U.S. patent application number 16/435257 was filed with the patent office on 2019-12-26 for treatment of cancer using anti-cd19 chimeric antigen receptor.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Novartis AG, The Trustees of the University of Pennsylvania. Invention is credited to Jennifer Brogdon, John Byrd, Jason Dubovsky, Joseph A. Fraietta, Saar Gill, David Jonathan Glass, Amy Johnson, Carl H. June, Saad Kenderian, Joan Mannick, Marcela Maus, Leon Murphy, Natarajan Muthusamy, David L. Porter, Marco Ruella, William Raj Sellers, Mariusz Wasik.
Application Number | 20190388471 16/435257 |
Document ID | / |
Family ID | 53008859 |
Filed Date | 2019-12-26 |
View All Diagrams
United States Patent
Application |
20190388471 |
Kind Code |
A1 |
June; Carl H. ; et
al. |
December 26, 2019 |
TREATMENT OF CANCER USING ANTI-CD19 CHIMERIC ANTIGEN RECEPTOR
Abstract
The invention provides compositions and methods for treating
diseases associated with expression of CD19, e.g., by administering
a recombinant T cell comprising the CD19 CAR as described herein,
in combination with a kinase inhibitor, e.g., a kinase inhibitor
described herein. The invention also provides kits and compositions
described herein.
Inventors: |
June; Carl H.; (Merion
Station, PA) ; Porter; David L.; (Springfield,
PA) ; Maus; Marcela; (Lexington, MA) ; Wasik;
Mariusz; (Ardmore, PA) ; Gill; Saar;
(Philadelphia, PA) ; Fraietta; Joseph A.;
(Williamstown, NJ) ; Ruella; Marco; (Ardmore,
PA) ; Byrd; John; (Columbus, OH) ; Dubovsky;
Jason; (Columbus, OH) ; Johnson; Amy; (Dublin,
OH) ; Muthusamy; Natarajan; (Galloway, OH) ;
Kenderian; Saad; (Philadelphia, PA) ; Mannick;
Joan; (Cambridge, MA) ; Glass; David Jonathan;
(Cambridge, MA) ; Murphy; Leon; (Cambridge,
MA) ; Brogdon; Jennifer; (Sudbury, MA) ;
Sellers; William Raj; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG
The Trustees of the University of Pennsylvania |
Basel
Philadelphia |
PA |
CH
US |
|
|
Assignee: |
Novartis AG
Basel
PA
The Trustees of the University of Pennsylvania
Philadelphia
PA
The Trustees of the University of Pennsylvania
Philadelphia
|
Family ID: |
53008859 |
Appl. No.: |
16/435257 |
Filed: |
June 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14680860 |
Apr 7, 2015 |
10357514 |
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16435257 |
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62097278 |
Dec 29, 2014 |
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62087888 |
Dec 5, 2014 |
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62076238 |
Nov 6, 2014 |
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62036493 |
Aug 12, 2014 |
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62007309 |
Jun 3, 2014 |
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61976396 |
Apr 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
C12N 2501/599 20130101; A61P 35/00 20180101; A61K 2039/505
20130101; A61K 35/17 20130101; A61K 31/53 20130101; C07K 16/2803
20130101; A61K 45/06 20130101; C12N 5/0636 20130101; A61K 2039/5156
20130101; C07K 2317/24 20130101; A61K 31/436 20130101; A61K 31/519
20130101; C07K 2317/565 20130101; A61K 2039/5158 20130101; C12N
2510/00 20130101; C07K 2317/14 20130101; A61K 39/3955 20130101;
C07K 2317/622 20130101; C07K 14/7051 20130101; A61K 39/39558
20130101; A61P 43/00 20180101; C12N 2501/727 20130101; C07K 2319/03
20130101; C07K 2317/73 20130101; A61K 31/436 20130101; A61K 2300/00
20130101; A61K 31/519 20130101; A61K 2300/00 20130101; A61K 31/53
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 39/395 20060101 A61K039/395; A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06; C07K 14/725 20060101
C07K014/725; A61K 31/436 20060101 A61K031/436; A61K 31/53 20060101
A61K031/53; C12N 5/0783 20060101 C12N005/0783; C07K 16/28 20060101
C07K016/28 |
Claims
1. A method of treating a mammal having a disease associated with
expression of CD19 comprising administering to the mammal an
effective amount of a population of cells that expresses a CAR
molecule that binds CD19 (a CAR19-expressing cell), in combination
with one or more kinase inhibitors chosen from a Bruton's tyrosine
kinase (BTK) inhibitor, a cyclin dependent kinase 4 (CDK4)
inhibitor, an mTOR inhibitor, or a mitogen activated protein kinase
interacting kinase (MNK) inhibitor.
2. (canceled)
3. The method of claim 1, wherein the CAR19-expressing cell is
administered to the mammal after administration of the kinase
inhibitor.
4. The method of claim 1, wherein the mammal is, or is identified
as being, a complete or partial responder to the BTK inhibitor, or
a complete or partial responder to the CAR19-expressing cell.
5. The method of claim 1, wherein the BTK inhibitor is chosen from
ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292,
ONO-4059, CNX-774, or LFM-A13.
6. The method of claim 1, wherein: (i) the CDK4 inhibitor is chosen
from: palbociclib, aloisine A, flavopiridol,
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4-chromenone; crizotinib (PF-02341066, P276-00, RAF265,
indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322,
AT7519, AZD5438, BMS908662; or ribociclib; (ii) the mTOR inhibitor
is chosen from: rapamycin, a rapamycin analog such as everolimus,
temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502,
SF1126, XL765, or OSI-027; and (iii) the MNK inhibitor is chosen
from: CGP052088, CGP57380, cercosporamide, ETC-1780445-2, or
4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.
7-8. (canceled)
9. The method of claim 1, wherein the kinase inhibitor is ibrutinib
and the ibrutinib has a dose of about 250 mg, 300 mg, 350 mg, 400
mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg,
580 mg, or 600 mg daily.
10. The method of claim 1, wherein the cell expresses a CAR
molecule comprising an anti-CD19 binding domain, a transmembrane
domain, and an intracellular signaling domain, optionally, wherein
the intracellular signaling domain comprises a costimulatory domain
and/or a primary signaling domain, wherein the anti-CD19 binding
domain comprises: (i) a light chain complementary determining
region 1 (LC CDR1), a light chain complementary determining region
2 (LC CDR2), a light chain complementary determining region 3 (LC
CDR3), a heavy chain complementary determining region 1 (HC CDR1),
a heavy chain complementary determining region 2 (HC CDR2), and a
heavy chain complementary determining region 3 (HC CDR3) of an
anti-CD19 binding domain; (ii) a murine light chain variable region
of Table 7, a murine heavy chain variable region of Table 7, or
both; (iii) a LC CDR1 of SEQ ID NO: 5, a LC CDR2 of SEQ ID NO: 26,
and a LC CDR3 of SEQ ID NO: 27; and/or wherein the anti-CD19
binding domain comprises a HC CDR1 of SEQ ID NO: 19, a LC CDR2 of
any of SEQ ID NOS: 20-23, and a HC CDR3 of SEQ ID NO: 24; (iv) a
sequence of SEQ ID NO:59, or a sequence with 95-99% identify
thereof; or (v) a sequence chosen from: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID
NO: 12, or a sequence with 95-99% identity thereof, and optionally,
the anti-CD19 binding domain is a scFv that comprises a light chain
variable region attached to a heavy chain variable via a linker,
wherein the linker comprises a sequence of SEQ ID NO: 53.
11-19. (canceled)
20. The method of claim 10, wherein the CAR molecule comprises a
transmembrane domain of a protein chosen from: the alpha, beta or
zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,
CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,
CD137 or CD154, optionally, wherein the transmembrane domain
comprises a sequence of SEQ ID NO: 15.
21. (canceled)
22. The method of claim 20, wherein the anti-CD19 binding domain is
connected to the transmembrane domain by a hinge region, wherein
the hinge region comprises a sequence of SEQ ID NO:14 or SEQ ID
NO:45.
23. The method of claim 10, wherein the CAR molecule comprises a
costimulatory domain, wherein the costimulatory domain comprises a
sequence of SEQ ID NO: 16 or SEQ ID NO:51.
24. The method of claim 10, wherein the CAR molecule comprises an
intracellular signaling domain, wherein the intracellular signaling
domain comprises: (i) a functional signaling domain of 4-1BB, a
functional signaling domain of CD3 zeta, or both, or wherein the
intracellular signaling domain comprises a sequence of CD27, a
functional signaling domain of CD3 zeta, or both; (ii) a sequence
of SEQ ID NO: 16, a sequence of SEQ ID NO: 17, or both; (iii) a
sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 43, or both;
(iv) a sequence of SEQ ID NO: 51, a sequence of SEQ ID NO: 17, or
both; or (v) a sequence of SEQ ID NO: 51, a sequence of SEQ ID NO:
43, or both.
25-26. (canceled)
27. The method of claim 10, wherein CAR molecule comprises an amino
acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID
NO:42.
28. The method of claim 1, further comprising administration of an
agent which inhibits an immune inhibitory molecule chosen from:
PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR
beta.
29. The method of claim 1, wherein the disease associated with
expression of CD19 is a cancer, optionally, a hematological cancer
chosen from a leukemia or lymphoma.
30. (canceled)
31. The method of claim 29, wherein the cancer is chosen from:
chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL),
multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma,
B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid
leukemia (TALL), small lymphocytic leukemia (SLL), B cell
prolymphocytic leukemia, blastic plasmacytoid dendritic cell
neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma
(DLBCL), DLBCL associated with chronic inflammation, follicular
lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small
cell- or a large cell-follicular lymphoma, malignant
lymphoproliferative conditions, MALT lymphoma (extranodal marginal
zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone
lymphoma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin
lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell
neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone
lymphoma, splenic lymphoma/leukemia, splenic diffuse red pulp small
B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic
lymphoma, a heavy chain disease, plasma cell myeloma, solitary
plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal
zone lymphoma, pediatric nodal marginal zone lymphoma, primary
cutaneous follicle center lymphoma, lymphomatoid granulomatosis,
primary mediastinal (thymic) large B-cell lymphoma, intravascular
large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell
lymphoma arising in HHV8-associated multicentric Castleman disease,
primary effusion lymphoma, B-cell lymphoma, or unclassifiable
lymphoma.
32. (canceled)
33. The method of claim 1, further comprising administration of a
cytokine chosen from IL-7, IL-15, or IL-21.
34. The method of claim 1, wherein the CAR is a regulatable CAR
(RCAR), wherein the RCAR comprises: an intracellular signaling
member comprising an intracellular signaling domain and a first
switch domain, an antigen binding member comprising an antigen
binding domain that binds CD19 and a second switch domain; and a
transmembrane domain.
35. (canceled)
36. The method of claim 1, wherein the CAR19-expressing cell is
administered in combination a second kinase inhibitor, wherein the
second kinase inhibitor is other than ibrutinib, when the mammal
is, or is identified as being, a non-responder or relapser to
ibrutinib, wherein second kinase inhibitor is chosen from one or
more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292,
ONO-4059, CNX-774, or LFM-A13, or a combination thereof.
37. The method of claim 1, wherein the mammal is, or is identified
as being, a partial responder to the kinase inhibitor, and the
mammal is administered the CAR19-expressing cell, alone or in
combination with the BTK inhibitor, during the period of partial
response.
38. The method of claim 1, wherein the kinase inhibitor is
ibrutinib and the ibrutinib is formulated for administration for 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles, wherein cycle
length is 21 or 28 days.
39. (canceled)
40. The method of claim 1, wherein the mammal has undergone
lymphodepletion, wherein the lymphodepletion comprises
administration of one or more of melphalan, cytoxan,
cyclophosphamide, and fludarabine.
41. (canceled)
42. A method of making a CAR-expressing immune effector population
of cells, comprising: contacting the population of cells with a BTK
inhibitor; and introducing a nucleic acid encoding a CAR molecule
into the population of cells under conditions such that the CAR
molecule is expressed.
43. (canceled)
44. The method of claim 42, which comprises contacting the
population of cells with the BTK inhibitor for 10-20, 20-30, 30-40,
40-60, or 60-120 minutes and subsequently removing most or all of
the BTK inhibitor from the population of cells, wherein the BTK
inhibitor is chosen from: ibrutinib, GDC-0834, RN-486, CGI-560,
CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13.
45. (canceled)
46. A reaction mixture comprising a population of immune effector
cells, a BTK inhibitor, and a CAR molecule or a nucleic acid
encoding a CAR molecule, wherein the BTK inhibitor is chosen from
ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292,
ONO-4059, CNX-774, or LFM-A13.
47. A composition comprising a population of cells that expresses a
CAR molecule that binds CD19, and one or more kinase inhibitors,
wherein the kinase inhibitor is chosen from a Bruton's tyrosine
kinase (BTK) inhibitor, a cyclin dependent kinase 4 (CDK4)
inhibitor, an mTOR inhibitor, or a mitogen activated protein kinase
interacting kinase (MNK) inhibitor, wherein the CAR19-expressing
cell and the one or more kinase inhibitors are present in a single
dose form, or as two or more dose forms.
Description
[0001] This application is a divisional of U.S. Ser. No.
14/680,860, filed Apr. 7, 2015, which claims priority to U.S. Ser.
No. 61/976,396 filed Apr. 7, 2014, U.S. Ser. No. 62/007,309 filed
Jun. 3, 2014, U.S. Ser. No. 62/036,493 filed Aug. 12, 2014, U.S.
Ser. No. 62/076,238 filed Nov. 6, 2014, U.S. Ser. No. 62/087,888
filed Dec. 5, 2014, and U.S. Ser. No. 62/097,278 filed Dec. 29,
2014, the contents of which are incorporated herein by reference in
their entireties. International Application Number PCT/US15/24671,
filed Apr. 7, 2015, is also incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 6, 2015, is named N2067-7051US_SL.txt and is 249,332 bytes
in size.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the use of T
cells engineered to express a Chimeric Antigen Receptor (CAR),
e.g., in combination with another agent such as, e.g., a kinase
inhibitor and/or a cytokine, to treat a disease associated with
expression of the Cluster of Differentiation 19 protein (CD19).
BACKGROUND OF THE INVENTION
[0004] Many patients with B cell malignancies are incurable with
standard therapy. In addition, traditional treatment options often
have serious side effects. Attempts have been made in cancer
immunotherapy, however, several obstacles render this a very
difficult goal to achieve clinical effectiveness. Although hundreds
of so-called tumor antigens have been identified, these are
generally derived from self and thus are poorly immunogenic.
Furthermore, tumors use several mechanisms to render themselves
hostile to the initiation and propagation of immune attack.
[0005] Recent developments using chimeric antigen receptor (CAR)
modified autologous T cell (CART) therapy, which relies on
redirecting T cells to a suitable cell-surface molecule on cancer
cells such as B cell malignancies, show promising results in
harnessing the power of the immune system to treat B cell
malignancies and other cancers (see, e.g., Sadelain et al., Cancer
Discovery 3:388-398 (2013)). The clinical results of the murine
derived CART19 (i.e., "CTL019") have shown promise in establishing
complete remissions in patients suffering with CLL as well as in
childhood ALL (see, e.g., Kalos et al., Sci Transl Med 3:95ra73
(2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM
368:1509-1518 (2013)). Besides the ability for the chimeric antigen
receptor on the genetically modified T cells to recognize and
destroy the targeted cells, a successful therapeutic T cell therapy
needs to have the ability to proliferate and persist over time, and
to further monitor for leukemic cell escapees. The variable quality
of T cells whether it's a result of anergy, suppression or
exhaustion will have effects on CAR-transformed T cells'
performance but for which skilled practitioners have limited
control over at this time. To be effective, CAR transformed patient
T cells need to persist and maintain the ability to proliferate in
response to the CAR's antigen. It has been shown that ALL patient T
cells perform can do this with CART19 comprising a murine scFv
(see, e.g., Grupp et al., NEJM 368:1509-1518 (2013)).
SUMMARY OF THE INVENTION
[0006] The disclosure features, at least in part, compositions and
methods of treating disorders such as cancer (e.g., hematological
cancers or other B-cell malignancies) using immune effector cells
(e.g., T cells or NK cells) that express a Chimeric Antigen
Receptor (CAR) molecule (e.g., a CAR that binds to a B-cell
antigen, e.g., Cluster of Differentiation 19 protein (CD19) (e.g.,
OMIM Acc. No. 107265, Swiss Prot. Acc No. P15391). The compositions
include, and the methods include administering, immune effector
cells (e.g., T cells or NK cells) expressing a B cell targeting
CAR, in combination with a kinase inhibitor (e.g., one or more of a
CDK4/6 inhibitor, a BTK inhibitor, an mTOR inhibitor, a MNK
inhibitor, a dual PI3K/mTOR inhibitor, or a combination thereof).
In some embodiments, the combination maintains, or has better
clinical effectiveness, as compared to either therapy alone. The
invention further pertains to the use of engineered cells, e.g.,
immune effector cells (e.g., T cells or NK cells), to express a CAR
molecule that binds to a B-cell antigen, e.g., CD19, in combination
with a kinase inhibitor (e.g., a kinase inhibitor chosen from one
or more of a cyclin dependent kinase 4 (CDK4) inhibitor, a Bruton's
tyrosine kinase (BTK) inhibitor, an mTOR inhibitor, a mitogen
activated protein kinase interacting kinase (MNK) inhibitor, a dual
phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, or a
combination thereof) to treat a disorder associated with expression
of a B-cell antigen, e.g., CD19 (e.g., a cancer, e.g., a
hematological cancer).
[0007] Accordingly, in one aspect, the invention pertains to a
method of treating a subject, e.g., a mammal, having a disease
associated with expression of a B-cell antigen, e.g., CD19. The
method comprises administering to the mammal an effective amount of
a cell e.g., an immune effector cell (e.g., a T cell or NK cell)
that expresses a CAR molecule that binds the B-cell antigen, in
combination with a kinase inhibitor, e.g., a kinase inhibitor
described herein. In one embodiment, the CAR molecule binds to
CD19, e.g., a CAR molecule that binds CD19 described herein. In
other embodiments, the CAR molecule binds to one or more of CD20,
CD22 or ROR1.
[0008] In one embodiment, the disease associated with expression of
a B-cell antigen (e.g., expression of one or more of CD19, CD20,
CD22 or ROR1), is selected from a proliferative disease such as a
cancer, a malignancy, or a precancerous condition such as a
myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is
a non-cancer related indication associated with expression of the
B-cell antigen, e.g., one or more of CD19, CD20, CD22 or ROR1. In
one embodiment, the disease is a solid or liquid tumor. In one
embodiment, the cancer is pancreatic cancer. In one embodiment, the
disease is a hematologic cancer. In one embodiment, the
hematological cancer is leukemia. In one embodiment, the cancer is
selected from the group consisting of one or more acute leukemias
including but not limited to B-cell acute lymphoid leukemia (BALL),
T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia
(SLL), acute lymphoid leukemia (ALL); one or more chronic leukemias
including but not limited to chronic myelogenous leukemia (CML),
chronic lymphocytic leukemia (CLL). Additional hematological
cancers or hematologic conditions include, but are not limited to,
mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic
plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse
large B cell lymphoma (DLBCL), follicular lymphoma, hairy cell
leukemia, small cell- or a large cell-follicular lymphoma,
malignant lymphoproliferative conditions, MALT lymphoma, Marginal
zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic
syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic
lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom
macroglobulinemia. In certain embodiments, the disease associated
with B-cell antigen (e.g., e.g., one or more of CD19, CD20, CD22 or
ROR1) expression is a "preleukemia" which is a diverse collection
of hematological conditions united by ineffective production (or
dysplasia) of myeloid blood cells. In some embodiments, the disease
associated with B-cell antigen (e.g., one or more of CD19, CD20,
CD22 or ROR1) expression includes, but is not limited to atypical
and/or non-classical cancers, malignancies, precancerous conditions
or proliferative diseases expressing the B-cell antigen (e.g., one
or more of CD19, CD20, CD22 or ROR1). Any combination of the
diseases associated with B-cell antigen (e.g., one or more of CD19,
CD20, CD22 or ROR1) expression described herein can be treated with
the methods and compositions described herein.
[0009] In one embodiment, the disease associated with expression of
the B-cell antigen (e.g., one or more of CD19, CD20, CD22 or ROR1)
is a lymphoma, e.g., MCL, Hodgkin lymphoma, or DLBCL. In one
embodiment, the disease associated with expression of the B-cell
antigen (e.g., one or more of CD19, CD20, CD22 or ROR1) is
leukemia, e.g., SLL, CLL and/or ALL. In one embodiment, the disease
associated with expression of the B-cell antigen is multiple
myeloma (e.g., a multiple myeloma that is CD19-negative, e.g.,
having a vast majority (99.95%) of the neoplastic plasma cells with
a CD19-negative phenotype, e.g., as detected by both flow cytometry
and RT-PCR.
[0010] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., a CDK4 inhibitor described herein, e.g., a CD4/6 inhibitor,
such as, e.g.,
6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylami-
no)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred
to as palbociclib or PD0332991). In one embodiment, the kinase
inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described
herein, such as, e.g., ibrutinib. In one embodiment, the kinase
inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described
herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The
mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2
inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor
described herein. In one embodiment, the kinase inhibitor is a MNK
inhibitor, e.g., a MNK inhibitor described herein, such as, e.g.,
4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d]pyrimidine. The MNK
inhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b
inhibitor. In one embodiment, the inhibitor can be a dual PI3K/mTOR
inhibitor, e.g., PF-04695102.
[0011] In one embodiment, the kinase inhibitor is a CDK4 inhibitor
selected from aloisine A; flavopiridol or HMR-1275,
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4-chromenone; crizotinib (PF-02341066);
2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3--
pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00);
1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N--
[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265);
indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991);
dinaciclib (SCH727965);
N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-car-
boxamide (BMS 387032);
4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]-
amino]-benzoic acid (MLN8054);
5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methy-
l-3-pyridinemethanamine (AG-024322);
4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid
N-(piperidin-4-yl)amide (AT7519);
4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phen-
yl]-2-pyrimidinamine (AZD5438); XL281 (BMS908662); and
ribociclib.
[0012] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., palbociclib (PD0332991), and the palbociclib is administered
at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100
mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g.,
75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily
for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21
day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
or more cycles of palbociclib are administered.
[0013] In one embodiment, the kinase inhibitor is a BTK inhibitor
selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560;
CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In a
preferred embodiment, the BTK inhibitor does not reduce or inhibit
the kinase activity of interleukin-2-inducible kinase (ITK), and is
selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;
CC-292; ONO-4059; CNX-774; and LFM-A13.
[0014] In one embodiment, the kinase inhibitor is a BTK inhibitor,
e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a
dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460
mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g.,
250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily
for 21 day cycle cycle, or daily for 28 day cycle. In one
embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of
ibrutinib are administered.
[0015] In one embodiment, the kinase inhibitor is an mTOR inhibitor
selected from temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2
[(1R,9S,12S, 15R, 16E, 18R,
19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17-
,21,23,
29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[3-
0.3.1.0.sup.4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxyc-
yclohexyl dimethylphosphinate, also known as AP23573 and MK8669;
everolimus (RAD001); rapamycin (AY22989); semapimod;
(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-me-
thoxyphenyl)methanol (AZD8055);
2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-
-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and
N.sup.2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholiniu-
m-4-yl]methoxy]butyl]-L-arginylglycyl-L-.alpha.-aspartylL-serine-,
inner salt (SF1126); and XL765.
[0016] In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., rapamycin, and the rapamycin is administered at a
dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg
(e.g., 6 mg) daily for a period of time, e.g., daily for 21 day
cycle cycle, or daily for 28 day cycle. In one embodiment, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are
administered. In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., everolimus and the everolimus is administered at a
dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9
mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily
for a period of time, e.g., daily for 28 day cycle. In one
embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of
everolimus are administered.
[0017] In one embodiment, the kinase inhibitor is an MNK inhibitor
selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo
[3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and
4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d]pyrimidine.
[0018] In one embodiment, the kinase inhibitor is a dual
phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected
from
2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-
-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502);
N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-mo-
rpholinyl-1,3,5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587);
2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,-
5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib
(GDC-0980, RG7422);
2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
-3-pyridinyl}benzenesulfonamide (GSK2126458);
8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluorometh-
yl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one Maleic acid
(NVP-BGT226);
3-[4-(4-Morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol
(PI-103);
5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2--
amine (VS-5584, SB2343); and
N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyp-
henyl)carbonyl]aminophenylsulfonamide (XL765).
[0019] In one embodiment, the cell expresses a CAR molecule
comprising an anti-CD19 binding domain (e.g., a murine or humanized
antibody or antibody fragment that specifically binds to CD19), a
transmembrane domain, and an intracellular signaling domain (e.g.,
an intracellular signaling domain comprising a costimulatory domain
and/or a primary signaling domain). In one embodiment, the CAR
comprises an antibody or antibody fragment which includes an
anti-CD19 binding domain described herein (e.g., a murine or
humanized antibody or antibody fragment that specifically binds to
CD19 as described herein), a transmembrane domain described herein,
and an intracellular signaling domain described herein (e.g., an
intracellular signaling domain comprising a costimulatory domain
and/or a primary signaling domain described herein).
[0020] In one embodiment, the CAR molecule is capable of binding
CD19 (e.g., wild-type or mutant human CD19). In one embodiment, the
CAR molecule comprises an anti-CD19 binding domain comprising one
or more (e.g., all three) light chain complementary determining
region 1 (LC CDR1), light chain complementary determining region 2
(LC CDR2), and light chain complementary determining region 3 (LC
CDR3) of an anti-CD19 binding domain described herein, and one or
more (e.g., all three) heavy chain complementary determining region
1 (HC CDR1), heavy chain complementary determining region 2 (HC
CDR2), and heavy chain complementary determining region 3 (HC CDR3)
of an anti-CD19 binding domain described herein, e.g., an anti-CD19
binding domain comprising one or more, e.g., all three, LC CDRs and
one or more, e.g., all three, HC CDRs. In one embodiment, the
anti-CD19 binding domain comprises one or more (e.g., all three)
heavy chain complementary determining region 1 (HC CDR1), heavy
chain complementary determining region 2 (HC CDR2), and heavy chain
complementary determining region 3 (HC CDR3) of an anti-CD19
binding domain described herein, e.g., the anti-CD19 binding domain
has two variable heavy chain regions, each comprising a HC CDR1, a
HC CDR2 and a HC CDR3 described herein. In one embodiment, the
anti-CD19 binding domain comprises a murine light chain variable
region described herein (e.g., in Table 7) and/or a murine heavy
chain variable region described herein (e.g., in Table 7). In one
embodiment, the anti-CD19 binding domain is a scFv comprising a
murine light chain and a murine heavy chain of an amino acid
sequence of Table 7. In an embodiment, the anti-CD19 binding domain
(e.g., an scFv) comprises: a light chain variable region comprising
an amino acid sequence having at least one, two or three
modifications (e.g., substitutions) but not more than 30, 20 or 10
modifications (e.g., substitutions) of an amino acid sequence of a
light chain variable region provided in Table 7, or a sequence with
95-99% identity with an amino acid sequence of Table 7; and/or a
heavy chain variable region comprising an amino acid sequence
having at least one, two or three modifications (e.g.,
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions) of an amino acid sequence of a heavy chain variable
region provided in Table 7, or a sequence with 95-99% identity to
an amino acid sequence of Table 7. In one embodiment, the anti-CD19
binding domain comprises a sequence of SEQ ID NO:59, or a sequence
with 95-99% identify thereof. In one embodiment, the anti-CD19
binding domain is a scFv, and a light chain variable region
comprising an amino acid sequence described herein, e.g., in Table
7, is attached to a heavy chain variable region comprising an amino
acid sequence described herein, e.g., in Table 7, via a linker,
e.g., a linker described herein. In one embodiment, the anti-CD19
binding domain includes a (Gly.sub.4-Ser)n linker, wherein n is 1,
2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light
chain variable region and heavy chain variable region of a scFv can
be, e.g., in any of the following orientations: light chain
variable region-linker-heavy chain variable region or heavy chain
variable region-linker-light chain variable region.
[0021] In one embodiment, the CAR molecule comprises a humanized
anti-CD19 binding domain that includes one or more (e.g., all
three) light chain complementary determining region 1 (LC CDR1),
light chain complementary determining region 2 (LC CDR2), and light
chain complementary determining region 3 (LC CDR3) of a humanized
anti-CD19 binding domain described herein, and one or more (e.g.,
all three) heavy chain complementary determining region 1 (HC
CDR1), heavy chain complementary determining region 2 (HC CDR2),
and heavy chain complementary determining region 3 (HC CDR3) of a
humanized anti-CD19 binding domain described herein, e.g., a
humanized anti-CD19 binding domain comprising one or more, e.g.,
all three, LC CDRs and one or more, e.g., all three, HC CDRs. In
one embodiment, the humanized anti-CD19 binding domain comprises at
least HC CDR2. In one embodiment, the humanized anti-CD19 binding
domain comprises one or more (e.g., all three) heavy chain
complementary determining region 1 (HC CDR1), heavy chain
complementary determining region 2 (HC CDR2), and heavy chain
complementary determining region 3 (HC CDR3) of a humanized
anti-CD19 binding domain described herein, e.g., the humanized
anti-CD19 binding domain has two variable heavy chain regions, each
comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In
one embodiment, the humanized anti-CD19 binding domain comprises at
least HC CDR2. In one embodiment, the light chain variable region
comprises one, two, three or all four framework regions of VK3_L25
germline sequence. In one embodiment, the light chain variable
region has a modification (e.g., substitution, e.g., a substitution
of one or more amino acid found in the corresponding position in
the murine light chain variable region of SEQ ID NO: 58, e.g., a
substitution at one or more of positions 71 and 87). In one
embodiment, the heavy chain variable region comprises one, two,
three or all four framework regions of VH4_4-59 germline sequence.
In one embodiment, the heavy chain variable region has a
modification (e.g., substitution, e.g., a substitution of one or
more amino acid found in the corresponding position in the murine
heavy chain variable region of SEQ ID NO: 58, e.g., a substitution
at one or more of positions 71, 73 and 78). In one embodiment, the
humanized anti-CD19 binding domain comprises a light chain variable
region described herein (e.g., in Table 3) and/or a heavy chain
variable region described herein (e.g., in Table 3). In one
embodiment, the humanized anti-CD19 binding domain is a scFv
comprising a light chain and a heavy chain of an amino acid
sequence of Table 3. In an embodiment, the humanized anti-CD19
binding domain (e.g., an scFv) comprises: a light chain variable
region comprising an amino acid sequence having at least one, two
or three modifications (e.g., substitutions) but not more than 30,
20 or 10 modifications (e.g., substitutions) of an amino acid
sequence of a light chain variable region provided in Table 3, or a
sequence with 95-99% identity with an amino acid sequence of Table
3; and/or a heavy chain variable region comprising an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions) of an amino acid sequence of a heavy chain variable
region provided in Table 3, or a sequence with 95-99% identity to
an amino acid sequence of Table 3. In one embodiment, the humanized
anti-CD19 binding domain comprises a sequence selected from a group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO: 12, or a sequence with
95-99% identify thereof. In one embodiment, the humanized anti-CD19
binding domain is a scFv, and a light chain variable region
comprising an amino acid sequence described herein, e.g., in Table
3, is attached to a heavy chain variable region comprising an amino
acid sequence described herein, e.g., in Table 3, via a linker,
e.g., a linker described herein. In one embodiment, the humanized
anti-CD19 binding domain includes a (Gly.sub.4-Ser)n linker,
wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:
53). The light chain variable region and heavy chain variable
region of a scFv can be, e.g., in any of the following
orientations: light chain variable region-linker-heavy chain
variable region or heavy chain variable region-linker-light chain
variable region.
[0022] In one embodiment, the CAR molecule comprises a
transmembrane domain of a protein selected from the group
consisting of the alpha, beta or zeta chain of the T-cell receptor,
CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,
CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment,
the transmembrane domain comprises a sequence of SEQ ID NO: 15. In
one embodiment, the transmembrane domain comprises an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions) but not more than 20, 10 or 5 modifications (e.g.,
substitutions) of an amino acid sequence of SEQ ID NO: 15, or a
sequence with 95-99% identity to an amino acid sequence of SEQ ID
NO: 15.
[0023] In one embodiment, the anti-CD19 binding domain is connected
to the transmembrane domain by a hinge region, e.g., a hinge region
described herein. In one embodiment, the encoded hinge region
comprises SEQ ID NO:14 or SEQ ID NO:45, or a sequence with 95-99%
identity thereof.
[0024] In one embodiment, the CAR molecule further comprises a
sequence encoding a costimulatory domain, e.g., a costimulatory
domain described herein. In one embodiment, the costimulatory
domain comprises a functional signaling domain of a protein
selected from the group consisting of OX40, CD2, CD27, CD28, CDS,
ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In one embodiment,
the costimulatory domain comprises a sequence of SEQ ID NO: 16. In
one embodiment, the costimulatory domain comprises a sequence of
SEQ ID NO:51. In one embodiment, the costimulatory domain comprises
an amino acid sequence having at least one, two or three
modifications (e.g., substitutions) but not more than 20, 10 or 5
modifications (e.g., substitutions) of an amino acid sequence of
SEQ ID NO: 16 or SEQ ID NO:51, or a sequence with 95-99% identity
to an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:51.
[0025] In one embodiment, the CAR molecule further comprises a
sequence encoding an intracellular signaling domain, e.g., an
intracellular signaling domain described herein. In one embodiment,
the intracellular signaling domain comprises a functional signaling
domain of 4-1BB and/or a functional signaling domain of CD3 zeta.
In one embodiment, the intracellular signaling domain comprises the
sequence of SEQ ID NO: 16 and/or the sequence of SEQ ID NO: 17. In
one embodiment, the intracellular signaling domain comprises the
sequence of SEQ ID NO: 16 and/or the sequence of SEQ ID NO:43. In
one embodiment, the intracellular signaling domain comprises a
functional signaling domain of CD27 and/or a functional signaling
domain of CD3 zeta. In one embodiment, the intracellular signaling
domain comprises the sequence of SEQ ID NO: 51 and/or the sequence
of SEQ ID NO:17. In one embodiment, the intracellular signaling
domain comprises the sequence of SEQ ID NO:51 and/or the sequence
of SEQ ID NO:43. In one embodiment, the intracellular signaling
domain comprises an amino acid sequence having at least one, two or
three modifications (e.g., substitutions) but not more than 20, 10
or 5 modifications (e.g., substitutions) of an amino acid sequence
of SEQ ID NO:16 or SEQ ID NO:51 and/or an amino acid sequence of
SEQ ID NO:17 or SEQ ID NO:43, or a sequence with 95-99% identity to
an amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and/or an
amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43. In one
embodiment, the intracellular signaling domain comprises the
sequence of SEQ ID NO:16 or SEQ ID NO:51 and the sequence of SEQ ID
NO: 17 or SEQ ID NO:43, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain.
[0026] In one embodiment, the CAR molecule further comprises a
leader sequence, e.g., a leader sequence described herein. In one
embodiment, the leader sequence comprises an amino acid sequence of
SEQ ID NO: 13, or a sequence with 95-99% identity to an amino acid
sequence of SEQ ID NO:13.
[0027] In one embodiment, the CAR molecule comprises a leader
sequence, e.g., a leader sequence described herein, e.g., a leader
sequence of SEQ ID NO: 13, or having 95-99% identity thereof; an
anti-CD19 binding domain described herein, e.g., an anti-CD19
binding domain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC
CDR1, a HC CDR2 and a HC CDR3 described herein, e.g., a murine
anti-CD19 binding domain described in Table 7, a humanized
anti-CD19 binding domain described in Table 3, or a sequence with
95-99% identify thereof; a hinge region, e.g., a hinge region
described herein, e.g., a hinge region of SEQ ID NO: 14 or having
95-99% identity thereof; a transmembrane domain, e.g., a
transmembrane domain described herein, e.g., a transmembrane domain
having a sequence of SEQ ID NO:15 or a sequence having 95-99%
identity thereof; an intracellular signaling domain, e.g., an
intracellular signaling domain described herein (e.g., an
intracellular signaling domain comprising a costimulatory domain
and/or a primary signaling domain). In one embodiment, the
intracellular signaling domain comprises a costimulatory domain,
e.g., a costimulatory domain described herein, e.g., a 4-1BB
costimulatory domain having a sequence of SEQ ID NO:16 or SEQ ID
NO:51, or having 95-99% identity thereof, and/or a primary
signaling domain, e.g., a primary signaling domain described
herein, e.g., a CD3 zeta stimulatory domain having a sequence of
SEQ ID NO: 17 or SEQ ID NO:43, or having 95-99% identity
thereof.
[0028] In one embodiment, the CAR molecule comprises (e.g.,
consists of) an amino acid sequence of SEQ ID NO:58, SEQ ID NO:31,
SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41 or SEQ ID NO:42, or an amino acid sequence having at least
one, two, three, four, five, 10, 15, 20 or 30 modifications (e.g.,
substitutions) but not more than 60, 50 or 40 modifications (e.g.,
substitutions) of an amino acid sequence of SEQ ID NO:58, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ
ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,
SEQ ID NO:41 or SEQ ID NO:42, or an amino acid sequence having 85%,
90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence
of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.
[0029] In one embodiment, the cell expressing the CAR molecule
comprises a vector that includes a nucleic acid sequence encoding
the CAR molecule. In one embodiment, the vector is selected from
the group consisting of a DNA, a RNA, a plasmid, a lentivirus
vector, adenoviral vector, or a retrovirus vector. In one
embodiment, the vector is a lentivirus vector. In one embodiment,
the vector further comprises a promoter. In one embodiment, the
promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter
comprises a sequence of SEQ ID NO: 100. In one embodiment, the
vector is an in vitro transcribed vector, e.g., a vector that
transcribes RNA of a nucleic acid molecule described herein. In one
embodiment, the nucleic acid sequence in the in vitro vector
further comprises a poly(A) tail, e.g., a poly A tail described
herein, e.g., comprising about 150 adenosine bases (SEQ ID NO:
104). In one embodiment, the nucleic acid sequence in the in vitro
vector further comprises a 3'UTR, e.g., a 3' UTR described herein,
e.g., comprising at least one repeat of a 3'UTR derived from human
beta-globulin. In one embodiment, the nucleic acid sequence in the
in vitro vector further comprises promoter, e.g., a T2A
promoter.
[0030] In certain embodiments of the compositions and methods
disclosed herein, the cell expressing the CAR molecule (also
referred to herein as a "CAR-expressing cell") is a cell or
population of cells as described herein, e.g., a human immune
effector cell or population of cells (e.g., a human T cell or a
human NK cell, e.g., a human T cell described herein or a human NK
cell described herein). In one embodiment, the human T cell is a
CD8+ T cell. In one embodiment, the cell is an autologous T cell.
In one embodiment, the cell is an allogeneic T cell. In one
embodiment, the cell is a T cell and the T cell is diaglycerol
kinase (DGK) deficient. In one embodiment, the cell is a T cell and
the T cell is Ikaros deficient. In one embodiment, the cell is a T
cell and the T cell is both DGK and Ikaros deficient. It shall be
understood that the compositions and methods disclosed herein
reciting the term "cell" encompass compositions and methods
comprising one or more cells, e.g., a population of cells.
[0031] In another embodiment, the cell expressing the CAR molecule,
e.g., as described herein, can further express another agent, e.g.,
an agent which enhances the activity of a CAR-expressing cell.
[0032] In one embodiment, the method further includes administering
a cell expressing the CAR molecule, as described herein, optionally
in combination with a kinase inhibitor, e.g., a BTK inhibitor such
as ibrutinib, in combination with an agent which enhances the
activity of a CAR-expressing cell. In certain embodiments, the
agent is a cytokine, e.g., IL-7, IL-15, IL-21, or a combination
thereof. In one embodiment, the method includes administering IL-7
to the subject. The cytokine can be delivered in combination with,
e.g., simultaneously or shortly after, administration of the
CAR-expressing cell. Alternatively, the cytokine can be delivered
after a prolonged period of time after administration of the
CAR-expressing cell, e.g., after assessment of the subject's
response to the CAR-expressing cell.
[0033] In other embodiments, the agent which enhances the activity
of a CAR-expressing cell can be an agent which inhibits an immune
inhibitory molecule. Examples of immune inhibitory molecules
include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3
and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and
TGFR beta. In one embodiment, the agent which inhibits an immune
inhibitory molecule comprises a first polypeptide, e.g., an immune
inhibitory molecule, associated with a second polypeptide that
provides a positive signal to the cell, e.g., an intracellular
signaling domain described herein. In one embodiment, the agent
comprises a first polypeptide, e.g., of an inhibitory molecule such
as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR
beta, or a fragment of any of these (e.g., at least a portion of
the extracellular domain of any of these), and a second polypeptide
which is an intracellular signaling domain described herein (e.g.,
comprising a costimulatory domain (e.g., 41BB, CD27 or CD28, e.g.,
as described herein) and/or a primary signaling domain (e.g., a CD3
zeta signaling domain described herein). In one embodiment, the
agent comprises a first polypeptide of PD1 or a fragment thereof
(e.g., at least a portion of the extracellular domain of PD1), and
a second polypeptide of an intracellular signaling domain described
herein (e.g., a CD28 signaling domain described herein and/or a CD3
zeta signaling domain described herein).
[0034] In one embodiment, lymphocyte infusion, for example
allogeneic lymphocyte infusion, is used in the treatment of the
cancer, wherein the lymphocyte infusion comprises at least one
CAR-expressing cell that binds toa B-cell antigen (e.g., CD19)
(also referred to herein as CD19 CAR-expressing cell), as described
herein. In one embodiment, autologous lymphocyte infusion is used
in the treatment of the cancer, wherein the autologous lymphocyte
infusion comprises at least one CD19-expressing cell.
[0035] In one embodiment, the CD19 CAR expressing cell, e.g., T
cell, is administered to a subject that has received a previous
stem cell transplantation, e.g., autologous stem cell
transplantation.
[0036] In one embodiment, the CD19 CAR expressing cell, e.g., T
cell, is administered to a subject that has received a previous
dose of melphalan.
[0037] In one embodiment, the cell expressing the CAR molecule,
e.g., a CAR molecule described herein, is administered in
combination with an agent that ameliorates one or more side effect
associated with administration of a cell expressing a CAR molecule,
e.g., an agent described herein.
[0038] In one embodiment, the kinase inhibitor, is administered in
combination with an agent that ameliorates one or more side effect
associated with administration of the kinase inhibitor, e.g., an
agent described herein.
[0039] In one embodiment, the cell expressing the CAR molecule,
e.g., a CAR molecule described herein, and the kinase inhibitor are
administered in combination with an additional agent that treats
the disease associated with CD19, e.g., an additional agent
described herein.
[0040] In one embodiment, the cells expressing a CAR molecule,
e.g., a CAR molecule described herein, are administered at a dose
and/or dosing schedule described herein.
[0041] In one embodiment, the CAR molecule is introduced into T
cells, e.g., using in vitro transcription, and the subject (e.g.,
human) receives an initial administration of cells comprising a CAR
molecule, and one or more subsequent administrations of cells
comprising a CAR molecule, wherein the one or more subsequent
administrations are administered less than 15 days, e.g., 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous
administration. In one embodiment, more than one administration of
cells comprising a CAR molecule are administered to the subject
(e.g., human) per week, e.g., 2, 3, or 4 administrations of cells
comprising a CAR molecule are administered per week. In one
embodiment, the subject (e.g., human subject) receives more than
one administration of cells comprising a CAR molecule per week
(e.g., 2, 3 or 4 administrations per week) (also referred to herein
as a cycle), followed by a week of no administration of cells
comprising a CAR molecule, and then one or more additional
administration of cells comprising a CAR molecule (e.g., more than
one administration of the cells comprising a CAR molecule per week)
is administered to the subject. In another embodiment, the subject
(e.g., human subject) receives more than one cycle of cells
comprising a CAR molecule, and the time between each cycle is less
than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the cells
comprising a CAR molecule are administered every other day for 3
administrations per week. In one embodiment, the cells comprising a
CAR molecule are administered for at least two, three, four, five,
six, seven, eight or more weeks.
[0042] In one embodiment, the combination of the kinase inhibitor
and the cells expressing a CAR molecule, e.g., a CAR molecule
described herein, are administered as a first line treatment for
the disease, e.g., the cancer, e.g., the cancer described herein.
In another embodiment, the combination of the kinase inhibitor and
the cells expressing a CAR molecule, e.g., a CAR molecule described
herein, are administered as a second, third, fourth line treatment
for the disease, e.g., the cancer, e.g., the cancer described
herein.
[0043] In one embodiment, a cell (e.g., a population of cells)
described herein is administered to the subject.
[0044] In one embodiment, the method includes administering a
population of cells, a plurality of which comprise a CAR molecule
described herein. In some embodiments, the population of
CAR-expressing cells comprises a mixture of cells expressing
different CARs. For example, in one embodiment, the population of
CAR-expressing cells can include a first cell expressing a CAR
having an anti-CD19 binding domain described herein, and a second
cell expressing a CAR having a different anti-CD19 binding domain,
e.g., an anti-CD19 binding domain described herein that differs
from the anti-CD19 binding domain in the CAR expressed by the first
cell. As another example, the population of CAR-expressing cells
can include a first cell expressing a CAR that includes an
anti-CD19 binding domain, e.g., as described herein, and a second
cell expressing a CAR that includes an antigen binding domain to a
target other than CD19 (e.g., CD123 or mesothelin). In one
embodiment, the population of CAR-expressing cells includes, e.g.,
a first cell expressing a CAR that includes a primary intracellular
signaling domain, and a second cell expressing a CAR that includes
a secondary signaling domain.
[0045] In one embodiment, the method includes administering a
population of cells wherein at least one cell in the population
expresses a CAR having an anti-CD19 domain described herein, and an
agent which enhances the activity of a CAR-expressing cell, e.g., a
second cell expressing the agent which enhances the activity of a
CAR-expressing cell. For example, in one embodiment, the agent can
be an agent which inhibits an immune inhibitory molecule. Examples
of immune inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3,
CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,
BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment,
the agent which inhibits an immune inhibitory molecule comprises a
first polypeptide, e.g., an inhibitory molecule, associated with a
second polypeptide that provides a positive signal to the cell,
e.g., an intracellular signaling domain described herein. In one
embodiment, the agent comprises a first polypeptide, e.g., of an
inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,
CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT,
LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these
(e.g., at least a portion of an extracellular domain of any of
these), and a second polypeptide which is an intracellular
signaling domain described herein (e.g., comprising a costimulatory
domain (e.g., 41BB, CD27 or CD28, e.g., as described herein) and/or
a primary signaling domain (e.g., a CD3 zeta signaling domain
described herein). In one embodiment, the agent comprises a first
polypeptide of PD1 or a fragment thereof (e.g., at least a portion
of the extracellular domain of PD1), and a second polypeptide of an
intracellular signaling domain described herein (e.g., a CD28
signaling domain described herein and/or a CD3 zeta signaling
domain described herein).
[0046] In another aspect, the invention pertains to a cell
expressing a CAR molecule described herein for use as a medicament
in combination with a kinase inhibitor, e.g., a kinase inhibitor
described herein (e.g., a BTK inhibitor such as ibrutinib). In
another aspect, the invention pertains to a kinase inhibitor
described herein (e.g., a BTK inhibitor such as ibrutinib) for use
as a medicament in combination with a cell expressing a CAR
molecule described herein.
[0047] In another aspect, the invention pertains to a cell
expressing a CAR molecule described herein for use in combination
with a kinase inhibitor, e.g., a kinase inhibitor described herein
(e.g., a BTK inhibitor such as ibrutinib), in the treatment of a
disease expressing the B-cell antigen (e.g., CD19). In another
aspect, the invention pertains to a kinase inhibitor described
herein (e.g., a BTK inhibitor such as ibrutinib), for use in
combination with a cell expressing a CAR molecule described herein,
in the treatment of a disease expressing the B-cell antigen (e.g.,
CD19). The disease may be, e.g., a cancer such as a hematologic
cancer. The cancer may be, e.g., a lymphoma, CLL, MCL, ALL, DLBCL,
multiple myeloma, or another cancer described herein.
[0048] In another aspect, the invention pertains to a cell
expressing a CAR molecule described herein for use as a medicament
in combination with a cytokine, e.g., IL-7, IL-15 and/or IL-21 as
described herein. In another aspect, the invention pertains to a
cytokine described herein for use as a medicament in combination
with a cell expressing a CAR molecule described herein.
[0049] In another aspect, the invention pertains to a cell
expressing a CAR molecule described herein for use in combination
with a cytokine, e.g., IL-7, IL-15 and/or IL-21 as described
herein, in the treatment of a disease expressing CD19. In another
aspect, the invention pertains to a cytokine described herein for
use in combination with a cell expressing a CAR molecule described
herein, in the treatment of a disease expressing CD19.
[0050] In another aspect, the invention pertains to a method of
treating a mammal having Hodgkin lymphoma, comprising administering
to the mammal an effective amount of the cell (e.g., cells)
expressing a CAR molecule, e.g., a CAR molecule described
herein.
[0051] In one embodiment, the cell expressing a CAR molecule, e.g.,
a CAR molecule described herein, is administered in combination
with an agent that increases the efficacy of a cell expressing a
CAR molecule, e.g., an agent described herein.
[0052] In one embodiment, the cell expressing a CAR molecule, e.g.,
a CAR molecule described herein, is administered in combination
with an agent that ameliorates one or more side effect associated
with administration of a cell expressing a CAR molecule, e.g., an
agent described herein.
[0053] In one embodiment, the cell expressing a CAR molecule, e.g.,
a CAR molecule described herein, is administered in combination
with an agent that treats Hodgkin lymphoma, e.g., an agent
described herein.
[0054] In one embodiment, the cell expressing a CAR molecule, e.g.,
a CAR molecule described herein, is administered in combination
with a low, immune enhancing dose of an mTOR inhibitor, e.g., an
mTOR inhibitor described herein. While not wishing to be bound by
theory, it is believed that treatment with a low, immune enhancing,
dose (e.g., a dose that is insufficient to completely suppress the
immune system but sufficient to improve immune function) is
accompanied by a decrease in PD-1 positive T cells or an increase
in PD-1 negative cells. PD-1 positive T cells, but not PD-1
negative T cells, can be exhausted by engagement with cells which
express a PD-1 ligand, e.g., PD-L1 or PD-L2.
[0055] In an embodiment this approach can be used to optimize the
performance of a CAR cell described herein in the subject. While
not wishing to be bound by theory, it is believed that, in an
embodiment, the performance of endogenous, non-modified immune
effector cells, e.g., T cells, is improved. While not wishing to be
bound by theory, it is believed that, in an embodiment, the
performance of a CD19 CAR expressing cell is improved. In other
embodiments, cells, e.g., T cells, which have, or will be
engineered to express a CAR, can be treated ex vivo by contact with
an amount of an mTOR inhibitor that increases the number of PD1
negative immune effector cells, e.g., T cells or increases the
ratio of PD1 negative immune effector cells, e.g., T cells/PD1
positive immune effector cells, e.g., T cells.
[0056] In an embodiment, administration of a low, immune enhancing,
dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g.,
RAD001, or a catalytic inhibitor, is initiated prior to
administration of an CAR expressing cell described herein, e.g., T
cells. In an embodiment, the mTOR inhibitor is RAD001 or rapamycin.
In an embodiment, the CAR cells are administered after a sufficient
time, or sufficient dosing, of an mTOR inhibitor, such that the
level of PD1 negative immune effector cells, e.g., T cells, or the
ratio of PD1 negative immune effector cells, e.g., T cells/PD1
positive immune effector cells, e.g., T cells, has been, at least
transiently, increased.
[0057] In an embodiment, the cell, e.g., an immune effector cell
(e.g., a T cell or NK cell), to be engineered to express a CAR, is
harvested after a sufficient time, or after sufficient dosing of
the low, immune enhancing, dose of an mTOR inhibitor, such that the
level of PD1 negative immune effector cells, e.g., T cells, or the
ratio of PD1 negative immune effector cells, e.g., T cells/PD1
positive immune effector cells, e.g., T cells, in the subject or
harvested from the subject has been, at least transiently,
increased.
[0058] In embodiments, any of the methods described herein further
comprise performing lymphodepletion on a subject, e.g., prior to
administering the one or more cells that express a CAR molecule
described herein, e.g., a CAR molecule that binds CD19. The
lymphodepletion can comprise, e.g., administering one or more of
melphalan, cytoxan, cyclophosphamide, and fludarabine.
[0059] In some embodiments, the CAR-expressing cell that is
administered comprises a regulatable CAR (RCAR), e.g., an RCAR as
described herein. The RCAR may comprise, e.g., an intracellular
signaling member comprising an intracellular signaling domain and a
first switch domain, an antigen binding member comprising an
antigen binding domain that binds CD19 and a second switch domain;
and a transmembrane domain. The method may further comprise
administering a dimerization molecule, e.g., in an amount
sufficient to cause dimerization of the first switch and second
switch domains.
[0060] In some embodiments, the CAR-expressing cell and the kinase
inhibitor are administered simultaneously or substantially
simultaneously, e.g., as a first line of therapy. In some
embodiments, the method comprises administering a combination of
the BTK inhibitor (e.g., ibrutinib) and the CAR-expressing cell
(e.g., a CAR19-expressing cell) to the subject, as a first line
therapy.
[0061] In other embodiments, the CAR-expressing cell and the kinase
inhibitor are administered sequentially. For example, the kinase
inhibitor is administered before the CAR-expressing cell, or the
CAR-expressing cell is administered before the kinase
inhibitor.
[0062] In some embodiments, the disease associated with expression
of CD19 is a hematological cancer (e.g., a hematological cancer
described herein such as CLL, MCL, or ALL) and the subject is, or
is identified as, a partial responder, non-responder, or relapser
to one or more therapies for the hematological cancer, e.g., to a
BTK inhibitor such as ibrutinib. In some embodiments, the subject
has, or is identified as having, a BTK mutation. The mutation may
be, e.g., a point mutation, an insertion, or a deletion. The
mutation may be, e.g., a mutation at the binding site for the BTK
inhibitor, e.g., at or near the ATP-binding pocket. The mutation
may confer a decreased response (e.g., resistance) to the BTK
inhibitor.
