U.S. patent application number 16/102194 was filed with the patent office on 2018-12-06 for compositions and methods for treating peritoneal cancers.
The applicant listed for this patent is Prospect CharterCare RWMC, LLC d/b/a Roger Williams Medical Center, Prospect CharterCare RWMC, LLC d/b/a Roger Williams Medical Center. Invention is credited to Richard JUNGHANS, Steven C. KATZ.
Application Number | 20180344769 16/102194 |
Document ID | / |
Family ID | 57757735 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180344769 |
Kind Code |
A1 |
KATZ; Steven C. ; et
al. |
December 6, 2018 |
COMPOSITIONS AND METHODS FOR TREATING PERITONEAL CANCERS
Abstract
The present disclosure provides compositions and methods for
treating a peritoneal cancer in a subject. The methods include
administering a T cell which is genetically modified to express a
chimeric T cell receptor protein. The chimeric T cell receptor
protein may include a T cell receptor signaling domain fused to the
tumor associated antigen-binding fragment of an antibody or a T
cell receptor signaling domain fused to a naturally occurring
ligand which specifically binds to a tumor cell surface protein.
The compositions and methods disclosed herein are therapeutically
effective to reduce, for example, tumor burden, abdominal ascites,
peritoneal mucin, or serum tumor marker levels.
Inventors: |
KATZ; Steven C.;
(Providence, RI) ; JUNGHANS; Richard; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Prospect CharterCare RWMC, LLC d/b/a Roger Williams Medical
Center |
Providence |
RI |
US |
|
|
Family ID: |
57757735 |
Appl. No.: |
16/102194 |
Filed: |
August 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15210818 |
Jul 14, 2016 |
10071118 |
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16102194 |
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62193217 |
Jul 16, 2015 |
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62298980 |
Feb 23, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/675 20130101;
C07K 16/3007 20130101; A61K 31/10 20130101; A61K 2039/54 20130101;
A61K 2039/5156 20130101; A61K 2039/5158 20130101; A61P 35/00
20180101; A61K 39/001182 20180801; A61K 35/17 20130101; C07K
2319/03 20130101; C07K 2317/622 20130101; C07K 2319/33 20130101;
A61K 39/001182 20180801; A61K 2300/00 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 31/675 20060101 A61K031/675; A61K 31/10 20060101
A61K031/10 |
Claims
1. A method of treating a peritoneal cancer in a subject,
comprising: infusing into the abdominal cavity of the subject a
composition comprising a substantially pure population of
genetically engineered T cells which express a chimeric antigen or
a chimeric ligand T cell receptor protein, wherein the chimeric
antigen or chimeric ligand T cell receptor protein binds to an
antigen expressed on malignant cells.
2. The method of claim 1, wherein the malignant cells are present
in the abdominal cavity.
3. The method of claim 1, wherein the malignant cells are present
outside of the abdominal cavity.
4. The method of claim 1, further comprising infusing a second
therapeutic agent into the abdominal cavity of the subject.
5. The method of claim 4, wherein the infusing the second
therapeutic agent is performed before, during or after the infusion
of the composition comprising the genetically engineered T
cells.
6. The method of claim 4, wherein the second therapeutic agent is
an inhibitor of GM-CSF, STAT3, PD-1, PD-L1, IL10 or TGF.beta.
activity.
7. The method of claim 1, wherein the composition is infused into
the abdominal cavity of the subject once every 1 week, once every 2
weeks, once every 3 weeks, or once every 4 weeks.
8. The method of claim 1, wherein the infusing into the abdominal
cavity of the subject the composition infusing 10.sup.6-10.sup.11
genetically engineered T cells.
9. The method of claim 1, wherein the infusing the composition
results in a decrease in the number and/or size of peritoneal
tumors, abdominal ascites, peritoneal mucin, and/or serum tumor
marker levels.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional application No. 62/193,217, filed Jul. 16, 2015 and
U.S. provisional application No. 62/298,980, filed Feb. 23, 2016,
each of which are hereby incorporated by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING
[0002] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Jul. 13, 2016, and named
"0962010241SEQ.txt" (2,234 bytes), the contents of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] The subject matter described herein relates to the design
and use of T cells engineered to express on its surface a receptor
protein which binds a tumor antigen and which activates activities
of the T cell. Methods include the intraperitoneal administration
of chimeric antigen receptor T cells (CAR-T cells) to inhibit
growth and/or survival of tumor cells in the peritoneal cavity.
BACKGROUND
[0004] Pseudomyxoma peritonei (PMP) and peritoneal carcinomatosis
(PC) are rare diseases with an estimated incidence of 1-2 per
million per year worldwide. PC affects 15% of all colorectal cancer
patients at initial presentation with devastating effects
(Coccolini et al, 2013, World J Gastroenterol, 19:6979-6994). These
patients typically have a very poor prognosis and suffer from
numerous complications of their disease, including progressive
bowel obstruction. Optimal treatment involves cytoreductive surgery
with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) which
has been used with modest success in highly selected patients with
limited disease burdens. During CRS-HIPEC, all visible
intraperitoneal tumor is debulked and residual microscopic disease
is treated with regionally delivered chemotherapy. CRS-HIPEC is
most effective when the tumor burden is small following CRS to
eliminate any tumor nodules larger than 2.5 mm. Outcomes are
dependent on tumor grade, with 5-year survival rates of 63-10% for
low grade, and 0%-65% for high grade disease (Sugarbaker et al.,
1999, Ann Surg Oncol, 6:727-731). A randomized controlled trial
demonstrated that CRS-HIPEC for patients with colorectal cancer PC
resulted in significantly improved survival compared to systemic
chemotherapy (Verwaal et al., 2003, J Clin Oncol, 21:3737-3743,
Verwaal et al., 2008, Ann Surg Oncol, 15:2426-2432). Unfortunately,
most PC patients are not candidates for CRS-HIPEC and ultimately
progress and die of disease (Coccolini et al, 2013, World J
Gastroenterol, 19:6979-6994; Cao et al., 2009, Ann Surg Oncol,
16:2152-2165). Even so, results with CRS-HIPEC for PC suggest that
regionally delivered therapeutics are a promising approach to
address this large unmet clinical need.
[0005] Immunotherapy for advanced solid tumors has gained
considerable traction in recent years (Hodi et al., 2010, N Engl J
Med, 363:711-723; Kantoff et al., 2010, N Engl J Med, 363:411-422;
Khan et al., 2014, J Surg Res, 191:189-195; Saied et al., 2014, J
Surg Res, 187:525-535). Several types of immunotherapy exist,
including vaccines, antibodies, and immune cell infusions. Cellular
immunotherapy for solid tumors has advanced largely through
application of chimeric antigen receptor T cells (CAR-Ts). CAR-Ts
are of particular interest based in part on their broad
applicability since they can be produced for almost any patient and
are not restricted by major histocompatibility complex types
(Eshhar, 2010, Curr Opin Mol Ther, 12:55-63).
[0006] CAR-T targeting carcinoembryonic antigen (CEA) was recently
tested in Phase I Hepatic Immunotherapy for Metastases (HITM)
clinical trials (NCT01373047, NCT02416466) examining the safety and
clinical activity of these cells against colorectal cancer LM (Katz
et al., 2015, Clin Cancer Res, 21:3149-3159). As the peritoneal
cavity is another common site of failure in stage IV CRC patients,
it was worthwhile to test regional CAR-T delivery for PC. While
regional delivery may enhance the anti-tumor efficacy of CAR-Ts,
intratumoral immunosuppression will likely present additional
challenges. The metastatic solid tumor microenvironment contains
many immunosuppressive cell types that inhibit CAR-Ts, including
myeloid-derived suppressor cells (MDSC) and regulatory T cells
(Treg) (Kershaw et al., 2013, Nat Rev Cancer, 13:525-541). It has
been previously shown that MDSC suppress CAR-T cells, and inhibit
the antigen presentation functions of liver B cells (Thorn et al.,
2014, J Leukoc Biol, 96:883-894). MDSC accomplish this
immunosuppressive function through the PD-1/PD-L1 axis and IDO
(Burga et al., 2015, Cancer Immunol Immunother, 64:817-829). Treg
are also well studied in tumor microenvironments and have been
shown to suppress CAR-Ts via PD-L1 and CTLA4 (Lee et al., 2011,
Cancer Res, 71:2871-2881).
[0007] Accordingly, provided herein is a method for infusing
immunoresponsive cells expressing chimeric T cell receptors to
treat subjects diagnosed with PMP/PC. Data are provided which
indicate that these genetically programmed cells attack tumors
expressing specific antigens, such as antigens expressed or
specifically expressed on adenocarcinoma cells present in PMP or
PC. Moreover, the data support the idea that effective IP CAR-T
therapy for PC will be further enhanced through inhibition of
immunosuppressive cell populations.
[0008] The foregoing examples of the related art and limitations
related therewith are intended to be illustrative and not
exclusive. Other limitations of the related art will become
apparent to those of skill in the art upon a reading of the
specification and a study of the drawings.
BRIEF SUMMARY
[0009] The following aspects and embodiments thereof described and
illustrated below are meant to be exemplary and illustrative, not
limiting in scope.
[0010] In one aspect, a method of treating an intraperitoneal tumor
or cancer in a subject is provided, comprising infusing into the
abdominal cavity of the subject a population of genetically
engineered lymphocytes which express a chimeric T cell receptor
which binds to a tumor associated antigen on malignant cells in the
abdominal cavity.
[0011] In some embodiments, the population of lymphocytes comprises
T cells, B cells and/or NK cells. In other embodiments, the T cells
comprise CD4+ cells, CD8+ cells, gamma delta T cells
(.gamma..delta. T cells), NK T cells and/or regulatory T cells
(Treg).
