formulated together with one or more
agents.
[0208] The agent may be without limitation a chemical entity, a
protein, a polypeptide, a peptide, a nucleic acid, a virus-like
particle, a steroid, a proteoglycan, a lipid, a carbohydrate, and
analogs, derivatives, mixtures, fusions, combinations or conjugates
thereof. The agent may be a pro-drug that is metabolized and thus
converted in vivo to its active (and/or stable) form.
[0209] The agents may be naturally occurring or non-naturally
occurring. Naturally occurring agents include those capable of
being synthesized by the subjects to whom the particles are
administered. Non-naturally occurring are those that do not exist
in nature normally, whether produced by plant, animal, microbe or
other living organism.
[0210] One class of agents that can be delivered in a localized
manner using nanostructures includes chemical compounds that are
non-naturally occurring, or chemical compounds that are not
naturally synthesized by mammalian (and in particular human)
cells.
[0211] A variety of agents that are currently used for therapeutic
purposes can be delivered according to the present disclosure and
these include without limitation immunomodulatory agents such as
immunostimulatory agents, antigens (e.g., HPV protein), adjuvants,
imaging agents, anti-cancer agents, anti-infective agents, and the
like.
[0212] One particular class of agents is inhibitors of
immunosuppression. Examples include Shp1/2 protein tyrosine
phosphatase (PTPase) inhibitor (NSC-87877; CAS 56932-43-5),
sunitinib, or other inhibitors of receptor tyrosine kinases, or p38
MAPK inhibitors including MAPK pathway inhibitors.
[0213] The p38 MAPK pathway inhibitor may be a RAF inhibitor such
as a pan-RAF inhibitor or a selective RAF inhibitor. Examples of
RAF inhibitors include RAF265, sorafenib, dabrafenib (GSK2118436),
SB590885, PLX 4720, PLX4032, GDC-0879 and ZM 336372. The p38 MAPK
pathway inhibitor may be a MEK inhibitor. Examples of MEK
inhibitors include CI-1040/PD184352, AZD6244, PD318088, PD98059,
PD334581, RDEA119,
6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinolin-
e-3-carbonitrile and
4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy--
7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile, trametinib
(GSK1120212), and ARRY-438162.
[0214] The p38 MAPK pathway inhibitor may be an ERK inhibitor.
Examples of ERK inhibitors include VTX11e, AEZS-131, PD98059,
FR180204, and FR148083. Still other p38 MAPK inhibitors are
Tocriset, SB239063, SB203580, pamapimod, dilmapimod, and
PH797804.
[0215] Imaging or Diagnostic Agents. As used herein, an imaging
agent is an agent that emits signal directly or indirectly thereby
allowing its detection in vivo. Imaging agents such as contrast
agents and radioactive agents that can be detected using medical
imaging techniques, such as nuclear medicine scans and magnetic
resonance imaging (MRI). Imaging agents for magnetic resonance
imaging (MRI) include Gd(DOTA), iron oxide or gold nanoparticles;
imaging agents for nuclear medicine include 201T1, gamma-emitting
radionuclide 99 mTc; imaging agents for positron-emission
tomography (PET) include positron-emitting isotopes,
(18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,
gadoamide, and radioisotopes of Pb(II) such as 203 Pb, and 11In;
imaging agents for in vivo fluorescence imaging such as fluorescent
dyes or dye-conjugated nanoparticles. In other embodiments, the
agent to be delivered is conjugated, or fused to, or mixed or
combined with an imaging agent.
[0216] Immunostimulatory Agents. As used herein, an
immunostimulatory agent is an agent that stimulates an immune
response (including enhancing a pre-existing immune response) in a
subject to whom it is administered, whether alone or in combination
with another agent. Examples include antigens, adjuvants (e.g., TLR
ligands such as imiquimod, imidazoquinoline, nucleic acids
comprising an unmethylated CpG dinucleotide, monophosphoryl lipid A
or other lipopolysaccharide derivatives, single-stranded or
double-stranded RNA, flagellin, muramyl dipeptide), cytokines
including interleukins (e.g., IL-2, IL-7, IL-15 (or
superagonist/mutant forms of these cytokines), IL-12, IFN-gamma,
IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies
(e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibody
fragments of these molecules), and the like.
[0217] Immunoinhibitory Agents. As used herein, an immunoinhibitory
agent is an agent that inhibits an immune response in a subject to
whom it is administered, whether alone or in combination with
another agent. Examples include steroids, retinoic acid,
dexamethasone, cyclophosphamide, anti-CD3 antibody or antibody
fragment, and other immunosuppressants.
[0218] Anti-Cancer Agents. As used herein, an anti-cancer agent is
an agent that at least partially inhibits the development or
progression of a cancer, including inhibiting in whole or in part
symptoms associated with the cancer even if only for the short
term. Several anti-cancer agents can be categorized as DNA damaging
agents and these include topoisomerase inhibitors (e.g., etoposide,
ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating
agents (e.g., cisplatin, mechlorethamine, cyclophosphamide,
ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine,
lomustine, carboplatin, dacarbazine, procarbazine), DNA strand
break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin,
idarubicin, mitomycin C), anti-microtubule agents (e.g.,
vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine,
methotrexate, hydroxyurea, 5-fluorouracil, floxuridine,
6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin,
chlorodeoxyadenosine), anthracyclines, vinca alkaloids. or
epipodophyllotoxins.
[0219] Examples of anti-cancer agents include without limitation
Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;
Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone
Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;
Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin;
Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride;
Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib
(VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;
Calusterone; Caracemide; Carbetimer; Carboplatin (a
platinum-containing regimen); Carmustine; Carubicin Hydrochloride;
Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (a
platinum-containing regimen); Cladribine; Crisnatol Mesylate;
Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin;
Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone;
Docetaxel (TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone;
Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin;
Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib
(TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide;
Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine;
Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin;
Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin;
Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC); Interferon
alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon
alpha-n3; Interferon beta-I a; Interferon gamma-I b; Iproplatin;
Irinotecan; Lanreotide; Lenalidomide (REVLIMID, REVIMID);
Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine;
Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol;
Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane;
Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA),
Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin;
Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate;
Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin;
Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine;
Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside;
Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin;
Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin;
Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone;
Thalidomide (THALOMID) and derivatives thereof; Thiamiprine;
Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan;
Toremifene; Trestolone; Triciribine; Trimetrexate; Triptorelin;
Tubulozole; Uracil Mustard; Uredepa; Vapreotide; Verteporfin;
Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate;
Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole;
Zeniplatin; Zinostatin; Zorubicin.
[0220] The anti-cancer agent may be an enzyme inhibitor including
without limitation tyrosine kinase inhibitor, a CDK inhibitor, a
MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase
inhibitor may be without limitation Genistein
(4',5,7-trihydroxyisoflavone), Tyrphostin 25
(3,4,5-trihydroxyphenyl), methylene]-propanedinitrile, Herbimycin
A, Daidzein (4',7-dihydroxyisoflavone), AG-126,
trans-1-(3'-carboxy-4'-hydroxyphenyl)-2-(2'',5''-dihydroxy-phenyl)ethane,
or HDBA (2-Hydroxy5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic
acid. The CDK inhibitor may be without limitation p21, p27, p57,
p15, p16, p18, or p19. The MAP kinase inhibitor may be without
limitation KY12420 (C23H2408), CNI-1493, PD98059, or
4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)
1H-imidazole. The EGFR inhibitor may be without limitation
erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline
derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF,
lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
[0221] The anti-cancer agent may be a VEGF inhibitor including
without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS),
pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib,
ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and
semaphorin.
[0222] The anti-cancer agent may be an antibody or an antibody
fragment including without limitation an antibody or an antibody
fragment including but not limited to bevacizumab (AVASTIN),
trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell
chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6,
anti-CD33, indicated for leukemia such as acute myeloid leukemia),
rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for
B cell malignancy), MDX-210 (bispecific antibody that binds
simultaneously to HER-2/neu oncogene protein product and type I Fc
receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab
(OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX),
daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for
respiratory conditions such as RSV infection), ibritumomab tiuxetan
(ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab
(ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-05, IOR-T6
(anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT OV103), epratuzumab
(LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for
brain cancer, melanoma).
[0223] Protein Agents. Examples of protein agents for use in
accordance with the present disclosure include, without limitation,
antibodies, single chain antibodies, antibody fragments, enzymes,
co-factors, receptors, ligands, transcription factors and other
regulatory factors, some antigens (as discussed below), cytokines,
chemokines, and the like. These protein agents may or may not be
naturally occurring. Other proteins are contemplated and may be
used in accordance with the disclosure.
[0224] In some embodiments, the protein agents of the disclosure
are fusion proteins of a biologically active protein fused to an
immunoglobulin Fc domain, referred to as an Fc fusion protein
(e.g., human IgG1 Fc fusion protein). In some embodiments, the
biologically active protein of the nanogel is a cytokine,
including, without limitation, IL-12, IL-15, IL-15-Sa, IL-18, IL-2,
and CCLS. In some embodiments the proteins of the nanogel are
cytokines fused to Fc domains (e.g., human IgG1 Fc domains).
[0225] In some embodiments, protein agents of the disclosure are
immunomodulatory proteins (e.g., immunostimulatory or
immunoinhibitory proteins). As used herein, an immunomodulatory
protein is a protein that modulates (e.g., stimulates or inhibits)
an immune response (including enhancing or reducing a pre-existing
immune response) in a subject to whom it is administered, whether
alone or in combination with another protein or agent. In certain
embodiments of the disclosure immunomodulatory proteins are PD-L1,
CTLA-4, IL-10 or TGF-beta.
[0226] In some embodiments, protein agents of the disclosure are
immunostimulatory proteins. As used herein, an immunostimulatory
protein is a protein that stimulates an immune response (including
enhancing a pre-existing immune response) in a subject to whom it
is administered, whether alone or in combination with another
protein or agent. Examples of immunostimulatory proteins that may
be used in accordance with the disclosure include, without
limitation, antigens, adjuvants (e.g., flagellin, muramyl
dipeptide), cytokines including interleukins (e.g., IL-2, IL-7,
IL-15 (or superagonist/mutant forms of these cytokines), IL-15SA,
IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand), and
immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28,
anti-CD3, or single chain/antibody fragments of these molecules).
Cytokines are a class of small proteins (.about.5-20 kDa) that are
released by cells and affect the behavior of cells via cell
signaling. Cytokines are produced by various cell types, including,
without limitation, immune cells such as macrophages, B
lymphocytes, T lymphocytes, and mast cells, and endothelial cells,
fibroblasts, and a variety of stromal cells. A cytokine may be
produced by more than cell type. Cytokines include, without
limitation, chemokines, interferons, interleukins, lymphokines, and
tumour necrosis factor. Other immunostimulatory proteins are
contemplated and may be used in accordance with the disclosure.
[0227] In some embodiments the immunostimulatory protein is
IL-15SA. The combination of human IL-1.5 with soluble human
IL-15R.alpha. generates a complex termed IL-15 superagonist
(IL-15SA) that possesses greater biological activity than human
IL-15 alone.
[0228] Soluble human IL-15R.alpha., as well as truncated versions
of the extracellular domain, has been described. in the art (Wei et
al., 2001 J of Immumol. 167: 277-282). The amino acid sequence of
human IL-15R.alpha. is set forth in SEQ ID NO: 9. Accordingly, some
aspects of the disclosure relate to IL-15SA comprising a complex of
human IL-15 and soluble human IL-15R.alpha. molecules. In some
aspects of the disclosure, IL-15SA comprises a complex of human
IL-15 and soluble human 1L-15R.alpha. comprising all or a portion
of the extracellular domain, without the transmembrane or
cytoplasmic domain. In some aspects of the disclosure, IL-15SA
comprises a complex of human IL-15 and soluble human IL-15R.alpha.
comprising the full extracellular domain or a truncated form of the
extracellular domain which retains IL-15 binding activity. Some
aspects of the disclosure relate to IL-15SA comprising a complex of
human IL-15 and soluble human IL-15R.alpha. comprising a truncated
form of the extracellular domain which retains IL-15 binding
activity, such as amino acids 1-60, 1-61, 1-62, 1-63, 1-64 or 1-65
of human IL-15R.alpha.. In some aspects of the disclosure, IL-15SA
comprises a complex of human IL-15 and soluble human IL-15R.alpha.
comprising a truncated form of the extracellular domain which
retains IL-15 binding activity, such as amino acids 1-80, 1-81,
1-82, 1-83, 1-84 or 1-85 of human IL-15R.alpha.. In some aspects of
the disclosure, IL-15SA comprises a complex of human IL-15 and
soluble human IL-15R.alpha. comprising a truncated form of the
extracellular domain which retains IL-15 binding activity, such as
amino acids 1-180, 1-181, or 1-182 of human IL-15R.alpha..
[0229] Some aspects of the disclosure relate to IL-15SA comprising
a complex of human IL-15 and soluble human IL-15R.alpha. comprising
a truncated form of the extracellular domain which retains IL-15
binding activity and comprises a Sushi domain. The Sushi domain of
IL-15R.alpha., is described in the art as approximately 60 amino
acids in length and comprises 4 cysteines. (Wei et al., 2001).
Truncated forms of soluble human IL-15R.alpha. which retain IL-15
activity and comprise a Sushi domain are useful in IL-15SA of the
present disclosure.
