U.S. patent application number 16/597798 was filed with the patent office on 2020-05-07 for hepatic arterial infusion of car-t cells.
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 | 20200138863 16/597798 |
Document ID | / |
Family ID | 57126310 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200138863 |
Kind Code |
A1 |
Katz; Steven C. ; et
al. |
May 7, 2020 |
HEPATIC ARTERIAL INFUSION OF CAR-T CELLS
Abstract
Disclosed herein are compositions and methods for the treatment
of liver metastases in a subject. The methods include hepatic
arterial infusion (HAI) of chimeric antigen receptor modified T
cells (CAR-T) which are highly specific for tumor antigens such as
carcinoembryonic antigen (CEA). The HAI method is optimized to
maximize exposure of the modified cells to the metastatic cells
while minimizing exposure to healthy cells. The methods include
co-administration of a second therapeutic agent, such as IL-2.
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: |
57126310 |
Appl. No.: |
16/597798 |
Filed: |
October 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15099370 |
Apr 14, 2016 |
10471098 |
|
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16597798 |
|
|
|
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62147793 |
Apr 15, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/00 20130101;
C12N 2510/00 20130101; A61K 9/0019 20130101; A61K 35/17 20130101;
A61P 1/16 20180101; C07K 14/7051 20130101; C07K 2319/03 20130101;
A61P 35/04 20180101; A61P 37/04 20180101; A61K 38/2013 20130101;
C07K 2319/33 20130101; A61K 45/06 20130101; A61P 35/00 20180101;
C12N 5/0636 20130101; A61K 38/2013 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783; C07K 14/725 20060101
C07K014/725; C07K 14/00 20060101 C07K014/00; A61K 9/00 20060101
A61K009/00; A61K 38/20 20060101 A61K038/20; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT INTEREST
[0002] This invention was made with Government support under
contract K08CA160662 awarded by the National Institutes of Health.
The government has certain rights in this invention.
Claims
1. A method for treating a liver metastasis in a subject,
comprising: infusing into the hepatic artery of the subject a
composition comprising a chimeric antigen receptor T cell (CAR-T
cell) which expresses a chimeric antigen receptor protein, wherein
the chimeric antigen receptor protein binds to an antigen expressed
on metastatic cells in the liver.
2. The method according to claim 1, further comprising performing
angiography to mapthe hepatic artery and nearby vessels prior to
the infusing step.
3. The method according to claim 2, further comprising occluding
vessels which do not feed into the liver.
4. The method according to claim 1, further comprising performing
angiography during the infusing step, wherein the angiography
monitors intrahepatic hemodynamic integrity during the infusing
step.
5. The method according to any one of the preceding claims, wherein
the chimeric antigen receptor protein specifically binds
carcinoembryonic antigen (CEA) expressed on the surface of the
metastatic cells.
6. The method according to claim 1, wherein the chimeric antigen
receptor protein comprises SEQ ID NO: 1.
7. The method according to claim 1, further comprising
administering a second therapeutic agent into the hepatic artery of
the subject.
8. The method according to claim 7, wherein the second therapeutic
agent is IL-2.
9. The method according to claim 8, wherein the administering the
second therapeutic agentis performed before, during or after the
infusion of the composition comprising the immunoresponsive
cell.
10. The method according to claim 1, wherein the composition
comprising the CAR-T cells is infused into the hepatic artery of
the subject once every 1 week, once every 2 weeks, once every 3
weeks, or once every 4 weeks.
11. The method according to claim 1, wherein the infusion into the
hepatic artery of the subject the composition comprising the CAR-T
cells comprises infusing 10.sup.8 to 10.sup.10 CAR-T cells into the
hepatic artery.
12. The method according to claim 1, wherein the infusion the
composition results in a 40% to 50% decrease in serum CEA as
compared to the level of CEA in the serum prior to the
infusion.
13. A method for treating a liver metastasis in a subject,
comprising: infusing into the hepatic artery of the subject a
composition comprising a CAR-T cell which expresses a chimeric
antigen receptor protein (CAR), wherein the CAR protein binds to
CEA.
14. The method according to claim 13, wherein the chimeric antigen
receptor protein comprises SEQ ID NO:1.
15. The method according to claim 14, wherein the CAR comprises in
an N-terminal to C-terminal direction: an scFv which binds CEA, a
CD8 hinge domain, a CD28 extracellular domain, a CD28 transmembrane
domain, a CD28 cytoplasmic domain, and a CD3 zeta cytoplasmic
domain.
16. The method according to claim 15, wherein the infusing
comprises infusing 10.sup.8 to 10.sup.10 CAR-T cells into the
hepatic artery.
17. The method according to claim 15, further comprising
administering a composition comprising a second therapeutic agent
to the subject.
18. The method according to claim 17, wherein the second
therapeutic agent is IL-2 oryttrium-90.
19. A method for decreasing CEA in the serum of a subject diagnosed
with liver metastases, wherein the method comprises infusing into
the hepatic artery of the subject a composition comprising a
chimeric antigen receptor T cell (CAR-T cell) which expresses a
chimeric antigen receptor protein, wherein the chimeric antigen
receptor protein binds to an antigen expressed on metastatic cells
in the liver.
20. The method according to claim 19, wherein the chimeric antigen
receptor protein binds CEA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/099,370, filed Apr. 14, 2016, which claims the benefit of
U.S. Provisional Application No. 62/147,793, filed Apr. 15, 2015,
the contents of which are incorporated by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING
[0003] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Apr. 14, 2016, and named
"096201-0261.txt" (12,553 bytes), the contents of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0004] The subject matter described herein relates to a method for
treating liver-associated cancers and metastases via hepatic artery
infusion of genetically modified or chimeric antigen receptor T
cells (e.g., CAR-T) expressing a receptor protein which binds a
tumor-specific antigen and which activates activities of the
modified T-cells.
BACKGROUND
[0005] Liver metastases (LM) are cancerous tumors that have
metastasized from another part of the body to the liver. Most cases
of liver metastases develop from colon or rectal cancers, with
approximately 60 to 70 percent of people with colorectal cancer
eventually developing a liver tumor. Liver metastases are a
significant cause of morbidity and mortality in patients with
gastrointestinal adenocarcinoma. While hepatic resection has been
considered the standard of care for patients who have resectable
hepatic metastases, many patients are not candidates for resection
of liver metastases. Chemotherapy is not curative for liver
metastases, creating a large unmet clinical need.
[0006] Tumor infiltrating lymphocyte (TIL) studies have revealed
that host T cell responses to LM are significant correlates of
patient survival (Katz et al., 2013, Ann Surg Oncol, 20:946-955;
Katz et al., 2010, HPB (Oxford), 12:674-683; Katz et al., Ann Surg
Oncol, 16:2524-2530; Wagner et al., 2008, Ann Surg Oncol,
15:2310-2317; Turcotte et al., Canc Immunol Res, 2:530-537). While
those who mount effective immune responses to LM tend to have
prolonged survival, the vast majority of patients succumb to their
disease in the context of endogenous immune failure. The
immunosuppressive nature of the intrahepatic milieu (Cantor et al.,
1967, Nature, 215:744-745; Katz et al., 2005, Hepatol, 42:293-300;
Katz et al., 2004, J Immunol, 173:230-235; Katz et al., 2011, J
Immunol, 187:1150-1156) may promote the development of LM and
contribute to aggressive disease biology.
[0007] Accordingly, there is a need for therapeutic strategies
which can facilitate host or provide immunological responses to the
presence of liver metastases. Given the favorable effects of robust
liver TIL responses and the inherent suppressive nature of the
intrahepatic space, delivery of highly specific immunoresponsive
cells for the treatment of LM is a rational approach. Described
herein are compositions and methods for hepatic artery infusion
(HAI) of anti-CEA CAR-Ts which can both limit extrahepatic toxicity
and optimize efficacy for treatment of liver metastases.
BRIEF SUMMARY
[0008] The following aspects and embodiments thereof described and
illustrated below are meant to be exemplary and illustrative, not
limiting in scope.
[0009] In one aspect, a method of treating liver metastases in a
subject diagnosed with having liver metastases is provided,
comprising infusing into the hepatic artery of the subject a
composition comprising an immunoresponsive cell which expresses a
chimeric antigen T cell receptor protein (CAR), wherein the
chimeric T cell receptor protein binds to an antigen expressed on
metastatic cells in the liver.
[0010] In some embodiments, the immunoresponsive cell expressing
the CAR is selected from the group consisting of a T cell, a
hematopoietic stem cell, a natural killer cell, anatural killer T
cell, a B cell and a cell of monocytic lineage. In a particular
embodiment, the immunoresponsive cell is a T cell.
[0011] In some embodiments, the immunoresponsive cell is autologous
to the subject. In another embodiment, the immunoresponsive cell is
not autologous to the subject.
[0012] In some embodiments, the immunoresponsive cell is a T cell
and the method comprises harvesting cells from the blood serum of
the subject. In other embodiments, a minimum of 10.sup.8, 10.sup.9
or 10.sup.10 cells are harvested from the blood serum of the
subject. In still other embodiments, the method further comprises
isolating and activating peripheral blood mononuclear cells (PBMC)
from the harvested cells to generate a population of autologous T
cells.
[0013] In some embodiments, the method comprises transfecting the
immunoresponsive cells with a nucleic acid vector which comprises a
nucleic acid sequence encoding the CAR sequence to generate a
population of immunoresponsive cells which expresses the CAR
protein. In other embodiments, the method further comprises
selecting and expanding the population of immunoresponsive cells
which expresses the CAR protein. In some embodiments, the
immunoresponsive cells are the population of autologous T
cells.
[0014] In some embodiments, the method comprises infusing a dose of
the immunoresponsive cells which express the CAR protein into the
patient over a treatment period ranging from about 1 to 8 weeks, 2
to 8 weeks, 2 to 6 weeks, 2 to 4 weeks, 1 to 4 weeks, 1 to 3 weeks,
1 to 2 weeks, 3 to 6 weeks, or 4 to 6 weeks. In other embodiments,
the infusing the immunoresponsive cells which express the CAR
comprises infusing the cells every week, 2 weeks, 3 weeks or 4
weeks over the treatment period. In a preferred embodiment, the
infusion the immunoresponsive cells which express the CAR comprises
infusing CEA CAR-T cells once per week for 3 weeks.
[0015] In some embodiments, the immunoresponsive cells which
express the CAR (CAR-T cells) and the CAR binds to CEA are
autologous T cells. In an alternative embodiment, the
immunoresponsive cells which express the CAR (CAR-T cells) and the
CAR binds to CEA are nonautologous T cells.
[0016] In some embodiments, the dose of immunoresponsive cells
infused into the patient is about 10.sup.7-10.sup.10 or
10.sup.8-10.sup.9 CAR-T cells. In other embodiments, the dose of
immunoresponsive cells infused into the patient is about 10.sup.7,
10.sup.8, 10.sup.9 or 10.sup.10 immunoresponsive cells. In a
preferred embodiment, the immunoresponsive cells are T cells and
the CAR binds to CEA.
[0017] In some embodiments, the method comprises infusing a
composition comprising the immunoresponsive cells and a
pharmaceutically compatible solution, wherein the total volume of
the composition ranges from about 25 ml to 125 ml, 50 ml to 75 ml,
75 ml to 100 ml, or 50 ml to 100 ml. In a preferred embodiment, the
immunoresponsive cells are T cells and the CAR binds to CEA.