[0063] In some embodiments of any of the methods disclosed herein,
the method comprises administering the BTK inhibitor (e.g.,
ibrutinib) to the subject, reducing the amount (e.g., ceasing
administration) of the BTK inhibitor, and subsequently
administering the CAR-expressing cell (e.g., a CAR19-expressing
cell) to the subject.
[0064] In some embodiments, the method comprises administering the
BTK inhibitor (e.g., ibrutinib) to the subject and subsequently
administering a combination of the BTK inhibitor and the
CAR-expressing cell (e.g., a CAR19-expressing cell) to the
subject.
[0065] In some embodiments, the method comprises administering the
BTK inhibitor (e.g., ibrutinib) to the subject, reducing the amount
(e.g., ceasing or discontinuing administration) of the BTK
inhibitor, and subsequently administering a combination of the
CAR-expressing cell (e.g., a CAR19-expressing cell) and a second
BTK inhibitor (e.g., a BTK inhibitor other than the first BTK
inhibitor, e.g., other than ibrutinib) to the subject. In some
embodiments, the second BTK inhibitor is chosen from one or more of
GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059,
CNX-774, or LFM-A13, or a combination thereof.
[0066] In some embodiments, the disease associated with expression
of the B-cell antigen (e.g., CD19) is a hematological cancer (e.g.,
a hematological cancer described herein, e.g., CLL, MCL, or ALL),
and the method delays or decreases resistance to the kinase
inhibitor (e.g., a BTK inhibitor such as ibrutinib), the the
CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject,
or both. In some embodiments, the disease associated with
expression of CD19 is a hematological cancer (e.g., a hematological
cancer described herein, e.g., CLL, MCL, or ALL), and wherein the
method prolongs remission or delays relapse of the hematological
cancer. For example, remission can be prolonged, relapse can be
delayed, resistance can be delayed, or resistance can be decreased,
compared to the expected course of disease when treated with a
monotherapy of the kinase inhibitor or the CAR-expressing cell.
[0067] Exemplary treatment regimens that can be used in any of the
aforesaid methods include one or more of the following:
[0068] In one embodiment, the kinase inhibitor and the
CAR-expressing cell (e.g., the CAR19-expressing cell) are
administered to the subject, e.g., mammal, as a first line of
therapy.
[0069] In another embodiment, the CAR-expressing cell (e.g., the
CAR19-expressing cell) is administered to the subject, e.g.,
mammal, after administration of the kinase inhibitor.
[0070] In other embodiments, the CAR-expressing cell (e.g., the
CAR19-expressing cell) is administered after ceasing administration
of the kinase inhibitor.
[0071] In other embodiments, administration of the kinase inhibitor
is begun prior to administration of the CAR19-expressing cell, and
the CAR19-expressing cell is administered in combination with
continued administration of the kinase inhibitor.
[0072] In one embodiment, a subject is administered a kinase
inhibitor (e.g., a BTK inhibitor such as ibrutinib), e.g., as a
first line therapy. After a predetermined time interval, (e.g., 1
or 2 months but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2
months, 3 months, 4 months, 6 months, 9 months, 12 months, 15
months, or 18 months), a CAR-expressing cell (e.g., a
CAR19-expressing cell) is administered to the subject alone, or in
combination with the kinase inhibitor. In some embodiments, the
subject's response to the treatment is assessed at predetermined
time intervals, e.g., before or during treatment with the kinase
inhibitor and/or CAR-expressing cell. If the assessment shows that
the subject is a complete responder, the CAR-expressing cell (e.g.,
a CAR19-expressing cell) is not administered. If the assessment
shows that the subject is a partial responder, or has stable
disease in response, to the kinase inhibitor, the CAR-expressing
cell (e.g., a CAR19-expressing cell) is administered in combination
with the kinase inhibitor e.g., as described herein. If the
assessment shows that the subject is a non-responder or relapser,
the CAR-expressing cell (e.g., a CAR19-expressing cell) is
administered in combination with the kinase inhibitor or a second
kinase inhibitor, e.g., a second kinase inhibitor as described
herein.
[0073] In other embodiments, the subject, e.g., mammal, is, or is
identified as being, a complete or partial responder to the BTK
inhibitor (e.g., ibrutinib), or a complete or partial responder to
the CAR19-expressing cell.
[0074] In some embodiments, when a subject is (or is identified as
being) a complete responder to the kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib), the subject is not administered a
CAR-expressing cell (e.g., a CAR19-expressing cell) during the
period of complete response. In other embodiments, when a subject
is (or is identified as being) a complete responder (e.g., a
complete responder to ibrutinib) to the kinase inhibitor, the
subject is administered a CAR-expressing cell (e.g., a
CAR19-expressing cell) during the period of complete response. In
an embodiment, after the CAR-expressing cell (e.g., a
CAR19-expressing cell), the subject experiences a prolonged
response or delayed relapse (e.g., compared to the expected course
of disease when treated without the CAR therapy).
[0075] In some embodiments, when a subject is (or is identified as
being) a partial responder to the kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib), the subject is not administered a
CAR-expressing cell (e.g., a CAR19-expressing cell) during the
period of partial response. In other embodiments, when a subject is
(or is identified as being) a partial responder to the kinase
inhibitor, the subject is administered a CAR-expressing cell (e.g.,
a CAR19-expressing cell) (alone or in combination with the BTK
inhibitor) during the period of partial response. In an embodiment,
after the CAR therapy, the subject experiences a complete response
and/or prolonged response or delayed relapse (e.g., compared to the
expected course of disease when treated without CAR therapy).
[0076] In some embodiments, when a subject has (or is identified as
having) stable disease after treatment with the kinase inhibitor
(e.g., a BTK inhibitor such as ibrutinib), the subject is not
administered a CAR therapy during the period of stable disease. In
other embodiments, when a subject has (or is identified as having)
stable disease after treatment with the kinase inhibitor, the
subject is administered a CAR therapy during the period of stable
disease. In an embodiment, after the CAR therapy, the subject
experiences a partial response, a complete response and/or
prolonged response or delayed relapse (e.g., compared to the
expected course of disease when treated without CAR therapy).
[0077] In some embodiments, when a subject has (or is identified as
having) progressive disease after treatment with the kinase
inhibitor (e.g., a BTK inhibitor such as ibrutinib), the subject is
not administered a CAR-expressing cell (e.g., a CAR19-expressing
cell) during the period of progressive disease. In other
embodiments, when a subject has (or is identified as having)
progressive disease after treatment with the kinase inhibitor, the
subject is administered a CAR-expressing cell (e.g., a
CAR19-expressing cell) during the period of progressive disease. In
an embodiment, after the CAR therapy, the subject experiences
stable disease, a partial response, a complete response and/or
prolonged response or delayed relapse (e.g., compared to the
expected course of disease when treated without CAR therapy).
[0078] In other embodiments, the CAR-expressing cell is
administered in combination a second kinase inhibitor, wherein the
second kinase inhibitor is other than ibrutinib, when the mammal
is, or is identified as being, a non-responder or relapser to
ibrutinib. The second kinase inhibitor can be chosen from one or
more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292,
ONO-4059, CNX-774, or LFM-A13, or a combination thereof.
[0079] In other embodiments, the subject, e.g., the mammal, is (or
is identified as being) a partial responder to the kinase
inhibitor, and the subject is administered the CAR-expressing cell
(e.g., the CAR19-expressing cell), alone or in combination with the
BTK inhibitor, during the period of partial response.
[0080] In other embodiments, the subject, e.g., the mammal, is (or
has identified as being) a non-responder having progressive or
stable disease after treatment with ibrutinib, and the subject is
administered the CAR-expressing cell (e.g., the CAR19-expressing
cell), alone or in combination with a second BTK inhibitor, during
the period of progressive or stable disease, wherein the second
kinase inhibitor is other than ibrutinib.
[0081] In another aspect, provided herein is a method of treating a
subject, e.g., a mammal, having a disease associated with
expression of the B-cell antigen (e.g., CD19). The method comprises
administering to the subject an effective amount of a kinase
inhibitor as described herein (e.g., a BTK kinase inhibitor
described herein, e.g., ibrutinib) and a CAR-expressing cell (e.g.,
a CAR19-expressing cell) in combination (e.g. simultaneously (or
substantially simultaneously), or sequentially).
[0082] In some embodiments, the kinase inhibitor and the
CAR-expressing cell (e.g., a CAR19 cell) are administered, in
combination, e.g., as a first line of therapy,
[0083] In some embodiments, the kinase inhibitor is administered
initially, e.g., a monotherapy or first line of therapy; after
reducing the amount (e.g., ceasing or discontinuing administration)
of the kinase inhibitor, administering the CAR-expressing cell
(e.g., a CAR19-expressing cell) to the subject.
[0084] In other embodiments, the kinase inhibitor is administered
initially, e.g., a monotherapy or first line of therapy; and
subsequently administering a combination of the kinase inhibitor
and the CAR-expressing cell (e.g., a CAR19-expressing cell) to the
subject.
[0085] In other embodiments, the kinase inhibitor is administered
initially, e.g., a monotherapy or first line of therapy; after
reducing the amount (e.g., ceasing or discontinuing administration)
of the kinase inhibitor, administering a combination of a second
kinase inhibitor and the CAR-expressing cell (e.g., a
CAR19-expressing cell) to the subject.
[0086] In some embodiments, the subject's response to the treatment
is assessed at predetermined time intervals, e.g., before or during
treatment with the kinase inhibitor and/or CAR-expressing cell. If
the assessment shows that the subject is a complete responder, the
CAR-expressing cell (e.g., a CAR19-expressing cell) is not
administered. If the assessment shows that the subject is a partial
responder, or has stable disease in response, to the kinase
inhibitor, the CAR-expressing cell (e.g., a CAR19-expressing cell)
is administered in combination with the kinase inhibitor e.g., as
described herein. If the assessment shows that the subject is a
non-responder or relapser, the CAR-expressing cell (e.g., a
CAR19-expressing cell) is administered in combination with the
kinase inhibitor or a second kinase inhibitor, e.g., a second
kinase inhibitor as described herein.
[0087] In some embodiments, the disease associated with expression
of a B-cell antigen (e.g., CD19) is a hematological cancer,
leukemia, lymphoma, MCL, CLL, ALL, Hodgkin lymphoma, or multiple
myeloma.
[0088] In some embodiments, the kinase inhibitor is a BTK inhibitor
chosen from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764,
HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13; a CDK4 inhibitor
chosen from palbociclib, aloisine A, flavopiridol,
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4-chromenone; crizotinib (PF-02341066, P276-00, RAF265,
indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322,
AT7519, AZD5438, BMS908662; or ribociclib; a mTOR inhibitor chosen
from rapamycin, a rapamycin analog such as everolimus,
temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502,
SF1126, XL765, or OSI-027; or a MNK inhibitor is chosen from:
CGP052088, CGP57380, cercosporamide, or ETC-1780445-2, or
4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.
[0089] In some aspects, the invention features a method of treating
or providing an anti-tumor immunity in a subject, e.g., mammal,
having Hodgkin lymphoma. The method comprises administering to the
subject an effective amount of a cell that expresses a CAR molecule
that binds CD19, alone or in combination with a second therapy.
[0090] In another aspect, the invention features a method of
treating, or providing anti-tumor immunity to a subject, e.g., a
mammal, having a multiple myeloma (e.g., a CD19-positive multiple
myeloma, or a CD19-negative myeloma). In one embodiment, the
multiple myeloma is CD19-negative, e.g., has a vast majority
(99.95%) of the neoplastic plasma cells with a CD19-negative
phenotype, e.g., as detected by both flow cytometry and RT-PCR. The
method comprises administering to the subject an effective amount
of a cell that expresses a CAR molecule that binds CD19, alone or
in combination with a second therapy (e.g., a standard of care
therapy for multiple myeloma). The method may further comprise
administering a kinase inhibitor as described herein.
[0091] In embodiments of the methods related to Hodgkin lymphoma or
multiple myeloma, the CAR molecule is a humanized CAR molecule,
e.g., as described herein. In embodiments, the CAR molecule is a
CAR molecule as described herein. For instance, in embodiments the
CAR molecule comprises an anti-CD19 binding domain that comprises a
one or more of (e.g., 2, 3, 4, 5, or all of) LC CDR1 of SEQ ID NO:
5, a LC CDR2 of SEQ ID NO: 26, and a LC CDR3 of SEQ ID NO: 27; a HC
CDR1 of SEQ ID NO: 19, a LC CDR2 of any of SEQ ID NOS: 20-23, and a
HC CDR3 of SEQ ID NO: 24.
[0092] In some embodiments of the methods related to Hodgkin
lymphoma or multiple myeloma, the CAR molecule (e.g., CART19 or
CTL019) is administered as a monotherapy. In some embodiments, the
method further comprises administering a kinase inhibitor, e.g., a
BTK inhibitor (such as ibrutinib), a CDK4 inhibitor, an mTOR
inhibitor, or a MNK inhibitor.
[0093] In some embodiments of the methods related to multiple
myeloma, the CAR molecule (e.g., CART19 or CTL019) is administered
in combination a standard of care therapy for multiple myeloma,
e.g., with myeloablative chemotherapy and/or autologous stem cell
transplant rescue (e.g., after melphalan administration (e.g., high
dose melphalan)).
[0094] In another aspect, the invention features a composition
comprising a cell that expresses a CAR molecule that binds a B cell
antigen (e.g., one or more of CD19, CD20. CD22 or ROR1), and one or
more kinase inhibitors, wherein the kinase inhibitor is chosen from
a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin dependent
kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a mitogen
activated protein kinase interacting kinase (MNK) inhibitor. The
CAR-expressing cell and the one or more kinase inhibitors can be
present in a single dose form, or as two or more dose forms.
[0095] In embodiments, the compositions disclosed herein are for
use as a medicament.
[0096] In embodiments, the compositions disclosed herein are use in
the treatment of a disease associated with expression of a B-cell
antigen (e.g., CD19).
Methods and Compositions for Producing CAR-Expressing Cells
[0097] The present disclosure also provides, in certain aspects, a
method of making a population of immune effector cells (e.g., T
cells or NK cells) that can be engineered to express a CAR (e.g., a
CAR described herein), the method comprising: providing a
population of immune effector cells; and contacting the immune
effector cells with a kinase inhibitor (e.g., a BTK inhibitor such
as ibrutinib) under conditions sufficient to inhibit a target of
the kinase inhibitor (e.g., BTK and/or ITK). The method can further
comprise contacting, e.g., transducing, the immune effector cells
with a nucleic acid encoding a CAR molecule.
[0098] In some aspects, the disclosure provides a method of making
a CAR-expressing cell (e.g., a CAR-expressing immune effector cell
or population of cells), comprising: contacting the cell or
population of cells with a kinase inhibitor, e.g., a BTK inhibitor
such as ibrutinib; and introducing (e.g., transducing) a nucleic
acid encoding a CAR molecule into the cell or population of cells
under conditions such that the CAR molecule is expressed.
[0099] In certain embodiments of the methods of producing
CAR-expressing cells, the CAR molecule encoded by the nucleic acid
is a CAR molecule that binds CD19. In embodiments, the method
further comprises culturing the cell or cells under conditions that
allow the cell or at least a sub-population of the cells to express
the CAR molecule. In embodiments, the cell is a T cell or NK cell,
or the population of cells includes T cells, NK cells, or both. In
embodiments, the method comprises contacting the cell or cells with
the kinase inhibitor (e.g., for 10-20, 20-30, 30-40, 40-60, or
60-120 minutes) and subsequently removing most or all of the kinase
inhibitor from the cell or cells. In embodiments, the kinase
inhibitor is added after the cell or cells are harvested or before
the cell or cells are stimulated. In embodiments, the kinase
inhibitor is a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor,
or a MNK inhibitor. In embodiments, the kinase inhibitor is
ibrutinib. In embodiments, the population of cells also comprises
cancer cells, e.g., leukemia or lymphoma cells. The cancer cells
may be, e.g., CLL, MCL, or ALL cells. In embodiments, the kinase
inhibitor inhibits a target (e.g., BTK) in the cancer cells, e.g.,
reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99%. In embodiments, the kinase inhibitor
inhibits a target (e.g., ITK) in the immune effector cells, e.g.,
reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99%.
[0100] In some aspects, the present disclosure also provides a
reaction mixture comprising a kinase inhibitor (e.g., a BTK
inhibitor) and a CAR molecule or a nucleic acid encoding a CAR
molecule. In some embodiments, the reaction mixture further
comprises a population of immune effector cells.
[0101] In some embodiments, one or more of the immune effector
cells expresses the CAR molecule or comprises the nucleic acid
encoding the CAR molecule. In some embodiments, the kinase
inhibitor is chosen from a BTK inhibitor, a CDK4 inhibitor, an mTOR
inhibitor, or a MNK inhibitor. In some embodiments, the BTK
inhibitor is chosen from: ibrutinib, GDC-0834, RN-486, CGI-560,
CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13. In
embodiments, the reaction mixture comprises cancer cells, e.g.,
haematological cancer cells. The cancer cells may be, e.g., cells
that were harvested from the subject when the immune effector cells
were harvested from the subject.
[0102] In certain aspects, the present disclosure also provides a
reaction mixture comprising a population of immune effector cells,
and a CAR molecule or a nucleic acid encoding a CAR molecule,
wherein the immune effector cells comprise covalently inactivated
ITK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% of ITK is covalently inactivated. In some
embodiments, the reaction mixture further comprises cancer cells.
In embodiments, the cancer cells comprise covalently inactivated
BTK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 99% of BTK is covalently inactivated. In
embodiments, the BTK or ITK forms a covalent bond at or near its
ATP binding domain to a small molecule such as ibrutinib. In
embodiments, the BTK forms a covalent bond at or near its
cysteine-481 to a small molecule such as ibrutinib.
[0103] In embodiments, a reaction mixture as described herein
further comprises a buffer or other reagent, e.g., a PBS containing
solution. In embodiments, the reaction mixture further comprises an
agent that activates and/or expands to cells of the population,
e.g., an agent that stimulates a CD3/TCR complex associated signal
and/or a ligand that stimulates a costimulatory molecule on the
surface of the cells. In embodiments, the agent is a bead
conjugated with anti-CD3 antibody, or a fragment thereof, and/or
anti-CD28 antibody, or a fragment thereof. In embodiments, the
reaction mixture further comprises one or more factors for
proliferation and/or viability, including serum (e.g., fetal bovine
or human serum), interleukin-2 (IL-2), insulin, IFN-.gamma., IL-4,
IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF.beta., and TNF-.alpha. or
any other additives for the growth of cells. In embodiments, the
reaction mixture further comprises IL-15 and/or IL-7. In
embodiments, a plurality of the cells of the population in the
reaction mixture comprise a nucleic acid molecule, e.g., a nucleic
acid molecule described herein, that comprises a CAR encoding
sequence, e.g., a CD19 CAR encoding sequence, e.g., as described
herein. In embodiments, a plurality of the cells of the population
in the reaction mixture comprise a vector comprising a nucleic acid
sequence encoding a CAR, e.g., a CAR described herein, e.g., a CD19
CAR described herein. In embodiments, the vector is a vector
described herein, e.g., a vector selected from the group consisting
of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector,
or a retrovirus vector. In embodiments, the reaction mixture
further comprises a cryoprotectant or stabilizer such as, e.g., a
saccharide, an oligosaccharide, a polysaccharide and a polyol
(e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose and
dextran), salts and crown ethers. In one embodiment, the
cryoprotectant is dextran.
[0104] In some embodiments, the method of making disclosed herein
further comprises contacting the population of immune effector
cells with a nucleic acid encoding a telomerase subunit, e.g.,
hTERT. The the nucleic acid encoding the telomerase subunit can be
DNA.
[0105] In some embodiments, the method of making disclosed herein
further comprises culturing the population of immune effector cells
in serum comprising 2% hAB serum.
[0106] Headings, sub-headings or numbered or lettered elements,
e.g., (a), (b), (i) etc, are presented merely for ease of reading.
The use of headings or numbered or lettered elements in this
document does not require the steps or elements be performed in
alphabetical order or that the steps or elements are necessarily
discrete from one another.
[0107] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0108] Other features, objects, and advantages of the invention
will be apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] FIG. 1A, 1B and 1C are graphic representations of
cytotoxicity as assayed in ND317 (normal donor) T cell transduced
with mouse anti-CD19 CAR or the humanized anti-CD19 CARs of the
invention and cultured with either control K562 cells that do not
express CD19 (K562cc) as shown in FIG. 1A, K562 cells transformed
with CD19 (K562.CD19) as shown in FIG. 1B or malignant B cells
isolated from a CLL patient (Pt 14 B cell isolate) as shown in FIG.
1C.
[0110] FIGS. 2A and 2B are graphs showing the proliferative
response of humanized and mouse anti-CD19 CAR-expressing cells to
CD19+ cells, where higher number of viable CAR+ T cells correlates
with populations showing maximal CD4+ and CD8+ T cell proliferation
to primary CLL cells.
[0111] FIG. 3 is a graphic representation of the deconvoluted HPLC
mass spectra for scFvs of the invention, where the top row depicts
untreated scFv and the bottom row depicts the cognate
deglycosylated scFv.
[0112] FIG. 4 is a graphic representation of the conformation
stability as measured by Differential Scanning Fluorimetry. The Tm
of mouse scFv was 57.degree. C. (thick line). All humanized scFv
variants show higher Tm at around 70.degree. C. as compared to the
parental mouse scFv. The residues introduced by humanization have
improved the Tm by more than 10.degree. C.
[0113] FIG. 5 is a graphic representation of CD19 CAR transduced T
cell proliferation, wherein the CART19 cells are directed either
towards (a) a chronic myelogenous leukemia ("CML") cell line that
is negative for the expression of CD19, and hence used as a
negative control; (b) recombinant K562 cells positive for
expression of CD19, and hence used as a positive control; or (c) to
Pt14 B cells isolated from a CLL patient and which expresses CD19
on the cell surface.
[0114] FIGS. 6A and 6B are schematics of representative CARs.
[0115] FIG. 7 depicts HALLX5447 primary ALL disease progression in
NSG mice after treatment with CD19 transduced CAR T cells. The
growth of primary human ALL cells in NSG mice after treatment with
CAR T cells specific for CD19 demonstrated control of disease
progression. Mean percentage of CD19.sup.+ human ALL cells was an
indicator of disease burden in the peripheral blood in NSG mice to
day 65 post tumor implant. Black circles: mice treated with 100 ul
of PBS via the tail vein; red squares: mice treated with mock
transduced T cells; blue triangles: mice treated with murine CD19
CAR transduced T cells; and inverted purple triangles: mice treated
with humanized CD19 CAR transduced T cells. Significance calculated
by ANOVA; * denotes P<0.01.
[0116] FIG. 8 depicts CD19 expression in a patient's tumor cells.
CD138.sup.+ CD45.sup.dim tumor cells were stained for CD19 (x-axis)
and CD38 (y-axis). Approximately 1-2% of the tumor cells expressed
the CD19 antigen.
[0117] FIGS. 9A and 9B are two graphs showing the cell
proliferation and cell size of CART19 cells when treated with
increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000
nM).
[0118] FIGS. 10A-1, 10A-2, 10A-3, 10A-4, and 10B shows the
proliferation of CART19 cells stimulated with MCL cell lines, while
in the presence or absence of ibrutinib. FIGS. 10A-1, 10A-2, 10A-3,
and 10A-4 is a series of histograms showing the proliferation of
CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and
RL, in the presence or absence of increasing concentrations of
ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained by CFSE
and analyzed by flow cytometry to determine the percentage of
proliferating cells, designated by the bar in each histogram. FIG.
10B is a quantification of representative histograms in FIG. 10A-,
10A-2, 10A-3, and 10A-4.
[0119] FIG. 11A-1, 11A-2, 11A-3, 11A-4, 11A-5, 11A-6, and 11B shows
CD107a degranulation of CART19 cells stimulated with MCL cell lines
in the presence or absence of ibrutinib. FIGS. 11A-1, 11A-2, 11A-3,
11A-4, 11A-5, and 11A-6 is a series of flow cytometry profiles
showing CD107a degranulation of CART19 cells stimulated with tumor
cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of
increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000
nM). CD107a expression is measured in the y-axis. FIG. 11B is the
quantification of the results from FIGS. 11A-1, 11A-2, 11A-3,
11A-4, 11A-5, and 11A-6.
[0120] FIGS. 12-1, 12-1, 12-3, 12-4, 12-5, and 12-6 is a series of
flow cytometry profiles showing intra-cytoplasmatic IL-2 production
by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1,
and RL) in the presence or absence of increasing concentrations of
ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2
expression.
[0121] FIGS. 13-1, 13-2, 13-3, 13-4, 13-5, and 13-6 is a series of
flow cytometry profiles showing intra-cytoplasmatic TNF-.alpha.
production by CART19 cells stimulated with tumor cell lines
(MOLM14, JEKO-1, and RL) in the presence or absence of increasing
concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The
y-axis represents TNF-.alpha. expression.
[0122] FIGS. 14-1, 14-2, 14-3, 14-4, 14-5, and 14-6 is a series of
flow cytometry profiles showing intra-cytoplasmatic IFN-g
production by CART19 cells stimulated with tumor cell lines
(MOLM14, JEKO-1, and RL) in the presence or absence of increasing
concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The
y-axis represents IFN-g expression.
[0123] FIG. 15-1, 15-2, 15-3, 15-4, 15-5, 15-6, 15-7, 15-8, 15-9,
and 15-10 are a series of graphs showing cytokine secretion from
CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and
RL) in the presence or absence of increasing concentrations of
ibrutinib (10 nM, 100 nM, and 1000 nM).
[0124] FIGS. 16A, 16B, 16C, 16D, 16E, and 16 F are graphs showing
CART19 killing of tumor cells, MOLM14 (FIGS. 16A and 16D), JEKO
(FIGS. 16B and 16E), and RL (FIGS. 16C and 16F), alone or in the
presence of increasing concentrations of ibrutinib. Untransduced
(UTD) or CART19 cells were incubated with tumor cells at varying
ratios and the total flux of cells (FIGS. 16A, 16B, and 16C) and
percentage of dead cells was assessed (16D, 16E, and 16F).
[0125] FIGS. 17A, 17B, and 17C are graphic representations of
CART19 killing of tumor cells after 24 hours as measured by flow
cytometry to count the total number of cells. Tumor cell lines
MOLM14 (FIG. 17A), JEKO (FIG. 17B), and RL (FIG. 17C) were
incubated with untransduced (UTD) or CART19 cells alone (ALONE), or
in combination with varying concentrations of ibrutinib.
[0126] FIGS. 18A, 18B, 18C, and 18D are graphic representations of
CART19 dose finding in the RL MCL mouse model. Tumor burden was
monitored by bioluminescence imaging (BLI) over time (FIGS. 18A and
18B). Overall survival was monitored over time (FIG. 18C).
[0127] FIGS. 19A and 19B are graphic representations of CART19 dose
finding in the JEKO-1 MCL mouse model. Tumor size is monitored by
bioluminescence imaging (BLI) over time (FIG. 19A) and overall
survival was also monitored over time (FIG. 19B).
[0128] FIG. 20 is a schematic showing the protocol for
administering and assessing CART19 and ibrutinib combination
therapy in in vivo mouse models.
[0129] FIGS. 21A, 21B, 21C, and 21D is a graphic representation
showing the transduction efficiency of PBMCs for generating CART19
T cells when cells are treated with ibrutinib prior to
transduction. Untreated PBMCs were analyzed for CAR19 expression
before transduction (FIG. 21A) and after transduction (FIG. 21C).
Ibrutinib-treated PBMCs were analyzed for CAR19 expression before
transduction (FIG. 21B) and after transduction (FIG. 21D).
[0130] FIGS. 22A, 22B, 22C, 22D, and 22E is a graphic
representation of the effect of ibrutinib treatment on
CD3/CD28-stimulated T cell proliferation. Increasing concentrations
of ibrutinib was assessed: untreated (FIG. 22A); 0.1 .mu.M
ibrutinib (FIG. 22B); 0.5 .mu.M ibrutinib (FIG. 22C), 1 .mu.M
ibrutinib (FIG. 22D), and 5 .mu.M ibrutinib (FIG. 22E).
[0131] FIG. 23 is a graphic representation demonstrating that
ibrutinib does not affect CART19 cytotoxicity.
[0132] FIG. 24 is a series of graphic representations that
demonstrate that ibrutinib treatment does not promote skewing of
T.sub.H1/T.sub.H2 cytokines in CART19 cells.
[0133] FIGS. 25A and 25B are graphic representations showing that
continuous administration of ibrutinib does not affect CART19
function in clearing tumor cells in vivo. FIG. 25A shows the Nalm/6
cells detected in peripheral blood during each treatment regimen.
FIG. 25B shows a Kaplan-Meier survival curve comparing the survival
of mice receiving CART19 with or without ibrutinib dosing.
[0134] FIGS. 26A, 26B, 26C, 26D, 26E, and 26F are graphic
representations that showing the efficiency of CAR19 transduction
in CLL patient cells at the indicated timepoints during ibrutinib
treatment. Cells were not transduced (FIGS. 26A, 26B, and 26C), or
were transduced with CAR19 (FIGS. 26D, 26E, and 26F). Cells stained
with GAM express CAR19 and are present in the boxes in each
profile.
[0135] FIGS. 27-1, 27-2, and 27-3 are a series of graphic
representations depicting the proliferation rate of untransduced
cells compared to cells that were transduced with CAR19 at the
indicated timepoints during ibrutinib treatment from a panel of
patients.
[0136] FIGS. 28A, 28B, and 28C are graphic representations
demonstrating that ibrutinib treatment in CLL patients induces
lymphocytosis. Cells from the patient were isolated at the
indicated timepoints: baseline (FIG. 28A); cycle 2, day 1 (FIG.
28B); and cycle 12, day 1 (FIG. 28C).
[0137] FIGS. 29A, 29B, and 29C are graphic representations
demonstrating that ibrutinib treatment in three CLL patients
reduces CD200 expression on tumor cells over time in CLL. Each
profile contains an overlay of CD200 expression histograms from
cells isolated at the indicated timepoints: baseline (screen);
cycle 2, day 1; and cycle 12, day 1.
[0138] FIGS. 30A, 30B, and 30C are graphic representations
demonstrating that ibrutinib treatment in CLL patients decreases
the frequency of PD1+ T cells over time. Cells from the patient
were isolated at the indicated timepoints: baseline (FIG. 30A);
cycle 2, day 1 (FIG. 30B); and cycle 12, day 1 (FIG. 30C).
[0139] FIGS. 31A and 31B is a graphic representation demonstrating
the sensitivity of MCL cell lines RL (FIG. 31A) and JEKO-1 (FIG.
31B) to ibrutinib treatment.
[0140] FIG. 32 is a graphic representation demonstrating the effect
of ibrutinib treatment in an in vivo model of MCL.
[0141] FIG. 33 (left panel and right panel) are images of
immunohistochemical analysis of a Hogdkin's lymphoma showing CD19
expressing cells present in the tumor. FIG. 33 (left panel) is at
1.times. magnification and FIG. 33 (right panel) is at 20.times.
magnification.
[0142] FIG. 34 is a schematic diagram of the experimental set-up
for a study to assess the therapeutic efficacy of CART19 treatment
in patients with Hodgkin lymphoma.
[0143] FIGS. 35A, 35B, 35C, and 35D show flow cytometry analysis of
PD1 and CAR19 expression on T cells. FIGS. 35A and 35B are
representative flow cytometry profiles demonstrating the
distribution of PD-1 and CAR19 expression on CD4+ T cells from
subjects that are complete responders (CR) or non-responders (NR)
to CART therapy. FIG. 35C is a graph showing the percent of PD1
cells in the CD4+ T cell population from groups of subjects with
different responses to CART therapy. FIG. 35D is a graph showing
the percent of PD1 cells in the CD8+ T cell population from groups
of subjects with different responses to CART therapy.
[0144] FIGS. 36A and 36B show the distribution of PD1 expression in
CD4 and CAR19-expressing cells (FIG. 36A) or CD8 and
CAR19-expressing cells (FIG. 36B) from groups of subjects with
different responses to CART therapy.
[0145] FIG. 37 shows flow cytometry analysis of PD1, CAR19, LAG3,
and TIM3 expression on T cells from subjects that are complete
responders (CR) or non-responders (NR) to CART therapy.
[0146] FIGS. 38A and 38B show the distribution of PD1 and LAG3
expression (FIG. 38A) or PD1 and TIM3 expression (FIG. 38B) from
groups of subjects with different responses to CART therapy.
[0147] FIG. 39 shows the plasma cell IgA immunophenotyping from a
myeloma patient who received CART19, demonstrating the response to
CART19 therapy.
[0148] FIGS. 40A and 40B are graphs showing an increase in titers
to influenza vaccine strains as compared to placebo. In FIG. 40A,
the increase above baseline in influenza geometric mean titers to
each of the 3 influenza vaccine strains (H1N1 A/California/07/2009,
H3N2 A/Victoria/210/2009, B/Brisbane/60/2008) relative to the
increase in the placebo cohort 4 weeks after vaccination is shown
for each of the RAD001 dosing cohorts in the intention to treat
population. The bold black line indicates the 1.2 fold increase in
titers relative to placebo that is required to be met for 2 out of
3 influenza vaccine strains to meet the primary endpoint of the
study. The star "*" indicates that the increase in GMT titer
relative to placebo exceeds 1 with posterior probability of at
least 80%. FIG. 40B is a graph of the same data as in FIG. 40A for
the subset of subjects with baseline influenza titers
<=1:40.
[0149] FIG. 41 shows a scatter plot of RAD001 concentration versus
fold increase in geometric mean titer to each influenza vaccine
strain 4 weeks after vaccination. RAD001 concentrations (1 hour
post dose) were measured after subjects had been dosed for 4 weeks.
All subjects who had pharmacokinetic measurements were included in
the analysis set. The fold increase in geometric mean titers at 4
weeks post vaccination relative to baseline is shown on the y
axis.
[0150] FIG. 42 is a graphic representation showing increase in
titers to heterologous influenza strains as compared to placebo.
The increase above baseline in influenza geometric mean titers to 2
heterologous influenza strains (A/H1N1 strain A/New Jersey/8/76 and
A/H3N2 strain A/Victoria/361/11) not contained in the influenza
vaccine relative to the increase in the placebo cohort 4 weeks
after vaccination is shown for each of the RAD001 dosing cohorts in
the intention to treat population. * indicates increase in titer
relative to placebo exceeds 1 with a posterior probability of at
least 80%.
[0151] FIGS. 43A and 43B are graphic representations of IgG and IgM
levels before and after influenza vaccination. Levels of
anti-A/H1N1/California/07/2009 influenza IgG and IgM were measured
in serum obtained from subjects before and 4 weeks post influenza
vaccination. No significant difference in the change from baseline
to 4 weeks post vaccination in anti-H1N1 influenza IgG and IgM
levels were detected between the RAD001 and placebo cohorts (all p
values >0.05 by Kruskal-Wallis rank sum test).
[0152] FIGS. 44A, 44B, and 44C are graphic representations of the
decrease in percent of PD-1-positive CD4 and CD8 and increase in
PD-1-negative CD4 T cells after RAD001 treatment. The percent of
PD-1-positive CD4, CD8 and PD-1-negative CD4 T cells was determined
by FACS analysis of PBMC samples at baseline, after 6 weeks of
study drug treatment (Week 6) and 6 weeks after study drug
discontinuation and 4 weeks after influenza vaccination (Week 12).
FIG. 44A shows there was a significant decrease (-37.1--28.5%) in
PD-1-positive CD4 T cells at week 12 in cohorts receiving RAD001 at
dose levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week
(n=30) as compared to the placebo cohort (n=25) with p=0.002
(0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05) respectively. FIG.
44B shows there was a significant decrease (-43.3--38.5%) in
PD-1-positive CD8 T cells at week 12 in cohorts receiving RAD001
(n=109) at dose levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20
mg/Week (n=30) as compared to the placebo cohort (n=25) with p=0.01
(0.05), p=0.007 (q=0.04), and p=0.01 (q=0.05) respectively. FIG.
44C shows was a significant increase (3.0-4.9%) in PD-1-negative
CD4 T cells at week 12 in cohorts receiving RAD001 (n=109) at dose
levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week (n=30) as
compared to the placebo cohort (n=25) with p=0.0007 (0.02), p=0.03
(q=0.07), and p=0.03 (q=0.08) respectively.
[0153] FIGS. 45A and 45B are graphic representations of the
decrease in percent of PD-1-positive CD4 and CD8 and increase in
PD-1-negative CD4 T cells after RAD001 treatment adjusted for
differences in baseline PD-1 expression. The percent of
PD-1-positive CD4, CD8 and PD-1-negative CD4 T cells was determined
by FACS analysis of PBMC samples at baseline, after 6 weeks of
study drug treatment (Week 6) and 6 weeks after study drug
discontinuation and 4 weeks after influenza vaccination (Week 12).
FIG. 45A shows a significant decrease of 30.2% in PD-1+CD4 T cells
at week 6 in the pooled RAD cohort (n=84) compared to placebo
cohort (n=25) with p=0.03 (q=0.13). The decrease in PD-1-positive
CD4 T cells at week 12 in the pooled RAD as compared to the placebo
cohort is 32.7% with p=0.05 (q=0.19). FIG. 45B shows a significant
decrease of 37.4% in PD-1-positive CD8 T cells at week 6 in the
pooled RAD001 cohort (n=84) compared to placebo cohort (n=25) with
p=0.008 (q=0.07). The decrease in PD-1-positive CD8 T cells at week
12 in the pooled RAD001 as compared to the placebo cohort is 41.4%
with p=0.066 (q=0.21). FIGS. 45A and 45B represent the data in
FIGS. 44A, 44B, and 44C but with the different RAD001 dosage groups
of FIGS. 44A, 44B, and 44C pooled into the single RAD001-treated
group in FIGS. 45A and 45B.
[0154] FIG. 46 depicts increases in exercise and energy in elderly
subjects in response to RAD001.
[0155] FIGS. 47A and 47B depict the predicted effect of RAD001 on
P70 S6K activity in cells. FIG. 47A depicts P70 S6 kinase
inhibition with higher doses of weekly and daily RAD001; FIG. 47B
depicts P70 S6 kinase inhibition with lower doses of weekly
RAD001.
[0156] FIGS. 48A and 48B show IL-7 receptor (CD127) expression on
cancer cell lines and CART cells. Expression of CD127 was measured
by flow cytometry analysis in three cancer cell lines: RL (mantle
cell lymphoma), JEKO (also known as Jeko-1, mantle cell lymphoma),
and Nalm-6 (B-ALL) (FIG. 48A). CD127 expression was measured by
flow cytometry analysis on CD3 positive (CART) cells that had been
infused and circulating in NSG mice (FIG. 48B).
[0157] FIGS. 49A, 49B, and 49C show the anti-tumor response after
CART19 treatment and subsequent IL-7 treatment. NSG mice engrafted
with a luciferase-expressing mantle lymphoma cell line (RL-luc) at
Day 0 were treated with varying dosages of CART19 cells at Day 6,
and tumor burden was monitored. Mice were divided into 4 groups and
received no CART19 cells, 0.5.times.10.sup.6 CART19 cells (CART19
0.5E6), 1.times.10.sup.6 CART19 cells (CART19 1E6), or
2.times.10.sup.6 CART19 cells (CART19 2E6). Tumor burden after CART
treatment was measured by detection of bioluminescence (mean BLI)
(FIG. 49A). Mice receiving 0.5.times.10.sup.6 CART19 cells (CART19
0.5E6) or 1.times.10.sup.6 CART19 cells (CART19 1E6) were
randomized to receive recominbant human IL-7 (rhIL-7) or not. Tumor
burden, represented here by mean bioluminescence (BLI), was
monitored for the three mice (#3827, #3829, and #3815, receiving
the indicated initial CART19 dose) from FIG. 49A that were treated
with IL-7 starting at Day 85 (FIG. 49B). IL-7 was administered
through IP injection 3 times weekly. Tumor burden, represented here
by mean bioluminescence (BLI) before Day 85 (PRE) and after Day 115
(POST) was compared between mice that did not receive IL-7 (CTRL)
and mice that received IL-7 treatment (IL-7) (FIG. 49C).
[0158] FIGS. 50A and 50B show the T cell dynamics after IL-7
treatment. The level of human T cells detected in the blood was
monitored for each of the mice receiving IL-7 or control mice (FIG.
50A). The level of CART19 cells (CD3+ cells) detected in the blood
was measured before (PRE) and 14 days after (Day 14) initiation of
IL-7 treatment (FIG. 50B).
[0159] FIG. 51 depicts the structures of two exemplary RCAR
configurations. The antigen binding members comprise an antigen
binding domain, a transmembrane domain, and a switch domain. The
intracellular binding members comprise a switch domain, a
co-stimulatory signaling domain and a primary signaling domain. The
two configurations demonstrate that the first and second switch
domains described herein can be in different orientations with
respect to the antigen binding member and the intracellular binding
member. Other RCAR configurations are further described herein.
[0160] FIG. 52A is an image of a RL cell line.
[0161] FIG. 52B is a set of flow cytometry scatterplots showing the
expression of CD19 and CD5 in RL primary and RL cell lines.
[0162] FIG. 52C is an image showing t(11;14) translocation by
fluorescence in-situ hybridization (FISH).
[0163] FIG. 52D is a graph showing the IC50 (by percentage MTT
conversion) of ibrutinib inhibition in different cell lines.
[0164] FIG. 52E is a set of images and graphs showing engraftment
of RL cells in NOD-SCID-gamma chain knockout (NSG) mice and the
resulting tumor burden.
[0165] FIG. 52F is a set of histological images showing
localization of MCL cells to various organs in mice.
[0166] FIG. 52G is a set of histological images of mice that have
been injected with MCL-RL cells.
[0167] FIG. 53A is a set of graphs showing the number of CD107a+
CART19 cells when exposed to various MCL cell lines.
[0168] FIG. 53B is a set of graphs showing the amount of IL-2 and
TNF-alpha produced by CART19 cells when exposed to various MCL cell
lines.
[0169] FIG. 53C is a graph showing the percent killing of various
MCL cell lines by CART19 cells at various effector:target cell
ratios.
[0170] FIG. 53D is a graph showing the amount of carboxyfluorescein
succinimidyl ester (CFSE), a measure of proliferation, in CART19
cells exposed to various MCL cell lines.
[0171] FIG. 53E is a set of graphs showing the percentage of T
cells before and after expansion.
[0172] FIG. 53F is a set of graphs showing the percentage of
untranduced or CAR-19 transduced T cells that express or produce
various biomolecules (e.g., cytokines).
[0173] FIG. 54A is a set of images showing the activation of
interleukin-2-inducible T-cell kinase (ITK) when CART19 cells were
stimulated specifically or non-specifically.
[0174] FIG. 54B is a set of graphs showing CD107a surface
expression (a measure of degranulation), IL-2 production, and
TNF-alpha production by CART19 cells with various concentrations
with ibrutinib.
[0175] FIG. 54C is a set of histograms showing the amount of CFSE
in CART19 cells with various concentrations of ibrutinib and
exposed to various MCL cell lines.
[0176] FIGS. 54D-1 and 54D-2 is a set of graphs showing the
expression or production of various cytokines and biomarkers as
indicators of the Th1 or Th2 state of CART19 cells when combined
with different concentrations of ibrutinib.
[0177] FIG. 54E is a set of graphs showing the percentage killing
by CART19 cells of various MCL cell lines when combined with
different concentrations of ibrutinib.
[0178] FIG. 54F is a bar graph showing the expression of various
markers of intrinsic cytotoxic function of CART19 cells when
combined with various concentrations of ibrutinib.
[0179] FIG. 55 is a schematic of an in vivo mouse model
experimental setup to test the effect of CART19 and/or ibrutinib on
MCL-RL-injected mice, with a readout being luminescence (a measure
of the number of tumor cells).
[0180] FIG. 56 is a schematic of an in vivo mouse model
experimental setup to test the effect of CART19 and/or ibrutinib on
MCL-RL-injected mice, with a readout being luminescence (a measure
of the number of tumor cells).
[0181] FIG. 57 is a set of graphs showing the luminescence (a
measure of the measure of tumor cell number) in mice treated with
ibrutinib at different concentrations and their overall survival
after treatment.
[0182] FIG. 58 is a set of graphs showing the luminescence (a
measure of tumor cell number) in mice treated with ibrutinib or
CART19 cells as well as their overall survival after treatment.
[0183] FIG. 59 is a graph showing the luminescence (a measure of
tumor cell number) in mice after treatment with ibrutinib,
untransduced T cells, ibrutinib with untransduced T cells, CART19
cells, and CART19 cells with ibrutinib.
[0184] FIG. 60 is a graph showing the luminescence (a measure of
tumor cell number) in mice after treatment with ibrutinib alone,
CART19 cells alone, or the combination of ibrutinib with CART19
cells.
[0185] FIG. 61A is a set of graphs showing the level of Th1
cytokines produced in mice treated with ibrutinib and/or CART19
cells. FIG. 61B is a set of graphs showing the level of Th2
cytokines produced in mice treated with ibrutinib and/or CART19
cells.
[0186] FIG. 61C is a graph showing the percentage of cells
expressing the proliferation marker Ki67 in mice treated with
CART19 cells or CART19 cells plus ibrutinib.
[0187] FIG. 61D is a graph showing the percentage of cells
expressing the anti-apoptotic marker BCL-2 in mice treated with
CART19 cells or CART19 cells plus ibrutinib.
DETAILED DESCRIPTION
Definitions
[0188] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains.
[0189] The term "a" and "an" refers to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
By way of example, "an element" means one element or more than one
element.
[0190] The term "about" when referring to a measurable value such
as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or in some instances.+-.10%, or in
some instances.+-.5%, or in some instances.+-.1%, or in some
instances.+-.0.1% from the specified value, as such variations are
appropriate to perform the disclosed methods.
[0191] The term "Chimeric Antigen Receptor" or alternatively a
"CAR" refers to a set of polypeptides, typically two in the
simplest embodiments, which when in an immune effector cell,
provides the cell with specificity for a target cell, typically a
cancer cell, and with intracellular signal generation. In some
embodiments, a CAR comprises at least an extracellular antigen
binding domain, a transmembrane domain and a cytoplasmic signaling
domain (also referred to herein as "an intracellular signaling
domain") comprising a functional signaling domain derived from a
stimulatory molecule and/or costimulatory molecule as defined
below. In some aspects, the set of polypeptides are contiguous with
each other, e.g., are in the same polypeptide chain (e.g., comprise
a chimeric fusion protein). In some embodiments, the set of
polypeptides are not contiguous with each other, e.g., are in
different polypeptide chains. In some embodiments, the set of
polypeptides include a dimerization switch that, upon the presence
of a dimerization molecule, can couple the polypeptides to one
another, e.g., can couple an antigen binding domain to an
intracellular signaling domain. In one aspect, the stimulatory
molecule is the zeta chain associated with the T cell receptor
complex. In one aspect, the cytoplasmic signaling domain further
comprises one or more functional signaling domains derived from at
least one costimulatory molecule as defined below. In one aspect,
the costimulatory molecule is chosen from the costimulatory
molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and/or
CD28. In one aspect, the CAR comprises a chimeric fusion protein
comprising an extracellular antigen binding domain, a transmembrane
domain and an intracellular signaling domain comprising a
functional signaling domain derived from a stimulatory molecule. In
one aspect, the CAR comprises a chimeric fusion protein comprising
an extracellular antigen binding domain, a transmembrane domain and
an intracellular signaling domain comprising a functional signaling
domain derived from a costimulatory molecule and a functional
signaling domain derived from a stimulatory molecule. In one
aspect, the CAR comprises a chimeric fusion protein comprising an
extracellular antigen binding domain, a transmembrane domain and an
intracellular signaling domain comprising two functional signaling
domains derived from one or more costimulatory molecule(s) and a
functional signaling domain derived from a stimulatory molecule. In
one aspect, the CAR comprises a chimeric fusion protein comprising
an extracellular antigen binding domain, a transmembrane domain and
an intracellular signaling domain comprising at least two
functional signaling domains derived from one or more costimulatory
molecule(s) and a functional signaling domain derived from a
stimulatory molecule. In one aspect the CAR comprises an optional
leader sequence at the amino-terminus (N-ter) of the CAR fusion
protein. In one aspect, the CAR further comprises a leader sequence
at the N-terminus of the extracellular antigen binding domain,
wherein the leader sequence is optionally cleaved from the antigen
binding domain (e.g., a scFv) during cellular processing and
localization of the CAR to the cellular membrane.
[0192] The term "signaling domain" refers to the functional portion
of a protein which acts by transmitting information within the cell
to regulate cellular activity via defined signaling pathways by
generating second messengers or functioning as effectors by
responding to such messengers.
[0193] As used herein, the term "CD19" refers to the Cluster of
Differentiation 19 protein, which is an antigenic determinant
detectable on leukemia precursor cells. The human and murine amino
acid and nucleic acid sequences can be found in a public database,
such as GenBank, UniProt and Swiss-Prot. For example, the amino
acid sequence of human CD19 can be found as UniProt/Swiss-Prot
Accession No. P15391 and the nucleotide sequence encoding of the
human CD19 can be found at Accession No. NM_001178098. As used
herein, "CD19" includes proteins comprising mutations, e.g., point
mutations, fragments, insertions, deletions and splice variants of
full length wild-type CD19. CD19 is expressed on most B lineage
cancers, including, e.g., acute lymphoblastic leukaemia, chronic
lymphocyte leukaemia and non-Hodgkin lymphoma. Other cells with
express CD19 are provided below in the definition of "disease
associated with expression of CD19." It is also an early marker of
B cell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34
(16-17): 1157-1165 (1997). In one aspect the antigen-binding
portion of the CART recognizes and binds an antigen within the
extracellular domain of the CD19 protein. In one aspect, the CD19
protein is expressed on a cancer cell.
[0194] As used herein, the term "CD20" refers to an antigenic
determinant known to be detectable on B cells. Human CD20 is also
called membrane-spanning 4-domains, subfamily A, member 1 (MS4A1).
The human and murine amino acid and nucleic acid sequences can be
found in a public database, such as GenBank, UniProt and
Swiss-Prot. For example, the amino acid sequence of human CD20 can
be found at Accession Nos. NP_690605.1 and NP_068769.2, and the
nucleotide sequence encoding transcript variants 1 and 3 of the
human CD20 can be found at Accession No. NM_152866.2 and
NM_021950.3, respectively. In one aspect the antigen-binding
portion of the CAR recognizes and binds an antigen within the
extracellular domain of the CD20 protein. In one aspect, the CD20
protein is expressed on a cancer cell.
[0195] As used herein, the term "CD22," refers to an antigenic
determinant known to be detectable on leukemia precursor cells. The
human and murine amino acid and nucleic acid sequences can be found
in a public database, such as GenBank, UniProt and Swiss-Prot. For
example, the amino acid sequences of isoforms 1-5 human CD22 can be
found at Accession Nos. NP 001762.2, NP 001172028.1, NP
001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and
the nucleotide sequence encoding variants 1-5 of the human CD22 can
be found at Accession No. NM 001771.3, NM 001185099.1, NM
001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In
one aspect the antigen-binding portion of the CAR recognizes and
binds an antigen within the extracellular domain of the CD22
protein. In one aspect, the CD22 protein is expressed on a cancer
cell.
[0196] As used herein, the term "ROR1" refers to an antigenic
determinant known to be detectable on leukemia precursor cells. The
human and murine amino acid and nucleic acid sequences can be found
in a public database, such as GenBank, UniProt and Swiss-Prot. For
example, the amino acid sequences of isoforms land 2 precursors of
human ROR1 can be found at Accession Nos. NP_005003.2 and
NP_001077061.1, respectively, and the mRNA sequences encoding them
can be found at Accession Nos. NM_005012.3 and NM_001083592.1,
respectively. In one aspect the antigen-binding portion of the CAR
recognizes and binds an antigen within the extracellular domain of
the ROR1 protein. In one aspect, the ROR1 protein is expressed on a
cancer cell.
[0197] The term "antibody," as used herein, refers to a protein, or
polypeptide sequence derived from an immunoglobulin molecule which
specifically binds with an antigen. Antibodies can be polyclonal or
monoclonal, multiple or single chain, or intact immunoglobulins,
and may be derived from natural sources or from recombinant
sources. Antibodies can be tetramers of immunoglobulin
molecules.
[0198] The term "antibody fragment" refers to at least one portion
of an antibody, that retains the ability to specifically interact
with (e.g., by binding, steric hinderance,
stabilizing/destabilizing, spatial distribution) an epitope of an
antigen. Examples of antibody fragments include, but are not
limited to, Fab, Fab', F(ab').sub.2, Fv fragments, scFv antibody
fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of
the VH and CH1 domains, linear antibodies, single domain antibodies
such as sdAb (either VL or VH), camelid VHH domains, multi-specific
antibodies formed from antibody fragments such as a bivalent
fragment comprising two Fab fragments linked by a disulfide brudge
at the hinge region, and an isolated CDR or other epitope binding
fragments of an antibody. An antigen binding fragment can also be
incorporated into single domain antibodies, maxibodies, minibodies,
nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR
and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology
23:1126-1136, 2005). Antigen binding fragments can also be grafted
into scaffolds based on polypeptides such as a fibronectin type III
(Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin
polypeptide minibodies).
[0199] The term "scFv" refers to a fusion protein comprising at
least one antibody fragment comprising a variable region of a light
chain and at least one antibody fragment comprising a variable
region of a heavy chain, wherein the light and heavy chain variable
regions are contiguously linked, e.g., via a synthetic linker,
e.g., a short flexible polypeptide linker, and capable of being
expressed as a single chain polypeptide, and wherein the scFv
retains the specificity of the intact antibody from which it is
derived. Unless specified, as used herein an scFv may have the VL
and VH variable regions in either order, e.g., with respect to the
N-terminal and C-terminal ends of the polypeptide, the scFv may
comprise VL-linker-VH or may comprise VH-linker-VL.