[0012] In some embodiments, the chimeric receptor is comprised of
the antigen-binding domain of an immunoglobulin and a T-cell
receptor signaling domain. In other embodiments, the chimeric
receptor is comprised of a natural ligand to a protein expressed on
the cell surface of the malignant cell and a T-cell receptor
signaling domain.
[0013] In some embodiments, the method comprises administering the
genetically engineered lymphocytes in an amount effective to reduce
the number of malignant cells in the abdominal cavity of the
subject. In other embodiments, the method comprises administering
genetically engineered lymphocytes in an amount effective to reduce
the mass of malignant cells in the abdominal cavity of the subject.
In still other embodiments, the number and/or mass of malignant
cells in the abdominal cavity is measured by imaging.
[0014] In some embodiments, the method comprises administering the
genetically engineered lymphocytes in an amount effective to reduce
the number of malignant cells outside of the abdominal cavity of
the subject. In other embodiments, the method comprises
administering genetically engineered lymphocytes in an amount
effective to reduce the mass of malignant cells outside of the
abdominal cavity of the subject. In still other embodiments, the
number and/or mass of malignant cells outside the abdominal cavity
is measured by imaging.
[0015] In some embodiments, the method comprising infusing the
genetically engineered lymphocytes results in a decrease in the
number of peritoneal tumor cells. In other embodiments, the method
results in a decrease of at least 30%, 40%, 50%, 60%, 70%, 80%, or
90% of the tumor size at or before the time of the first
administration of the genetically engineered lymphocytes.
[0016] In some embodiments, the method comprising infusing the
genetically engineered lymphocytes results in a decrease in the
size of peritoneal tumors. In other embodiments, the method results
in a decrease of at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of
the size of the peritoneal tumors at or before the time of the
first administration of the chimeric receptor T cells.
[0017] In some embodiments, the method comprising infusing the
genetically engineered lymphocytes results in a decrease of at
least 300%, 400%, 50%, 60%, 70%, 80% or 90% of the peritoneal
volume as determined at or before the time of the first
administration of the genetically engineered lymphocytes.
[0018] In some embodiments, the genetically engineered lymphocytes
are infused into the abdominal cavity of the subject once every 1
week, once every 2 weeks, once every 3 weeks, or once every 4
weeks.
[0019] In some embodiments the genetically engineered lymphocytes
are autologous to the subject. In other embodiments, the
genetically engineered lymphocytes are not autologous to the
subject.
[0020] In some embodiments, the infusing into the abdominal cavity
of the subject the genetically engineered lymphocytes comprises
infusing 10.sup.6-10.sup.11 genetically engineered lymphocytes.
[0021] In some embodiments, the method comprises infusing a
composition the genetically engineered lymphocytes and a
pharmaceutically compatible solution comprising the chimeric
receptor T cells in normal saline with or without 10% DMSO, wherein
the total volume of the composition ranges from about 100 ml to 500
ml.
[0022] In some embodiments, the chimeric T cell receptor protein
comprises an extracellular domain which specifically binds to a
tumor associated antigen expressed on the surface of an
adenocarcinoma, sarcoma or neuroendocrine tumor cell. In other
embodiments, the adenocarcinoma, sarcoma or neuroendocrine tumor
cell is present in the peritoneal cavity of the subject. In other
embodiments, the adenocarcinoma, sarcoma or neuroendocrine tumor
cell is present outside of the peritoneal cavity of the
subject.
[0023] In some embodiments, the method further comprises infusing a
second therapeutic agent into the abdominal cavity of the subject.
In other embodiments, the second therapeutic agent is an immune
suppressive cell inhibitor that blocks an immunoinhibitory pathway
within a suppressive cell. In still other embodiments, the
suppressive cell is a myeloid-derived suppressor cell (MDSC) or a
regulatory T cell (Treg). In some embodiments, the second
therapeutic agent inhibits immunosuppression mediated by PD-1,
PD-L1, PD-L2, IDO, STAT3, GM-CSF, IL10 or TGF.beta.. In yet other
embodiments, the second therapeutic agent is an antibody or
fragment thereof that binds PD-1, PD-L1, PD-L2, IDO, STAT3, GM-CSF,
IL10 or TGF.beta..
[0024] In some embodiments, the infusing the second therapeutic
agent is performed before, during or after the infusion of the
lymphocyte which expresses a chimeric receptor protein. In other
embodiments, the second therapeutic agent is infused into the
abdominal cavity or intravenously.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIGS. 1A and 1B provide schematics of various anti-CEA CAR-T
constructs.
[0026] FIG. 2 shows lysis by untransduced splenic cells and
chimeric receptor transduced lymphocytes.
[0027] FIG. 3A shows luminescence in animals harboring tumors and
which had been administered chimeric receptor transduced
lymphocytes by intraperitoneal (IP) or tail vein (TV)
injections.
[0028] FIG. 3B shows reduction in tumor volume in animals harboring
tumors and which had been administered chimeric receptor transduced
lymphocytes by intraperitoneal (IP) or tail vein (TV)
injections.
[0029] FIG. 4A shows luminescence in animals harboring tumors which
had been treated with chimeric receptor transduced lymphocytes by
intraperitoneal (IP) or tail vein (TV) injections and which were
rechallenged with tumor cells.
[0030] FIGS. 4B and 4C show infiltration of tumors in vivo by
leukocytes expressing the chimeric receptor protein (FIG. 4B) or by
leukocytes having an effector memory phenotype (FIG. 4C).
[0031] FIG. 5A illustrates therapeutic efficacy of IP chimeric
receptor T cell infusion on tumors outside of the peritoneal
cavity.
[0032] FIG. 5B shows IP tumor reduction via bioluminescence after
TV vs. IP administration of chimeric receptor T cells.
[0033] FIG. 5C shows reduced flank tumor burden via measurement
with calipers after TV vs. IP administration of chimeric receptor T
cells.
[0034] FIG. 5D shows systemic IFN.gamma. levels after IP
administration of chimeric receptor T cells.
[0035] FIGS. 6A and 6B show the presence of CD11b+ and MDSC (Ly6G+)
cells within IP tumor and spleen.
[0036] FIGS. 7A and 7B show the presence of MDSC Ly6G+ and MDSC
PD-L1+ cells within IP tumor and spleen.
[0037] FIGS. 8A and 8B show the presence of Treg (FoxP3+) and CD4 T
cells within IP tumor and spleen.
[0038] FIG. 9A shows the effects of TV and IP chimeric receptor T
cell infusion on tumor burden on Day 8 after infusion.
[0039] FIG. 9B shows the effects of administration of antibodies
that bind PD-L1, Gr-1 or GITR on efficacy of IP chimeric receptor T
cell infusion on Day 8 after infusion.
[0040] FIG. 10A shows the effects of TV and IP chimeric receptor T
cell infusion on tumor burden on Day 14 after infusion.
[0041] FIG. 10B shows the effects of administration of antibodies
that bind PD-L1, Gr-1 or GITR on efficacy of IP chimeric receptor T
cell infusion on Day 14 after infusion.
[0042] FIG. 11 shows the effects of administration of antibodies
that bind PD-L1, Gr-1 or GITR on efficacy of IP chimeric receptor T
cell infusion over a 14-day period after infusion.
DETAILED DESCRIPTION
[0043] Various aspects now will be described more fully
hereinafter. Such aspects may, however, be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey its scope to those skilled in the art.
I. DEFINITIONS
[0044] As used in this specification, the singular forms "a," "an,"
and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to a "polymer"
includes a single polymer as well as two or more of the same or
different polymers, reference to an "excipient" includes a single
excipient as well as two or more of the same or different
excipients, and the like.
[0045] Where a range of values is provided, it is intended that
each intervening value between the upper and lower limit of that
range and any other stated or intervening value in that stated
range is encompassed within the disclosure. For example, if a range
of 1 .mu.m to 8 .mu.m is stated, it is intended that 2 .mu.m, 3
.mu.m, 4 .mu.m, 5 .mu.m, 6 .mu.m, and 7 .mu.m are also explicitly
disclosed, as well as the range of values greater than or equal to
1 .mu.m and the range of values less than or equal to 8 .mu.m.
[0046] The term "substantially pure" or "substantially purified" as
used herein means that the CAR-T cells are as pure as it is
possible to obtain by standard techniques and methods commonly
known to one of ordinary skill in the art to which this invention
pertains. However, a purity of 70%, 80%, 90% or greater is
necessary for the monocytes to be substantially pure.
[0047] The term "peritoneal cavity" as used herein refers to the
hollow or space, or a potential space, between the parietal and the
visceral peritoneum.
[0048] The term "intraperitoneal cancer," "intraperitoneal tumor,"
"intraperitoneal malignancy" or the like as used herein refers to a
malignancy including for example a tumor mass or one or more tumor
cells, which is located within the peritoneal cavity. A peritoneal
cancer, malignancy or tumor is a malignancy which originated in the
peritoneum or peritoneal cavity.
[0049] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0050] As used herein the term "therapeutically effective" applied
to dose or amount refers to that quantity of a compound or
pharmaceutical composition (e.g., a composition comprising immune
cells such as T lymphocytes and/or NK cells) comprising a chimeric
receptor of the disclosure, and further comprising a drug
resistance polypeptide that is sufficient to result in a desired
activity upon administration to a subject in need thereof. Within
the context of the present disclosure, the term "therapeutically
effective" refers to that quantity of a compound or pharmaceutical
composition that is sufficient to delay the manifestation, arrest
the progression, relieve or alleviate at least one symptom of a
disorder treated by the methods of the present disclosure. Note
that when a combination of active ingredients is administered the
effective amount of the combination may or may not include amounts
of each ingredient that would have been effective if administered
individually.