[0230] Mutant forms of human. IL-15 are known in the art,
Accordingly, the present disclosure provides any of the foregoing
IL-15SA complexes in which human IL-15 is wild-type or mutant IL-15
comprising one or more mutations (e.g., one or more amino acid
substitutions, additions or deletions). An exemplary IL-15 mutant
having increased biological activity relative to wild-type IL-15
for use in the IL-15SA of the present disclosure comprises an
asparagine tin aspartic acid substitution at amino acid 72 (N72D).
(Zhu et al., 2009 J of Immunol. 183:3598.)
[0231] In any of the foregoing embodiments, the present disclosure
relates to a complex comprising soluble human IL-15R.alpha.
expressed as a fusion protein, such as an Fc fusion as described
herein (e.g., human IgG1 Fc), with IL-15. In some embodiments,
IL-15SA comprises a dimeric human IL-15R.alpha.Fc fusion protein
(e.g., human IgG1 Fc) complexed with two human IL-15 molecules.
[0232] In some embodiments an IL-15SA cytokine complex comprises an
IL-15 molecule comprising an amino acid sequence set forth in SEQ
ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some
embodiments, an IL-15SA cytokine complex comprises a soluble
IL-15R.alpha. molecule comprising a sequence of SEQ ID NO: 3, SEQ
ID NO: 7 or SEQ ID NO: 8.
[0233] In some embodiments the IL-15SA is a cytokine complex
comprising a dimeric IL-15R.alpha.Fc fusion protein complexed with
two IL-15 molecules. In some embodiments, IL-15-SA comprises a
dimeric IL-15R.alpha.Su (Sushi domain)/Fc (SEQ ID NO: 2) and two
IL-15N72D (SEQ ID NO: 1) molecules (also known as ALT-803), as
described in US20140134128, incorporated herein by reference. In
some embodiments, the IL-15SA comprises a dimeric
IL-15R.alpha.Su/Fc molecule (SEQ ID NO: 2) and two IL-15 molecules
(SEQ ID NO: 4). In some embodiments, the IL-15SA comprises a
dimeric IL-15R.alpha.Su/Fc molecule (SEQ ID NO: 2) and two IL-15
molecules (SEQ ID NO: 5). In some embodiments, the IL-15SA
comprises a dimeric IL-15R.alpha.Su/Fc molecule (SEQ ID NO: 2) and
two IL-15 molecules (SEQ ID NO: 6).
[0234] In some embodiments, the IL-15SA comprises a dimeric
IL-15R.alpha.Su/Fc molecule (SEQ ID NO: 2) and two IL-15 molecules
comprising sequences selected from SEQ ID NO: 1, 4, 5, and 6.
[0235] In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 3) and two IL-15 molecules (SEQ
ID NO: 1). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 3) and two IL-15 molecules (SEQ
ID NO: 4). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 3) and two IL-15 molecules (SEQ
ID NO: 5). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 3) and two IL-15 molecules (SEQ
ID NO: 6).
[0236] In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 7) and two IL-15 molecules (SEQ
ID NO: 1). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 7) and two IL-15 molecules (SEQ
ID NO: 4). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 7) and two IL-15 molecules (SEQ
ID NO: 5). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 7) and two IL-15 molecules (SEQ
ID NO: 6).
[0237] In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 8) and two IL-15 molecules (SEQ
ID NO: 1). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 8) and two IL-15 molecules (SEQ
ID NO: 4). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 8) and two IL-15 molecules (SEQ
ID NO: 5). In some embodiments, the IL-15SA comprises a soluble
IL-15R.alpha. molecule (SEQ ID NO: 8) and two IL-15 molecules (SEQ
ID NO: 6).
[0238] In some embodiments, the IL-15SA comprises a dimeric
IL-15R.alpha.Su/Fc (SEQ ID NO: 2) molecule and two IL-15 molecules
(SEQ ID NO: 5). In some embodiments, the IL-15SA comprises a
dimeric IL-15R.alpha.Su/Fc (SEQ ID NO: 2) molecule and two IL-15
molecules (SEQ ID NO: 6).
[0239] In some embodiments, the IL-15SA comprises SEQ ID NO: 1 and
SEQ ID NO: 3. In some embodiments IL-15SA comprises SEQ ID NO: 4 or
SEQ ID NO: 5. In some embodiments the IL-15SA comprises SEQ ID NO:
4 and SEQ ID NO: 2. In some embodiments the IL-15SA comprises SEQ
ID NO: 5 and SEQ ID NO: 2 In some embodiments the IL-15SA comprises
SEQ ID NO: 6 and SEQ ID NO: 2. In some embodiments, the IL-15SA
comprises SEQ ID NO: 4 and SEQ ID NO: 3. In some embodiments the
IL-15SA comprises SEQ ID NO: 5 and SEQ ID NO: 3.
[0240] In some embodiments, protein agents of the disclosure are
cancer antigens. A cancer antigen is an antigen that is expressed
preferentially by cancer cells (i.e., it is expressed at higher
levels in cancer cells than on non-cancer cells) and, in some
instances, it is expressed solely by cancer cells. Cancer antigens
may be expressed within a cancer cell or on the surface of the
cancer cell. Cancer antigens that may be used in accordance with
the disclosure include, without limitation, MART-1/Melan-A, gp100,
adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b,
colorectal associated antigen (CRC)-0017-1A/GA733, carcinoembryonic
antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen
(PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen
(PSMA), T cell receptor/CD3-zeta chain and CD20. The cancer antigen
may be selected from the group consisting of MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,
MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4
and MAGE-05. The cancer antigen may be selected from the group
consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, GAGE-8 and GAGE-9. The cancer antigen may be selected from
the group consisting of BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1,
CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,
.alpha.-fetoprotein, E-cadherin, .alpha.-catenin, .beta.-catenin,
.gamma.-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27,
adenomatous polyposis coli protein (APC), fodrin, Connexin 37,
Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human
papilloma virus proteins, Smad family of tumor antigens, lmp-1,
P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen
phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,
SCP-1 and CT-7, CD20 and c-erbB-2. Other cancer antigens are
contemplated and may be used in accordance with the disclosure.
[0241] In some embodiments, the protein of the nanogel is human
papillomavirus (HPV) protein.
[0242] In some embodiments, protein agents of the disclosure are
antibodies or antibody fragments including, without limitation,
bevacizumab (AVASTIN.RTM.), trastuzumab (HERCEPTIN.RTM.),
alemtuzumab (CAMPATH.RTM., indicated for B cell chronic lymphocytic
leukemia,), gemtuzumab (MYLOTARG.RTM., hP67.6, anti-CD33, indicated
for leukemia such as acute myeloid leukemia), rituximab
(RITUXAN.RTM.), tositumomab (BEXXAR.RTM., anti-CD20, indicated for
B cell malignancy), MDX-210 (bispecific antibody that binds
simultaneously to HER-2/neu oncogene protein product and type I Fc
receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab
(OVAREX.RTM., indicated for ovarian cancer), edrecolomab
(PANOREX.RTM.), daclizumab (ZENAPAX.RTM.), palivizumab
(SYNAGIS.RTM., indicated for respiratory conditions such as RSV
infection), ibritumomab tiuxetan (ZEVALIN.RTM., indicated for
Non-Hodgkin's lymphoma), cetuximab (ERBITUX.RTM.), MDX-447, MDX-22,
MDX-220 (anti-TAG-72), IOR-05, IOR-T6 (anti-CD1), IOR EGF/R3,
celogovab (ONCOSCINT.RTM. OV103), epratuzumab (LYMPHOCIDE.RTM.),
pemtumomab (THERAGYN.RTM.) and Gliomab-H (indicated for brain
cancer, melanoma). Other antibodies and antibody fragments are
contemplated and may be used in accordance with the disclosure.
[0243] Anti-Infective Agents. The agent may be a prophylactic agent
or an anti-infective agent including without limitation an
anti-bacterial agent, an anti-viral agent, an anti-parasitic agent,
an anti-fungal agent, and an anti-mycobacterial agent.
[0244] Anti-bacterial agents may be without limitation
.beta.-lactam antibiotics, penicillins (such as natural
penicillins, aminopenicillins, penicillinase-resistant penicillins,
carboxy penicillins, ureido penicillins), cephalosporins (first
generation, second generation, and third generation
cephalosporins), other .beta.-lactams (such as imipenem,
monobactams), .beta.-lactamase inhibitors, vancomycin,
aminoglycosides and spectinomycin, tetracyclines, chloramphenicol,
erythromycin, lincomycin, clindamycin, rifampin, metronidazole,
polymyxins, sulfonamides and trimethoprim, or quinolines.
[0245] Other anti-bacterials may be without limitation Acedapsone;
Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin;
Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin
Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;
Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin;
Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin;
Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin;
Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride;
Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc;
Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate;
Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione
Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate;
Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium;
Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam
Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;
Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;
Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime;
Cefepime Hydrochloride; Cefetecol; Cefixime; Cefmenoxime
Hydrochloride; Cefmetazole; Cefmetazole Sodium; Cefonicid
Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide;
Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam
Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole;
Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome
Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin
Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone
Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;
Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin
Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;
Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride;
Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate;
Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium
Succinate; Chlorhexidine Phosphanilate; Chloroxylenol;
Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride;
Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin;
Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;
Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;
Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine;
Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin
Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine;
Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline
Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin;
Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione;
Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline
Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin;
Epicillin; Epitetracycline Hydrochloride; Erythromycin;
Erythromycin Acistrate; Erythromycin Estolate; Erythromycin
Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate;
Erythromycin Propionate; Erythromycin Stearate; Ethambutol
Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;
Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;
Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic
Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin;
Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem;
Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate;
Kitasamycin; Levofuraltadone; Levopropylcillin Potassium;
Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin;
Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef;
Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin
Potassium Phosphate; Mequidox; Meropenem; Methacycline;
Methacycline Hydrochloride; Methenamine; Methenamine Hippurate;
Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole
Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin
Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin
Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium;
Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin
Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin
Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel;
Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol;
Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide;
Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin
Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline;
Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin;
Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate;
Penamecillin; Penicillin G Benzathine; Penicillin G Potassium;
Penicillin G Procaine; Penicillin G Sodium; Penicillin V;
Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin
Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin
Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;
Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin;
Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate;
Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin;
Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin;
Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate;
Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate;
Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin;
Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium;
Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin
Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin;
Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate;
Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;
Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine
Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter;
Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran;
Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet;
Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;
Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium;
Talampicillin Hydrochloride; Teicoplanin; Temafloxacin
Hydrochloride; Temocillin; Tetracycline; Tetracycline
Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim;
Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium;
Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium
Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin;
Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines;
Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin;
Vancomycin Hydrochloride; Virginiamycin; or Zorbamycin.
[0246] Anti-mycobacterial agents may be without limitation
Myambutol (Ethambutol Hydrochloride), Dapsone
(4,4'-diaminodiphenylsulfone), Paser Granules (aminosalicylic acid
granules), Priftin (rifapentine), Pyrazinamide, Isoniazid, Rifadin
(Rifampin), Rifadin IV, Rifamate (Rifampin and Isoniazid), Rifater
(Rifampin, Isoniazid, and Pyrazinamide), Streptomycin Sulfate or
Trecator-SC (Ethionamide).
[0247] Anti-viral agents may be without limitation amantidine and
rimantadine, ribivarin, acyclovir, vidarabine, trifluorothymidine,
ganciclovir, zidovudine, retinovir, and interferons.
[0248] Anti-viral agents may be without limitation further include
Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine;
Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone;
Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine
Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;
Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine
Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet
Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium;
Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine
Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;
Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;
Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;
Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;
Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate;
Viroxime; Zalcitabine; Zidovudine; Zinviroxime or integrase
inhibitors.
[0249] Anti-fungal agents may be without limitation imidazoles and
triazoles, polyene macrolide antibiotics, griseofulvin,
amphotericin B, and flucytosine. Antiparasites include heavy
metals, antimalarial quinolines, folate antagonists,
nitroimidazoles, benzimidazoles, avermectins, praxiquantel,
ornithine decarboxylase inhibitors, phenols (e.g., bithionol,
niclosamide); synthetic alkaloid (e.g., dehydroemetine);
piperazines (e.g., diethylcarbamazine); acetanilide (e.g.,
diloxanide furonate); halogenated quinolines (e.g., iodoquinol
(diiodohydroxyquin)); nitrofurans (e.g., nifurtimox); diamidines
(e.g., pentamidine); tetrahydropyrimidine (e.g., pyrantel pamoate);
or sulfated naphthylamine (e.g., suramin)
[0250] Other anti-infective agents may be without limitation
Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam
Disodium; Ornidazole; Pentisomicin; Sarafloxacin Hydrochloride;
Protease inhibitors of HIV and other retroviruses; Integrase
Inhibitors of HIV and other retroviruses; Cefaclor (Ceclor);
Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin (Mefoxin);
Cefuroxime axetil (Ceftin); Ciprofloxacin (Cipro); Aminacrine
Hydrochloride; Benzethonium Chloride: Bithionolate Sodium;
Bromchlorenone; Carbamide Peroxide; Cetalkonium Chloride;
Cetylpyridinium Chloride: Chlorhexidine Hydrochloride; Clioquinol;
Domiphen Bromide; Fenticlor; Fludazonium Chloride; Fuchsin, Basic;
Furazolidone; Gentian Violet; Halquinols; Hexachlorophene: Hydrogen
Peroxide; Ichthammol; Imidecyl Iodine; Iodine; Isopropyl Alcohol;
Mafenide Acetate; Meralein Sodium; Mercufenol Chloride; Mercury,
Ammoniated; Methylbenzethonium Chloride; Nitrofurazone;
Nitromersol; Octenidine Hydrochloride; Oxychlorosene; Oxychlorosene
Sodium; Parachlorophenol, Camphorated; Potassium Permanganate;
Povidone-Iodine; Sepazonium Chloride; Silver Nitrate; Sulfadiazine,
Silver; Symclosene; Thimerfonate Sodium; Thimerosal; or Troclosene
Potassium.