[0200] The portion of the CAR of the invention comprising an
antibody or antibody fragment thereof may exist in a variety of
forms where the antigen binding domain is expressed as part of a
contiguous polypeptide chain including, for example, a single
domain antibody fragment (sdAb), a single chain antibody (scFv), a
humanized antibody or bispecific antibody (Harlow et al., 1999, In:
Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A
Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426). In one aspect, the antigen binding domain of a CAR
composition of the invention comprises an antibody fragment. In a
further aspect, the CAR comprises an antibody fragment that
comprises a scFv. The precise amino acid sequence boundaries of a
given CDR can be determined using any of a number of well-known
schemes, including those described by Kabat et al. (1991),
"Sequences of Proteins of Immunological Interest," 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,
927-948 ("Chothia" numbering scheme), or a combination thereof.
[0201] As used herein, the term "binding domain" or "antibody
molecule" refers to a protein, e.g., an immunoglobulin chain or
fragment thereof, comprising at least one immunoglobulin variable
domain sequence. The term "binding domain" or "antibody molecule"
encompasses antibodies and antibody fragments. In an embodiment, an
antibody molecule is a multispecific antibody molecule, e.g., it
comprises a plurality of immunoglobulin variable domain sequences,
wherein a first immunoglobulin variable domain sequence of the
plurality has binding specificity for a first epitope and a second
immunoglobulin variable domain sequence of the plurality has
binding specificity for a second epitope. In an embodiment, a
multispecific antibody molecule is a bispecific antibody molecule.
A bispecific antibody has specificity for no more than two
antigens. A bispecific antibody molecule is characterized by a
first immunoglobulin variable domain sequence which has binding
specificity for a first epitope and a second immunoglobulin
variable domain sequence that has binding specificity for a second
epitope.
[0202] The portion of the CAR of the invention comprising an
antibody or antibody fragment thereof may exist in a variety of
forms where the antigen binding domain is expressed as part of a
contiguous polypeptide chain including, for example, a single
domain antibody fragment (sdAb), a single chain antibody (scFv), a
humanized antibody, or bispecific antibody (Harlow et al., 1999,
In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A
Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426). In one aspect, the antigen binding domain of a CAR
composition of the invention comprises an antibody fragment. In a
further aspect, the CAR comprises an antibody fragment that
comprises a scFv.
[0203] The term "antibody heavy chain," refers to the larger of the
two types of polypeptide chains present in antibody molecules in
their naturally occurring conformations, and which normally
determines the class to which the antibody belongs.
[0204] The term "antibody light chain," refers to the smaller of
the two types of polypeptide chains present in antibody molecules
in their naturally occurring conformations. Kappa (K) and lambda
(.lamda.) light chains refer to the two major antibody light chain
isotypes.
[0205] The term "recombinant antibody" refers to an antibody which
is generated using recombinant DNA technology, such as, for
example, an antibody expressed by a bacteriophage or yeast
expression system. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using recombinant DNA or amino acid sequence technology which is
available and well known in the art.
[0206] The term "antigen" or "Ag" refers to a molecule that
provokes an immune response. This immune response may involve
either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. Furthermore, one skilled in the art will understand that an
antigen need not be encoded solely by a full length nucleotide
sequence of a gene. It is readily apparent that the present
invention includes, but is not limited to, the use of partial
nucleotide sequences of more than one gene and that these
nucleotide sequences are arranged in various combinations to encode
polypeptides that elicit the desired immune response. Moreover, a
skilled artisan will understand that an antigen need not be encoded
by a "gene" at all. It is readily apparent that an antigen can be
generated synthesized or can be derived from a biological sample,
or might be macromolecule besides a polypeptide. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a fluid with other biological components.
[0207] The term "anti-cancer effect" refers to a biological effect
which can be manifested by various means, including but not limited
to, e.g., a decrease in tumor volume, a decrease in the number of
cancer cells, a decrease in the number of metastases, an increase
in life expectancy, decrease in cancer cell proliferation, decrease
in cancer cell survival, or amelioration of various physiological
symptoms associated with the cancerous condition. An "anti-cancer
effect" can also be manifested by the ability of the peptides,
polynucleotides, cells and antibodies in prevention of the
occurrence of cancer in the first place. The term "anti-tumor
effect" refers to a biological effect which can be manifested by
various means, including but not limited to, e.g., a decrease in
tumor volume, a decrease in the number of tumor cells, a decrease
in tumor cell proliferation, or a decrease in tumor cell
survival.
[0208] The term "autologous" refers to any material derived from
the same individual to whom it is later to be re-introduced into
the individual.
[0209] The term "allogeneic" refers to any material derived from a
different animal of the same species as the individual to whom the
material is introduced. Two or more individuals are said to be
allogeneic to one another when the genes at one or more loci are
not identical. In some aspects, allogeneic material from
individuals of the same species may be sufficiently unlike
genetically to interact antigenically
[0210] The term "xenogeneic" refers to a graft derived from an
animal of a different species.
[0211] The term "cancer" refers to a disease characterized by the
uncontrolled growth of aberrant cells. Cancer cells can spread
locally or through the bloodstream and lymphatic system to other
parts of the body. Examples of various cancers are described herein
and include but are not limited to, breast cancer, prostate cancer,
ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,
colorectal cancer, renal cancer, liver cancer, brain cancer,
lymphoma, leukemia, lung cancer and the like. The terms "tumor" and
"cancer" are used interchangeably herein, e.g., both terms
encompass solid and liquid, e.g., diffuse or circulating, tumors.
As used herein, the term "cancer" or "tumor" includes premalignant,
as well as malignant cancers and tumors.
[0212] The phrase "disease associated with expression of CD19"
includes, but is not limited to, a disease associated with
expression of CD19 or condition associated with cells which
express, or at any time expressed, CD19 including, e.g.,
proliferative diseases such as a cancer or malignancy or a
precancerous condition such as a myelodysplasia, a myelodysplastic
syndrome or a preleukemia; or a noncancer related indication
associated with cells which express CD19. For the avoidance of
doubt, a disease associated with expression of CD19 may include a
condition associated with cells which do not presently express
CD19, e.g., because CD19 expression has been downregulated, e.g.,
due to treatment with a molecule targeting CD19, e.g., a CD19 CAR,
but which at one time expressed CD19. In one aspect, a cancer
associated with expression of CD19 is a hematological cancer. In
one aspect, the hematolical cancer is a leukemia or a lymphoma. In
one aspect, a cancer associated with expression of CD19 includes
cancers and malignancies including, but not limited to, e.g., one
or more acute leukemias including but not limited to, e.g., B-cell
acute Lymphoid Leukemia (BALL), T-cell acute Lymphoid Leukemia
(TALL), acute lymphoid leukemia (ALL); one or more chronic
leukemias including but not limited to, e.g., chronic myelogenous
leukemia (CML), Chronic Lymphoid Leukemia (CLL). Additional cancers
or hematologic conditions associated with expression of CD19
comprise, but are not limited to, e.g., B cell prolymphocytic
leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's
lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy
cell leukemia, small cell- or a large cell-follicular lymphoma,
malignant lymphoproliferative conditions, MALT lymphoma, mantle
cell lymphoma (MCL), Marginal zone lymphoma, multiple myeloma,
myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma,
Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic
cell neoplasm, Waldenstrom macroglobulinemia, and "preleukemia"
which are a diverse collection of hematological conditions united
by ineffective production (or dysplasia) of myeloid blood cells,
and the like. Further diseases associated with expression of CD19
expression include, but not limited to, e.g., atypical and/or
non-classical cancers, malignancies, precancerous conditions or
proliferative diseases associated with expression of CD19.
Non-cancer related indications associated with expression of CD19
include, but are not limited to, e.g., autoimmune disease, (e.g.,
lupus), inflammatory disorders (allergy and asthma) and
transplantation. In some embodiments, the tumor antigen-expressing
cells express, or at any time expressed, mRNA encoding the tumor
antigen. In an embodiment, the tumor antigen-expressing cells
produce the tumor antigen protein (e.g., wild-type or mutant), and
the tumor antigen protein may be present at normal levels or
reduced levels. In an embodiment, the tumor antigen-expressing
cells produced detectable levels of a tumor antigen protein at one
point, and subsequently produced substantially no detectable tumor
antigen protein.
[0213] The phrase "disease associated with expression of a B-cell
antigen" includes, but is not limited to, a disease associated with
expression of one or more of CD19, CD20, CD22 or ROR1, or a
condition associated with cells which express, or at any time
expressed, one or more of CD19, CD20, CD22 or ROR1, including,
e.g., proliferative diseases such as a cancer or malignancy or a
precancerous condition such as a myelodysplasia, a myelodysplastic
syndrome or a preleukemia; or a noncancer related indication
associated with cells which express one or more of CD19, CD20, CD22
or ROR1. For the avoidance of doubt, a disease associated with
expression of the B-cell antigen may include a condition associated
with cells which do not presently express the B-cell antigen, e.g.,
because the antigen expression has been downregulated, e.g., due to
treatment with a molecule targeting the B-cell antigen, e.g., a
B-cell targeting CAR, but which at one time expressed the antigen.
The phrase "disease associated with expression of a B-cell antigen"
includes a disease associated with expression of CD19, as described
herein.
[0214] The term "conservative sequence modifications" refers to
amino acid modifications that do not significantly affect or alter
the binding characteristics of the antibody or antibody fragment
containing the amino acid sequence. Such conservative modifications
include amino acid substitutions, additions and deletions.
Modifications can be introduced into an antibody or antibody
fragment of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within a CAR of the invention can be
replaced with other amino acid residues from the same side chain
family and the altered CAR can be tested using the functional
assays described herein.
[0215] The term "stimulation," refers to a primary response induced
by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or
CAR) with its cognate ligand (or tumor antigen in the case of a
CAR) thereby mediating a signal transduction event, such as, but
not limited to, signal transduction via the TCR/CD3 complex or
signal transduction via the appropriate NK receptor or signaling
domains of the CAR. Stimulation can mediate altered expression of
certain molecules.
[0216] The term "stimulatory molecule," refers to a molecule
expressed by an immune cell (e.g., T cell, NK cell, B cell) that
provides the cytoplasmic signaling sequence(s) that regulate
activation of the immune cell in a stimulatory way for at least
some aspect of the immune cell signaling pathway. In one aspect,
the signal is a primary signal that is initiated by, for instance,
binding of a TCR/CD3 complex with an MHC molecule loaded with
peptide, and which leads to mediation of a T cell response,
including, but not limited to, proliferation, activation,
differentiation, and the like. A primary cytoplasmic signaling
sequence (also referred to as a "primary signaling domain") that
acts in a stimulatory manner may contain a signaling motif which is
known as immunoreceptor tyrosine-based activation motif or ITAM.
Examples of an ITAM containing cytoplasmic signaling sequence that
is of particular use in the invention includes, but is not limited
to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc
gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3
epsilon, CD79a, CD79b, DAP10, and DAP12. In a specific CAR of the
invention, the intracellular signaling domain in any one or more
CARS of the invention comprises an intracellular signaling
sequence, e.g., a primary signaling sequence of CD3-zeta. In a
specific CAR of the invention, the primary signaling sequence of
CD3-zeta is the sequence provided as SEQ ID NO: 17, or the
equivalent residues from a non-human species, e.g., mouse, rodent,
monkey, ape and the like. In a specific CAR of the invention, the
primary signaling sequence of CD3-zeta is the sequence as provided
in SEQ ID NO: 43, or the equivalent residues from a non-human
species, e.g., mouse, rodent, monkey, ape and the like.
[0217] The term "antigen presenting cell" or "APC" refers to an
immune system cell such as an accessory cell (e.g., a B-cell, a
dendritic cell, and the like) that displays a foreign antigen
complexed with major histocompatibility complexes (MHC's) on its
surface. T-cells may recognize these complexes using their T-cell
receptors (TCRs). APCs process antigens and present them to
T-cells.
[0218] An "intracellular signaling domain," as the term is used
herein, refers to an intracellular portion of a molecule. The
intracellular signaling domain generates a signal that promotes an
immune effector function of the CAR containing cell, e.g., a CART
cell. Examples of immune effector function, e.g., in a CART cell,
include cytolytic activity and helper activity, including the
secretion of cytokines.
[0219] In an embodiment, the intracellular signaling domain can
comprise a primary intracellular signaling domain. Exemplary
primary intracellular signaling domains include those derived from
the molecules responsible for primary stimulation, or antigen
dependent simulation. In an embodiment, the intracellular signaling
domain can comprise a costimulatory intracellular domain. Exemplary
costimulatory intracellular signaling domains include those derived
from molecules responsible for costimulatory signals, or antigen
independent stimulation. For example, in the case of a CART, a
primary intracellular signaling domain can comprise a cytoplasmic
sequence of a T cell receptor, and a costimulatory intracellular
signaling domain can comprise cytoplasmic sequence from co-receptor
or costimulatory molecule.
[0220] A primary intracellular signaling domain can comprise a
signaling motif which is known as an immunoreceptor tyrosine-based
activation motif or ITAM. Examples of ITAM containing primary
cytoplasmic signaling sequences include, but are not limited to,
those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma
RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon,
CD79a, CD79b, DAP10, and DAP12.
[0221] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or
"TCR-zeta" is defined as the protein provided as GenBan Acc. No.
BAG36664.1, or the equivalent residues from a non-human species,
e.g., mouse, rodent, monkey, ape and the like, and a "zeta
stimulatory domain" or alternatively a "CD3-zeta stimulatory
domain" or a "TCR-zeta stimulatory domain" is defined as the amino
acid residues from the cytoplasmic domain of the zeta chain, or
functional derivatives thereof, that are sufficient to functionally
transmit an initial signal necessary for T cell activation. In one
aspect the cytoplasmic domain of zeta comprises residues 52 through
164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from
a non-human species, e.g., mouse, rodent, monkey, ape and the like,
that are functional orthologs thereof. In one aspect, the "zeta
stimulatory domain" or a "CD3-zeta stimulatory domain" is the
sequence provided as SEQ ID NO: 17. In one aspect, the "zeta
stimulatory domain" or a "CD3-zeta stimulatory domain" is the
sequence provided as SEQ ID NO:43.
[0222] The term "costimulatory molecule" refers to the cognate
binding partner on a T cell that specifically binds with a
costimulatory ligand, thereby mediating a costimulatory response by
the T cell, such as, but not limited to, proliferation.
Costimulatory molecules are cell surface molecules other than
antigen receptors or their ligands that are contribute to an
efficient immune response. Costimulatory molecules include, but are
not limited to an MHC class I molecule, BTLA and a Toll ligand
receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1
1a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such
costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM
(LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,
CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4,
VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d,
ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b, ITGAX, CD11c,
ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,
TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96
(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100
(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150, IPO-3),
BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp,
CD19a, and a ligand that specifically binds with CD83.
[0223] A costimulatory intracellular signaling domain can be the
intracellular portion of a costimulatory molecule. A costimulatory
molecule can be represented in the following protein families: TNF
receptor proteins, Immunoglobulin-like proteins, cytokine
receptors, integrins, signaling lymphocytic activation molecules
(SLAM proteins), and activating NK cell receptors. Examples of such
molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30,
CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated
antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D,
SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that
specifically binds with CD83, and the like.
[0224] The intracellular signaling domain can comprise the entire
intracellular portion, or the entire native intracellular signaling
domain, of the molecule from which it is derived, or a functional
fragment or derivative thereof.
[0225] The term "4-1BB" refers to a member of the TNFR superfamily
with an amino acid sequence provided as GenBank Acc. No.
AAA62478.2, or the equivalent residues from a non-human species,
e.g., mouse, rodent, monkey, ape and the like; and a "4-1BB
costimulatory domain" is defined as amino acid residues 214-255 of
GenBank accno. AAA62478.2, or the equivalent residues from a
non-human species, e.g., mouse, rodent, monkey, ape and the like.
In one aspect, the "4-1BB costimulatory domain" is the sequence
provided as SEQ ID NO: 16 or the equivalent residues from a
non-human species, e.g., mouse, rodent, monkey, ape and the
like.
[0226] "Immune effector cell," as that term is used herein, refers
to a cell that is involved in an immune response, e.g., in the
promotion of an immune effector response. Examples of immune
effector cells include T cells, e.g., alpha/beta T cells and
gamma/delta T cells, B cells, natural killer (NK) cells, natural
killer T (NKT) cells, mast cells, and myeloic-derived
phagocytes.
[0227] "Immune effector function or immune effector response," as
that term is used herein, refers to function or response, e.g., of
an immune effector cell, that enhances or promotes an immune attack
of a target cell. E.g., an immune effector function or response
refers a property of a T or NK cell that promotes killing or the
inhibition of growth or proliferation, of a target cell. In the
case of a T cell, primary stimulation and co-stimulation are
examples of immune effector function or response.
[0228] The term "encoding" refers to the inherent property of
specific sequences of nucleotides in a polynucleotide, such as a
gene, a cDNA, or an mRNA, to serve as templates for synthesis of
other polymers and macromolecules in biological processes having
either a defined sequence of nucleotides (e.g., rRNA, tRNA and
mRNA) or a defined sequence of amino acids and the biological
properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes
a protein if transcription and translation of mRNA corresponding to
that gene produces the protein in a cell or other biological
system. Both the coding strand, the nucleotide sequence of which is
identical to the mRNA sequence and is usually provided in sequence
listings, and the non-coding strand, used as the template for
transcription of a gene or cDNA, can be referred to as encoding the
protein or other product of that gene or cDNA.
[0229] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or a RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0230] The term "effective amount" or "therapeutically effective
amount" are used interchangeably herein, and refer to an amount of
a compound, formulation, material, or composition, as described
herein effective to achieve a particular biological result.
[0231] The term "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0232] The term "exogenous" refers to any material introduced from
or produced outside an organism, cell, tissue or system.
[0233] The term "expression" refers to the transcription and/or
translation of a particular nucleotide sequence driven by a
promoter.
[0234] The term "transfer vector" refers to a composition of matter
which comprises an isolated nucleic acid and which can be used to
deliver the isolated nucleic acid to the interior of a cell.
Numerous vectors are known in the art including, but not limited
to, linear polynucleotides, polynucleotides associated with ionic
or amphiphilic compounds, plasmids, and viruses. Thus, the term
"transfer vector" includes an autonomously replicating plasmid or a
virus. The term should also be construed to further include
non-plasmid and non-viral compounds which facilitate transfer of
nucleic acid into cells, such as, for example, a polylysine
compound, liposome, and the like. Examples of viral transfer
vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, lentiviral
vectors, and the like.
[0235] The term "expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, including cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses (e.g.,
lentiviruses, retroviruses, adenoviruses, and adeno-associated
viruses) that incorporate the recombinant polynucleotide.
[0236] The term "lentivirus" refers to a genus of the Retroviridae
family. Lentiviruses are unique among the retroviruses in being
able to infect non-dividing cells; they can deliver a significant
amount of genetic information into the DNA of the host cell, so
they are one of the most efficient methods of a gene delivery
vector. HIV, SIV, and FIV are all examples of lentiviruses.
[0237] The term "lentiviral vector" refers to a vector derived from
at least a portion of a lentivirus genome, including especially a
self-inactivating lentiviral vector as provided in Milone et al.,
Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus
vectors that may be used in the clinic, include but are not limited
to, e.g., the LENTIVECTOR.RTM. gene delivery technology from Oxford
BioMedica, the LENTIMAX.TM. vector system from Lentigen and the
like. Nonclinical types of lentiviral vectors are also available
and would be known to one skilled in the art.
[0238] The term "homologous" or "identity" refers to the subunit
sequence identity between two polymeric molecules, e.g., between
two nucleic acid molecules, such as, two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit; e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous or identical at
that position. The homology between two sequences is a direct
function of the number of matching or homologous positions; e.g.,
if half (e.g., five positions in a polymer ten subunits in length)
of the positions in two sequences are homologous, the two sequences
are 50% homologous; if 90% of the positions (e.g., 9 of 10), are
matched or homologous, the two sequences are 90% homologous.
[0239] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies and antibody fragments thereof are human immunoglobulins
(recipient antibody or antibody fragment) in which residues from a
complementary-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, a
humanized antibody/antibody fragment can comprise residues which
are found neither in the recipient antibody nor in the imported CDR
or framework sequences. These modifications can further refine and
optimize antibody or antibody fragment performance. In general, the
humanized antibody or antibody fragment thereof will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or a
significant portion of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody or antibody
fragment can also comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For
further details, see Jones et al., Nature, 321: 522-525, 1986;
Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op.
Struct. Biol., 2: 593-596, 1992.
[0240] "Fully human" refers to an immunoglobulin, such as an
antibody or antibody fragment, where the whole molecule is of human
origin or consists of an amino acid sequence identical to a human
form of the antibody or immunoglobulin.
[0241] The term "isolated" means altered or removed from the
natural state. For example, a nucleic acid or a peptide naturally
present in a living animal is not "isolated," but the same nucleic
acid or peptide partially or completely separated from the
coexisting materials of its natural state is "isolated." An
isolated nucleic acid or protein can exist in substantially
purified form, or can exist in a non-native environment such as,
for example, a host cell.
[0242] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0243] The term "operably linked" or "transcriptional control"
refers to functional linkage between a regulatory sequence and a
heterologous nucleic acid sequence resulting in expression of the
latter. For example, a first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Operably linked
DNA sequences can be contiguous with each other and, e.g., where
necessary to join two protein coding regions, are in the same
reading frame.
[0244] The term "parenteral" administration of an immunogenic
composition includes, e.g., subcutaneous (s.c.), intravenous
(i.v.), intramuscular (i.m.), or intrasternal injection,
intratumoral, or infusion techniques.
[0245] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)).
[0246] The terms "peptide," "polypeptide," and "protein" are used
interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide
must contain at least two amino acids, and no limitation is placed
on the maximum number of amino acids that can comprise a protein's
or peptide's sequence. Polypeptides include any peptide or protein
comprising two or more amino acids joined to each other by peptide
bonds. As used herein, the term refers to both short chains, which
also commonly are referred to in the art as peptides, oligopeptides
and oligomers, for example, and to longer chains, which generally
are referred to in the art as proteins, of which there are many
types. "Polypeptides" include, for example, biologically active
fragments, substantially homologous polypeptides, oligopeptides,
homodimers, heterodimers, variants of polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, among others.
A polypeptide includes a natural peptide, a recombinant peptide, or
a combination thereof.
[0247] The term "promoter" refers to a DNA sequence recognized by
the synthetic machinery of the cell, or introduced synthetic
machinery, required to initiate the specific transcription of a
polynucleotide sequence.
[0248] The term "promoter/regulatory sequence" refers to a nucleic
acid sequence which is required for expression of a gene product
operably linked to the promoter/regulatory sequence. In some
instances, this sequence may be the core promoter sequence and in
other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue specific manner.
[0249] The term "constitutive" promoter refers to a nucleotide
sequence which, when operably linked with a polynucleotide which
encodes or specifies a gene product, causes the gene product to be
produced in a cell under most or all physiological conditions of
the cell.
[0250] The term "inducible" promoter refers to a nucleotide
sequence which, when operably linked with a polynucleotide which
encodes or specifies a gene product, causes the gene product to be
produced in a cell substantially only when an inducer which
corresponds to the promoter is present in the cell.
[0251] The term "tissue-specific" promoter refers to a nucleotide
sequence which, when operably linked with a polynucleotide encodes
or specified by a gene, causes the gene product to be produced in a
cell substantially only if the cell is a cell of the tissue type
corresponding to the promoter.
[0252] The term "flexible polypeptide linker" or "linker" as used
in the context of a scFv refers to a peptide linker that consists
of amino acids such as glycine and/or serine residues used alone or
in combination, to link variable heavy and variable light chain
regions together. In one embodiment, the flexible polypeptide
linker is a Gly/Ser linker and comprises the amino acid sequence
(Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or
greater than 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7,
n=8, n=9 and n=10 (SEQ ID NO:105). In one embodiment, the flexible
polypeptide linkers include, but are not limited to, (Gly.sub.4
Ser).sub.4 (SEQ ID NO: 106) or (Gly.sub.4 Ser).sub.3 (SEQ ID NO:
107). In another embodiment, the linkers include multiple repeats
of (Gly.sub.2Ser), (GlySer) or (Gly.sub.3Ser) (SEQ ID NO: 108).
Also included within the scope of the invention are linkers
described in WO2012/138475, incorporated herein by reference).
[0253] As used herein, a 5' cap (also termed an RNA cap, an RNA
7-methylguanosine cap or an RNA m.sup.7G cap) is a modified guanine
nucleotide that has been added to the "front" or 5' end of a
eukaryotic messenger RNA shortly after the start of transcription.
The 5' cap consists of a terminal group which is linked to the
first transcribed nucleotide. Its presence is critical for
recognition by the ribosome and protection from RNases. Cap
addition is coupled to transcription, and occurs
co-transcriptionally, such that each influences the other. Shortly
after the start of transcription, the 5' end of the mRNA being
synthesized is bound by a cap-synthesizing complex associated with
RNA polymerase. This enzymatic complex catalyzes the chemical
reactions that are required for mRNA capping. Synthesis proceeds as
a multi-step biochemical reaction. The capping moiety can be
modified to modulate functionality of mRNA such as its stability or
efficiency of translation.
[0254] As used herein, "in vitro transcribed RNA" refers to RNA,
preferably mRNA, that has been synthesized in vitro. Generally, the
in vitro transcribed RNA is generated from an in vitro
transcription vector. The in vitro transcription vector comprises a
template that is used to generate the in vitro transcribed RNA.
[0255] As used herein, a "poly(A)" is a series of adenosines
attached by polyadenylation to the mRNA. In the preferred
embodiment of a construct for transient expression, the polyA is
between 50 and 5000 (SEQ ID NO: 109), preferably greater than 64,
more preferably greater than 100, most preferably greater than 300
or 400. poly(A) sequences can be modified chemically or
enzymatically to modulate mRNA functionality such as localization,
stability or efficiency of translation.
[0256] As used herein, "polyadenylation" refers to the covalent
linkage of a polyadenylyl moiety, or its modified variant, to a
messenger RNA molecule. In eukaryotic organisms, most messenger RNA
(mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A)
tail is a long sequence of adenine nucleotides (often several
hundred) added to the pre-mRNA through the action of an enzyme,
polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is
added onto transcripts that contain a specific sequence, the
polyadenylation signal. The poly(A) tail and the protein bound to
it aid in protecting mRNA from degradation by exonucleases.
Polyadenylation is also important for transcription termination,
export of the mRNA from the nucleus, and translation.
Polyadenylation occurs in the nucleus immediately after
transcription of DNA into RNA, but additionally can also occur
later in the cytoplasm. After transcription has been terminated,
the mRNA chain is cleaved through the action of an endonuclease
complex associated with RNA polymerase. The cleavage site is
usually characterized by the presence of the base sequence AAUAAA
near the cleavage site. After the mRNA has been cleaved, adenosine
residues are added to the free 3' end at the cleavage site.
[0257] As used herein, "transient" refers to expression of a
non-integrated transgene for a period of hours, days or weeks,
wherein the period of time of expression is less than the period of
time for expression of the gene if integrated into the genome or
contained within a stable plasmid replicon in the host cell.
[0258] The term "signal transduction pathway" refers to the
biochemical relationship between a variety of signal transduction
molecules that play a role in the transmission of a signal from one
portion of a cell to another portion of a cell. The phrase "cell
surface receptor" includes molecules and complexes of molecules
capable of receiving a signal and transmitting signal across the
membrane of a cell.
[0259] The term "subject" is intended to include living organisms
in which an immune response can be elicited (e.g., mammals,
human).
[0260] The term, a "substantially purified" cell refers to a cell
that is essentially free of other cell types. A substantially
purified cell also refers to a cell which has been separated from
other cell types with which it is normally associated in its
naturally occurring state. In some instances, a population of
substantially purified cells refers to a homogenous population of
cells. In other instances, this term refers simply to cell that
have been separated from the cells with which they are naturally
associated in their natural state. In some aspects, the cells are
cultured in vitro. In other aspects, the cells are not cultured in
vitro.
[0261] The term "therapeutic" as used herein means a treatment. A
therapeutic effect is obtained by reduction, suppression,
remission, or eradication of a disease state.
[0262] The term "prophylaxis" as used herein means the prevention
of or protective treatment for a disease or disease state.
[0263] In the context of the present invention, "tumor antigen" or
"hyperproliferative disorder antigen" or "antigen associated with a
hyperproliferative disorder" refers to antigens that are common to
specific hyperproliferative disorders. In certain aspects, the
hyperproliferative disorder antigens of the present invention are
derived from, cancers including but not limited to primary or
metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver
cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine
cancer, cervical cancer, bladder cancer, kidney cancer and
adenocarcinomas such as breast cancer, prostate cancer, ovarian
cancer, pancreatic cancer, and the like.
[0264] The term "transfected" or "transformed" or "transduced"
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.
[0265] The term "specifically binds," refers to an antibody, or a
ligand, which recognizes and binds with a binding partner (e.g., a
stimulatory tumor antigen) protein present in a sample, but which
antibody or ligand does not substantially recognize or bind other
molecules in the sample.
[0266] "Regulatable chimeric antigen receptor (RCAR)," as that term
is used herein, refers to a set of polypeptides, typically two in
the simplest embodiments, which when in a RCARX cell, provides the
RCARX cell with specificity for a target cell, typically a cancer
cell, and with regulatable intracellular signal generation or
proliferation, which can optimize an immune effector property of
the RCARX cell. An RCARX cell relies at least in part, on an
antigen binding domain to provide specificity to a target cell that
comprises the antigen bound by the antigen binding domain. In an
embodiment, an RCAR includes a dimerization switch that, upon the
presence of a dimerization molecule, can couple an intracellular
signaling domain to the antigen binding domain.
[0267] "Membrane anchor" or "membrane tethering domain", as that
term is used herein, refers to a polypeptide or moiety, e.g., a
myristoyl group, sufficient to anchor an extracellular or
intracellular domain to the plasma membrane.
[0268] "Switch domain," as that term is used herein, e.g., when
referring to an RCAR, refers to an entity, typically a
polypeptide-based entity, that, in the presence of a dimerization
molecule, associates with another switch domain. The association
results in a functional coupling of a first entity linked to, e.g.,
fused to, a first switch domain, and a second entity linked to,
e.g., fused to, a second switch domain. A first and second switch
domain are collectively referred to as a dimerization switch. In
embodiments, the first and second switch domains are the same as
one another, e.g., they are polypeptides having the same primary
amino acid sequence, and are referred to collectively as a
homodimerization switch. In embodiments, the first and second
switch domains are different from one another, e.g., they are
polypeptides having different primary amino acid sequences, and are
referred to collectively as a heterodimerization switch. In
embodiments, the switch is intracellular. In embodiments, the
switch is extracellular. In embodiments, the switch domain is a
polypeptide-based entity, e.g., FKBP or FRB-based, and the
dimerization molecule is small molecule, e.g., a rapalogue. In
embodiments, the switch domain is a polypeptide-based entity, e.g.,
an scFv that binds a myc peptide, and the dimerization molecule is
a polypeptide, a fragment thereof, or a multimer of a polypeptide,
e.g., a myc ligand or multimers of a myc ligand that bind to one or
more myc scFvs. In embodiments, the switch domain is a
polypeptide-based entity, e.g., myc receptor, and the dimerization
molecule is an antibody or fragments thereof, e.g., myc
antibody.
[0269] "Dimerization molecule," as that term is used herein, e.g.,
when referring to an RCAR, refers to a molecule that promotes the
association of a first switch domain with a second switch domain.
In embodiments, the dimerization molecule does not naturally occur
in the subject, or does not occur in concentrations that would
result in significant dimerization. In embodiments, the
dimerization molecule is a small molecule, e.g., rapamycin or a
rapalogue, e.g, RAD001.
[0270] The term "bioequivalent" refers to an amount of an agent
other than the reference compound (e.g., RAD001), required to
produce an effect equivalent to the effect produced by the
reference dose or reference amount of the reference compound (e.g.,
RAD001). In an embodiment the effect is the level of mTOR
inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as
evaluated in an in vivo or in vitro assay, e.g., as measured by an
assay described herein, e.g., the Boulay assay, or measurement of
phosphorylated S6 levels by western blot. In an embodiment, the
effect is alteration of the ratio of PD-1 positive/PD-1 negative T
cells, as measured by cell sorting. In an embodiment a
bioequivalent amount or dose of an mTOR inhibitor is the amount or
dose that achieves the same level of P70 S6 kinase inhibition as
does the reference dose or reference amount of a reference
compound. In an embodiment, a bioequivalent amount or dose of an
mTOR inhibitor is the amount or dose that achieves the same level
of alteration in the ratio of PD-1 positive/PD-1 negative T cells
as does the reference dose or reference amount of a reference
compound.
[0271] The term "low, immune enhancing, dose" when used in
conjuction with an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR
inhibitor, refers to a dose of mTOR inhibitor that partially, but
not fully, inhibits mTOR activity, e.g., as measured by the
inhibition of P70 S6 kinase activity. Methods for evaluating mTOR
activity, e.g., by inhibition of P70 S6 kinase, are discussed
herein. The dose is insufficient to result in complete immune
suppression but is sufficient to enhance the immune response. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in a decrease in the number of PD-1 positive T cells and/or
an increase in the number of PD-1 negative T cells, or an increase
in the ratio of PD-1 negative T cells/PD-1 positive T cells. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in an increase in the number of naive T cells. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in one or more of the following:
[0272] an increase in the expression of one or more of the
following markers: CD62L.sup.highCD127.sup.high, CD27.sup.+, and
BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0273] a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; and
[0274] an increase in the number of memory T cell precursors, e.g.,
cells with any one or combination of the following characteristics:
increased CD62L.sup.high, increased CD127.sup.high increased CD27+,
decreased KLRG1, and increased BCL2;
wherein any of the changes described above occurs, e.g., at least
transiently, e.g., as compared to a non-treated subject.
[0275] "Refractory" as used herein refers to a disease, e.g.,
cancer, that does not respond to a treatment. In embodiments, a
refractory cancer can be resistant to a treatment before or at the
beginning of the treatment. In other embodiments, the refractory
cancer can become refractory during a treatment.
[0276] A "complete responder" as used herein refers to a subject
having a disease, e.g., a cancer, who exhibits a complete response,
e.g., a complete remission, to a treatment. A complete response may
be identified, e.g., using the Cheson criteria as described
herein.
[0277] A "partial responder" as used herein refers to a subject
having a disease, e.g., a cancer, who exhibits a partial response,
e.g., a partial remission, to a treatment. A partial response may
be identified, e.g., using the Cheson criteria.
[0278] A "non-responder" as used herein refers to a subject having
a disease, e.g., a cancer, who does not exhibit a response to a
treatment, e.g., the patient has stable disease or progressive
disease. A non-responder may be identified, e.g., using the Cheson
criteria as described herein.
[0279] The term "relapse" as used herein refers to reappearance of
a disease (e.g., cancer) after an initial period of responsiveness
(e.g., complete response or partial response). The initial period
of responsiveness may involve the level of cancer cells falling
below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%,
2%, or 1%. The reappearance may involve the level of cancer cells
rising above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%,
3%, 2%, or 1%. Relapse may be identified, e.g., using the Cheson
criteria as described herein.
[0280] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as
95-99% identity, includes something with 95%, 96%, 97%, 98% or 99%
identity, and includes subranges such as 96-99%, 96-98%, 96-97%,
97-99%, 97-98% and 98-99% identity. This applies regardless of the
breadth of the range.
DESCRIPTION
[0281] Provided herein are compositions of matter and methods of
use for the treatment of a disease such as cancer (e.g.,
hematological cancers or other B cell malignancies) using immune
effector cells (e.g., T cells or NK cells) that express a chimeric
antigen receptor (CAR) (e.g., a CAR that targets a B-cell marker,
such as CD19). The methods include, inter alia, administering
immune effector cells (e.g., T cells or NK cells) expressing a B
cell targeting CAR described herein in combination with another
agent such as a kinase inhibitor, e.g., a kinase inhibitor
described herein.
[0282] The present invention provides, at least in part,
experiments supporting the high efficacy of a combination of a CAR
therapy (e.g., a B-cell targeting CAR therapy) and a kinase
inhibitor, e.g., a BTK inhibitor such as ibrutinib. The combination
of a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib,
with a CAR therapy can increase efficacy of the combination therapy
relative to a monotherapy of the kinase inhibitor, or a dose of
CAR-expressing cells, or both. These beneficial effects can, for
example, allow for a lower dose of the kinase inhibitor or the
CAR-expressing cells, or both, while maintaining efficacy. The
results herein are applicable to a wide range of cancers, e.g.,
hematological cancers and other B cell malignancies. For example,
ibrutinib inhibits BTK, which is elevated in most lymphomas. An
immune effector cell (e.g., T cell or NK cell) that expresses CAR19
targets cancers with CD19 surface expression, which is expressed in
most B cell malignancies. Alternatively or in combination with
CAR19, any other B-cell targeting CAR (e.g., a CAR targeting one or
more of: CD20, CD22, or ROR1) can be used in the combination
therapies described herein. Therefore, the combination of a CAR
therapy (e.g., one or more of a CD19 CAR, CD20 CAR, CD22 CAR or
ROR1 CAR therapy) with a BTK inhibitor (e.g., ibrutinib) is
suitable for treating a wide range of cancers involving
overproliferation of B cells, including lymphomas (e.g., Hodgkin
lymphoma), MCL, CLL, DLBCL, and multiple myeloma.
[0283] According to the present invention, ibrutinib can reduce
tumor masses and mobilize neoplastic B cells in the peripheral
blood (see e.g., Example 8 herein). Without wishing to be bound by
theory, certain lymphomas, such as MCL, are characterized by masses
of cancerous cells in proliferation centers in lymph nodes.
CAR-expressing immune effector cells sometimes have difficulty
penetrating these densely packed masses. Thus, a BTK inhibitor,
such as ibrutinib, can reduce tumor masses and mobilize neoplastic
B cells in the peripheral blood, making the lymphoma cells more
vulnerable to the CAR-expressing cells.
[0284] Alternatively or in combination, BTK inhibitors, such as
ibrutinib, can also affect the CAR-expressing cells. The present
invention demonstrates that ibrutinib treatment increases the level
of circulating CART19 cells (see e.g., data shown in Example 8).
Without wishing to be bound by theory, the increase in the level of
circulating CART19 cells may be a result of, for example, increased
proliferation, alteration of T cell phenotype, or other factors.
For example, ibrutinib can inhibit ITK, a kinase with homology to
BTK. ITK is expressed in T cells, and its inhibition may alter the
T cell phenotype. Treatment with a kinase inhibitor, such as
ibrutinib, can alter the T cell phenotype from a Th2 phenotype to a
Th1 phenotype, and thus increase the T cell proliferative capacity.
Pre-treatment, or co-administration, to a subject, of a BTK
inhibitor may increase the T cell proliferative capacity in the
subject, thus increasing the level of circulating CAR-expressing
cells. In addition, a subject pre-treated with a BTK inhibitor,
e.g., ibrutinib, can have a T cell population with a higher
proliferative capacity in their apheresis for CAR
manufacturing.
[0285] In one aspect, the invention provides a number of chimeric
antigen receptors (CAR) comprising an antibody or antibody fragment
engineered for specific binding to a B-cell antigen (e.g., chosen
from one or more of CD19, CD20, CD22 or ROR1 protein). In one
aspect, the invention provides a cell (e.g., T cell) engineered to
express a CAR, wherein the CAR T cell ("CART") exhibits an
anticancer property. In one aspect a cell is transformed with the
CAR and the CAR is expressed on the cell surface. In some
embodiments, the cell (e.g., T cell) is transduced with a viral
vector encoding a CAR. In some embodiments, the viral vector is a
retroviral vector. In some embodiments, the viral vector is a
lentiviral vector. In some such embodiments, the cell may stably
express the CAR. In another embodiment, the cell (e.g., T cell) is
transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a
CAR. In some such embodiments, the cell may transiently express the
CAR.
[0286] In one aspect, the anti-CD19 protein binding portion of the
CAR is a scFv antibody fragment. In one aspect such antibody
fragments are functional in that they retain the equivalent binding
affinity, e.g., they bind the same antigen with comparable
affinity, as the IgG antibody from which it is derived. In one
aspect such antibody fragments are functional in that they provide
a biological response that can include, but is not limited to,
activation of an immune response, inhibition of signal-transduction
origination from its target antigen, inhibition of kinase activity,
and the like, as will be understood by a skilled artisan. In one
aspect, the anti-CD19 antigen binding domain of the CAR is a scFv
antibody fragment that is humanized compared to the murine sequence
of the scFv from which it is derived. In one aspect, the parental
murine scFv sequence is the CAR19 construct provided in PCT
publication WO2012/079000 (incorporated herein by reference) and
provided herein as SEQ ID NO:59. In one embodiment, the anti-CD19
binding domain is a scFv described in WO2012/079000 and provided in
SEQ ID NO:59.
[0287] In some aspects, the antibodies of the invention are
incorporated into a chimeric antigen receptor (CAR). In one aspect,
the CAR comprises the polypeptide sequence provided as SEQ ID NO:
12 in PCT publication WO2012/079000, and provided herein as SEQ ID
NO: 58, wherein the scFv domain is substituted by one or more
sequences selected from SEQ ID NOS: 1-12. In one aspect, the scFv
domains of SEQ ID NOS:1-12 are humanized variants of the scFv
domain of SEQ ID NO:59, which is an scFv fragment of murine origin
that specifically binds to human CD19. Humanization of this mouse
scFv may be desired for the clinical setting, where the
mouse-specific residues may induce a human-anti-mouse antigen
(HAMA) response in patients who receive CART19 treatment, e.g.,
treatment with T cells transduced with the CAR19 construct.
[0288] In one aspect, the anti-CD19 binding domain, e.g., humanized
scFv, portion of a CAR of the invention is encoded by a transgene
whose sequence has been codon optimized for expression in a
mammalian cell. In one aspect, entire CAR construct of the
invention is encoded by a transgene whose entire sequence has been
codon optimized for expression in a mammalian cell. Codon
optimization refers to the discovery that the frequency of
occurrence of synonymous codons (i.e., codons that code for the
same amino acid) in coding DNA is biased in different species. Such
codon degeneracy allows an identical polypeptide to be encoded by a
variety of nucleotide sequences. A variety of codon optimization
methods is known in the art, and include, e.g., methods disclosed
in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.
[0289] In one aspect, the humanized CAR19 comprises the scFv
portion provided in SEQ ID NO: 1. In one aspect, the humanized
CAR19 comprises the scFv portion provided in SEQ ID NO:2. In one
aspect, the humanized CAR19 comprises the scFv portion provided in
SEQ ID NO:3. In one aspect, the humanized CAR19 comprises the scFv
portion provided in SEQ ID NO:4. In one aspect, the humanized CAR19
comprises the scFv portion provided in SEQ ID NO:5. In one aspect,
the humanized CAR19 comprises the scFv portion provided in SEQ ID
NO:6. In one aspect, the humanized CAR19 comprises the scFv portion
provided in SEQ ID NO:7. In one aspect, the humanized CAR19
comprises the scFv portion provided in SEQ ID NO:8. In one aspect,
the humanized CAR19 comprises the scFv portion provided in SEQ ID
NO:9. In one aspect, the humanized CAR19 comprises the scFv portion
provided in SEQ ID NO:10. In one aspect, the humanized CAR19
comprises the scFv portion provided in SEQ ID NO:11. In one aspect,
the humanized CAR19 comprises the scFv portion provided in SEQ ID
NO:12.
[0290] In one aspect, the CARs of the invention combine an antigen
binding domain of a specific antibody with an intracellular
signaling molecule. For example, in some aspects, the intracellular
signaling molecule includes, but is not limited to, CD3-zeta chain,
4-1BB and CD28 signaling modules and combinations thereof. In one
aspect, the CD19 CAR comprises a CAR selected from the sequence
provided in one or more of SEQ ID NOS: 31-42. In one aspect, the
CD19 CAR comprises the sequence provided in SEQ ID NO:31. In one
aspect, the CD19 CAR comprises the sequence provided in SEQ ID
NO:32. In one aspect, the CD19 CAR comprises the sequence provided
in SEQ ID NO:33. In one aspect, the CD19 CAR comprises the sequence
provided in SEQ ID NO:34. In one aspect, the CD19 CAR comprises the
sequence provided in SEQ ID NO:35. In one aspect, the CD19 CAR
comprises the sequence provided in SEQ ID NO:36. In one aspect, the
CD19 CAR comprises the sequence provided in SEQ ID NO:37. In one
aspect, the CD19 CAR comprises the sequence provided in SEQ ID
NO:38. In one aspect, the CD19 CAR comprises the sequence provided
in SEQ ID NO:39. In one aspect, the CD19 CAR comprises the sequence
provided in SEQ ID NO:40. In one aspect, the CD19 CAR comprises the
sequence provided in SEQ ID NO:41. In one aspect, the CD19 CAR
comprises the sequence provided in SEQ ID NO:42.
[0291] Furthermore, the present invention provides CD19 CAR
compositions and their use in medicaments or methods for treating,
among other diseases, cancer or any malignancy or autoimmune
diseases involving cells or tissues which express CD19.
[0292] In one aspect, the CAR of the invention can be used to
eradicate CD19-expressing normal cells, thereby applicable for use
as a cellular conditioning therapy prior to cell transplantation.
In one aspect, the CD19-expressing normal cell is a CD19-expressing
normal stem cell and the cell transplantation is a stem cell
transplantation.
[0293] In one aspect, the invention provides a cell (e.g., T cell)
engineered to express a chimeric antigen receptor (CAR), wherein
the CAR-expressing cell, e.g., CAR T cell ("CART"), exhibits an
anticancer property. A preferred antigen is CD19. In one aspect,
the antigen binding domain of the CAR comprises a partially
humanized anti-CD19 antibody fragment. In one aspect, the antigen
binding domain of the CAR comprises a partially humanized anti-CD19
antibody fragment comprising a scFv. Accordingly, the invention
provides a CD19-CAR that comprises a humanized anti-CD19 binding
domain and is engineered into an immune effector cell, e.g., a T
cell or an NK cell, and methods of their use for adoptive
therapy.
[0294] In one aspect, the CD19-CAR comprises at least one
intracellular domain selected from the group of a CD137 (4-1BB)
signaling domain, a CD28 signaling domain, a CD3zeta signal domain,
and any combination thereof. In one aspect, the CD19-CAR comprises
at least one intracellular signaling domain is from one or more
co-stimulatory molecule(s) other than a CD137 (4-1BB) or CD28.
Chimeric Antigen Receptor (CAR)
[0295] The present invention encompasses a recombinant DNA
construct comprising sequences encoding a CAR, wherein the CAR
comprises an antibody or antibody fragment that binds specifically
to a B-cell antigen (e.g., CD19, e.g., human CD19), wherein the
sequence of the antibody fragment is contiguous with and in the
same reading frame as a nucleic acid sequence encoding an
intracellular signaling domain. The intracellular signaling domain
can comprise a costimulatory signaling domain and/or a primary
signaling domain, e.g., a zeta chain. The costimulatory signaling
domain refers to a portion of the CAR comprising at least a portion
of the intracellular domain of a costimulatory molecule. In one
embodiment, the antigen binding domain is a murine antibody or
antibody fragment described herein. In one embodiment, the antigen
binding domain is a humanized antibody or antibody fragment.
[0296] In specific aspects, a CAR construct of the invention
comprises a scFv domain selected from the group consisting of SEQ
ID NOS:1-12 or an scFV domain of SEQ ID NO:59, wherein the scFv may
be preceded by an optional leader sequence such as provided in SEQ
ID NO: 13, and followed by an optional hinge sequence such as
provided in SEQ ID NO: 14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID
NO:49, a transmembrane region such as provided in SEQ ID NO: 15, an
intracellular signalling domain that includes SEQ ID NO: 16 or SEQ
ID NO:51 and a CD3 zeta sequence that includes SEQ ID NO:17 or SEQ
ID NO:43, wherein the domains are contiguous with and in the same
reading frame to form a single fusion protein. Also included in the
invention is a nucleotide sequence that encodes the polypeptide of
each of the scFv fragments selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59. Also included in the
invention is a nucleotide sequence that encodes the polypeptide of
each of the scFv fragments selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59, and each of the
domains of SEQ ID NOS: 13-17, plus the encoded CD19CAR fusion
protein of the invention. In one aspect an exemplary CD19CAR
constructs comprise an optional leader sequence, an extracellular
antigen binding domain, a hinge, a transmembrane domain, and an
intracellular stimulatory domain. In one aspect an exemplary
CD19CAR construct comprises an optional leader sequence, an
extracellular antigen binding domain, a hinge, a transmembrane
domain, an intracellular costimulatory domain and an intracellular
stimulatory domain. Specific CD19 CAR constructs containing
humanized scFv domains of the invention are provided as SEQ ID NOS:
31-42, or a murine scFv domain as provided as SEQ ID NO:59.
[0297] Full-length CAR sequences are also provided herein as SEQ ID
NOS: 31-42 and 58, as shown in Table 7 and Table 3.
[0298] An exemplary leader sequence is provided as SEQ ID NO: 13.
An exemplary hinge/spacer sequence is provided as SEQ ID NO: 14 or
SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49. An exemplary
transmembrane domain sequence is provided as SEQ ID NO:15. An
exemplary sequence of the intracellular signaling domain of the
4-1BB protein is provided as SEQ ID NO: 16. An exemplary sequence
of the intracellular signaling domain of CD27 is provided as SEQ ID
NO:51. An exemplary CD3zeta domain sequence is provided as SEQ ID
NO: 17 or SEQ ID NO:43.
[0299] In one aspect, the present invention encompasses a
recombinant nucleic acid construct comprising a nucleic acid
molecule encoding a CAR, wherein the nucleic acid molecule
comprises the nucleic acid sequence encoding an anti-CD19 binding
domain, e.g., described herein, that is contiguous with and in the
same reading frame as a nucleic acid sequence encoding an
intracellular signaling domain. In one aspect, the anti-CD19
binding domain is selected from one or more of SEQ ID NOS:1-12 and
58. In one aspect, the anti-CD 19 binding domain is encoded by a
nucleotide residues 64 to 813 of the sequence provided in one or
more of SEQ ID NOS:61-72 and 59. In one aspect, the anti-CD19
binding domain is encoded by a nucleotide residues 64 to 813 of SEQ
ID NO:61. In one aspect, the anti-CD19 binding domain is encoded by
a nucleotide residues 64 to 813 of SEQ ID NO:62. In one aspect, the
anti-CD19 binding domain is encoded by a nucleotide residues 64 to
813 of SEQ ID NO:63. In one aspect, the anti-CD19 binding domain is
encoded by a nucleotide residues 64 to 813 of SEQ ID NO:64. In one
aspect, the anti-CD19 binding domain is encoded by a nucleotide
residues 64 to 813 of SEQ ID NO:65. In one aspect, the anti-CD19
binding domain is encoded by a nucleotide residues 64 to 813 of SEQ
ID NO:66. In one aspect, the anti-CD19 binding domain is encoded by
a nucleotide residues 64 to 813 of SEQ ID NO:67. In one aspect, the
anti-CD19 binding domain is encoded by a nucleotide residues 64 to
813 of SEQ ID NO:68. In one aspect, the anti-CD19 binding domain is
encoded by a nucleotide residues 64 to 813 of SEQ ID NO:69. In one
aspect, the anti-CD19 binding domain is encoded by a nucleotide
residues 64 to 813 of SEQ ID NO:70. In one aspect, the anti-CD19
binding domain is encoded by a nucleotide residues 64 to 813 of SEQ
ID NO:71. In one aspect, the anti-CD19 binding domain is encoded by
a nucleotide residues 64 to 813 of SEQ ID NO:72.
[0300] In one aspect, the present invention encompasses a
recombinant nucleic acid construct comprising a transgene encoding
a CAR, wherein the nucleic acid molecule comprises a nucleic acid
sequence encoding an anti-CD19 binding domain selected from one or
more of SEQ ID NOS:61-72, wherein the sequence is contiguous with
and in the same reading frame as the nucleic acid sequence encoding
an intracellular signaling domain. An exemplary intracellular
signaling domain that can be used in the CAR includes, but is not
limited to, one or more intracellular signaling domains of, e.g.,
CD3-zeta, CD28, 4-1BB, and the like. In some instances, the CAR can
comprise any combination of CD3-zeta, CD28, 4-1BB, and the like. In
one aspect the nucleic acid sequence of a CAR construct of the
invention is selected from one or more of SEQ ID NOS:85-96. In one
aspect the nucleic acid sequence of a CAR construct is SEQ ID
NO:85. In one aspect the nucleic acid sequence of a CAR construct
is SEQ ID NO:86. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:87. In one aspect the nucleic acid sequence
of a CAR construct is SEQ ID NO:88. In one aspect the nucleic acid
sequence of a CAR construct is SEQ ID NO:89. In one aspect the
nucleic acid sequence of a CAR construct is SEQ ID NO:90. In one
aspect the nucleic acid sequence of a CAR construct is SEQ ID
NO:91. In one aspect the nucleic acid sequence of a CAR construct
is SEQ ID NO:92. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:93. In one aspect the nucleic acid sequence
of a CAR construct is SEQ ID NO:94. In one aspect the nucleic acid
sequence of a CAR construct is SEQ ID NO:95. In one aspect the
nucleic acid sequence of a CAR construct is SEQ ID NO:96. In one
aspect the nucleic acid sequence of a CAR construct is SEQ ID
NO:97. In one aspect the nucleic acid sequence of a CAR construct
is SEQ ID NO:98. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:99.
[0301] The nucleic acid sequences coding for the desired molecules
can be obtained using recombinant methods known in the art, such
as, for example by screening libraries from cells expressing the
gene, by deriving the gene from a vector known to include the same,
or by isolating directly from cells and tissues containing the
same, using standard techniques. Alternatively, the nucleic acid of
interest can be produced synthetically, rather than cloned.
[0302] The present invention includes retroviral and lentiviral
vector constructs expressing a CAR that can be directly transduced
into a cell.
[0303] The present invention also includes an RNA construct that
can be directly transfected into a cell. A method for generating
mRNA for use in transfection involves in vitro transcription (IVT)
of a template with specially designed primers, followed by polyA
addition, to produce a construct containing 3' and 5' untranslated
sequence ("UTR"), a 5' cap and/or Internal Ribosome Entry Site
(IRES), the nucleic acid to be expressed, and a polyA tail,
typically 50-2000 bases in length (SEQ ID NO: 118). RNA so produced
can efficiently transfect different kinds of cells. In one
embodiment, the template includes sequences for the CAR. In an
embodiment, an RNA CAR vector is transduced into a T cell by
electroporation.