[0018] In some embodiments, the composition is administered to the
hepatic artery by a surgical technique. In other embodiments, the
composition is administered to the hepatic artery by a percutaneous
technique. In still other embodiments, administering by the
percutaneous technique is preceded embolization of the
gastroduodenal artery and/or gastric artery.
[0019] In some embodiments, the method further comprises using
angiography to confirm intrahepatic hemodynamic integrity during
the infusion process.
[0020] In some embodiments, the method comprises infusing a
composition comprising the immunoresponsive cells and a
pharmaceutically compatible solution via a percutaneous catheter
and performing liver volumetric calculations to divide the hepatic
arterial dosing to reflect aberrant anatomical considerations.
[0021] In some embodiments, the method further comprises infusing a
second therapeutic agent into the hepatic artery of the subject. In
other embodiments, the second therapeutic agent is interleukin-2
(IL-2). In other embodiments, the second therapeutic agent inhibits
suppression of the immunoresponsive cell in the subject as compared
to suppression of the immunoresponsive cell in a patient not
administered IL-2.
[0022] In some embodiments, the method results in a 15-50%, 20-50%,
30-50%, 40-50%, or 19 to 48% decrease in serum CEA as compared to
CEA levels prior to the administration of the IL-2 to the subject.
In another embodiment, the decrease in serum CEA occurs with 1, 2,
3, 4 or 5 days after the infusion.
[0023] In some embodiments, the IL-2 is administered in a
continuous systemic dose ranging from about 25,000 to 150,000
IU/kg/day, 25,000 to 75,000 IU/kg/day, 50,000 to 100,000 IU/kg/day
for the duration of the CAR-T treatment period. In other
embodiments, the IL-2 is administered in a continuous systemic dose
of about 25,000, 35,000, 40,000, 50,000, 60,000, 75,000, 85,000 or
100,000 IU/kg/day. In a preferred embodiment, the IL-2 is
administered in a continuous systemic dose of about 50,000
IU/kg/day.
[0024] In some embodiments, the infusing the second therapeutic
agent is performed before, during or after the infusion of the
immunoresponsive cell which expresses a chimeric T cell receptor
protein.
[0025] In some embodiments, the method further comprises
administering to the subject radiation therapy into the hepatic
artery of the subject. In other embodiments, the radiation therapy
comprises administration of a plurality of microspheres containing
yttrium-90 (.sup.90Y). In still other embodiments, the
administering radiation therapy comprises administering about 1 to
4 gigabecquerels (GBq), 1 to 3 GBq, 2 to 4 GBq, 3 to 4 GBq, or 2 to
3 GBq of radioactivity. In yet other embodiments, the administering
radiation therapy comprises administering about 1 GBq, 1.5 GBq, 2
GBq, 2.5 GBq, 3 GBq, 3.5 GBq, or 4 GBq of radioactivity.
[0026] In some embodiments, the administering the radiation therapy
comprising administering the radiation therapy about 1 week, 2
weeks, or 3 weeks after the last of the CAR-T infusions.
[0027] In some embodiments, the subject is diagnosed with a
metastatic disease localized to the liver. In other embodiments,
the metastatic disease is a cancer. In still other embodiments, the
cancer metastasized from a primary tumor in the breast, colon,
rectum, esophagus, lung, pancreas and/or stomach. In still other
embodiments, the subject is diagnosed with unresectable metastatic
liver tumors. In yet other embodiments, the subject is diagnosed
with unresectable metastatic liver tumors from primary colorectal
cancer. In some embodiments, the subject is diagnosed with
hepatocellular carcinoma.
[0028] In some embodiments, the subject is diagnosed with a liver
metastases, wherein the malignant cells of the liver metastases
have been demonstrated to express the carcinoembryonic antigen
(CEA) protein.
[0029] In some embodiments, the method results in a decrease in
tumor burden in the liver of the subject. In other embodiments, the
decrease in tumor burden is measured using positron emission
tomography (PET), magnetic resonance imaging (MRI) or biopsy. In
still other embodiments, wherein the tumor burden is measured 1-8
weeks, or about 1, 2, 3, 4, 5, 6, 7 or 8 weeks after the treatment
period. In yet other embodiments, the decrease is measured relative
to the tumor burden prior to infusing the dose of CAR-T cells.
[0030] In some embodiments, the tumor burden measured 1-8 weeks, or
about 1, 2, 3, 4, 5, 6, 7 or 8 weeks after the treatment period is
no more than 50% to 90% of the tumor burden prior to the infusion
of the first dose of CAR-T cells.
[0031] In some embodiments, a method for decreasing the tumor
burden in a subject diagnosed with liver metastases is provided
comprising administering to the subject a CAR-T cells as described
herein. In other embodiments, the tumor burden is decreased to an
amount that is less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or 90% of the tumor burden prior to the administering the CAR-T
cells to the subject.
[0032] In some embodiments, a method for decreasing amounts of CEA
in the blood serum of a patient diagnosed with liver metastases is
provided comprising administering to the subject a CAR-T cells as
described herein. In other embodiments, the amount of CEA is
decreased to an amount which is less than about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80% or 90% of the amount of CEA in the subject prior
to administering the CAR-T cells.
[0033] In some embodiments, the chimeric T cell receptor protein
comprises an extracellular domain which specifically binds to a
tumor antigen expressed on the surface of the metastatic cells in
the liver. In other embodiments, the chimeric T cell receptor
protein comprises an extracellular domain which specifically binds
to the carcinoembryonic antigen (CEA) protein.
[0034] In some embodiments, the chimeric T cell receptor protein
comprises, in an N-terminal to C-terminal direction, a CEA-binding
IgG immunoglobulin domain, a CD8 hinge domain, a CD28 extracellular
domain, a CD28 transmembrane domain, a CD28 cytoplasmic domain and
a CD3 zeta cytoplasmic domain.
[0035] In some embodiments, the CEA-binding IgG immunoglobulin
domain comprises SEQ ID NO:1.
[0036] In some embodiments, the CD8 hinge region comprises a
sequence which is 12 amino acids in length and which is at least
75%, 83%, 91%, or 100% identical to the sequence of residues
169-180 of SEQ ID NO:2.
[0037] In some embodiments the CD28 extracellular domain comprises
a sequence which is 40 amino acids in length and which is at least
92%, 95%, 97%, or 100% identical to the sequence of residues
113-152 of SEQ ID NO:4.
[0038] In some embodiments the CD28 transmembrane domain comprises
a sequence which is 27 amino acids in length and which is at least
88%, 92%, 96%, or 100% identical to the sequence of residues
153-179 of SEQ ID NO:4.
[0039] In some embodiments the CD28 signaling domain comprises a
sequence which is 41 amino acids in length and which is at least
90%, 92%, 95%, 97%, or 100% identical to the sequence of residues
180-220 of SEQ ID NO:4.
[0040] In some embodiments, the zeta cytoplasmic domain comprises a
sequence which is 113 amino acids in length and which is at least
90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of
residues 52-164 of SEQ ID NO:3.
[0041] In some embodiments, the chimeric T cell receptor protein
further comprises a signal sequence at the N-terminus of the T cell
receptor protein. In other embodiments, the signal peptide is at
least 84%, 89%, 94% or 100% identical to SEQ ID NO:6.
[0042] In some embodiments, the method is HITM.RTM. HEPATIC
IMMUNOTHERAPY FOR METASTASES.
BRIEF DESCRIPTION OF DRAWINGS
[0043] FIG. 1 illustrates mean percentages of CD3+ and CAR+ cells
in patients prior to infusion of modified immunoresponsive
cells.
[0044] FIG. 2 illustrates results of an assay to test patient
product killing of CEA+ target cells.
[0045] FIG. 3 illustrates a phase 1 clinical trial protocol to test
therapeutic efficacy of modified immunoresponsive cells as
described herein.
[0046] FIG. 4 illustrates serum CEA levels in patients treated
according to methods described herein.
[0047] FIGS. 5A-5B illustrate LM fibrosis (FIG. 5A) and LM necrosis
(FIG. 5B) in patients treated according to methods described
herein.
[0048] FIG. 6 illustrates IFN.gamma. levels in patients treated
according to methods described herein.
[0049] FIG. 7 illustrates peak and mean IFN.gamma. levels in
patients treated according to methods described herein.
DETAILED DESCRIPTION
[0050] 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
[0051] 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.
[0052] 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 mL to 8 mL is stated, it is intended that 2 mL, 3 mL, 4 mL, 5
mL, 6 mL, and 7 mL are also explicitly disclosed, as well as the
range of values greater than or equal to 1 mL and the range of
values less than or equal to 8 mL.
[0053] 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.
[0054] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
[0055] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject. The term
"transfected" or "transformed" or "transduced" as used herein
refers to a process by which exogenous nucleic acid is transferred
or introduced into the host cell. A "transfected" or "transformed"
or "transduced" cell is one which has been transfected, transformed
or transduced with exogenous nucleic acid. The cell includes the
primary subject cell and its progeny.
[0056] 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.
[0057] 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.
[0058] 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.
[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).
II. HEPATIC ARTERY INFUSIONS OF CAR-T CELLS
[0060] Studies have demonstrated that liver metastases (LM)
patients with robust T cell responses have significantly improved
outcomes, however, most LM patients fail to mount effective
intrahepatic anti-tumor immunity (Katz et al., 2003, Ann Surg Onc,
20:946-955). Chimeric antigen receptor modified T cells (CAR-T),
highly specific immunotherapeutic products designed to target
specific tumor antigens, hold promise in providing needed
anti-tumor immunity, especially in patients diagnosed with
unresectable tumors. While administration of antigen-specific CAR-T
cells have demonstrated encouraging results in early-phase clinical
trials for leukemia, successful adaptation of CAR-T technology for
CEA-expressing adenocarcinoma LM, a major cause of death in
patients with gastrointestinal cancers, has yet to be achieved.
Accordingly, a phase I clinical trial was designed and conducted as
described herein to show that CAR-T technology is a viable option
for the treatment of LM. Moreover, the clinical studies address
inefficient intrahepatic delivery of CAR-T via systemic infusion
which can limit the effectiveness of CAR-T treatments forLM.
Described below are studies to test CAR-T hepatic artery infusions
(HAI) to show that direct regional delivery of CAR-T to LM is safe
and effective in treating LM. In summary, the data demonstrate that
CAR-T HAIs are well tolerated and associated with evidence of tumor
cell killing, showing that the hepatic artery infusion (HAI) of
CAR-T to LM can indeed effectively treat LM in patients in need
thereof.
Chimeric Antigen Receptor T Cells
[0061] T cells engineered with chimeric antigen receptors (CAR) 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). Reprogramming T cells with CAR genes provides an
MHC-independent mechanism for docking with and lysing tumor cells.
Such modified T cells have been alternatively termed "designer T
cells," "T-bodies," or "CAR-T cells" (Ma et al., 2002, Cancer
Chemotherapy & Biological Response Modifiers: Elsevier Science,
pp. 319-345; Park et al., 2011, Trends Biotech, 29:550-557; Ma et
al., 2014, Prostate, 74:286-296). Carcinoembryonic antigen (CEA) is
an attractive target for CAR-T treatment of adenocarcinoma LM given
high levels of CEA expression and the ability to measure CEA in
serum (Blumenthal et al., 2007, BMC Cancer, 7:2; Midiri et al.,
1985, Cancer, 55:2624-2629). Upon antigen recognition, anti-CEA
CAR-Ts proliferate, produce cytokines, and kill target cells
(Emtage et al., Clin Canc Res, 14:8112-8122).