[0051] The term "chimeric receptor" as used herein is defined as a
cell-surface receptor comprising an extracellular ligand binding
domain, a transmembrane domain and one or more cytoplasmic
co-stimulatory signaling domains in a combination that is not
naturally found together on a single protein. This particularly
includes receptors wherein the extracellular domain and the
cytoplasmic domain are not naturally found together on a single
receptor protein. The chimeric receptors of the present disclosure
are intended primarily for use with T cells and natural killer (NK)
cells. A chimeric receptor described herein may also be referred to
herein as a chimeric antigen receptor (CAR), a chimeric ligand
receptor, or a chimeric T cell receptor.
[0052] The term "tumor associated antigen" or "antigen" as used
herein refers to an antigen which is specifically expressed by
tumor cells or expressed at a higher frequency or density by tumor
cells than by non-tumor cells of the same tissue type.
Tumor-associated antigens may be antigens not normally expressed by
the host; they may be mutated, truncated, misfolded, or otherwise
abnormal manifestations of molecules normally expressed by the
host; they may be identical to molecules normally expressed but
expressed at abnormally high levels; or they may be expressed in a
context or milieu that is abnormal. Tumor-associated antigens may
be, for example, proteins or protein fragments, complex
carbohydrates, gangliosides, haptens, nucleic acids, or any
combination of these or other biological molecules.
[0053] The term "immune suppressive cell inhibitor" refers to a
substance capable of reducing or suppressing the number or function
of immune suppressive cells of a mammal. Examples of immune
suppressive cells include regulatory T cells ("T regs"), myeloid
derived suppressor cells (MDSCs), and tumor-associated
macrophages.
[0054] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins. The antibodies in the present
invention may exist in a variety of forms including, for example,
polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2,
as well as single chain antibodies and humanized antibodies (Harlow
et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A
Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al, 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426).
[0055] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody.
[0056] The term "antibody-derived targeting domain" "or antigen
binding domain" as used herein refers to the minimum antibody
fragment which contains a complete antigen-recognition and binding
site. An "Fv" domain also refers to the minimum antibody fragment
which contains a complete antigen-recognition and -binding site and
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0057] The term "natural ligand" as used herein refers to a
naturally occurring protein which binds specifically to another
naturally occurring protein. "Natural ligand" encompasses both the
full-length protein and fragments thereof which bind specifically
to the same naturally occurring protein. A natural ligand as used
herein can be recombinantly produced or synthetic.
[0058] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein.
[0059] As used herein, the expression "specifically binds" in
reference to a chimeric T cell receptor means that the chimeric T
cell receptor binds to its target protein with greater affinity
that it does to a structurally different protein(s).
[0060] As used herein, the expression "tumor load" or "tumor
burden" refers to the number of cancer cells, the size of a tumor,
or the amount of cancer in the body of a subject.
Intraperitoneal Administration of Chimeric Receptor Immune
Cells
[0061] In developing therapies for treatment of disseminated tumors
such as intraperitoneal tumors, it is advantageous to utilize a
tumor-selective therapeutic. Immunotherapeutic cells engineered to
express chimeric receptors (e.g., CAR T cells) that recognize and
bind to tumor associated antigens is increasingly being proven as a
promising approach to cancer treatment. Despite the ability of the
engineered cells to target the tumor cells, systemic intravascular
administration can nevertheless result in inadequate exposure of
tumor cells to the CAR-T cells and adverse side effects due to
binding of CAR-T cells to normal cells. Accordingly, it is
advantageous to provide a method for administering the CAR-T cells
directly to the organ or anatomic space containing the tumors. In
some aspects of the present disclosure, methods are provided
comprising intraperitoneal administration of chimeric receptor
lymphocytes as described herein. In some embodiments, the
lymphocytes are T cells.
[0062] Current CAR T therapies involve systemic infusion of the
engineered cells to the patient. Such administration methods,
however, may suffer from reduced concentrations of the cells at the
disease site or presentation of adverse side effects due to
activities of the cells. Provided herein are compositions and
methods for intraperitoneal (IP) infusion of engineered immune
cells to treat patients diagnosed with an intraperitoneal cancer as
experiments described below show that regional IP infusion of the
cells resulted in superior protection against peritoneal tumors
when compared to systemically infused cells. Moreover,
administration of immune pathway inhibitors to the patients
receiving the IP cell (IPC) therapy further improved therapeutic
efficacy for treating peritoneal metastases.
Chimeric Receptor Immune Cell Therapy
[0063] Cancer research is increasingly focused on the use of immune
system components to combat malignant disease. For example,
numerous therapeutic antibodies have proven successful in treating
cancers and are presently marketed throughout the world. More
recently, cell-based immunotherapy is emerging as a promising
approach to cancer treatment in which a patient's own immune cells
are engineered to recognize and attack tumors in their body.
Diagnosis of a subject as having malignant tumors may include
determining what tumor antigen proteins (tumor associated antigens)
are expressed on the tumor cell surface. The subject can then be
treated with anti-tumor immune cells which have been engineered to
target and bind to the tumor associated antigen, ultimately leading
to the killing of the tumor cells by the immune cell and possibly
other co-administered cells or therapeutic agents. Disclosed herein
are compositions and methods for treating tumors in the abdominal
cavity via intraperitoneal infusion of engineered immune cells.
[0064] In one aspect are lymphocytes which have been engineered to
express a chimeric receptor. The population of lymphocytes for use
according to the present methods include but are not limited to T
cells, B cells and NK cells. In some embodiments, the T cells
comprise CD4+ cells, CD8+ cells, gamma delta T cells
(.gamma..delta. T cells), NK T cells and/or regulatory T cells
(Treg). Of particular interest are T cells which express a chimeric
receptor ("chimeric receptor T cells). The chimeric receptor immune
cells are designed to bind, via the chimeric receptor protein, to
diseased or malignant cells which express a cell surface protein.
For example, malignant cells in the intraperitoneal cavity may
express the carcinoembryonic antigen (CEA, GenBank Acc. No.
NP_04354 and its related isoforms), the KIT tyrosine kinase
receptor protein (GenBank Acc. No. P10721), the epithelial cell
adhesion molecule protein (EpCAM; GenBank Acc. No. NP_002345 and
its related isoforms), or the mucin 1 protein (MUC1, GenBank Acc.
No. NP_001018016 and its related isoforms) (e.g., Yamamoto et al.,
2014, J Cancer Res Clin Oncol, 140:607-612; Joensuu, 2006, Ann
Oncol, 17:x280-x286; Chauhan et al., 2009, J Ovarian Res, 2:21-29;
Flatmark et al., 2013, Int J Cancer, 133:1497-1506). Other examples
of antigen targets expressed on cancer cells and that are currently
being studied for CAR-T cell therapy include CD20 or GD2
(follicular lymphoma), CD171 (neuroblastoma), CD20 (non-Hodgkin
lymphoma), CD19 (lymphoma), IL13R.alpha.2 (glioblastoma), and CD19
(chronic lymphocytic leukemia or CLL and acute lymphocytic leukemia
or ALL). Virus specific CAR-T cells have also been developed to
attack cells harboring virus such as HIV. For example, a clinical
trial was initiated using a CAR specific for Gp100 for treatment of
HIV (Chicaybam et al (2011) Int Rev Immunol 30:294-311). It is
understood that the present methods and compositions include, but
are not limited to, the antigen targets listed above.
[0065] Generation of chimeric receptor proteins and immune cells
expressing these proteins is well known in the art and combines the
targeting function and specificity of a ligand or antibody or
fragment thereof with the anti-tumor activity of an immune cell.
See for example, Sadelain et al., 2013, Cancer Discov, 3:388-398.
The chimeric receptor protein comprises in an N-terminal to
C-terminal direction a target binding domain which specifically
binds a protein expressed on the surface of a diseased target cell
(e.g., a cancer cell or malignant cell present in the peritoneal
cavity), a hinge domain, a transmembrane domain, and an
immunomodulatory signaling domain. In some embodiments, the
construct further comprises a signal peptide fused to the
N-terminus of the target binding domain.
[0066] In some embodiments, the target binding domain of the
chimeric receptor protein comprises the antigen-binding portion of
an immunoglobulin wherein the immunoglobulin binds a protein on the
surface of the diseased cell. This construct is alternatively
referred to herein as a chimeric antigen receptor (CAR). The
antigen binding domain can be any domain that binds to the cell
surface antigen including but not limited to monoclonal antibodies,
polyclonal antibodies, synthetic antibodies, human antibodies,
humanized antibodies, and fragments thereof. In preferred
embodiments, the antigen-binding domain of the CAR is a fragment of
an antibody that is able to specifically bind the antigen when part
of a CAR construct. In some instances, it is beneficial for the
antigen binding domain to be derived from the same species in which
the CAR will ultimately be used in. For example, for use in humans,
it may be beneficial for the antigen binding domain of the CAR to
comprise a fragment of a human or humanized antibody. Accordingly,
in some embodiments, the antigen binding domain portion of a CAR
comprises a tumor antigen binding fragment of a human or humanized
antibody. In each of these embodiments, the antigen-binding domain
of an antibody, such as the single-chain variable fragment (scFV or
Fab) or is fused to a transmembrane domain and a signaling
intracellular domain (endodomain) of a T cell receptor. Often, a
spacer or hinge is introduced between the extracellular antigen
binding domain and the transmembrane domain to provide flexibility
which allows the antigen-binding domain to orient in different
directions to facilitate antigen recognition and binding.