Antigen Binding Domain
[0304] In one aspect, the CAR of the invention comprises a
target-specific binding element otherwise referred to as an antigen
binding domain. The choice of moiety depends upon the type and
number of ligands that define the surface of a target cell. For
example, the antigen binding domain may be chosen to recognize a
ligand that acts as a cell surface marker on target cells
associated with a particular disease state. Thus examples of cell
surface markers that may act as ligands for the antigen binding
domain in a CAR of the invention include those associated with
viral, bacterial and parasitic infections, autoimmune disease and
cancer cells.
[0305] In one aspect, the CAR-mediated T-cell response can be
directed to an antigen of interest by way of engineering an antigen
binding domain that specifically binds a desired antigen into the
CAR.
[0306] In one aspect, the portion of the CAR comprising the antigen
binding domain comprises an antigen binding domain that targets
CD19. In one aspect, the antigen binding domain targets human CD19.
In one aspect, the antigen binding domain of the CAR has the same
or a similar binding specificity as the FMC63 scFv fragment
described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165
(1997). In one embodiment, the antigen binding domain of the CAR
includes the scFv fragment described in Nicholson et al. Mol.
Immun. 34 (16-17): 1157-1165 (1997).
[0307] The antigen binding domain can be any domain that binds to
the antigen including but not limited to a monoclonal antibody, a
polyclonal antibody, a recombinant antibody, a murine antibody, a
human antibody, a humanized antibody, and a functional fragment
thereof, including but not limited to a single-domain antibody such
as a heavy chain variable domain (VH), a light chain variable
domain (VL) and a variable domain (VHH) of camelid derived
nanobody, and to an alternative scaffold known in the art to
function as antigen binding domain, such as a recombinant
fibronectin domain, and the like.
[0308] In one embodiment, the CAR molecule comprises an anti-CD19
binding domain comprising one or more (e.g., all three) light chain
complementary determining region 1 (LC CDR1), light chain
complementary determining region 2 (LC CDR2), and light chain
complementary determining region 3 (LC CDR3) of an anti-CD19
binding domain described herein, and one or more (e.g., all three)
heavy chain complementary determining region 1 (HC CDR1), heavy
chain complementary determining region 2 (HC CDR2), and heavy chain
complementary determining region 3 (HC CDR3) of an anti-CD19
binding domain described herein, e.g., an anti-CD19 binding domain
comprising one or more, e.g., all three, LC CDRs and one or more,
e.g., all three, HC CDRs. In one embodiment, the anti-CD19 binding
domain comprises one or more (e.g., all three) heavy chain
complementary determining region 1 (HC CDR1), heavy chain
complementary determining region 2 (HC CDR2), and heavy chain
complementary determining region 3 (HC CDR3) of an anti-CD19
binding domain described herein, e.g., the anti-CD19 binding domain
has two variable heavy chain regions, each comprising a HC CDR1, a
HC CDR2 and a HC CDR3 described herein. In one embodiment, the
anti-CD19 binding domain comprises a murine light chain variable
region described herein (e.g., in Table 7) and/or a murine heavy
chain variable region described herein (e.g., in Table 7). In one
embodiment, the anti-CD19 binding domain is a scFv comprising a
murine light chain and a murine heavy chain of an amino acid
sequence of Table 7. In an embodiment, the anti-CD19 binding domain
(e.g., an scFv) comprises: a light chain variable region comprising
an amino acid sequence having at least one, two or three
modifications (e.g., substitutions) but not more than 30, 20 or 10
modifications (e.g., substitutions) of an amino acid sequence of a
light chain variable region provided in Table 7, or a sequence with
95-99% identity with an amino acid sequence of Table 7; and/or a
heavy chain variable region comprising an amino acid sequence
having at least one, two or three modifications (e.g.,
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions) of an amino acid sequence of a heavy chain variable
region provided in Table 7, or a sequence with 95-99% identity to
an amino acid sequence of Table 7. In one embodiment, the anti-CD19
binding domain comprises a sequence of SEQ ID NO:59, or a sequence
with 95-99% identify thereof. In one embodiment, the anti-CD19
binding domain is a scFv, and a light chain variable region
comprising an amino acid sequence described herein, e.g., in Table
7, is attached to a heavy chain variable region comprising an amino
acid sequence described herein, e.g., in Table 7, via a linker,
e.g., a linker described herein. In one embodiment, the anti-CD19
binding domain includes a (Gly.sub.4-Ser)n linker, wherein n is 1,
2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light
chain variable region and heavy chain variable region of a scFv can
be, e.g., in any of the following orientations: light chain
variable region-linker-heavy chain variable region or heavy chain
variable region-linker-light chain variable region.
[0309] 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
human or humanized residues for the antigen binding domain of an
antibody or antibody fragment.
[0310] Thus, in one aspect, the antigen binding domain comprises a
humanized antibody or an antibody fragment. In one embodiment, the
humanized anti-CD19 binding domain comprises one or more (e.g., all
three) light chain complementary determining region 1 (LC CDR1),
light chain complementary determining region 2 (LC CDR2), and light
chain complementary determining region 3 (LC CDR3) of a murine or
humanized anti-CD19 binding domain described herein, and/or one or
more (e.g., all three) heavy chain complementary determining region
1 (HC CDR1), heavy chain complementary determining region 2 (HC
CDR2), and heavy chain complementary determining region 3 (HC CDR3)
of a murine or humanized anti-CD19 binding domain described herein,
e.g., a humanized anti-CD19 binding domain comprising one or more,
e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
In one embodiment, the humanized anti-CD19 binding domain comprises
one or more (e.g., all three) heavy chain complementary determining
region 1 (HC CDR1), heavy chain complementary determining region 2
(HC CDR2), and heavy chain complementary determining region 3 (HC
CDR3) of a murine or humanized anti-CD19 binding domain described
herein, e.g., the humanized anti-CD19 binding domain has two
variable heavy chain regions, each comprising a HC CDR1, a HC CDR2
and a HC CDR3 described herein. In one embodiment, the humanized
anti-CD19 binding domain comprises a humanized light chain variable
region described herein (e.g., in Table 3) and/or a humanized heavy
chain variable region described herein (e.g., in Table 3). In one
embodiment, the humanized anti-CD19 binding domain comprises a
humanized heavy chain variable region described herein (e.g., in
Table 3), e.g., at least two humanized heavy chain variable regions
described herein (e.g., in Table 3). In one embodiment, the
anti-CD19 binding domain is a scFv comprising a light chain and a
heavy chain of an amino acid sequence of Table 3. In an embodiment,
the anti-CD19 binding domain (e.g., an scFv) comprises: a light
chain variable region comprising an amino acid sequence having at
least one, two or three modifications (e.g., substitutions) but not
more than 30, 20 or 10 modifications (e.g., substitutions) of an
amino acid sequence of a light chain variable region provided in
Table 3, or a sequence with 95-99% identity with an amino acid
sequence of Table 3; and/or a heavy chain variable region
comprising an amino acid sequence having at least one, two or three
modifications (e.g., substitutions) but not more than 30, 20 or 10
modifications (e.g., substitutions) of an amino acid sequence of a
heavy chain variable region provided in Table 3, or a sequence with
95-99% identity to an amino acid sequence of Table 3. In one
embodiment, the humanized anti-CD19 binding domain comprises a
sequence selected from a group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ
ID NO:12, or a sequence with 95-99% identify thereof. In one
embodiment, the nucleic acid sequence encoding the humanized
anti-CD19 binding domain comprises a sequence selected from a group
consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID
NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ
ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, or a sequence with 95-99%
identify thereof. In one embodiment, the humanized anti-CD19
binding domain is a scFv, and a light chain variable region
comprising an amino acid sequence described herein, e.g., in Table
3, is attached to a heavy chain variable region comprising an amino
acid sequence described herein, e.g., in Table 3, via a linker,
e.g., a linker described herein. In one embodiment, the humanized
anti-CD19 binding domain includes a (Gly.sub.4-Ser)n linker,
wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:53).
The light chain variable region and heavy chain variable region of
a scFv can be, e.g., in any of the following orientations: light
chain variable region-linker-heavy chain variable region or heavy
chain variable region-linker-light chain variable region.
[0311] In one aspect, the antigen binding domain portion comprises
one or more sequence selected from SEQ ID NOS:1-12. In one aspect
the humanized CAR is selected from one or more sequence selected
from SEQ ID NOS: 31-42. In some aspects, a non-human antibody is
humanized, where specific sequences or regions of the antibody are
modified to increase similarity to an antibody naturally produced
in a human or fragment thereof.
[0312] A humanized antibody can be produced using a variety of
techniques known in the art, including but not limited to,
CDR-grafting (see, e.g., European Patent No. EP 239,400;
International Publication No. WO 91/09967; and U.S. Pat. Nos.
5,225,539, 5,530,101, and 5,585,089, each of which is incorporated
herein in its entirety by reference), veneering or resurfacing
(see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan,
1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al.,
1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994,
PNAS, 91:969-973, each of which is incorporated herein by its
entirety by reference), chain shuffling (see, e.g., U.S. Pat. No.
5,565,332, which is incorporated herein in its entirety by
reference), and techniques disclosed in, e.g., U.S. Patent
Application Publication No. US2005/0042664, U.S. Patent Application
Publication No. US2005/0048617, U.S. Pat. Nos. 6,407,213,
5,766,886, International Publication No. WO 9317105, Tan et al., J.
Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng.,
13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000),
Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et
al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res.,
55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res.,
55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and
Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which
is incorporated herein in its entirety by reference. Often,
framework residues in the framework regions will be substituted
with the corresponding residue from the CDR donor antibody to
alter, for example improve, antigen binding. These framework
substitutions are identified by methods well-known in the art,
e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323,
which are incorporated herein by reference in their
entireties.)
[0313] A humanized antibody or antibody fragment has one or more
amino acid residues remaining in it from a source which is
nonhuman. These nonhuman amino acid residues are often referred to
as "import" residues, which are typically taken from an "import"
variable domain. As provided herein, humanized antibodies or
antibody fragments comprise one or more CDRs from nonhuman
immunoglobulin molecules and framework regions wherein the amino
acid residues comprising the framework are derived completely or
mostly from human germline. Multiple techniques for humanization of
antibodies or antibody fragments are well-known in the art and can
essentially be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody, i.e.,
CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S.
Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089;
6,548,640, the contents of which are incorporated herein by
reference herein in their entirety). In such humanized antibodies
and antibody fragments, substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a nonhuman species. Humanized antibodies are often human
antibodies in which some CDR residues and possibly some framework
(FR) residues are substituted by residues from analogous sites in
rodent antibodies. Humanization of antibodies and antibody
fragments can also be achieved by veneering or resurfacing (EP
592,106; EP 519,596; Padlan, 1991, Molecular Immunology,
28(4/5):489-498; Studnicka et al., Protein Engineering,
7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994))
or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which
are incorporated herein by reference herein in their entirety.
[0314] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is to reduce
antigenicity. According to the so-called "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of
which are incorporated herein by reference herein in their
entirety). Another method uses a particular framework derived from
the consensus sequence of all human antibodies of a particular
subgroup of light or heavy chains. The same framework may be used
for several different humanized antibodies (see, e.g., Nicholson et
al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc.
Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.,
151:2623 (1993), the contents of which are incorporated herein by
reference herein in their entirety). In some embodiments, the
framework region, e.g., all four framework regions, of the heavy
chain variable region are derived from a VH4_4-59 germline
sequence. In one embodiment, the framework region can comprise,
one, two, three, four or five modifications, e.g., substitutions,
e.g., from the amino acid at the corresponding murine sequence
(e.g., of SEQ ID NO:59). In one embodiment, the framework region,
e.g., all four framework regions of the light chain variable region
are derived from a VK3_1.25 germline sequence. In one embodiment,
the framework region can comprise, one, two, three, four or five
modifications, e.g., substitutions, e.g., from the amino acid at
the corresponding murine sequence (e.g., of SEQ ID NO:59).
[0315] In some aspects, the portion of a CAR composition of the
invention that comprises an antibody fragment is humanized with
retention of high affinity for the target antigen and other
favorable biological properties. According to one aspect of the
invention, humanized antibodies and antibody fragments are prepared
by a process of analysis of the parental sequences and various
conceptual humanized products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, e.g., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind the target antigen. In this way, FR residues
can be selected and combined from the recipient and import
sequences so that the desired antibody or antibody fragment
characteristic, such as increased affinity for the target antigen,
is achieved. In general, the CDR residues are directly and most
substantially involved in influencing antigen binding.
[0316] A humanized antibody or antibody fragment may retain a
similar antigenic specificity as the original antibody, e.g., in
the present invention, the ability to bind human CD19. In some
embodiments, a humanized antibody or antibody fragment may have
improved affinity and/or specificity of binding to human CD19.
[0317] In one aspect, the anti-CD19 binding domain is characterized
by particular functional features or properties of an antibody or
antibody fragment. For example, in one aspect, the portion of a CAR
composition of the invention that comprises an antigen binding
domain specifically binds human CD19. In one aspect, the antigen
binding domain has the same or a similar binding specificity to
human CD19 as the FMC63 scFv described in Nicholson et al. Mol.
Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention
relates to an antigen binding domain comprising an antibody or
antibody fragment, wherein the antibody binding domain specifically
binds to a CD19 protein or fragment thereof, wherein the antibody
or antibody fragment comprises a variable light chain and/or a
variable heavy chain that includes an amino acid sequence of SEQ ID
NO: 1-12 or SEQ ID NO:59. In one aspect, the antigen binding domain
comprises an amino acid sequence of an scFv selected from SEQ ID
NOs: 1-12 or SEQ ID NO:59. In certain aspects, the scFv is
contiguous with and in the same reading frame as a leader sequence.
In one aspect the leader sequence is the polypeptide sequence
provided as SEQ ID NO:13.
[0318] In one aspect, the anti-CD19 binding domain is a fragment,
e.g., a single chain variable fragment (scFv). In one aspect, the
anti-CD19 binding domain is a Fv, a Fab, a (Fab')2, or a
bi-functional (e.g. bi-specific) hybrid antibody (e.g.,
Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one
aspect, the antibodies and fragments thereof of the invention binds
a CD19 protein with wild-type or enhanced affinity.
[0319] In some instances, scFvs can be prepared according to method
known in the art (see, for example, Bird et al., (1988) Science
242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883). ScFv molecules can be produced by linking VH and VL
regions together using flexible polypeptide linkers. The scFv
molecules comprise a linker (e.g., a Ser-Gly linker) with an
optimized length and/or amino acid composition. The linker length
can greatly affect how the variable regions of a scFv fold and
interact. In fact, if a short polypeptide linker is employed (e.g.,
between 5-10 amino acids) intrachain folding is prevented.
Interchain folding is also required to bring the two variable
regions together to form a functional epitope binding site. For
examples of linker orientation and size see, e.g., Hollinger et al.
1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent
Application Publication Nos. 2005/0100543, 2005/0175606,
2007/0014794, and PCT publication Nos.
[0320] WO2006/020258 and WO2007/024715, is incorporated herein by
reference.
[0321] A scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, or more amino acid residues between its VL and VH
regions. The linker sequence may comprise any naturally occurring
amino acid. In some embodiments, the linker sequence comprises
amino acids glycine and serine. In another embodiment, the linker
sequence comprises sets of glycine and serine repeats such as
(Gly4Ser)n, where n is a positive integer equal to or greater than
1 (SEQ ID NO:18). In one embodiment, the linker can be
(Gly4Ser).sub.4 (SEQ ID NO: 106) or (Gly4Ser).sub.3 (SEQ ID NO:
107). Variation in the linker length may retain or enhance
activity, giving rise to superior efficacy in activity studies.
[0322] In some embodiments, the amino acid sequence of the antigen
binding domain (or other portions or the entire CAR) can be
modified, e.g., an amino acid sequence described herein can be
modified, e.g., by a conservative substitution. Families of amino
acid residues having similar side chains have been defined in the
art, including basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0323] Percent identity in the context of two or more nucleic acids
or polypeptide sequences, refers to two or more sequences that are
the same. Two sequences are "substantially identical" if two
sequences have a specified percentage of amino acid residues or
nucleotides that are the same (e.g., 60% identity, optionally 70%,
71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% identity over a specified region, or, when not
specified, over the entire sequence), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection.
Optionally, the identity exists over a region that is at least
about 50 nucleotides (or 10 amino acids) in length, or more
preferably over a region that is 100 to 500 or 1000 or more
nucleotides (or 20, 50, 200 or more amino acids) in length.
[0324] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. Methods of alignment of sequences for
comparison are well known in the art. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
2:482c, by the homology alignment algorithm of Needleman and
Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity
method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA
85:2444, by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by manual alignment and visual inspection (see, e.g., Brent et
al., (2003) Current Protocols in Molecular Biology).
[0325] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al.,
(1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J.
Mol. Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information.
[0326] The percent identity between two amino acid sequences can
also be determined using the algorithm of E. Meyers and W. Miller,
(1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated
into the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4. In
addition, the percent identity between two amino acid sequences can
be determined using the Needleman and Wunsch (1970) J. Mol. Biol.
48:444-453) algorithm which has been incorporated into the GAP
program in the GCG software package (available at www.gcg.com),
using either a Blossom 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,
3, 4, 5, or 6.
[0327] In one aspect, the present invention contemplates
modifications of the starting antibody or fragment (e.g., scFv)
amino acid sequence that generate functionally equivalent
molecules. For example, the VH or VL of an anti-CD19 binding
domain, e.g., scFv, comprised in the CAR can be modified to retain
at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL
framework region of the anti-CD19 binding domain, e.g., scFv. The
present invention contemplates modifications of the entire CAR
construct, e.g., modifications in one or more amino acid sequences
of the various domains of the CAR construct in order to generate
functionally equivalent molecules. The CAR construct can be
modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of
the starting CAR construct.
[0328] Bispecific CARs
[0329] In an embodiment a multispecific antibody molecule is a
bispecific antibody molecule. A bispecific antibody has specificity
for no more than two antigens. A bispecific antibody molecule is
characterized by a first immunoglobulin variable domain sequence
which has binding specificity for a first epitope and a second
immunoglobulin variable domain sequence that has binding
specificity for a second epitope. In an embodiment the first and
second epitopes are on the same antigen, e.g., the same protein (or
subunit of a multimeric protein). In an embodiment the first and
second epitopes overlap. In an embodiment the first and second
epitopes do not overlap. In an embodiment the first and second
epitopes are on different antigens, e.g., different proteins (or
different subunits of a multimeric protein). In an embodiment a
bispecific antibody molecule comprises a heavy chain variable
domain sequence and a light chain variable domain sequence which
have binding specificity for a first epitope and a heavy chain
variable domain sequence and a light chain variable domain sequence
which have binding specificity for a second epitope. In an
embodiment a bispecific antibody molecule comprises a half antibody
having binding specificity for a first epitope and a half antibody
having binding specificity for a second epitope. In an embodiment a
bispecific antibody molecule comprises a half antibody, or fragment
thereof, having binding specificity for a first epitope and a half
antibody, or fragment thereof, having binding specificity for a
second epitope. In an embodiment a bispecific antibody molecule
comprises a scFv, or fragment thereof, have binding specificity for
a first epitope and a scFv, or fragment thereof, have binding
specificity for a second epitope.
Transmembrane Domain
[0330] With respect to the transmembrane domain, in various
embodiments, a CAR can be designed to comprise a transmembrane
domain that is attached to the extracellular domain of the CAR. A
transmembrane domain can include one or more additional amino acids
adjacent to the transmembrane region, e.g., one or more amino acid
associated with the extracellular region of the protein from which
the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
up to 15 amino acids of the extracellular region) and/or one or
more additional amino acids associated with the intracellular
region of the protein from which the transmembrane protein is
derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids
of the intracellular region). In one aspect, the transmembrane
domain is one that is associated with one of the other domains of
the CAR, e.g., in one embodiment, the transmembrane domain may be
from the same protein that the signaling domain, costimulatory
domain or the hinge domain is derived from. In another aspect, the
transmembrane domain is not derived from the same protein that any
other domain of the CAR is derived from. In some instances, the
transmembrane domain can be selected or modified by amino acid
substitution to avoid binding of such domains to the transmembrane
domains of the same or different surface membrane proteins, e.g.,
to minimize interactions with other members of the receptor
complex. In one aspect, the transmembrane domain is capable of
homodimerization with another CAR on the cell surface of a
CAR-expressing cell. In a different aspect the amino acid sequence
of the transmembrane domain may be modified or substituted so as to
minimize interactions with the binding domains of the native
binding partner present in the same CAR-expressing cell.
[0331] The transmembrane domain may be derived either from a
natural or from a recombinant source. Where the source is natural,
the domain may be derived from any membrane-bound or transmembrane
protein. In one aspect the transmembrane domain is capable of
signaling to the intracellular domain(s) whenever the CAR has bound
to a target. A transmembrane domain of particular use in this
invention may include at least the transmembrane region(s) of e.g.,
the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3
epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,
CD80, CD86, CD134, CD137, CD154. In some embodiments, a
transmembrane domain may include at least the transmembrane
region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18),
ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR),
SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta,
IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,
VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,
ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,
TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),
CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D),
SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150, IPO-3), BLAME (SLAMF8),
SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.
[0332] In some instances, the transmembrane domain can be attached
to the extracellular region of the CAR, e.g., the antigen binding
domain of the CAR, via a hinge, e.g., a hinge from a human protein.
For example, in one embodiment, the hinge can be a human Ig
(immunoglobulin) hinge, e.g., an IgG4 hinge, an IgD hinge), a GS
linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a
CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g.,
consists of) the amino acid sequence of SEQ ID NO: 14. In one
aspect, the transmembrane domain comprises (e.g., consists of) a
transmembrane domain of SEQ ID NO: 15.
[0333] In one aspect, the hinge or spacer comprises an IgG4 hinge.
For example, in one embodiment, the hinge or spacer comprises a
hinge of the amino acid sequence
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK
TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID
NO:45). In some embodiments, the hinge or spacer comprises a hinge
encoded by a nucleotide sequence of
TABLE-US-00001 (SEQ ID NO: 46)
GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTC
CTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACC
CTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTG
TCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTG
GAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGC
ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTG
AACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGC
AGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCC
CAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC
CCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTG
ACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCC
GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGC
CTGTCCCTGGGCAAGATG.
[0334] In one aspect, the hinge or spacer comprises an IgD hinge.
For example, in one embodiment, the hinge or spacer comprises a
hinge of the amino acid sequence
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERET
KTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTG
GVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQA
PVKLSLNLLAS SDPPEAAS WLLCEVS GFSPPNILLMWLEDQREVNTS GFAPARPPPQPG
STTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:47).
In some embodiments, the hinge or spacer comprises a hinge encoded
by a nucleotide sequence of
TABLE-US-00002 (SEQ ID NO: 48)
AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACA
GCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTA
CGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAA
GAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATAC
CCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGC
TTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAG
GATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGT
TGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACT
CAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACA
TGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAG
AGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCA
GTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGC
TTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGT
GAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTA
CCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCC
CAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCT
GCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT.
[0335] In one aspect, the transmembrane domain may be recombinant,
in which case it will comprise predominantly hydrophobic residues
such as leucine and valine. In one aspect a triplet of
phenylalanine, tryptophan and valine can be found at each end of a
recombinant transmembrane domain.
[0336] Optionally, a short oligo- or polypeptide linker, between 2
and 10 amino acids in length may form the linkage between the
transmembrane domain and the cytoplasmic region of the CAR. A
glycine-serine doublet provides a particularly suitable linker. For
example, in one aspect, the linker comprises the amino acid
sequence of GGGGSGGGGS (SEQ ID NO:49). In some embodiments, the
linker is encoded by a nucleotide sequence of
TABLE-US-00003 (SEQ ID NO: 50) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC.
[0337] In one aspect, the hinge or spacer comprises a KIR2DS2
hinge.
Cytoplasmic Domain
[0338] The cytoplasmic domain or region of the CAR includes an
intracellular signaling domain. An intracellular signaling domain
is generally responsible for activation of at least one of the
normal effector functions of the immune cell in which the CAR has
been introduced. The term "effector function" refers to a
specialized function of a cell. Effector function of a T cell, for
example, may be cytolytic activity or helper activity including the
secretion of cytokines. Thus the term "intracellular signaling
domain" refers to the portion of a protein which transduces the
effector function signal and directs the cell to perform a
specialized function. While usually the entire intracellular
signaling domain can be employed, in many cases it is not necessary
to use the entire chain. To the extent that a truncated portion of
the intracellular signaling domain is used, such truncated portion
may be used in place of the intact chain as long as it transduces
the effector function signal. The term intracellular signaling
domain is thus meant to include any truncated portion of the
intracellular signaling domain sufficient to transduce the effector
function signal.
[0339] Examples of intracellular signaling domains for use in the
CAR of the invention include the cytoplasmic sequences of the T
cell receptor (TCR) and co-receptors that act in concert to
initiate signal transduction following antigen receptor engagement,
as well as any derivative or variant of these sequences and any
recombinant sequence that has the same functional capability.
[0340] It is known that signals generated through the TCR alone are
insufficient for full activation of the T cell and that a secondary
and/or costimulatory signal is also required. Thus, T cell
activation can be said to be mediated by two distinct classes of
cytoplasmic signaling sequences: those that initiate
antigen-dependent primary activation through the TCR (primary
intracellular signaling domains) and those that act in an
antigen-independent manner to provide a secondary or costimulatory
signal (secondary cytoplasmic domain, e.g., a costimulatory
domain).
[0341] A primary signaling domain regulates primary activation of
the TCR complex either in a stimulatory way, or in an inhibitory
way. Primary intracellular signaling domains that act in a
stimulatory manner may contain signaling motifs which are known as
immunoreceptor tyrosine-based activation motifs or ITAMs.
[0342] Examples of ITAM containing primary intracellular signaling
domains that are of particular use in the invention include those
of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc
Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b,
DAP10, and DAP12. In one embodiment, a CAR of the invention
comprises an intracellular signaling domain, e.g., a primary
signaling domain of CD3-zeta.
[0343] In one embodiment, a primary signaling domain comprises a
modified ITAM domain, e.g., a mutated ITAM domain which has altered
(e.g., increased or decreased) activity as compared to the native
ITAM domain. In one embodiment, a primary signaling domain
comprises a modified ITAM-containing primary intracellular
signaling domain, e.g., an optimized and/or truncated
ITAM-containing primary intracellular signaling domain. In an
embodiment, a primary signaling domain comprises one, two, three,
four or more ITAM motifs.
[0344] Further examples of molecules containing a primary
intracellular signaling domain that are of particular use in the
invention include those of DAP10, DAP12, and CD32.
[0345] The intracellular signalling domain of the CAR can comprise
the CD3-zeta signaling domain by itself or it can be combined with
any other desired intracellular signaling domain(s) useful in the
context of a CAR of the invention. For example, the intracellular
signaling domain of the CAR can comprise a CD3 zeta chain portion
and a costimulatory signaling domain. The costimulatory signaling
domain refers to a portion of the CAR comprising the intracellular
domain of a costimulatory molecule. A costimulatory molecule is a
cell surface molecule other than an antigen receptor or its ligands
that is required for an efficient response of lymphocytes to an
antigen. Examples of such molecules include CD27, CD28, 4-1BB
(CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds with CD83, and the
like. For example, CD27 costimulation has been demonstrated to
enhance expansion, effector function, and survival of human CART
cells in vitro and augments human T cell persistence and antitumor
activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further
examples of such costimulatory molecules include CDS, ICAM-1, GITR,
BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46,
CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R
alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,
ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b,
ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,
TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96
(Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,
CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150,
IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,
PAG/Cbp, and CD19a.
[0346] The intracellular signaling sequences within the cytoplasmic
portion of the CAR of the invention may be linked to each other in
a random or specified order. Optionally, a short oligo- or
polypeptide linker, for example, between 2 and 10 amino acids
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may
form the linkage between intracellular signaling sequence. In one
embodiment, a glycine-serine doublet can be used as a suitable
linker. In one embodiment, a single amino acid, e.g., an alanine, a
glycine, can be used as a suitable linker.
[0347] In one aspect, the intracellular signaling domain is
designed to comprise two or more, e.g., 2, 3, 4, 5, or more,
costimulatory signaling domains. In an embodiment, the two or more,
e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are
separated by a linker molecule, e.g., a linker molecule described
herein. In one embodiment, the intracellular signaling domain
comprises two costimulatory signaling domains. In some embodiments,
the linker molecule is a glycine residue. In some embodiments, the
linker is an alanine residue.
[0348] In one aspect, the intracellular signaling domain is
designed to comprise the signaling domain of CD3-zeta and the
signaling domain of CD28. In one aspect, the intracellular
signaling domain is designed to comprise the signaling domain of
CD3-zeta and the signaling domain of 4-1BB. In one aspect, the
signaling domain of 4-1BB is a signaling domain of SEQ ID NO: 16.
In one aspect, the signaling domain of CD3-zeta is a signaling
domain of SEQ ID NO: 17.
[0349] In one aspect, the intracellular signaling domain is
designed to comprise the signaling domain of CD3-zeta and the
signaling domain of CD27. In one aspect, the signaling domain of
CD27 comprises an amino acid sequence of
QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:51). In
one aspect, the signalling domain of CD27 is encoded by a nucleic
acid sequence of
TABLE-US-00004 (SEQ ID NO: 52)
AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCC
CCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCAC
GCGACTTCGCAGCCTATCGCTCC.
[0350] In one aspect, the CAR-expressing cell described herein can
further comprise a second CAR, e.g., a second CAR that includes a
different antigen binding domain, e.g., to the same target (CD19)
or a different target (e.g., CD123 or mesothelin). In one
embodiment, when the CAR-expressing cell comprises two or more
different CARs, the antigen binding domains of the different CARs
can be such that the antigen binding domains do not interact with
one another. For example, a cell expressing a first and second CAR
can have an antigen binding domain of the first CAR, e.g., as a
fragment, e.g., an scFv, that does not form an association with the
antigen binding domain of the second CAR, e.g., the antigen binding
domain of the second CAR is a VHH.
[0351] In another aspect, the CAR-expressing cell described herein
can further express another agent, e.g., an agent which enhances
the activity of a CAR-expressing cell. For example, in one
embodiment, the agent can be an agent which inhibits an inhibitory
molecule. Inhibitory molecules, e.g., PD1, can, in some
embodiments, decrease the ability of a CAR-expressing cell to mount
an immune effector response. Examples of inhibitory molecules
include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3
and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and
TGFR beta. In one embodiment, the agent which inhibits an
inhibitory molecule comprises a first polypeptide, e.g., an
inhibitory molecule, associated with a second polypeptide that
provides a positive signal to the cell, e.g., an intracellular
signaling domain described herein. In one embodiment, the agent
comprises a first polypeptide, e.g., of an inhibitory molecule such
as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR
beta, or a fragment of any of these (e.g., at least a portion of an
extracellular domain of any of these), and a second polypeptide
which is an intracellular signaling domain described herein (e.g.,
comprising a costimulatory domain (e.g., 41BB, CD27 or CD28, e.g.,
as described herein) and/or a primary signaling domain (e.g., a CD3
zeta signaling domain described herein). In one embodiment, the
agent comprises a first polypeptide of PD1 or a fragment thereof
(e.g., at least a portion of an extracellular domain of PD1), and a
second polypeptide of an intracellular signaling domain described
herein (e.g., a CD28 signaling domain described herein and/or a CD3
zeta signaling domain described herein). PD1 is an inhibitory
member of the CD28 family of receptors that also includes CD28,
CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T
cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).
Two ligands for PD1, PD-L1 and PD-L2 have been shown to
downregulate T cell activation upon binding to PD1 (Freeman et a.
2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol
2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is
abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7;
Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et
al. 2004 Clin Cancer Res 10:5094). Immune suppression can be
reversed by inhibiting the local interaction of PD1 with PD-L1.
[0352] In one embodiment, the agent comprises the extracellular
domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1
(PD1), can be fused to a transmembrane domain and intracellular
signaling domains such as 41BB and CD3 zeta (also referred to
herein as a PD1 CAR). In one embodiment, the PD1 CAR, when used in
combinations with a CD19 CAR described herein, improves the
persistence of the T cell. In one embodiment, the CAR is a PD1 CAR
comprising the extracellular domain of PD1 indicated as underlined
in SEQ ID NO: 121. In one embodiment, the PD1 CAR comprises the
amino acid sequence of SEQ ID NO:121.
TABLE-US-00005 (SEQ ID NO: 121)
Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdn
atftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtq
lpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterra
evptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrp
aaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyi
fkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkma
eayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.
[0353] In one embodiment, the PD1 CAR comprises the amino acid
sequence provided below (SEQ ID NO:119).
TABLE-US-00006 (SEQ ID NO: 119)
pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrm
spsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgt
ylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv
tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwa
plagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscr
fpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrr
grdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdgl
yqglstatkdtydalhmqalppr.
[0354] In one embodiment, the agent comprises a nucleic acid
sequence encoding the PD1 CAR, e.g., the PD1 CAR described herein.
In one embodiment, the nucleic acid sequence for the PD1 CAR is
shown below, with the PD1 ECD underlined below in SEQ ID NO:
120
TABLE-US-00007 (SEQ ID NO: 120)
atggccctccctgtcactgccctgcttctccccctcgcactcctgctcca
cgccgctagaccacccggatggtttctggactctccggatcgcccgtgga
atcccccaaccttctcaccggcactcttggttgtgactgagggcgataat
gcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaa
ctggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttc
cggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaa
ctgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaa
cgactccgggacctacctgtgcggagccatctcgctggcgcctaaggccc
aaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagct
gaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtt
tcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccc
caactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccct
gccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacat
ctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccc
tggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacatt
ttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacgg
ttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcg
tgaagttctcccggagcgccgacgcccccgcctataagcagggccagaac
cagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgct
ggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaa
agaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggcc
gaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggg
gcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacg
atgccctgcacatgcaggcccttccccctcgc.
[0355] In another aspect, the present invention provides a
population of CAR-expressing cells, e.g., CART cells. In some
embodiments, the population of CAR-expressing cells comprises a
mixture of cells expressing different CARs. For example, in one
embodiment, the population of CAR-expressing cells can include a
first cell expressing a CAR having an anti-CD19 binding domain
described herein, and a second cell expressing a CAR having a
different anti-CD19 binding domain, e.g., an anti-CD19 binding
domain described herein that differs from the anti-CD19 binding
domain in the CAR expressed by the first cell. As another example,
the population of CAR-expressing cells can include a first cell
expressing a CAR that includes an anti-CD19 binding domain, e.g.,
as described herein, and a second cell expressing a CAR that
includes an antigen binding domain to a target other than CD19
(e.g., CD123). In one embodiment, the population of CAR-expressing
cells includes, e.g., a first cell expressing a CAR that includes a
primary intracellular signaling domain, and a second cell
expressing a CAR that includes a secondary signaling domain.
[0356] In another aspect, the present invention provides a
population of cells wherein at least one cell in the population
expresses a CAR having an anti-CD19 binding domain described
herein, and a second cell expressing another agent, e.g., an agent
which enhances the activity of a CAR-expressing cell. For example,
in one embodiment, the agent can be an agent which inhibits an
inhibitory molecule. Inhibitory molecules, e.g., can, in some
embodiments, decrease the ability of a CAR-expressing cell to mount
an immune effector response. Examples of inhibitory molecules
include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3
and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or
TGFR beta. In one embodiment, the agent which inhibits an
inhibitory molecule comprises a first polypeptide, e.g., an
inhibitory molecule, associated with a second polypeptide that
provides a positive signal to the cell, e.g., an intracellular
signaling domain described herein. In one embodiment, the agent
comprises a first polypeptide, e.g., of an inhibitory molecule such
as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR
beta, or a fragment of any of these (e.g., at least a portion of an
extracellular domain of any of these), and a second polypeptide
which is an intracellular signaling domain described herein (e.g.,
comprising a costimulatory domain (e.g., 41BB, CD27 or CD28, e.g.,
as described herein) and/or a primary signaling domain (e.g., a CD3
zeta signaling domain described herein). In one embodiment, the
agent comprises a first polypeptide of PD1 or a fragment thereof
(e.g., at least a portion of the extracellular domain of PD1), and
a second polypeptide of an intracellular signaling domain described
herein (e.g., a CD28 signaling domain described herein and/or a CD3
zeta signaling domain described herein).
Regulatable Chimeric Antigen Receptors
[0357] In some embodiments, a regulatable CAR (RCAR) where the CAR
activity can be controlled is desirable to optimize the safety and
efficacy of a CAR therapy. There are many ways CAR activities can
be regulated. For example, inducible apoptosis using, e.g., a
caspase fused to a dimerization domain (see, e.g., Di Stasa et al.,
N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a
safety switch in the CAR therapy of the instant invention. In one
embodiment, the cells (e.g., T cells or NK cells) expressing a CAR
of the present invention further comprise an inducible apoptosis
switch, wherein a human caspase (e.g., caspase 9) or a modified
version is fused to a modification of the human FKB protein that
allows conditional dimerization. In the presence of a small
molecule, such as a rapalog (e.g., AP 1903, AP20187), the inducible
caspase (e.g., caspase 9) is activated and leads to the rapid
apoptosis and death of the cells (e.g., T cells or NK cells)
expressing a CAR of the present invention. Examples of a
caspase-based inducible apoptosis switch (or one or more aspects of
such a switch) have been described in, e.g., US2004040047;
US20110286980; US20140255360; WO1997031899; WO2014151960;
WO2014164348; WO2014197638; WO2014197638; all of which are
incorporated by reference herein.
[0358] In an aspect, a RCAR comprises a set of polypeptides,
typically two in the simplest embodiments, in which the components
of a standard CAR described herein, e.g., an antigen binding domain
and an intracellular signaling domain, are partitioned on separate
polypeptides or members. In some embodiments, the set of
polypeptides include a dimerization switch that, upon the presence
of a dimerization molecule, can couple the polypeptides to one
another, e.g., can couple an antigen binding domain to an
intracellular signaling domain. In one embodiment, the CARs of the
present invention utilizes a dimerization switch as those described
in, e.g., WO2014127261, which is incorporated by reference
herein.
[0359] In an aspect, an RCAR comprises two polypeptides or members:
1) an intracellular signaling member comprising an intracellular
signaling domain, e.g., a primary intracellular signaling domain
described herein, and a first switch domain; 2) an antigen binding
member comprising an antigen binding domain, e.g., that targets
CD19, as described herein and a second switch domain. Optionally,
the RCAR comprises a transmembrane domain described herein. In an
embodiment, a transmembrane domain can be disposed on the
intracellular signaling member, on the antigen binding member, or
on both. (Unless otherwise indicated, when members or elements of
an RCAR are described herein, the order can be as provided, but
other orders are included as well. In other words, in an
embodiment, the order is as set out in the text, but in other
embodiments, the order can be different. E.g., the order of
elements on one side of a transmembrane region can be different
from the example, e.g., the placement of a switch domain relative
to a intracellular signaling domain can be different, e.g.,
reversed).
[0360] In an embodiment, the first and second switch domains can
form an intracellular or an extracellular dimerization switch. In
an embodiment, the dimerization switch can be a homodimerization
switch, e.g., where the first and second switch domain are the
same, or a heterodimerization switch, e.g., where the first and
second switch domain are different from one another.
[0361] In embodiments, an RCAR can comprise a "multi switch." A
multi switch can comprise heterodimerization switch domains or
homodimerization switch domains. A multi switch comprises a
plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, switch domains,
independently, on a first member, e.g., an antigen binding member,
and a second member, e.g., an intracellular signaling member. In an
embodiment, the first member can comprise a plurality of first
switch domains, e.g., FKBP-based switch domains, and the second
member can comprise a plurality of second switch domains, e.g.,
FRB-based switch domains. In an embodiment, the first member can
comprise a first and a second switch domain, e.g., a FKBP-based
switch domain and a FRB-based switch domain, and the second member
can comprise a first and a second switch domain, e.g., a FKBP-based
switch domain and a FRB-based switch domain.
[0362] In an embodiment, the intracellular signaling member
comprises one or more intracellular signaling domains, e.g., a
primary intracellular signaling domain and one or more
costimulatory signaling domains.
[0363] In an embodiment, the antigen binding member may comprise
one or more intracellular signaling domains, e.g., one or more
costimulatory signaling domains. In an embodiment, the antigen
binding member comprises a plurality, e.g., 2 or 3 costimulatory
signaling domains described herein, e.g., selected from 41BB, CD28,
CD27, ICOS, and OX40, and in embodiments, no primary intracellular
signaling domain. In an embodiment, the antigen binding member
comprises the following costimulatory signaling domains, from the
extracellular to intracellular direction: 41BB-CD27; 41BB-CD27;
CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB;
or 41BB-CD28. In such embodiments, the intracellular binding member
comprises a CD3zeta domain. In one such embodiment the RCAR
comprises (1) an antigen binding member comprising, an antigen
binding domain, a transmembrane domain, and two costimulatory
domains and a first switch domain; and (2) an intracellular
signaling domain comprising a transmembrane domain or membrane
tethering domain and at least one primary intracellular signaling
domain, and a second switch domain.
[0364] An embodiment provides RCARs wherein the antigen binding
member is not tethered to the surface of the CAR cell. This allows
a cell having an intracellular signaling member to be conveniently
paired with one or more antigen binding domains, without
transforming the cell with a sequence that encodes the antigen
binding member. In such embodiments, the RCAR comprises: 1) an
intracellular signaling member comprising: a first switch domain, a
transmembrane domain, an intracellular signaling domain, e.g., a
primary intracellular signaling domain, and a first switch domain;
and 2) an antigen binding member comprising: an antigen binding
domain, and a second switch domain, wherein the antigen binding
member does not comprise a transmembrane domain or membrane
tethering domain, and, optionally, does not comprise an
intracellular signaling domain. In some embodiments, the RCAR may
further comprise 3) a second antigen binding member comprising: a
second antigen binding domain, e.g., a second antigen binding
domain that binds a different antigen than is bound by the antigen
binding domain; and a second switch domain.
[0365] Also provided herein are RCARs wherein the antigen binding
member comprises bispecific activation and targeting capacity. In
this embodiment, the antigen binding member can comprise a
plurality, e.g., 2, 3, 4, or 5 antigen binding domains, e.g.,
scFvs, wherein each antigen binding domain binds to a target
antigen, e.g. different antigens or the same antigen, e.g., the
same or different epitopes on the same antigen. In an embodiment,
the plurality of antigen binding domains are in tandem, and
optionally, a linker or hinge region is disposed between each of
the antigen binding domains. Suitable linkers and hinge regions are
described herein.
[0366] An embodiment provides RCARs having a configuration that
allows switching of proliferation. In this embodiment, the RCAR
comprises: 1) an intracellular signaling member comprising:
optionally, a transmembrane domain or membrane tethering domain;
one or more co-stimulatory signaling domain, e.g., selected from
41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an
antigen binding member comprising: an antigen binding domain, a
transmembrane domain, and a primary intracellular signaling domain,
e.g., a CD3zeta domain, wherein the antigen binding member does not
comprise a switch domain, or does not comprise a switch domain that
dimerizes with a switch domain on the intracellular signaling
member. In an embodiment, the antigen binding member does not
comprise a co-stimulatory signaling domain. In an embodiment, the
intracellular signaling member comprises a switch domain from a
homodimerization switch. In an embodiment, the intracellular
signaling member comprises a first switch domain of a
heterodimerization switch and the RCAR comprises a second
intracellular signaling member which comprises a second switch
domain of the heterodimerization switch. In such embodiments, the
second intracellular signaling member comprises the same
intracellular signaling domains as the intracellular signaling
member. In an embodiment, the dimerization switch is intracellular.
In an embodiment, the dimerization switch is extracellular.
[0367] In any of the RCAR configurations described here, the first
and second switch domains comprise a FKBP-FRB based switch as
described herein.
[0368] Also provided herein are cells comprising an RCAR described
herein. Any cell that is engineered to express a RCAR can be used
as a RCARX cell. In an embodiment the RCARX cell is a T cell, and
is referred to as a RCART cell. In an embodiment the RCARX cell is
an NK cell, and is referred to as a RCARN cell.
[0369] Also provided herein are nucleic acids and vectors
comprising RCAR encoding sequences. Sequence encoding various
elements of an RCAR can be disposed on the same nucleic acid
molecule, e.g., the same plasmid or vector, e.g., viral vector,
e.g., lentiviral vector. In an embodiment, (i) sequence encoding an
antigen binding member and (ii) sequence encoding an intracellular
signaling member, can be present on the same nucleic acid, e.g.,
vector. Production of the corresponding proteins can be achieved,
e.g., by the use of separate promoters, or by the use of a
bicistronic transcription product (which can result in the
production of two proteins by cleavage of a single translation
product or by the translation of two separate protein products). In
an embodiment, a sequence encoding a cleavable peptide, e.g., a P2A
or F2A sequence, is disposed between (i) and (ii). In an
embodiment, a sequence encoding an IRES, e.g., an EMCV or EV71
IRES, is disposed between (i) and (ii). In these embodiments, (i)
and (ii) are transcribed as a single RNA. In an embodiment, a first
promoter is operably linked to (i) and a second promoter is
operably linked to (ii), such that (i) and (ii) are transcribed as
separate mRNAs.
[0370] Alternatively, the sequence encoding various elements of an
RCAR can be disposed on the different nucleic acid molecules, e.g.,
different plasmids or vectors, e.g., viral vector, e.g., lentiviral
vector. E.g., the (i) sequence encoding an antigen binding member
can be present on a first nucleic acid, e.g., a first vector, and
the (ii) sequence encoding an intracellular signaling member can be
present on the second nucleic acid, e.g., the second vector.
Dimerization Switches
[0371] Dimerization switches can be non-covalent or covalent. In a
non-covalent dimerization switch, the dimerization molecule
promotes a non-covalent interaction between the switch domains. In
a covalent dimerization switch, the dimerization molecule promotes
a covalent interaction between the switch domains.
[0372] In an embodiment, the RCAR comprises a FKBP/FRAP, or
FKBP/FRB,-based dimerization switch. FKBP12 (FKBP, or FK506 binding
protein) is an abundant cytoplasmic protein that serves as the
initial intracellular target for the natural product
immunosuppressive drug, rapamycin. Rapamycin binds to FKBP and to
the large PI3K homolog FRAP (RAFT, mTOR). FRB is a 93 amino acid
portion of FRAP, that is sufficient for binding the FKBP-rapamycin
complex (Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S.
L. (1995) Identification of an 11-kDa FKBP12-rapamycin-binding
domain within the 289-kDa FKBP12-rapamycin-associated protein and
characterization of a critical serine residue. Proc Natl Acad Sci
USA 92: 4947-51.)
[0373] In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based
switch can use a dimerization molecule, e.g., rapamycin or a
rapamycin analog.
[0374] The amino acid sequence of FKBP is as follows:
TABLE-US-00008 (SEQ ID NO: 122) D V P D Y A S L G G P S S P K K K R
K V S R G V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L
E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A
Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V
F D V E L L K L E T S Y
[0375] In embodiments, an FKBP switch domain can comprise a
fragment of FKBP having the ability to bind with FRB, or a fragment
or analog thereof, in the presence of rapamycin or a rapalog, e.g.,
the underlined portion of SEQ ID NO: 122, which is:
TABLE-US-00009 (SEQ ID NO: 123) V Q V E T I S P G D G R T F P K R G
Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q
E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H
P G I I P P H A T L V F D V E L L K L E T S
[0376] The amino acid sequence of FRB is as follows:
TABLE-US-00010 (SEQ ID NO: 124) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV
LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR
ISK
[0377] "FKBP/FRAP, e.g., an FKBP/FRB, based switch" as that term is
used herein, refers to a dimerization switch comprising: a first
switch domain, which comprises an FKBP fragment or analog thereof
having the ability to bind with FRB, or a fragment or analog
thereof, in the presence of rapamycin or a rapalog, e.g., RAD001,
and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%
identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4,
3, 2, or 1 amino acid residues from, the FKBP sequence of SEQ ID
NO: 122 or 123; and a second switch domain, which comprises an FRB
fragment or analog thereof having the ability to bind with FRB, or
a fragment or analog thereof, in the presence of rapamycin or a
rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or
99% identity with, or differs by no more than 30, 25, 20, 15, 10,
5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ
ID NO: 124. In an embodiment, a RCAR described herein comprises one
switch domain comprises amino acid residues disclosed in SEQ ID NO:
122 (or SEQ ID NO: 123), and one switch domain comprises amino acid
residues disclosed in SEQ ID NO: 124.
[0378] In embodiments, the FKBP/FRB dimerization switch comprises a
modified FRB switch domain that exhibits altered, e.g., enhanced,
complex formation between an FRB-based switch domain, e.g., the
modified FRB switch domain, a FKBP-based switch domain, and the
dimerization molecule, e.g., rapamycin or a rapalogue, e.g.,
RAD001. In an embodiment, the modified FRB switch domain comprises
one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more,
selected from mutations at amino acid position(s) L2031, E2032,
S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108,
where the wild-type amino acid is mutated to any other
naturally-occurring amino acid. In an embodiment, a mutant FRB
comprises a mutation at E2032, where E2032 is mutated to
phenylalanine (E2032F), methionine (E2032M), arginine (E2032R),
valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ
ID NO: 125, or leucine (E2032L), e.g., SEQ ID NO: 126. In an
embodiment, a mutant FRB comprises a mutation at T2098, where T2098
is mutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ
ID NO: 127. In an embodiment, a mutant FRB comprises a mutation at
E2032 and at T2098, where E2032 is mutated to any amino acid, and
where T2098 is mutated to any amino acid, e.g., SEQ ID NO: 128. In
an embodiment, a mutant FRB comprises an E2032I and a T2098L
mutation, e.g., SEQ ID NO: 129. In an embodiment, a mutant FRB
comprises an E2032L and a T2098L mutation, e.g., SEQ ID NO:
130.
TABLE-US-00011 TABLE 14 Exemplary mutant FRB having increased
affinity for a dimerization molecule. SEQ ID FRB mutant Amino Acid
Sequence NO: E2032I mutant
ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 125
DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS E2032L mutant
ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 126
DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 12098L mutant
ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 127
DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032, T2098
ILWHEMWHEGLXEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 128 mutant
DLMEAQEWCRKYMKSGNVKDLXQAWDLYYHVFRRISKTS E2032I, T2098L
ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 129 mutant
DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032L, T2098L
ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR 130 mutant
DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS
[0379] Other suitable dimerization switches include a GyrB-GyrB
based dimerization switch, a Gibberellin-based dimerization switch,
a tag/binder dimerization switch, and a halo-tag/snap-tag
dimerization switch. Following the guidance provided herein, such
switches and relevant dimerization molecules will be apparent to
one of ordinary skill.
Dimerization Molecule
[0380] Association between the switch domains is promoted by the
dimerization molecule. In the presence of dimerization molecule
interaction or association between switch domains allows for signal
transduction between a polypeptide associated with, e.g., fused to,
a first switch domain, and a polypeptide associated with, e.g.,
fused to, a second switch domain. In the presence of non-limiting
levels of dimerization molecule signal transduction is increased by
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100
fold, e.g., as measured in a system described herein.
[0381] Rapamycin and rapamycin analogs (sometimes referred to as
rapalogues), e.g., RAD001, can be used as dimerization molecules in
a FKBP/FRB-based dimerization switch described herein. In an
embodiment the dimerization molecule can be selected from rapamycin
(sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus,
AP-23573 (ridaforolimus), biolimus and AP21967. Additional
rapamycin analogs suitable for use with FKBP/FRB-based dimerization
switches are further described in the section entitled "Combination
Therapies", or in the subsection entitled "Exemplary mTOR
inhibitors".
Split CAR
[0382] In some embodiments, the CAR-expressing cell uses a split
CAR. The split CAR approach is described in more detail in
publications WO2014/055442 and WO2014/055657. Briefly, a split CAR
system comprises a cell expressing a first CAR having a first
antigen binding domain and a costimulatory domain (e.g., 41BB), and
the cell also expresses a second CAR having a second antigen
binding domain and an intracellular signaling domain (e.g., CD3
zeta). When the cell encounters the first antigen, the
costimulatory domain is activated, and the cell proliferates. When
the cell encounters the second antigen, the intracellular signaling
domain is activated and cell-killing activity begins. Thus, the
CAR-expressing cell is only fully activated in the presence of both
antigens.
RNA Transfection
[0383] Disclosed herein are methods for producing an in vitro
transcribed RNA CAR. The present invention also includes a CAR
encoding RNA construct that can be directly transfected into a
cell. A method for generating mRNA for use in transfection can
involve in vitro transcription (IVT) of a template with specially
designed primers, followed by polyA addition, to produce a
construct containing 3' and 5' untranslated sequence ("UTR"), a 5'
cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to
be expressed, and a polyA tail, typically 50-2000 bases in length
(SEQ ID NO: 118). RNA so produced can efficiently transfect
different kinds of cells. In one aspect, the template includes
sequences for the CAR.
[0384] In one aspect the anti-CD19 CAR is encoded by a messenger
RNA (mRNA). In one aspect, the mRNA encoding the anti-CD19 CAR is
introduced into an immune effector cell, e.g., a T cell or a NK
cell, for production of a CAR-expressing cell, e.g., a CART cell or
a CAR NK cell.
[0385] In one embodiment, the in vitro transcribed RNA CAR can be
introduced to a cell as a form of transient transfection. The RNA
is produced by in vitro transcription using a polymerase chain
reaction (PCR)-generated template. DNA of interest from any source
can be directly converted by PCR into a template for in vitro mRNA
synthesis using appropriate primers and RNA polymerase. The source
of the DNA can be, for example, genomic DNA, plasmid DNA, phage
DNA, cDNA, synthetic DNA sequence or any other appropriate source
of DNA. The desired temple for in vitro transcription is a CAR of
the present invention. For example, the template for the RNA CAR
comprises an extracellular region comprising a single chain
variable domain of an anti-tumor antibody; a hinge region, a
transmembrane domain (e.g., a transmembrane domain of CD8a); and a
cytoplasmic region that includes an intracellular signaling domain,
e.g., comprising the signaling domain of CD3-zeta and the signaling
domain of 4-1BB.