[0062] Generation of chimeric antigen receptor (CAR) proteins and
immune cells (e.g., immunoresponsive or T 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 Discovery, 3:388-398. The
chimeric antigen receptor protein generally 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.
[0063] In a preferred embodiment, the target binding domain of the
CAR protein binds to the CEA protein. This CEA-binding protein was
generated from a humanized monoclonal antibody (U.S. Pat. No.
6,676,924; Akamatsu et al., 1998, Clin Cancer Res, 4:2825-2832;
Nolan et al., 1999, Clin Cancer Res, 5:3928-3941). In generating
the anti-CEA CAR construct, the scFV construct was generated from
the heavy and light chain variable domains using methods routine in
the art and then the scFV fragment (disclosed herein as SEQ ID
NO:1) was fused to other receptor domains to generate the CAR for
use in the treatment methods presently described.
[0064] Preferred embodiments for each of the hinge domain,
transmembrane domain and signaling domain(s) are provided in Table
1 below. In some embodiments, the CAR construct comprises in an
N-terminal to C-terminal direction a CEA-binding domain, a CD8
hinge domain, a CD3 zeta chain transmembrane domain, and a CD3 zeta
chain cytoplasmic domain. In preferred embodiments, the CAR
construct comprises in an N-terminal to C-terminal direction a
CEA-binding domain, a CD8 hinge domain, a CD28 extracellular
domain, a CD28 transmembrane domain, a CD28 signaling domain and a
CD3 zeta chain cytoplasmic domain. In some embodiments, the
construct further comprises a signal peptide fused to the
N-terminus of the target binding domain. It is understood that the
signal peptide is not present in the CAR protein expressed on the
administered immunoresponsive cells as it has been cleaved in
vivo.
TABLE-US-00001 TABLE 1 Parent Sequence Sequence CAR Domain CD8
MALPVTALLLPLALLLHAARPSQFRVSP CD8 Hinge domain (SwissProt/GenBankA
LDRTWNLGETVELKCQVLLSNPTSGCS (bold, underlined cc. No. P01732;
WLFQPRGAAASPTFLLYLSQNKPKAAEG sequence represents a SEQ ID NO: 2)
LDTQRFSGKRLGDTFVLTLSDFRRENEG preferred hinge domain)
YYFCSALSNSIMYFSHFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNHRNRRRVCKCPRPVVKS
GDKPSLSARYV CD3 Zeta chain MKWKALFTAAILQAQLPITEAQSFGLLD CD3 Zeta
domain (bold, (SwissProt/GenBank PKLCYLLDGILFIYGVILTALFLRVKFSRSA
underlined sequence Acc. No. P20963; DAPAYQQGQNQLYNELNLGRREEYDV
represents a preferred SEQ ID NO: 3) LDKRRGRDPEMGGKPQRRKNPQEGL zeta
chain cytoplasmic YNELQKDKMAEAYSEIGMKGERRRG domain; italics
KGHDGLYQGLSTATKDTYDALHMQA represents a preferred LPPR transmembrane
domain) CD28 MLRLLLALNLFPSIQVTGNKILVKQSPML CD28 TM domain (bold,
(SwissProt/GenBank VAYDNAVNLSCKYSYNLFSREFRASLHK underlined
represents a Acc. No. P10747; GLDSAVEVCVVYGNYSQQLQVYSKTGF preferred
extracellular SEQ ID NO: 4) NCDGKLGNESVTFYLQNLYVNQTDIYFC domain;
italics KIEVMYPPPYLDNEKSNGTIIHVKGKH represents a preferred
LCPSPLFPGPSKPFWVLVVVGGVLACYSL transmembrane domain;
LVTVAFIIFWVRSKRSRLLHSDYMNMTPR underline only represents
RPGPTRKHYQPYAPPRDFAAYRS a preferred signaling domain)
[0065] 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. The antigen binding domain can be any
domain that binds to the cell surface antigen including but not
limited to ligands to the receptor or immunoglobulin proteins such
as monoclonal antibodies, polyclonal antibodies, synthetic
antibodies, human antibodies, humanized antibodies, and fragments
thereof. In preferred embodiments, the antigen-binding domain of
the CAR is constructed from the variable domains 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 an
Fab fragment 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.
[0066] 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 an amino acid sequence which is at
least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
1 shown below:
TABLE-US-00002 (SEQ ID NO: 1)
DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYW
TSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0067] In some embodiments, the CEA CAR-T construct further
comprises a signal peptide 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. Signal sequences are well known to the ordinarily
skill artisan and functions to direct the translated protein to the
cell surface. The signal sequence is cleaved after passage from the
endoplasmic reticulum during translocation of the CAR protein to
the cell surface. In other embodiments, the signal peptide has the
sequence MGWSCIILFLVATATGVHS (SEQ ID NO:6). In still other
embodiments, a linkersequence comprising 1, 2, 3, 4, 5 or 6 amino
acids is present between the signal peptide and the CEA-binding
domain.
[0068] The Fab or scFv domain can then be fused at its C-terminus
via a peptide bond to a hinge domain such as that from the CD8
hinge domain (see SwissProt/GenBank Acc. No. P01732; SEQ ID NO:2).
In a preferred embodiment, the hinge domain comprises a sequence
which is 12 amino acids in length and is at least 83%, 91% or 100%
identical to the sequence of residues 169-180 of SEQ ID NO:2. In
other embodiments, the hinge domain comprises a sequence which is
at least 95%, 96%, 97%, 98%, 99% or 100% identical to a continguous
sequence of 10-20, 10-30, 10-40 or 10-50 residues present in
residues 111-190 of SEQ ID NO:2.
[0069] The hinge domain can then be fused at its C-terminus to a
transmembrane domain. In some embodiments, the transmembrane domain
comprises a sequence which is from the CD3 zeta chain
(GenBank/SwissProt Acc. No. P20963; SEQ ID NO:3). In this
embodiment, the transmembrane domain comprises a sequence which is
20-30 amino acids or 20, 21, 22, 23 amino acids in length and is at
least 85%, 90%, 95% or 100% identical to a contiguous sequence of
15-25 or 20-30 residues present in residues 31-51 of SEQ ID NO:3.
In some embodiments, the transmembrane domain from the CD3 zeta
chain comprises the sequence of amino acids at positions 31-51 of
SEQ ID NO:3. In a preferred embodiment, the transmembrane domain of
the CAR is from the CD28 protein (e.g., GenBank/SwissProt Acc. No.
P10747; SEQ ID NO:4). The transmembrane domain can comprise a
sequence which is 27 amino acids in length and is at least 88%, 92%
or 100% identical to the sequence of residues 153-179 of SEQ ID
NO:4. In other embodiments, the transmembrane domain comprises a
sequence which is at least 95%, 96%, 97%, 98%, 99% or 100%
identical to a contiguous sequence of 20-30, 20-40, or 20-50
residues present in the sequence of residues 150-190 of SEQ ID
NO:4. When the CAR transmembrane domain as is a CD28 transmembrane
domain as described above, the CAR can further comprise a CD28
extracellular domain, wherein the extracellular domain is
positioned between the CD8 hinge domain and the CD28 transmembrane
domain. The CD28 extracellular domain is 35-45, or about 38, 39,
40, 41, or 42 amino acids in length and is at least 92%, 95%, 97%
or 100% identical to a contiguous sequence of 35-45 residues
present in the sequence of residues 110-160 of SEQ ID NO:4.
[0070] The CD3 zeta chain cytoplasmic domain is present at the
C-terminus of the CAR construct and comprises a sequence which is
at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identical to a
contiguous sequence of 100-120 amino acids present in the sequence
of residues 40-164 of SEQ ID NO:3. In a preferred embodiment, the
zeta chain domain comprises a sequence which is at least 97%, 98%,
99% or 100% identical to residues 52-164 of SEQ ID NO:3.
[0071] In a preferred embodiment, the CAR further comprises a CD28
signaling domain C-terminal to the transmembrane domain and
N-terminal to the CD3 zeta chain cytoplasmic domain. In some
embodiments, the CD28 signaling domain comprises a sequence which
is at least 92%, 95% or 100% identical to a contiguous sequence of
35-45 amino acids present in the sequence of residues 175-220 of
SEQ ID NO:4. In a preferred embodiment, the CD28 signaling domain
comprises a sequence which is at least 92%, 95% or 100% identical
to residues 180-220 of SEQ ID NO:4.
[0072] In a preferred embodiment, the CAR polypeptide sequence
comprises in an N-terminal to C-terminal direction: a CEA-binding
domain comprising SEQ ID NO:1, a CD8 hinge domain as described
above, a CD28 extracellular domain as described above, a CD28
transmembrane domain as described above, a CD28 signaling domain as
described above, and a CD3 zeta cytoplasmic domain as described
above. In one embodiment, this CAR sequence comprises in an
N-terminal to C-terminal domain each of the following segments: SEQ
ID NO:1 (CEA binding domain), residues 169-180 of SEQ ID NO:2 (CD8
hinge), residues 113-152 of SEQ ID NO:4 (CD28 extracellular
domain), residues 153-179 of SEQ ID NO:4 (CD28 transmembrane
domain), residues 180-220 of SEQ ID NO:4 (CD28 cytoplasmic domain),
and residues 52-164 of SEQ ID NO:3 (CD3 zeta chain cytoplasmic
domain) (alternatively referred to herein as "anti-CEA
scfv-CD8.alpha.-CD28/CD3.zeta."). Construction of the CAR construct
using routine methods can involve the use of PCR amplification of
full-length gene sequences with introduction of restriction
endonuclease sites that allow digestion and ligation of the various
domains to generate the desired fusion construct, but which also my
encode one or more amino acids between each of the domains of the
chimeric construct. Accordingly, in some embodiments, the CAR
sequence comprises a linker sequence of 1, 2 or 3 amino acids
between the binding and CD8 hinge domains, between the hinge and
CD28 extracellular domains, between the extracellular domain and
the transmembrane domains, between the transmembrane and signaling
domains, between the transmembrane and zeta cytoplasmic domains,
and/or between the CD28 signaling and zeta cytoplasmic domains.
Preparation of CAR-T Cells
[0073] Prior to expansion and genetic modification, a source of T
cells is obtained from a subject in need of treatment for liver
metastases. T cells can be obtained from a number of sources,
including peripheral blood mononuclear cells, bone marrow, lymph
node tissue, cord blood, thymus tissue, tissue from a site of
infection, ascites, pleural effusion, spleen tissue, and tumors. In
certain aspects of the present disclosure, any number of T cell
lines available in the art may be used. T cells can be obtained
from a unit of blood collected from a subject using any number of
techniques known to the skilled artisan, such as Ficoll.TM.
separation. In a preferred embodiment, cells from the circulating
blood of an individual are obtained by apheresis. The apheresis
product typically contains lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood
cells, red blood cells, and platelets. The cells collected by
apheresis can be washed to remove the plasma fraction and to place
the cells in an appropriate buffer or media for subsequent
processing steps. As those of ordinary skill in the art would
readily appreciate a washing step may be accomplished by methods
known to those in the art, such as by using a semi-automated
flow-through centrifuge according to the manufacturer's
instructions. After washing, the cells may be resuspended in a
variety of biocompatible buffers, such as, for example, Ca-free,
Mg-free PBS, or other saline solution with or without buffer.