[0251] Adjuvants. The adjuvant may be without limitation alum
(e.g., aluminum hydroxide, aluminum phosphate); saponins purified
from the bark of the Q. saponaria tree such as QS21 (a glycolipid
that elutes in the 21st peak with HPLC fractionation; Antigenics,
Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene
(PCPP polymer; Virus Research Institute, USA), Flt3 ligand,
Leishmania elongation factor (a purified Leishmania protein; Corixa
Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes
which contain mixed saponins, lipids and form virus-sized particles
with pores that can hold antigen; CSL, Melbourne, Australia),
Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which
contains alum and MPL; SBB, Belgium), non-ionic block copolymers
that form micelles such as CRL 1005 (these contain a linear chain
of hydrophobic polyoxypropylene flanked by chains of
polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS
(e.g., IMS 1312, water-based nanoparticles combined with a soluble
immunostimulant, Seppic).
[0252] Adjuvants may be Toll-like receptor (TLR) ligands. Adjuvants
that act through TLR3 include without limitation double-stranded
RNA. Adjuvants that act through TLR4 include without limitation
derivatives of lipopolysaccharides such as monophosphoryl lipid A
(MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl
dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi);
OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma
SA, Meyrin, Switzerland). Adjuvants that act through TLR5 include
without limitation flagellin. Adjuvants that act through TLR7
and/or TLR8 include single-stranded RNA, oligoribonucleotides
(ORN), synthetic low molecular weight compounds such as
imidazoquinolinamines (e.g., imiquimod, resiquimod). Adjuvants
acting through TLR9 include DNA of viral or bacterial origin, or
synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another
adjuvant class is phosphorothioate containing molecules such as
phosphorothioate nucleotide analogs and nucleic acids containing
phosphorothioate backbone linkages.
[0253] Compositions and Methods of Administration
[0254] Compositions provided herein comprise a nucleated carrier
cell that homes to a tumor and is coupled to a nanostructure (e.g.,
a protein nanogel or a liposome) comprising an agent (e.g., a
biologically active protein), wherein the carrier cell comprises a
cell surface coupling receptor (e.g., CD45) and is coupled to the
nanostructure with a ligand that binds the receptor or the carrier
cell comprises a negatively charged cell membrane and the
nanostructure comprises a polycation surface which interacts
electrostatically with the cell membrane.
[0255] In certain aspects the composition comprises a carrier cell
having a CD45 receptor and the carrier cell is coupled to the
nanostructure having a ligand that binds the CD45 receptor. In
certain aspects the ligand is an anti-CD45 monoclonal antibody.
[0256] In certain aspects the carrier cell comprises a negatively
charged cell membrane and the nanostructure comprises a polycation
surface which interacts electrostatically with the cell membrane.
In certain aspects, the polycation is polylysine. In certain
aspects, the polycation is polyethylene glycol-b-polylysine
(PEG-PLL).
[0257] In some embodiments, the composition comprises a protein
nanogel. In certain aspects the protein nanogel comprises a
plurality of biologically active proteins reversibly and covalently
crosslinked to each other through a degradable linker. In some
embodiments, the degradable linker is a redox responsive linker
that comprises a disulfide bond.
[0258] In some embodiments, the composition is a liposome. In
certain embodiments the liposome comprises a plurality of
biologically active proteins. In certain aspects the liposome is an
interbilayer-crosslinked multilamellar or unilamellar vesicle.
[0259] In some embodiments, the composition comprises a carrier
cell wherein the carrier cell is a T cell, a B cell a Natural
Killer (NK) cell or a stem cell. In some aspects, the carrier cell
is a T cell. In some aspects, the T cell is a CD8.sup.+ T cell or a
CD4.sup.+ T cell. In some aspects, the T cell is an adoptively
transferred T cell. In some aspects, the T cell is a chimeric
antigen receptor (CAR) T cell.
[0260] In some embodiments, the composition comprises a
nanostructure comprising biologically active protein wherein the
biologically active protein is selected from the group consisting
of antibodies, antibody fragments, soluble protein receptors and
cytokines. In some aspects, the cytokine is IL-2, IL-15 or IL-15SA.
In some aspects, the cytokine is IL-15-Sa. In some aspects, the
IL-15Sa comprises a complex comprising a dimeric IL-15R.alpha.Su/Fc
and two IL-15N72D molecules. In some aspects, the dimeric
IL-15R.alpha.Su/Fc comprises an amino acid sequence set forth in
SEQ ID NO: 1 and the IL-15N72D molecule comprises an amino acid
sequence set forth in SEQ ID NO: 2.
[0261] In some embodiments the composition comprises a
pharmaceutically acceptable carrier. In some aspects the
composition is useful as a medicament for delivering a biologically
active protein to a subject having a tumor.
[0262] Certain aspects of the disclosure provide methods of
treating cancer in a subject comprising administering to a subject
in need thereof a composition as described herein.
[0263] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) that homes to
a tumor and is coupled to a nanostructure comprising a biologically
active protein, wherein (a) the carrier cell comprises a CD45
receptor and is coupled to the nanostructure with a ligand that
binds the CD45 receptor; or (b) the carrier cell comprises a
negatively charged cell membrane and the nanostructure comprises a
polycation surface which interacts electrostatically with the cell
membrane; or (c) the carrier cell comprises a CD45 receptor and is
coupled to the nanostructure with a ligand that binds the CD45
receptor, and the carrier cell comprises a negatively charged cell
membrane and the nanostructure comprises a polycation surface which
interacts electrostatically with the cell membrane.
[0264] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) comprising a
CD45 receptor and a nanostructure comprising a biologically active
protein, wherein carrier cell is coupled to the nanostructure with
a ligand that binds the CD45 receptor.
[0265] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) that homes to
a tumor and is coupled to a nanostructure comprising a biologically
active protein, wherein the carrier cell comprises a negatively
charged cell membrane and the nanostructure comprises a polycation
surface which interacts electrostatically with the cell
membrane.
[0266] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell comprising a CD45 receptor and
a protein nanogel, wherein the carrier cell is coupled to the
protein nanogel with a ligand that binds the CD45 receptor, and
wherein the protein nanogel comprises a plurality of biologically
active proteins reversibly and covalently crosslinked to each other
through a degradable linker.
[0267] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) that homes to
a tumor and is coupled to a protein nanogel, wherein the carrier
cell comprises a negatively charged cell membrane and the protein
nanogel comprises a polycation surface which interacts
electrostatically with the cell membrane, and wherein the protein
nanogel comprises a plurality of biologically active proteins
reversibly and covalently crosslinked to each other through a
degradable linker.
[0268] Certain aspects of the disclosure provide a composition
comprising a nucleated carrier cell (e.g.: a T cell) comprising a
CD45 receptor and a liposome comprises a plurality of biologically
active proteins, wherein the carrier cell is coupled to the
liposome with a ligand that binds the CD45 receptor.
[0269] The compositions provided herein may be used for a variety
of biomedical and pharmaceutical applications. In some embodiments,
the compositions are used for agent (e.g., drug) delivery in vivo
for targeted immunotherapy. In some embodiments, the compositions
are used in adoptive cell therapy. Adoptive cell therapy (ACT) is a
highly personalized cancer therapy that typically involves
administration to the cancer-bearing host of immune cells with
direct anticancer activity. The invention contemplates
administration of the compositions of the invention to subjects
having or at risk of developing a cancer including for example a
solid tumor cancer. Compositions, including pharmaceutical
compositions, comprising protein nanostructures (e.g., protein
nanogels) are provided herein. Also provided herein are
compositions, including pharmaceutical composition, comprising a
protein nanostructure (e.g., protein nanogels) coupled to a carrier
cell. A composition can be administered to a subject in
pharmaceutically-acceptable amounts and in
pharmaceutically-acceptable compositions. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredients (e.g., biologically-active proteins of the
nanostructures). Such compositions may, in some embodiments,
contain salts, buffering agents, preservatives, and optionally
other therapeutic agents.
[0270] Pharmaceutical compositions also may contain, in some
embodiments, suitable preservatives.
[0271] Pharmaceutical compositions may, in some embodiments, be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy.
[0272] Pharmaceutical compositions suitable for parenteral
administration, in some embodiments, comprise a sterile aqueous or
non-aqueous preparation of the nanostructures, which is, in some
embodiments, isotonic with the blood of the recipient subject. This
preparation may be formulated according to known methods. A sterile
injectable preparation also may be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or
solvent.
[0273] Pharmaceutical compositions of the present disclosure are
administered, in some embodiments, by a conventional route,
including injection or by gradual infusion over time.
Administration may, for example, be oral, intravenous,
intraperitoneal, intramuscular, intracavity, intratumor, or
transdermal.
[0274] Pharmaceutical compositions of the present disclosure are
administered, in some embodiments, in effective amounts. An
"effective amount" is that amount of any of the nanostructure
provided herein that alone, or together with further doses and/or
other therapeutic agents, produces a desired response (e.g.,
pseudoautocrine stimulation, augment T cell expansion and minimize
systemic side effects of adjuvant drugs in vivo).
[0275] Pharmaceutical compositions of the present disclosure, in
some embodiments, may be sterile and contain an effective amount of
a nanostructure (e.g., nanogel), alone or in combination with
another agent, for producing the desired response in a unit of
weight or volume suitable for administration to a subject (e.g.,
human subject). The response can, for example, be measured by
determining the physiological effects of the nanostructure
composition.
[0276] The doses of compositions administered to a subject may be
chosen in accordance with different parameters, in particular in
accordance with the mode of administration used and the state of
the subject. Other factors include the desired period of treatment.
In the event that a response in a subject is insufficient at the
initial doses applied, higher doses (or effectively higher doses by
a different, more localized delivery route) may be employed to the
extent that subject/patient tolerance permits.
[0277] Methods of Administration
[0278] The disclosure also provides methods of administering
compositions comprising carrier cells coupled to nanostructures in
vivo to subjects. The methods of the disclosure can be practiced in
a subject that is likely to benefit from delivery of an agent
(e.g., a biologically active protein) as described herein. Human
subjects are preferred subjects in some embodiments. Subjects also
include animals such as household pets (e.g., dogs, cats, rabbits,
ferrets), livestock or farm animals (e.g., cows, horses, pigs,
sheep, chickens and other poultry), laboratory animals (e.g., mice,
rats, rabbits), and the like. Subjects also include fish and other
aquatic species.
[0279] The subjects to whom compositions are delivered may be
normal, or healthy, subjects. Alternatively they may have or may be
at risk of developing a condition that can be diagnosed or that can
benefit from delivery of one or more agents as disclosed herein.
Such conditions include cancer (e.g., solid tumor cancers),
autoimmune disorders, allergies or allergic conditions, asthma,
transplant rejection, and the like.
[0280] Tests for diagnosing various conditions embraced by the
present disclosure are known in the art and will be familiar to the
ordinary medical practitioner. These laboratory tests include
without limitation microscopic analyses, cultivation dependent
tests (such as cultures), and nucleic acid detection tests. These
include wet mounts, stain-enhanced microscopy, immune microscopy
(e.g., FISH), hybridization microscopy, particle agglutination,
enzyme-linked immunosorbent assays, urine screening tests, DNA
probe hybridization, serologic tests, etc. The medical practitioner
will generally also take a full history and conduct a complete
physical examination in addition to running the laboratory tests
listed above.
[0281] A subject having a cancer is a subject who has detectable
cancer cells. A subject at risk of developing a cancer is a subject
who has a higher than normal probability of developing cancer.
These subjects include, for instance, subjects having a genetic
abnormality that has been demonstrated to be associated with a
higher likelihood of developing a cancer, subjects having a
familial disposition to cancer, subjects exposed to cancer causing
agents (e.g., carcinogens) such as tobacco, asbestos, or other
chemical toxins, and subjects previously treated for cancer and in
apparent remission.
[0282] Subjects having an infection are those that exhibit symptoms
thereof including without limitation fever, chills, myalgia,
photophobia, pharyngitis, acute lymphadenopathy, splenomegaly,
gastrointestinal upset, leukocytosis or leukopenia, and/or those in
whom infectious pathogens or byproducts thereof can be
detected.
[0283] A subject at risk of developing an infection is one that is
at risk of exposure to an infectious pathogen. Such subjects
include those that live in an area where such pathogens are known
to exist and where such infections are common. These subjects also
include those that engage in high risk activities such as sharing
of needles, engaging in unprotected sexual activity, routine
contact with infected samples of subjects (e.g., medical
practitioners), people who have undergone surgery, including but
not limited to abdominal surgery, etc.
[0284] The subject may have or may be at risk of developing an
infection such as a bacterial infection, a viral infection, a
fungal infection, a parasitic infection or a mycobacterial
infection. In these embodiments, the nanostructures may comprise an
anti-microbial agent such as an anti-bacterial agent, an anti-viral
agent, an anti-fungal agent, an anti-parasitic agent, or an
anti-mycobacterial agent and the cell carriers (e.g., the T cells)
may be genetically engineered to produce another agent useful in
stimulating an immune response against the infection, or
potentially treating the infection.