[0067] In some embodiments, the antigen binding moiety portion of
the chimeric antigen T cell receptor targets the CEA antigen and
comprises the CEA-binding domain of an antibody which has been
shown to bind CEA expressed on a cell surface. The chimeric
receptor construct can be generated according to methods and
compositions known to the ordinarily skilled artisan. For example,
a CEA CAR-T construct used in the Examples below comprises portions
of the variable domain of a humanized MN14 antibody (described in
U.S. Pat. No. 5,874,540, the contents of which are incorporated
herein by reference it their entirety). A Fab or scFv construct can
be generated from a CEA antibody according to the methods of Nolan
et al. (1999, Clinical Canc Res, 5:3928-3941) to include the
CEA-binding domains of the CEA antibody. In some embodiments, the
CEA CAR-T construct comprises the amino acid sequence of SEQ ID NO:
1 shown below:
TABLE-US-00001 (SEQ ID NO: 1)
DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYW
TSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0068] In some embodiments, the CEA CAR-T construct further
comprises a signal sequence at the N-terminus of SEQ ID NO:1 which
is cleaved from the construct after in vivo expression of the CEA
CAR-T construct. In other embodiments, the signal sequence has the
sequence MGWSCIILFLVATATGVHS (SEQ ID NO:2). The Fab or scFv domain
can then be fused to a hinge domain such as that from the CD8 hinge
domain (see GenBank Acc. No. NP_001759). The hinge domain can then
be fused at its C-terminus to a transmembrane domain. In one
embodiment, the transmembrane domain is from the CD3 zeta chain
(e.g., GenBank Acc. No. NP_000725 or from the CD28 protein (e.g.,
GenBank Acc. No. NP_006130). The transmembrane domain of the
chimeric construct can then be fused at its C-terminus to the
signaling domain of the CD3 zeta chain (e.g., GenBank Acc. No.
NP_000725).
[0069] In some embodiments, the CEA-binding domain is a scFv or Fab
domain from an antibody that binds CEA and the chimeric receptor
construct comprises, in an N-terminal to C-terminal direction: the
CEA-binding domain (e.g., SEQ ID NO: 1), a CD8 hinge domain, a zeta
transmembrane domain and a zeta cytoplasmic signaling domain. In
other embodiments, the chimeric receptor construct comprises, in an
N-terminal to C-terminal direction: the CEA-binding domain (e.g.,
SEQ ID NO:1), the CD8 hinge domain, a domain comprising (in an
N-terminal to C-terminal direction) a portion of the CD28
extracellular domain, the CD28 transmembrane domain, and the CD28
cytoplasmic co-stimulatory domain, and a zeta cytoplasmic signaling
domain.
[0070] In alternative embodiments, a known ligand to a protein
expressed on the surface of a tumor cell is fused to a T cell
receptor signaling domain to produce what is alternatively referred
to herein as a "chimeric ligand T cell receptor" or "chimeric
ligand receptor." As with CAR-T cells, T cells that express a
chimeric ligand T cell receptor protein become activated in the
presence of a cell expressing the target ligand receptor protein,
resulting in the attack on the targeted cell by the activated
T-cell in a non-MHC dependent manner. In some embodiments, a
chimeric ligand receptor is specifically designed to include the
extracellular domain of the KIT-ligand, a cytokine that binds to
tyrosine-protein kinase KIT protein (cKIT receptor or CD117)
expressed on the surface of gastrointestinal stromal tumor (GIST)
cells. A chimeric T cell receptor was engineered as described in
PCT Pub. No. WO 2014/121264 (see also Katz et al., J Transl Med.,
2013, 11:46). The anti-KIT chimeric receptor was expressed on the
surface of the T cells and the engineered cells were able to
proliferate when co-cultured with KIT+ tumor cells and produce
IFN.gamma.. Moreover, mice with established GIST xenografts and
treated with the anti-KIT chimeric ligand receptor T cells showed
significant reductions in tumor growth rates. Accordingly, it is
understood that such chimeric ligand receptor T cells can be used
to treat intraperitoneal cancers according to the methods described
herein. A schematic of two alternative CAR-T constructs for use in
the methods as described herein are provided in FIGS. 1A and
1B.
Chimeric Receptor Intracellular Domain
[0071] The intracellular signaling domain of the chimeric T cell
receptor is activated upon binding of the target antigen by the
antigen-binding domain of the CAR or by the ligand portion of the
chimeric ligand receptor. Generally, the domain of the endogenous
CD3 T cell receptor is used as the signaling domain. More recently,
however, second generation CAR molecules have been designed to
further include another intracellular signaling domain from a
costimulatory receptor such as CD28, 41BB, or ICOS to provide
additional signals to the engineered T cell which may improve its
efficacy and/or viability. Third generation chimeric T cell
receptors combine multiple signaling domains or accessory regions
to provide novel functionality. Accordingly in some embodiments,
the cytoplasmic domain further comprises one or more co-stimulatory
domains selected from the group consisting of an OX-40
costimulatory domain, an HVEM co-stimulatory domain, a 41BB
co-stimulatory domain, an ICOS co-stimulatory domain, an OX40
co-stimulatory domain and a CD27 co-stimulatory domain. In one
embodiment, the additional co-stimulatory domain is positioned
between a CD28 co-stimulatory domain and a CD3-zeta signaling
domain.
Chimeric Receptor Lymphocytes for IP Infusion
[0072] Lymphocytes engineered with chimeric receptors to enable
highly specific tumor recognition and killing have gained
considerable attention following promising clinical results (Grupp
et al., 2013, N Eng J Med, 368:1509-1518; Porter et al., 2011, N
Eng J Med, 365:725-733, Sadelain et al., 2009, Curr Opin Immunol,
21:215-223). Types of lymphocytes that can be used in the methods
of the present disclosure include, without limitation, peripheral
donor lymphocytes genetically modified to express chimeric
receptors (Sadelain, M., et al. 2003, Nat Rev Cancer 3:35-45),
lymphocyte cultures derived from tumor infiltrating lymphocytes
(TILs) in tumor biopsies (Panelli, M. C., et al. 2000 J Immunol
164:495-504; Panelli, M. C., et al. 2000 J Immunol 164:4382-4392),
and selectively in vitro-expanded antigen-specific peripheral blood
leukocytes employing artificial antigen-presenting cells (AAPCs) or
pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res
65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood
102:2498-2505). The T cells may be autologous, non-autologous
(e.g., allogeneic), or derived in vitro from engineered progenitor
or stem cells. T cells may prepared in bulk as commonly performed
with Peripheral blood lymphocytes (PBL), or tumor infiltrating
lymphocytes (TILs), T cells may be purified by using, e.g. CD4,
CD8, CD62L.
[0073] Genetic modification of immunoresponsive cells (e.g., T
cells, CTL cells, NK cells) can be accomplished by transducing a
substantially homogeneous cell composition with a recombinant DNA
or RNA construct. Preferably, a retroviral vector (either gamma
retroviral or lentiviral) is employed for the introduction of the
DNA or RNA construct into the host cell genome. For example, a
polynucleotide encoding a receptor that binds an antigen (e.g., a
tumor antigen, or a variant, or a fragment thereof), can be cloned
into a retroviral vector and expression can be driven from its
endogenous promoter, from the retroviral long terminal repeat, or
from an alternative internal promoter. Non-viral vectors or RNA may
be used as well. Random chromosomal integration, or targeted
integration (e.g., using a nuclease, transcription activator-like
effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and/or
clustered regularly interspaced short palindromic repeats
(CRISPRs), or transgene expression (e.g., using a natural or
chemically modified RNA) can be used.
[0074] For initial genetic modification of the cells to provide
chimeric receptor-expressing cells, a retroviral vector is
generally employed for transduction, however any other suitable
viral vector or non-viral delivery system can be used. For
subsequent genetic modification of the cells to provide cells
comprising an antigen presenting complex comprising at least two
co-stimulatory ligands, retroviral gene transfer (transduction)
likewise proves effective. Combinations of retroviral vector and an
appropriate packaging line are also suitable, where the capsid
proteins will be functional for infecting human cells.
[0075] In yet another aspect, the disclosure is directed to
pharmaceutical compositions to facilitate administration of
transduced T cells as described herein to a subject in need. The
transduced T cells according to the disclosure can be made into a
pharmaceutical composition or made implant appropriate for
administration in vivo, with appropriate carriers or diluents,
which further can be pharmaceutically acceptable. The means of
making such a composition or an implant have been described in the
art (see, for instance, Remington's Pharmaceutical Sciences, 16th
Ed., Mack, ed. (1980)). Where appropriate, the transduced T cells
can be formulated into a preparation in semisolid or liquid form,
such as a capsule, solution, injection, inhalant, or aerosol, in
the usual ways for their respective route of administration. Means
known in the art can be utilized to prevent or minimize release and
absorption of the composition until it reaches the target tissue or
organ, or to ensure timed-release of the composition. Desirably,
however, a pharmaceutically acceptable form is employed which does
not ineffectuate the cells expressing the chimeric receptor. Thus,
desirably the transduced T cells can be made into a pharmaceutical
composition containing a balanced salt solution, preferably Hanks'
balanced salt solution, or normal saline. For instance, the
compositions can be formulated with a physiologically acceptable
carrier or excipient to prepare a pharmaceutical composition. The
carrier and composition can be sterile. The formulation should suit
the mode of administration.
[0076] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, dextrose, magnesium stearate,
talc, silicic acid, viscous paraffin, perfume oil, fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well
as combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like that do not deleteriously react
with the active compounds.
[0077] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0078] The composition can be formulated in accordance with the
routine procedures as a pharmaceutical composition adapted for
administration to human beings. For example, compositions for
intravenous administration typically are solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also
include a solubilizing agent and a local anesthetic to ease pain at
the site of the injection. Generally, the ingredients are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampule or sachette
indicating the quantity of active compound. Where the composition
is to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water,
saline or dextrose/water. Where the composition is administered by
injection, an ampule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0079] Compositions of the invention comprising genetically
modified immunoresponsive cells can be conveniently provided as
sterile liquid preparations, e.g., isotonic aqueous solutions,
suspensions, emulsions, dispersions, or viscous compositions, which
may be buffered to a selected pH. Liquid preparations are normally
easier to prepare than gels, other viscous compositions, and solid
compositions. Additionally, liquid compositions are somewhat more
convenient to administer, especially by injection. Viscous
compositions, on the other hand, can be formulated within the
appropriate viscosity range to provide longer contact periods with
specific tissues. Liquid or viscous compositions can comprise
carriers, which can be a solvent or dispersing medium containing,
for example, water, saline, phosphate buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol,
and the like) and suitable mixtures thereof.