Alternatively, the undesirable components of the apheresis sample
may be removed and the cells directly resuspended in culture
media.
[0074] A specific subpopulation of T cells, such as CD3+, CD28+,
CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be isolated and/or
enriched by positive or negative selection techniques. For example,
in some embodiments, T cells are isolated by incubation with
anti-CD3/anti-CD28-conjugated beads, for a time period sufficient
for positive selection of the desired T cells. The skilled artisan
would recognize that multiple rounds of selection can also be used
in the context of this disclosure. In certain aspects, it may be
desirable to perform the selection procedure and use the unselected
cells in the activation and expansion process. Unselected cells can
also be subjected to further rounds of selection.
[0075] In some embodiments, a T cell population can be selected
that expresses one or more of IFN-.gamma., TNF.alpha., IL-17A,
IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin,
or other appropriate molecules, e.g., other cytokines. Methods for
screening for cell expression can be determined, e.g., by the
methods described in PCT Publication No.: WO 2013/126712. T cells
may be activated and expanded generally using methods as described,
for example, in U.S. Pat. Nos. 6,352,694; 7,232,566; 6,797,514;
6,867,041; and U.S. Patent Application Publication No.
20060121005.
[0076] For the preparation of CAR-T cells for use in treating
subjects diagnosed with liver metastases, patients undergo
leukapheresis to harvest a minimum of 4.times.10.sup.9 T cells,
with an ideal target of about 6.times.10.sup.9 T cells. The initial
assessment of T cell numbers in the leukapheresis product can be
performed at 2 hours and repeated thereafter as deemed necessary.
Cells are purified to retrieve peripheral blood mononuclear cells
(PBMC), alternatively referred to herein as lymphocyte-rich (PBL).
The lymphocytes are activated by exposure to 50 ng/ml OKT3 and 3000
IU/mL IL-2 (Walker et al., 1993, 4:659-680). The activated cells
are transduced with high titer supernatant of retrovirus containing
recombinant chimeric CAR as described above with 10 .mu.g/mL
protamine (Cornetta et al., 1989, 23:187-194). On the following
day, the procedure is repeated to increase the fraction of
transduced cells. Two days following transduction a small aliquot
of the cells is analyzed by flow cytometry for expression of the
transgene. If the fraction of transduced cells is less than 10%,
the cells can be allowed to undergo two more rounds of transduction
and again analyzed by flow cytometry for percentage of transduced
cells. When adequate numbers of T cells have been transduced (e.g.,
>10%) the cells are cultured, e.g., at 1.2-1.5.times.10.sup.6/mL
under activation conditions (above). The remainder of activated
cells is maintained until the transduction results are known, and
used for a second attempt if necessary. The transduced T cells are
expanded in culture and monitored for growth/transduction
parameters (doubling time, total cell numbers, % transduction, T
cell activation indicators). Prior to freeze storage of the cells,
10% DMSO, 20% human serum and 3000 IU/ml of IL-2 is added. The
decision on when to harvest cells is based on the presence of
sufficient total cells, such that enough cells will remain in
culture after harvest to be expanded for future infusions.
Typically, the interval from T cell activation to dose harvesting
ranges from 2-3 weeks. During expansion, flow cytometry is
performed to document the presence of T cells expressing chimeric
receptor. Other tests will include viability, sterility, and
standard cytotoxicity assays against CEA+ and CEA-targets. Up to
three attempts are made with patient T cells to achieve adequate
transduction efficiencies and cell numbers to constitute a dose. In
some embodiments, cells expanded from different transductions are
pooled to achieve the dose.
[0077] Therapeutic CAR-T cells can be engineered to express CAR
nucleic acid constructs by transfecting a population of lymphocytes
with an expression vector encoding the CAR construct. Appropriate
means for preparing a transduced population of lymphocytes
expressing a selected CAR construct are well known to the skilled
artisan, and include but are not limited to retrovirus, MFG
vectors, adenovirus-based vectors, adeno-associated virus
(AAV)-based vectors, retroviral vectors, retroviral-adenoviral
vectors, and vectors derived from herpes simplex viruses (HSVs).
Example 1 below describes the method used to generate the CAR-T
cells administered to the patients.
[0078] In a preferred embodiment, the T cells are transfected with
a retrovirus harboring the desired CAR construct. The MFG
retrovirus and its use in transfecting eukaryotic cells are
well-known to the person having ordinary skill in the art. For
generation of the CAR-T cells, an expression cassette encoding the
CEA CAR can be inserted between the NcoI-BamHI sites of an MFG
retroviral vector backbone. The initiation codon of the inserted
sequences is located precisely at the position of the viral env
initiation codon. The MFG vector, referred to as MPSV/PBSQ, can be
used in which the MoMLV LTRs have been replaced with the homologous
sequences from the myeloproliferative virus, and the 5' primer
binding site has been replaced with the homologous sequences from a
variant that utilizes tRNAglu rather than tRNApro as a positive
strand primer. In some embodiments, the retroviral vector does not
contain a selectable marker gene. Retroviral vector supernatant
from the transfected cells can be produced using the PG13 packaging
cell line. The PG13 cell line was produced by stably introducing a
gibbon ape leukemia virus (GALV) helper packaging system into mouse
3T3 cells. The vector producer cell (VPC) line is made by a
two-step process. First, the CAR-MFG vector is transfected into the
GP+E86 ecotropic packaging cell line. Transient viral supernatant
from the transfected cells is collected and used to infect PG13
cells. After infection, the PG13 cells are assayed for expression
of the CAR transgene by flow cytometry. The cells are then sorted
for stable expression of the CAR transgene by FACS to establish a
master working cell bank, and are tested for safety, sterility,
identity and absence of replication competent retrovirus (RCR). The
transduced cells are frozen into quantities to make up the patient
dose. In some embodiments, the appropriate quantity of CAR-T cells
is stored in liquid nitrogen vapor phase in one bag containing
about 50-100 mL, 75-100 mL, 75-125 mL, or about 50 mL, 75 mL, 100
mL, or 135 mL of a solution which is isotonic and pharmaceutically
acceptable. In some embodiments, the solution contains about
15-25%, 15%, 20% or 25% albumin. In other embodiments, the solution
contains about 5-15%, 5%, 10% or 15% dimethyl sulfoxide (DMSO). In
a preferred embodiment, the solution volume is about 100 mL and
contains the appropriate number of cells, about 20% albumin and
about 10% DMSO. In some embodiments, about 400,000-500,000 IU or
about 450,000 IU IL-2 is added to the bag prior to freezing in
order to maintain cell viability prior to administration. The
solution is thawed prior to administration.
Hepatic Artery Infusion (HAI) of CAR-T Cells
[0079] The studies and results disclosed herein demonstrate that
administration of CAR-expressing immunoresponsive cells via hepatic
artery infusion can provide therapeutic efficacy in treating
cancers which have metastasized to the liver. Administration of
cells which have been modified as described herein to target liver
metastases, specifically metastatic cells expressing CEA, is a
significantly more complex process compared with infusion of, for
example, a small molecule chemotherapeutic. Normal cells, such as
those in the colon, express CEA. It is important to minimize or
eliminate contact of normal cells by the modified CAR-T cells to
minimize or prevent destruction of healthy cells. CAR-T cells are
immunologic cells which can secrete various cytokines, contributing
to adverse side effects. Described below are methods to address
this problem, specifically, means for minimizing exposure of the
modified immunoresponsive cells to healthy cells while optimizing
contact of the modified cells with diseased cells.
[0080] In a preferred embodiment, the CAR expressing
immunoresponsive cells are anti-CEA CAR-T cells which were
generated using T cells obtained from the subject to be treated
with the engineered cells. HAI of CAR-T cells to treat patients
diagnosed with liver metastases will be most effective when the
modified cells are efficiently directed to the liver, where the
metastatic cells are present. HAI of CAR-T was also chosen in order
to minimize immune mediated damage to CEA-expressing extrahepatic
tissues. Patients most likely to benefit from the CAR-T therapies
are those diagnosed with liver metastases such as colorectal cancer
liver metastases. However, it is understood that the therapeutic
methods disclosed herein can be effective in treating patients in
which the liver tumor cells or liver metastases cells express one
or more proteins which are recognized by an engineered and
administered CAR-T cell. Such tumor antigens include but are not
limited to carbohydrate antigen (CA)19-9, carbohydrate antigen (CA)
125, and thymidine kinase. In a preferred embodiment, the chimeric
receptor expressed on the surface of the CAR-T cell recognizes and
binds to the well-known cancer antigen carcinoembryonic antigen.
The carcinoembryonic antigen (CEA) is an oncofetal cell surface
glycoprotein expressed by many malignant cell types, including but
not limited to colorectal, gastric, pancreatic, lung, breast and
medullary thyroid carcinoma cells. Such malignant cells that
metastasize to another organ such as the liver maintain their
phenotypes including the expression of CEA and are thus targets for
anti-CEA immunotherapies. CEA has multiple isoforms which are well
known and well-characterized in structure and sequence (e.g.,
GenBank Accession Numbers NP_001171742, NP_001171744, NP_001020083,
and SwissProt Accession Number P13688). The present methods employ
the use of immunoresponsive cells which are engineered to express a
chimeric receptor protein that specifically recognizes a CEA
protein as known in the art. In some embodiments, the chimeric
receptor protein specifically binds to a CEA protein which
comprises an amino acid sequence that is at least 90%, 95%, 98% or
99% identical to SEQ ID NO:5).
[0081] Patients treated with the CAR-T cells have been diagnosed
with a cancer in the liver. In some embodiments, the patients have
detectable unresectable CEA-positive liver metastasis or detectable
serum CEA levels. In other embodiments, the patients failed one or
more lines of conventional systemic therapy or chemotherapy. Liver
MRI and PET examinations are performed prior to CAR-T treatment to
determine the location and extent of the cancer or metastasis.
[0082] While infusing the modified cells directly into the hepatic
artery will direct most of the modified cells to the liver, many
cells will be diverted due to variations in hepatic arterial
anatomy observed in as many as 40-45% of people. Branching of the
proper hepatic artery into right and left hepatic arteries to
supply the entire liver is seen in about 80-85% of the population.
In the remainder of the population, for example, the hepatic artery
may branch only to a right or only to a left hepatic artery
(feeding the right or left lobe of the liver, respectively) or the
proper hepatic artery may be replaced by aberrant vessels diverting
some blood flow to organs other than the liver. Accordingly, it is
important to map the vessels leading to the right and left lobes of
the liver prior to performing a HAI and, when necessary, occluding
vessels which do not lead to the liver. In some embodiments, prior
to infusion, patients undergo a mapping angiogram, e.g., via a
common femoral artery approach.
[0083] Methods for mapping vessels in the body are well known to
the ordinarily skilled artisan. Once such mapping is completed
prior to treatment, a practitioner will determine which vessels
will be occluded. Occlusion is achieved, for example, through the
use of microcoil embolization, which allows the practitioner to
block off-target arteries or vessels, thereby optimizing delivery
of the modified cells to the liver. Microcoil embolization can be
performed as needed prior to administering the first dose of CAR-T
cells to facilitate optimal infusion of the pharmaceutical
composition comprising the CAR-T cells.