[0285] In some instances, the subjects to whom the compositions are
administered are in need of hematopoietic reconstitution. Such
subjects may have been exposed to a deliberate or accidental
myeloablative event, including without limitation myeloablative
chemotherapy and/or whole body radiation, as may be given as part
of a therapeutic regimen for non-solid cancers or metastatic
cancers. The disclosure provides method of administering to such
subjects hematopoietic progenitor cells conjugated to
nanostructures that comprise agents capable of stimulating the
proliferation of the progenitor cells. In some instances, the
agents may also be differentiating agents (i.e., agents that drive
the progenitor cells and their progeny to differentiate, optionally
towards all lineages or a subset of lineages. In other instances,
the agents may be self-renewal agents (i.e., agents that drive the
progenitor cells to self-renew). In yet other instances, the
carrier cells may be conjugated to nanostructures that comprise
both types of agents, whether such agents be in the same or
different nanostructures. Moreover, the disclosure provides that
exposure of the subject to these different agents may be staggered
(e.g., exposure to the self-renewing agents may occur before
exposure to the differentiating agents).
[0286] Methods of Treatment
[0287] Methods of Treating Cancer
[0288] The disclosure provides administration of compositions as
described herein to subjects having or at risk of developing a
cancer including for example a solid tumor cancer. The cancer may
be carcinoma, sarcoma or melanoma. Carcinomas include without
limitation to basal cell carcinoma, biliary tract cancer, bladder
cancer, breast cancer, cervical cancer, choriocarcinoma, CNS
cancer, colon and rectum cancer, kidney or renal cell cancer,
larynx cancer, liver cancer, small cell lung cancer, non-small cell
lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell
carcinoma, and squamous cell carcinoma), oral cavity cancer,
ovarian cancer, pancreatic cancer, prostate cancer, skin cancer
(including basal cell cancer and squamous cell cancer), stomach
cancer, testicular cancer, thyroid cancer, uterine cancer, rectal
cancer, cancer of the respiratory system, and cancer of the urinary
system.
[0289] Sarcomas are rare mesenchymal neoplasms that arise in bone
(osteosarcomas) and soft tissues (fibrosarcomas). Sarcomas include
without limitation liposarcomas (including myxoid liposarcomas and
pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas,
malignant peripheral nerve sheath tumors (also called malignant
schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's
tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not
bone) Ewing's sarcoma, and primitive neuroectodermal tumor),
synovial sarcoma, angiosarcomas, hemangiosarcomas,
lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, desmoid
tumor (also called aggressive fibromatosis), dermatofibrosarcoma
protuberans (DFSP), malignant fibrous histiocytoma (MFH),
hemangiopericytoma, malignant mesenchymoma, alveolar soft-part
sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic
small cell tumor, gastrointestinal stromal tumor (GIST) (also known
as GI stromal sarcoma), and chondrosarcoma.
[0290] Melanomas are tumors arising from the melanocytic system of
the skin and other organs. Examples of melanoma include without
limitation lentigo maligna melanoma, superficial spreading
melanoma, nodular melanoma, and acral lentiginous melanoma.
[0291] The cancer may be a solid tumor lymphoma. Examples include
Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B cell
lymphoma.
[0292] The cancer may be without limitation bone cancer, brain
cancer, breast cancer, colorectal cancer, connective tissue cancer,
cancer of the digestive system, endometrial cancer, esophageal
cancer, eye cancer, cancer of the head and neck, gastric cancer,
intra-epithelial neoplasm, melanoma neuroblastoma, Non-Hodgkin's
lymphoma, non-small cell lung cancer, prostate cancer,
retinoblastoma, or rhabdomyosarcoma.
[0293] Method of Treating Infection
[0294] The disclosure also provides methods of administrating
compositions described herein to subjects having or at risk of
developing an infection such as a bacterial infection, a viral
infection, a fungal infection, a parasitic infection or a
mycobacterial infection. The bacterial infection may be without
limitation an E. coli infection, a Staphylococcal infection, a
Streptococcal infection, a Pseudomonas infection, Clostridium
difficile infection, Legionella infection, Pneumococcus infection,
Haemophilus infection, Klebsiella infection, Enterobacter
infection, Citrobacter infection, Neisseria infection, Shigella
infection, Salmonella infection, Listeria infection, Pasteurella
infection, Streptobacillus infection, Spirillum infection,
Treponema infection, Actinomyces infection, Borrelia infection,
Corynebacterium infection, Nocardia infection, Gardnerella
infection, Campylobacter infection, Spirochaeta infection, Proteus
infection, Bacteriodes infection, H. pylori infection, or anthrax
infection.
[0295] The mycobacterial infection may be without limitation
tuberculosis or leprosy respectively caused by the M. tuberculosis
and M. leprae species.
[0296] The viral infection may be without limitation a Herpes
simplex virus 1 infection, a Herpes simplex virus 2 infection,
cytomegalovirus infection, hepatitis A virus infection, hepatitis B
virus infection, hepatitis C virus infection, human papilloma virus
infection, Epstein Barr virus infection, rotavirus infection,
adenovirus infection, influenza A virus infection, H1N1 (swine flu)
infection, respiratory syncytial virus infection, varicella-zoster
virus infections, small pox infection, monkey pox infection, SARS
infection or avian flu infection.
[0297] The fungal infection may be without limitation candidiasis,
ringworm, histoplasmosis, blastomycosis, paracoccidioidomycosis,
crytococcosis, aspergillosis, chromomycosis, mycetoma infections,
pseudallescheriasis, or tinea versicolor infection.
[0298] The parasite infection may be without limitation amebiasis,
Trypanosoma cruzi infection, Fascioliasis, Leishmaniasis,
Plasmodium infections, Onchocerciasis, Paragonimiasis, Trypanosoma
brucei infection, Pneumocystis infection, Trichomonas vaginalis
infection, Taenia infection, Hymenolepsis infection, Echinococcus
infections, Schistosomiasis, neurocysticercosis, Necator americanus
infection, or Trichuris trichuria infection.
[0299] Methods of Treating Allergy and Asthma
[0300] The disclosure further provides administration of the
compositions described herein to subjects having or at risk of
developing an allergy or asthma. An allergy is an acquired
hypersensitivity to an allergen. Allergic conditions include but
are not limited to eczema, allergic rhinitis or coryza, hay fever,
bronchial asthma, urticaria (hives) and food allergies, and other
atopic conditions. Allergies are generally caused by IgE antibody
generation against harmless allergens. Asthma is a disorder of the
respiratory system characterized by inflammation, narrowing of the
airways and increased reactivity of the airways to inhaled agents.
Asthma is frequently, although not exclusively, associated with
atopic or allergic symptoms. Administration of Th1 cytokines, such
as IL-12 and IFN-gamma, according to the disclosure can be used to
treat allergy or asthma.
[0301] Methods of Treating Autoimmune Disease
[0302] The disclosure provides administration of the compositions
described herein to subjects having or at risk of developing an
autoimmune disease. Autoimmune disease is a class of diseases in
which a subject's own antibodies react with host tissue or in which
immune effector T cells are autoreactive to endogenous self
peptides and cause destruction of tissue. Thus an immune response
is mounted against a subject's own antigens, referred to as self
antigens. Autoimmune diseases are generally considered to be Th1
biased. As a result, induction of a Th2 immune response or Th2 like
cytokines can be beneficial. Such cytokines include IL-4, IL-5 and
IL-10.
[0303] Autoimmune diseases include but are not limited to
rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic
lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia
gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome,
pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune
hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma
with anti-collagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
[0304] Transplant Therapy
[0305] The methods provided herein may also be used to modulate
immune responses following transplant therapy. Transplant success
is often limited by rejection of the transplanted tissue by the
body's immune system. As a result, transplant recipients are
usually immunosuppressed for extended periods of time in order to
allow the transplanted tissue to survive. The disclosure provides
localized delivery of immunomodulators, and particularly
immunoinhibitory agents, to transplant sites in order to minimize
transplant rejection. Thus, the disclosure provides administration
of the compositions to subjects that are going to undergo, are
undergoing, or have undergone a transplant. The foregoing lists are
not intended to be exhaustive but rather exemplary. Those of
ordinary skill in the art will identify other examples of each
condition type that are amenable to prevention and treatment using
the methods of the disclosure.
[0306] Effective Amounts, Regimens, Formulations
[0307] The compositions described herein are administered in
effective amounts. An effective amount is a dosage of the agent
sufficient to provide a medically desirable result. The effective
amount will vary with the particular condition being treated, the
age and physical condition of the subject being treated, the
severity of the condition, the duration of the treatment, the
nature of the concurrent or combination therapy (if any), the
specific route of administration and like factors within the
knowledge and expertise of the health practitioner. It is preferred
generally that a maximum dose be used, that is, the highest safe
dose according to sound medical judgment.
[0308] For example, if the subject has a tumor, an effective amount
may be that amount that reduces the tumor volume or load (as for
example determined by imaging the tumor). Effective amounts may
also be assessed by the presence and/or frequency of cancer cells
in the blood or other body fluid or tissue (e.g., a biopsy). If the
tumor is impacting the normal functioning of a tissue or organ,
then the effective amount may be assessed by measuring the normal
functioning of the tissue or organ.
[0309] The disclosure provides pharmaceutical compositions.
Pharmaceutical compositions are sterile compositions that comprise
cells, nanostructures and/or agent(s), preferably in a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" means one or more compatible
solid or liquid filler, diluents or encapsulating substances which
are suitable for administration to a human or other subject as
described herein. The term "carrier" denotes an organic or
inorganic ingredient, natural or synthetic, with which the cells,
nanostructures and agent(s) are combined to facilitate
administration. The components of the pharmaceutical compositions
are commingled in a manner that precludes interaction that would
substantially impair their desired pharmaceutical efficiency.
[0310] The compositions, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers. Pharmaceutical
parenteral formulations include aqueous solutions of the
ingredients. Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Alternatively,
suspensions of ingredients may be prepared as oil-based
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes.
[0311] Other Embodiments
[0312] In a first aspect, the present disclosure provides a
composition comprising a nucleated carrier cell that homes to a
tumor and is coupled to a nanostructure comprising an agent,
wherein the carrier cell comprises a cell surface coupling
receptor, and wherein the nanostructure is coupled to the carrier
cell with a ligand that binds to the cell surface coupling
receptor. In some embodiments of the first aspect, the ligand is
selected from the group consisting of antibodies, antibody
fragments, soluble protein receptors, cytokines, peptides, small
molecules, co-factors, hormones and neurotransmitters. In some
embodiments, the cell surface receptor is CD45, such as a receptor
that binds or is bound by an anti-CD45 monoclonal antibody (e.g., a
human anti-CD45 antibody or a humanized anti-CD45 antibody). In
some embodiments, the anti-CD45 monoclonal antibody is selected
from the group consisting of BC8, 4B2, 9.4 and GAP8.3. In any of
the foregoing embodiments of the first aspect, the carrier cell is
a T cell, a B cell or a Natural Killer (NK) cell. In some
embodiments, the carrier cell is a T cell, such as a CD8.sup.+ T
cell or a CD4.sup.+ T cell. In some embodiments, the T cell is an
adoptively transferred T cell. In some embodiments, the T cell is a
chimeric antigen receptor (CAR) T cell.
[0313] In any of the foregoing embodiments of the first aspect, the
nanostructure is selected from the group consisting of: liposomes,
protein nanogels, nucleic acid nanogels and solidified polymers. In
some embodiments, the nanostructure is a liposome. In some
embodiments, the liposome is an interbilayer-crosslinked
multilamellar vesicle (ICMV). In some embodiments, the
nanostructure is a protein nanogel. In any of the foregoing
embodiments of the first aspect, the nanostructure has a diameter
of 1 to 1000 nanometers (nm). In some embodiments, the
nanostructure has a diameter of 50 to 500 nm. In any of the
foregoing embodiments of the first aspect, the ligand is covalently
conjugated to the nanostructure. In some embodiments, the ligand is
covalently conjugated to the nanostructure via a maleimide-thiol
interaction.
[0314] In any of the foregoing embodiments of the first aspect, the
agent is selected from the group consisting of a therapeutic agent,
a prophylactic agent, a diagnostic agent and an imaging agent. In
some embodiments, the agent is selected from the group consisting
of proteins, nucleic acids and small molecule drugs. In some
embodiments, the agent is a protein. In some embodiments, the
protein is a cytokine. In some embodiments, the cytokine is IL-2,
IL-15 or IL-15-Sa. In any of the foregoing embodiments of the first
aspect, the nanostructure comprises on its surface a polycation. In
some embodiments, the polycation is polylysine. In some
embodiments, the polycation is polyethylene glycol-b-polylysine
(PEG-PLL).
[0315] In a second aspect, the present disclosure provides a
composition comprising a nucleated carrier cell that homes to a
tumor coupled to a nanostructure comprising an agent, wherein the
nanostructure comprises a surface associated with a polycation. In
some embodiments, the carrier cell is a T cell, a B cell or a
Natural Killer (NK) cell. In some embodiments, the carrier cell is
a T cell. In some embodiments, the T cell is a CD8.sup.+T cell or a
CD4.sup.+ T cell. In some embodiments, the T cell is an adoptively
transferred T cell. In some embodiments, the T cell is a chimeric
antigen receptor (CAR) T cell. In any of the foregoing embodiments
of the second aspect, the nanostructure is selected from the group
consisting of: liposomes, protein nanogels, nucleic acid nanogels
and solidified polymers. In some embodiments, the nanostructure is
a liposome. In some embodiments, the liposome is an
interbilayer-crosslinked multilamellar vesicle (ICMV). In some
embodiments, the nanostructure is a protein nanogel. In some
embodiments, the nanostructure has a diameter of 1 to 1000
nanometers (nm). In some embodiments, the nanostructure has a
diameter of 50 to 500 nm. In any of the foregoing embodiments of
the second aspect, the carrier cell is covalently conjugated to the
nanostructure via a maleimide-thiol interaction.