[0080] Those skilled in the art will recognize that the components
of the compositions should be selected to be chemically inert and
will not affect the viability or efficacy of the genetically
modified immunoresponsive cells as described in the present
invention. This will present no problem to those skilled in
chemical and pharmaceutical principles, or problems can be readily
avoided by reference to standard texts or by simple experiments
(not involving undue experimentation), from this disclosure and the
documents cited herein.
Therapeutic Methods
[0081] The present disclosure describes compositions and methods
for intraperitoneal infusion of lymphocytes which express chimeric
receptor T cells and which thereby target and bind malignant cells
in the peritoneal cavity, leading to inhibition of tumor cell
growth or death of tumor cells. Intraperitoneal administration
provides a higher concentration of therapeutic agents to the tumor
location to maximize therapeutic efficacy and minimize systemic
toxicity of the therapeutic cells. Data provided herein shows that
genetically engineered lymphocytes have significantly greater
efficacy when administered via IP infusion as compared to systemic
infusion. The therapeutic efficacy of these cells in enhanced by
use of inhibitors of immune suppressor cells.
[0082] Therapeutic use of chimeric receptor lymphocytes involves
harvesting white blood cells from a subject diagnosed with cancer,
isolating and culturing the lymphocytes, transforming the
lymphocytes with a vector containing the chimeric receptor gene,
and administering to the subject the resultant engineered
lymphocytes. Cells prepared for administration to a subject can
comprise a purified population of cells, for example CD4+ T cells.
Those having ordinary skill in the art can readily determine the
percentage of genetically modified lymphocytes in a population
using various well-known methods, such as fluorescence activated
cell sorting (FACS).
[0083] The chimeric receptor T cells can be administered in any
physiologically acceptable vehicle. In some embodiments, a dose of
about 1.times.10.sup.6 to 1.times.10.sup.11, 1.times.10.sup.6 to
1.times.10.sup.10, 1.times.10.sup.6 to 1.times.10.sup.9,
1.times.10.sup.7 to 1.times.10.sup.11, 1.times.10.sup.7 to
1.times.10.sup.10, 1.times.10.sup.7 to 1.times.10.sup.9 or
1.times.10.sup.8 to 1.times.10.sup.9 cells are administered. In
other embodiments, a dose of about 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, or 1.times.10.sup.11 cells are administered. The
precise determination of what would be considered an effective dose
may be based on factors individual to each subject, including their
size, age, sex, weight, and condition of the particular subject.
Dosages can be readily ascertained and readily adjusted by those
skilled in the art from this disclosure and the knowledge in the
art. Preferable ranges of purity in populations comprising chimeric
receptor T cells are about 70 to about 75%, about 75 to about 80%,
about 80 to about 85%; and still more preferably the purity is
about 85 to about 90%, about 90 to about 95%, and about 95 to about
100%. The cells can be administered by, for example, injection or
catheter. Cells may also be administered by minimally invasive
surgical techniques.
[0084] The chimeric receptor T cells are administered to the
patient via intraperitoneal infusion once, twice, 3 times, 4 times
or 5 times over a period of time. The period of time may be about 1
month, 2 months, 3 months, 4 months or 5 months. For example, a
dose of the chimeric receptor T cells are administered once, twice,
3 times or 4 times in a one-week period. Furthermore, the one-week
dosing regimen is performed every week, every other week, or 3
weeks or every month. Alternatively, the one-week dosing regimen is
performed every other week. In one embodiment, the dose of the
chimeric receptor T cells is administered 3 times per week, every
other week. The dosing regimen is continued until the tumor load is
reduced by at least 5%, 10%, 15%, 20%, 25%, 50%, 60%, 70%, 80%, 90%
or 95% relative to the tumor load prior to administration of the
first dose of chimeric receptor T cells.
[0085] In some embodiments, the chimeric receptor T cells are
administered to a patient who has undergone debulking surgery to
render the patient as disease-free as is surgically possible.
Immediately following surgery, or within 1, 2 or 5 days following
surgery, the patient receives intraperitoneal infusion of the CAR-T
cells.
[0086] Effective chimeric receptor T cell therapy is achieved in
part by determining an optimal dose of the chimeric receptor T
cells. A therapeutically effective dose for chimeric receptor T
cell treatment can be determined, for example, by imaging the
abdomen of the patient by CT or PET scans or MRI imaging. A
therapeutically effective dose will decrease the volume and/or
number of malignant tumors as determined by imagine by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or by 100%. A
therapeutically effective dose would be expected to decrease the
volume and/or number of malignant tumors in the abdomen within
about 5 days, 1 week, 2 weeks, 4 weeks, 6 weeks or 10 weeks after
the first administered dose of chimeric receptor T cells.
Alternatively, a therapeutically effective dose will decrease the
amount or volume of malignant ascites and/or intraperitoneal mucin
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over the
dosing period. A therapeutically effective dose will also decrease
serum tumor markers if available for the targeted tumor type by at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over the
dosing period.
Intraperitoneal Infusion of CEA Car-T Cells
[0087] The efficacy of chimeric receptor T cells by IP infusion of
chimeric receptor lymphocytes was shown using methods described
herein. In mice treated by IP infusion of unmodified T cells or
anti-CEA CAR-T cells, there was a significant reduction in tumor
load as compared to animals untreated or treated with unmodified T
cells. The ordinarily skilled artisan would understand that the
methods described herein are useful for reducing tumor load using
any chimeric receptor T cell (e.g., CAR-T cell or chimeric ligand
receptor T cell) which has been engineered to specifically bind via
the chimeric T cell receptor to the target protein or antigen
expressed on the surface of the tumor cell.
[0088] As shown in the Examples below, direct IP infusion of CAR-Ts
in mice with PC was more effective at controlling tumor than
systemic infusion. CAR-Ts within peritoneal tumors were detected
following IP infusion, whereas CAR-Ts were not present in
peritoneal tumors following systemic injection. Treatment of
malignancies using IP CAR-T infusion methods as described herein
results in a reduction in adverse side effects as well.
[0089] The compositions and methods described herein are used for
treating patients diagnosed with intraperitoneal tumors. The
patient first undergoes diagnostic laparoscopy to lyse any
peritoneal adhesions in order to ensure optimal CAR-T distribution
following IP infusion of the CAR-T. This diagnostic laparoscopy can
also be used to assess the disease, acquire pre-treatment cell or
tissue specimens, and/or for placement of a peritoneal dialysis
catheter. The IP CAR-T infusion can be performed later the same day
or on a following day.
[0090] IP infusion of CAR-T comprises infusion of an initial dose
of about 1.times.10.sup.9 to 1.times.10.sup.11, or about
1.times.10.sup.10 cells into the peritoneal cavity. The CAR-T cells
are suspended in a physiological solution such as normal saline. In
some embodiments, the solution contains about 5% to 15% or about
10% dimethyl sulfoxide (DMSO). In some embodiments, immediately
prior to IP CAR-T infusion, ascites fluid is drained from the
peritoneal cavity. In other embodiments, aspiration is performed
prior to injection of the dose of CAR-T cells to confirm the
absence of blood and/or intestinal contents.
[0091] The IP infusion of a dose of CAR-T cells can be carried out
manually and at room temperature. In some embodiments, the dose is
infused over a time period of about 5 min to 60 min, about 30 min
to 120 min, about 5 min to 30 min, about 5 min to 20 min, or about
10 min, 15 min, 20 min, 25 min, 30 min, 45 min or 60 min. The
infusion can be carried out in an out-patient setting.
[0092] One or more additional doses of the CAR-T cells can be
administered after the initial IP infusion. For example, an
additional dose can be administered weekly, every 3 days or every 5
days wherein the additional dose is administered once, twice, or
three times. In other embodiments an additional dose is
administered weekly, every 3 days or every 5 days until a
post-infusion assessment fails to detect malignancy in the
peritoneal cavity. In some embodiments, the additional dose is
equal to the initial dose. In other embodiments, each of the
additional doses is about 75%, 90%, 120% or 150% of the initial
dose in terms of the number of CAR-T cells. In a preferred
embodiment, an additional dose of about 1.times.10.sup.10 is
administered to the patient IP once per week for 2 or 3 weeks.
[0093] In some embodiments, a method for treating malignancies in
which tumor cells are located outside of the peritoneal cavity is
provided. Studies were done to determine if IP CAR-T infusions
could reduce or inhibit the growth of flank tumors in mice with
synchronous PC. IP CAR-T infusions were able to significantly limit
the growth of distant flank tumors while inducing marked IP
responses (see Example 6). CAR-Ts were not detected within the
flank tumors, suggesting that the flank tumor responses were due to
IFN.gamma. surges which were detected 4 days following IP CAR-T
treatment (FIG. 7D). IP infusion of CAR-Ts with profound
destruction of peritoneal tumors may have induced a phenomenon
similar to the abscopal effect seen with radiation therapy (Park et
al., 2015, Cancer Immunol Res, 3:610-619). Alternatively, CAR-Ts
may have infiltrated the flank tumor at earlier time points.