[0084] The methods described involve administration of the
therapeutic CAR-T cells through a catheter which is placed directly
into the hepatic artery. For example, a pharmaceutical composition
comprising the therapeutic CAR-T cells are hand-injected into the
hepatic artery via a syringe at a rate of <1 mL/second, <2
mL/second, or at a rate of about 1 to 2 mL/second, 1 to 3
mL/second, 1 to 5 mL/second, 1 mL/second, 2 mL/second, or 3
mL/second. Alternatively, the cells are injected by use of an
infusion pump as readily known in the art at the same rates as
described for hand injection. The pharmaceutical composition
comprising a dose of the therapeutic CAR-T cells (e.g., about
10.sup.8, 10.sup.9 or 10.sup.10 cells) has a total volume of about
25 to 100 mL, 25 to 75 mL, 40 to 60 mL, 50 to 75 mL or 50 to 100
mL, or has a total volume of about 25 mL, 40 mL, 50 mL, 60 mL, 75
mL or 100 mL. The method as disclosed herein may further comprise
performing liver volumetric calculations to ensure that the dose of
modified cells administered to the patient is directed to the right
and left hepatic arteries, thereby providing a therapeutically
effective dose throughout the liver. Liver volumetric calculations
are performed according to standard methods known to the ordinarily
skilled artisan and include but are not limited to scintigraphy,
ultrasound, single-photon emission computed tomography, computed
tomography (CT) and magnetic resonance imaging. Once the volumetric
calculations for a patient are complete, the dose or number of
modified cells is divided for infusion into the right and left
hepatic arteries such that the number of modified cells delivered
to the right and left lobes is proportional to the volume of the
right and left lobes, respectively.
[0085] In some embodiments, 50% of the total volume is infused into
the patient, the CAR-T solution is agitated to ensure complete cell
suspension, then the final 50% of the total volume is infused into
the patient. Infusion can be performed using an 18-guage
needle.
Co-Administration of IL-2
[0086] The patients receive the CAR-T infusions, e.g., weekly or
every 2 weeks for the duration of the CAR-T treatment period. The
CAR-T treatment period can be 2, 3, 4, 5, 6 7, 8, 9, or 10 weeks or
can last from 2-10, 4-9, 2-8, 2-6, 2-4, 3-6, 4-8 or 4-6 weeks. The
start of the CAR-T treatment period (Day 0) is the day that blood
cells are harvested from the patient to be treated (or,
alternatively, from a subject who is not the patient to be
treated). Patients receiving the infusions of CAR-T cells can also
be administered IL-2. IL-2 facilitates viability of the CAR-T cells
after infusion, however, it is preferable to use a dose of IL-2
that does not cause or enhance adverse side effects such as fever,
nausea, emesis, and/or tachycardia. In some embodiments, the IL-2
is administered continuously during the full span of the CAR-T
treatment period. Such continuous infusions can be carried out
using a pump reservoir and administered through a central venous
catheter or other method which allows the patient to be ambulatory.
IL-2 infusion can be initiated less than 1, 2 or 3 hours prior to
the start of the CAR-T infusion, at the time of or during the first
CAR-T infusion is started, or within about 1, 2 or 3 hours after
the completion of the first CAR-T infusion.
Phase I Trial for CEA CAR-T HAI
[0087] A Phase 1 clinical trial was performed in which 8 patients
with liver metastases (LM) were initially enrolled and treated with
anti-CEA CAR-T cells as described herein. Six of the patients
completed the protocol. The patients were divided into Cohort 1 and
Cohort 2. Cells were harvested from all patients on Day 0 and used
to generated anti-CEA CAR-T cells (anti-CEA
scfv-CD8.alpha.-CD28/CD3.zeta. CAR-T cells). On Day 14, 28, and 42,
each of the patients in Cohort 1 received an infusion of 10.sup.8
cells on Day 14, 10.sup.9 cells on Day 28 and 10.sup.10 cells on
Day 42. For Cohort 2, on Days 14, 28, and 42, each of the patients
received an infusion of 10.sup.10 cells. The patients in Cohort 2
also received continuous infusion of a dose of 75,000 IU/kg/day of
IL-2 beginning at Day 14 and ending on Day 55 or Day 56. On about
Day 56. MRI and PET analysis of the patients in Cohorts 1 and 2 was
done on Day 56. Data from the 6 patients that completed the
protocol demonstrated that HAIs of anti-CEA CAR-Ts are well
tolerated with and without systemic IL-2 infusion. Spikes in
IFN.gamma. were noted to occur 24 to 48 hours after doses in all
patients, with or without system IL-2 (see FIG. 6). Although there
were no radiographic partial or complete responses, 1 of 6 patients
had stable disease and was alive for at least 24 months
follow-up.
[0088] The studies described herein established the safety of
anti-CEA CAR-T HAIs with and without systemic IL-2 support,
reaching the maximum planned dose of 10.sup.10 cells. Accordingly,
with respect to the safety and efficacy of CAR-T HAIs, the findings
support use of CAR-T HAIs for treatment of LM. The limited systemic
exposure of CAR-T in the study subjects likely accounted for the
favorable adverse event profile. Systemic IL-2 support was
associated with increased serum IFN.gamma. levels and improved CEA
responses, at the expense of more severe but manageable adverse
events. As shown in Example 4, HAI led to preferential accumulation
of CAR-T within liver metastases in 5 of 6 of the patients,
compared with normal liver and peripheral blood. CAR-Ts were not
detected in the peripheral blood in 4 of the 6 patients and only
transiently in patients 7 and 8. Importantly, histologic evidence
of increased LM necrosis and fibrosis were seen in the majority of
subjects following CAR-T HAI (see FIG. 5). These data all show that
effective delivery of the CEA CAR-T cells to the CEA+ tumor
deposits correlates well with histologic evidence of tumor killing
and serum cytokine surges, supporting the therapeutic efficacy of
the CAR-T cells (e.g., anti-CEA scfv-CD8.alpha.-CD28/CD3.zeta.
CAR-T cells) for the treatment of liver metastases via hepatic
arterial infusion.
[0089] HAI led to preferential accumulation of CAR-T within LM in 5
of 6 HITM patients, compared to normal liver and peripheral blood.
CAR-Ts were not detected in the peripheral blood in 4 of 6 patients
and only transiently in patients 7 and 8. Moderate elevations of
liver function test values (e.g., transient elevations of alkaline
phosphatase, total bilirubin and aspartate aminotransferase levels)
were likely related to the CAR-T HAI but did not result in
clinically significant consequences. Systemic infusion of T cells
expressing anti-CEA CAR-T was previously reported to result in
dose-limiting toxicity (Parkhurst et al., 2011, Mol Ther,
19:620-626). Similar toxicities were seen in the present study with
the anti-CEA CAR-T when systemically infused, particularly with
IL-2 support (not shown). Continuous ambulatory infusion dose of
IL-2, 75,000 IU/kg/day, is several-fold lower than what is given in
other protocols (Rosenberg et al., 1999, J Clin Oncol, 17:968-975.
Despite the low daily dose of the IL-2 in this study, 2 patients
experienced grade 3 events requiring IL-2 dose reductions. These
adverse events, including severe pyrexia and colitis, can be
attributed to the IL-2 based upon the fact that the symptoms
resolved promptly upon IL-2 dose reduction. In one subject, it is
possible that the IL-2 activated a small number of systemically
circulating anti-CEA CAR-T that mediated fever and colitis.
Overall, the IL-2 infusion administration was well tolerated and
the adverse events easily managed by dose reductions.
[0090] In one aspect of the present disclosure, the dose level of
CEA CAR-T cells for each patient is 10.sup.10 cells administered
via HAI once per week. Therapeutic efficacy of a once-per-week
infusion of the cells is supported by the serum liver chemistry and
cytokine data from the phase 1 trial disclosed herein. In the phase
1 study in which infusions were performed every 2 weeks, most
patients demonstrated transient but clinically insignificant
elevations in serum alkaline phosphatase, bilirubin, and aspartate
aminotransferase (AST) levels following CEA CAR-T HAI (see Example
7). In nearly all cases, levels normalized within 3-4 days.
Likewise, patients demonstrated surges in serum IFN.gamma. and IL-6
levels following CAR-T HAI, which returned to baseline or
near-baseline levels within 1 week. These data suggest that a
1-week interval is sufficient to allow for resolution of the acute
inflammatory response to CAR-T HAI, and to permit safe repeat
CAR-T. Changing the CAR-T interval from 2 weeks to 1 week will also
minimize IL-2 exposure for the patients and allow for a more rapid
return to systemic therapy for those in whom this is clinically
indicated. In a preferred embodiment, patients being administered
CEA CAR-T cell infusions once per week receive continuous systemic
infusion of IL-2 at a dose of 50,000 IU/kg/day. In some
embodiments, a patient diagnosed with unresectable liver metastases
is administered a dose of 50,000 IU/kg/day IL-2 for 28 days by
continuous intravenous infusion during the CAR-T infusion treatment
period, inclusive of the 14 days after the final CAR-T
infusion.
[0091] Therapeutic efficacy of the CAR-T treatment of liver
metastases can be determined by routine imaging techniques such as
magnetic resonance imaging (MRI), positron emission tomography
(PET), or ultrasound. Such imaging procedures can measure the tumor
burden or extent of tumor in the liver before, during and after
treatment. A therapeutically effective dosing regimen for HAI of
CAR-T cells can reduce tumor burden relative to the tumor burden
prior to the first infusion of cells by about 10% to 100%, 10% to
80%, 10% to 60%, 10% to 40%, 20% to 40%, 20% to 60%, 20% to
80%.
CAR-T HAI with Selective Internal Radiation Therapy (SIRT)
[0092] Selective Internal Radiation Therapy (SIRT) is a form of
radiation therapy generally used for patient diagnosed with
unresectable cancers. SIRT is administered as radioactive
microspheres into a target such as an organ, tissue or tumor in
order to effectively deliver a therapeutic dose of ionizing
radiation to that target resulting in damage or death of that
target organ, tissue or tumor.
[0093] Radioactive microspheres for therapeutic application
typically comprise a matrix material that can act as a carrier for
a radionuclide material which emits ionizing radiation. In
particular, it has previously been shown that a number of beta
radiation emitting radionuclides, such as Phosphorus-32,
Holmium-166 or Yttrium-90, can be attached to matrix microspheres
such as polymeric resin or glass microspheres for injection into
the blood stream of a cancer patient with therapeutic effect.
[0094] The radioactive microspheres are generally delivered via the
arterial blood supply of the target tissue or tumor. To this end, a
catheter is guided to the branch of the blood vessel that feeds the
target tissue or tumor to infuse the microspheres into the
circulation. The radioactive microspheres can be introduced into
the arterial blood supply of either the whole liver, a section of
the liver, or into the arterial blood supply of that part of the
liver containing the tumor that is to be treated, by injection of
the radioactive microspheres into the hepatic artery, the portal
vein, or a branch of either of these vessels. The radioactive
microspheres become trapped in the capillary beds of target tissue
or tumor providing for the selective delivery of a dose of
radiation to the target tissue ortumor.
[0095] Two commercially-available products available for SIRT
treatment of liver cancer include TheraSphere.RTM. (MDS Nordion,
Inc.), and SIR-Spheres.RTM. (SIRTeX.RTM. Medical Ltd.). Both
products are Yttrium-90 labelled microspheres: TheraSpheres.RTM.
being glass microspheres having a diameter of 25.+-.10 .mu.m; and
SIR-Spheres.RTM. being resin-based microspheres that having a
diameter of 32.+-.2.5 .mu.m.