[0316] In any of the foregoing embodiments of the second aspect,
the agent is selected from the group consisting of a therapeutic
agent, a prophylactic agent, a diagnostic agent and an imaging
agent. In some embodiments, the agent is selected from the group
consisting of proteins, nucleic acids and small molecule drugs. In
some embodiments, the agent is a protein. In some embodiments, the
protein is a cytokine. In some embodiments, the cytokine is IL-2,
IL-15 or IL-15-Sa. In any of the foregoing embodiments of the
second aspect, the polycation is polylysine. In some embodiments,
the polycation is polyethylene glycol-b-polylysine (PEG-PLL).
[0317] In a third aspect, the present disclosure provides a
composition comprising a T cell having a CD45 receptor coupled to a
protein nanogel that comprises a polycation, wherein the T cell is
coupled to the protein nanogel with a ligand that binds the CD45
receptor. In some embodiments, the ligand is selected from the
group consisting of antibodies, soluble protein receptors,
cytokines, peptides, small molecules, co-factors, hormones and
neurotransmitters. In some embodiments, the ligand is an anti-CD45
monoclonal antibody, such as a human anti-CD45 antibody or a
humanized anti-CD45 antibody. In some embodiments, the anti-CD45
monoclonal antibody is selected from the group consisting of BC8,
4B2, 9.4 and GAP8.3. In any of the foregoing embodiments of the
third aspect, the T cell is a CD8.sup.+T cell or a CD4.sup.+ T
cell. In some embodiments, the T cell is an adoptively transferred
T cell. In some embodiments, the T cell is a chimeric antigen
receptor (CAR) T cell. In any of the foregoing embodiments of the
third aspect, the protein nanogel has a diameter of 1 to 1000
nanometers (nm). In some embodiments, the protein nanogel has a
diameter of 50 to 500 nm. In any of the foregoing embodiments of
the third aspect, the ligand is covalently conjugated to the
protein nanogel. In some embodiments, the anti-CD45 antibody is
covalently conjugated to the protein nanogel via a linker that
contains two N-hydroxysulfosuccinimide groups.
[0318] In any of the foregoing embodiments of the third aspect, the
protein nanogel comprises a protein selected from the group
consisting of therapeutic proteins, prophylactic proteins,
diagnostic proteins and imaging proteins. In some embodiments, the
protein nanogel comprises a cytokine. In some embodiments, the
cytokine is IL-2, IL-15 or IL-15-Sa. In any of the foregoing
embodiments of the third aspect, the polycation is polylysine. In
some embodiments, the polycation is polyethylene
glycol-b-polylysine (PEG-PLL).
[0319] The invention is further illustrated by the following
Examples, which in no way should be construed as further limiting.
The entire contents of all of the references (including literature
references, issued patents, published patent applications, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated by reference, in particular for
the teachings that are referenced herein.
TABLE-US-00001 SEQUENCE TABLE A SEQ ID NO: Name* Sequence 1 Human
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK IL-15 (N72D
CFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESG mutant)
CKECEELEEKNIKEFLQSFVHIVQMFINTS 2 Human IL- ITCPPPMSVE HADIWVKSYS
LYSRERYICN SGFKRKAGTS 15R.alpha.Su/Fc SLTECVLNKA TNVAHWTTPS
LKCIREPKSC DKTHTCPPCP (IgG1 CH2- APELLGGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSHED CH3 (Fc PEVKFNWYVD GVEVHNAKTK PREEQYNSTY domain))
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK 3 Human ITCPPPMSVE HADIWVKSYS
LYSRERYICN SGFKRKAGTS IL-15R.alpha.Su SLTECVLNKA TNVAHWTTPS LKCIR
(65 aa truncated extracellular domain) 4 Human
MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIEDLIQSMH IL-15
IDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD isoform 2
TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSF VHIVQMFINTS 5 Human
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGL IL-15
PKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK isoform 1
VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNG
NVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 6 Human
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAM IL-15
KCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES (without
GCKECEELEEKNIKEFLQSFVHIVQMFINTS signal peptide) 7 Human
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS IL-15R.alpha.
SLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT (85 aa TAGV truncated
extracellular domain) 8 Human
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS IL-15R.alpha.
SLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT (182 aa -
TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL truncated
MPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPP extracellular
GVYPQGHSDTTVAISTST domain) 9 Human IL-
MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEH 15R.alpha.
ADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT Full length
NVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESL (Swiss prot:
SPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTE Q13261.1)
ISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT
VAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEA LPVTWGTSSRDEDLENCSHHL
*Su, refers to sushi domain.
EXAMPLES
[0320] While many nanostructures close to the cell surface are
internalized rapidly via endocytosis or passive membrane
permeation, some can retain on cell surface for a longer period of
time. The reasons for the differences in internalization kinetics
still remain elusive. Thus, studying the slowly-internalizing cell
surface proteins bound by nanostructures provides insight into
designing nanostructures with long surface retention. Currently,
most effort is devoted to design internalizing drug conjugates to
deliver intracellular drugs. However, if cell surface receptors
with minimal internalization can be employed to design new
structures with prolonged stability and residence time on cell
surface, expanded drug libraries (e.g.: extracellular drugs such as
cytokines and small molecule drugs) can be delivered to cells. The
prolonged stability and maintenance of nanostructures on the cell
surface also improves the efficacy and efficiency of nanostructures
for use in drug delivery, imaging, tracking and diagnostic
purposes.
Example 1
Identification of CD45 as Stable Cell Surface Anchor for Maximizing
Liposome Loading
[0321] An interbilayer-crosslinked multilamellar vesicle (ICMV)
(Nature Mater. 2011, 10, 243-251) is one type of particle that can
remain on the cell surface (e.g., T cell surface) for more than
four days. A candidate pool of slowly-internalizing receptors was
obtained by mass spectrometry analysis of the total surface
proteins ICMVs bound most abundantly. The screening for the main
contributors for ICMVs' long surface retention was conducted by
using liposomes which are surface coupled with an antibody against
top candidate surface receptors such as CD2, CD11 and CD45.
Antibody-conjugated liposomes coupled to carrier cells.
[0322] Liposome Synthesis
[0323] Antibody-conjugated liposomes were produced by hydrating
dried high-T.sub.M phospholipid films containing 2.5% PEG-maleimide
and 1% biotin head groups. Specifically, vacuum dried lipid films
composed of 1,2-distearoyl-sn-glycero-3-phospho
ethanolamine-N-[maleimide(polyethylene glycol)-2000
(maleimide-PEG2000-DSPE)/cholesterol/hydrogenated Soy
L-.alpha.-phosphatidylcholine
(HSPC)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyet-
hylene glycol)-2000 (biotin-PEG.sub.2000-DSPE) (Avanti Polar
Lipids, Alabaster, Ala., USA) in a molar ratio of 2.5/27/68/1.5
together with 1% of a fluorescent lipophilic tracer dye DiD were
rehydrated in 250 .mu.L of 50-mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/150-mM
NaCl-buffer (pH=6.5). Lipids were vortexed every 10 mM for 1 h at
62.degree. C. to form vesicles and size extruded through a
polycarbonate membrane (0.2 .mu.m). After washing in excess PBS and
spinning down by ultracentrifugation at 110,000.times.g for 4 h,
liposomes were re-suspended in 100 .mu.l PBS per 1.4 mg of lipids.
For coupling to maleimide groups of the liposomes, antibody,
cytokine and antibody/cytokine mixtures at different molar ratios
(2-5 mg/mL) were treated with 1.8 mM dithiothreitol (DTT) in the
presence of 10 mM EDTA at 25.degree. C. for 20 mM to expose hinge
region free thiols. DTT was subsequently removed by using Zeba
desalting columns before mixing with maleimide-bearing liposomes
(1/1 wt./wt. for protein/lipid) in PBS. After incubation for 18 h
at 25.degree. C. on a rotator, excess protein was removed by
ultracentrifugation in excess PBS. Unbound liposomes were washed
off before T cells were incubated in Roswell
[0324] Park Memorial Institute (RPMI) media at 37.degree. C.
[0325] Antibodies (Ab) against surface receptors CD2, CD11, CD45 or
a 1:1 ratio mixture of antibodies against two surface markers
(e.g.: CD2/CD45 or CD11/CD45) were reduced by DTT to expose hinge
region free thiol and then coupled to the liposome surface via a
maleimide-thiol reaction, as described above. Antibodies were
obtained from BioXcell (West Lebanon, N.H., USA), including
anti-mouse CD45 antibody, clone number: MB23G2.
[0326] Liposome Coupling to Carrier Cells
[0327] Antibody-conjugated liposomes (0.7 mg lipids) in 100 .mu.L
PBS were incubated with 20.times.10.sup.6 primed pmel-1 Thy
1.1.sup.+ CD8.sup.+ T-cells in 0.5 ml complete RPMI supplemented
with 10% fetal calf serum (FCS) for 30 min at 37.degree. C. with
gentle agitation every 15 min. Conjugated T-cells were washed with
PBS (20 mL.times.2) to remove unbound liposomes and incubated in
RPMI media with recombinant IL-7 (1.5 ng/mL) and 10% FCS at
0.5.times.10.sup.6 cells/mL at 37.degree. C.
[0328] Liposome Characterization
[0329] Unbound liposomes were washed off and cells were incubated
at 37.degree. C. in complete RPMI. Cells were stained by
streptavidin-fluorophore and analyzed by flow cytometer at zero (0)
hours (hr), 6 hr, 24 hr and 48 hr post conjugation (FIG. 1A) or
zero (0) hr, 19 hr, 45 hr and 69 hrs after conjugation (FIG. 1B and
FIG. 1C). The percentage of fluorescently labeled cells was plotted
over time. Only T cells with surface liposomes would be
fluorescently labeled as internalized liposomes did not have access
to the streptavidin-dye.
[0330] After 48 hrs, almost 100% of cells still had anti-CD45
(.alpha.-CD45) antibody decorated liposomes anchoring on their
surface while surface anti-CD11 (.alpha.-CD11) antibody and
anti-CD2 (.alpha.-CD2) antibody liposomes were only found on 30%
and 70% of cells respectively (FIG. 1A). Unexpectedly, CD45
exhibited prolonged cell surface residence time over the faster
internalization receptors CD11 and CD2. When .alpha.-CD45 was used
in combination with .alpha.-CD2 or .alpha.-CD11 to surface decorate
liposomes, liposomes anchored on both CD45 and CD2 (or CD45 and
CD11) still remained on 100% of cells after two days regardless of
the binding to the faster internalization receptor CD2 and CD11
(FIG. 1A). Anti-CD45 liposomes exhibited prolonged cell surface
retention not only compared to CD2 and CD11, but also CD8 and
Thy1.1 (FIG. 1B).
[0331] To further confirm the non-internalizing property of CD45 on
liposomes, CD45 was tested together with a well-known internalizing
IL-2 receptor and a congenic marker Thy1. Antibodies against Thy1
(.alpha.-Thy1.1) or engineered IL-2-Fc protein composed of IL-2
fused to a mouse Fc antibody framework (prepared as previously
described, (J Controlled Release 2013, 172, 426-435) were coupled
to the liposome surface as described above. A ligand mixture
containing fixed amounts of different mole ratios of .alpha.-Thy1.1
and a-CD45 (or IL-2-Fc and .alpha.-CD45) was also used to decorate
the liposome surface. After conjugation to activated CD8.sup.+ T
cells, 95% of IL-2-Fc liposomes and 70% of .alpha.-Thy1.1 liposomes
were internalized within 20 hours respectively (FIG. 1C). Only 1
mol % of .alpha.-CD45 in the ligand cocktail assured 67% of cells
had .alpha.-Thy1.1/.alpha.-CD45 liposomes on the surface even after
70 hours and 5% of .alpha.-CD45 assured almost 100% of cells with
surface liposomes after 3 days. Similarly, 5% of .alpha.-CD45
reversed the fast internalization of IL-2 receptor and almost 90%
of cells with surface retained IL-2-Fc/.alpha.-CD45 liposomes after
3 days. 100% of cells had IL-2-Fc/.alpha.-CD45 liposomes on surface
when 10% .alpha.-CD45 was used (FIG. 1C). Surprisingly, binding of
CD45 is safe to T cells despite the fact that CD45 is one of the
most abundant glycoproteins on the lymphoid cells. When T cells
were incubated with IL-2-Fc liposomes, the presence of .alpha.-CD45
liposomes (0.2 .mu.g/ml) did not change the IL-2 signaling or
affect T cell survival and proliferation (data not shown).
Example 2
Preparation and Surface Modification of Cytokine Nanogels for
Efficient and Stable T Cell Surface Coupling
[0332] Cytokine nanogels (NGs) were prepared as described in
International Publication Number WO 2015/048498 A2, published on 2
Apr. 2015, incorporated by reference herein. Further modifications
we made to the cytokine nanogels as described herein. Generally,
the cytokines were chemically crosslinked with a reversible linker
(NHS-SS-NHS) to form crosslinked nano-structure of proteins, named
protein nanogel (NG, FIG. 2). After completion the crosslinking
reaction of cytokines, anti-CD45 antibody and/or polycations are
added in situ for modifying the NG surface (FIG. 2). Following
synthesis, the nanogels were characterized and coupled to carrier
cells as described further below.