Surprisingly, systemic infusion also did not lead to a meaningful
flank tumor response, which may reflect inadequate CAR-T dosing by
this route, as most cells likely traffic to nodes, lung, and
spleen. Importantly, the response of distant subcutaneous tumors
was less durable than the response of IP tumors in accordance with
the brief surge in serum IFN.gamma. levels. Sequential regional and
systemic therapy may offer improvements in efficacy for PC in the
context of extra-abdominal disease. Accordingly, in some
embodiments, a method of treatment is provided wherein a subject
diagnosed with a peritoneal malignancy is treated with IP infusion
of a chimeric receptor lymphocyte followed by treatment with
systemic infusion of the chimeric receptor lymphocyte.
[0094] As PC can have a prolonged natural history, the durability
of protection from IP tumor growth following IP CAR-T infusion was
examined (see Example 4). Following repeated IP CAR-T dosing, mice
were protected from repeat IP tumor challenge for up to 10
additional days. CAR-Ts were detectable within the PC as late as 28
days. This finding suggests persistence of CAR-Ts in the peritoneal
space, potentially with the CAR-Ts acquiring effector memory
features. CAR-Ts with an effector memory phenotype
(CD44+CD62L-CCR7-) were detected within IP tumors in greater
proportion at day 28 compared to day 10. These data suggest that
following initial IP infusion, CAR-Ts undergo effector memory
programming, which may have accounted for the prolonged anti-tumor
protection in the peritoneal space.
Immunosuppressor Agents
[0095] Therapeutic efficacy of chimeric receptor T cell infusions
is likely to be affected by factors that lead to immunosuppression,
e.g., suppression of tumor-killing cells or decreased expression of
anti-tumor cytokines. It is important to consider the effects of
immune environment of the intraperitoneal space in the presence of
a carcinoma and to treat a patient undergoing chimeric receptor T
cell therapy accordingly.
[0096] The accumulation of immunosuppressive regulatory T cells
(Tregs) and myeloid derived suppressor cells (MDSCs) within the
tumor microenvironment represents a potential major obstacle for
the development of effective antitumor immunotherapies (Weiss et
al., 2014, J Immunol., 192:5821-5829). Elimination of MDSCs has
been shown to significantly improve immune responses in
tumor-bearing mice and in cancer patients (Ostrong-Rosenberg et
al., 2009, J Immunol, 182:4499-4506), Talmadge, 2007, Clin Cancer
Rres, 13:5243-5248). Provided herein are methods for inhibiting
immunosuppression by, for example, Treg and MDSC, in a patient
undergoing chimeric receptor T cell therapy, wherein the patient is
also administered an agent which inhibits functions of
immunosuppressive cells.
[0097] To examine the extent of immunosuppressive activity upon
treatment with chimeric receptor T cells, Treg and MDSC were
characterized in C57BL/6 mice bearing MC38 tumor cells.
Specifically, Treg and MDSC are characterized in terms of their
cell surface markers, cytokines and enzymes believed to play a role
in suppressive activity. As shown in Example 7 below, studies
showed that both MIDSC and Treg could be detected within IP tumors.
Both MDSC and Treg have been well described as inhibitors of
endogenous T cell and CAR-T anti-tumor responses (Khaled et al.,
2013, Immunol Cell Biol, 91:493-502; Burkholder et al., 2014,
Biochim, Biophys Acta, 1845:182-201). IP MDSC also expressed high
levels of PD-L1 (programmed death-1 receptor ligand), which was
previously demonstrated to be an important mediator of CAR-T
suppression (Burga et al., 2015, 64:817-829). Addition of an MDSC
depletion antibody which binds Gr1 (granulocytic myeloid marker
protein) or a PD-L1 blocking antibody treatment enhanced IP CAR-T
performance in terms of tumor killing. The encouraging additive
effects of IP CAR-T and suppressor cell targeting provide
justification for combinatorial strategies in developing solid
tumor immunotherapy. Accordingly, in some embodiments, a method for
treating a subject diagnosed with a peritoneal cancer is provided,
wherein the subject is administered a population of lymphocytes
expressing a chimeric receptor as described herein via IP infusion
and wherein the subject is also administered an immunosuppressing
agent which suppresses the activity of suppressor T cells such as
MDSCs or Tregs.
[0098] In some embodiments, the immunosuppressing agent is an
antibody that binds IL10, PD-1 (programmed death-1 receptor), PD-L
(programmed death-1 receptor ligand 1), PD-L2 (programmed death-1
receptor ligand 2), IDO (Indolamine 2,3-dexoygenase), STAT3 (signal
transducer and activator of transcription 3), GM-CSF, CD25, GITR
(glucocorticoid-induced TNFR-related protein), TGF-.beta., or
CTLA4. In other embodiments, the immunosuppressing agent is
administered to the subject before IP administration of chimeric
receptor lymphocytes. In still other embodiments, the
immunosuppressing agent is administered to the subject after IP
administration of chimeric receptor lymphocytes. The
immunosuppressing agent can be administered multiple times, for
example, every day, every 2 days, every 3 days, every 4 days, every
5 days, every 6 days or once per week (every 7 days) after IP
administration of the chimeric receptor lymphocytes. The
immunosuppressing agent can be administered on the same day as the
IP administration of the chimeric receptor lymphocytes. The
immunosuppressing agent can be administered 1 day, 2 days, 3 days,
4 days, 5 days, 6 days, 7 days or more prior to IP administration
of the chimeric receptor lymphocytes. More than one
immunosuppressing agent can be administered to the patient, for
example, the subject may be co-administered or serially
administered antibodies which bind CD25 and antibodies which bind
GR1.
Additional Therapeutic Agents
[0099] The chimeric receptor T cells of the present disclosure can
be used alone or in combination with other therapies.
Immunomodulatory agents may include but are not limited to
interleukins, e.g. IL-2, IL-3, IL-6, IL-1, IL7, IL12, IL21, as well
as the other 10 interleukins, the colony stimulating factors, such
as granulocyte colony stimulating factor (G-CSF), and macrophage
colony stimulating factor (M-CSF), and interferons, such as
.gamma.-interferon and erythropoietin. Other immunomodulatory
agents may include monoclonal antibodies or small molecules
designed to target immunoinhibitory pathways such as an antibody or
fragment thereof which binds TGF.beta. or IL10, thereby blocking
the function of TGF.beta. or IL10, respectively.
[0100] In a preferred embodiment, administration of the chimeric
receptor T cells is coupled with administration of one or more
agents as listed above which inhibit chimeric receptor T cell
suppressor pathways. For example, a patient in need thereof
receives intraperitoneal infusion of both chimeric receptor T cells
and an agent which increases in situ viability of the chimeric
receptor T cells after intraperitoneal infusion. In a preferred
embodiment, the patient is administered chimeric receptor T cells
and a dose of IL2. Administration of the agent which increases
viability of the chimeric receptor T cells may be performed before,
during or after administration of the chimeric receptor T
cells.
IV. EXAMPLES
[0101] The following examples are illustrative in nature and are in
no way intended to be limiting.
Example 1
Preparation of CEA Car-T Cells
[0102] The anti-CEA scfv-CD28/CD3.zeta. (Tandem) chimeric antigen
receptor used in the examples described herein was previously
generated according to the method of Emtage et al. (2008, Clin
Cancer Res, 14:8112-8122). Briefly, a tandem molecule was generated
by molecularly fusing an hMN14 sFv-CD8 hinge segment of a
monoclonal antibody which specifically binds CEA upstream of a
construct encoding a cytoplasmic domain comprising in an N-terminal
to C-terminal direction, a human CD28 extracellular domain, the
CD28 cytoplasmic domain, and the cytoplasmic domain. The resultant
chimeric construct was cloned into a retroviral vector and verified
by restriction digestion and sequencing.
[0103] For the present studies, 6-8 week old B6.SJL-Ptprca
Pepcb/BoyJ (CD45.1) mice were purchased from Jackson for the
purpose of generating distinguishable CAR-Ts when isolated from
tissues ex vivo. Mice were housed in the animal facility at Roger
Williams Medical Center in pathogen-free conditions under
guidelines from the Institutional Care and Use Committee. CD45.1
mouse spleens were harvested in sterile fashion then pulverized.
Red blood cells were lysed and T cells were isolated using MACS
immunomagnetic bead isolation (Miltenyi). T cells were cultured in
complete media with IL-2 (500 IU/mL) and anti-CD3/CD28 T-activator
Dynabeads (Life Technologies) for 48 hours to achieve activation.
Phoenix Ecotropic cells harboring a hMN14
sFv-CD8.alpha.-CD28/CD3.zeta. CAR (Emtage et al., 2008, Clin Cancer
Res, 14:8112-8122) were used to produce supernatant for
transduction. Activated T cells were cultured in the retroviral
supernatant and underwent two spinfections. Transduced T cells were
cultured and expanded in the presence of IL-2 (500 IU/mL), and CAR
expression levels were checked 48 hours after transduction.
[0104] Transduction of murine splenocytes was confirmed 48 hours
after transduction by measuring CAR expression on CD3+ cells using
flow cytometry and an antibody which specifically binds the sFv
portion of the CEA CAR-T molecule. A standard gating strategy was
used to identify viable, single cells expressing CD3 and the
chimeric anti-CEA CAR-T. The results showed that viral transduction
efficiency was about 73% (data not shown).
Example 2
In Vitro Killing of Tumor Cells
[0105] Killing by the transduced CAR-T cells was tested in vitro
using as target cells MC38 cells which were stably transfected with
a gene encoding human CEA and firefly luciferase. MC38CEA+ cells
were first generated by stably transfecting MC38 cells with the
human CEA gene. MC38-luc was generated by transfecting MC38CEA+
cells with pLenti-III-UbC-Luciferase (Applied Biological Materials
Inc, Richmond, BC Canada). Effector cells were either CEA CAR-T
cells generated as described in Example 1 or untransduced splenic T
cells which were used as a negative control for the effector cells.