[0096] Despite the promising results of the phase 1 trial disclosed
herein and the therapeutic efficacy of the CEA CAR-T HAI, it is
always best to optimize treatment efficacy and convenience wherever
possible. Combinatorial strategies can often maximize the benefit
for patients diagnosed with an incurable disease. Radiotherapy
induces immunogenic tumor cell death through antigen release and
recruitment of effector T cells. Radiotherapy alone is only rarely
capable of generating effective anti-tumor immunity. However, when
combined with targeted immunotherapy agents, radiotherapy
significantly contributes to a therapeutically effective anti-tumor
immune response. A "HITM-SIR" trial has been designed to test the
safety and potential increase in tumor killing by using
SIR-Spheres.RTM. following CAR-T HAI. HITM-SIR is a novel iteration
of established principles and approaches with proven safety. This
novel trial has the potential to generate paradigm-changing data
for the management of LM.
[0097] In one aspect, a method of treating subjects diagnosed with
a liver metastasis is provided wherein the subject is treated with
CAR-T HAI as described above, with or without systemic IL-2
administration, followed by administration of SIRT. Patients are
dosed with SIR-Spheres equivalent to either 2GBq, 2.5GBq, or 3GBq
of .sup.90Y activity based on the volume of the tumor. In some
embodiments, patients with a tumor volume that is less than 25%,
about 25%-50% or greater than 50% of the total liver volume are
given SIR-Spheres equivalent to 2GBq, 2.5GBq, or 3GBq of .sup.90Y
activity, respectively. A dose of SIR-Spheres is administered to
the patient about 1 week, 2 weeks, or 3 weeks after the final CAR-T
HAI infusion. In a preferred embodiment, the dose of SIR-Spheres is
administered about 1 week after the final CAR-T HAI infusion.
Immunosuppressor Agents
[0098] Therapeutic efficacy of chimeric antigen 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.
[0099] 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. 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). Accordingly, in one aspect of the present disclosure,
the patient receiving CAR-T HAI is also administered an
immunosuppressing agent which suppresses the activity of suppressor
T cells such as MDSCs or Tregs. In some embodiments, the
immunosuppressing agent is an MDSC depletion antibody which binds
Gr1 (granulocytic myeloid marker protein) or a PD-L1 blocking
antibody.
[0100] In some embodiments, the immunosuppressing agent is an
antibody that binds IL-10, PD-1 (programmed death-1 receptor),
PD-L1 (programed death-1 receptor ligand 1), PD-L2 (programed
death-1 receptor ligand 2), 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
infusion of the CAR-T cells. In still other embodiments, the
immunosuppressing agent is administered to the subject after
infusion of the CAR-T cells. 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 a CAR-T HAI. The immunosuppressing agent
can be administered on the same day as the infusion or can be
administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days
or more prior to the first CAR-T hepatic artery infusion. 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.
[0101] 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.
IV. EXAMPLES
[0102] The following examples are illustrative in nature and are in
no way intended to be limiting.
Example 1
Human CAR-T Cell Production
[0103] As described in more detail below, six patients (referred to
herein as Patient numbers 1, 4, 5, 6, 7 and 8) were treated with
hepatic infusions of CAR-T cells which specifically target
metastatic cells expressing the CEA antigen on their surface. The
anti-CEA scfv-CD8.alpha.-CD28/CD3.zeta. (Tandem) chimeric antigen
receptor was cloned into the MFG retroviral backbone as previously
described (FDA BB IND 10791) (Emtage et al., Clin Canc Res,
14:8112-8122, incorporated herein by reference in its entirety).
Briefly, a tandem molecule was generated by molecularly fusing in
an N-terminal to C-terminal direction, a hMN14 sFv (SEQ ID NO:1) of
a monoclonal antibody which specifically binds CEA, a CD8 hinge
segment, a CD28 extracellular domain, transmembrane domain and
cytoplasmic domain and a .zeta. cytoplasmic domain. The resultant
chimeric construct was cloned into a retroviral vector and verified
by restriction digestion and sequencing. The clinical retroviral
vector supernatant was produced using PG13 cells to generate gibbon
ape leukemia virus pseudotyped viral particles as previously
described (Beaudoin et al., 2008, J Virol Methods, 148:253-259).
All clinical batches were prepared at Indiana University vector
production facility (Indianapolis, Ind.) and stored at -80.degree.
C. until used.
[0104] Rhode Island Blood Center personnel performed leukapheresis
at the Roger Williams Medical Center (RWMC, Providence, R.I.).
Anti-CEA CAR-Ts were prepared at the RWMC Cell Immunotherapy and
Gene Therapy (CITGT) 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) were isolated from leukapheresis product using Ficoll
(Sigma, St; Louis, Mo.). We then activated PBMCs for 48-72 hours in
tissue culture flasks (BD Falcon, Franklin Lakes, N.J.) containing
AIM V media (Life Technologies, Grand Island, N.Y.) supplemented
with 5% sterile human AB serum (Valley Biomedical, Winchester,
Va.), 50 ng/mL of anti-CD3 monoclonal antibody (OKT3; Ortho
Biotech, Horsham, Pa.) and 3000 U/mL of IL-2 (Prometheus, San
Diego, Calif.).
[0105] Using the spinoculation method (Quintas-Cardama et al.,
2007, Hum Gene Ther, 18:1253-1260), 7.2-14.4.times.10.sup.8T cells
obtained from patients were transduced in retronectin (Takara Bio
Inc, Japan) coated 6-well plates in AIM V media with 5% human AB
serum, 3000 U/mL of IL-2, and protamine sulfate (MP Biomedicals) at
low speed centrifugation for 1 hour at room temperature. The
transduction step was carried out a total of three times over
24-hrs. After transduction, cells were washed in media and
incubated for 48-72 hours at 37.degree. C. CAR-Ts were further
expanded in Lifecell tissue culture bags (Baxter, Deerfield, Ill.)
for 10-14 days. CAR-T growth curves and cell viability were
examined periodically and cell growth media was replaced as
required. CAR-Ts were examined by flow cytometry with fluorescently
labeled antibodies specific for CD3 (UCHT1, Invitrogen, Frederick,
Md.), CD4 (SK3, BD Biosciences, San Jose, Calif.), CD8 (3B5,
Invitrogen), and CAR expression (WI2 antibody, Immunomedics, Norris
Plains, N.J.). The WI2 antibody was prepared as an APC conjugate
(WI2-APC; Molecular Probes). Flow cytometry was performed on a CyAn
(Beckman Coulter, Brea, Calif.) or LSR-II (BD Biosciences, San
Jose, Calif.) machine. In vitro activity of patient products was
measured by bioluminescence cytotoxicity assay.
Luciferase-expressing CEA+ tumor cells were mixed with anti-CEA
CAR-T at various ratios in 96-well round bottom plates and loss of
bioluminescence from each well measured (Karimi et al., 2014, PLoS
One, 9:e89357). 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.
[0106] To assess the CAR-T cells, the leukapheresis product from
each patient was analyzed by flow cytometry prior to and following
transduction with anti-CEA CAR construct. For Patients 1, 4, 5, 6,
7 and 8, the mean percentage of CD3+ cells following leukapheresis
was 55% (range, 12.0-82.0) and increased to 91% (range, 72-97)
following activation and transduction (FIG. 1). The mean CD4:CD8
ratio was 2.4 (range, 1.4-4.7) in the leukapheresis samples and 0.8
(0.2-2.2) in the final products (not shown). The transduction
efficiency (CAR+) ranged from 10% to 64%, with a mean of 45% (FIG.
1). Negligible FoxP3 staining was detected among CAR+ T cells prior
to infusion (not shown). Cells in the final products were 85%
viable prior to infusion (range, 71-95). In vitro cytotoxicity
assays were performed to test patient product killing of CEA+
target cells. Anti-CEA CAR-T cells were cultured with CEA+MC38
colorectal carcinoma target cells. Target cell killing was
quantified by loss of bioluminescence following addition of
luciferin. Specific lysis was calculated based on residual photon
counts. These assays confirmed that patient products specifically
lysed CEA+ target cells (FIG. 2).
[0107] Clinical doses were prepared using a Fenwal cell harvester
system (Baxter, Deerfield, Ill.) in freezing media containing
PlasmaLyte (Baxter), 20% human bovine albumin (Valley Biomedicals),
10% DMSO (Bioniche Pharma, Lake Forrest, Ill.) and IL-2. Bacterial
and fungal cultures were monitored for 14 and 28 days respectively.
Assays for bacterial endotoxin were performed using LAL Endotoxin
assay kits (Lonza, Walkersville, Md.). The clinical dose was stored
in liquid nitrogen and thawed immediately prior to infusion.
Example 2
Clinical Study Design
[0108] A phase I clinical study (NCT01373047, RWH 11-335-99) was
performed. The study enrolled eight patients with unresectable CEA+
adenocarcinoma LM who progressed on an average of 2.5 (range 2-4)
lines of conventional systemic therapy (Table 2).
TABLE-US-00003 TABLE 2 Patient Characteristics Size CEA CAR-T ID
Sex Age Dx Chemo DFI EHD #LM (cm) (ng/ml) IL-2 Doses 1 F 56 Colon 4
0 None >10 14.4 3265 No 3 2 M 52 Colon 2 0 Lungs >15 12.6 352
No 0{circumflex over ( )} 3 M 52 Gastric 1 0 None 1 5.7 29.9 No
0{circumflex over ( )}{circumflex over ( )} 4 M 55 Ampullary* 2 9
Lungs, RPN 1 1.7 362 No 3 5 M 63 Colon 3 37 None 2 5.7 2** No 3 6 M
51 Colon 3 36 Lungs >10 10.5 1112 Yes 3 7 F 53 Colon 3 0 Lungs
>10 8.0 32 Yes 3 8 M 66 Colon 2 0 Lungs >10 9.8 72 Yes 3 Mean
= 57 Mean = 2.5 Mean = 8.4 Mean = 807.2 DFI = disease free interval
from diagnosis of primary to liver metastases; LM = liver
metastases; SIZE = largest LM prior to CAR-T treatment; IL-2 =
continuous IL-2 infusion with CAR-T; RPN = retroperitoneal nodes;
*= pancreatobiliary subtype of ampullary carcinoma; {circumflex
over ( )}= withdrew after 2 doses due to extrahepatic progression:
{circumflex over ( )}{circumflex over ( )}= withdrew due to
unrelated medical condition; **= CEA expression confirmed in tumor
specimen by immunohistochemistry.
[0109] Six patients completed the protocol (FIG. 3), one patient
withdrew due to an unrelated infection prior to treatment, and
another patient withdrew due to extrahepatic disease progression
prior to his third CAR-T HAI. Of the patients that completed the
protocol, 4 were male and 2 were female. Five patients had stage IV
colorectal carcinoma and one patient had pancreatobiliary ampullary
carcinoma. The average age was 57 (range, 51-66). Patients
presented with substantial disease burdens, with the average size
of the largest LM being 8.4 cm (range, 1.7-14.4) and five patients
having more than 10 LM. The mean CEA level upon enrollment was 807
ng/ml (range, 2-3265). Five of eight patients had synchronous
colorectal LM and the mean disease-free interval was 27.3 months
(range, 9 to 37) for patients with metachronous LM. All further
analyses include only the six patients who completed the study.