[0333] Synthesis of Nanogels
[0334] Synthesis of NHS-SS-NHS crosslinker: In a 125 mL
round-bottom flask, 2-hydroxyethyl disulfide (1.54 g, 10 mmol) was
dissolved in tetrahydrofuran (THF, 30 mL, anhydrous) and added
dropwise to the solution of phosgene (15 mL, 15 wt. % in toluene,
22 mmol). The mixture was stirred at 25.degree. C. for 10 h
followed by the removal of the solvent under vacuum.
N-hydroxysuccinimide (NHS) (2.3 g, 22 mmol) was dissolved in THF
(30 mL, anhydrous) and added as one portion, and then dry
triethylamine (1.57 mL, 11 mmol) was injected. The reaction was
carried at 40.degree. C. for 16 h. The solvent was removed under
vacuum and the mixture was filtered to remove precipitates. The
crude product was purified by silica gel column chromatography
(dichloromethane/methanol=10/1) and recrystallized with icy hexane
(80 mL). The resulting white solid was dried under vacuum (3.1 g,
yield 71%). .sup.1H-NMR (CDCl.sub.3, 500 MHz): .delta. 4.58 (t,
4H), 3.05 (t, 4H), 2.84 (s, 8H). .sup.13C-NMR (CDCl.sub.3, 500
MHz): .delta. 168.77, 151.66, 68.84, 36.68, 25.69. ESI (m/z): calcd
for C.sub.14H.sub.16N.sub.2O.sub.10S.sub.2, 436.4 [M]; found, 459.0
[M+Na].sup.+.
[0335] Nanogel Assembly
[0336] Human IL-15Sa nanogels: NHS-SS-NHS (93.5 .mu.g, 0.214
.mu.mol) was dissolved in 9.35 .mu.L DMSO was added to
IL-15Sa(ALT-803 (1320 .mu.g, 0.0143 .mu.mol), Altor BioScience
Corporation (Miramar, Fla., USA.) See also US2014/0134128 and
Cytokine 2011, 56, 804-810) solution in 132 .mu.L phosphate
buffered saline (PBS) pH 7.4. The mixture was rotated at 25.degree.
C. for 30 min followed by the addition of 1188 .mu.L PBS buffer.
For nanogels (NGs) incorporating the CD45 targeting antibody,
anti-CD45 (215 .mu.g, 0.0014 .mu.mol) in 31.7 .mu.L PBS buffer was
then added to the diluted solution. Either anti-mouse CD45RB
(clone: MB23G2: purchased from BioXCell (West Lebanon, N.H., USA))
or anti-human CD45 (clone: MEM-28: purchased from Abcam (Cambridge,
United Kingdom)) was used. The reaction mixture was rotated at
25.degree. C. for another 30 min. The preparation of IL-15Sa-NG
without anti-CD45 was similar except that anti-CD45 was replaced by
the permanent linker, NH.sub.2-PEG.sub.10k-NH.sub.2 (715 .mu.g,
0.0715 .mu.mol) (Laysan Bio: Arab, Ala., USA) in 35.8 .mu.L PBS
buffer. Non-degradable NGs (e.g., .alpha.CD45/IL-15Sa-NG
(non-deg.)) were prepared using a permanent linker,
bis(sulfosuccinimidyl) substrate (Thermo Fisher Scientific,
Waltham, Mass., USA) in lieu of NHS-SS-NHS.
[0337] Other protein nanogels: IL-2-Fc protein NGs were prepared
with similar protein concentrations and the same
crosslinker/protein mole ratio. IL-2-Fc is a bivalent fision
protein of the C-terminus of murine wild type IL-2 linked to a
mouse IgG2a backbone. IL-2Fc was prepared as previously described
(J Controlled Release 2013, 172, 426-435). Bovine Serum Albumin
(BSA) (Sigma-Aldrich, St Louis, Mo., USA) and Human IgG (Jackson
Immuno Research Labs, West Grove, Pa., USA) nanogel controls were
also prepared with similar protein concentrations and the same
crosslinker/protein mole ratios as described above. The resultant
NGs were then washed with PBS (1.5 mL.times.3) in an Amicon
centrifugal filter (molecular weight cut-off=100 kDa, Millipore,
Billerica, Mass., USA). For example, in the preparation of mouse
IL-2Fc nanogels, reversible disulfide crosslinker (107.9 .mu.g,
0.247 .mu.mol) dissolved in 10.8 .mu.L DMSO was added to IL-2-Fc
(1650 .mu.g, 0.0165 .mu.mol) solution in 165 .mu.L PBS buffer. The
mixture was rotated at room temperature (rt) for 30 min followed by
the addition of 1485 .mu.L PBS buffer. Anti-CD45 (247.5 .mu.g,
0.0016 .mu.mol) in 43 .mu.L PBS buffer was then added to the
diluted solution. The reaction mixture was rotated at room
temperature for another 30 min. The resultant IL-2-Fc NGs was
collected with Millipore Amicon ultra-centrifugal filter (molecular
weight cutoff=100,000 Da) and washed with PBS (1.5 mL.times.3). The
final concentration of IL-2-Fc NG was determined with Nanodrop
(A280). The purified IL-2-Fc NG was stored in PBS at 4.degree. C.
before use. The preparation of IL-2Fc nanogels without anti-CD45
was similar except that anti-CD45 was replaced by the permanent
linker, NH.sub.2-PEG.sub.10k-NH.sub.2 (715 .mu.g, 0.0715 .mu.mol)
(Laysan Bio: Arab, Ala., USA) in 35.8 .mu.L PBS buffer.
[0338] Polylysine Modification of Nanogels
[0339] In order to enrich more NGs onto T cell surface, a
polycation (e.g., polyethylene glycol-b-polylysine (PEG-PLL)) was
added in situ to some samples prior to carrier cell coupling. For
example, freshly prepared anti-CD45/IL-15Sa-NG solution was diluted
to 1 .mu.g/.mu.L followed by the addition of polyethylene
glycol-b-polylysine (PEG.sub.5K-PLKC.sub.200, 19 .mu.g,
0.0005.mu.mol or 43.6 .mu.g, 0.0011 .mu.mol)(Alamanda Polymers,
Huntsville, Ala., USA) in 43.6-.mu.L PBS prior to T cell coupling
described below. The mixture was then rotated at 25.degree. C. for
30 min and used without further purification.
[0340] Fluorescence and Biotin Labelling of NGs.
[0341] To prepare fluorescently-labeled NGs, cytokine cargos were
fluorescently labeled with Alexa Fluor 647 NHS ester (Thermo Fisher
Scientific) and purified with Amicon ultra-centrifugal filters
(molecular weight cut-off 50 kDa). Fluorescent cytokine was mixed
with non-labelled cytokine (10 mol % labelled cytokine) for the
preparation of fluorescent NGs following the same procedure as
described above. For the preparation of biotinylated NGs,
NHS-SS-NHS (93.5 .mu.g, 0.214 .mu.mol) dissolved in 9.35 .mu.L DMSO
was added to IL-15Sa (1320 .mu.g, 0.0143 .mu.mol) solution in 132
.mu.L PBS buffer. The mixture was rotated at 25.degree. C. for 20
min followed by the addition of EZ-Link NHS-LC-LC-Biotin (40.6
.mu.g, 0.072 .mu.mol, Thermo Fisher Scientific) in 7.5 .mu.L DMSO.
The mixture was rotated at 25.degree. C. for another 20 min and
then diluted with 1188 .mu.L PBS buffer followed by the addition of
anti-CD45 (215 .mu.g, 0.0014 .mu.mol) in 31.7 .mu.L PBS buffer. The
rest procedure was the same as described above.
[0342] Coupling of NGs to Carrier Cells
[0343] In a typical experiment, aCD45/IL-15Sa-NG (950 .mu.g, 0.010
.mu.mol) labelled with Alexa Fluor 647 in 950 .mu.L PBS was added
to mouse CD8.sup.+ T-cells (95.times.10.sup.6) in 475 .mu.L HBSS
followed by incubation at 37.degree. C. for 1 h. The T-cells with
surface coupled NGs were collected by centrifugation at 800.times.g
for 5 min, washed with PBS (1.0 mL.times.2), and resuspended in
buffer or media at desired concentrations for in vitro or in vivo
studies. For measurements of total NG coupling,
fluorescently-labelled NGs were coupled to T-cells, and
supernatants were collected and measured for fluorescence intensity
at excitation/emission wavelengths of 640/680 nm using a plate
reader (Tecan Infinite.RTM. M1000 PRO). Fluorescence readings were
converted to NG concentrations using standard curves prepared from
serial dilutions of NG stock solutions. The amount of coupled NG
was calculated by subtracting the unbound NG from the total added
amount. NG loading per cell was controlled by varying the mass of
NGs added to cells for coupling. For the conjugation of NGs lacking
anti-CD45 to T-cells, IL-15Sa-NG (950 .mu.g, 0.010 .mu.mol) in PBS
(950 .mu.L) was first activated with sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (218 .mu.g, 0.50
.mu.mol) or bis(sulfosuccinimidyl) substrate (286 .mu.g, 0.50
.mu.mol), collected with Amicon ultra-centrifugal filter (molecular
weight cut-off 50 kDa) and washed with PBS (1.5 mL.times.3), and
then added to CD8.sup.+ T-cells (95.times.10.sup.6) in 475 .mu.L
HBSS followed by incubation at 37.degree. C. for 1 hr. Cells were
washed and collected similarly. The amount of conjugated NG was
determined similarly as described above. Coupling of NGs with or
without anti-human CD45 to human CD8.sup.+ T-cells followed the
similar procedures as described above. Similar assays were
performed with anti-CD3/CD28-activated human CD8.sup.+ T cells
isolated from peripheral blood mononuclear cells.
[0344] The modified cytokine NGs showed higher efficiency for T
cell coupling compared with non-modified NGs (Tables 1-3). The
amount of PEG-PLL added in the coupling reaction was titrated, and
it was found that the trend of increased coupling efficiency with
increased amount of PEG-PLL added (Table 1). Unexpectedly, the
coupling efficiency to T cells can be improved to 40.4% when only
0.05 equiv. PEG-PLL was added to the IL-2-Fc NGs with slightly
increased particle size (180.0.+-.2.8 nm) compared with coupling
efficiency of 13.2% for unmodified NGs. Surface density of NGs of
4.04 .mu.g/1.times.10.sup.6 cells were easily achieved, which
permits high loading of cytokine drug to adoptively transferred T
cells for prolonged expansion and anticancer activity. Due to the
different coupling efficiency, T cells with different surface
density of IL-2-Fc NGs were obtained, which was verified with flow
cytometry (data not shown). A similar study was performed with
human IL-15Sa NGs. Coupling efficiency of 77.0% was achieved when
0.05 equiv. PEG-PLL was added to human IL-15Sa NGs (Table 2). T
cells with different surface density of human IL-15Sa NGs were also
obtained and verified with flow cytometry (data not shown).
TABLE-US-00002 TABLE 1 Polycations improve the coupling efficiency
of mouse IL-2-Fc nanogels on T cell surface. PEG-PLL (equiv. of
Surface-bound IL2-Fc, Size Coupling nanogel Entry mol/mol).sup.[a]
(nm).sup.[b] efficiency.sup.[c] (.mu.g/1 .times. 10.sup.6
cells).sup.[d] 1 0 85.1 .+-. 2.6 13.2% 0.66 2 0.01 101.2 .+-. 4.9
33.1% 1.66 3 0.02 111.3 .+-. 1.2 33.3% 1.66 4 0.05 180.0 .+-. 2.8
40.4% 2.02 5 0.08 601.9 .+-. 16.5 80.8% 4.04 Nanogels contained
fixed amounts of CD45 to IL-2Fc (1:10 moles) .sup.[a]Different
amount of PEG-PLL was mixed with mouse IL-2-Fc nanogels right
before the coupling reaction; .sup.[b]The size of the nanogel was
measured with dynamic light scattering after adding PEG-PLL;
.sup.[c]Coupling efficiency = conjugated nanogel/total nanogel
added to T cells; .sup.[d]The amount of surface-bound nanogel was
calculated based on the feeding amount of nanogel (5 .mu.g/100,000
cells) and coupling efficiency.
TABLE-US-00003 TABLE 2 Polycations improve the coupling efficiency
of human IL-15Sa nanogels on the T cell surface. PEG-PLL (equiv. of
Surface-bound IL-15Sa, Size Coupling nanogel Entry mol/mol).sup.[a]
(nm).sup.[b] efficiency.sup.[c] (.mu.g/1 .times. 10.sup.6
cells).sup.[d] 1 0 71.1 .+-. 5.3 25.6% 1.28 2 0.02 297.8 .+-. 11.4
35.4% 1.77 3 0.05 280.8 .+-. 13.1 77.0% 3.85 4 0.08 301.5 .+-. 15.5
89.6% 4.48 Nanogels contained fixed amounts of CD45 to IL-15Sa
(1:10 moles). .sup.[a]Different amount of PEG-PLL was mixed with
human IL-15Sa nanogel right before the coupling reaction;
.sup.[b]The size of the nanogel was measured with dynamic light
scattering after adding PEG-PLL; .sup.[c]Coupling efficiency =
conjugated nanogel/total nanogel added to T cells; .sup.[d]The
amount of surface-bound nanogel was calculated based on the feeding
amount of nanogel (5 .mu.g/100,000 cells) and coupling
efficiency.