[0386] In one embodiment, the DNA to be used for PCR contains an
open reading frame. The DNA can be from a naturally occurring DNA
sequence from the genome of an organism. In one embodiment, the
nucleic acid can include some or all of the 5' and/or 3'
untranslated regions (UTRs). The nucleic acid can include exons and
introns. In one embodiment, the DNA to be used for PCR is a human
nucleic acid sequence. In another embodiment, the DNA to be used
for PCR is a human nucleic acid sequence including the 5' and 3'
UTRs. The DNA can alternatively be an artificial DNA sequence that
is not normally expressed in a naturally occurring organism. An
exemplary artificial DNA sequence is one that contains portions of
genes that are ligated together to form an open reading frame that
encodes a fusion protein. The portions of DNA that are ligated
together can be from a single organism or from more than one
organism.
[0387] PCR is used to generate a template for in vitro
transcription of mRNA which is used for transfection. Methods for
performing PCR are well known in the art. Primers for use in PCR
are designed to have regions that are substantially complementary
to regions of the DNA to be used as a template for the PCR.
"Substantially complementary," as used herein, refers to sequences
of nucleotides where a majority or all of the bases in the primer
sequence are complementary, or one or more bases are
non-complementary, or mismatched. Substantially complementary
sequences are able to anneal or hybridize with the intended DNA
target under annealing conditions used for PCR. The primers can be
designed to be substantially complementary to any portion of the
DNA template. For example, the primers can be designed to amplify
the portion of a nucleic acid that is normally transcribed in cells
(the open reading frame), including 5' and 3' UTRs. The primers can
also be designed to amplify a portion of a nucleic acid that
encodes a particular domain of interest. In one embodiment, the
primers are designed to amplify the coding region of a human cDNA,
including all or portions of the 5' and 3' UTRs. Primers useful for
PCR can be generated by synthetic methods that are well known in
the art. "Forward primers" are primers that contain a region of
nucleotides that are substantially complementary to nucleotides on
the DNA template that are upstream of the DNA sequence that is to
be amplified. "Upstream" is used herein to refer to a location 5,
to the DNA sequence to be amplified relative to the coding strand.
"Reverse primers" are primers that contain a region of nucleotides
that are substantially complementary to a double-stranded DNA
template that are downstream of the DNA sequence that is to be
amplified. "Downstream" is used herein to refer to a location 3' to
the DNA sequence to be amplified relative to the coding strand.
[0388] Any DNA polymerase useful for PCR can be used in the methods
disclosed herein. The reagents and polymerase are commercially
available from a number of sources.
[0389] Chemical structures with the ability to promote stability
and/or translation efficiency may also be used. The RNA preferably
has 5' and 3' UTRs. In one embodiment, the 5' UTR is between one
and 3000 nucleotides in length. The length of 5' and 3' UTR
sequences to be added to the coding region can be altered by
different methods, including, but not limited to, designing primers
for PCR that anneal to different regions of the UTRs. Using this
approach, one of ordinary skill in the art can modify the 5' and 3'
UTR lengths required to achieve optimal translation efficiency
following transfection of the transcribed RNA.
[0390] The 5' and 3' UTRs can be the naturally occurring,
endogenous 5' and 3' UTRs for the nucleic acid of interest.
Alternatively, UTR sequences that are not endogenous to the nucleic
acid of interest can be added by incorporating the UTR sequences
into the forward and reverse primers or by any other modifications
of the template. The use of UTR sequences that are not endogenous
to the nucleic acid of interest can be useful for modifying the
stability and/or translation efficiency of the RNA. For example, it
is known that AU-rich elements in 3' UTR sequences can decrease the
stability of mRNA. Therefore, 3' UTRs can be selected or designed
to increase the stability of the transcribed RNA based on
properties of UTRs that are well known in the art.
[0391] In one embodiment, the 5' UTR can contain the Kozak sequence
of the endogenous nucleic acid. Alternatively, when a 5' UTR that
is not endogenous to the nucleic acid of interest is being added by
PCR as described above, a consensus Kozak sequence can be
redesigned by adding the 5' UTR sequence. Kozak sequences can
increase the efficiency of translation of some RNA transcripts, but
does not appear to be required for all RNAs to enable efficient
translation. The requirement for Kozak sequences for many mRNAs is
known in the art. In other embodiments the 5' UTR can be 5'UTR of
an RNA virus whose RNA genome is stable in cells. In other
embodiments various nucleotide analogues can be used in the 3' or
5' UTR to impede exonuclease degradation of the mRNA.
[0392] To enable synthesis of RNA from a DNA template without the
need for gene cloning, a promoter of transcription should be
attached to the DNA template upstream of the sequence to be
transcribed. When a sequence that functions as a promoter for an
RNA polymerase is added to the 5' end of the forward primer, the
RNA polymerase promoter becomes incorporated into the PCR product
upstream of the open reading frame that is to be transcribed. In
one preferred embodiment, the promoter is a T7 polymerase promoter,
as described elsewhere herein. Other useful promoters include, but
are not limited to, T3 and SP6 RNA polymerase promoters. Consensus
nucleotide sequences for T7, T3 and SP6 promoters are known in the
art.
[0393] In a preferred embodiment, the mRNA has both a cap on the 5'
end and a 3' poly(A) tail which determine ribosome binding,
initiation of translation and stability mRNA in the cell. On a
circular DNA template, for instance, plasmid DNA, RNA polymerase
produces a long concatameric product which is not suitable for
expression in eukaryotic cells. The transcription of plasmid DNA
linearized at the end of the 3' UTR results in normal sized mRNA
which is not effective in eukaryotic transfection even if it is
polyadenylated after transcription.
[0394] On a linear DNA template, phage T7 RNA polymerase can extend
the 3' end of the transcript beyond the last base of the template
(Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985);
Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65
(2003).
[0395] The conventional method of integration of polyA/T stretches
into a DNA template is molecular cloning. However polyA/T sequence
integrated into plasmid DNA can cause plasmid instability, which is
why plasmid DNA templates obtained from bacterial cells are often
highly contaminated with deletions and other aberrations. This
makes cloning procedures not only laborious and time consuming but
often not reliable. That is why a method which allows construction
of DNA templates with polyA/T 3' stretch without cloning highly
desirable.
[0396] The polyA/T segment of the transcriptional DNA template can
be produced during PCR by using a reverse primer containing a polyT
tail, such as 100T tail (SEQ ID NO: 110) (size can be 50-5000 T
(SEQ ID NO: 111)), or after PCR by any other method, including, but
not limited to, DNA ligation or in vitro recombination. Poly(A)
tails also provide stability to RNAs and reduce their degradation.
Generally, the length of a poly(A) tail positively correlates with
the stability of the transcribed RNA. In one embodiment, the
poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO:
112).
[0397] Poly(A) tails of RNAs can be further extended following in
vitro transcription with the use of a poly(A) polymerase, such as
E. coli polyA polymerase (E-PAP). In one embodiment, increasing the
length of a poly(A) tail from 100 nucleotides to between 300 and
400 nucleotides (SEQ ID NO: 113) results in about a two-fold
increase in the translation efficiency of the RNA. Additionally,
the attachment of different chemical groups to the 3' end can
increase mRNA stability. Such attachment can contain
modified/artificial nucleotides, aptamers and other compounds. For
example, ATP analogs can be incorporated into the poly(A) tail
using poly(A) polymerase. ATP analogs can further increase the
stability of the RNA.
[0398] 5' caps on also provide stability to RNA molecules. In a
preferred embodiment, RNAs produced by the methods disclosed herein
include a 5' cap. The 5' cap is provided using techniques known in
the art and described herein (Cougot, et al., Trends in Biochem.
Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001);
Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966
(2005)).
[0399] The RNAs produced by the methods disclosed herein can also
contain an internal ribosome entry site (IRES) sequence. The IRES
sequence may be any viral, chromosomal or artificially designed
sequence which initiates cap-independent ribosome binding to mRNA
and facilitates the initiation of translation. Any solutes suitable
for cell electroporation, which can contain factors facilitating
cellular permeability and viability such as sugars, peptides,
lipids, proteins, antioxidants, and surfactants can be
included.
[0400] RNA can be introduced into target cells using any of a
number of different methods, for instance, commercially available
methods which include, but are not limited to, electroporation
(Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM
830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser
II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg
Germany), cationic liposome mediated transfection using
lipofection, polymer encapsulation, peptide mediated transfection,
or biolistic particle delivery systems such as "gene guns" (see,
for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70
(2001).
Non-Viral Delivery Methods
[0401] In some aspects, non-viral methods can be used to deliver a
nucleic acid encoding a CAR described herein into a cell or tissue
or a subject.
[0402] In some embodiments, the non-viral method includes the use
of a transposon (also called a transposable element). In some
embodiments, a transposon is a piece of DNA that can insert itself
at a location in a genome, for example, a piece of DNA that is
capable of self-replicating and inserting its copy into a genome,
or a piece of DNA that can be spliced out of a longer nucleic acid
and inserted into another place in a genome. For example, a
transposon comprises a DNA sequence made up of inverted repeats
flanking genes for transposition.
[0403] Exemplary methods of nucleic acid delivery using a
transposon include a Sleeping Beauty transposon system (SBTS) and a
piggyBac (PB) transposon system. See, e.g., Aronovich et al. Hum.
Mol. Genet. 20.R1 (2011):R14-20; Singh et al. Cancer Res.
15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589;
Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al.
Blood. 122.21(2013):166; Williams. Molecular Therapy
16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65;
and Ding et al. Cell. 122.3(2005):473-83, all of which are
incorporated herein by reference.
[0404] The SBTS includes two components: 1) a transposon containing
a transgene and 2) a source of transposase enzyme. The transposase
can transpose the transposon from a carrier plasmid (or other donor
DNA) to a target DNA, such as a host cell chromosome/genome. For
example, the transposase binds to the carrier plasmid/donor DNA,
cuts the transposon (including transgene(s)) out of the plasmid,
and inserts it into the genome of the host cell. See, e.g.,
Aronovich et al. supra.
[0405] Exemplary transposons include a pT2-based transposon. See,
e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and
Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are
incorporated herein by reference. Exemplary transposases include a
Tc1/mariner-type transposase, e.g., the SB10 transposase or the
SB11 transposase (a hyperactive transposase which can be expressed,
e.g., from a cytomegalovirus promoter). See, e.g., Aronovich et
al.; Kebriaei et al.; and Grabundzija et al., all of which are
incorporated herein by reference.
[0406] Use of the SBTS permits efficient integration and expression
of a transgene, e.g., a nucleic acid encoding a CAR described
herein. Provided herein are methods of generating a cell, e.g., T
cell or NK cell, that stably expresses a CAR described herein,
e.g., using a transposon system such as SBTS.
[0407] In accordance with methods described herein, in some
embodiments, one or more nucleic acids, e.g., plasmids, containing
the SBTS components are delivered to a cell (e.g., T or NK cell).
For example, the nucleic acid(s) are delivered by standard methods
of nucleic acid (e.g., plasmid DNA) delivery, e.g., methods
described herein, e.g., electroporation, transfection, or
lipofection. In some embodiments, the nucleic acid contains a
transposon comprising a transgene, e.g., a nucleic acid encoding a
CAR described herein. In some embodiments, the nucleic acid
contains a transposon comprising a transgene (e.g., a nucleic acid
encoding a CAR described herein) as well as a nucleic acid sequence
encoding a transposase enzyme. In other embodiments, a system with
two nucleic acids is provided, e.g., a dual-plasmid system, e.g.,
where a first plasmid contains a transposon comprising a transgene,
and a second plasmid contains a nucleic acid sequence encoding a
transposase enzyme. For example, the first and the second nucleic
acids are co-delivered into a host cell.
[0408] In some embodiments, cells, e.g., T or NK cells, are
generated that express a CAR described herein by using a
combination of gene insertion using the SBTS and genetic editing
using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription
Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system,
or engineered meganuclease re-engineered homing endonucleases).
[0409] In some embodiments, use of a non-viral method of delivery
permits reprogramming of cells, e.g., T or NK cells, and direct
infusion of the cells into a subject. Advantages of non-viral
vectors include but are not limited to the ease and relatively low
cost of producing sufficient amounts required to meet a patient
population, stability during storage, and lack of
immunogenicity.
Nucleic Acid Constructs Encoding a CAR
[0410] The present invention also provides nucleic acid molecules
encoding one or more CAR constructs described herein. In one
aspect, the nucleic acid molecule is provided as a messenger RNA
transcript. In one aspect, the nucleic acid molecule is provided as
a DNA construct.
[0411] Accordingly, in one aspect, the invention pertains to an
isolated nucleic acid molecule encoding a chimeric antigen receptor
(CAR), wherein the CAR comprises a anti-CD19 binding domain (e.g.,
a humanized anti-CD19 binding domain), a transmembrane domain, and
an intracellular signaling domain comprising a stimulatory domain,
e.g., a costimulatory signaling domain and/or a primary signaling
domain, e.g., zeta chain. In one embodiment, the anti-CD19 binding
domain is an anti-CD19 binding domain described herein, e.g., an
anti-CD19 binding domain which comprises a sequence selected from a
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59, or
a sequence with 95-99% identify thereof. In one embodiment, the
transmembrane domain is transmembrane domain of a protein selected
from the group consisting of the alpha, beta or zeta chain of the
T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,
CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one
embodiment, the transmembrane domain comprises a sequence of SEQ ID
NO: 15, or a sequence with 95-99% identity thereof. In one
embodiment, the anti-CD19 binding domain is connected to the
transmembrane domain by a hinge region, e.g., a hinge described
herein. In one embodiment, the hinge region comprises SEQ ID NO: 14
or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49, or a sequence with
95-99% identity thereof. In one embodiment, the isolated nucleic
acid molecule further comprises a sequence encoding a costimulatory
domain. In one embodiment, the costimulatory domain is a functional
signaling domain of a protein selected from the group consisting of
OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278),
and 4-1BB (CD137). In one embodiment, the costimulatory domain
comprises a sequence of SEQ ID NO: 16, or a sequence with 95-99%
identity thereof. In one embodiment, the intracellular signaling
domain comprises a functional signaling domain of 4-1BB and a
functional signaling domain of CD3 zeta. In one embodiment, the
intracellular signaling domain comprises the sequence of SEQ ID NO:
16 or SEQ ID NO:51, or a sequence with 95-99% identity thereof, and
the sequence of SEQ ID NO: 17 or SEQ ID NO:43, or a sequence with
95-99% identity thereof, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain.
[0412] In another aspect, the invention pertains to an isolated
nucleic acid molecule encoding a CAR construct comprising a leader
sequence of SEQ ID NO: 13, a scFv domain having a sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and
SEQ ID NO:59, (or a sequence with 95-99% identify thereof), a hinge
region of SEQ ID NO: 14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID
NO:49 (or a sequence with 95-99% identity thereof), a transmembrane
domain having a sequence of SEQ ID NO: 15 (or a sequence with
95-99% identity thereof), a 4-1BB costimulatory domain having a
sequence of SEQ ID NO: 16 or a CD27 costimulatory domain having a
sequence of SEQ ID NO:51 (or a sequence with 95-99% identity
thereof), and a CD3 zeta stimulatory domain having a sequence of
SEQ ID NO: 17 or SEQ ID NO:43 (or a sequence with 95-99% identity
thereof).
[0413] In another aspect, the invention pertains to an isolated
polypeptide molecule encoded by the nucleic acid molecule. In one
embodiment, the isolated polypeptide molecule comprises a sequence
selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:59 or a sequence with 95-99% identify
thereof.
[0414] In another aspect, the invention pertains to a nucleic acid
molecule encoding a chimeric antigen receptor (CAR) molecule that
comprises an anti-CD19 binding domain, a transmembrane domain, and
an intracellular signaling domain comprising a stimulatory domain,
and wherein said anti-CD19 binding domain comprises a sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ
ID NO:59, or a sequence with 95-99% identify thereof.
[0415] In one embodiment, the encoded CAR molecule further
comprises a sequence encoding a costimulatory domain. In one
embodiment, the costimulatory domain is a functional signaling
domain of a protein selected from the group consisting of OX40,
CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In
one embodiment, the costimulatory domain comprises a sequence of
SEQ ID NO: 16. In one embodiment, the transmembrane domain is a
transmembrane domain of a protein selected from the group
consisting of the alpha, beta or zeta chain of the T-cell receptor,
CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,
CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment,
the transmembrane domain comprises a sequence of SEQ ID NO: 15. In
one embodiment, the intracellular signaling domain comprises a
functional signaling domain of 4-1BB and a functional signaling
domain of zeta. In one embodiment, the intracellular signaling
domain comprises the sequence of SEQ ID NO: 16 and the sequence of
SEQ ID NO: 17, wherein the sequences comprising the intracellular
signaling domain are expressed in the same frame and as a single
polypeptide chain. In one embodiment, the anti-CD19 binding domain
is connected to the transmembrane domain by a hinge region. In one
embodiment, the hinge region comprises SEQ ID NO: 14. In one
embodiment, the hinge region comprises SEQ ID NO:45 or SEQ ID NO:47
or SEQ ID NO:49.
[0416] In another aspect, the invention pertains to an encoded CAR
molecule comprising a leader sequence of SEQ ID NO: 13, a scFv
domain having a sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO: 12, and SEQ ID NO:59, or a sequence with 95-99%
identify thereof, a hinge region of SEQ ID NO:14 or SEQ ID NO:45 or
SEQ ID NO:47 or SEQ ID NO:49, a transmembrane domain having a
sequence of SEQ ID NO: 15, a 4-1BB costimulatory domain having a
sequence of SEQ ID NO: 16 or a CD27 costimulatory domain having a
sequence of SEQ ID NO:51, and a CD3 zeta stimulatory domain having
a sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the
encoded CAR molecule comprises a sequence selected from a group
consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID
NO:59, or a sequence with 95-99% identify thereof.
[0417] The nucleic acid sequences coding for the desired molecules
can be obtained using recombinant methods known in the art, such
as, for example by screening libraries from cells expressing the
gene, by deriving the gene from a vector known to include the same,
or by isolating directly from cells and tissues containing the
same, using standard techniques. Alternatively, the gene of
interest can be produced synthetically, rather than cloned.
[0418] The present invention also provides vectors in which a DNA
of the present invention is inserted. Vectors derived from
retroviruses such as the lentivirus are suitable tools to achieve
long-term gene transfer since they allow long-term, stable
integration of a transgene and its propagation in daughter cells.
Lentiviral vectors have the added advantage over vectors derived
from onco-retroviruses such as murine leukemia viruses in that they
can transduce non-proliferating cells, such as hepatocytes. They
also have the added advantage of low immunogenicity. A retroviral
vector may also be, e.g., a gammaretroviral vector. A
gammaretroviral vector may include, e.g., a promoter, a packaging
signal (W), a primer binding site (PBS), one or more (e.g., two)
long terminal repeats (LTR), and a transgene of interest, e.g., a
gene encoding a CAR. A gammaretroviral vector may lack viral
structural gens such as gag, pol, and env. Exemplary
gammaretroviral vectors include Murine Leukemia Virus (MLV),
Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma
Virus (MPSV), and vectors derived therefrom. Other gammaretroviral
vectors are described, e.g., in Tobias Maetzig et al.,
"Gammaretroviral Vectors: Biology, Technology and Application"
Viruses. 2011 June; 3(6): 677-713.
[0419] In another embodiment, the vector comprising the nucleic
acid encoding the desired CAR of the invention is an adenoviral
vector (A5/35). In another embodiment, the expression of nucleic
acids encoding CARs can be accomplished using of transposons such
as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See
below June et al. 2009Nature Reviews Immunology 9.10: 704-716, is
incorporated herein by reference.
[0420] In brief summary, the expression of natural or synthetic
nucleic acids encoding CARs is typically achieved by operably
linking a nucleic acid encoding the CAR polypeptide or portions
thereof to a promoter, and incorporating the construct into an
expression vector. The vectors can be suitable for replication and
integration eukaryotes. Typical cloning vectors contain
transcription and translation terminators, initiation sequences,
and promoters useful for regulation of the expression of the
desired nucleic acid sequence.
[0421] The expression constructs of the present invention may also
be used for nucleic acid immunization and gene therapy, using
standard gene delivery protocols. Methods for gene delivery are
known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,
5,589,466, incorporated by reference herein in their entireties. In
another embodiment, the invention provides a gene therapy
vector.
[0422] The nucleic acid can be cloned into a number of types of
vectors. For example, the nucleic acid can be cloned into a vector
including, but not limited to a plasmid, a phagemid, a phage
derivative, an animal virus, and a cosmid. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors, and sequencing vectors.
[0423] Further, the expression vector may be provided to a cell in
the form of a viral vector. Viral vector technology is well known
in the art and is described, for example, in Sambrook et al., 2012,
MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring
Harbor Press, NY), and in other virology and molecular biology
manuals. Viruses, which are useful as vectors include, but are not
limited to, retroviruses, adenoviruses, adeno-associated viruses,
herpes viruses, and lentiviruses. In general, a suitable vector
contains an origin of replication functional in at least one
organism, a promoter sequence, convenient restriction endonuclease
sites, and one or more selectable markers, (e.g., WO 01/96584; WO
01/29058; and U.S. Pat. No. 6,326,193).
[0424] A number of viral based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene delivery systems. A selected gene can
be inserted into a vector and packaged in retroviral particles
using techniques known in the art. The recombinant virus can then
be isolated and delivered to cells of the subject either in vivo or
ex vivo. A number of retroviral systems are known in the art. In
some embodiments, adenovirus vectors are used. A number of
adenovirus vectors are known in the art. In one embodiment,
lentivirus vectors are used.
[0425] Additional promoter elements, e.g., enhancers, regulate the
frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription. Exemplary promoters
include the CMV IE gene, EF-1.alpha., ubiquitin C, or
phosphoglycerokinase (PGK) promoters.
[0426] An example of a promoter that is capable of expressing a CAR
transgene in a mammalian T cell is the EF1a promoter. The native
EF1a promoter drives expression of the alpha subunit of the
elongation factor-1 complex, which is responsible for the enzymatic
delivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has
been extensively used in mammalian expression plasmids and has been
shown to be effective in driving CAR expression from transgenes
cloned into a lentiviral vector. See, e.g., Milone et al., Mol.
Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter
comprises the sequence provided as SEQ ID NO: 100.
[0427] Another example of a promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. However, other constitutive promoter sequences may
also be used, including, but not limited to the simian virus 40
(SV40) early promoter, mouse mammary tumor virus (MMTV), human
immunodeficiency virus (HIV) long terminal repeat (LTR) promoter,
MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr
virus immediate early promoter, a Rous sarcoma virus promoter, as
well as human gene promoters such as, but not limited to, the actin
promoter, the myosin promoter, the elongation factor-1.alpha.
promoter, the hemoglobin promoter, and the creatine kinase
promoter. Further, the invention should not be limited to the use
of constitutive promoters. Inducible promoters are also
contemplated as part of the invention. The use of an inducible
promoter provides a molecular switch capable of turning on
expression of the polynucleotide sequence which it is operatively
linked when such expression is desired, or turning off the
expression when expression is not desired. Examples of inducible
promoters include, but are not limited to a metallothionine
promoter, a glucocorticoid promoter, a progesterone promoter, and a
tetracycline promoter.
[0428] A vector may also include, e.g., a signal sequence to
facilitate secretion, a polyadenylation signal and transcription
terminator (e.g., from Bovine Growth Hormone (BGH) gene), an
element allowing episomal replication and replication in
prokaryotes (e.g. SV40 origin and ColE1 or others known in the art)
and/or elements to allow selection (e.g., ampicillin resistance
gene and/or zeocin marker).
[0429] In order to assess the expression of a CAR polypeptide or
portions thereof, the expression vector to be introduced into a
cell can also contain either a selectable marker gene or a reporter
gene or both to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In other aspects,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, for example, antibiotic-resistance genes, such as neo and
the like.
[0430] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
that encodes a polypeptide whose expression is manifested by some
easily detectable property, e.g., enzymatic activity. Expression of
the reporter gene is assayed at a suitable time after the DNA has
been introduced into the recipient cells. Suitable reporter genes
may include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription.
[0431] Methods of introducing and expressing genes into a cell are
known in the art. In the context of an expression vector, the
vector can be readily introduced into a host cell, e.g., mammalian,
bacterial, yeast, or insect cell by any method in the art. For
example, the expression vector can be transferred into a host cell
by physical, chemical, or biological means.
[0432] Physical methods for introducing a polynucleotide into a
host cell include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells comprising vectors and/or
exogenous nucleic acids are well-known in the art. See, for
example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY
MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred
method for the introduction of a polynucleotide into a host cell is
calcium phosphate transfection
[0433] Biological methods for introducing a polynucleotide of
interest into a host cell include the use of DNA and RNA vectors.
Viral vectors, and especially retroviral vectors, have become the
most widely used method for inserting genes into mammalian, e.g.,
human cells. Other viral vectors can be derived from lentivirus,
poxviruses, herpes simplex virus I, adenoviruses and
adeno-associated viruses, and the like. See, for example, U.S. Pat.
Nos. 5,350,674 and 5,585,362.
[0434] Chemical means for introducing a polynucleotide into a host
cell include colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. An exemplary colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (e.g., an artificial
membrane vesicle). Other methods of state-of-the-art targeted
delivery of nucleic acids are available, such as delivery of
polynucleotides with targeted nanoparticles or other suitable
sub-micron sized delivery system.
[0435] In the case where a non-viral delivery system is utilized,
an exemplary delivery vehicle is a liposome. The use of lipid
formulations is contemplated for the introduction of the nucleic
acids into a host cell (in vitro, ex vivo or in vivo). In another
aspect, the nucleic acid may be associated with a lipid. The
nucleic acid associated with a lipid may be encapsulated in the
aqueous interior of a liposome, interspersed within the lipid
bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
oligonucleotide, entrapped in a liposome, complexed with a
liposome, dispersed in a solution containing a lipid, mixed with a
lipid, combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. Lipid, lipid/DNA or lipid/expression vector associated
compositions are not limited to any particular structure in
solution. For example, they may be present in a bilayer structure,
as micelles, or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates that are
not uniform in size or shape. Lipids are fatty substances which may
be naturally occurring or synthetic lipids. For example, lipids
include the fatty droplets that naturally occur in the cytoplasm as
well as the class of compounds which contain long-chain aliphatic
hydrocarbons and their derivatives, such as fatty acids, alcohols,
amines, amino alcohols, and aldehydes.
[0436] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). However, compositions that have different structures in
solution than the normal vesicular structure are also encompassed.
For example, the lipids may assume a micellar structure or merely
exist as nonuniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
[0437] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present invention, in order to confirm the presence of the
recombinant DNA sequence in the host cell, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays well known to those of skill in the art, such as
Southern and Northern blotting, RT-PCR and PCR; "biochemical"
assays, such as detecting the presence or absence of a particular
peptide, e.g., by immunological means (ELISAs and Western blots) or
by assays described herein to identify agents falling within the
scope of the invention.
[0438] The present invention further provides a vector comprising a
CAR encoding nucleic acid molecule. In one aspect, a CAR vector can
be directly transduced into a cell, e.g., a T cell. In one aspect,
the vector is a cloning or expression vector, e.g., a vector
including, but not limited to, one or more plasmids (e.g.,
expression plasmids, cloning vectors, minicircles, minivectors,
double minute chromosomes), retroviral and lentiviral vector
constructs. In one aspect, the vector is capable of expressing the
CAR construct in mammalian T cells. In one aspect, the mammalian T
cell is a human T cell.
Sources of Cells
[0439] Prior to expansion and genetic modification or other
modification, a source of cells, e.g., T cells or natural killer
(NK) cells, can be obtained from a subject. The term "subject" is
intended to include living organisms in which an immune response
can be elicited (e.g., mammals). Examples of subjects include
humans, monkeys, chimpanzees, dogs, cats, mice, rats, and
transgenic species thereof. 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.
[0440] In certain aspects of the present disclosure, immune
effector cells, e.g., 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 one
preferred aspect, 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.
In one aspect, the cells collected by apheresis may be washed to
remove the plasma fraction and, optionally, to place the cells in
an appropriate buffer or media for subsequent processing steps. In
one embodiment, the cells are washed with phosphate buffered saline
(PBS). In an alternative embodiment, the wash solution lacks
calcium and may lack magnesium or may lack many if not all divalent
cations.
[0441] Initial activation steps in the absence of calcium can lead
to magnified activation. 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 (for example, the Cobe
2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell
Saver 5) 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, PlasmaLyte A,
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.
[0442] In one aspect, T cells are isolated from peripheral blood
lymphocytes by lysing the red blood cells and depleting the
monocytes, for example, by centrifugation through a PERCOLL.TM.
gradient or by counterflow centrifugal elutriation.
[0443] The methods described herein can include, e.g., selection of
a specific subpopulation of immune effector cells, e.g., T cells,
that are a T regulatory cell-depleted population, CD25+ depleted
cells, using, e.g., a negative selection technique, e.g., described
herein. Preferably, the population of T regulatory depleted cells
contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of
CD25+ cells.
[0444] In one embodiment, T regulatory cells, e.g., CD25+ T cells,
are removed from the population using an anti-CD25 antibody, or
fragment thereof, or a CD25-binding ligand, IL-2. In one
embodiment, the anti-CD25 antibody, or fragment thereof, or
CD25-binding ligand is conjugated to a substrate, e.g., a bead, or
is otherwise coated on a substrate, e.g., a bead. In one
embodiment, the anti-CD25 antibody, or fragment thereof, is
conjugated to a substrate as described herein.
[0445] In one embodiment, the T regulatory cells, e.g., CD25+ T
cells, are removed from the population using CD25 depletion reagent
from Miltenyi.TM.. In one embodiment, the ratio of cells to CD25
depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to 15 uL, or
1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL,
or 1e7 cells to 1.25 uL. In one embodiment, e.g., for T regulatory
cells, e.g., CD25+ depletion, greater than 500 million cells/ml is
used. In a further aspect, a concentration of cells of 600, 700,
800, or 900 million cells/ml is used.
[0446] In one embodiment, the population of immune effector cells
to be depleted includes about 6.times.10.sup.9 CD25+ T cells. In
other aspects, the population of immune effector cells to be
depleted include about 1.times.10.sup.9 to 1.times.10.sup.10 CD25+
T cell, and any integer value in between. In one embodiment, the
resulting population T regulatory depleted cells has
2.times.10.sup.9 T regulatory cells, e.g., CD25+ cells, or less
(e.g., 1.times.10.sup.9, 5.times.10.sup.8, 1.times.10.sup.8,
5.times.10.sup.7, 1.times.10.sup.7, or less CD25+ cells).
[0447] In one embodiment, the T regulatory cells, e.g., CD25+
cells, are removed from the population using the CliniMAC system
with a depletion tubing set, such as, e.g., tubing 162-01. In one
embodiment, the CliniMAC system is run on a depletion setting such
as, e.g., DEPLETION2.1.
[0448] Without wishing to be bound by a particular theory,
decreasing the level of negative regulators of immune cells (e.g.,
decreasing the number of unwanted immune cells, e.g., T.sub.REG
cells), in a subject prior to apheresis or during manufacturing of
a CAR-expressing cell product can reduce the risk of subject
relapse. For example, methods of depleting T.sub.REG cells are
known in the art. Methods of decreasing T.sub.REG cells include,
but are not limited to, cyclophosphamide, anti-GITR antibody (an
anti-GITR antibody described herein), CD25-depletion, and
combinations thereof.
[0449] In some embodiments, the manufacturing methods comprise
reducing the number of (e.g., depleting) T.sub.REG cells prior to
manufacturing of the CAR-expressing cell. For example,
manufacturing methods comprise contacting the sample, e.g., the
apheresis sample, with an anti-GITR antibody and/or an anti-CD25
antibody (or fragment thereof, or a CD25-binding ligand), e.g., to
deplete T.sub.REG cells prior to manufacturing of the
CAR-expressing cell (e.g., T cell, NK cell) product.
[0450] In an embodiment, a subject is pre-treated with one or more
therapies that reduce T.sub.REG cells prior to collection of cells
for CAR-expressing cell product manufacturing, thereby reducing the
risk of subject relapse to CAR-expressing cell treatment. In an
embodiment, methods of decreasing T.sub.REG cells include, but are
not limited to, administration to the subject of one or more of
cyclophosphamide, anti-GITR antibody, CD25-depletion, or a
combination thereof. Administration of one or more of
cyclophosphamide, anti-GITR antibody, CD25-depletion, or a
combination thereof, can occur before, during or after an infusion
of the CAR-expressing cell product.
[0451] In an embodiment, a subject is pre-treated with
cyclophosphamide prior to collection of cells for CAR-expressing
cell product manufacturing, thereby reducing the risk of subject
relapse to CAR-expressing cell treatment. In an embodiment, a
subject is pre-treated with an anti-GITR antibody prior to
collection of cells for CAR-expressing cell product manufacturing,
thereby reducing the risk of subject relapse to CAR-expressing cell
treatment.
[0452] In one embodiment, the population of cells to be removed are
neither the regulatory T cells or tumor cells, but cells that
otherwise negatively affect the expansion and/or function of CART
cells, e.g. cells expressing CD14, CD11b, CD33, CD15, or other
markers expressed by potentially immune suppressive cells. In one
embodiment, such cells are envisioned to be removed concurrently
with regulatory T cells and/or tumor cells, or following said
depletion, or in another order.
[0453] The methods described herein can include more than one
selection step, e.g., more than one depletion step. Enrichment of a
T cell population by negative selection can be accomplished, e.g.,
with a combination of antibodies directed to surface markers unique
to the negatively selected cells. One method is cell sorting and/or
selection via negative magnetic immunoadherence or flow cytometry
that uses a cocktail of monoclonal antibodies directed to cell
surface markers present on the cells negatively selected. For
example, to enrich for CD4+ cells by negative selection, a
monoclonal antibody cocktail can include antibodies to CD14, CD20,
CD11b, CD16, HLA-DR, and CD8.
[0454] The methods described herein can further include removing
cells from the population which express a tumor antigen, e.g., a
tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38,
CD123, CD20, CD14 or CD11b, to thereby provide a population of T
regulatory depleted, e.g., CD25+ depleted, and tumor antigen
depleted cells that are suitable for expression of a CAR, e.g., a
CAR described herein. In one embodiment, tumor antigen expressing
cells are removed simultaneously with the T regulatory, e.g., CD25+
cells. For example, an anti-CD25 antibody, or fragment thereof, and
an anti-tumor antigen antibody, or fragment thereof, can be
attached to the same substrate, e.g., bead, which can be used to
remove the cells or an anti-CD25 antibody, or fragment thereof, or
the anti-tumor antigen antibody, or fragment thereof, can be
attached to separate beads, a mixture of which can be used to
remove the cells. In other embodiments, the removal of T regulatory
cells, e.g., CD25+ cells, and the removal of the tumor antigen
expressing cells is sequential, and can occur, e.g., in either
order.
[0455] Also provided are methods that include removing cells from
the population which express a check point inhibitor, e.g., a check
point inhibitor described herein, e.g., one or more of PD1+ cells,
LAG3+ cells, and TIM3+ cells, to thereby provide a population of T
regulatory depleted, e.g., CD25+ depleted cells, and check point
inhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted
cells. Exemplary check point inhibitors include B7-H1, B7-1, CD160,
P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment,
check point inhibitor expressing cells are removed simultaneously
with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25
antibody, or fragment thereof, and an anti-check point inhibitor
antibody, or fragment thereof, can be attached to the same bead
which can be used to remove the cells, or an anti-CD25 antibody, or
fragment thereof, and the anti-check point inhibitor antibody, or
fragment there, can be attached to separate beads, a mixture of
which can be used to remove the cells. In other embodiments, the
removal of T regulatory cells, e.g., CD25+ cells, and the removal
of the check point inhibitor expressing cells is sequential, and
can occur, e.g., in either order.
[0456] Methods described herein can include a positive selection
step. For example, T cells can isolated by incubation with
anti-CD3/anti-CD28 (e.g., 3.times.28)-conjugated beads, such as
DYNABEADS.RTM. M-450 CD3/CD28 T, for a time period sufficient for
positive selection of the desired T cells. In one embodiment, the
time period is about 30 minutes. In a further embodiment, the time
period ranges from 30 minutes to 36 hours or longer and all integer
values there between. In a further embodiment, the time period is
at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the
time period is 10 to 24 hours, e.g., 24 hours. Longer incubation
times may be used to isolate T cells in any situation where there
are few T cells as compared to other cell types, such in isolating
tumor infiltrating lymphocytes (TIL) from tumor tissue or from
immunocompromised individuals. Further, use of longer incubation
times can increase the efficiency of capture of CD8+ T cells. Thus,
by simply shortening or lengthening the time T cells are allowed to
bind to the CD3/CD28 beads and/or by increasing or decreasing the
ratio of beads to T cells (as described further herein),
subpopulations of T cells can be preferentially selected for or
against at culture initiation or at other time points during the
process. Additionally, by increasing or decreasing the ratio of
anti-CD3 and/or anti-CD28 antibodies on the beads or other surface,
subpopulations of T cells can be preferentially selected for or
against at culture initiation or at other desired time points.
[0457] In one embodiment, a T cell population can be selected that
expresses one or more of IFN-.sup..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.
[0458] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain aspects,
it may be desirable to significantly decrease the volume in which
beads and cells are mixed together (e.g., increase the
concentration of cells), to ensure maximum contact of cells and
beads. For example, in one aspect, a concentration of 10 billion
cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml,
or 5 billion/ml is used. In one aspect, a concentration of 1
billion cells/ml is used. In yet one aspect, a concentration of
cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In
further aspects, concentrations of 125 or 150 million cells/ml can
be used.
[0459] Using high concentrations can result in increased cell
yield, cell activation, and cell expansion. Further, use of high
cell concentrations allows more efficient capture of cells that may
weakly express target antigens of interest, such as CD28-negative T
cells, or from samples where there are many tumor cells present
(e.g., leukemic blood, tumor tissue, etc.). Such populations of
cells may have therapeutic value and would be desirable to obtain.
For example, using high concentration of cells allows more
efficient selection of CD8+ T cells that normally have weaker CD28
expression.
[0460] In a related aspect, it may be desirable to use lower
concentrations of cells. By significantly diluting the mixture of T
cells and surface (e.g., particles such as beads), interactions
between the particles and cells is minimized. This selects for
cells that express high amounts of desired antigens to be bound to
the particles. For example, CD4+ T cells express higher levels of
CD28 and are more efficiently captured than CD8+ T cells in dilute
concentrations. In one aspect, the concentration of cells used is
5.times.10.sup.6/ml. In other aspects, the concentration used can
be from about 1.times.10.sup.5/ml to 1.times.10.sup.6/ml, and any
integer value in between.
[0461] In other aspects, the cells may be incubated on a rotator
for varying lengths of time at varying speeds at either
2-10.degree. C. or at room temperature.
[0462] T cells for stimulation can also be frozen after a washing
step. Wishing not to be bound by theory, the freeze and subsequent
thaw step provides a more uniform product by removing granulocytes
and to some extent monocytes in the cell population. After the
washing step that removes plasma and platelets, the cells may be
suspended in a freezing solution. While many freezing solutions and
parameters are known in the art and will be useful in this context,
one method involves using PBS containing 20% DMSO and 8% human
serum albumin, or culture media containing 10% Dextran 40 and 5%
Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25%
Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5%
Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable
cell freezing media containing for example, Hespan and PlasmaLyte
A, the cells then are frozen to -80.degree. C. at a rate of 10 per
minute and stored in the vapor phase of a liquid nitrogen storage
tank. Other methods of controlled freezing may be used as well as
uncontrolled freezing immediately at -20.degree. C. or in liquid
nitrogen.
[0463] In certain aspects, cryopreserved cells are thawed and
washed as described herein and allowed to rest for one hour at room
temperature prior to activation using the methods of the present
invention.
[0464] Also contemplated in the context of the invention is the
collection of blood samples or apheresis product from a subject at
a time period prior to when the expanded cells as described herein
might be needed. As such, the source of the cells to be expanded
can be collected at any time point necessary, and desired cells,
such as T cells, isolated and frozen for later use in immune
effector cell therapy for any number of diseases or conditions that
would benefit from immune effector cell therapy, such as those
described herein. In one aspect a blood sample or an apheresis is
taken from a generally healthy subject. In certain aspects, a blood
sample or an apheresis is taken from a generally healthy subject
who is at risk of developing a disease, but who has not yet
developed a disease, and the cells of interest are isolated and
frozen for later use. In certain aspects, the T cells may be
expanded, frozen, and used at a later time. In certain aspects,
samples are collected from a patient shortly after diagnosis of a
particular disease as described herein but prior to any treatments.
In a further aspect, the cells are isolated from a blood sample or
an apheresis from a subject prior to any number of relevant
treatment modalities, including but not limited to treatment with
agents such as natalizumab, efalizumab, antiviral agents,
chemotherapy, radiation, immunosuppressive agents, such as
cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,
antibodies, or other immunoablative agents such as CAMPATH,
anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506,
rapamycin, mycophenolic acid, steroids, FR901228, and
irradiation.
[0465] In a further aspect of the present invention, T cells are
obtained from a patient directly following treatment that leaves
the subject with functional T cells. In this regard, it has been
observed that following certain cancer treatments, in particular
treatments with drugs that damage the immune system, shortly after
treatment during the period when patients would normally be
recovering from the treatment, the quality of T cells obtained may
be optimal or improved for their ability to expand ex vivo.
Likewise, following ex vivo manipulation using the methods
described herein, these cells may be in a preferred state for
enhanced engraftment and in vivo expansion. Thus, it is
contemplated within the context of the present invention to collect
blood cells, including T cells, dendritic cells, or other cells of
the hematopoietic lineage, during this recovery phase. Further, in
certain aspects, mobilization (for example, mobilization with
GM-CSF) and conditioning regimens can be used to create a condition
in a subject wherein repopulation, recirculation, regeneration,
and/or expansion of particular cell types is favored, especially
during a defined window of time following therapy. Illustrative
cell types include T cells, B cells, dendritic cells, and other
cells of the immune system.
[0466] In one embodiment, the immune effector cells expressing a
CAR molecule, e.g., a CAR molecule described herein, are obtained
from a subject that has received a low, immune enhancing dose of an
mTOR inhibitor. In an embodiment, the population of immune effector
cells, e.g., T cells, to be engineered to express a CAR, are
harvested after a sufficient time, or after sufficient dosing of
the low, immune enhancing, dose of an mTOR inhibitor, such that the
level of PD1 negative immune effector cells, e.g., T cells, or the
ratio of PD1 negative immune effector cells, e.g., T cells/PD1
positive immune effector cells, e.g., T cells, in the subject or
harvested from the subject has been, at least transiently,
increased.
[0467] In other embodiments, population of immune effector cells,
e.g., T cells, which have, or will be engineered to express a CAR,
can be treated ex vivo by contact with an amount of an mTOR
inhibitor that increases the number of PD1 negative immune effector
cells, e.g., T cells or increases the ratio of PD1 negative immune
effector cells, e.g., T cells/PD1 positive immune effector cells,
e.g., T cells.
[0468] In one embodiment, a T cell population is diaglycerol kinase
(DGK)-deficient. DGK-deficient cells include cells that do not
express DGK RNA or protein, or have reduced or inhibited DGK
activity. DGK-deficient cells can be generated by genetic
approaches, e.g., administering RNA-interfering agents, e.g.,
siRNA, shRNA, miRNA, to reduce or prevent DGK expression.
Alternatively, DGK-deficient cells can be generated by treatment
with DGK inhibitors described herein.
[0469] In one embodiment, a T cell population is Ikaros-deficient.
Ikaros-deficient cells include cells that do not express Ikaros RNA
or protein, or have reduced or inhibited Ikaros activity,
Ikaros-deficient cells can be generated by genetic approaches,
e.g., administering RNA-interfering agents, e.g., siRNA, shRNA,
miRNA, to reduce or prevent Ikaros expression. Alternatively,
Ikaros-deficient cells can be generated by treatment with Ikaros
inhibitors, e.g., lenalidomide.
[0470] In embodiments, a T cell population is DGK-deficient and
Ikaros-deficient, e.g., does not express DGK and Ikaros, or has
reduced or inhibited DGK and Ikaros activity. Such DGK and
Ikaros-deficient cells can be generated by any of the methods
described herein.
[0471] In an embodiment, the NK cells are obtained from the
subject. In another embodiment, the NK cells are an NK cell line,
e.g., NK-92 cell line (Conkwest).
Allogeneic CAR
[0472] In embodiments described herein, the immune effector cell
can be an allogeneic immune effector cell, e.g., T cell or NK cell.
For example, the cell can be an allogeneic T cell, e.g., an
allogeneic T cell lacking expression of a functional T cell
receptor (TCR) and/or human leukocyte antigen (HLA), e.g., HLA
class I and/or HLA class II.
[0473] A T cell lacking a functional TCR can be, e.g., engineered
such that it does not express any functional TCR on its surface,
engineered such that it does not express one or more subunits that
comprise a functional TCR or engineered such that it produces very
little functional TCR on its surface. Alternatively, the T cell can
express a substantially impaired TCR, e.g., by expression of
mutated or truncated forms of one or more of the subunits of the
TCR. The term "substantially impaired TCR" means that this TCR will
not elicit an adverse immune reaction in a host.
[0474] A T cell described herein can be, e.g., engineered such that
it does not express a functional HLA on its surface. For example, a
T cell described herein, can be engineered such that cell surface
expression HLA, e.g., HLA class 1 and/or HLA class II, is
downregulated.
[0475] In some embodiments, the T cell can lack a functional TCR
and a functional HLA, e.g., HLA class I and/or HLA class II.
[0476] Modified T cells that lack expression of a functional TCR
and/or HLA can be obtained by any suitable means, including a knock
out or knock down of one or more subunit of TCR or HLA. For
example, the T cell can include a knock down of TCR and/or HLA
using siRNA, shRNA, clustered regularly interspaced short
palindromic repeats (CRISPR) transcription-activator like effector
nuclease (TALEN), or zinc finger endonuclease (ZFN).
[0477] In some embodiments, the allogeneic cell can be a cell which
does not express or expresses at low levels an inhibitory molecule,
e.g. by any method described herein. For example, the cell can be a
cell that does not express or expresses at low levels an inhibitory
molecule, e.g., that can decrease the ability of a CAR-expressing
cell to mount an immune effector response. Examples of inhibitory
molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4 and TGFR beta. Inhibition of an inhibitory molecule, e.g., by
inhibition at the DNA, RNA or protein level, can optimize a
CAR-expressing cell performance. In embodiments, an inhibitory
nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA,
e.g., an siRNA or shRNA, a clustered regularly interspaced short
palindromic repeats (CRISPR), a transcription-activator like
effector nuclease (TALEN), or a zinc finger endonuclease (ZFN),
e.g., as described herein, can be used.
siRNA and shRNA to Inhibit TCR or HLA
[0478] In some embodiments, TCR expression and/or HLA expression
can be inhibited using siRNA or shRNA that targets a nucleic acid
encoding a TCR and/or HLA in a T cell.
[0479] Expression of siRNA and shRNAs in T cells can be achieved
using any conventional expression system, e.g., such as a
lentiviral expression system.
[0480] Exemplary shRNAs that downregulate expression of components
of the TCR are described, e.g., in US Publication No.:
2012/0321667. Exemplary siRNA and shRNA that downregulate
expression of HLA class I and/or HLA class II genes are described,
e.g., in U.S. publication No.: US 2007/0036773.
CRISPR to Inhibit TCR or HLA
[0481] "CRISPR" or "CRISPR to TCR and/or HLA" or "CRISPR to inhibit
TCR and/or HLA" as used herein refers to a set of clustered
regularly interspaced short palindromic repeats, or a system
comprising such a set of repeats. "Cas", as used herein, refers to
a CRISPR-associated protein. A "CRISPR/Cas" system refers to a
system derived from CRISPR and Cas which can be used to silence or
mutate a TCR and/or HLA gene.
[0482] Naturally-occurring CRISPR/Cas systems are found in
approximately 40% of sequenced eubacteria genomes and 90% of
sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172.
This system is a type of prokaryotic immune system that confers
resistance to foreign genetic elements such as plasmids and phages
and provides a form of acquired immunity. Barrangou et al. (2007)
Science 315: 1709-1712; Marragini et al. (2008) Science 322:
1843-1845.
[0483] The CRISPR/Cas system has been modified for use in gene
editing (silencing, enhancing or changing specific genes) in
eukaryotes such as mice or primates. Wiedenheft et al. (2012)
Nature 482: 331-8. This is accomplished by introducing into the
eukaryotic cell a plasmid containing a specifically designed CRISPR
and one or more appropriate Cas.
[0484] The CRISPR sequence, sometimes called a CRISPR locus,
comprises alternating repeats and spacers. In a naturally-occurring
CRISPR, the spacers usually comprise sequences foreign to the
bacterium such as a plasmid or phage sequence; in the TCR and/or
HLA CRISPR/Cas system, the spacers are derived from the TCR or HLA
gene sequence.
[0485] RNA from the CRISPR locus is constitutively expressed and
processed by Cas proteins into small RNAs. These comprise a spacer
flanked by a repeat sequence. The RNAs guide other Cas proteins to
silence exogenous genetic elements at the RNA or DNA level. Horvath
et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology
Direct 1: 7. The spacers thus serve as templates for RNA molecules,
analogously to siRNAs. Pennisi (2013) Science 341: 833-836.
[0486] As these naturally occur in many different types of
bacteria, the exact arrangements of the CRISPR and structure,
function and number of Cas genes and their product differ somewhat
from species to species. Haft et al. (2005) PLoS Comput. Biol. 1:
e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005)
J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151:
2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern
et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas
subtype, E. coli) proteins (e.g., CasA) form a functional complex,
Cascade, that processes CRISPR RNA transcripts into spacer-repeat
units that Cascade retains. Brouns et al. (2008) Science 321:
960-964. In other prokaryotes, Cas6 processes the CRISPR
transcript. The CRISPR-based phage inactivation in E. coli requires
Cascade and Cas3, but not Cas 1 or Cas2. The Cmr (Cas RAMP module)
proteins in Pyrococcus furiosus and other prokaryotes form a
functional complex with small CRISPR RNAs that recognizes and
cleaves complementary target RNAs. A simpler CRISPR system relies
on the protein Cas9, which is a nuclease with two active cutting
sites, one for each strand of the double helix. Combining Cas9 and
modified CRISPR locus RNA can be used in a system for gene editing.
Pennisi (2013) Science 341: 833-836.
[0487] The CRISPR/Cas system can thus be used to edit a TCR and/or
HLA gene (adding or deleting a basepair), or introducing a
premature stop which thus decreases expression of a TCR and/or HLA.
The CRISPR/Cas system can alternatively be used like RNA
interference, turning off TCR and/or HLA gene in a reversible
fashion. In a mammalian cell, for example, the RNA can guide the
Cas protein to a TCR and/or HLA promoter, sterically blocking RNA
polymerases.
[0488] Artificial CRISPR/Cas systems can be generated which inhibit
TCR and/or HLA, using technology known in the art, e.g., that
described in U.S. Publication No. 20140068797, and Cong (2013)
Science 339: 819-823. Other artificial CRISPR/Cas systems that are
known in the art may also be generated which inhibit TCR and/or
HLA, e.g., that described in Tsai (2014) Nature Biotechnol., 32:6
569-576, U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945;
and 8,697,359.
TALEN to Inhibit TCR and/or HLA
[0489] "TALEN" or "TALEN to HLA and/or TCR" or "TALEN to inhibit
HLA and/or TCR" refers to a transcription activator-like effector
nuclease, an artificial nuclease which can be used to edit the HLA
and/or TCR gene.
[0490] TALENs are produced artificially by fusing a TAL effector
DNA binding domain to a DNA cleavage domain. Transcription
activator-like effects (TALEs) can be engineered to bind any
desired DNA sequence, including a portion of the HLA or TCR gene.
By combining an engineered TALE with a DNA cleavage domain, a
restriction enzyme can be produced which is specific to any desired
DNA sequence, including a HLA or TCR sequence. These can then be
introduced into a cell, wherein they can be used for genome
editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al.
(2009) Science 326: 1509-12; Moscou et al. (2009) Science 326:
3501.
[0491] TALEs are proteins secreted by Xanthomonas bacteria. The DNA
binding domain contains a repeated, highly conserved 33-34 amino
acid sequence, with the exception of the 12th and 13th amino acids.
These two positions are highly variable, showing a strong
correlation with specific nucleotide recognition. They can thus be
engineered to bind to a desired DNA sequence.
[0492] To produce a TALEN, a TALE protein is fused to a nuclease
(N), which is a wild-type or mutated FokI endonuclease. Several
mutations to FokI have been made for its use in TALENs; these, for
example, improve cleavage specificity or activity. Cermak et al.
(2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature
Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29:
731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010)
Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25:
786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.
[0493] The FokI domain functions as a dimer, requiring two
constructs with unique DNA binding domains for sites in the target
genome with proper orientation and spacing. Both the number of
amino acid residues between the TALE DNA binding domain and the
FokI cleavage domain and the number of bases between the two
individual TALEN binding sites appear to be important parameters
for achieving high levels of activity. Miller et al. (2011) Nature
Biotech. 29: 143-8.
[0494] A HLA or TCR TALEN can be used inside a cell to produce a
double-stranded break (DSB). A mutation can be introduced at the
break site if the repair mechanisms improperly repair the break via
non-homologous end joining. For example, improper repair may
introduce a frame shift mutation. Alternatively, foreign DNA can be
introduced into the cell along with the TALEN; depending on the
sequences of the foreign DNA and chromosomal sequence, this process
can be used to correct a defect in the HLA or TCR gene or introduce
such a defect into a wt HLA or TCR gene, thus decreasing expression
of HLA or TCR.
[0495] TALENs specific to sequences in HLA or TCR can be
constructed using any method known in the art, including various
schemes using modular components. Zhang et al. (2011) Nature
Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509.