[0110] In the study, two cohorts of three patients were treated
with anti-CEA CAR-T HAIs without or with systemic IL-2 support
(FIG. 3). Cohort 1 (Patients 1, 4 and 5) was treated with CAR-T
HAIs in intrapatient dose escalation fashion (10.sup.8, 10.sup.9,
and 10.sup.10 cells) without IL-2. Specifically, T cells were
collected from each patient on Day 0, the first infusion was on Day
14 during which 10.sup.8 cells were infused, the second infusion
was on Day 28 during which 10.sup.9 cells were infused and the
third infusion was on Day 44 during which 10.sup.10 cells were
infused. Those in the cohort 2 (Patients 6, 7 and 8) received 3 HAI
of 10.sup.10 CAR-Ts on Days 14, 28 and 44 in addition to continuous
systemic IL-2 infusion at 75,000 U/kg/day via an ambulatory
infusion pump for 6 weeks beginning at the time of the first
infusion on Day 14.
[0111] Eligible patients had measurable unresectable CEA-positive
LM or detectable serum CEA levels and failed one or more lines of
conventional systemic therapy. Minimal extra-hepatic disease in the
lungs or abdomen was permitted. Clinical assessments were performed
at baseline, on infusion days, and 1, 2, 4, and 7 days
post-infusion. Planned imaging assessments with liver MRI and PET
examinations were scheduled within one month prior to the first
infusion and then within one month following the third CAR-T HAI.
The study radiologist (BS) graded responses according to modified
RECIST (mRECIST) and immune related response criteria (Wolchok et
al., 2009, Clin Cancer Res, 15:7412-7420). A blinded pathologist
scored tumor necrosis and fibrosis on slides from percutaneous
biopsies performed prior to treatment and two weeks following the
second dose. Safety evaluation was performed per protocol. Severity
of adverse events was graded using the National Cancer Institute
Common Terminology Criteria for Adverse Events version 3.0.
Example 3
CAR-T Cell Hepatic Arterial Infusion
[0112] At baseline, a mapping angiogram was performed via a right
common femoral artery approach. The gastroduodenal and right
gastric arteries, in addition to other potential sources of
extrahepatic perfusion, were embolized with microcoils. For CAR-T
infusions, the same arterial access procedure was carried out and
the cells were hand-injected via a 60cc syringe at a rate of <2
cc/second with a total volume of 100 cc. Angiography with
calibrated contrast rate was performed after the first 50 cc and at
completion of the CAR-T infusion to confirm preserved arterial
flow. Infusions were delivered into the proper hepatic artery when
possible. In cases of aberrant hepatic arterial anatomy, where
either the right or left hepatic artery did not arise from the
proper hepatic artery, the dose was split based upon lobar volume
calculations. In such cases, split doses were delivered separately
into the right and left hepatic arteries to ensure proportionate
CAR-T delivery to bothlobes.
[0113] The anti-CEA scfv-CD8.alpha.-CD28/CD3.zeta. (Tandem)
chimeric antigen receptor used in the Examples below was cloned
into the MFG retroviral backbone as previously described (FDA BB
IND 10791) (Nolan, et al., 1999, Clin Cancer Res, 5:3928-394;
Emtage et al., 2008, Clin Cancer Res, 14:8112-8122; see Example 1
above). Briefly, the tandem molecule was generated by molecularly
fusing a fragment encoding the hMN14 sFv-CD8 hinge segment in the
MFG retroviral backbone with a hybrid CD28/CD3.zeta. molecule. The
construct was verified by restriction digestion and sequencing. The
clinical retroviral vector supernatant was produced using PG13
cells to generate gibbon ape leukemia virus pseudotyped viral
particles as previously described (Beaudoin et al., 2008, J Virol
Methods, 148:253-259). All clinical batches were prepared at
Indiana University vector production facility (Indianapolis, Ind.)
and are stored at -80.degree. C. until use.
Example 4
CAR-T Cell Trafficking Following Infusion
[0114] CT guided percutaneous biopsies were obtained in order to
sample LM and normal liver prior to the first CAR-T HAI and at the
time of the final HAI. The proportions of CAR-T (CAR+/total
lymphocyte %) in LM biopsy, normal liver biopsy, and peripheral
blood samples were determined by flow cytometry. For example,
samples from patient 7 demonstrated that 1.8% of normal liver
lymphocytes were CAR+ following HAI of CAR-T and 7.6% of
intratumoral lymphocytes were CAR+. It was confirmed that that CAR+
cells in the post-infusion LM biopsy specimen were CD3+. CAR-T
population data in peripheral blood, normal liver, and LM were
determined for all patients. CAR-Ts were more abundant in the LM
compared to normal liver in 5 of 6 patients. In patient 5, CAR-Ts
were found to comprise 1.4% of LM lymphocytes in a sample obtained
during a microwave ablation procedure 12 weeks following his final
CAR-T infusion. In 4 patients, CAR-Ts were not detectable in
peripheral blood but were transiently present in patient 7 and
patient 8 at the time of the final infusion, and the levels dropped
below detection 3 days later. Quantitative PCR was performed on
peripheral blood samples taken at day 2 following the final
infusion; only patient 7 had a measurable increase (1.1-fold) in
CAR DNA relative to baseline. Anti-CAR antibodies were not detected
in patient sera following CAR-T infusion.
Example 5
Therapeutic Activity
[0115] At last follow-up, 5 of the 6 heavily pre-treated patients
who completed the trial died due to disease progression (Table
3).
TABLE-US-00004 TABLE 3 Patient Outcomes ID IL-2 CAR+ % MRI PET
.DELTA.CEA %{circumflex over ( )} OS(weeks) Status 1 NO 10.4 PD PD
-1 30 DOD 4 NO 27.2 +401 8 DOD 5 NO 48.9 SD SD +63 140 AWD -
Residual disease treated with microwave ablation and further
systemic therapy 6 YES 63.5 PD PD -19 13.0 DOD 7 YES 57.4 PD PD -48
17 DOD -Underwent resection of obstructing primary- right colon
tumor after final CAR-T infusion 8 YES 61.9 PD PD -43 19 DOD
{circumflex over ( )}Fold change from baseline at time of 2.sup.nd
biopsy or IL-2 infusion disruption; SAE: serious adverse events;
DOD: dead of disease; AWD: alive with disease; PD: progressive
disease; SD: stable Patient 2 withdrawn after 2 CAR-T doses due to
extrahepatic progression and was DOD 23 days after 2.sup.nd CAR-T
infusion. Patient 3 withdrawn after cell collection due to
unrelated medical condition.
[0116] MRI and PET scans were performed in 5 of 6 patients at
baseline and 2-4 weeks following the third CAR-T HAI. Patient 8 did
not obtain final imaging following a return to his native country
and ultimately died of disease progression. All patients except
Patient 5 were determined to have radiographic disease progression
by mRECIST and criteria. Patient 5 was found to have stable disease
by MRI and PET. Patient 7 developed new lesions and demonstrated an
increase in size of some pre-existing lesions, while other lesions
decreased in size. The lesion in the posterior sector of Patient 7
that decreased in size on MRI was not visible on PET. More medial
disease that was decreased in size on MRI was noted to become
hypometabolic on the post-infusion PET for Patient 7.
[0117] Given the limited utility for short follow-up conventional
imaging following infusion of CAR-T, we measured serum CEA levels
at multiple time points following each of the three HAIs for each
patient. Among the patients in cohort 1, transient decreases in
serum CEA were demonstrated in two patients following each CAR-T
HAI (FIG. 4, Patients 1 and 5). CEA kinetics were closely
paralleled by changes in serum CA19-9 levels (not shown). Patient
4, who presented with hepatobiliary subtype ampullary carcinoma,
was the only patient without a CEA decrease at any point during the
trial and he also had the shortest survival time.
[0118] The patients in cohort 2 who received systemic IL-2 along
with anti-CEA CAR-T had more favorable CEA responses to treatment.
As each of the three patients in cohort 2 required an IL-2
interruption or dose reduction, which would likely impact CAR-T
function, we compared CEA levels at baseline with the time point
just prior to IL-2 dose change (indicated by the arrows in FIG. 4).
When using these time points, all three patients in cohort 2 had
decreases in serum CEA concentrations (FIG. 4 and Table 3).
Patients 7 and 8 had a 48% and 43% decrease in serum CEA
concentrations, respectively, prior to IL-2 dose interruption or
reduction. The mean overall survival time for the 6 patients who
completed the trial was 30 weeks with a median of 15 weeks (range,
8-73). Patient 5 is alive with disease at 24 months following his
final CAR-T HAI. Following completion of the HITM trial, Patient 5
was determined to have stable disease and we performed a microwave
ablation of residual unresectable tumor.
[0119] Detecting radiographic responses in heavily pre-treated
patients with advanced metastatic disease is challenging, and even
more so with immunotherapy where intratumoral inflammation and
edema may minimize the relevance of standard RECIST criteria
(Wolchok et al., 2009, Clin Canc Res, 15:7412-7420). As such, we
obtained LM biopsies prior to and following CAR-T HAIs to assess
degrees of intratumoral necrosis and fibrosis. Normal liver and
liver metastasis core needle (16-gauge) biopsies were obtained
under sonographic guidance at baseline and at the time of the third
CAR-T HAI. Three cores were obtained for normal liver and liver
metastases, with each confirmed by cytology. For each case, 4- to
5-mm sections were stained with hematoxylin and eosin (H&E) and
additional unstained slides were stained with anti-CEA antibody (TF
3H8-1; Ventana). All immunohistochemical stains were performed on
the Ventana Medical System at Our Lady of Fatima Hospital
(Providence, R.I.). All slides were reviewed in blinded fashion and
graded for necrosis and fibrosis. Fibrosis was scored as follows:
0%, grade 0; 5% to 10%, grade 1; 11% to 50%, grade 2; >50%,
grade 3. Necrosis was scored as follows: 0%, grade 0; 0% to 10%,
grade 1; 11% to 50%, grade 2; >50%, grade 3. Flow cytometry was
performed on fresh biopsy tissue for CAR-T cells and peripheral
blood as described above. After review by a blinded pathologist, 4
patients had an increase in intratumoral fibrosis and 3 patients
were scored as having an increase in necrosis within their LM (FIG.
5). For each patient, baseline and post infusion scores are shown
from left to right in FIG. 5. Patients 1, 4, 7 and 8 showed an
increase in fibrosis while patients 4 and 5 showed an increase in
necrosis.
Example 6
Serum IFN.gamma. Concentration and CEA Response Correlation with
IL-2 Administration
[0120] Serum IFN.gamma. levels were measured by ELISA at multiple
time points. Spikes in IFN.gamma. were noted to occur 24-48 hours
after doses in all patients, without or with systemic IL-2 (FIG. 6;
dotted vertical lines indicate CAR-T infusion time points and the
first data point represents the baseline value prior to CAR-T
infusion). Serum CEA changes were compared to peak change in
IFN.gamma. for each patient (FIG. 7, top). The inverse correlation
between peak IFN.gamma. levels and CEA change was significant
(R=-0.94, p=0.02). All patient HAI CAR-T doses contained a quantity
of IL-2 (600,000 IU). The three patients (Patient numbers 6, 7, and
8) with continuous systemic IL-2 exposure and largest CAR-T doses
had the best CEA responses and the highest mean IFN.gamma. levels
(P=0.03, FIG. 7, bottom).