TABLE-US-00004 TABLE 3 Hydrodynamic size and .xi.-potential of
IL-15Sa-NGs, before and after surface modification with anti-CD45
and PEG-PLL. surface modification Size (nm) zeta potential (mV)
before 89.6 .+-. 9.3 -10.3 after 121.0 .+-. 13.1 7.03
[0345] To evaluate the TCR signaling responsiveness of protein
nanogels resting on primed mouse T cells we isolated using the
following protocol. Spleens from C57B1/6 or pmel-1 Thy 1.1.sup.+
mice (Jackson Laboratory) were ground through a 70-.mu.m cell
strainer and red blood cells were removed by incubating with ACK
lysis buffer (2 mL per spleen) for 5 min at 25.degree. C. Resting
CD4.sup.+ or CD5.sup.+ T-cells were isolated from splenocytes
directly via magnetic negative selection using an EasySep.TM. Mouse
CD4.sup.+ or CD8.sup.+T-cell Enrichment Kit (Stemcell Technologies,
Vancouver, Canada) respectively. For activated CD8.sup.+ T-cells,
the splenocytes were washed with PBS and then cultured in RPMI 1640
medium containing 10% FCS, con-A (2 .mu.g/mL) and IL-7 (1 ng/mL) at
37.degree. C. for activation. After 2-day incubation, dead cells
were removed by Ficoll-Pague Plus gradient separation and
CD8.sup.+T-cells were isolated using an EasySep.TM. Mouse CD8.sup.+
T-cell Enrichment Kit. Purified CD8.sup.+ T-cells were re-suspended
at 1.5.times.10.sup.6 per mL in RPMI containing 10 ng/mL
recombinant murine IL-2. After 24 h, cells were washed 3 times in
PBS and re-suspended in buffer or media for in vitro and in vivo
studies.
[0346] Human CD8.sup.+ T-cells were also isolated for parallel
experiments. Total peripheral blood mononuclear cells (PBMCs) were
obtained from healthy donors (New York Blood Center, Long Island
City, N.Y., USA). Resting CD5.sup.+ T-cells were isolated directly
using an RosetteSep.TM. Human CD8.sup.+ T-Cell Enrichment Cocktail
(Stemcell). The human CD8.sup.+ T-cells were activated in
non-tissue culture plated coated with anti-human CD3 (2.5 .mu.g/mL)
and anti-human CD28 (1.0 .mu.g/mL) in the presence of human IL-2
(50 UI/mL) for 2 days. Cells were washed 3 times in PBS and
re-suspended in buffer or media for in vitro studies.
[0347] T-cell surface reduction activity was determined using a
commercial WST-1 assay kit containing WST-1 and an electron
coupling reagent (Roche, Basel, Switzerland). Resting or
concanavalin A (con-A)-primed CD8.sup.+ T-cells from C57Bl/6 mice
were suspended in Hank's Balanced Salt Solution (HBSS) at
1.times.10.sup.6/mL. The commercial WST-1 reagent mixture (10
.mu.L) was added to the T-cell suspension (200 .mu.L). The cells
were incubated at 37.degree. C. for 1 h. WST-1 formazan production
rate was measured with a plate reader (Tecan Infinite.RTM. M1000
PRO, Tecan, Mannedorf, Switzerland) for increased absorbance at 450
nm during the incubation. For the measure of cell surface reduction
in response to TCR triggering, resting or con-A-activated CD8.sup.+
T-cells were incubated with anti-CD3/CD28-coated beads (1:1
cell:bead ratio) or gp100 peptide (10 .mu.g/mL) in the presence in
IL-7 (1 ng/mL) at 37.degree. C. for 24 h. Cells were washed and
resuspended in HBSS (1.times.10.sup.6/mL) and measured for surface
reduction with the same commercial WST-1 reagent mixture after 1-h
incubation at 37.degree. C.
[0348] Primed CD8.sup.+ T-cells showed markedly elevated cell
surface reduction rates compared with resting T-cells as measure by
the WST-1 assay (FIG. 3A). T-cell surface redox activity further
increased following stimulation with antigen or
anti-CD3/CD28-coated beads. We reasoned that increased redox
activity at the T-cell surface could be exploited to obtain
antigen-triggered adjuvant protein release using
reduction-responsive nanoparticles bound to the plasma membrane of
T-cells. Thus, the disulphide crosslinker was designed to cleave in
response to reducing conditions at the T-cell surface, followed by
release of un-adducted protein cargo through a self-immolative
reaction. We focused on promising therapeutic cytokine cargos.
Primed pmel-s CD8.sup.+ T-cells were conjugated with
anti-CD45/cytokine- or cytokine only-biotinylated NGs, incubated
for indicated periods of time, then stained with streptavidin for
analysis of cell-surface NGs by flow cytometry. With anti-CD45 on
the nanogel surface, both the IL-2-Fc NG and IL-15Sa NG showed
increase coupling on the T cell surface. Cytokine release from the
nanogel also occurred at the expect molecular weight indicating the
intact cytokine without residual chemical groups.
[0349] In addition, anti-CD45 NGs coupled to human primed T cells
also releases protein much faster when the cells were stimulated
with anti-CD3/CD28 beads (FIG. 3B). Results surprisingly show that
surface modified anti-CD45 and polycations, the cytokine NGs can be
efficiently coupled to and stably anchored on the T cell surface
enabling TCR-responsive protein cargo release in vitro.
Example 3
Cytokine Nanogels Promote Effector T Cell Expansion In Vitro and In
Vivo
[0350] In vitro proliferation assays of T cells were conducted to
determine the effect of anti-CD45 on T cell expansion. Naive pmel-1
CD8.sup.+ T-cells were labelled with carboxyfluorescein
succinimidyl ester (CFSE) and then conjugated with
aCD45/IL-15Sa-NG, IL-15Sa-NG, or aCD45/IL-15Sa-NG (non-deg.)
respectively as described above. After removing unbound NGs,
T-cells were resuspended in RPMI with 10% FCS
(5.0.times.10.sup.5/mL) and added to anti-CD3/CD28 coated beads at
a 1:2 bead:T-cell ratio. Free IL-15Sa was added to the cells in
control groups at equivalent dose (pulsed or continuous). Cell
media were replaced every 3 days and free IL-15Sa was replenished
in the continuous treatment group. At selected time points,
replicates of T-cells were added with counting beads and washed
with flow cytometry buffer (PBS with 2% FCS) followed by aqua
live/dead staining. Cells were stained for surface markers (CD8,
CD122) with antibodies followed by fixation and permeabilization
with Intracellular Fixation & Permeabilization Buffer Set
(eBioscience). Cells were then stained intracellularly for pSTAT5
and Ki67, and analysed with a flow cytometer (BD Canto, BD
Biosciences).
[0351] Anti-CD45/IL-15Sa-NG coupled to T-cells stimulated with
anti-CD3/CD28 beads proliferated extensively, expanding
.about.100-fold in 5 days, much greater than T-cells pulsed with
the same total quantity of free IL-15Sa for 1 hr. NG coupled
T-cells also expanded more than cells cultured continuously with
free IL-15Sa, suggesting cell surface localization of the NGs
enhanced receptor engagement (FIG. 4). Nanogels linked to T-cells
covalently rather than via anti-CD45 (IL-15Sa-NGs) and NGs formed
with a non-degradable crosslinker (aCD45/IL-15Sa-NGs (non-deg.))
stimulated weaker T-cell expansion than redox-responsive,
aCD45/IL-15Sa-NGs (FIG. 4); thus, prolonged cell surface retention
and release of cytokine from NGs were both important for maximal
stimulation. NGs enhanced T-cell proliferative responses to
anti-CD3/CD28 beads at doses as low as .about.30 ng
IL-15Sa/10.sup.6 cells. IL-15Sa-coupled T-cells maintained
approximately constant levels of IL-15.beta. (CD122) and maintained
stimulation of T-cells for at least a week in culture, as evidenced
by elevated levels of pSTAT5 and Ki67 over 9 days. Addition of a
CD45 inhibitor did not alter the proliferative response to the NGs,
implying that NG anchoring did not trigger suppressive CD45
phosphatase activity. Thus, IL-15Sa NG backpacks provide sustained
and potent stimulation of T-cells in the presence of TCR
activation.
[0352] This might be due to the combined effect of (1) prolonged
surface retention of IL-15Sa/a-CD45 NG thus more IL-15Sa released
before internalization and (2) enhanced loading of the cytokine
nanogel to the cell surface, thus enhanced IL-15Sa loading on the
cell surface as additional anchor CD45 facilitates binding of
IL-15Sa/a-CD45 NG.
[0353] To determine the In vivo effect of IL-Sa/anti-CD45 NGs on T
cell expansion in tumor bearing mice using the syngeneic B16F10
melanoma mouse model (Proc. Natl. Acad. Sci. U.S.A., 2004, 101,
1969-1974). B16F10 melanoma cells (American Tissue Culture
Collection (Manassas, Va., USA)(5.0.times.10.sup.5) were injected
subcutaneously (s.c.) in the flanks of C57B1/6 mice on day 0.
Animals were sublethally lymphodepleted by total body irradiation
(5 Gy) 6 days post tumour inoculation. Primed pmel-1 CD8.sup.+
T-cells (1.0.times.10.sup.7) alone or with surface coupled NGs in
200 .mu.l PBS were administered intravenously (i.v.) at day 7.
Treatment groups, included T cell alone, T cells followed by a
systemic injection of free IL-15Sa (40 ug), and T cells coupled
with an anti-CD45IL-15Sa-NG (40 ug). Tumour area (product of 2
measured orthogonal diameters) and body weight were measured every
two days. To monitor in vivo T-cell expansion and function, mice
were sacrificed on day 14 for necropsy and flow cytometry analyses,
unless body weight loss was beyond 20% of predosing weight, or
tumour area reached 150 mm.sup.2 (as predetermined endpoint), or
the animal had become moribund at which point mice were
euthanized.
[0354] Inguinal lymph nodes (distal or tumour draining lymph node)
and spleens were ground through a 70-.mu.m cell strainer.
Splenocytes were then lysed with ACK lysis buffer (2 mL per spleen)
for 5 min at 25.degree. C. to remove red blood cells. Blood samples
(200 .mu.L) were lysed with ACK lysis buffer (1 mL.times.2) for 5
min at 25.degree. C. Tumours were weighed and ground through a
70-.mu.m cell strainer. All cells were added with counting beads
and washed with flow cytometry buffer (PBS with 2% FCS) followed by
aqua live/dead staining. Cells were stained for surface markers
(CD8, Thy1.1, CD4, NK1.1) with antibodies followed by fixation and
permeabilization with Cytofix/Cytoperm (BD Biosciences). Cell were
then stained intracellularly for Ki67. After washing with FACS
buffer, cells were re-suspended in FACS buffer and analysed by flow
cytometry. For intracellular cytokine staining, samples in
single-cell suspensions were incubated with gp100 peptide (10
.mu.g/mL) at 37.degree. C. for 2 h followed by the addition of
brefeldin A (eBioscience, San Diego, Calif., USA) and incubation
for another 4 h. Following surface staining as described above,
samples were fixed and permeabilized in the same manner and stained
with antibodies against IFN-.gamma., TNF-.alpha. and IL-2. Flow
cytometric analysis was carried out using a BD Fortessa (BD
Biosciences), and data analysis was performed using FlowJo software
(Tree Star, Oreg., USA).
[0355] ACT adjuvanted by free systemic IL-15Sa led to substantial
expansion of both transferred pmel-1 T-cells and endogenous T-cells
in the blood (FIG. 5A), and also expanded NK cells and CD4.sup.+
T-cells in the systemic circulation. Systemic IL-15Sa also expanded
endogenous T-cells in tumour-draining lymph nodes (TDLNs) (data not
shown) and tumours (FIG. 5B). By contrast, IL-15Sa delivered as
backpacks expanded the transferred CD8.sup.+ T-cells but did not
expand endogenous T-cells in any compartment (FIG. 5A-5B). In
tumours, where we expected antigen recognition to accelerate
IL-15Sa release from the NGs, IL-15Sa-coupled T-cells expanded
16-fold more than pmel-1 cells in the soluble IL-15Sa-adjuvanted
group and 1000-fold more than the T-cells without cytokine support
(FIG. 5B). Ranking tissues in order of expected increasing antigen
concentration (blood<distal LN<TDLN<tumour), we observed a
corresponding increasing ratio of ACT T-cell counts in the NG group
vs. ACT cells in the free IL-15Sa-adjuvanted group (FIG. 5C).
Backpacked T-cells in the tumour were also proliferating and
producing effector cytokines (FIG. 5D-5E). Even at high doses of
II-15Sa/anti-CD45 NGs still did not inflict toxicity (e.g.: 57 ug
per mouse) while systemic IL15Sa caused a significant (18%) drop in
body weight. Nanogel IL-15Sa delivery thus focused cytokine action
on the transferred T-cells, and preferentially in antigen-bearing
microenvironments in vivo with no toxic effects.