Zinc Finger Nuclease to Inhibit HLA and/or TCR
[0496] "ZFN" or "Zinc Finger Nuclease" or "ZFN to HLA and/or TCR"
or "ZFN to inhibit HLA and/or TCR" refer to a zinc finger nuclease,
an artificial nuclease which can be used to edit the HLA and/or TCR
gene.
[0497] Like a TALEN, a ZFN comprises a FokI nuclease domain (or
derivative thereof) fused to a DNA-binding domain. In the case of a
ZFN, the DNA-binding domain comprises one or more zinc fingers.
Carroll et al. (2011) Genetics Society of America 188: 773-782; and
Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.
[0498] A zinc finger is a small protein structural motif stabilized
by one or more zinc ions. A zinc finger can comprise, for example,
Cys2His2, and can recognize an approximately 3-bp sequence. Various
zinc fingers of known specificity can be combined to produce
multi-finger polypeptides which recognize about 6, 9, 12, 15 or
18-bp sequences. Various selection and modular assembly techniques
are available to generate zinc fingers (and combinations thereof)
recognizing specific sequences, including phage display, yeast
one-hybrid systems, bacterial one-hybrid and two-hybrid systems,
and mammalian cells.
[0499] Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a
pair of ZFNs are required to target non-palindromic DNA sites. The
two individual ZFNs must bind opposite strands of the DNA with
their nucleases properly spaced apart. Bitinaite et al. (1998)
Proc. Natl. Acad. Sci. USA 95: 10570-5.
[0500] Also like a TALEN, a ZFN can create a double-stranded break
in the DNA, which can create a frame-shift mutation if improperly
repaired, leading to a decrease in the expression and amount of HLA
and/or TCR in a cell. ZFNs can also be used with homologous
recombination to mutate in the HLA or TCR gene.
[0501] ZFNs specific to sequences in HLA AND/OR TCR can be
constructed using any method known in the art. See, e.g., Provasi
(2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122:
1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et
al. (2010) J. Mol. Biol. 400: 96; U.S. Patent Publication
2011/0158957; and U.S. Patent Publication 2012/0060230.
Telomerase Expression
[0502] While not wishing to be bound by any particular theory, in
some embodiments, a therapeutic T cell has short term persistence
in a patient, due to shortened telomeres in the T cell;
accordingly, transfection with a telomerase gene can lengthen the
telomeres of the T cell and improve persistence of the T cell in
the patient. See Carl June, "Adoptive T cell therapy for cancer in
the clinic", Journal of Clinical Investigation, 117:1466-1476
(2007). Thus, in an embodiment, an immune effector cell, e.g., a T
cell, ectopically expresses a telomerase subunit, e.g., the
catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. In some
aspects, this disclosure provides a method of producing a
CAR-expressing cell, comprising contacting a cell with a nucleic
acid encoding a telomerase subunit, e.g., the catalytic subunit of
telomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with
the nucleic acid before, simultaneous with, or after being
contacted with a construct encoding a CAR.
[0503] In one aspect, the disclosure features a method of making a
population of immune effector cells (e.g., T cells or NK cells). In
an embodiment, the method comprises: providing a population of
immune effector cells (e.g., T cells or NK cells), contacting the
population of immune effector cells with a nucleic acid encoding a
CAR; and contacting the population of immune effector cells with a
nucleic acid encoding a telomerase subunit, e.g., hTERT, under
conditions that allow for CAR and telomerase expression.
[0504] In an embodiment, the nucleic acid encoding the telomerase
subunit is DNA. In an embodiment, the nucleic acid encoding the
telomerase subunit comprises a promoter capable of driving
expression of the telomerase subunit.
[0505] In an embodiment, hTERT has the amino acid sequence of
GenBank Protein ID AAC51724.1 (Meyerson et al., "hEST2, the
Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated
in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4,
22 Aug. 1997, Pages 785-795) as follows:
TABLE-US-00012 (SEQ ID NO: 131)
MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRAL
VAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFG
FALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLV
HLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCE
RAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTP
VGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVG
RQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSL
RPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNH
AQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQ
LLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKH
AKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMS
VYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRE
LSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKR
AERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQ
DPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKA
AHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNE
ASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDME
NKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNL
RKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYA
RTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTN
IYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAK
NAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQ
TQLSRKLPGTTLTALEAAANPALPSDFKTILD
[0506] In an embodiment, the hTERT has a sequence at least 80%,
85%, 90%, 95%, 96{circumflex over ( )}, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 131. In an embodiment, the hTERT has
a sequence of SEQ ID NO: 131. In an embodiment, the hTERT comprises
a deletion (e.g., of no more than 5, 10, 15, 20, or 30 amino acids)
at the N-terminus, the C-terminus, or both. In an embodiment, the
hTERT comprises a transgenic amino acid sequence (e.g., of no more
than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, the
C-terminus, or both.
[0507] In an embodiment, the hTERT is encoded by the nucleic acid
sequence of GenBank Accession No. AF018167 (Meyerson et al.,
"hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is
Up-Regulated in Tumor Cells and during Immortalization" Cell Volume
90, Issue 4, 22 Aug. 1997, Pages 785-795):
TABLE-US-00013 (SEQ ID NO: 132) 1 caggcagcgt ggtcctgctg cgcacgtggg
aagccctggc cccggccacc cccgcgatgc 61 cgcgcgctcc ccgctgccga
gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 121 tgccgctggc
cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 181
gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg
241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag
gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa
cgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc gggggccccc
ccgaggcctt caccaccagc gtgcgcagct 421 acctgcccaa cacggtgacc
gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 481 gccgcgtggg
cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 541
tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca
601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga
tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg
cctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc
tgccgttgcc caagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg
acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 841 cgcgtggacc
gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 901
ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc
961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac
acgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc
aggcgacaag gagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc
ccagcctgac tggcgctcgg aggctcgtgg 1141 agaccatctt tctgggttcc
aggccctgga tgccagggac tccccgcagg ttgccccgcc 1201 tgccccagcg
ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1261
agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag
1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc
gaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca
cagcagcccc tggcaggtgt 1441 acggcttcgt gcgggcctgc ctgcgccggc
tggtgccccc aggcctctgg ggctccaggc 1501 acaacgaacg ccgcttcctc
aggaacacca agaagttcat ctccctgggg aagcatgcca 1561 agctctcgct
gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1621
ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg
1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg
tctttctttt 1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt
ctaccggaag agtgtctgga 1801 gcaagttgca aagcattgga atcagacagc
acttgaagag ggtgcagctg cgggagctgt 1861 cggaagcaga ggtcaggcag
catcgggaag ccaggcccgc cctgctgacg tccagactcc 1921 gcttcatccc
caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1981
ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt
2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc
tctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct
gcgtgtgcgg gcccaggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg
atgtgacggg cgcgtacgac accatccccc 2221 aggacaggct cacggaggtc
atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 2281 gtcggtatgc
cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2341
acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg
2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg
aatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca
ccacgccgtg cgcatcaggg 2521 gcaagtccta cgtccagtgc caggggatcc
cgcagggctc catcctctcc acgctgctct 2581 gcagcctgtg ctacggcgac
atggagaaca agctgtttgc ggggattcgg cgggacgggc 2641 tgctcctgcg
tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2701
ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga
2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct
tttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct
ggatacccgg accctggagg 2881 tgcagagcga ctactccagc tatgcccgga
cctccatcag agccagtctc accttcaacc 2941 gcggcttcaa ggctgggagg
aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3001 gtcacagcct
gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3061
acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc
3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac
acggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc
gctgggggcc aagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt
ggctgtgcca ccaagcattc ctgctcaagc 3301 tgactcgaca ccgtgtcacc
tacgtgccac tcctggggtc actcaggaca gcccagacgc 3361 agctgagtcg
gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3421
cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg
3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg
gaggggcggc 3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag
tgagtgtttg gccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc
tgaggcctga gcgagtgtcc agccaagggc 3661 tgagtgtcca gcacacctgc
cgtcttcact tccccacagg ctggcgctcg gctccacccc 3721 agggccagct
tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3781
cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc
3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga
ccaaaggtgt 3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt
ccctgtgggt caaattgggg 3961 ggaggtgctg tgggagtaaa atactgaata
tatgagtttt tcagttttga aaaaaaaaaa 4021 aaaaaaa
[0508] In an embodiment, the hTERT is encoded by a nucleic acid
having a sequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 132. In an embodiment, the
hTERT is encoded by a nucleic acid of SEQ ID NO: 132.
Activation and Expansion of Immune Effector Cells (e.g., T
Cells)
[0509] Immune effector cells such as T cells may be activated and
expanded generally using methods as described, for example, in U.S.
Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;
6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;
7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S.
Patent Application Publication No. 20060121005.
[0510] The procedure for ex vivo expansion of hematopoietic stem
and progenitor cells is described in U.S. Pat. No. 5,199,942,
incorporated herein by reference, can be applied to the cells of
the present invention. Other suitable methods are known in the art,
therefore the present invention is not limited to any particular
method of ex vivo expansion of the cells. Briefly, ex vivo culture
and expansion of T cells can comprise: (1) collecting CD34+
hematopoietic stem and progenitor cells from a mammal from
peripheral blood harvest or bone marrow explants; and (2) expanding
such cells ex vivo. In addition to the cellular growth factors
described in U.S. Pat. No. 5,199,942, other factors such as flt3-L,
IL-1, IL-3 and c-kit ligand, can be used for culturing and
expansion of the cells.
[0511] Generally, a population of immune effector cells e.g., T
regulatory cell depleted cells, may be expanded by contact with a
surface having attached thereto an agent that stimulates a CD3/TCR
complex associated signal and a ligand that stimulates a
costimulatory molecule on the surface of the T cells. In
particular, T cell populations may be stimulated as described
herein, such as by contact with an anti-CD3 antibody, or
antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C
activator (e.g., bryostatin) in conjunction with a calcium
ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule
is used. For example, a population of T cells can be contacted with
an anti-CD3 antibody and an anti-CD28 antibody, under conditions
appropriate for stimulating proliferation of the T cells. To
stimulate proliferation of either CD4+ T cells or CD8+ T cells, an
anti-CD3 antibody and an anti-CD28 antibody can be used. Examples
of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,
Besancon, France) can be used as can other methods commonly known
in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998;
Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al.,
J. Immunol Meth. 227(1-2):53-63, 1999).
[0512] In certain aspects, the primary stimulatory signal and the
costimulatory signal for the T cell may be provided by different
protocols. For example, the agents providing each signal may be in
solution or coupled to a surface. When coupled to a surface, the
agents may be coupled to the same surface (i.e., in "cis"
formation) or to separate surfaces (i.e., in "trans" formation).
Alternatively, one agent may be coupled to a surface and the other
agent in solution. In one aspect, the agent providing the
costimulatory signal is bound to a cell surface and the agent
providing the primary activation signal is in solution or coupled
to a surface. In certain aspects, both agents can be in solution.
In one aspect, the agents may be in soluble form, and then
cross-linked to a surface, such as a cell expressing Fc receptors
or an antibody or other binding agent which will bind to the
agents. In this regard, see for example, U.S. Patent Application
Publication Nos. 20040101519 and 20060034810 for artificial antigen
presenting cells (aAPCs) that are contemplated for use in
activating and expanding T cells in the present invention.
[0513] In one aspect, the two agents are immobilized on beads,
either on the same bead, i.e., "cis," or to separate beads, i.e.,
"trans." By way of example, the agent providing the primary
activation signal is an anti-CD3 antibody or an antigen-binding
fragment thereof and the agent providing the costimulatory signal
is an anti-CD28 antibody or antigen-binding fragment thereof; and
both agents are co-immobilized to the same bead in equivalent
molecular amounts. In one aspect, a 1:1 ratio of each antibody
bound to the beads for CD4+ T cell expansion and T cell growth is
used. In certain aspects of the present invention, a ratio of anti
CD3:CD28 antibodies bound to the beads is used such that an
increase in T cell expansion is observed as compared to the
expansion observed using a ratio of 1:1. In one particular aspect
an increase of from about 1 to about 3 fold is observed as compared
to the expansion observed using a ratio of 1:1. In one aspect, the
ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to
1:100 and all integer values there between. In one aspect, more
anti-CD28 antibody is bound to the particles than anti-CD3
antibody, i.e., the ratio of CD3:CD28 is less than one. In certain
aspects, the ratio of anti CD28 antibody to anti CD3 antibody bound
to the beads is greater than 2:1. In one particular aspect, a 1:100
CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a
1:75 CD3:CD28 ratio of antibody bound to beads is used. In a
further aspect, a 1:50 CD3:CD28 ratio of antibody bound to beads is
used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to
beads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio of
antibody bound to beads is used. In one aspect, a 1:3 CD3:CD28
ratio of antibody bound to the beads is used. In yet one aspect, a
3:1 CD3:CD28 ratio of antibody bound to the beads is used.
[0514] Ratios of particles to cells from 1:500 to 500:1 and any
integer values in between may be used to stimulate T cells or other
target cells. As those of ordinary skill in the art can readily
appreciate, the ratio of particles to cells may depend on particle
size relative to the target cell. For example, small sized beads
could only bind a few cells, while larger beads could bind many. In
certain aspects the ratio of cells to particles ranges from 1:100
to 100:1 and any integer values in-between and in further aspects
the ratio comprises 1:9 to 9:1 and any integer values in between,
can also be used to stimulate T cells. The ratio of anti-CD3- and
anti-CD28-coupled particles to T cells that result in T cell
stimulation can vary as noted above, however certain preferred
values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7,
1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,
9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1
particles per T cell. In one aspect, a ratio of particles to cells
of 1:1 or less is used. In one particular aspect, a preferred
particle: cell ratio is 1:5. In further aspects, the ratio of
particles to cells can be varied depending on the day of
stimulation. For example, in one aspect, the ratio of particles to
cells is from 1:1 to 10:1 on the first day and additional particles
are added to the cells every day or every other day thereafter for
up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell
counts on the day of addition). In one particular aspect, the ratio
of particles to cells is 1:1 on the first day of stimulation and
adjusted to 1:5 on the third and fifth days of stimulation. In one
aspect, particles are added on a daily or every other day basis to
a final ratio of 1:1 on the first day, and 1:5 on the third and
fifth days of stimulation. In one aspect, the ratio of particles to
cells is 2:1 on the first day of stimulation and adjusted to 1:10
on the third and fifth days of stimulation. In one aspect,
particles are added on a daily or every other day basis to a final
ratio of 1:1 on the first day, and 1:10 on the third and fifth days
of stimulation. One of skill in the art will appreciate that a
variety of other ratios may be suitable for use in the present
invention. In particular, ratios will vary depending on particle
size and on cell size and type. In one aspect, the most typical
ratios for use are in the neighborhood of 1:1, 2:1 and 3:1 on the
first day.
[0515] In further aspects, the cells, such as T cells, are combined
with agent-coated beads, the beads and the cells are subsequently
separated, and then the cells are cultured. In an alternative
aspect, prior to culture, the agent-coated beads and cells are not
separated but are cultured together. In a further aspect, the beads
and cells are first concentrated by application of a force, such as
a magnetic force, resulting in increased ligation of cell surface
markers, thereby inducing cell stimulation.
[0516] By way of example, cell surface proteins may be ligated by
allowing paramagnetic beads to which anti-CD3 and anti-CD28 are
attached (3.times.28 beads) to contact the T cells. In one aspect
the cells (for example, 10.sup.4 to 10.sup.9 T cells) and beads
(for example, DYNABEADS.RTM. M-450 CD3/CD28 T paramagnetic beads at
a ratio of 1:1) are combined in a buffer, for example PBS (without
divalent cations such as, calcium and magnesium). Again, those of
ordinary skill in the art can readily appreciate any cell
concentration may be used. For example, the target cell may be very
rare in the sample and comprise only 0.01% of the sample or the
entire sample (i.e., 100%) may comprise the target cell of
interest. Accordingly, any cell number is within the context of the
present invention. In certain aspects, it may be desirable to
significantly decrease the volume in which particles and cells are
mixed together (i.e., increase the concentration of cells), to
ensure maximum contact of cells and particles. For example, in one
aspect, a concentration of about 10 billion cells/ml, 9 billion/ml,
8 billion/ml, 7 billion/ml, 6 billion/ml, 5 billion/ml, or 2
billion cells/ml is used. In one aspect, greater than 100 million
cells/ml is used. In a further aspect, a concentration of cells of
10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In
yet one aspect, a concentration of cells from 75, 80, 85, 90, 95,
or 100 million cells/ml is used. In further aspects, concentrations
of 125 or 150 million cells/ml can be used. Using high
concentrations can result in increased cell yield, cell activation,
and cell expansion. Further, use of high cell concentrations allows
more efficient capture of cells that may weakly express target
antigens of interest, such as CD28-negative T cells. Such
populations of cells may have therapeutic value and would be
desirable to obtain in certain aspects. For example, using high
concentration of cells allows more efficient selection of CD8+ T
cells that normally have weaker CD28 expression.
[0517] In one embodiment, cells transduced with a nucleic acid
encoding a CAR, e.g., a CAR described herein, are expanded, e.g.,
by a method described herein. In one embodiment, the cells are
expanded in culture for a period of several hours (e.g., about 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one
embodiment, the cells are expanded for a period of 4 to 9 days. In
one embodiment, the cells are expanded for a period of 8 days or
less, e.g., 7, 6 or 5 days. In one embodiment, the cells, e.g., a
CD19 CAR cell described herein, are expanded in culture for 5 days,
and the resulting cells are more potent than the same cells
expanded in culture for 9 days under the same culture conditions.
Potency can be defined, e.g., by various T cell functions, e.g.
proliferation, target cell killing, cytokine production,
activation, migration, or combinations thereof. In one embodiment,
the cells, e.g., a CD19 CAR cell described herein, expanded for 5
days show at least a one, two, three or four fold increase in cells
doublings upon antigen stimulation as compared to the same cells
expanded in culture for 9 days under the same culture conditions.
In one embodiment, the cells, e.g., the cells expressing a CD19 CAR
described herein, are expanded in culture for 5 days, and the
resulting cells exhibit higher proinflammatory cytokine production,
e.g., IFN-.gamma. and/or GM-CSF levels, as compared to the same
cells expanded in culture for 9 days under the same culture
conditions. In one embodiment, the cells, e.g., a CD19 CAR cell
described herein, expanded for 5 days show at least a one, two,
three, four, five, ten fold or more increase in pg/ml of
proinflammatory cytokine production, e.g., IFN-.gamma. and/or
GM-CSF levels, as compared to the same cells expanded in culture
for 9 days under the same culture conditions.
[0518] Several cycles of stimulation may also be desired such that
culture time of T cells can be 60 days or more. Conditions
appropriate for T cell culture include an appropriate media (e.g.,
Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza))
that may contain factors necessary for proliferation and viability,
including serum (e.g., fetal bovine or human serum), interleukin-2
(IL-2), insulin, IFN-.gamma., IL-4, IL-7, GM-CSF, IL-10, IL-12,
IL-15, TGF.beta., and TNF-.alpha. or any other additives for the
growth of cells known to the skilled artisan. Other additives for
the growth of cells include, but are not limited to, surfactant,
plasmanate, and reducing agents such as N-acetyl-cysteine and
2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,
.alpha.-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added
amino acids, sodium pyruvate, and vitamins, either serum-free or
supplemented with an appropriate amount of serum (or plasma) or a
defined set of hormones, and/or an amount of cytokine(s) sufficient
for the growth and expansion of T cells. Antibiotics, e.g.,
penicillin and streptomycin, are included only in experimental
cultures, not in cultures of cells that are to be infused into a
subject. The target cells are maintained under conditions necessary
to support growth, for example, an appropriate temperature (e.g.,
37.degree. C.) and atmosphere (e.g., air plus 5% CO.sub.2).
[0519] In one embodiment, the cells are expanded in an appropriate
media (e.g., media described herein) that includes one or more
interleukin that result in at least a 200-fold (e.g., 200-fold,
250-fold, 300-fold, 350-fold) increase in cells over a 14 day
expansion period, e.g., as measured by a method described herein
such as flow cytometry. In one embodiment, the cells are expanded
in the presence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7).
[0520] In embodiments, methods described herein, e.g.,
CAR-expressing cell manufacturing methods, comprise removing T
regulatory cells, e.g., CD25+ T cells, from a cell population,
e.g., using an anti-CD25 antibody, or fragment thereof, or a
CD25-binding ligand, IL-2. Methods of removing T regulatory cells,
e.g., CD25+ T cells, from a cell population are described herein.
In embodiments, the methods, e.g., manufacturing methods, further
comprise contacting a cell population (e.g., a cell population in
which T regulatory cells, such as CD25+ T cells, have been
depleted; or a cell population that has previously contacted an
anti-CD25 antibody, fragment thereof, or CD25-binding ligand) with
IL-15 and/or IL-7. For example, the cell population (e.g., that has
previously contacted an anti-CD25 antibody, fragment thereof, or
CD25-binding ligand) is expanded in the presence of IL-15 and/or
IL-7.
[0521] In some embodiments a CAR-expressing cell described herein
is contacted with a composition comprising a interleukin-15 (IL-15)
polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide,
or a combination of both a IL-15 polypeptide and a IL-15Ra
polypeptide e.g., hetIL-15, during the manufacturing of the
CAR-expressing cell, e.g., ex vivo. In embodiments, a
CAR-expressing cell described herein is contacted with a
composition comprising a IL-15 polypeptide during the manufacturing
of the CAR-expressing cell, e.g., ex vivo. In embodiments, a
CAR-expressing cell described herein is contacted with a
composition comprising a combination of both a IL-15 polypeptide
and a IL-15 Ra polypeptide during the manufacturing of the
CAR-expressing cell, e.g., ex vivo. In embodiments, a
CAR-expressing cell described herein is contacted with a
composition comprising hetIL-15 during the manufacturing of the
CAR-expressing cell, e.g., ex vivo.
[0522] In one embodiment the CAR-expressing cell described herein
is contacted with a composition comprising hetIL-15 during ex vivo
expansion. In an embodiment, the CAR-expressing cell described
herein is contacted with a composition comprising an IL-15
polypeptide during ex vivo expansion. In an embodiment, the
CAR-expressing cell described herein is contacted with a
composition comprising both an IL-15 polypeptide and an IL-15Ra
polypeptide during ex vivo expansion. In one embodiment the
contacting results in the survival and proliferation of a
lymphocyte subpopulation, e.g., CD8+ T cells.
[0523] T cells that have been exposed to varied stimulation times
may exhibit different characteristics. For example, typical blood
or apheresed peripheral blood mononuclear cell products have a
helper T cell population (TH, CD4+) that is greater than the
cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo
expansion of T cells by stimulating CD3 and CD28 receptors produces
a population of T cells that prior to about days 8-9 consists
predominately of TH cells, while after about days 8-9, the
population of T cells comprises an increasingly greater population
of TC cells. Accordingly, depending on the purpose of treatment,
infusing a subject with a T cell population comprising
predominately of TH cells may be advantageous. Similarly, if an
antigen-specific subset of TC cells has been isolated it may be
beneficial to expand this subset to a greater degree.
[0524] Further, in addition to CD4 and CD8 markers, other
phenotypic markers vary significantly, but in large part,
reproducibly during the course of the cell expansion process. Thus,
such reproducibility enables the ability to tailor an activated T
cell product for specific purposes.
[0525] In other embodiments, the method of making disclosed herein
further comprises contacting the population of immune effector
cells with a nucleic acid encoding a telomerase subunit, e.g.,
hTERT. The the nucleic acid encoding the telomerase subunit can be
DNA.
[0526] In some embodiments, a kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib) is added during the CAR cell
manufacturing process. According to the non-limiting theory herein,
the kinase inhibitor can improve the quality of the population of
cells produced. For instance, CAR-expressing cells are often
produced from a cancer patient's own plasma apheresis sample, which
can contain cancer cells, and the kinase inhibitor can alter
signalling in those cancer cells (e.g., a BTK-expressing cancer
such as CLL or MCL), e.g., reducing their proliferation or
increasing levels of apoptosis. As another example, the kinase
inhibitor may alter signalling in the CAR-expressing cells (or
immune effector cells before they express CAR), e.g., by inhibiting
ITK in T cells. The kinase inhibitor may shift the balance of T
cells from TH2 cells towards TH1 cells.
[0527] The kinase inhibitor (e.g., a BTK inhibitor such as
ibrutinib) can be added to the reaction mixture in a level
sufficient to inhibit its target, e.g., BTK. In some embodiments,
the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is
added at a concentration of about 0.1-0.2, 0.2-0.5, 0.5-1, 1-2,
2-5, or 5-10 .mu.M. In some embodiments, the kinase inhibitor is a
covalent inhibitor (such as ibrutinib) and a short pulse is
sufficient to irreversibly inactivate the target while avoiding
nonspecific toxicity. Consequently, the kinase inhibitor may be
added for, e.g., 10-20, 20-30, 30-40, 40-60, or 60-120 minutes. The
kinase inhibitor may also be added for longer periods of time, for
instance if the kinase inhibitor has a noncovalent mode of action.
Thus, the kinase inhibitor may be added for, e.g., 2-4, 4-6, 6-8,
8-12, 12-18, or 18-24 hours, or for 1-2, 2-3, 3-4, 4-6, 6-8, 8-10
days, or for the entire length of time the cells are being
cultured. The kinase inhibitor may be added at various points
during the manufacturing process, for example, after harvesting the
cells, before stimulating with beads, after stimulating with beads,
before transduction, after transduction, or before administration
of the cells to the patient. In some embodiments, the kinase
inhibitor (e.g., a BTK inhibitor such as ibrutinib) is added after
harvesting the cells or before stimulating, e.g., with beads.
Before and after, in this context, can refer to, e.g., about 1, 5,
15, 30, 45, or 60 minutes before or after, or 1, 2, 3, 4, 5, or 6
hours before or after.
[0528] Once a CD19 CAR is constructed, various assays can be used
to evaluate the activity of the molecule, such as but not limited
to, the ability to expand T cells following antigen stimulation,
sustain T cell expansion in the absence of re-stimulation, and
anti-cancer activities in appropriate in vitro and animal models.
Assays to evaluate the effects of a CD19 CAR are described in
further detail below
[0529] Western blot analysis of CAR expression in primary T cells
can be used to detect the presence of monomers and dimers. See,
e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
Very briefly, T cells (1:1 mixture of CD4.sup.+ and CD8.sup.+ T
cells) expressing the CARs are expanded in vitro for more than 10
days followed by lysis and SDS-PAGE under reducing conditions. CARs
containing the full length TCR-.zeta. cytoplasmic domain and the
endogenous TCR-.zeta. chain are detected by western blotting using
an antibody to the TCR-.zeta. chain. The same T cell subsets are
used for SDS-PAGE analysis under non-reducing conditions to permit
evaluation of covalent dimer formation.
[0530] In vitro expansion of CAR.sup.+ T cells following antigen
stimulation can be measured by flow cytometry. For example, a
mixture of CD4.sup.+ and CD8.sup.+ T cells are stimulated with
.alpha.CD3/.alpha.CD28 beads followed by transduction with
lentiviral vectors expressing GFP under the control of the
promoters to be analyzed. Exemplary promoters include the CMV IE
gene, EF-1.alpha., ubiquitin C, or phosphoglycerokinase (PGK)
promoters. GFP fluorescence is evaluated on day 6 of culture in the
CD4.sup.+ and/or CD8.sup.+ T cell subsets by flow cytometry. See,
e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
Alternatively, a mixture of CD4.sup.+ and CD8.sup.+ T cells are
stimulated with .alpha.CD3/.alpha.CD28 coated magnetic beads on day
0, and transduced with CAR on day 1 using a bicistronic lentiviral
vector expressing CAR along with eGFP using a 2A ribosomal skipping
sequence. Cultures are re-stimulated with either CD19.sup.+ K562
cells (K562-CD19), wild-type K562 cells (K562 wild type) or K562
cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and
anti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous
IL-2 is added to the cultures every other day at 100 IU/ml.
GFP.sup.+ T cells are enumerated by flow cytometry using bead-based
counting. See, e.g., Milone et al., Molecular Therapy 17(8):
1453-1464 (2009).
[0531] Sustained CAR.sup.+ T cell expansion in the absence of
re-stimulation can also be measured. See, e.g., Milone et al.,
Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell
volume (fl) is measured on day 8 of culture using a Coulter
Multisizer particle counter, a Nexcelom Cellometer Vision or
Millipore Scepter, following stimulation with
.alpha.CD3/.alpha.CD28 coated magnetic beads on day 0, and
transduction with the indicated CAR on day 1.
[0532] Animal models can also be used to measure a CART activity.
For example, xenograft model using human CD19-specific CAR.sup.+ T
cells to treat a primary human pre-B ALL in immunodeficient mice
can be used. See, e.g., Milone et al., Molecular Therapy 17(8):
1453-1464 (2009). Very briefly, after establishment of ALL, mice
are randomized as to treatment groups. Different numbers of
.alpha.CD19-.zeta. and .alpha.CD19-BB-.zeta. engineered T cells are
coinjected at a 1:1 ratio into NOD-SCID-.gamma..sup.-/- mice
bearing B-ALL. The number of copies of .alpha.CD19-.zeta. and
.alpha.CD19-BB-.zeta. vector in spleen DNA from mice is evaluated
at various times following T cell injection. Animals are assessed
for leukemia at weekly intervals. Peripheral blood CD19.sup.+ B-ALL
blast cell counts are measured in mice that are injected with
.alpha.CD19-.zeta. CAR.sup.+ T cells or mock-transduced T cells.
Survival curves for the groups are compared using the log-rank
test. In addition, absolute peripheral blood CD4.sup.+ and
CD8.sup.+ T cell counts 4 weeks following T cell injection in
NOD-SCID-.gamma..sup.-/- mice can also be analyzed. Mice are
injected with leukemic cells and 3 weeks later are injected with T
cells engineered to express CAR by a bicistronic lentiviral vector
that encodes the CAR linked to eGFP. T cells are normalized to
45-50% input GFP.sup.+ T cells by mixing with mock-transduced cells
prior to injection, and confirmed by flow cytometry. Animals are
assessed for leukemia at 1-week intervals. Survival curves for the
CAR.sup.+ T cell groups are compared using the log-rank test.
[0533] Dose dependent CAR treatment response can be evaluated. See,
e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For
example, peripheral blood is obtained 35-70 days after establishing
leukemia in mice injected on day 21 with CAR T cells, an equivalent
number of mock-transduced T cells, or no T cells. Mice from each
group are randomly bled for determination of peripheral blood
CD19.sup.+ ALL blast counts and then killed on days 35 and 49. The
remaining animals are evaluated on days 57 and 70.
[0534] Assessment of cell proliferation and cytokine production has
been previously described, e.g., at Milone et al., Molecular
Therapy 17(8): 1453-1464 (2009). Briefly, assessment of
CAR-mediated proliferation is performed in microtiter plates by
mixing washed T cells with K562 cells expressing CD19 (K19) or CD32
and CD137 (KT32-BBL) for a final T-cell:K562 ratio of 2:1. K562
cells are irradiated with gamma-radiation prior to use. Anti-CD3
(clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are
added to cultures with KT32-BBL cells to serve as a positive
control for stimulating T-cell proliferation since these signals
support long-term CD8.sup.+ T cell expansion ex vivo. T cells are
enumerated in cultures using CountBright.TM. fluorescent beads
(Invitrogen, Carlsbad, Calif.) and flow cytometry as described by
the manufacturer. CAR.sup.+ T cells are identified by GFP
expression using T cells that are engineered with eGFP-2A linked
CAR-expressing lentiviral vectors. For CAR+ T cells not expressing
GFP, the CAR+ T cells are detected with biotinylated recombinant
CD19 protein and a secondary avidin-PE conjugate. CD4+ and
CD8.sup.+ expression on T cells are also simultaneously detected
with specific monoclonal antibodies (BD Biosciences). Cytokine
measurements are performed on supernatants collected 24 hours
following re-stimulation using the human TH1/TH2 cytokine
cytometric bead array kit (BD Biosciences, San Diego, Calif.)
according the manufacturer's instructions. Fluorescence is assessed
using a FACScalibur flow cytometer, and data is analyzed according
to the manufacturer's instructions.
[0535] Cytotoxicity can be assessed by a standard .sup.51Cr-release
assay. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464
(2009). Briefly, target cells (K562 lines and primary pro-B-ALL
cells) are loaded with .sup.51Cr (as NaCrO.sub.4, New England
Nuclear, Boston, Mass.) at 37.degree. C. for 2 hours with frequent
agitation, washed twice in complete RPMI and plated into microtiter
plates. Effector T cells are mixed with target cells in the wells
in complete RPMI at varying ratios of effector cell:target cell
(E:T). Additional wells containing media only (spontaneous release,
SR) or a 1% solution of triton-X 100 detergent (total release, TR)
are also prepared. After 4 hours of incubation at 37.degree. C.,
supernatant from each well is harvested. Released 51Cr is then
measured using a gamma particle counter (Packard Instrument Co.,
Waltham, Mass.). Each condition is performed in at least
triplicate, and the percentage of lysis is calculated using the
formula: % Lysis=(ER-SR)/(TR-SR), where ER represents the average
51Cr released for each experimental condition.
[0536] Imaging technologies can be used to evaluate specific
trafficking and proliferation of CARs in tumor-bearing animal
models. Such assays have been described, for example, in Barrett et
al., Human Gene Therapy 22:1575-1586 (2011). Briefly,
NOD/SCID/.gamma.c.sup.-/- (NSG) mice are injected IV with Nalm-6
cells followed 7 days later with T cells 4 hour after
electroporation with the CAR constructs. The T cells are stably
transfected with a lentiviral construct to express firefly
luciferase, and mice are imaged for bioluminescence. Alternatively,
therapeutic efficacy and specificity of a single injection of
CAR.sup.+ T cells in Nalm-6 xenograft model can be measured as the
following: NSG mice are injected with Nalm-6 transduced to stably
express firefly luciferase, followed by a single tail-vein
injection of T cells electroporated with CD19 CAR 7 days later.
Animals are imaged at various time points post injection. For
example, photon-density heat maps of firefly luciferasepositive
leukemia in representative mice at day 5 (2 days before treatment)
and day 8 (24 hr post CAR.sup.+ PBLs) can be generated.
[0537] Other assays, including those described in the Example
section herein as well as those that are known in the art can also
be used to evaluate the CD19 CAR constructs of the invention.
Kinase Inhibitor
[0538] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., a CDK4 inhibitor described herein, e.g., a CDK4/6 inhibitor,
such as, e.g.,
6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylami-
no)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred
to as palbociclib or PD0332991). In one embodiment, the kinase
inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described
herein, such as, e.g., ibrutinib. In one embodiment, the kinase
inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described
herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The
mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2
inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor
described herein. In one embodiment, the kinase inhibitor is a MNK
inhibitor, e.g., a MNK inhibitor described herein, such as, e.g.,
4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d]pyrimidine. The MNK
inhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b
inhibitor.
[0539] In one embodiment, the kinase inhibitor is a CDK4 inhibitor
selected from aloisine A; flavopiridol or HMR-1275,
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4-chromenone; crizotinib (PF-02341066;
2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3--
pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00);
1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N--
[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265);
indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991);
dinaciclib (SCH727965);
N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-car-
boxamide (BMS 387032);
4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]-
amino]-benzoic acid (MLN8054);
5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methy-
l-3-pyridinemethanamine (AG-024322);
4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid
N-(piperidin-4-yl)amide (AT7519);
4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phen-
yl]-2-pyrimidinamine (AZD5438); and XL281 (BMS908662).
[0540] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., palbociclib (PD0332991), and the palbociclib is administered
at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100
mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g.,
75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily
for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21
day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
or more cycles of palbociclib are administered.
[0541] An exemplary CDK4/6 inhibitor is LEE011 (also called
ribociclib), the structure of which is shown below.
##STR00001##
Without being bound by theory, it is believed that administration
of a CAR-expressing cell described herein with a CDK4/6 inhibitor
(e.g., LEE011 or other CDK4/6 inhibitor described herein) can
achieve higher responsiveness, e.g., with higher remission rates
and/or lower relapse rates, e.g., compared to a CDK4/6 inhibitor
alone.
[0542] While not wishing to be bound by theory, in some embodiments
the CDK4/6 inhibitor acts to reduce cyclin D1 activity in cancer
cells, e.g., MCL cells. Some cancer cells are characterized by
elevated cyclin D1 levels due to a translocation. CDK4 complexes
with cyclin D to promote cell cycle progression. Accordingly, in
some embodiments, administration of the CK4/6 inhibitor reduces
cancer cell proliferation. See, e.g., Marzec et al., "Mantle cell
lymphoma cells express predominantly cyclin D1a isoform and are
highly sensitive to selective inhibition of CDK4 kinase activity."
Blood. 2006 Sep. 1;108(5):1744-50. Epub 2006 May 11.
[0543] In one embodiment, the kinase inhibitor is a BTK inhibitor
selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560;
CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In an
embodiment, the BTK inhibitor does not reduce or inhibit the kinase
activity of interleukin-2-inducible kinase (ITK), and is selected
from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292;
ONO-4059; CNX-774; and LFM-A13.
[0544] In one embodiment, the kinase inhibitor is a BTK inhibitor,
e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a
dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460
mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g.,
250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily
for 21 day cycle cycle, or daily for 28 day cycle. In one
embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of
ibrutinib are administered.
[0545] In embodiments, the BTK inhibitor (e.g., ibrutinib) is
administered to a subject that has CLL, mantle cell lymphoma (MCL),
or small lymphocytic lymphoma (SLL). For example, the subject to
whom the BTK inhibitor is administered has a deletion in the short
arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In other
examples, the subject to whom the BTK inhibitor is administered
does not have a del(17p). In embodiments, the subject to whom the
BTK inhibitor is administered has relapsed CLL or SLL, e.g., the
subject has previously been administered a cancer therapy (e.g.,
previously been administered one, two, three, or four prior cancer
therapies). In embodiments, the subject to whom the BTK inhibitor
is administered has refractory CLL or SLL. In other embodiments,
the subject to whom the BTK inhibitor is administered has
follicular lymphoma, e.g., relapse or refractory follicular
lymphoma.
[0546] The structure of ibrutinib
(1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-
piperidin-1-yl]prop-2-en-1-one) is shown below.
##STR00002##
[0547] Concentrations of ibrutinib exceeding approximately 400 nM
are not clinically relevant. In the study by Advani et al (J Clin
Oncol 2012; 31:88), the mean peak serum concentration of ibrutinib
was about 130 ng/ml, which is equivalent to 295 nM (based on the
molecular weight of ibrutinib of 440.5). Therefore, data showing
the effect of ibrutinib on T cells at a concentration of 1 uM
significantly exceeds the clinically relevant concentration in
vivo.
[0548] In one embodiment, the kinase inhibitor is an mTOR inhibitor
selected from temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2
[(1R,9S,12S,15R,16E, 18R,
19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17-
,21,23,
29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[3-
0.3.1.0.sup.4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxyc-
yclohexyl dimethylphosphinate, also known as AP23573 and MK8669;
everolimus (RAD001); rapamycin (AY22989); simapimod;
(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-me-
thoxyphenyl)methanol (AZD8055);
2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-
-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and
N.sup.2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholiniu-
m-4-yl]methoxy]butyl]-L-arginylglycyl-L-.alpha.-aspartylL-serine-,
inner salt (SF1126); and XL765.
[0549] In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., rapamycin, and the rapamycin is administered at a
dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg
(e.g., 6 mg) daily for a period of time, e.g., daily for 21 day
cycle cycle, or daily for 28 day cycle. In one embodiment, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are
administered. In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., everolimus and the everolimus is administered at a
dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9
mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily
for a period of time, e.g., daily for 28 day cycle. In one
embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of
everolimus are administered.
[0550] In one embodiment, the kinase inhibitor is an MNK inhibitor
selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo
[3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and
4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d]pyrimidine.
[0551] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with a phosphoinositide 3-kinase (PI3K) inhibitor
(e.g., a PI3K inhibitor described herein, e.g., idelalisib or
duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with
idelalisib and rituximab. In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with
duvelisib and rituximab. Idelalisib (also called GS-1101 or
CAL-101; Gilead) is a small molecule that blocks the delta isoform
of PI3K. The structure of idelalisib
(5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolin-
one) is shown below.
##STR00003##
Duvelisib is a small molecule that blocks PI3K-.delta.,.gamma.. The
structure of duvelisib
(8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolin-
one) is shown below.
##STR00004##
In embodiments, the subject has CLL. In embodiments, the subject
has relapsed CLL, e.g., the subject has previously been
administered a cancer therapy (e.g., previously been administered
an anti-CD20 antibody or previously been administered ibrutinib).
For example, the subject has a deletion in the short arm of
chromosome 17 (del(17p), e.g., in a leukemic cell). In other
examples, the subject does not have a del(17p). In embodiments, the
subject comprises a leukemic cell comprising a mutation in the
immunoglobulin heavy-chain variable-region (IgV.sub.H) gene. In
other embodiments, the subject does not comprise a leukemic cell
comprising a mutation in the immunoglobulin heavy-chain
variable-region (IgV.sub.H) gene. In embodiments, the subject has a
deletion in the long arm of chromosome 11 (del(11q)). In other
embodiments, the subject does not have a del(11q). In embodiments,
idelalisib is administered at a dosage of about 100-400 mg (e.g.,
100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275,
275-300, 325-350, 350-375, or 375-400 mg), e.g., BID. In
embodiments, duvelisib is administered at a dosage of about 15-100
mg (e.g., about 15-25, 25-50, 50-75, or 75-100 mg), e.g., twice a
day. In embodiments, rituximab is administered at a dosage of about
350-550 mg/m.sup.2 (e.g., 350-375, 375-400, 400-425, 425-450,
450-475, or 475-500 mg/m.sup.2), e.g., intravenously.
[0552] In one embodiment, the kinase inhibitor is a dual
phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected
from
2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-
-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502);
N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-m-
orpholinyl-1,3,5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587);
2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,-
5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib
(GDC-0980, RG7422);
2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
-3-pyridinyl}benzenesulfonamide (GSK2126458);
8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluorometh-
yl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one Maleic acid
(NVP-BGT226);
3-[4-(4-Morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol
(PI-103);
5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2--
amine (VS-5584, SB2343); and
N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyp-
henyl)carbonyl]aminophenylsulfonamide (XL765).
[0553] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with an anaplastic lymphoma kinase (ALK) inhibitor.
Exemplary ALK kinase inhibitors include but are not limited to
crizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai),
brigatinib (also called AP26113; Ariad), entrectinib (Ignyta),
PF-06463922 (Pfizer), TSR-O11 (Tesaro) (see, e.g., Clinical Trial
Identifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery).
In some embodiments, the subject has a solid cancer, e.g., a solid
cancer described herein, e.g., lung cancer.
[0554] The chemical name of crizotinib is
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-
-4-yl)pyridin-2-amine. The chemical name of ceritinib is
5-Chloro-N.sup.2-[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N.sup.4--
[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical
name of alectinib is
9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5-
H-benzo[b]carbazole-3-carbonitrile. The chemical name of brigatinib
is
5-Chloro-N.sup.2-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N.-
sup.4-[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine. The
chemical name of entrectinib is
N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-(-
(tetrahydro-2H-pyran-4-yl)amino)benzamide. The chemical name of
PF-06463922 is
(10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2-
H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carb-
onitrile. The chemical structure of CEP-37440 is
(S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8-
,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methyl-
benzamide. The chemical name of X-396 is
(R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiper-
azine-1-carbonyl)phenyl)pyridazine-3-carboxamide.
[0555] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with an indoleamine 2,3-dioxygenase (IDO) inhibitor.
IDO is an enzyme that catalyzes the degradation of the amino acid,
L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g.,
prostatic, colorectal, pancreatic, cervical, gastric, ovarian,
head, and lung cancer. pDCs, macrophages, and dendritic cells (DCs)
can express IDO. Without being bound by theory, it is thought that
a decrease in L-tryptophan (e.g., catalyzed by IDO) results in an
immunosuppressive milieu by inducing T-cell anergy and apoptosis.
Thus, without being bound by theory, it is thought that an IDO
inhibitor can enhance the efficacy of a CAR-expressing cell
described herein, e.g., by decreasing the suppression or death of a
CAR-expressing immune cell. In embodiments, the subject has a solid
tumor, e.g., a solid tumor described herein, e.g., prostatic,
colorectal, pancreatic, cervical, gastric, ovarian, head, or lung
cancer. Exemplary inhibitors of IDO include but are not limited to
1-methyl-tryptophan, indoximod (NewLink Genetics) (see, e.g.,
Clinical Trial Identifier Nos. NCT01191216; NCT01792050), and
INCB024360 (Incyte Corp.) (see, e.g., Clinical Trial Identifier
Nos. NCT01604889; NCT01685255)
[0556] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with a modulator of myeloid-derived suppressor cells
(MDSCs). MDSCs accumulate in the periphery and at the tumor site of
many solid tumors. These cells suppress T cell responses, thereby
hindering the efficacy of CAR-expressing cell therapy. Without
being bound by theory, it is thought that administration of a MDSC
modulator enhances the efficacy of a CAR-expressing cell described
herein. In an embodiment, the subject has a solid tumor, e.g., a
solid tumor described herein, e.g., glioblastoma. Exemplary
modulators of MDSCs include but are not limited to MCS110 and
BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage
colony-stimulating factor (M-CSF). See, e.g., Clinical Trial
Identifier No. NCT00757757. BLZ945 is a small molecule inhibitor of
colony stimulating factor 1 receptor (CSF1R). See, e.g., Pyonteck
et al. Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown
below.
##STR00005##
[0557] In some embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with a interleukin-15 (IL-15) polypeptide, a
interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a
combination of both a IL-15 polypeptide and a IL-15Ra polypeptide
e.g., hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a
heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15
is described in, e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598,
U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311,
incorporated herein by reference. In embodiments, het-IL-15 is
administered subcutaneously. In embodiments, the subject has a
cancer, e.g., solid cancer, e.g., melanoma or colon cancer. In
embodiments, the subject has a metastatic cancer.
Therapeutic Application
[0558] CD19 Associated Diseases and/or Disorders
[0559] In one aspect, the invention provides methods for treating a
disease associated with CD19 expression. In one aspect, the
invention provides methods for treating a disease wherein part of
the tumor is negative for CD19 and part of the tumor is positive
for CD19. For example, the CAR of the invention is useful for
treating subjects that have undergone treatment for a disease
associated with elevated expression of CD19, wherein the subject
that has undergone treatment for elevated levels of CD19 exhibits a
disease associated with elevated levels of CD19.
[0560] The therapies described herein can be used to treat, e.g.,
subjects who respond to a kinase inhibitor such as ibrutinib (e.g.,
partial response or complete response) or subjects who do not
(e.g., non-responders or relapsers). Without wishing to be bound by
theory, a number of patients undergoing treatment with kinase
inhibitors (e.g., BTK inhibitors such as ibrutinib) may show a
reduced response to the treatment (e.g., are partial or
non-responders to the treatment, or relapse during treatment).
According, administration of the CAR-therapies disclosed herein, in
combination with the kinase inhibitors can result in beneficial
effects.
[0561] Exemplary therapeutic regimens for these subjects are
described below.
[0562] In some cases, when the subject is a non-responder or
relapser to a kinase inhibitor (e.g. a BTK inhibitor such as
ibrutinib), the kinase inhibitor is withdrawn and CAR therapy is
administered. In other cases, when the subjects does not respond to
a kinase inhibitor (e.g. a BTK inhibitor such as ibrutinib), the
kinase inhibitor therapy is continued and CAR therapy is added to
the regimen. This use is supported, e.g., by experiments in Example
8 herein which indicate that CAR therapy is effective as a
monotherapy in ibrutinib-resistant cells. Without wishing to be
bound by theory, continuing kinase inhibitor therapy can improve
the efficacy of the CAR therapy, e.g., by increasing the number of
CAR-expressing cells in the bloodstream (see Example 8 herein).
[0563] Without being bound by theory, a subject who is a
non-responder or relapser to a kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib) can be non-responsive for at least two
reasons: the subjects may have a mutation in the drug target (e.g.,
BTK, e.g., a C481S mutation) that prevents target inhibition, or
can have alterations in other pathways that can drive proliferation
even when the target is adequately inhibited (e.g., a mutation in
PLC.gamma., such as an activating mutation in PLC.gamma. resulting
in constitutive BTK-independent cell signaling). The treatment can
be altered depending on the reason for non-responsiveness. For
instance, in the first situation (in some embodiments), if the
subjects has (or is identified as having) a mutation that prevents
the kinase inhibitor from inhibiting its target, a second kinase
inhibitor (e.g., directed against the same target) can be
substituted for (or administered in combination with) the kinase
inhibitor. More specifically, in some embodiments where the patient
has (or is identified as having) a mutation that prevents ibrutinib
from inhibiting BTK, a second BTK inhibitor, e.g., a BTK inhibitor
described herein such as GDC-0834, RN-486, CGI-560, CGI-1764,
HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13) can be substituted
for ibrutinib. Without wishing to be bound by theory, the second
kinase inhibitor may act on a region of the target that is not
disrupted by the mutation, and therefore the subject is sensitive
to the second kinase inhibitor. In other embodiments, the original
kinase inhibitor (e.g. a BTK inhibitor such as ibrutinib) is
maintained. According to the non-limiting theory here, the original
kinase inhibitor may have useful activity on the CAR-expressing
cells, e.g., promoting a TH1 phenotype, promoting proliferation, or
otherwise increasing levels or activity of the cells.
[0564] As noted above, in some cases a subject is non-responsive
because the subject has an alteration (e.g., a mutation) in another
pathway that can drive proliferation even when the target is
adequately inhibited. Accordingly, if the subject has (or is
identified has having) an alteration in a pathway that makes the
kinase inhibitor's activity ineffectual, the kinase inhibitor
therapy can be maintained. Without wishing to be bound by theory,
the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib)
activity can promote useful biological changes in the cancer cells
even if the kinase inhibitor alone is not sufficient to slow
proliferation. For instance, the kinase inhibitor can be sufficient
to mobilize cancer cells out of the lymph nodes, making them more
vulnerable to the CAR therapy.
[0565] Turning now to subjects who respond to a kinase inhibitor
(e.g., a BTK inhibitor such as ibrutinib), various therapeutic
regimens are now described. In some embodiments, when a subject is
(or is identified as being) a complete responder to the kinase
inhibitor, the subject is not administered a CAR therapy during the
period of complete response. In other embodiments, when a subject
is (or is identified as being) a complete responder to the kinase
inhibitor, the subject is administered a CAR therapy during the
period of complete response. In an embodiment, after the CAR
therapy, the subject experiences a prolonged response or delayed
relapse (e.g., compared to the expected course of disease when
treated without CAR therapy). For instance, MCL treated with
ibrutinib monotherapy has a median duration of response of about
17.5 months.
[0566] In some embodiments, when a subject is (or is identified as
being) a partial responder to the kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib), the subject is not administered a CAR
therapy during the period of partial response. In other
embodiments, when a subject is (or is identified as being) a
partial responder to the kinase inhibitor, the subject is
administered a CAR therapy during the period of partial response.
In an embodiment, after the CAR therapy, the subject experiences a
complete response and/or prolonged response or delayed relapse
(e.g., compared to the expected course of disease when treated
without CAR therapy).
[0567] In some embodiments, when a subject has (or is identified as
having) stable disease after the beginning of treatment with the
kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the
subject is not administered a CAR therapy during the period of
stable disease. In other embodiments, when a subject has (or is
identified as having) stable disease after the beginning of
treatment with the kinase inhibitor, the subject is administered a
CAR therapy during the period of stable disease. In an embodiment,
after the CAR therapy, the subject experiences a partial response,
a complete response and/or prolonged response or delayed relapse
(e.g., compared to the expected course of disease when treated
without CAR therapy).
[0568] In some embodiments, when a subject has (or is identified as
having) progressive disease after the beginning of treatment with
the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the
subject is not administered a CAR therapy during the period of
progressive disease. In other embodiments, when a subject has (or
is identified as having) progressive disease after the beginning of
treatment with the kinase inhibitor, the subject is administered a
CAR therapy during the period of progressive disease. In an
embodiment, after the CAR therapy, the subject experiences stable
disease, a partial response, a complete response and/or prolonged
response or delayed relapse (e.g., compared to the expected course
of disease when treated without CAR therapy).
[0569] Thus, one or more disease assessment steps can be performed
before or during treatment, to determine which course of treatment
is suitable for a given patient. For instance, a subject can be
administered a kinase inhibitor (e.g., a BTK inhibitor such as
ibrutinib) as a first line therapy. Then, after a period of time
(e.g., 1 or 2 months but also 2 weeks, 3 weeks, 1 month, 1.5
months, 2 months, 3 months, 4 months, 6 months, 9 months, 12
months, 15 months, or 18 months) the patient's response can be
assessed. If the assessment shows that the subject is a complete
responder, in some embodiments CAR therapy is not administered,
e.g., as described above. If the assessment shows that the subject
is a partial responder or has stable disease, in some embodiments
CAR therapy is administered in combination with the kinase
inhibitor e.g., as described above. If the assessment shows that
the subject is a non-responder or relapser, in some embodiments CAR
therapy is administered in combination with the kinase inhibitor or
a second kinase inhibitor, e.g., as described above. In some
embodiments, the kinase inhibitor controls the disease while a
CAR-expressing cell is being manufactured, e.g., while the
patient's own T cells are being engineered to express a CAR and/or
other factors.
[0570] Clinical standards for classifying a patient's responder
status or relapser status are known in the art. As an example, for
malignant lymphoma, standardized response criteria are described in
Cheson et al, J Clin Oncol 17:1244 (1999) and Cheson et al.,
"Revised Response Criteria for Malignant Lymphoma", J Clin Oncol
25:579-586 (2007) (both of which are incorporated by reference
herein in their entireties). Accordingly, in some embodiments, a
subject is considered a complete responder, partial responder,
having stable disease, a non-responder, or a relapser according to
Cheson criteria or modified Cheson criteria. Criteria for
classifying other hematological malignancies are known in the
art.
[0571] According to the criteria in Table 2 of Cheson 2007, a
complete responder has disappearance of all evidence of disease; a
partial responder has regression of measurable disease and no new
sites; a patient with stable disease has a failure to attain CR/PR
or PD; and a patient with relapsed disease or progressive disease
has any new lesion or increase by greater than or equal to 50% of
previously involved sites from nadir. The assessment can involve a
determination of whether the disease is FDG-avid, PET positive or
negative, whether nodules are present e.g., palpable in the liver
or spleen, and whether bone marrow is cleared or shows
involvement.