Bioluminescence assays were performed in which CAR-Ts or
untransduced T cells were co-cultured with MC38CEA-luc at various
Effector:Target ratios. Effectors were cultured in complete media
with IL-2 (500 IU/mL) prior to the assays. Cells were plated in
complete media in 96 well optical plates at varying Effector:Target
ratios and incubated overnight. After incubation, media was
discarded and luciferin (150 .mu.g/mL) was added to the wells.
Plates were analyzed in an IVIS 100. Supernatants were collected
and measured for luminescence activity and Specific Lysis % was
calculated as 100.times.[(experimental killing-spontaneous
luminescence)/(maximal killing-spontaneous luminescence)]. As seen
in FIG. 2, transduced CAR-T cells caused lysis at a significantly
higher rate than untransfected cells. At an Effector:Target ratio
as low as 0.03:1, specific lysis was 40% and significantly higher
than activated untransduced T cells (p=0.02).
Example 3
CAR-T Cell Delivery and Killing of Tumor Cells
[0106] To show that IP delivery improves CAR-T efficacy in mice
with peritoneal cancers (PC) compared to systemic tail vein (TV)
infusion, both infusion methods were studied in mice with
established IP tumors. Mice harboring CEA+PC were generated by the
IP injection of the MC38CEA-luc cells.
[0107] Six to eight week old C57Bl/6J mice were purchased from
Jackson Laboratories (Bar Harbor, Me.) and were used in all in vivo
models. Mice were injected intraperitoneally with
2.5.times.10.sup.6 MC38CEA-luc cells on day 0 using a 26
gauge.times.1/2'' needle attached to a 1 ml syringe. Cells had been
resuspended in normal saline for injection and the injection was
performed at room temperature. The needle traverses the midline
fascia 2-3 mm superior to the pubic symphysis and aspiration was
performed prior to injection to confirm absence of blood or
intestinal contents. In vivo work was carried out over the span of
14 days. On days 3 and 6, tumor-bearing mice were treated with
CAR-Ts (2.5.times.10.sup.6 cells), either via IP or TV infusion.
All mice were administered IL-2 (1000 IU/injection) daily beginning
with the first CAR-T injection on day 3. Control mice were treated
with untransduced splenic cells on days 3 and 6 or treated with
IL-2 alone. For the bioluminescent mice were imaged on an IVIS 100
imaging station (Caliper Life Sciences) on even days during in vivo
studies after being injected with 200 .mu.L of 15 mg/mL
luciferin.
[0108] Data are presented in FIGS. 3A and 3B. In FIG. 3A each line
on the plot is representative of the average of 4 mice. Fold
reduction in tumor luminescence was calculated between days 4 and
14 of the in vivo study, comparing TV to IP CAR-T delivery and the
results are shown in FIG. 3B. Error bars in FIGS. 3A and 3B are
representative of SEM values. P values were calculated using
Student's t test.
[0109] A single treatment of regionally delivered IP CAR-Ts
resulted in significantly reduced tumor burden (p<0.01), and
this remained significant compared to untreated animals at each
subsequent time point. IP infusion of CAR-Ts remained more
efficacious than systemic TV CAR-Ts for up to 8 days following the
second CAR-T treatment. In contrast to IP CAR-Ts, TV CAR-Ts did not
have a significant impact on tumor growth until day 14 when
compared to untreated animals (p=0.04). IP CAR-T treated mice
exhibited a 37-fold reduction in tumor burden between days 4 and
14, whereas TV CAR-T treated mice exhibited only a 3-fold reduction
in tumor burden over the same time period (p=0.05) (FIG. 3B). In 4
mice treated with regionally delivered IP CAR-Ts, there was no
detectable tumor upon necroscopy at day 14. Microscopic tumor was,
however, still detectable by bioluminescence monitoring on the same
day. In contrast, all of the TV treated animals had grossly visible
IP tumor upon necroscopy.
Example 4
Durable Protection by Car-T Cells
[0110] Having confirmed that IP CAR-T infusions are superior to
systemic administration, studies were performed to assess the
durability of the protection against IP tumor challenge. Following
IP CAR-T infusion treatment, mice were re-challenged with IP tumor
injections and tumor progression was monitored by bioluminescence.
In this study, mice received CAR-Ts on days 2, 4, 6 and 8, and
received a rechallenge dose of 2.5.times.10.sup.6 MC38CEA-luc on
Day 10. Tumor growth was measured by bioluminescence as described
in Example 3.
[0111] Mice that had received prior CAR-T IP infusions demonstrated
a significant decrease in tumor growth compared to mice with no
prior CAR-T treatment (p=0.02). Protection from IP tumor growth
extended for up to 10 days following tumor re-challenge (p=0.01)
(FIG. 4A). The frequencies of CAR+ lymphocytes recovered from IP
tumor tissue at both day 10 (n=5) and day 28 (n=3) time points were
compared. Small amounts of visible tumor were harvested and CAR-Ts
were found to comprise 69% of intratumoral leukocytes on day 10,
and 47% on day 28 (FIG. 4B). Memory phenotypes of CAR+ phenotypes
were examined at both day 10 (n=5) and day 28 (n=3) time points
using flow cytometry in which intratumoral CAR-Ts were
immunophenotyped. A standard gating strategy was used with
antibodies to CD62L (MEL-14, BD Bioscience), CCR7 (4B12, BD
Bioscience) and CD44 (1M7, BD Bioscience). An increase in the
proportion of CAR-Ts with an effector memory phenotype
(CAR+CD44+CD62L-CCR7-) was detected in the intratumoral CAR-T cells
(FIG. 4C), suggesting that following initial IP infusion, CAR-T
cells undergo effector memory programming.
Example 5
Protection Against Extra-Abdominal Tumor Growth by IP Car-T
Infusion
[0112] Considering that patients with IP tumors may have disease at
other anatomic sites, studies were performed to determine if IP
CAR-T infusions protected against subcutaneous flank tumor growth.
Mice were simultaneously injected with 1.0.times.10.sup.6
MC38CEA-luc cells IP and in the left flank. Flank tumor size was
measured in two dimensions (mm.sup.2) with calipers. Mice were
imaged on an IVIS 100 on even days during in vivo studies, after
being injected with 200 .mu.L of 15 mg/mL luciferin as described in
Example 3.
[0113] Following two treatments on days 3 and 6, IP CAR-Ts led to
decreased IP and flank tumor burden compared to untreated animals
(p<0.05), as well as animals receiving untransduced splenic T
cells (data not shown). Tumor reduction also trended favorably when
compared to mice that received CAR-Ts via TV and mice that received
IL-2 support only. This corresponded with a significantly less
flank tumor area in IP CAR-T treated mice when compared to
untreated animals on the same day (p=0.03, FIGS. 5A, 5B and 5C).
CAR-Ts were not recovered after flow cytometry staining for
trafficking in whole blood, flank tumor tissue, or left inguinal
lymph nodes. However, IP CAR-T infusions did lead to high levels of
systemic IFN at 4 days following treatment (FIG. 5D).
Example 6
IP Tumor Infiltration by Immunosuppressive Cells
[0114] Although IP CAR-T infusions mediated durable responses in
mice with PC, it was worthwhile to consider that immunosuppressive
cells could limit CAR-T function. MDSC and Treg, which we have
previously shown to suppress CAR-Ts in colorectal cancer LM models
(Burga et al., 2015, Cancer Immunol Immunother, 64:817-829), were
detected within IP tumors.
[0115] Tumor leukocyte contents were immunophenotyped using flow
cytometry as described in Example 3 to detect the presence of
suppressive cell populations. Antibodies used for these surface
markers: CD4 (RM4-5, BD Bioscience), CD11b (MI/17, BD Bioscience),
Ly6C (AL-21, BD Bioscience), Ly6G (1AB, BD Bioscience), PD-L (MIH5,
BD Bioscience). Intracellular FoxP3 staining was performed with
Mouse FoxP3 Permeabilization Kit (BD Bioscience). Single stain and
isotype controls were used for each experiment. Analysis of
acquired flow samples was performed with FlowJo software (Tree Star
Inc., Ashland Oreg.).
[0116] Tumor leukocyte contents were immunophenotyped to detect the
presence of suppressive cell populations. MDSC were found in the
tumors after staining for CD11b, Ly6C and Ly6G. Representative dot
plots show MDSC from the IP tumors, along with bar graphs comparing
MDSC populations from the tumors and spleens of the same untreated
animals. The percentages of CD11b+ cells among all live cells and
MDSC (Gr-1+) among CD11b+ cells are shown in FIGS. 6A and 6B). MDSC
were also immunophenotyped for the expression of the
immunosuppressive marker PD-L1 (FIGS. 7A and 7B). Representative
tumor dot plots show that Treg, expressed as the percentage of
FoxP3+ cells among CD3+CD4+ T cells, were also found within the IP
tumors. Smaller populations were found within the spleens of the
same animals (FIGS. 8A and 8B). Bars are representative of 3 mice
per group. Error bars are representative of SEM values. P values
were calculated using Student's t test.
[0117] On average, CD11b+ cells represented 57% of leukocytes in IP
tumors, compared to 11% from the spleens of the same animals
(p<0.01). Both Ly6G+ granulocytic MDSC (gMDSC) and Ly6C+
monocytic MDSC (mMDSC) were found within IP tumor (43%) and spleen
(41%) (FIGS. 6A and 6B). The immunosuppressive marker PD-L1 was
expressed on both MDSC subsets, and was expressed at equally high
levels, whether they were derived from the tumor or the spleen
(FIGS. 7A and 7B). Treg (FoxP3+) were found to comprise 82% of CD4
T cells within the tumors, compared with 7% in spleens from the
same animals (p<0.01) (FIGS. 8A and 8B).