Example 7
Safety Data
[0121] Adverse events (AE) of any grade attributable to any cause
were observed in all patients who completed the trial (Table 4).
The dose in cohort 1 reached the planned maximal HAI CAR-T infusion
level at 10.sup.10 cells. No CAR-T dose reductions were required in
cohort 1 and therefore, all patients in cohort 2 received 3 doses
at the 10.sup.10 level with IL-2 support. There were no grade 4 or
5 adverse events. Febrile AEs were observed in 4 patients. Patient
7 experienced grade 3 fever and tachycardia, with a temperature
peak of 104.degree. F. The fever and tachycardia resolved in
Patient 7 after a 50% dose reduction in her systemic IL-2 infusion.
Of note, Patient 7 also experienced an increase in her peripheral
eosinophil count with a peak of 20% and absolute count of 3,740/ml.
Given the reported association between IL-2 infusion and cardiac
thrombosis with other features of Loeffler's syndrome (Junghans et
al., 2001, New Eng J Med, 344:859-860), we obtained an
echocardiogram and electrocardiogram which were normal. Her
eosinophil count returned to normal limits without specific
intervention.
TABLE-US-00005 TABLE 4 Adverse Events ID IL-2 Grade # Description 1
NO 1 12 Fever, mylagias, abdominal pain, nausea, emesis, and
tachycardia 2 2 Abdominal wall muscle spasm and .uparw.ALT 3 2
.uparw.AST and .uparw.alk phos 4* 1 5 Ascites, edema,
thrombocytopenia, .uparw.ALT, .uparw.AST 2 5 .uparw.alk phos,
leukopenia, dyspnea 3 2 Pleural effusion, anorexia 5 1 2 Fever,
rash 3 1 Emesis 6 YES 1 5 .uparw.AST, .uparw.ALT, thrombocytopenia,
dyspnea, rash 2 1 Lower extremity edema 3 3 Emesis, subscapular
liver hematoma, .uparw.alk phos 7 1 7 Eosinophilia, chills, fever,
abdominal pain, .uparw.bilirubin 2 2 Emesis, diarrhea 3 3
Tachycardia with fever (104.degree. F.){circumflex over ( )},
emesis, abdominal pain 8 2 6 Fever, tachycardia, diarrhea,
dehydration, lower extremity edema 3 3 Anemia, abdominal pain,
colitis{circumflex over ( )} *Death due to disease progression 28
days after third infusion. {circumflex over ( )}Led to IL-2 dose
reduction.
Patient #2 experienced grade 3 abdominal pain and dehydration; he
was taken off protocol after the 2nd HAI and died due to disease
progression 23 days later. Patient #3 was withdrawn prior to CAR-T
infusion due to an unrelated medical condition. Liver function test
adverse events reflect values outside of normal range and not
necessarily change from baseline.
[0122] Normal liver parenchyma and biliary structures were well
preserved following CAR-T HAIs. Biopsies from normal liver did not
demonstrate increased levels of inflammation or fibrosis following
CAR-T HAI whether or not systemic IL-2 was administered. While all
patients experienced transient elevations of alkaline phosphatase
(alk phos), total bilirubin, and aspartate aminotransferase levels
(AST), only Patient 1 experienced grade 3 elevations and the
majority of values did not deviate significantly from baseline
levels. Portal pressures and liver synthetic function were not
adversely affected by the CAR-T HAIs, as reflected by no patient
becoming thrombocytopenic or coagulopathic.
Example 8
Safety Data
[0123] A phase I clinical study is conducted wherein patients are
each administered 10.sup.10 anti-CEA scfv-CD8.alpha.-CD28/CD3.zeta.
CAR-T cells via HAI. was performed (as described in the Examples
above). T cells will be isolated from each patient on Day 0,
transfected and selected to express the anti-CEA
scfv-CD8.alpha.-CD28/CD3.zeta. construct. Each patient is then
administered 10.sup.10 cells on Days 14, 21 and 28. On Day 44
(after a 2-week break following the final cell infusion), a dose of
SIR-Spheres.RTM. containing is infused into each patient.
[0124] For dosing SIR-Spheres.RTM., patients will receive a
predetermined quantity of SIR-Spheres.RTM. that will vary depending
on the size of the tumor volume relative to normal liver volume.
Patients with tumor that was either <25%, 25%-50% or >50% of
the total liver volume are given SIR-Spheres.RTM. equivalent to
either 2 gigabecquerels (GBq), 2.5 GBq, or 3 GBq of .sup.90Y
activity. Provisions are made for patients in whom the lung-liver
breaththrough scan indicatied that more than 10% of the
microspheres passed through the liver and lodged in the lungs. The
amount of .sup.90Y activity to be administered is reduced by 2% for
each 1% that the lung-liver breakthrough percentage is greater than
10%. Patients are discharged either the same day or the following
day after the SIR-Spheres.RTM. treatment.
[0125] Patients are evaluated as having a complete response, a
partial response, stable disease or progressive disease at one
month after the final infusion by appropriate clinical and
radiologic studies. Circulating levels of modified T cells are
monitored by flow cytometry to detect anti-CEA
scfv-CD8.alpha.-CD28/CD3.zeta. and CD4/CD8 and by PCR. LM and
normal liver biopsy specimens are used to determine the degree of
CAR-T tumor infiltration. Biopsy samples are analyzed by flow
cytometry and/or immunohistochemistry to quantify the presence of
anti-CEA scfv-CD8.alpha.-CD28/CD3.zeta. CAR-T. Samples are drawn
for serum cytokine levels and changes in normal liver T cell
populations. ELISA assays are performed to measure serum IL-2,
IFN.gamma., IL6, IL17, and IL10. Neutrophil:lymphocyte ratios are
determined at the same time points from CBCs, as well as a detailed
assessment of hepatic and peripheral T cell populations by flow
cytometry. Intrahepatic and peripheral T cells are stained with
antibodies specific for CD3, CD4, CD8, FOXP3, PD-1, CD25, CTLA4,
and CD69.
[0126] 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
61213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Gly Thr Ser 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
2102235PRTHomo sapiens 2Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro
Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Ser Gln Phe Arg Val
Ser Pro Leu Asp Arg Thr 20 25 30Trp Asn Leu Gly Glu Thr Val Glu Leu
Lys Cys Gln Val Leu Leu Ser 35 40 45Asn Pro Thr Ser Gly Cys Ser Trp
Leu Phe Gln Pro Arg Gly Ala Ala 50 55 60Ala Ser Pro Thr Phe Leu Leu
Tyr Leu Ser Gln Asn Lys Pro Lys Ala65 70 75 80Ala Glu Gly Leu Asp
Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95Thr Phe Val Leu
Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110Tyr Phe
Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120
125Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg
130 135 140Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
Leu Arg145 150 155 160Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
Val His Thr Arg Gly 165 170 175Leu Asp Phe Ala Cys Asp Ile Tyr Ile
Trp Ala Pro Leu Ala Gly Thr 180 185 190Cys Gly Val Leu Leu Leu Ser
Leu Val Ile Thr Leu Tyr Cys Asn His 195 200 205Arg Asn Arg Arg Arg
Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220Gly Asp Lys
Pro Ser Leu Ser Ala Arg Tyr Val225 230 2353164PRTHomo sapiens 3Met
Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu1 5 10
15Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys
20 25 30Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr
Ala 35 40 45Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
Ala Tyr 50 55 60Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg65 70 75 80Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu Met 85 90 95Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro
Gln Glu Gly Leu Tyr Asn 100 105 110Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala145 150 155 160Leu
Pro Pro Arg4220PRTHomo sapiens 4Met Leu Arg Leu Leu Leu Ala Leu Asn
Leu Phe Pro Ser Ile Gln Val1 5 10 15Thr Gly Asn Lys Ile Leu Val Lys
Gln Ser Pro Met Leu Val Ala Tyr 20 25 30Asp Asn Ala Val Asn Leu Ser
Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45Arg Glu Phe Arg Ala Ser
Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60Val Cys Val Val Tyr
Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser65 70 75 80Lys Thr Gly
Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95Phe Tyr
Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105
110Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
115 120 125Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro
Ser Pro 130 135 140Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu
Val Val Val Gly145 150 155 160Gly Val Leu Ala Cys Tyr Ser Leu Leu
Val Thr Val Ala Phe Ile Ile 165 170 175Phe Trp Val Arg Ser Lys Arg
Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190Asn Met Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205Tyr Ala Pro
Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 2205526PRTHomo sapiens
5Met Gly His Leu Ser Ala Pro Leu His Arg Val Arg Val Pro Trp Gln1 5
10 15Gly Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro
Thr 20 25 30Thr Ala Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala
Glu Gly 35 40 45Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Gln
Leu Phe Gly 50 55 60Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn
Arg Gln Ile Val65 70 75 80Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr
Pro Gly Pro Ala Asn Ser 85 90 95Gly Arg Glu Thr Ile Tyr Pro Asn Ala
Ser Leu Leu Ile Gln Asn Val 100 105 110Thr Gln Asn Asp Thr Gly Phe
Tyr Thr Leu Gln Val Ile Lys Ser Asp 115 120 125Leu Val Asn Glu Glu
Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu 130 135 140Pro Lys Pro
Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys145 150 155
160Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr
165 170 175Leu Trp Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg
Leu Gln 180 185 190Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser
Val Thr Arg Asn 195 200 205Asp Thr Gly Pro Tyr Glu Cys Glu Ile Gln
Asn Pro Val Ser Ala Asn 210 215 220Arg Ser Asp Pro Val Thr Leu Asn
Val Thr Tyr Gly Pro Asp Thr Pro225 230 235 240Thr Ile Ser Pro Ser
Asp Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser 245 250 255Leu Ser Cys
Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu 260 265 270Ile
Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280
285Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser
290 295 300Val Thr Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val
Thr Glu305 310 315 320Leu Ser Pro Val Val Ala Lys Pro Gln Ile Lys
Ala Ser Lys Thr Thr 325 330 335Val Thr Gly Asp Lys Asp Ser Val Asn
Leu Thr Cys Ser Thr Asn Asp 340 345 350Thr Gly Ile Ser Ile Arg Trp
Phe Phe Lys Asn Gln Ser Leu Pro Ser 355 360 365Ser Glu Arg Met Lys
Leu Ser Gln Gly Asn Thr Thr Leu Ser Ile Asn 370 375 380Pro Val Lys
Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn385 390 395
400Pro Ile Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr
405 410 415Asn Ala Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly Ala Ile
Ala Gly 420 425 430Ile Val Ile Gly Val Val Ala Leu Val Ala Leu Ile
Ala Val Ala Leu 435 440 445Ala Cys Phe Leu His Phe Gly Lys Thr Gly
Arg Ala Ser Asp Gln Arg 450 455 460Asp Leu Thr Glu His Lys Pro Ser
Val Ser Asn His Thr Gln Asp His465 470 475 480Ser Asn Asp Pro Pro
Asn Lys Met Asn Glu Val Thr Tyr Ser Thr Leu 485 490 495Asn Phe Glu
Ala Gln Gln Pro Thr Gln Pro Thr Ser Ala Ser Pro Ser 500 505 510Leu
Thr Ala Thr Glu Ile Ile Tyr Ser Glu Val Lys Lys Gln 515 520
525619PRTHomo sapiens 6Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser
* * * * *