[0572] The CAR therapy and the kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib) can be administered, e.g.,
simultaneously or sequentially. In some embodiments, the CAR
therapy is begun at substantially the same time as kinase inhibitor
therapy begins. In some embodiments, the CAR therapy is begun
before the kinase inhibitor therapy begins. In some embodiments,
the CAR therapy is begun after the kinase inhibitor therapy begins.
For instance, the CAR therapy can be begun, e.g., at least 1, 2, 3,
or 4 weeks, or 1, 2, 3, 4, 6, 9, 12, 15, 18, or 24 months after the
kinase inhibitor therapy begins. In some embodiments, the CAR
therapy is begun while a patient has physiologically relevant
levels of the kinase inhibitor in their body.
[0573] When administered in combination, the CAR therapy and the
kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or
both, can be administered in an amount or dose that is higher,
lower or the same than the amount or dosage of each agent used
individually, e.g., as a monotherapy. In certain embodiments, the
administered amount or dosage of the CAR therapy, the kinase
inhibitor, or both, is lower (e.g., at least 20%, at least 30%, at
least 40%, or at least 50%) than the amount or dosage of each agent
used individually, e.g., as a monotherapy. In other embodiments,
the amount or dosage of the CAR therapy, the kinase inhibitor, or
both, that results in a desired effect (e.g., treatment of cancer)
is lower (e.g., at least 20%, at least 30%, at least 40%, or at
least 50% lower) than the amount or dosage of each agent used
individually, e.g., as a monotherapy, required to achieve the same
therapeutic effect.
[0574] When administered in combination, the CAR therapy and the
kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or
both, can be administered with a duration that is longer, shorter,
or the same than the duration of each agent used individually,
e.g., as a monotherapy. In certain embodiments, the duration of
administration of the CAR therapy, the kinase inhibitor, or both,
is shorter (e.g., at least 20%, at least 30%, at least 40%, or at
least 50%) than the duration of each agent used individually, e.g.,
as a monotherapy. In other embodiments, the duration of
administration of the CAR therapy, the kinase inhibitor, or both,
that results in a desired effect (e.g., treatment of cancer) is
shorter (e.g., at least 20%, at least 30%, at least 40%, or at
least 50% shorter) than the duration of each agent used
individually, e.g., as a monotherapy, required to achieve the same
therapeutic effect. In some embodiment, the patient is administered
an abbreviated course of the kinase inhibitor (e.g., a BTK
inhibitor such as ibrutinib). For instance, the abbreviated course
of the kinase inhibitor may last about 0-2, 2-4, 4-6, 6-8, 8-10,
10-12, 12-15, 15-18, 18-21, or 21-24 months total or may last about
0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-15, 15-18, 18-21, or 21-24
months after administration of the CAR therapy. In embodiments, the
abbreviated course of the kinase inhibitor ends before relapse. In
embodiments, the kinase inhibitor is administered at normal (e.g.,
monotherapy) levels during the abbreviated course.
[0575] In embodiments, a single dose of CAR-expressing cells
comprises about 5.times.10.sup.8 CD19 CART cells. A dose of
CAR-expressing cells may also comprise about 5.times.10.sup.6,
1.times.10.sup.7, 2.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 2.times.10.sup.8, 5.times.10.sup.8,
1.times.10.sup.9, 2.times.10.sup.9, or 5.times.10.sup.9 cells,
e.g., CD19 CAR cells, e.g., CD19 CART cells.
[0576] In one aspect, the invention pertains to a vector comprising
CD19 CAR operably linked to promoter for expression in mammalian
cells, e.g., T cells. In one aspect, the invention provides a
recombinant cell, e.g., a T cell, expressing the CD19 CAR for use
in treating CD19-expressing tumors, wherein the recombinant T cell
expressing the CD19 CAR is termed a CD19 CART. In one aspect, the
CD19 CART described herein, is capable of contacting a tumor cell
with at least one CD19 CAR expressed on its surface such that the
CART targets the tumor cell and growth of the tumor is
inhibited.
[0577] In one aspect, the invention pertains to a method of
inhibiting growth of a CD19-expressing tumor cell, comprising
contacting the tumor cell with a CD19 CAR expressing cell, e.g., a
CD19 CART cell, described herein such that the CART is activated in
response to the antigen and targets the cancer cell, wherein the
growth of the tumor is inhibited. The CD19 CAR-expressing cell,
e.g., T cell, is administered in combination with a kinase
inhibitor, e.g., a kinase inhibitor described herein.
[0578] Administered "in combination", as used herein, means that
two (or more) different treatments are delivered to the subject
during the course of the subject's affliction with the disorder,
e.g., the two or more treatments are delivered after the subject
has been diagnosed with the disorder and before the disorder has
been cured or eliminated or treatment has ceased for other reasons.
In some embodiments, the delivery of one treatment is still
occurring when the delivery of the second begins, so that there is
overlap in terms of administration. This is sometimes referred to
herein as "simultaneous" or "concurrent delivery". In other
embodiments, the delivery of one treatment ends before the delivery
of the other treatment begins. In some embodiments of either case,
the treatment is more effective because of combined administration.
For example, the second treatment is more effective, e.g., an
equivalent effect is seen with less of the second treatment, or the
second treatment reduces symptoms to a greater extent, than would
be seen if the second treatment were administered in the absence of
the first treatment, or the analogous situation is seen with the
first treatment. In some embodiments, delivery is such that the
reduction in a symptom, or other parameter related to the disorder
is greater than what would be observed with one treatment delivered
in the absence of the other. The effect of the two treatments can
be partially additive, wholly additive, or greater than additive.
The delivery can be such that an effect of the first treatment
delivered is still detectable when the second is delivered. In one
embodiment, the CAR-expressing cell is administered at a dose
and/or dosing schedule described herein, and the kinase inhibitor
or agent that enhances the activity of the CAR-expressing cell is
administered at a dose and/or dosing schedule described herein.
[0579] The invention includes a type of cellular therapy where T
cells are genetically modified to express a chimeric antigen
receptor (CAR) and the CAR T cell is infused to a recipient in need
thereof. The infused cell is able to kill tumor cells in the
recipient. Unlike antibody therapies, CAR-modified T cells are able
to replicate in vivo resulting in long-term persistence that can
lead to sustained tumor control. In various aspects, the T cells
administered to the patient, or their progeny, persist in the
patient for at least four months, five months, six months, seven
months, eight months, nine months, ten months, eleven months,
twelve months, thirteen months, fourteen month, fifteen months,
sixteen months, seventeen months, eighteen months, nineteen months,
twenty months, twenty-one months, twenty-two months, twenty-three
months, two years, three years, four years, or five years after
administration of the T cell to the patient.
[0580] The invention also includes a type of cellular therapy where
T cells are modified, e.g., by in vitro transcribed RNA, to
transiently express a chimeric antigen receptor (CAR) and the CAR T
cell is infused to a recipient in need thereof. The infused cell is
able to kill tumor cells in the recipient. Thus, in various
aspects, the T cells administered to the patient, is present for
less than one month, e.g., three weeks, two weeks, one week, after
administration of the T cell to the patient.
[0581] Without wishing to be bound by any particular theory, the
anti-tumor immunity response elicited by the CAR-modified T cells
may be an active or a passive immune response, or alternatively may
be due to a direct vs indirect immune response. In one aspect, the
CAR transduced T cells exhibit specific proinflammatory cytokine
secretion and potent cytolytic activity in response to human cancer
cells expressing the CD19, resist soluble CD19 inhibition, mediate
bystander killing and mediate regression of an established human
tumor. For example, antigen-less tumor cells within a heterogeneous
field of CD19-expressing tumor may be susceptible to indirect
destruction by CD19-redirected T cells that has previously reacted
against adjacent antigen-positive cancer cells.
[0582] In one aspect, the fully-human CAR-modified T cells of the
invention may be a type of vaccine for ex vivo immunization and/or
in vivo therapy in a mammal. In one aspect, the mammal is a
human.
[0583] With respect to ex vivo immunization, at least one of the
following occurs in vitro prior to administering the cell into a
mammal: i) expansion of the cells, ii) introducing a nucleic acid
encoding a CAR to the cells or iii) cryopreservation of the
cells.
[0584] Ex vivo procedures are well known in the art and are
discussed more fully below. Briefly, cells are isolated from a
mammal (e.g., a human) and genetically modified (i.e., transduced
or transfected in vitro) with a vector expressing a CAR disclosed
herein. The CAR-modified cell can be administered to a mammalian
recipient to provide a therapeutic benefit. The mammalian recipient
may be a human and the CAR-modified cell can be autologous with
respect to the recipient. Alternatively, the cells can be
allogeneic, syngeneic or xenogeneic with respect to the recipient.
In addition to using a cell-based vaccine in terms of ex vivo
immunization, also included in the methods described herein are
compositions and methods for in vivo immunization to elicit an
immune response directed against an antigen in a patient.
[0585] Generally, the cells activated and expanded as described
herein may be utilized in the treatment and prevention of diseases
that arise in individuals who are immunocompromised. In particular,
the CAR-expressing cells described herein are used in the treatment
of diseases, disorders and conditions associated with expression of
CD19. In certain aspects, the cells are used in the treatment of
patients at risk for developing diseases, disorders and conditions
associated with expression of CD19. Thus, the present invention
provides methods for the treatment or prevention of diseases,
disorders and conditions associated with expression of CD19
comprising administering to a subject in need thereof, a
therapeutically effective amount of the CAR-expressing cells
described herein, in combination with a kinase inhibitor, e.g., a
kinase inhibitor described herein.
[0586] The present invention also provides methods for inhibiting
the proliferation or reducing a CD19-expressing cell population,
the methods comprising contacting a population of cells comprising
a CD19-expressing cell with an anti-CD19 CAR-expressing cell
described herein that binds to the CD19-expressing cell, and
contacting the population of CD19-expressing cells with a kinase
inhibitor, e.g., a kinase inhibitor described herein. In a specific
aspect, the present invention provides methods for inhibiting the
proliferation or reducing the population of cancer cells expressing
CD19, the methods comprising contacting the CD19-expressing cancer
cell population with an anti-CD19 CAR-expressing cell described
herein that binds to the CD19-expressing cell, and contacting the
CD19-expressing cell with a kinase inhibitor, e.g., a kinase
inhibitor described herein. In one aspect, the present invention
provides methods for inhibiting the proliferation or reducing the
population of cancer cells expressing CD19, the methods comprising
contacting the CD19-expressing cancer cell population with an
anti-CD19 CAR-expressing cell described herein that binds to the
CD19-expressing cell and contacting the CD19-expressing cell with a
kinase inhibitor, e.g., a kinase inhibitor described herein. In
certain aspects, the combination of the anti-CD19 CAR-expressing
cell described herein and the kinase inhibitor, e.g., a kinase
inhibitor described herein, reduces the quantity, number, amount or
percentage of cells and/or cancer cells by at least 25%, at least
30%, at least 40%, at least 50%, at least 65%, at least 75%, at
least 85%, at least 95%, or at least 99% in a subject with or
animal model for a hematological cancer or another cancer
associated with CD19-expressing cells relative to a negative
control. In one aspect, the subject is a human.
[0587] The present invention also provides methods for preventing,
treating and/or managing a disease associated with CD19-expressing
cells (e.g., a hematologic cancer or atypical cancer expressing
CD19), the methods comprising administering to a subject in need an
anti-CD19 CAR-expressing cell that binds to the CD19-expressing
cell and administering a kinase inhibitor, e.g., a kinase inhibitor
described herein. In one aspect, the subject is a human.
Non-limiting examples of disorders associated with CD19-expressing
cells include autoimmune disorders (such as lupus), inflammatory
disorders (such as allergies and asthma) and cancers (such as
hematological cancers or atypical cancers expressing CD19).
[0588] The present invention also provides methods for preventing,
treating and/or managing a disease associated with CD19-expressing
cells, the methods comprising administering to a subject in need an
anti-CD19 CART cell of the invention that binds to the
CD19-expressing cell. In one aspect, the subject is a human.
[0589] The present invention provides methods for preventing
relapse of cancer associated with CD19-expressing cells, the
methods comprising administering to a subject in need thereof an
anti-CD19 expressing cell (such as an anti-CD19 CART cell) of the
invention that binds to the CD19-expressing cell. In one aspect,
the methods comprise administering to the subject in need thereof
an effective amount of an anti-CD19 expressing cell (such as an
anti-CD19 CART cell) described herein that binds to the
CD19-expressing cell in combination with an effective amount of
another therapy.
[0590] In one aspect, the invention pertains to a method of
treating cancer in a subject. The method comprises administering to
the subject a cell (e.g., an immune effector cell) expressing a
B-cell targeting CAR, e.g., a T cell or NK cell, described herein,
in combination with a kinase inhibitor, e.g., a kinase inhibitor
described herein, such that the cancer is treated in the subject.
An example of a cancer that is treatable by the methods described
herein is a cancer associated with expression of the B-cell
antigen, e.g., CD19. In one embodiment, the disease is a solid or
liquid tumor. In one embodiment, the disease is a hematologic
cancer. In one embodiment, the hematologic cancer is leukemia. In
one embodiment, the hematologic cancer is a mature B cell neoplasm,
e.g., according to WHO classification. In one embodiment, the
hematologic cancer is a CD19+B-lymphocyte-derived malignancy. In
one embodiment, the cancer is selected from the group consisting of
one or more acute leukemias including but not limited to B-cell
acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia
(TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia
(ALL); one or more chronic leukemias including but not limited to
chronic myelogenous leukemia (CML), chronic lymphocytic leukemia
(CLL); additional hematologic cancers or hematologic conditions
including, but not limited to mantle cell lymphoma (MCL), B cell
prolymphocytic leukemia, blastic plasmacytoid dendritic cell
neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL)
(e.g., T-cell/histiocyte rich large B-cell lymphoma, primary DLCBL
of the CNS, primary cutaneous DLBCL leg type, or EBV+ DLBCL of the
elderly), DLBCL associated with chronic inflammation, follicular
lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small
cell- or a large cell-follicular lymphoma, malignant
lymphoproliferative conditions, MALT lymphoma (extranodal marginal
zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone
lymphoma, multiple myeloma, myelodysplasia and myelodysplastic
syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic
lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom
macroglobulinemia, splenic marginal zone lymphoma, splenic
lymphoma/leukemia (e.g., unclassifiable), splenic diffuse red pulp
small B-cell lymphoma, hairy cell leukemia-variant,
lymphoplasmacytic lymphoma, a heavy chain disease (e.g., alpha
heavy chain disease, gamma heavy chain disease, or mu heavy chain
disease), plasma cell myeloma, solitary plasmocytoma of bone,
extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric
nodal marginal zone lymphoma, primary cutaneous follicle center
lymphoma, lymphomatoid granulomatosis, primary mediastinal
(theymic) large B-cell lymphoma, intravascular large B-cell
lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising
in HHV8-associated multicenric Castleman disease, primary effusion
lymphoma, B-cell lymphoma, unclassifiable (e.g., with features
intermediate between DLBCL and Burkitt lymphoma or intermediate
between DLBCL and classical Hodgkin lymphoma), and "preleukemia"
which are a diverse collection of hematological conditions united
by ineffective production (or dysplasia) of myeloid blood cells,
and to disease associated with B-cell antigen- (e.g., CD19-)
expression include, but not limited to atypical and/or
non-classical cancers, malignancies, precancerous conditions or
proliferative diseases expressing B-cell antigen (e.g., CD19); and
any combination thereof.
[0591] In some embodiments, the cancer is Hodgkin lymphoma, and the
patient is treated with CAR expressing cells, e.g., as a
monotherapy, or in combination with one or more additional
therapeutics. In embodiments, the Hodgkin lymphoma is stage I, II,
III, or IV. The additional therapeutic may comprise, e.g., a kinase
inhibitor such as a BTK inhibitor like ibrutinib. The additional
therapeutic may comprise a treatment for Hodgkin lymphoma. The
additional therapeutic may comprise, e.g., radiation therapy, MOPP
(Mustargen, Oncovin, Prednisone, and Procarbazine), ABVD
(Adriamycin, bleomycin, vinblastine, and dacarbazine), Stanford V
(a regimen with chemotherapy and radiation treatment), or BEACOPP
(Bleomycin, Etoposide, Adriamycin, Cyclophosphamide, Oncovin,
Procarbazine, Prednisone). In some embodiments, the subject has
previously been treated with, or is resistant to, or is refractory
to, one or more of radiation therapy, MOPP, Stanford V, or
BEACOPP.
[0592] Non-cancer related indications associated with expression of
B-cell antigen, e.g., one or more of CD19, CD20, CD22 or ROR1,
include, but are not limited to, e.g., autoimmune disease, (e.g.,
lupus), inflammatory disorders (allergy and asthma) and
transplantation.
[0593] In some embodiments, a cancer that can be treated with the
combination described herein is multiple myeloma. Multiple myeloma
is a cancer of the blood, characterized by accumulation of a plasma
cell clone in the bone marrow. Current therapies for multiple
myeloma include, but are not limited to, treatment with
lenalidomide, which is an analog of thalidomide. Lenalidomide has
activities which include anti-tumor activity, angiogenesis
inhibition, and immunomodulation. In some embodiments, a CD19 CAR,
e.g., as described herein, may be used to target myeloma cells. In
some embodiments, the combination described herein can be used with
one or more additional therapies, e.g., lenalidomide treatment.
[0594] The CAR-expressing cells described herein may be
administered either alone, or as a pharmaceutical composition in
combination with diluents and/or with other components such as IL-2
or other cytokines or cell populations.
[0595] In embodiments, a lymphodepleting chemotherapy is
administered to the subject prior to, concurrently with, or after
administration (e.g., infusion) of CAR cells, e.g., CAR-expressing
cells described herein. In an example, the lymphodepleting
chemotherapy is administered to the subject prior to administration
of CAR cells. For example, the lymphodepleting chemotherapy ends
1-4 days (e.g., 1, 2, 3, or 4 days) prior to CAR cell infusion. In
embodiments, multiple doses of CAR cells are administered, e.g., as
described herein. For example, a single dose comprises about
5.times.10.sup.8 CAR cells. In embodiments, a lymphodepleting
chemotherapy is administered to the subject prior to, concurrently
with, or after administration (e.g., infusion) of a CAR-expressing
cell described herein.
Hematologic Cancer
[0596] Hematological cancer conditions are the types of cancer such
as leukemia, lymphoma and malignant lymphoproliferative conditions
that affect blood, bone marrow and the lymphatic system.
[0597] Leukemia can be classified as acute leukemia and chronic
leukemia. Acute leukemia can be further classified as acute
myelogenous leukemia (AML) and acute lymphoid leukemia (ALL).
Chronic leukemia includes chronic myelogenous leukemia (CML) and
chronic lymphoid leukemia (CLL). Other related conditions include
myelodysplastic syndromes (MDS, formerly known as "preleukemia")
which are a diverse collection of hematological conditions united
by ineffective production (or dysplasia) of myeloid blood cells and
risk of transformation to AML.
[0598] Lymphoma is a group of blood cell tumors that develop from
lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and
Hodgkin lymphoma.
Combination Therapies
[0599] The combination of a CAR-expressing cell described herein
(e.g., and a kinase inhibitor described herein) may be used in
combination with other known agents and therapies.
[0600] A CAR-expressing cell described herein, the kinase inhibitor
and/or the at least one additional therapeutic agent can be
administered simultaneously, in the same or in separate
compositions, or sequentially. For sequential administration, the
CAR-expressing cell described herein can be administered first, and
the additional agent can be administered second, or the order of
administration can be reversed.
[0601] The CAR therapy and/or other therapeutic agents, procedures
or modalities can be administered during periods of active
disorder, or during a period of remission or less active disease.
The CAR therapy can be administered before another treatment,
concurrently with the treatment, post-treatment, or during
remission of the disorder.
[0602] When administered in combination, the CAR therapy and one or
more additional agent (e.g., kinase inhibitor and/or a third
agent), or all, can be administered in an amount or dose that is
higher, lower or the same than the amount or dosage of each agent
used individually, e.g., as a monotherapy. In certain embodiments,
the administered amount or dosage of the CAR therapy, the
additional agent (e.g., kinase inhibitor and/or third agent), or
all, is lower (e.g., at least 20%, at least 30%, at least 40%, or
at least 50%) than the amount or dosage of each agent used
individually, e.g., as a monotherapy. In other embodiments, the
amount or dosage of the CAR therapy, the additional agent (e.g.,
kinase inhibitor and/or third agent), or all, that results in a
desired effect (e.g., treatment of cancer) is lower (e.g., at least
20%, at least 30%, at least 40%, or at least 50% lower) than the
amount or dosage of each agent used individually, e.g., as a
monotherapy, required to achieve the same therapeutic effect.
[0603] In further aspects, the combination of the CAR-expressing
cell described herein (e.g., and the kinase inhibitor) may be used
in a treatment regimen in combination with surgery, chemotherapy,
radiation, immunosuppressive agents, such as cyclosporin,
azathioprine, methotrexate, mycophenolate, and FK506, antibodies,
or other immunoablative agents such as CAMPATH, anti-CD3 antibodies
or other antibody therapies, cytoxin, fludarabine, cyclosporin,
FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines,
and irradiation. peptide vaccine, such as that described in Izumoto
et al. 2008 J Neurosurg 108:963-971.
[0604] In one embodiment, the combination of a CAR-expressing cell
described herein (e.g., and a kinase inhibitor described herein)
can be used in combination with another chemotherapeutic agent.
Exemplary chemotherapeutic agents include an anthracycline (e.g.,
doxorubicin (e.g., liposomal doxorubicin)); a vinca alkaloid (e.g.,
vinblastine, vincristine, vindesine, vinorelbine); an alkylating
agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide,
temozolomide); an immune cell antibody (e.g., alemtuzamab,
gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab); an
antimetabolite (including, e.g., folic acid antagonists, pyrimidine
analogs, purine analogs and adenosine deaminase inhibitors (e.g.,
fludarabine)); a TNFR glucocorticoid induced TNFR related protein
(GITR) agonist; a proteasome inhibitor (e.g., aclacinomycin A,
gliotoxin or bortezomib); an immunomodulator such as thalidomide or
a thalidomide derivative (e.g., lenalidomide).
[0605] General Chemotherapeutic agents considered for use in
combination therapies include anastrozole (Arimidex.RTM.),
bicalutamide (Casodex.RTM.), bleomycin sulfate (Blenoxane.RTM.),
busulfan (Myleran.RTM.), busulfan injection (Busulfex.RTM.),
capecitabine (Xeloda.RTM.),
N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin
(Paraplatin.RTM.), carmustine (BiCNU.RTM.), chlorambucil
(Leukeran.RTM.), cisplatin (Platinol.RTM.), cladribine
(Leustatin.RTM.), cyclophosphamide (Cytoxan.RTM. or Neosar.RTM.),
cytarabine, cytosine arabinoside (Cytosar-U.RTM.), cytarabine
liposome injection (DepoCyt.RTM.), dacarbazine (DTIC-Dome.RTM.),
dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride
(Cerubidine.RTM.), daunorubicin citrate liposome injection
(DaunoXome.RTM.), dexamethasone, docetaxel (Taxotere.RTM.),
doxorubicin hydrochloride (Adriamycin.RTM., Rubex.RTM.), etoposide
(Vepesid.RTM.), fludarabine phosphate (Fludara.RTM.),
5-fluorouracil (Adrucil.RTM., Efudex.RTM.), flutamide
(Eulexin.RTM.), tezacitibine, gemcitabine (difluorodeoxycitidine),
hydroxyurea (Hydrea.RTM.), Idarubicin (Idamycin.RTM.), ifosfamide
(IFEX.RTM.), irinotecan (Camptosar.RTM.), L-asparaginase
(ELSPAR.RTM.), leucovorin calcium, melphalan (Alkeran.RTM.),
6-mercaptopurine (Purinethol.RTM.), methotrexate (Folex.RTM.),
mitoxantrone (Novantrone.RTM.), mylotarg, paclitaxel (Taxol.RTM.),
phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with
carmustine implant (Gliadel.RTM.), tamoxifen citrate
(Nolvadex.RTM.), teniposide (Vumon.RTM.), 6-thioguanine, thiotepa,
tirapazamine (Tirazone.RTM.), topotecan hydrochloride for injection
(Hycamptin.RTM.), vinblastine (Velban.RTM.), vincristine
(Oncovin.RTM.), and vinorelbine (Navelbine.RTM.).
[0606] Exemplary alkylating agents include, without limitation,
nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,
nitrosoureas and triazenes): uracil mustard (Aminouracil
Mustard.RTM., Chlorethaminacil.RTM., Demethyldopan.RTM.,
Desmethyldopan.RTM., Haemanthamine.RTM., Nordopan.RTM., Uracil
nitrogen Mustard.RTM., Uracillost.RTM., Uracilmostaza.RTM.,
Uramustin.RTM., Uramustine.RTM.), chlormethine (Mustargen.RTM.),
cyclophosphamide (Cytoxan.RTM., Neosar.RTM., Clafen.RTM.,
Endoxan.RTM., Procytox.RTM., Revimmune.TM.), ifosfamide
(Mitoxana.RTM.), melphalan (Alkeran.RTM.), Chlorambucil
(Leukeran.RTM.), pipobroman (Amedel.RTM., Vercyte.RTM.),
triethylenemelamine (Hemel.RTM., Hexalen.RTM., Hexastat.RTM.),
triethylenethiophosphoramine, Temozolomide (Temodar.RTM.), thiotepa
(Thioplex.RTM.), busulfan (Busilvex.RTM., Myleran.RTM.), carmustine
(BiCNU.RTM.), lomustine (CeeNU.RTM.), streptozocin (Zanosar.RTM.),
and Dacarbazine (DTIC-Dome.RTM.). Additional exemplary alkylating
agents include, without limitation, Oxaliplatin (Eloxatin.RTM.);
Temozolomide (Temodar.RTM. and Temodal.RTM.); Dactinomycin (also
known as actinomycin-D, Cosmegen.RTM.); Melphalan (also known as
L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran.RTM.);
Altretamine (also known as hexamethylmelamine (HMM), Hexalen.RTM.);
Carmustine (BiCNU.RTM.); Bendamustine (Treanda.RTM.); Busulfan
(Busulfex.RTM. and Myleran.RTM.); Carboplatin (Paraplatin.RTM.);
Lomustine (also known as CCNU, CeeNU.RTM.); Cisplatin (also known
as CDDP, Platinol.RTM. and Platinol.RTM.-AQ); Chlorambucil
(Leukeran.RTM.); Cyclophosphamide (Cytoxan.RTM. and Neosar.RTM.);
Dacarbazine (also known as DTIC, DIC and imidazole carboxamide,
DTIC-Dome.RTM.); Altretamine (also known as hexamethylmelamine
(HMM), Hexalen.RTM.); Ifosfamide (Ifex.RTM.); Prednumustine;
Procarbazine (Matulane.RTM.); Mechlorethamine (also known as
nitrogen mustard, mustine and mechloroethamine hydrochloride,
Mustargen.RTM.); Streptozocin (Zanosar.RTM.); Thiotepa (also known
as thiophosphoamide, TESPA and TSPA, Thioplex.RTM.);
Cyclophosphamide (Endoxan.RTM., Cytoxan.RTM., Neosar.RTM.,
Procytox.RTM., Revimmune.RTM.); and Bendamustine HCl
(Treanda.RTM.).
[0607] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with fludarabine, cyclophosphamide, and/or rituximab.
In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with fludarabine,
cyclophosphamide, and rituximab (FCR). In embodiments, the subject
has CLL. For example, the subject has a deletion in the short arm
of chromosome 17 (del(17p), e.g., in a leukemic cell). In other
examples, the subject does not have a del(17p). In embodiments, the
subject comprises a leukemic cell comprising a mutation in the
immunoglobulin heavy-chain variable-region (IgV.sub.H) gene. In
other embodiments, the subject does not comprise a leukemic cell
comprising a mutation in the immunoglobulin heavy-chain
variable-region (IgV.sub.H) gene. In embodiments, the fludarabine
is administered at a dosage of about 10-50 mg/m.sup.2 (e.g., about
10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50
mg/m.sup.2), e.g., intravenously. In embodiments, the
cyclophosphamide is administered at a dosage of about 200-300
mg/m.sup.2 (e.g., about 200-225, 225-250, 250-275, or 275-300
mg/m.sup.2), e.g., intravenously. In embodiments, the rituximab is
administered at a dosage of about 400-600 mg/m.sup.2 (e.g.,
400-450, 450-500, 500-550, or 550-600 mg/m.sup.2), e.g.,
intravenously.
[0608] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with bendamustine and rituximab. In embodiments, the
subject has CLL. For example, the subject has a deletion in the
short arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In
other examples, the subject does not have a del(17p). In
embodiments, the subject comprises a leukemic cell comprising a
mutation in the immunoglobulin heavy-chain variable-region
(IgV.sub.H) gene. In other embodiments, the subject does not
comprise a leukemic cell comprising a mutation in the
immunoglobulin heavy-chain variable-region (IgV.sub.H) gene. In
embodiments, the bendamustine is administered at a dosage of about
70-110 mg/m.sup.2 (e.g., 70-80, 80-90, 90-100, or 100-110
mg/m.sup.2), e.g., intravenously. In embodiments, the rituximab is
administered at a dosage of about 400-600 mg/m.sup.2 (e.g.,
400-450, 450-500, 500-550, or 550-600 mg/m.sup.2), e.g.,
intravenously.
[0609] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with rituximab, cyclophosphamide, doxorubicine,
vincristine, and/or a corticosteroid (e.g., prednisone). In
embodiments, a CAR-expressing cell described herein is administered
to a subject in combination with rituximab, cyclophosphamide,
doxorubicine, vincristine, and prednisone (R-CHOP). In embodiments,
the subject has diffuse large B-cell lymphoma (DLBCL). In
embodiments, the subject has nonbulky limited-stage DLBCL (e.g.,
comprises a tumor having a size/diameter of less than 7 cm). In
embodiments, the subject is treated with radiation in combination
with the R-CHOP. For example, the subject is administered R-CHOP
(e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP),
followed by radiation. In some cases, the subject is administered
R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of
R-CHOP) following radiation.
[0610] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with etoposide, prednisone, vincristine,
cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a
CAR-expressing cell described herein is administered to a subject
in combination with etoposide, prednisone, vincristine,
cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In
embodiments, a CAR-expressing cell described herein is administered
to a subject in combination with dose-adjusted EPOCH-R
(DA-EPOCH-R). In embodiments, the subject has a B cell lymphoma,
e.g., a Myc-rearranged aggressive B cell lymphoma.
[0611] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with rituximab and/or lenalidomide. Lenalidomide
((RS)-3-(4-Amino-1-oxo
1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is an
immunomodulator. In embodiments, a CAR-expressing cell described
herein is administered to a subject in combination with rituximab
and lenalidomide. In embodiments, the subject has follicular
lymphoma (FL) or mantle cell lymphoma (MCL). In embodiments, the
subject has FL and has not previously been treated with a cancer
therapy. In embodiments, lenalidomide is administered at a dosage
of about 10-20 mg (e.g., 10-15 or 15-20 mg), e.g., daily. In
embodiments, rituximab is administered at a dosage of about 350-550
mg/m.sup.2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or
475-500 mg/m.sup.2), e.g., intravenously.
[0612] Exemplary immunomodulators include, e.g., afutuzumab
(available from Roche.RTM.); pegfilgrastim (Neulasta.RTM.);
lenalidomide (CC-5013, Revlimid.RTM.); thalidomide (Thalomid.RTM.),
pomelidomide, actimid (CC4047); and IRX-2 (mixture of human
cytokines including interleukin 1, interleukin 2, and interferon
.gamma., CAS 951209-71-5, available from IRX Therapeutics).
[0613] Exemplary anthracyclines include, e.g., doxorubicin
(Adriamycin.RTM. and Rubex.RTM.); bleomycin (Lenoxane.RTM.);
daunorubicin (dauorubicin hydrochloride, daunomycin, and
rubidomycin hydrochloride, Cerubidine.RTM.); daunorubicin liposomal
(daunorubicin citrate liposome, DaunoXome.RTM.); mitoxantrone
(DHAD, Novantrone.RTM.); epirubicin (Ellence.TM.); idarubicin
(Idamycin.RTM., Idamycin PFS.RTM.); mitomycin C (Mutamycin.RTM.);
geldanamycin; herbimycin; ravidomycin; and
desacetylravidomycin.
[0614] Exemplary vinca alkaloids include, e.g., vinorelbine
tartrate (Navelbine.RTM.), Vincristine (Oncovin.RTM.), and
Vindesine (Eldisine.RTM.)); vinblastine (also known as vinblastine
sulfate, vincaleukoblastine and VLB, Alkaban-AQ.RTM. and
Velban.RTM.); and vinorelbine (Navelbine.RTM.).
[0615] Exemplary proteosome inhibitors include bortezomib
(Velcade.RTM.); carfilzomib (PX-171-007,
(S)-4-Methyl-N--((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxope-
ntan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamid-
o)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib
citrate (MLN-9708); delanzomib (CEP-18770); and
O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1
S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide
(ONX-0912).
[0616] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with brentuximab. Brentuximab is an antibody-drug
conjugate of anti-CD30 antibody and monomethyl auristatin E. In
embodiments, the subject has Hodgkin lymphoma (HL), e.g., relapsed
or refractory HL. In embodiments, the subject comprises CD30+HL. In
embodiments, the subject has undergone an autologous stem cell
transplant (ASCT). In embodiments, the subject has not undergone an
ASCT. In embodiments, brentuximab is administered at a dosage of
about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg),
e.g., intravenously, e.g., every 3 weeks.
[0617] In embodiments, a CAR-expressing cell described herein,
optionally in combination with a kinase inhibitor e.g., a BTK
inhibitor such as ibrutinib, is administered to a subject in
combination with brentuximab and dacarbazine or in combination with
brentuximab and bendamustine. Dacarbazine is an alkylating agent
with a chemical name of
5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine
is an alkylating agent with a chemical name of
4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic
acid. In embodiments, the subject has Hodgkin lymphoma (HL). In
embodiments, the subject has not previously been treated with a
cancer therapy. In embodiments, the subject is at least 60 years of
age, e.g., 60, 65, 70, 75, 80, 85, or older. In embodiments,
dacarbazine is administered at a dosage of about 300-450 mg/m.sup.2
(e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or
425-450 mg/m.sup.2), e.g., intravenously. In embodiments,
bendamustine is administered at a dosage of about 75-125 mg/m.sup.2
(e.g., 75-100 or 100-125 mg/m.sup.2, e.g., about 90 mg/m.sup.2),
e.g., intravenously. In embodiments, brentuximab is administered at
a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or
2.5-3 mg/kg), e.g., intravenously, e.g., every 3 weeks.
[0618] In some embodiments, a CAR-expressing cell described herein
is administered to a subject in combination with a CD20 inhibitor,
e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific
antibody) or a fragment thereof. Exemplary anti-CD20 antibodies
include but are not limited to rituximab, ofatumumab, ocrelizumab,
veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals),
ocaratuzumab, and Pro131921 (Genentech). See, e.g., Lim et al.
Haematologica. 95.1(2010):135-43.
[0619] In some embodiments, the anti-CD20 antibody comprises
rituximab. Rituximab is a chimeric mouse/human monoclonal antibody
IgG1 kappa that binds to CD20 and causes cytolysis of a CD20
expressing cell, e.g., as described in
www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf.
In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with rituximab. In
embodiments, the subject has CLL or SLL.
[0620] In some embodiments, rituximab is administered
intravenously, e.g., as an intravenous infusion. For example, each
infusion provides about 500-2000 mg (e.g., about 500-550, 550-600,
600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950,
950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,
1500-1600, 1600-1700, 1700-1800, 1800-1900, or 1900-2000 mg) of
rituximab. In some embodiments, rituximab is administered at a dose
of 150 mg/m.sup.2 to 750 mg/m.sup.2, e.g., about 150-175
mg/m.sup.2, 175-200 mg/m.sup.2, 200-225 mg/m.sup.2, 225-250
mg/m.sup.2, 250-300 mg/m.sup.2, 300-325 mg/m.sup.2, 325-350
mg/m.sup.2, 350-375 mg/m.sup.2, 375-400 mg/m.sup.2, 400-425
mg/m.sup.2, 425-450 mg/m.sup.2, 450-475 mg/m.sup.2, 475-500
mg/m.sup.2, 500-525 mg/m.sup.2, 525-550 mg/m.sup.2, 550-575
mg/m.sup.2, 575-600 mg/m.sup.2, 600-625 mg/m.sup.2, 625-650
mg/m.sup.2, 650-675 mg/m.sup.2, or 675-700 mg/m.sup.2, where
m.sup.2 indicates the body surface area of the subject. In some
embodiments, rituximab is administered at a dosing interval of at
least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, or more. For
example, rituximab is administered at a dosing interval of at least
0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more. In
some embodiments, rituximab is administered at a dose and dosing
interval described herein for a period of time, e.g., at least 2
weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20 weeks, or greater. For example, rituximab is
administered at a dose and dosing interval described herein for a
total of at least 4 doses per treatment cycle (e.g., at least 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more doses per treatment
cycle).
[0621] In some embodiments, the anti-CD20 antibody comprises
ofatumumab. Ofatumumab is an anti-CD20 IgG1.kappa. human monoclonal
antibody with a molecular weight of approximately 149 kDa. For
example, ofatumumab is generated using transgenic mouse and
hybridoma technology and is expressed and purified from a
recombinant murine cell line (NS0). See, e.g.,
www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf;
and Clinical Trial Identifier number NCT01363128, NCT01515176,
NCT01626352, and NCT01397591. In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with
ofatumumab. In embodiments, the subject has CLL or SLL.
[0622] In some embodiments, ofatumumab is administered as an
intravenous infusion. For example, each infusion provides about
150-3000 mg (e.g., about 150-200, 200-250, 250-300, 300-350,
350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700,
700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1200,
1200-1400, 1400-1600, 1600-1800, 1800-2000, 2000-2200, 2200-2400,
2400-2600, 2600-2800, or 2800-3000 mg) of ofatumumab. In
embodiments, ofatumumab is administered at a starting dosage of
about 300 mg, followed by 2000 mg, e.g., for about 11 doses, e.g.,
for 24 weeks. In some embodiments, ofatumumab is administered at a
dosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35
days, or more. For example, ofatumumab is administered at a dosing
interval of at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 24, 26, 28, 20, 22, 24, 26, 28, 30 weeks, or more. In some
embodiments, ofatumumab is administered at a dose and dosing
interval described herein for a period of time, e.g., at least 1
week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or greater, or
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater, or 1, 2,
3, 4, 5 years or greater. For example, ofatumumab is administered
at a dose and dosing interval described herein for a total of at
least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or more doses per
treatment cycle).
[0623] In some cases, the anti-CD20 antibody comprises ocrelizumab.
Ocrelizumab is a humanized anti-CD20 monoclonal antibody, e.g., as
described in Clinical Trials Identifier Nos. NCT00077870,
NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et
al. Lancet. 19.378(2011):1779-87.
[0624] In some cases, the anti-CD20 antibody comprises veltuzumab.
Veltuzumab is a humanized monoclonal antibody against CD20. See,
e.g., Clinical Trial Identifier No. NCT00547066, NCT00546793,
NCT01101581, and Goldenberg et al. Leuk Lymphoma.
51(5)(2010):747-55.
[0625] In some cases, the anti-CD20 antibody comprises GA101. GA101
(also called obinutuzumab or R05072759) is a humanized and
glyco-engineered anti-CD20 monoclonal antibody. See, e.g., Robak.
Curr. Opin. Investig. Drugs. 10.6(2009):588-96; Clinical Trial
Identifier Numbers: NCT01995669, NCT01889797, NCT02229422, and
NCT01414205; and
www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf.
[0626] In some cases, the anti-CD20 antibody comprises AME-133v.
AME-133v (also called LY2469298 or ocaratuzumab) is a humanized
IgG1 monoclonal antibody against CD20 with increased affinity for
the Fc.gamma.RIIIa receptor and an enhanced antibody dependent
cellular cytotoxicity (ADCC) activity compared with rituximab. See,
e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et
al. Clin Cancer Res. 18.5(2012):1395-403.
[0627] In some cases, the anti-CD20 antibody comprises PRO131921.
PRO131921 is a humanized anti-CD20 monoclonal antibody engineered
to have better binding to Fc.gamma.RIIIa and enhanced ADCC compared
with rituximab. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25;
and Casulo et al. Clin Immunol. 154.1(2014):37-46; and Clinical
Trial Identifier No. NCT00452127.
[0628] In some cases, the anti-CD20 antibody comprises TRU-015.
TRU-015 is an anti-CD20 fusion protein derived from domains of an
antibody against CD20. TRU-015 is smaller than monoclonal
antibodies, but retains Fc-mediated effector functions. See, e.g.,
Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an
anti-CD20 single-chain variable fragment (scFv) linked to human
IgG1 hinge, CH2, and CH3 domains but lacks CH1 and CL domains.
[0629] In some embodiments, an anti-CD20 antibody described herein
is conjugated or otherwise bound to a therapeutic agent, e.g., a
chemotherapeutic agent (e.g., cytoxan, fludarabine, histone
deacetylase inhibitor, demethylating agent, peptide vaccine,
anti-tumor antibiotic, tyrosine kinase inhibitor, alkylating agent,
anti-microtubule or anti-mitotic agent), anti-allergic agent,
anti-nausea agent (or anti-emetic), pain reliever, or
cytoprotective agent described herein.
[0630] In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with a B-cell lymphoma 2
(BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or
GDC-0199) and/or rituximab. In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with
venetoclax and rituximab. Venetoclax is a small molecule that
inhibits the anti-apoptotic protein, BCL-2. The structure of
venetoclax
(4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazi-
n-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfon-
yl)-2-(1H-pyrrololfonyl)-2-([2,3-b]pyridin-5-yloxy)benzamide) is
shown below.
##STR00006##
[0631] In embodiments, the subject has CLL. In embodiments, the
subject has relapsed CLL, e.g., the subject has previously been
administered a cancer therapy. In embodiments, venetoclax is
administered at a dosage of about 15-600 mg (e.g., 15-20, 20-50,
50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg),
e.g., daily. In embodiments, rituximab is administered at a dosage
of about 350-550 mg/m2 (e.g., 350-375, 375-400, 400-425, 425-450,
450-475, or 475-500 mg/m2), e.g., intravenously, e.g., monthly.
[0632] In an embodiment, cells expressing a CAR described herein,
optionally in combination with a kinase inhibitor, e.g., a BTK
inhibitor such as ibrutinib, are administered to a subject in
combination with a molecule that decreases the Treg cell
population. Methods that decrease the number of (e.g., deplete)
Treg cells are known in the art and include, e.g., CD25 depletion,
cyclophosphamide administration, modulating GITR function. Without
wishing to be bound by theory, it is believed that reducing the
number of Treg cells in a subject prior to apheresis or prior to
administration of a CAR-expressing cell described herein reduces
the number of unwanted immune cells (e.g., Tregs) in the tumor
microenvironment and reduces the subject's risk of relapse. In one
embodiment, cells expressing a CAR described herein, optionally in
combination with a kinase inhibitor, e.g., a BTK inhibitor such as
ibrutinib, are administered to a subject in combination with a
molecule targeting GITR and/or modulating GITR functions, such as a
GITR agonist and/or a GITR antibody that depletes regulatory T
cells (Tregs). In embodiments, cells expressing a CAR described
herein, optionally in combination with a kinase inhibitor, e.g., a
BTK inhibitor such as ibrutinib, are administered to a subject in
combination with cyclophosphamide. In one embodiment, the GITR
binding molecules and/or molecules modulating GITR functions (e.g.,
GITR agonist and/or Treg depleting GITR antibodies) are
administered prior to administration of the CAR-expressing cell.
For example, in one embodiment, the GITR agonist can be
administered prior to apheresis of the cells. In embodiments,
cyclophosphamide is administered to the subject prior to
administration (e.g., infusion or re-infusion) of the
CAR-expressing cell or prior to aphersis of the cells. In
embodiments, cyclophosphamide and an anti-GITR antibody are
administered to the subject prior to administration (e.g., infusion
or re-infusion) of the CAR-expressing cell or prior to apheresis of
the cells. In one embodiment, the subject has cancer (e.g., a solid
cancer or a hematological cancer such as ALL or CLL). In an
embodiment, the subject has CLL. In embodiments, the subject has
ALL. In embodiments, the subject has a solid cancer, e.g., a solid
cancer described herein.
[0633] Exemplary GITR agonists include, e.g., GITR fusion proteins
and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such
as, e.g., a GITR fusion protein described in U.S. Pat. No.
6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023,
PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an
anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962,
European Patent No.: 1947183B1, U.S. Pat. Nos. 7,812,135,
8,388,967, 8,591,886, European Patent No.: EP 1866339, PCT
Publication No.: WO 2011/028683, PCT Publication No.: WO
2013/039954, PCT Publication No.: WO2005/007190, PCT Publication
No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT
Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720,
PCT Publication No.: WO99/20758, PCT Publication No.:
WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.
7,618,632, and PCT Publication No.: WO 2011/051726.
[0634] In one embodiment, the combination of a CAR expressing cell
described herein and a kinase inhibitor described herein is
administered to a subject in combination with a GITR agonist, e.g.,
a GITR agonist described herein. In one embodiment, the GITR
agonist is administered prior to the CAR-expressing cell. For
example, in one embodiment, the GITR agonist can be administered
prior to apheresis of the cells. In one embodiment, the subject has
CLL.
[0635] Drugs that inhibit either the calcium dependent phosphatase
calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase
that is important for growth factor induced signaling (rapamycin).
(Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.
73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,
1993) can also be used. In a further aspect, the cell compositions
of the present invention may be administered to a patient in
conjunction with (e.g., before, simultaneously or following) bone
marrow transplantation, T cell ablative therapy using chemotherapy
agents such as, fludarabine, external-beam radiation therapy (XRT),
cyclophosphamide, and/or antibodies such as OKT3 or CAMPATH. In one
aspect, the cell compositions of the present invention are
administered following B-cell ablative therapy such as agents that
react with CD20, e.g., Rituxan. For example, in one embodiment,
subjects may undergo standard treatment with high dose chemotherapy
followed by peripheral blood stem cell transplantation. In certain
embodiments, following the transplant, subjects receive an infusion
of the expanded immune cells of the present invention. In an
additional embodiment, expanded cells are administered before or
following surgery.
[0636] In one embodiment, the subject can be administered an agent
which reduces or ameliorates a side effect associated with the
administration of a CAR-expressing cell. Side effects associated
with the administration of a CAR-expressing cell include, but are
not limited to CRS, and hemophagocytic lymphohistiocytosis (HLH),
also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS
include high fevers, nausea, transient hypotension, hypoxia, and
the like. Accordingly, the methods described herein can comprise
administering a CAR-expressing cell described herein to a subject
and further administering an agent to manage elevated levels of a
soluble factor resulting from treatment with a CAR-expressing cell.
In one embodiment, the soluble factor elevated in the subject is
one or more of IFN-.gamma., TNF.alpha., IL-2 receptor and IL-6.
Therefore, an agent administered to treat this side effect can be
an agent that neutralizes one or more of these soluble factors.
Examples of such agents include, but are not limited to a steroid
(e.g., corticosteroid), an inhibitor of TNF.alpha., and an
inhibitor of IL-6. An example of a TNF.alpha. inhibitor is an
anti-TNF.alpha. antibody molecule such as, infliximab, adalimumab,
certolizumab pegol, and golimumab. Another example of a TNF.alpha.
inhibitor is a fusion protein such as entanercept. Small molecule
inhibitor of TNF.alpha. include, but are not limited to, xanthine
derivatives (e.g. pentoxifylline) and bupropion. An example of an
IL-6 inhibitor is an anti-IL-6 antibody molecule or anti-IL-6
receptor antibody molecule such as tocilizumab (toc), sarilumab,
elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364,
CDP6038, VX30, ARGX-109, FE301, and FM101. In one embodiment, the
anti-IL-6 receptor antibody molecule is tocilizumab. An example of
an IL-1R based inhibitor is anakinra.
[0637] In one embodiment, the subject can be administered an agent
which enhances the activity of a CAR-expressing cell. For example,
in one embodiment, the agent can be an agent which inhibits an
inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1
(PD1), can, in some embodiments, decrease the ability of a
CAR-expressing cell to mount an immune effector response. Examples
of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM
(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA,
TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition of an inhibitory
molecule, e.g., by inhibition at the DNA, RNA or protein level, can
optimize a CAR-expressing cell performance. In embodiments, an
inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a
dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced
short palindromic repeats (CRISPR), a transcription-activator like
effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can
be used to inhibit expression of an inhibitory molecule in the
CAR-expressing cell. In an embodiment the inhibitor is an shRNA. In
an embodiment, the inhibitory molecule is inhibited within a
CAR-expressing cell. In these embodiments, a dsRNA molecule that
inhibits expression of the inhibitory molecule is linked to the
nucleic acid that encodes a component, e.g., all of the components,
of the CAR. In one embodiment, the inhibitor of an inhibitory
signal can be, e.g., an antibody or antibody fragment that binds to
an inhibitory molecule. For example, the agent can be an antibody
or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4
(e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and
marketed as Yervoy.RTM.; Bristol-Myers Squibb; Tremelimumab (IgG2
monoclonal antibody available from Pfizer, formerly known as
ticilimumab, CP-675,206).). In an embodiment, the agent is an
antibody or antibody fragment that binds to TIM3. In an embodiment,
the agent is an antibody or antibody fragment that binds to LAG3.
In an embodiment, the agent is an antibody or antibody fragment
that binds to CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5).
[0638] PD1 is an inhibitory member of the CD28 family of receptors
that also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed
on activated B cells, T cells and myeloid cells (Agata et al. 1996
Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have
been shown to downregulate T cell activation upon binding to PD1
(Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001
Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).
PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med
81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314;
Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression
can be reversed by inhibiting the local interaction of PD1 with
PD-L. Antibodies, antibody fragments, and other inhibitors of PD1,
PD-L1 and PD-L2 are known and may be used combination with a CD19
CAR described herein. For example, nivolumab (also referred to as
BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4
monoclonal antibody which specifically blocks PD1. Nivolumab (clone
5C4) and other human monoclonal antibodies that specifically bind
to PD1 are disclosed in U.S. Pat. No. 8,008,449 and WO2006/121168.
Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal
antibody that binds to PD1. Pidilizumab and other humanized
anti-PD1 monoclonal antibodies are disclosed in WO2009/101611.
Pembrolizumab (formerly known as lambrolizumab, and also referred
to as Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal
antibody that binds to PD1. Pembrolizumab and other humanized
anti-PD1 antibodies are disclosed in U.S. Pat. No. 8,354,509 and
WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody
that binds to PDL1, and inhibits interaction of the ligand with
PD1. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1
monoclonal antibody that binds to PD-L. MDPL3280A and other human
monoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No.
7,943,743 and U.S Publication No.: 20120039906. Other anti-PD-L1
binding agents include YW243.55.570 (heavy and light chain variable
regions are shown in SEQ ID NOs 20 and 21 in WO2010/077634) and
MDX-1 105 (also referred to as BMS-936559, and, e.g., anti-PD-L1
binding agents disclosed in WO2007/005874). AMP-224 (B7-DCIg;
Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342), is
a PD-L2 Fc fusion soluble receptor that blocks the interaction
between PD1 and B7-H1. Other anti-PD1 antibodies include AMP 514
(Amplimmune), among others, e.g., anti-PD1 antibodies disclosed in
U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649.
[0639] TIM3 (T cell immunoglobulin-3) also negatively regulates T
cell function, particularly in IFN-g-secreting CD4+T helper 1 and
CD8+T cytotoxic 1 cells, and plays a critical role in T cell
exhaustion. Inhibition of the interaction between TIM3 and its
ligands, e.g., galectin-9 (Gal9), phosphotidylserine (PS), and
HMGB1, can increase immune response. Antibodies, antibody
fragments, and other inhibitors of TIM3 and its ligands are
available in the art and may be used combination with a CD19 CAR
described herein. For example, antibodies, antibody fragments,
small molecules, or peptide inhibitors that target TIM3 binds to
the IgV domain of TIM3 to inhibit interaction with its ligands.
Antibodies and peptides that inhibit TIM3 are disclosed in
WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include
humanized versions of RMT3-23 (disclosed in Ngiow et al., 2011,
Cancer Res, 71:3540-3551), and clone 8B. 2C12 (disclosed in Monney
et al., 2002, Nature, 415:536-541). Bi-specific antibodies that
inhibit TIM3 and PD-1 are disclosed in US20130156774.
[0640] In other embodiments, the agent which enhances the activity
of a CAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1,
CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the
inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary
anti-CEACAM-1 antibodies are described in WO 2010/125571, WO
2013/082366 WO 2014/059251 and WO 2014/022332, e.g., a monoclonal
antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as
described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO
99/052552. In other embodiments, the anti-CEACAM antibody binds to
CEACAM-5 as described in, e.g., Zheng et al. PLoS One. 2010 Sep.
2;5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146), or
crossreacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO
2013/054331 and US 2014/0271618.
[0641] Without wishing to be bound by theory, carcinoembryonic
antigen cell adhesion molecules (CEACAM), such as CEACAM-1 and
CEACAM-5, are believed to mediate, at least in part, inhibition of
an anti-tumor immune response (see e.g., Markel et al. J Immunol.
2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov.
1;177(9):6062-71; Markel et al. Immunology. 2009 February;
126(2):186-200; Markel et al. Cancer Immunol Immunother. 2010
February; 59(2):215-30; Ortenberg et al. Mol Cancer Ther. 2012
June; 11(6):1300-10; Stern et al. J Immunol. 2005 Jun.
1;174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2;5(9). pii:
e12529). For example, CEACAM-1 has been described as a heterophilic
ligand for TIM-3 and as playing a role in TIM-3-mediated T cell
tolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al.