Example 7
Car-T Administration Combined with Suppressor Cell Depletion
[0118] Tests were performed to study the potential therapeutic
efficacy of IP CAR-T infusions in combination with suppressor cell
depletion or blockade of the PD-1/PD-L1 immunoinhibitory pathway.
IP CAR-Ts combined with depleting antibodies against MDSC and Treg,
or blocking antibodies against the PD-L1 pathway, were administered
to mice that had been injected with MC38CEA-luc. The depleting
antibodies administered were anti-PD-L1 and anti-Gr1 antibodies
(which bind the PD-L1 and Gr1 proteins on the surface of MDSCs) and
anti-GITR antibodies (which bind the GITR protein on the surface of
Treg cells). Tumor reduction was monitored by bioluminescence over
14 days as described in Example 3.
[0119] Bar graphs compare the efficacy of regional IP CAR-Ts to
systemic TV CAR-Ts (FIG. 9A), and IP CAR-Ts alone to IP CAR-Ts with
antibodies on day 8 after the treatments (FIG. 9B) and the efficacy
of regional IP CAR-Ts to systemic TV CAR-Ts (FIG. 10A), and IP
CAR-Ts alone to IP CAR-Ts with antibodies at the end of the study
on day 14 (FIG. 10B). Bars are representative of 4 animals per
group. Error bars are representative of SEM values. P values were
calculated using Student's t test. Gross inspection images and
bioluminescence images were analyzed as well (data not shown).
[0120] IP CAR-Ts alone, and when used in combination with
anti-PD-L1, anti-Gr1, or anti-GITR antibodies, resulted in
significant reductions in tumor burden compared to untreated
animals. On day 14, CAR-Ts alone significantly diminished tumor
burden when compared to untreated mice, mice that received
untransduced T cells, and mice that received daily dose IL-2 alone
(FIG. 10A, p<0.05). CAR-Ts combined with the depletion of Treg
showed even further reduced burden from CAR-Ts alone (FIG. 10B,
p<0.01), as did the combination of CAR-Ts and MDSC depletion
(p=0.01.sup.7) (FIGS. 10A and 10B). Tumor burden was measured
through day 14 with results shown in FIG. 11. The combination of
CAR-Ts and anti-Gr-1 was the most efficacious overall, showing no
detectable bioluminescence on days 8 and 10. On day 14, there was
no detectable tumor found in any mouse that received IP CAR-T upon
gross inspection (data not shown).
Example 8
Patient Car-T Cell Production
[0121] Leukapheresis is performed at a validated blood center.
CAR-Ts are prepared at a Good Manufacturing Practice (GMP) facility
with standard operating procedures (SOPs) for processing,
manufacturing, expansion, dose harvesting, labeling, storage and
distribution. Briefly, patient peripheral blood mononuclear cells
(PBMCs) are isolated from leukapheresis product using Ficoll. PBMCs
are then activated for 48-72 hours in tissue culture flasks
containing AIM V media (Life Technologies, Grand Island, N.Y.)
supplemented with 5% sterile human AB serum, 50 ng/mL of anti-CD3
monoclonal antibody and 300-3000 U/mL of IL2.
[0122] Using the spinoculation method (e.g., Quintas-Cardama et
al., 2007, Hum Gene Ther, 18:1253-1260), 7.2-14.4.times.10.sup.8 T
cells are transduced in retronectin coated 6-well plates in AIM V
media with 5% human AB serum, 3000 U/mL of IL2, and protamine at
low speed centrifugation for 1 hour at room temperature. The
transduction step is carried out a total of two-three times over
24-hrs. After transduction, cells are washed in media and incubated
for 48-72 hours at 37.degree. C. CAR-Ts are further expanded in
Lifecell tissue culture bags (Baxter, Deerfield, Ill.) for 10-14
days. CAR-T growth curves and cell viability are examined
periodically and cell growth media is replaced as required. CAR-Ts
are examined by flow cytometry with fluorescently labeled
antibodies specific for CD3, CD4, CD8, and anti-CAR antibodies.
Flow cytometry is performed on a CyAn (Beckman Coulter, Brea,
Calif.) or LSR-II (BD Biosciences, San Jose, Calif.) machine. In
vitro activity of patient products is measured by bioluminescence
cytotoxicity assay. Luciferase-expressing tumor cells with the
appropriate target are mixed with specific CAR-T at various ratios
in 96-well round bottom plates and loss of bioluminescence from
each well is measured (Karimi et al., 2014, PLoS One,
9:e89357).
[0123] Clinical doses are prepared using a Fenwal cell harvester
system (Baxter, Deerfield, Ill.) in freezing media containing
PlasmaLyte (Baxter), 20% human bovine albumin, 10% DMSO and IL2.
Bacterial and fungal cultures are monitored for 14 and 28 days
respectively. Assays for bacterial endotoxin are performed using
LAL Endotoxin assay kits (Lonza, Walkersville, Md.). The clinical
dose is stored in liquid nitrogen and thawed immediately prior to
infusion.
Example 9
Dose Determination in a Mouse Model
[0124] Animal studies are performed to identify a minimal dose of
CAR-T cells necessary to achieve killing of IP tumor cells. A
murine model of carcinomatosis is generated by injecting C57BL/6
mice with tumor antigen-expressing tumor cells. The
antigen-expressing tumor cells are produced from the MC38 cell
line, a colorectal carcinoma cell line derived from primary mouse
colon carcinoma (Rosenberg et al., 1986, Science, 233:1318-1321).
MC38 cells are transduced with full length human antigen cDNA using
a retroviral expression vector. The MC38 cells are also stably
transfected with a luciferase gene. C57BL/6 mice are injected
intraperitoneally with 2.5.times.10.sup.6 murine colorectal
carcinoma cells. Seven days after injection of the tumor cells, the
mice are infused with 2.5.times.10.sup.6, 10.sup.7, or 10 specific
CAR-T cells using a needle inserted directly into the peritoneal
cavity. Each mouse receives a subcutaneous injection of IL2 (200
.mu.l of 1.5 .mu.g/mL) each day following the CAR-T infusion.
[0125] After infusion of the CAR-T cells, the mice are monitored
for tumor growth and response to treatment by measuring
bioluminescence using, e.g., an IVIS system (PerkinElmer). To
assess extraperitoneal or off-target CAR-T delivery, flow cytometry
is performed on peripheral blood, liver, lung, kidney, colon, and
stomach to measure the frequency of CAR+ T cells at these sites.
Animal survival is also carefully monitored and charted.
Example 10
Duration of Car-T Persistence and Multiple Car-T Infusions
[0126] If mice treated according to the study described in Example
3 fail to achieve a complete response to a single IP CAR-T
infusion, studies are done to determine the duration of CAR-T
persistence in IP tumors after a single IP infusion and to test the
therapeutic effect of multiple CAR-T infusions.
[0127] The duration of CAR-T persistence in IP tumors after a
single IP infusion is determined using the mouse model described in
Example 3. Using an optimal dose as determined in Example 3, ten
mice with established MC38 IP tumors are treated with an IP
infusion of the specific CAR-T. Tumors and ascites fluid are
analyzed by flow cytometry using a monoclonal antibody specific for
CAR at 1, 2, 4, 7, 14, and 21 days following treatment.
[0128] Based on the duration of CAR-T persistence and the effects
of the single dose of anti-CAR-T on tumor progression as determined
according to Example 3, a dosing schedule for multiple CAR-T
infusions is identified and used in the multiple infusion treatment
regimen.
[0129] If CAR-T persistence in IP tumors is particularly
short-lived (<2-3 days), a total body irradiation
preconditioning strategy is employed to promote CAR-T engraftment
in the host animal.
Example 11
IP Car-T Treatment with Chemotherapeutics
[0130] IP delivery of CAR-T to patients is conducted in compliance
with Good Clinical Practice guidelines. Patients first undergo a
diagnostic laparoscopy in the operating room for lysis of
peritoneal adhesions, disease assessment, acquisition of
pre-treatment biospecimens, and placement of a peritoneal dialysis
catheter. On postoperative Day 1, about 1.times.10.sup.10 CAR-T are
infused in 200 ml normal saline (NS) with 10% dimethyl sulfoxide
(DMSO). The infusion is carried out by manual injection at the
bedside over a 15 minute period with continuous vital sign
monitoring. Two additional CAR-T doses of 1.times.10.sup.10 cells
are given at 1-week intervals.
[0131] Six weeks following the first CAR-T dose, the patient is
returned to the operating room for a diagnostic laparoscopy to
assess disease response and to acquire post-treatment
biospecimens.
Example 12
IP Car-T Treatment with Chemotherapeutics
[0132] Effects of the chemotherapeutic agent cyclophosphamide on
therapeutic efficacy of CAR-T cells in the mouse model are studied
using methods similar to those described above. C57BL/6 mice are
injected intraperitoneally with 2.5.times.10.sup.6 tumor cells.
Seven days after this injection, the mice receive IP injections of
CAR-T cells generated as described in Example 1. Mice also receive
IP injections of cyclophosphamide. The cyclophosphamide is
administered 1 day prior to CAR-T infusion and then every 2 days
after CAR-T administration for a total of 4 doses of the antibody.
A control group of mice receive saline injection via the same
dosing schedule relative to the CAR-T infusion. Efficacy of each
treatment is measured by measuring bioluminescence and survival of
the mice.
[0133] While a number of exemplary aspects and embodiments have
been discussed above, those of skill in the art will recognize
certain modifications, permutations, additions and sub-combinations
thereof. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such modifications, permutations, additions and
sub-combinations as are within their true spirit and scope.
Sequence CWU 1
1
11213PRTartificialSynthetic 1Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Asp Val Gly Thr Ser 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr
Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg
Ser 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205 Asn Arg Gly Glu Cys 210
* * * * *