Example 4
Cytokine Delivery by Nanogel Backpacks Increases the Therapeutic
Window for Adjuvant Cytokine Therapy
[0356] The syngeneic B 16F10 melanoma mouse model described above
was used, however, the treatment groups were as follows. Animals
received sham injections of PBS, T cells only, T cells followed by
different doses of i.v. injected free IL-15Sa as a single dose
(immediately after adoptive transfer) or split into multiple doses
(days 7, 10, 13 and 16) or T cells coupled with anti-CD45/IL-15Sa
NG at different doses. Body weights and systemic cytokine,
chemokine and liver enzyme levels were analysed over time. Relative
body weigh was normalized to day 7 body weight. Counts of
cytokine+endogenous CD8.sup.+ T-cells and ACT CD8.sup.+ T-cells in
blood were analyzed by intracellular cytokine staining and flow
cytometry on day 14. Serum cytokine levels and liver enzymes were
measured from samples collected on day 17 or when animals were
euthanized due to toxicity. Cytokine levels were measured using
Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD
Biosciences). Serum samples were also sent to IDEXX Reference
Laboratories for analysis of alanine transaminase (ALT) and
aspartate transaminase (AST). Data represent the mean.+-.s.e.m.
(n=5/group) and are compared with control group (T cells only) for
statistical analyses. *, p<0.05; **, p<0.01; ***, p<0.001
by One-Way ANOVA and Tukey's tests; n.s., not significant; n.d.,
not detectable.
[0357] Animals receiving free high-dose IL-15Sa lost significant
weight following therapy, which prompted us to evaluate the
toxicity of IL-15Sa as a function of dose and delivery modality.
Animals receiving >10 .mu.g of free IL-15Sa steadily lost weight
and eventually succumbed to lethal immunotoxicity irrespective of
dosing regimen, setting the maximum tolerated dose (MTD) at 10
.mu.g in this model (FIG. 6A). In contrast, when administered in
the form of T-cell-bound NGs, no overt toxicity was observed up to
the maximum achievable IL-15Sa loading per cell (80 .mu.g
IL-15Sa/10.times.10.sup.6 T-cells, FIG. 6A). Free IL-15Sa
stimulated cytokine production from both pmel-1 and endogenous
T-cells in the blood, in contrast to IL-15Sa delivered by NGs,
where the majority of both backpacked and endogenous T-cells
remained quiescent in the systemic circulation (FIG. 6B and FIG.
6C). In lymphoreplete animals, >10 .mu.g free IL-15Sa does not
elicit high levels of serum cytokine induction (Biomaterials 2012,
33, 5776-5787). However, in this lymphodepletion setting, ACT with
>10 .mu.g free IL-15Sa induced systemic cytokine release of
TNF-.alpha. (FIG. 6D), IL-6 (FIG. 6E) and IL-10 (data not shown),
and elevated liver enzymes, ALT and AST (data not shown). In
contrast, backpacked T-cells (protein nanogel coupled T cells)
elicited basal levels of these biomarkers up to the maximum
administrable dosage. Thus, T-cell bound NGs limit stimulation of
lymphocytes in the systemic circulation, enabling significantly
greater IL-15Sa dosing.
[0358] To determine the impact of the increased therapeutic window
afforded by IL-15Sa-NGs, we compared the anti-tumor efficacy of ACT
with T-cells only, T-cells and free IL-15Sa (at the MTD of 10
.mu.g), or NG-backpacked T-cells following the same treatment
scheme as above. Tumor growth was significantly delayed in the 10
.mu.g IL-15Sa-NG group compared to T-cells with free IL-15Sa
support at the same dose. However, tumor suppression was further
substantially enhanced by increasing the cytokine-NG dose, with
animals treated at the maximal 80 .mu.g dose showing a 1.7-fold
increase in median survival relative to animals treated with the
MTD of free IL-15Sa, an impressive response in this challenging
tumor model. Notably, despite the use of a xenogeneic (human)
IL-15Sa cytokine, no anti-hIL-15Sa antibodies above background were
detected in serum following treatment in any of the
NG-backpack-treated groups.
Example 6
TCR Signaling-Responsive Cytokine Nanogels Improve Efficacy of
Human CAR T-Cell Therapy
[0359] We evaluated whether NG-delivered cytokine could also
positively impact the function of CAR T-cells, as an important
modality of T-cell therapy in the clinic. For this purpose, we
employed human CAR T-cells targeting EGFR in a
luciferase-expressing human glioblastoma model in immunodeficient
NSG mice. CAR T-cells were prepared using the huEGFRscFv-BBz
chimeric antigen receptor (Johnson et. Al., 2015, Sci Transl Med.,
7(275); US2014/0271635A1) designed based on the heavy and light
chains of cetuximab to form a single-chain variable fragment which
was fused to a portion of the extracellular and transmembrane
domains of human CD8.alpha., followed by the intracellular domains
of 4-1BB and CD3.zeta.. The Bicistronic vector also encoded
truncated human CD19 as a selectable marker, and was placed
following a T2A ribosomal skip sequence. The plasmid coding hu
EGFRscFv-BBz-CAR was synthesized and lentivirus packaging was
produced by VectorBuilder. Isolated T-cells were derived from
purchased leukapheresis products obtained from de-indentified
healthy donors under an IRB-approved protocol. T-cells were
stimulated with Dynabeads Human T Activator CD3/CD28 (Life
Technologies) at a bead to cell ratio 3:1. T-cells were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum, Hepes
Buffer (20 mM), penicillin and streptomycin (1%) and IL-2 (20
IU/mL). T-cells were transduced (TDN) with CAR lentivirus and or
left untransduced (UTD) one day following bead stimulation, and
then T-cells were expanded for 10 days and cryopreserved until
used. Surface expression of the CAR was confirmed and quantified
with biotinylated human EGFR protein (ACRO Biosystems).
[0360] To test the ability of NG-delivered cytokines to improve the
function of CAR T-cells, Luciferase-expressing U-87 MG human
glioblastoma cells (American Tissue Culture Collection, Manassas,
Va., USA) (1.0.times.10.sup.6) were injected s.c. in NSG mice
(n=5/group). Animals received i.v. adoptive transfer of human
T-cells (2.6.times.10.sup.6 total cells, 38% transduced with
EGFR-targeting CAR (1.0.times.10.sup.6 CAR-T-cells)) on day 7.
Animals were treated with sham saline injections, CAR-T alone,
CAR-T followed by 13.8 .mu.g of free IL-15Sa, or CAR-T-cells
coupled with aCD45/IL-15Sa-NGs (13.8 .mu.g). In vivo
bioluminescence imaging of luciferase-expressing U-87 MG tumors
over time was also measured. Individual tumour growth curves and
survival curves of treatment groups were generated. Statistical
analyses were performed using Two-Way ANOVA test for tumour growth
data and Log-rank test for survival curves. Data represent the
mean.+-.s.e.m. *, p<0.05; **, p<0.01; ***, p<0.001; n.s.,
not significant.
[0361] Tumor growth was significantly delayed in 10 .mu.g
IL-15Sa-NG group compared to T cells with free IL-15Sa support at
the same dose (FIG. 7A). However, tumor suppression was further
substantially enhanced by increasing the cytokine-NG dose (FIG.
7A), with animals treated at the maximal 80 .mu.g dose showing a
1.7 fold increase in median survival relative to animals treated
with the MTD of free IL-15Sa. CAR T-cells coupled with
aCD45/IL-15Sa-NGs were compared to CAR T-cells alone or T-cells
supplemented with an equivalent systemic dose of free IL-15Sa.
Transfer of 10.sup.6 CAR-T-cells alone had a small impact on tumor
growth and survival, which did not reach statistical significance;
responses were marginally improved by the addition of free IL-15Sa
(FIGS. 7B and 7C). By contrast, NG-coupled CAR T-cells eradicated
tumors in 4 of 5 animals and increase animal survival (FIG. 7C).
Thus, aCD45/IL-15Sa-NG delivery of cytokines was shown to enhance
CAR T-cell therapy.
[0362] To measure serum antibody, serum samples were collected from
treated mice in different groups at 30 days post ACT. Serum
concentration of anti-IL-15Sa antibody was measured by a standard
ELISA procedure with calibration of a monoclonal anti-human IL-15
antibody (eBioscience).
[0363] While several embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings is/are used. Those skilled in
the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific inventive
embodiments described herein. It is, therefore, to be understood
that the foregoing embodiments are presented by way of example only
and that, within the scope of the appended claims and equivalents
thereto, embodiments may be practiced otherwise than as
specifically described and claimed. Embodiments of the present
disclosure are directed to each individual feature, system,
article, material, kit, and/or method described herein. In
addition, any combination of two or more such features, systems,
articles, materials, kits, and/or methods, if such features,
systems, articles, materials, kits, and/or methods are not mutually
inconsistent, is included within the inventive scope of the present
disclosure.
[0364] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0365] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0366] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0367] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0368] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0369] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0370] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
91114PRTHomo sapiensmisc_feature(1)..(114)Human IL-15 (N72D mutant)
1Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile1 5
10 15Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val
His 20 25 30Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu
Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser
Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu
Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe Leu Gln Ser Phe Val His
Ile Val Gln Met Phe Ile Asn 100 105 110Thr Ser2297PRTHomo
sapiensmisc_feature(1)..(297)Human IL-15R-alpha-Su/Fc (IgG1 CH2-CH3
(Fc domain)) 2Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp
Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile
Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr
Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr
Pro Ser Leu Lys Cys Ile 50 55 60Arg Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro65 70 75 80Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys 85 90 95Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val 100 105 110Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 115 120 125Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 130 135
140Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His145 150 155 160Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 165 170 175Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 180 185 190Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu 195 200 205Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 210 215 220Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn225 230 235 240Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 245 250
255Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
260 265 270Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 275 280 285Lys Ser Leu Ser Leu Ser Pro Gly Lys 290
295365PRTHomo sapiensmisc_feature(1)..(65)Human IL-15R-alpha-Su
(65aa truncated extracellular domain) 3Ile Thr Cys Pro Pro Pro Met
Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr
Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala
Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn
Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55
60Arg654135PRTHomo sapiensmisc_feature(1)..(135)Human IL-15 isoform
2 4Met Val Leu Gly Thr Ile Asp Leu Cys Ser Cys Phe Ser Ala Gly Leu1
5 10 15Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys 20 25 30Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr 35 40 45Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe 50 55 60Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile65 70 75 80His Asp Thr Val Glu Asn Leu Ile Ile Leu
Ala Asn Asn Ser Leu Ser 85 90 95Ser Asn Gly Asn Val Thr Glu Ser Gly
Cys Lys Glu Cys Glu Glu Leu 100 105 110Glu Glu Lys Asn Ile Lys Glu
Phe Leu Gln Ser Phe Val His Ile Val 115 120 125Gln Met Phe Ile Asn
Thr Ser 130 1355162PRTHomo sapiensmisc_feature(1)..(162)Human IL-15
isoform 1 5Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln
Cys Tyr1 5 10 15Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala
Gly Ile His 20 25 30Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro
Lys Thr Glu Ala 35 40 45Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys
Ile Glu Asp Leu Ile 50 55 60Gln Ser Met His Ile Asp Ala Thr Leu Tyr
Thr Glu Ser Asp Val His65 70 75 80Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110Asn Leu Ile Ile Leu
Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125Thr Glu Ser
Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140Lys
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn145 150
155 160Thr Ser6114PRTHomo sapiensmisc_feature(1)..(114)Human IL-15
(without signal peptide) 6Asn Trp Val Asn Val Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile1 5 10 15Gln Ser Met His Ile Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His 20 25 30Pro Ser Cys Lys Val Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45Val Ile Ser Leu Glu Ser Gly
Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60Asn Leu Ile Ile Leu Ala
Asn Asn Ser Leu Ser Ser Asn Gly Asn Val65 70 75 80Thr Glu Ser Gly
Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95Lys Glu Phe
Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110Thr
Ser785PRTHomo sapiensmisc_feature(1)..(85)Human IL-15R-alpha (85aa
truncated extracellular domain) 7Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Asp Pro Ala
Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val65 70 75 80Thr Thr
Ala Gly Val 858182PRTHomo sapiensmisc_feature(1)..(182)Human
IL-15R-alpha (182aa truncated extracellular domain) 8Ile Thr Cys
Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser
Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe
Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40
45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr
Val65 70 75 80Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser
Pro Ser Gly 85 90 95Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn
Thr Ala Ala Thr 100 105 110Thr Ala Ala Ile Val Pro Gly Ser Gln Leu
Met Pro Ser Lys Ser Pro 115 120 125Ser Thr Gly Thr Thr Glu Ile Ser
Ser His Glu Ser Ser His Gly Thr 130 135 140Pro Ser Gln Thr Thr Ala
Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser145 150 155 160His Gln Pro
Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val 165 170 175Ala
Ile Ser Thr Ser Thr 1809267PRTHomo
sapiensmisc_feature(1)..(267)Human IL-15R-alpha Full length 9Met
Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala1 5 10
15Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr
20 25 30Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys
Ser 35 40 45Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
Phe Lys 50 55 60Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu
Asn Lys Ala65 70 75 80Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu
Lys Cys Ile Arg Asp 85 90 95Pro Ala Leu Val His Gln Arg Pro Ala Pro
Pro Ser Thr Val Thr Thr 100 105 110Ala Gly Val Thr Pro Gln Pro Glu
Ser Leu Ser Pro Ser Gly Lys Glu 115 120 125Pro Ala Ala Ser Ser Pro
Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 130 135 140Ala Ile Val Pro
Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr145 150 155 160Gly
Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 165 170
175Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln
180 185 190Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val
Ala Ile 195 200 205Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala
Val Ser Leu Leu 210 215 220Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro
Pro Leu Ala Ser Val Glu225 230 235 240Met Glu Ala Met Glu Ala Leu
Pro Val Thr Trp Gly Thr Ser Ser Arg 245 250 255Asp Glu Asp Leu Glu
Asn Cys Ser His His Leu 260 265
* * * * *