(2014) Nature doi:10.1038/nature13848). In embodiments, co-blockade
of CEACAM-1 and TIM-3 has been shown to enhance an anti-tumor
immune response in xenograft colorectal cancer models (see e.g., WO
2014/022332; Huang, et al. (2014), supra). In other embodiments,
co-blockade of CEACAM-1 and PD-1 reduce T cell tolerance as
described, e.g., in WO 2014/059251. Thus, CEACAM inhibitors can be
used with the other immunomodulators described herein (e.g.,
anti-PD-1 and/or anti-TIM-3 inhibitors) to enhance an immune
response against a cancer, e.g., a melanoma, a lung cancer (e.g.,
NSCLC), a bladder cancer, a colon cancer an ovarian cancer, and
other cancers as described herein.
[0642] LAG3 (lymphocyte activation gene-3 or CD223) is a cell
surface molecule expressed on activated T cells and B cells that
has been shown to play a role in CD8+ T cell exhaustion.
Antibodies, antibody fragments, and other inhibitors of LAG3 and
its ligands are available in the art and may be used combination
with a CD19 CAR described herein. For example, 3BMS-986016
(Bristol-Myers Squib) is a monoclonal antibody that targets LAG3.
IMP701 (Immutep) is an antagonist LAG3 antibody and IMP731 (Immutep
and GlaxoSmithKline) is a depleting LAG3 antibody. Other LAG3
inhibitors include IMP321 (Immutep), which is a recombinant fusion
protein of a soluble portion of LAG3 and Ig that binds to MHC class
II molecules and activates antigen presenting cells (APC). Other
antibodies are disclosed, e.g., in WO2010/019570.
[0643] In some embodiments, the CAR therapy and kinase inhibitor
are administered in combination with a toll like receptor (TLR)
agonist. The TLR agonist can be a TLR9 agonist. In some
embodiments, the TLR agonist is an oligodeoxynucleotide, e.g., a
CG-enriched oligodeoxynucleotide, e.g., an unmethylated CG-enriched
oligodeoxynucleotide. See, e.g., Sagiv-Barfi et al., "Ibrutinib
enhances the antitumor immune response induced by intratumoral
injection of a TLR9 ligand in syngeneic mouse lymphoma model."
Blood. 2015 Feb. 6. pii: blood-2014-08-593137, which is
incorporated herein by reference in its entirety. In some
embodiments, the TLR agonist is administered in combination with a
CAR-expressing NK cell. Without being bound by theory, the TLR
agonist may promote activation of NK cells such as CAR-expressing
NK cells. In some embodiments, the TLR agonist is administered by
injection, e.g., intrarumoral injection.
[0644] In some embodiments, the agent which enhances the activity
of a CAR-expressing cell can be, e.g., a fusion protein comprising
a first domain and a second domain, wherein the first domain is an
inhibitory molecule, or fragment thereof, and the second domain is
a polypeptide that is associated with a positive signal, e.g., a
polypeptide comprising an intracellular signaling domain as
described herein. In some embodiments, the polypeptide that is
associated with a positive signal can include a costimulatory
domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain
of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g.,
of CD3 zeta, e.g., described herein. In one embodiment, the fusion
protein is expressed by the same cell that expressed the CAR. In
another embodiment, the fusion protein is expressed by a cell,
e.g., a T cell that does not express an anti-CD19 CAR.
[0645] In one embodiment, the agent which enhances activity of a
CAR-expressing cell described herein is miR-17-92.
[0646] In one embodiment, the agent which enhances activity of a
CAR-described herein is a cytokine. Cytokines have important
functions related to T cell expansion, differentiation, survival,
and homeostatis. Cytokines that can be administered to the subject
receiving a CAR-expressing cell described herein include: IL-2,
IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21, or a combination
thereof. In preferred embodiments, the cytokine administered is
IL-7, IL-15, or IL-21, or a combination thereof. The cytokine can
be administered once a day or more than once a day, e.g., twice a
day, three times a day, or four times a day. The cytokine can be
administered for more than one day, e.g. the cytokine is
administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2
weeks, 3 weeks, or 4 weeks. For example, the cytokine is
administered once a day for 7 days.
[0647] In embodiments, the cytokine is administered in combination
with CAR-expressing cells. The cytokine can be administered
simultaneously or concurrently with the CAR-expressing cells, e.g.,
administered on the same day. The cytokine may be prepared in the
same pharmaceutical composition as the CAR-expressing cells, or may
be prepared in a separate pharmaceutical composition.
Alternatively, the cytokine can be administered shortly after
administration of the CAR-expressing T cells, e.g., 1 day, 2 days,
3 days, 4 days, 5 days, 6 days, or 7 days after administration of
the CAR-expressing cells. In embodiments where the cytokine is
administered in a dosing regimen that occurs over more than one
day, the first day of the cytokine dosing regimen can be on the
same day as administration with the CAR-expressing cells, or the
first day of the cytokine dosing regimen can be 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, or 7 days after administration of the
CAR-expressing T cells. In one embodiment, on the first day, the
CAR-expressing cells are administered to the subject, and on the
second day, a cytokine is administered once a day for the next 7
days. In a preferred embodiment, the cytokine to be administered in
combination with the CAR-expressing cells is IL-7, IL-15, and/or
IL-21.
[0648] In other embodiments, the cytokine is administered a
sufficient period of time after administration of the
CAR-expressing cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6
weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, or 1 year or more
after administration of CAR-expressing cells. In one embodiment,
the cytokine is administered after assessment of the subject's
response to the CAR-expressing cells. For example, the subject is
administered CAR-expressing cells according to the dosage and
regimens described herein. The response of the subject to CART
therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks,
10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8
months, 9 months, 10 months, 11 months, or 1 year or more after
administration of CAR-expressing cells, using any of the methods
described herein, including inhibition of tumor growth, reduction
of circulating tumor cells, or tumor regression. Subjects that do
not exhibit a sufficient response to CART therapy can be
administered a cytokine. Administration of the cytokine to the
subject that has sub-optimal response to the CART therapy improves
CART efficacy and/or anti-tumor activity. In a preferred
embodiment, the cytokine administered after administration of
CAR-expressing cells is IL-7.
[0649] Combination with a Low Dose of an mTOR Inhibitor
[0650] In one embodiment, the cells expressing a CAR molecule,
e.g., a CAR molecule described herein, are administered in
combination with a low, immune enhancing dose of an mTOR
inhibitor.
[0651] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
90%, at least 10 but no more than 90%, at least 15, but no more
than 90%, at least 20 but no more than 90%, at least 30 but no more
than 90%, at least 40 but no more than 90%, at least 50 but no more
than 90%, at least 60 but no more than 90%, or at least 70 but no
more than 90%.
[0652] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
80%, at least 10 but no more than 80%, at least 15, but no more
than 80%, at least 20 but no more than 80%, at least 30 but no more
than 80%, at least 40 but no more than 80%, at least 50 but no more
than 80%, or at least 60 but no more than 80%.
[0653] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
70%, at least 10 but no more than 70%, at least 15, but no more
than 70%, at least 20 but no more than 70%, at least 30 but no more
than 70%, at least 40 but no more than 70%, or at least 50 but no
more than 70%.
[0654] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
60%, at least 10 but no more than 60%, at least 15, but no more
than 60%, at least 20 but no more than 60%, at least 30 but no more
than 60%, or at least 40 but no more than 60%.
[0655] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
50%, at least 10 but no more than 50%, at least 15, but no more
than 50%, at least 20 but no more than 50%, at least 30 but no more
than 50%, or at least 40 but no more than 50%.
[0656] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
40%, at least 10 but no more than 40%, at least 15, but no more
than 40%, at least 20 but no more than 40%, at least 30 but no more
than 40%, or at least 35 but no more than 40%.
[0657] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
30%, at least 10 but no more than 30%, at least 15, but no more
than 30%, at least 20 but no more than 30%, or at least 25 but no
more than 30%.
[0658] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but
no more than 20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at
least 1, 2, 3, 4 or 5, but no more than 35, at least 1, 2, 3, 4 or
5 but no more than 40%, or at least 1, 2, 3, 4 or 5 but no more
than 45%.
[0659] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but
no more than 90%.
[0660] As is discussed herein, the extent of mTOR inhibition can be
expressed as the extent of P70 S6 kinase inhibition, e.g., the
extent of mTOR inhibition can be determined by the level of
decrease in P70 S6 kinase activity, e.g., by the decrease in
phosphorylation of a P70 S6 kinase substrate. The level of mTOR
inhibition can be evaluated by a method described herein, e.g. by
the Boulay assay, or measurement of phosphorylated S6 levels by
western blot.
Exemplary mTOR Inhibitors
[0661] As used herein, the term "mTOR inhibitor" refers to a
compound or ligand, or a pharmaceutically acceptable salt thereof,
which inhibits the mTOR kinase in a cell. In an embodiment an mTOR
inhibitor is an allosteric inhibitor. In an embodiment an mTOR
inhibitor is a catalytic inhibitor.
[0662] Allosteric mTOR inhibitors include the neutral tricyclic
compound rapamycin (sirolimus), rapamycin-related compounds, that
is compounds having structural and functional similarity to
rapamycin including, e.g., rapamycin derivatives, rapamycin analogs
(also referred to as rapalogs) and other macrolide compounds that
inhibit mTOR activity.
[0663] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygroscopicus having the structure shown in Formula
A.
##STR00007##
[0664] See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991)
44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113:
7433; U.S. Pat. No. 3,929,992. There are various numbering schemes
proposed for rapamycin. To avoid confusion, when specific rapamycin
analogs are named herein, the names are given with reference to
rapamycin using the numbering scheme of formula A.
[0665] Rapamycin analogs useful in the invention are, for example,
O-substituted analogs in which the hydroxyl group on the cyclohexyl
ring of rapamycin is replaced by OR.sub.1 in which R.sub.1 is
hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl;
e.g. RAD001, also known as, everolimus as described in U.S. Pat.
No. 5,665,772 and WO94/09010 the contents of which are incorporated
by reference. Other suitable rapamycin analogs include those
substituted at the 26- or 28-position. The rapamycin analog may be
an epimer of an analog mentioned above, particularly an epimer of
an analog substituted in position 40, 28 or 26, and may optionally
be further hydrogenated, e.g. as described in U.S. Pat. No.
6,015,815, WO95/14023 and WO99/15530 the contents of which are
incorporated by reference, e.g. ABT578 also known as zotarolimus or
a rapamycin analog described in U.S. Pat. No. 7,091,213, WO98/02441
and WO01/14387 the contents of which are incorporated by reference,
e.g. AP23573 also known as ridaforolimus.
[0666] Examples of rapamycin analogs suitable for use in the
present invention from U.S. Pat. No. 5,665,772 include, but are not
limited to, 40-O-benzyl-rapamycin,
40-O-(4'-hydroxymethyl)benzyl-rapamycin,
40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin,
40-O-allyl-rapamycin,
40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-en-1'-yl]-rapamycin,
(2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-en-1'-yl)-rapamycin,
40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,
40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,
40-O-(6-hydroxy)hexyl-rapamycin,
40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,
40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,
40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,
40-O-(2-acetoxy)ethyl-rapamycin,
40-O-(2-nicotinoyloxy)ethyl-rapamycin,
40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,
40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,
40-O-[2-(N-methyl-N'-piperazinyl)acetoxy]ethyl-rapamycin,
39-O-desmethyl-39,40-O,O-ethylene-rapamycin,
(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,
40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,
40-O-(2-nicotinamidoethyl)-rapamycin,
40-O-(2-(N-methyl-imidazo-2'-ylcarbethoxamido)ethyl)-rapamycin,
40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,
40-O-(2-tolylsulfonamidoethyl)-rapamycin and
40-O-[2-(4',5'-dicarboethoxy-1',2',3'-triazol-1'-yl)-ethyl]-rapamycin.
[0667] Other rapamycin analogs useful in the present invention are
analogs where the hydroxyl group on the cyclohexyl ring of
rapamycin and/or the hydroxy group at the 28 position is replaced
with an hydroxyester group are known, for example, rapamycin
analogs found in U.S. RE44,768, e.g. temsirolimus.
[0668] Other rapamycin analogs useful in the preset invention
include those wherein the methoxy group at the 16 position is
replaced with another substituent, preferably (optionally
hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl or
chlorobenzyl and/or wherein the methoxy group at the 39 position is
deleted together with the 39 carbon so that the cyclohexyl ring of
rapamycin becomes a cyclopentyl ring lacking the 39 position
methyoxy group; e.g. as described in WO95/16691 and WO96/41807 the
contents of which are incorporated by reference. The analogs can be
further modified such that the hydroxy at the 40-position of
rapamycin is alkylated and/or the 32-carbonyl is reduced.
[0669] Rapamycin analogs from WO95/16691 include, but are not
limited to, 16-demethoxy-16-(pent-2-ynyl)oxy-rapamycin,
16-demethoxy-16-(but-2-ynyl)oxy-rapamycin,
16-demethoxy-16-(propargyl)oxy-rapamycin,
16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,
16-demethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,
16-demethoxy-16-benzyloxy-rapamycin,
16-demethoxy-16-ortho-methoxybenzyl-rapamycin,
16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,
39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamy-
cin,
39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-[N-methyl,
N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and
39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapam-
ycin.
[0670] Rapamycin analogs from WO96/41807 include, but are not
limited to, 32-deoxo-rapamycin,
16-O-pent-2-ynyl-32-deoxo-rapamycin,
16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,
16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and
32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.
[0671] Another suitable rapamycin analog is umirolimus as described
in US2005/0101624 the contents of which are incorporated by
reference.
[0672] RAD001, otherwise known as everolimus (Afinitor.RTM.), has
the chemical name
(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydrox-
y-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methyl-
ethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tric-
yclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone
[0673] Further examples of allosteric mTOR inhibitors include
sirolimus (rapamycin, AY-22989),
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also
called temsirolimus or CCI-779) and ridaforolimus
(AP-23573/MK-8669). Other examples of allosteric mTor inhibitors
include zotarolimus (ABT578) and umirolimus.
[0674] Alternatively or additionally, catalytic, ATP-competitive
mTOR inhibitors have been found to target the mTOR kinase domain
directly and target both mTORC1 and mTORC2. These are also more
effective inhibitors of mTORC1 than such allosteric mTOR inhibitors
as rapamycin, because they modulate rapamycin-resistant mTORC1
outputs such as 4EBP1-T37/46 phosphorylation and cap-dependent
translation.
[0675] Catalytic inhibitors include: BEZ235 or
2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]q-
uinolin-1-yl)-phenyl]-propionitrile, or the monotosylate salt form.
the synthesis of BEZ235 is described in WO2006/122806; CCG168
(otherwise known as AZD-8055, Chresta, C. M., et al., Cancer Res,
2010, 70(1), 288-298) which has the chemical name
{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-m-
ethoxy-phenyl}-methanol;
3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-met-
hylbenzamide (WO09104019);
3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4--
amine (WO10051043 and WO2013023184); A
N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)
sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and
WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem., 2010, 53,
2636-2645) which has the chemical name
1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholi-
no-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem.
Lett., 2010, 1, 39-43) which has the chemical name
2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}-
benzenesulfonamide;
5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine
(WO10114484);
(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2--
yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamid-
e (WO12007926).
[0676] Further examples of catalytic mTOR inhibitors include
8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-
-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806)
and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J., 2009,
421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian
target of rapamycin (mTOR).) WYE-354 is another example of a
catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical,
Cellular, and In vivo Activity of Novel ATP-Competitive and
Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer
Res. 69(15): 6232-6240).
[0677] mTOR inhibitors useful according to the present invention
also include prodrugs, derivatives, pharmaceutically acceptable
salts, or analogs thereof of any of the foregoing.
[0678] mTOR inhibitors, such as RAD001, may be formulated for
delivery based on well-established methods in the art based on the
particular dosages described herein. In particular, U.S. Pat. No.
6,004,973 (incorporated herein by reference) provides examples of
formulations useable with the mTOR inhibitors described herein.
Evaluation of mTOR Inhibition
[0679] mTOR phosphorylates the kinase P70 S6, thereby activating
P70 S6 kinase and allowing it to phosphorylate its substrate. The
extent of mTOR inhibition can be expressed as the extent of P70 S6
kinase inhibition, e.g., the extent of mTOR inhibition can be
determined by the level of decrease in P70 S6 kinase activity,
e.g., by the decrease in phosphorylation of a P70 S6 kinase
substrate. One can determine the level of mTOR inhibition, by
measuring P70 S6 kinase activity (the ability of P70 S6 kinase to
phosphorylate a substrate), in the absence of inhibitor, e.g.,
prior to administration of inhibitor, and in the presences of
inhibitor, or after the administration of inhibitor. The level of
inhibition of P70 S6 kinase gives the level of mTOR inhibition.
Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as
measured by P70 S6 kinase activity, is inhibited by 40%. The extent
or level of inhibition referred to herein is the average level of
inhibition over the dosage interval. By way of example, if the
inhibitor is given once per week, the level of inhibition is given
by the average level of inhibition over that interval, namely a
week.
[0680] Boulay et al., Cancer Res, 2004, 64:252-61, hereby
incorporated by reference, teaches an assay that can be used to
assess the level of mTOR inhibition (referred to herein as the
Boulay assay). In an embodiment, the assay relies on the
measurement of P70 S6 kinase activity from biological samples
before and after administration of an mTOR inhibitor, e.g., RAD001.
Samples can be taken at preselected times after treatment with an
mTOR inhibitor, e.g., 24, 48, and 72 hours after treatment.
Biological samples, e.g., from skin or peripheral blood mononuclear
cells (PBMCs) can be used. Total protein extracts are prepared from
the samples. P70 S6 kinase is isolated from the protein extracts by
immunoprecipitation using an antibody that specifically recognizes
the P70 S6 kinase. Activity of the isolated P70 S6 kinase can be
measured in an in vitro kinase assay. The isolated kinase can be
incubated with 40S ribosomal subunit substrates (which is an
endogenous substrate of P70 S6 kinase) and gamma-.sup.32P under
conditions that allow phosphorylation of the substrate. Then the
reaction mixture can be resolved on an SDS-PAGE gel, and .sup.32P
signal analyzed using a PhosphorImager. A .sup.32P signal
corresponding to the size of the 40S ribosomal subunit indicates
phosphorylated substrate and the activity of P70 S6 kinase.
Increases and decreases in kinase activity can be calculated by
quantifying the area and intensity of the .sup.32P signal of the
phosphorylated substrate (e.g., using ImageQuant, Molecular
Dynamics), assigning arbitrary unit values to the quantified
signal, and comparing the values from after administration with
values from before administration or with a reference value. For
example, percent inhibition of kinase activity can be calculated
with the following formula: 1-(value obtained after
administration/value obtained before administration).times.100. As
described above, the extent or level of inhibition referred to
herein is the average level of inhibition over the dosage
interval.
[0681] Methods for the evaluation of kinase activity, e.g., P70 S6
kinase activity, are also provided in U.S. Pat. No. 7,727,950,
hereby incorporated by reference.
[0682] The level of mTOR inhibition can also be evaluated by a
change in the ration of PD1 negative to PD1 positive T cells. T
cells from peripheral blood can be identified as PD1 negative or
positive by art-known methods.
Low-Dose mTOR Inhibitors
[0683] Methods described herein use low, immune enhancing, dose
mTOR inhibitors, doses of mTOR inhibitors, e.g., allosteric mTOR
inhibitors, including rapalogs such as RAD001. In contrast, levels
of inhibitor that fully or near fully inhibit the mTOR pathway are
immunosuppressive and are used, e.g., to prevent organ transplant
rejection. In addition, high doses of rapalogs that fully inhibit
mTOR also inhibit tumor cell growth and are used to treat a variety
of cancers (See, e.g., Antineoplastic effects of mammalian target
of rapamycine inhibitors. Salvadori M. World J Transplant. 2012
Oct. 24;2(5):74-83; Current and Future Treatment Strategies for
Patients with Advanced Hepatocellular Carcinoma: Role of mTOR
Inhibition. Finn R S. Liver Cancer. 2012 November; 1(3-4):247-256;
Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A,
Cornella H, Villanueva A. Liver Cancer. 2012 September; 1(2):83-93;
Targeted cancer therapy--Are the days of systemic chemotherapy
numbered?Joo W D, Visintin I, Mor G. Maturitas. 2013 Sep. 20; Role
of natural and adaptive immunity in renal cell carcinoma response
to VEGFR-TKIs and mTOR inhibitor. Santoni M, Berardi R, Amantini C,
Burattini L, Santini D, Santoni G, Cascinu S. Int J Cancer. 2013
Oct. 2).
[0684] The present invention is based, at least in part, on the
surprising finding that doses of mTOR inhibitors well below those
used in current clinical settings had a superior effect in
increasing an immune response in a subject and increasing the ratio
of PD-1 negative T cells/PD-1 positive T cells. It was surprising
that low doses of mTOR inhibitors, producing only partial
inhibition of mTOR activity, were able to effectively improve
immune responses in human human subjects and increase the ratio of
PD-1 negative T cells/PD-1 positive T cells.
[0685] Alternatively, or in addition, without wishing to be bound
by any theory, it is believed that low, a low, immune enhancing,
dose of an mTOR inhibitor can increase naive T cell numbers, e.g.,
at least transiently, e.g., as compared to a non-treated subject.
Alternatively or additionally, again while not wishing to be bound
by theory, it is believed that treatment with an mTOR inhibitor
after a sufficient amount of time or sufficient dosing results in
one or more of the following:
[0686] an increase in the expression of one or more of the
following markers: CD62L.sup.highCD.sub.127.sup.high, CD27.sup.+,
and BCL2, e.g., on memory T cells, e.g., memory T cell
precursors;
[0687] a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; and
[0688] an increase in the number of memory T cell precursors, e.g.,
cells with any one or combination of the following characteristics:
increased CD62L.sup.high, increased CD127.sup.high, increased
CD27.sup.+, decreased KLRG1, and increased BCL2;
and wherein any of the changes described above occurs, e.g., at
least transiently, e.g., as compared to a non-treated subject
(Araki, K et al. (2009) Nature 460:108-112). Memory T cell
precursors are memory T cells that are early in the differentiation
program. For example, memory T cells have one or more of the
following characteristics: increased CD62L.sup.high, increased
CD127high, increased CD27.sup.+, decreased KLRG1, and/or increased
BCL2.
[0689] In an embodiment, the invention relates to a composition, or
dosage form, of an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., a rapalog, rapamycin, or RAD001, or a catalytic
mTOR inhibitor, which, when administered on a selected dosing
regimen, e.g., once daily or once weekly, is associated with: a
level of mTOR inhibition that is not associated with complete, or
significant immune suppression, but is associated with enhancement
of the immune response.
[0690] An mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g.,
a rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, can
be provided in a sustained release formulation. Any of the
compositions or unit dosage forms described herein can be provided
in a sustained release formulation. In some embodiments, a
sustained release formulation will have lower bioavailability than
an immediate release formulation. E.g., in embodiments, to attain a
similar therapeutic effect of an immediate release forlation a
sustained release formulation will have from about 2 to about 5,
about 2.5 to about 3.5, or about 3 times the amount of inhibitor
provided in the immediate release formulation.
[0691] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per week, having 0.1 to 20,
0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs per unit dosage
form, are provided. For once per week administrations, these
immediate release formulations correspond to sustained release
forms, having, respectively, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9
to 18, or about 15 mgs of an mTOR inhibitor, e.g., an allosteric
mTOR inhibitor, e.g., rapamycin or RAD001. In embodiments both
forms are administered on a once/week basis.
[0692] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per day, having having 0.005
to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to
1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5,
0.3 to 0.6, or about 0.5 mgs per unit dosage form, are provided.
For once per day administrations, these immediate release forms
correspond to sustained release forms, having, respectively, 0.015
to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to
4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5,
0.9 to 1.8, or about 1.5 mgs of an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per
week administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 0.1 to 30, 0.2 to
30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to
30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of an mTOR
inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or
RAD001.
[0693] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per day, having having 0.01
to 1.0 mgs per unit dosage form, are provided. For once per day
administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 0.03 to 3 mgs of an
mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin
or RAD001. For once per week administrations, these immediate
release forms correspond to sustained release forms, having,
respectively, 0.2 to 20 mgs of an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., rapamycin or RAD001.
[0694] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per week, having having 0.5
to 5.0 mgs per unit dosage form, are provided. For once per week
administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 1.5 to 15 mgs of an
mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin
or RAD001.
[0695] As described above, one target of the mTOR pathway is the
P70 S6 kinase. Thus, doses of mTOR inhibitors which are useful in
the methods and compositions described herein are those which are
sufficient to achieve no greater than 80% inhibition of P70 S6
kinase activity relative to the activity of the P70 S6 kinase in
the absence of an mTOR inhibitor, e.g., as measured by an assay
described herein, e.g., the Boulay assay. In a further aspect, the
invention provides an amount of an mTOR inhibitor sufficient to
achieve no greater than 38% inhibition of P70 S6 kinase activity
relative to P70 S6 kinase activity in the absence of an mTOR
inhibitor.
[0696] In one aspect the dose of mTOR inhibitor useful in the
methods and compositions of the invention is sufficient to achieve,
e.g., when administered to a human subject, 90+/-5% (i.e., 85-95%),
89+/-5%, 88+/-5%, 87+/-5%, 86+/-5%, 85+/-5%, 84+/-5%, 83+/-5%,
82+/-5%, 81+/-5%, 80+/-5%, 79+/-5%, 78+/-5%, 77+/-5%, 76+/-5%,
75+/-5%, 74+/-5%, 73+/-5%, 72+/-5%, 71+/-5%, 70+/-5%, 69+/-5%,
68+/-5%, 67+/-5%, 66+/-5%, 65+/-5%, 64+/-5%, 63+/-5%, 62+/-5%,
61+/-5%, 60+/-5%, 59+/-5%, 58+/-5%, 57+/-5%, 56+/-5%, 55+/-5%,
54+/-5%, 54+/-5%, 53+/-5%, 52+/-5%, 51+/-5%, 50+/-5%, 49+/-5%,
48+/-5%, 47+/-5%, 46+/-5%, 45+/-5%, 44+/-5%, 43+/-5%, 42+/-5%,
41+/-5%, 40+/-5%, 39+/-5%, 38+/-5%, 37+/-5%, 36+/-5%, 35+/-5%,
34+/-5%, 33+/-5%, 32+/-5%, 31+/-5%, 30+/-5%, 29+/-5%, 28+/-5%,
27+/-5%, 26+/-5%, 25+/-5%, 24+/-5%, 23+/-5%, 22+/-5%, 21+/-5%,
20+/-5%, 19+/-5%, 18+/-5%, 17+/-5%, 16+/-5%, 15+/-5%, 14+/-5%,
13+/-5%, 12+/-5%, 11+/-5%, or 10+/-5%, inhibition of P70 S6 kinase
activity, e.g., as measured by an assay described herein, e.g., the
Boulay assay.
[0697] P70 S6 kinase activity in a subject may be measured using
methods known in the art, such as, for example, according to the
methods described in U.S. Pat. No. 7,727,950, by immunoblot
analysis of phosphoP70 S6K levels and/or phosphoP70 S6 levels or by
in vitro kinase activity assays.
[0698] As used herein, the term "about" in reference to a dose of
mTOR inhibitor refers to up to a +/-10% variability in the amount
of mTOR inhibitor, but can include no variability around the stated
dose.
[0699] In some embodiments, the invention provides methods
comprising administering to a subject an mTOR inhibitor, e.g., an
allosteric inhibitor, e.g., RAD001, at a dosage within a target
trough level. In some embodiments, the trough level is
significantly lower than trough levels associated with dosing
regimens used in organ transplant and cancer patients. In an
embodiment mTOR inhibitor, e.g., RAD001, or rapamycin, is
administered to result in a trough level that is less than/2, 1/4,
1/10, or 1/20 of the trough level that results in immunosuppression
or an anticancer effect. In an embodiment mTOR inhibitor, e.g.,
RAD001, or rapamycin, is administered to result in a trough level
that is less than/2, 1/4, 1/10, or 1/20 of the trough level
provided on the FDA approved packaging insert for use in
immunosuppression or an anticancer indications.
[0700] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.1 to 10 ng/ml, 0.1 to 5 ng/ml, 0.1 to 3 ng/ml, 0.1 to 2
ng/ml, or 0.1 to 1 ng/ml.
[0701] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.2 to 10 ng/ml, 0.2 to 5 ng/ml, 0.2 to 3 ng/ml, 0.2 to 2
ng/ml, or 0.2 to 1 ng/ml.
[0702] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g. an, allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.3 to 10 ng/ml, 0.3 to 5 ng/ml, 0.3 to 3 ng/ml, 0.3 to 2
ng/ml, or 0.3 to 1 ng/ml.
[0703] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.4 to 10 ng/ml, 0.4 to 5 ng/ml, 0.4 to 3 ng/ml, 0.4 to 2
ng/ml, or 0.4 to 1 ng/ml.
[0704] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.5 to 10 ng/ml, 0.5 to 5 ng/ml, 0.5 to 3 ng/ml, 0.5 to 2
ng/ml, or 0.5 to 1 ng/ml.
[0705] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 1 to 10 ng/ml, 1 to 5 ng/ml, 1 to 3 ng/ml, or 1 to 2
ng/ml.
[0706] As used herein, the term "trough level" refers to the
concentration of a drug in plasma just before the next dose, or the
minimum drug concentration between two doses.
[0707] In some embodiments, a target trough level of RAD001 is in a
range of between about 0.1 and 4.9 ng/ml. In an embodiment, the
target trough level is below 3 ng/ml, e.g., is between 0.3 or less
and 3 ng/ml. In an embodiment, the target trough level is below 3
ng/ml, e.g., is between 0.3 or less and 1 ng/ml.
[0708] In a further aspect, the invention can utilize an mTOR
inhibitor other than RAD001 in an amount that is associated with a
target trough level that is bioequivalent to the specified target
trough level for RAD001. In an embodiment, the target trough level
for an mTOR inhibitor other than RAD001, is a level that gives the
same level of mTOR inhibition (e.g., as measured by a method
described herein, e.g., the inhibition of P70 S6 kinase) as does a
trough level of RAD001 described herein.
Pharmaceutical Compositions: mTOR Inhibitors
[0709] In one aspect, the present invention relates to
pharmaceutical compositions comprising an mTOR inhibitor, e.g., an
mTOR inhibitor as described herein, formulated for use in
combination with CAR cells described herein.
[0710] In some embodiments, the mTOR inhibitor is formulated for
administration in combination with an additional, e.g., as
described herein.
[0711] In general, compounds of the invention will be administered
in therapeutically effective amounts as described above via any of
the usual and acceptable modes known in the art, either singly or
in combination with one or more therapeutic agents.
[0712] The pharmaceutical formulations may be prepared using
conventional dissolution and mixing procedures. For example, the
bulk drug substance (e.g., an mTOR inhibitor or stabilized form of
the compound (e.g., complex with a cyclodextrin derivative or other
known complexation agent) is dissolved in a suitable solvent in the
presence of one or more of the excipients described herein. The
mTOR inhibitor is typically formulated into pharmaceutical dosage
forms to provide an easily controllable dosage of the drug and to
give the patient an elegant and easily handleable product.
[0713] Compounds of the invention can be administered as
pharmaceutical compositions by any conventional route, in
particular enterally, e.g., orally, e.g., in the form of tablets or
capsules, or parenterally, e.g., in the form of injectable
solutions or suspensions, topically, e.g., in the form of lotions,
gels, ointments or creams, or in a nasal or suppository form. Where
an mTOR inhibitor is administered in combination with (either
simultaneously with or separately from) another agent as described
herein, in one aspect, both components can be administered by the
same route (e.g., parenterally). Alternatively, another agent may
be administered by a different route relative to the mTOR
inhibitor. For example, an mTOR inhibitor may be administered
orally and the other agent may be administered parenterally.
Sustained Release
[0714] mTOR inhibitors, e.g., allosteric mTOR inhibitors or
catalytic mTOR inhibitors, disclosed herein can be provided as
pharmaceutical formulations in form of oral solid dosage forms
comprising an mTOR inhibitor disclosed herein, e.g., rapamycin or
RAD001, which satisfy product stability requirements and/or have
favorable pharmacokinetic properties over the immediate release
(IR) tablets, such as reduced average plasma peak concentrations,
reduced inter- and intra-patient variability in the extent of drug
absorption and in the plasma peak concentration, reduced
C.sub.max/C.sub.min ratio and/or reduced food effects. Provided
pharmaceutical formulations may allow for more precise dose
adjustment and/or reduce frequency of adverse events thus providing
safer treatments for patients with an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001.
[0715] In some embodiments, the present disclosure provides stable
extended release formulations of an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001, which are multi-particulate
systems and may have functional layers and coatings.
[0716] The term "extended release, multi-particulate formulation as
used herein refers to a formulation which enables release of an
mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, over an
extended period of time e.g. over at least 1, 2, 3, 4, 5 or 6
hours. The extended release formulation may contain matrices and
coatings made of special excipients, e.g., as described herein,
which are formulated in a manner as to make the active ingredient
available over an extended period of time following ingestion.
[0717] The term "extended release" can be interchangeably used with
the terms "sustained release" (SR) or "prolonged release". The term
"extended release" relates to a pharmaceutical formulation that
does not release active drug substance immediately after oral
dosing but over an extended in accordance with the definition in
the pharmacopoeias Ph. Eur. (7.sup.th edition) monograph for
tablets and capsules and USP general chapter <1151> for
pharmaceutical dosage forms. The term "Immediate Release" (IR) as
used herein refers to a pharmaceutical formulation which releases
85% of the active drug substance within less than 60 minutes in
accordance with the definition of "Guidance for Industry:
"Dissolution Testing of Immediate Release Solid Oral Dosage Forms"
(FDA CDER, 1997). In some embodiments, the term "immediate release"
means release of everolimus from tablets within the time of 30
minutes, e.g., as measured in the dissolution assay described
herein.
[0718] Stable extended release formulations of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, can be characterized
by an in-vitro release profile using assays known in the art, such
as a dissolution assay as described herein: a dissolution vessel
filled with 900 mL phosphate buffer pH 6.8 containing sodium
dodecyl sulfate 0.2% at 37.degree. C. and the dissolution is
performed using a paddle method at 75 rpm according to USP by
according to USP testing monograph 711, and Ph.Eur. testing
monograph 2.9.3. respectively.
[0719] In some embodiments, stable extended release formulations of
an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001,
release the mTOR inhibitor in the in-vitro release assay according
to following release specifications:
[0720] 0.5 h: <45%, or <40, e.g., <30%
[0721] 1 h: 20-80%, e.g., 30-60%
[0722] 2 h: >50%, or >70%, e.g., >75%
[0723] 3 h: >60%, or >65%, e.g., >85%, e.g., >90%.
[0724] In some embodiments, stable extended release formulations of
an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001,
release 50% of the mTOR inhibitor not earlier than 45, 60, 75, 90,
105 min or 120 min in the in-vitro dissolution assay.
Biopolymer Delivery Methods
[0725] In some embodiments, one or more CAR-expressing cells as
disclosed herein, optionally in combination with a kinase
inhibitor, e.g., a BTK inhibitor such as ibrutinib, can be
administered or delivered to the subject via a biopolymer scaffold,
e.g., a biopolymer implant. Biopolymer scaffolds can support or
enhance the delivery, expansion, and/or dispersion of the
CAR-expressing cells described herein. A biopolymer scaffold
comprises a biocompatible (e.g., does not substantially induce an
inflammatory or immune response) and/or a biodegradable polymer
that can be naturally occurring or synthetic.
[0726] Examples of suitable biopolymers include, but are not
limited to, agar, agarose, alginate, alginate/calcium phosphate
cement (CPC), beta-galactosidase (.beta.-GAL),
(1,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan,
collagen, elastin, gelatin, hyaluronic acid collagen,
hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate)
(PHBHHx), poly(lactide), poly(caprolactone) (PCL),
poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO),
poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO),
polyvinyl alcohol) (PVA), silk, soy protein, and soy protein
isolate, alone or in combination with any other polymer
composition, in any concentration and in any ratio. The biopolymer
can be augmented or modified with adhesion- or migration-promoting
molecules, e.g., collagen-mimetic peptides that bind to the
collagen receptor of lymphocytes, and/or stimulatory molecules to
enhance the delivery, expansion, or function, e.g., anti-cancer
activity, of the cells to be delivered. The biopolymer scaffold can
be an injectable, e.g., a gel or a semi-solid, or a solid
composition.
[0727] In some embodiments, CAR-expressing cells described herein
are seeded onto the biopolymer scaffold prior to delivery to the
subject. In embodiments, the biopolymer scaffold further comprises
one or more additional therapeutic agents described herein (e.g.,
another CAR-expressing cell, an antibody, or a small molecule) or
agents that enhance the activity of a CAR-expressing cell, e.g.,
incorporated or conjugated to the biopolymers of the scaffold. In
embodiments, the biopolymer scaffold is injected, e.g.,
intratumorally, or surgically implanted at the tumor or within a
proximity of the tumor sufficient to mediate an anti-tumor effect.
Additional examples of biopolymer compositions and methods for
their delivery are described in Stephan et al., Nature
Biotechnology, 2015, 33:97-101; and WO2014/110591.
Pharmaceutical Compositions and Treatments
[0728] Pharmaceutical compositions of the present invention may
comprise a CAR-expressing cell, e.g., a plurality of CAR-expressing
cells, as described herein, in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers such as
neutral buffered saline, phosphate buffered saline and the like;
carbohydrates such as glucose, mannose, sucrose or dextrans,
mannitol; proteins; polypeptides or amino acids such as glycine;
antioxidants; chelating agents such as EDTA or glutathione;
adjuvants (e.g., aluminum hydroxide); and preservatives.
Compositions of the present invention are in one aspect formulated
for intravenous administration.
[0729] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease, although
appropriate dosages may be determined by clinical trials.
[0730] In one embodiment, the pharmaceutical composition is
substantially free of, e.g., there are no detectable levels of a
contaminant, e.g., selected from the group consisting of endotoxin,
mycoplasma, replication competent lentivirus (RCL), p24, VSV-G
nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads,
mouse antibodies, pooled human serum, bovine serum albumin, bovine
serum, culture media components, vector packaging cell or plasmid
components, a bacterium and a fungus. In one embodiment, the
bacterium is at least one selected from the group consisting of
Alcaligenes faecalis, Candida albicans, Escherichia coli,
Haemophilus influenza, Neisseria meningitides, Pseudomonas
aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and
Streptococcus pyogenes group A.
[0731] When "an immunologically effective amount," "an anti-tumor
effective amount," "a tumor-inhibiting effective amount," or
"therapeutic amount" is indicated, the precise amount of the
compositions of the present invention to be administered can be
determined by a physician with consideration of individual
differences in age, weight, tumor size, extent of infection or
metastasis, and condition of the patient (subject). It can
generally be stated that a pharmaceutical composition comprising
the T cells described herein may be administered at a dosage of
10.sup.4 to 10.sup.9 cells/kg body weight, in some instances
10.sup.5 to 10.sup.6 cells/kg body weight, including all integer
values within those ranges. T cell compositions may also be
administered multiple times at these dosages. The cells can be
administered by using infusion techniques that are commonly known
in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.
319:1676, 1988).
[0732] In certain aspects, it may be desired to administer
activated T cells to a subject and then subsequently redraw blood
(or have an apheresis performed), activate T cells therefrom
according to the present invention, and reinfuse the patient with
these activated and expanded T cells. This process can be carried
out multiple times every few weeks. In certain aspects, T cells can
be activated from blood draws of from 10cc to 400cc. In certain
aspects, T cells are activated from blood draws of 20cc, 30cc,
40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.
[0733] The administration of the subject compositions may be
carried out in any convenient manner, including by aerosol
inhalation, injection, ingestion, transfusion, implantation or
transplantation. The compositions described herein may be
administered to a patient trans arterially, subcutaneously,
intradermally, intratumorally, intranodally, intramedullary,
intramuscularly, by intravenous (i.v.) injection, or
intraperitoneally. In one aspect, the T cell compositions of the
present invention are administered to a patient by intradermal or
subcutaneous injection. In one aspect, the T cell compositions of
the present invention are administered by i.v. injection. The
compositions of T cells may be injected directly into a tumor,
lymph node, or site of infection.
[0734] In a particular exemplary aspect, subjects may undergo
leukapheresis, wherein leukocytes are collected, enriched, or
depleted ex vivo to select and/or isolate the cells of interest,
e.g., T cells. These T cell isolates may be expanded by methods
known in the art and treated such that one or more CAR constructs
of the invention may be introduced, thereby creating a CAR T cell
of the invention. Subjects in need thereof may subsequently undergo
standard treatment with high dose chemotherapy followed by
peripheral blood stem cell transplantation. In certain aspects,
following or concurrent with the transplant, subjects receive an
infusion of the expanded CAR T cells of the present invention. In
an additional aspect, expanded cells are administered before or
following surgery.
[0735] The dosage of the above treatments to be administered to a
patient will vary with the precise nature of the condition being
treated and the recipient of the treatment. The scaling of dosages
for human administration can be performed according to art-accepted
practices. The dose for CAMPATH, for example, will generally be in
the range 1 to about 100 mg for an adult patient, usually
administered daily for a period between 1 and 30 days. The
preferred daily dose is 1 to 10 mg per day although in some
instances larger doses of up to 40 mg per day may be used
(described in U.S. Pat. No. 6,120,766).
[0736] In one embodiment, the CAR is introduced into T cells, e.g.,
using in vitro transcription, and the subject (e.g., human)
receives an initial administration of CAR T cells of the invention,
and one or more subsequent administrations of the CAR T cells of
the invention, wherein the one or more subsequent administrations
are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, or 2 days after the previous administration. In one
embodiment, more than one administration of the CAR T cells of the
invention are administered to the subject (e.g., human) per week,
e.g., 2, 3, or 4 administrations of the CAR T cells of the
invention are administered per week. In one embodiment, the subject
(e.g., human subject) receives more than one administration of the
CAR T cells per week (e.g., 2, 3 or 4 administrations per week)
(also referred to herein as a cycle), followed by a week of no CAR
T cells administrations, and then one or more additional
administration of the CAR T cells (e.g., more than one
administration of the CAR T cells per week) is administered to the
subject. In another embodiment, the subject (e.g., human subject)
receives more than one cycle of CAR T cells, and the time between
each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one
embodiment, the CAR T cells are administered every other day for 3
administrations per week. In one embodiment, the CAR T cells of the
invention are administered for at least two, three, four, five,
six, seven, eight or more weeks.
[0737] In one aspect, CAR-expressing cells are generated using
lentiviral viral vectors, such as lentivirus. Cells, e.g., CARTs
generated that way will have stable CAR expression.
[0738] In one aspect, CAR-expressing cells, e.g., CARTs, are
generated using a viral vector such as a gammaretroviral vector,
e.g., a gammaretroviral vector described herein. CARTs generated
using these vectors can have stable CAR expression.
[0739] In one aspect, CARTs transiently express CAR vectors for 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.
Transient expression of CARs can be effected by RNA CAR vector
delivery. In one aspect, the CAR RNA is transduced into the T cell
by electroporation.
[0740] A potential issue that can arise in patients being treated
using transiently expressing CAR T cells (particularly with murine
scFv bearing CARTs) is anaphylaxis after multiple treatments.
[0741] Without being bound by this theory, it is believed that such
an anaphylactic response might be caused by a patient developing
humoral anti-CAR response, i.e., anti-CAR antibodies having an
anti-IgE isotype. It is thought that a patient's antibody producing
cells undergo a class switch from IgG isotype (that does not cause
anaphylaxis) to IgE isotype when there is a ten to fourteen day
break in exposure to antigen.
[0742] If a patient is at high risk of generating an anti-CAR
antibody response during the course of transient CAR therapy (such
as those generated by RNA transductions), CART infusion breaks
should not last more than ten to fourteen days.
EXAMPLES
[0743] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0744] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples specifically point out various aspects
of the present invention, and are not to be construed as limiting
in any way the remainder of the disclosure.
Example 1: Humanization of Murine Anti-CD19 Antibody
[0745] A CD19 antibody molecule can be, e.g., an antibody molecule
(e.g., a humanized anti-CD19 antibody molecule) described in
WO2014/153270, which is incorporated herein by reference in its
entirety. Humanization of murine CD19 antibody is desired for the
clinical setting, where the mouse-specific residues may induce a
human-anti-mouse antigen (HAMA) response in patients who receive
CART19 treatment, i.e., treatment with T cells transduced with the
CAR19 construct. VH and VL sequences of hybridoma derived murine
CD19 antibody were extracted from published literature (Nicholson
et al, 1997, supra). Humanization was accomplished by grafting CDR
regions from murine CD19 antibody onto human germline acceptor
frameworks VH4_4-59 and VK3_L25 (vBASE database). In addition to
the CDR regions, five framework residues, i.e. VH #71, #73, #78 and
VL #71 #87, thought to support the structural integrity of the CDR
regions were retained from the murine sequence. Further, the human
J elements JH4 and JK2 were used for the heavy and light chain,
respectively. The resulting amino acid sequences of the humanized
antibody were designated FMC63_VL_hz and FMC63_VH_hz1,
respectively, and are shown below in Table 1. The residue numbering
follows Kabat (Kabat E. A. et al, 1991, supra). For CDR
definitions, both Kabat as well as Chothia et al, 1987 supra) were
used. Residues coming from mouse CD19 are shown in bold/italic.
Positions #60/61/62 boxed indicate potential post-translational
modification (PTM) site in CDR H2, also termed HCDR2.
TABLE-US-00014 TABLE 1 Amino acid sequences of humanized CD19
variable domains (SEQ ID NOs: 114-117, respectively, in order of
appearance). ##STR00008##
[0746] These humanized CD19 IgGs were used to generate soluble
scFvs to test for expression and scFvs for the full CART CD19
constructs (See Examples below). Of interest was that during
humanization, position 62 in the CDRH2 region prefers to be a
serine residue rather than the alanine present in the murine CDRH2.
The murine sequence lacks a post-translational modification (PTM),
and has asparagine-serine-alanine at positions 60/61/62,
respectively in CDRH2. This generates potential PTM motifs
(indicated as the boxed cite in CDRH2) during the course of
humanization. Whether the PTM site generated during humanization
process was actually a "true" PTM site or merely a theoretical one
was tested. It was hypothesized that the amino acid motif
asparagine followed by serine (NS) may be susceptible to
post-translational deamidation but not something that was readily
apparent. It was also hypothesized that asparagine followed by any
amino acid except proline and then followed by serine (N.times.S,
x.noteq.P) may be susceptible to post-translational
N-glycosylation. To test this hypothesis, two IgG variants, were
generated in which the asparagine at position 60 (known to be a
glycosylation site) was mutated to serine, or glutamine and
designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q),
respectively. These constructs were generated in order to eliminate
the potential post-translational modification site (PTM) and test
for retained activity (See Example 2 below).
Cloning:
[0747] DNA sequences coding for mouse and humanized VL and VH
domains were obtained, and the codons for the constructs were
optimized for expression in cells from Homo sapiens.
[0748] Sequences coding for VL and VH domain were subcloned from
the cloning vectors into expression vectors suitable for secretion
in mammalian cells. The heavy and light chains were cloned into
individual expression vectors to allow co-transfection. Elements of
the expression vector include a promoter (Cytomegalovirus (CMV)
enhancer-promoter), a signal sequence to facilitate secretion, a
polyadenylation signal and transcription terminator (Bovine Growth
Hormone (BGH) gene), an element allowing episomal replication and
replication in prokaryotes (e.g. SV40 origin and ColE1 or others
known in the art) and elements to allow selection (ampicillin
resistance gene and zeocin marker).
Expression:
[0749] Chimera and humanized IgG candidates were expressed in
HEK293F mammalian cells at 1 ml scale. Cleared supernatants were
used for FACS binding studies. More precisely, HEK293F cells were
diluted to 5E5 cells/ml in FreeStyle medium supplemented with
Pen/Strep and 1 ml transferred into 24 round bottom deep well
plate. 0.5 .mu.g of light and 0.5 .mu.g of heavy chain mammalian
expression plasmids were diluted in the same medium together with 4
.mu.l of FuGENE HD (Roche REF 04709705001). After 15 min RT
incubation, DNA/Fugene mix was added drop-wise to the cells and
placed in a 5% CO.sub.2 incubator at 250 rpm, 37.degree. C. for
five days. Supernatant were then separated from the cells by
centrifugation. To measure IgG content, aliquots of 200 .mu.L were
placed in the wells of 96-well microtiter plates. All samples and
standards were measured in duplicate using Protein A Dip and read
biosensors (Fortebio Cat No 18-5010). The plate was placed in an
Octet instrument (ForteBio) and allowed to equilibrate to
27.degree. C. in the thermostated chamber. Data were processed
automatically using the Octet User Software version 3.0 and
concentration determined by comparing to an IgG standard curve.
Binding Analysis by FACS:
[0750] Humanized and chimera antibodies were evaluated with a flow
cytometry binding assay using cell line 300.19-hsCD19FL. This cell
line was generated by transfecting the mouse preB cell line 300.19
with a vector (hCD19 FL/pEF4-myc-His A) encoding the full length
human CD19 encoding sequence and natural promoter as well as a
Zeocin resistance gene. In brief, 300.19 cells were electroporated
with the linearized plasmid and then cells expressing high levels
of hsCD19 were identified using an APC-conjugated anti-human CD19
Ab (clone HIB19 from BD 555415) and subsequently sorted using a
FACS Aria flow cytometer. The sorted hsCD19+ cells were cultured
and confirmed to stably express high levels of hsCD19.
[0751] The binding assay could be performed directly with the serum
free culture media containing the expressed IgG. All evaluated IgGs
were normalized to the same concentration (85 nM), before to be
diluted by a 3 fold serial dilution down to 1.4 pM. Then, in a
96-well plate, aliquots of 5.times.10.sup.5 cells/well were
incubated for 30 min at 4.degree. C. with diluted IgGs. Cells were
washed twice with FACS buffer (0.5% BSA in PBS) before addition of
the detection antibody, an APC conjugated goat anti-hu IgG, Fc
fragment specific (Dianova #109-136-098), diluted 1:1000 in FACS
buffer. Cells were incubated a further 30 min at 4.degree. C., then
washed twice in FACS buffer and assayed using FACS Calibur (BD
Bioscience). Binding curves plotting (median of fluorescence
intensity versus IgG concentration) and EC.sub.50 determination
were performed with GraphPad Prism.TM. 3.0 software with nonlinear
regression analysis, sigmoidal dose response (variable slope).
[0752] The FACS analyses show that apparent binding for all
evaluated IgGs can vary widely, with some constructs exhibiting a 5
to 10 fold shift in EC50 as an IgG versus a scFv. Based on
EC.sub.50 values, lead candidates are chosen that have a binding
affinity within a factor of 2 or better compared to the chimeric
reference.
Example 2: Characterization of Anti-CD19 Soluble scFv Fragments
Derived from Humanized CD19 IgG Antibodies
[0753] Soluble scFv fragments were generated from the humanized
CD19 IgGs described in Example 1 using standard molecule biology
techniques. These soluble scFvs were used in characterization
studies to examine the stability, cell surface expression, and
binding properties of the scFvs. Additionally, experiments were
also conducted to investigate the impact of the potential PTM
introduced during the humanization process.
scFv Expression and Purification
[0754] For transfection of each scFv construct, around 3e8 293F
cells were transfected with 100 .mu.g of plasmid using PEI as the
transfection reagent at the ratio of 3:1 (PEI:DNA). The cells were
grown in 100 ml EXPi293 Expression media (Invitrogen) in a shaker
flask at 37.degree. C., 125 rpm, 8% CO.sub.2. The culture was
harvested after six days and used for protein purification.
[0755] 293F cells were harvested by spinning down at 3500 g for 20
minutes. The supernatant was collected and filtered through
VacuCap90 PF Filter Unit (w/0.8/0.2 .mu.m Super Membrane, PALL).
Around 400 .mu.l 400 ul of Ni-NTA agarose beads (Qiagen) were added
to the supernatant. The mixture was rotated and incubated for 4 hrs
at 4.degree. C. It was loaded onto a purification column and washed
with washing buffer with 20 mM Histidine. The protein was eluted
with 500 .mu.l elution buffer with 300 mM Histidine. The samples
were dialyzed against PBS buffer at 4C overnight. Protein samples
were quantified using nanodrop 2000c.
scFv Conformation and Colloidal Stability Analysis
[0756] Thermostability of the scFv was determined by DSF: mix 10-20
.mu.l of protein sample with the dye Sypro Orange (Invitrogen
Cat#S6650) of a final dilution at 1:1000, in a total volume of 25
.mu.l in PBS, run BioRad CFX1000 (25 C for 2 min, then increment
0.5.degree. C. for 30 second, 25 to 95.degree. C.).
[0757] For analytical SEC experiment, around 15-20 .mu.g of scFv
protein sample in 20 .mu.l PBS was injected onto TSKgel Super
SW2000 at 0.3 ml/min flow rate on n Agilent 1100 series.
EC50 by FACS Binding
[0758] Mouse cell line 300.CD19 were grown in RPMI 1640 with 0.5
mg/ml Zeocin. Around 5e5 cells/per well were transferred to the BD
Falcon 96 well plate. The cells were spin down at 900 rpm (Sorval
Legend XT centrifuge) for 3 minutes. The supernatant were removed.
Anti-CD19 scFv protein samples were diluted in DPBS with 5% FBS.
The samples were added into the wells, mixed well with the cells
and incubated for 1 hour. The cells were washed twice in the DPBS
with 5% FBS. The cells were incubated with antipoly His PE
(R&D) for 1 hour, washed twice before FACS analysis (LSRII from
BD Biosciences).
Kinetic Analysis by Proteon
[0759] Kinetics were determined using Bio-Rad Proteon.
Immobilization was performed using standard amine coupling on a GLC
sensor chip. The scFv samples were diluted to 0.03 mg/mL in acetate
pH 4.5 and applied to the chip at a flow rate of 30 .mu.L/min for
300 seconds. The CD19 ligand was then serial diluted in PBS-Tween
and injected at a flow rate of 50 .mu.L/min for 120 seconds with a
dissociation time of 480 seconds. The chip surface was regenerated
with glycine pH 2.5. Data was fitted using a 1:1 Langmuir
model.
Surface Expression of CART19 Constructs and Staining by FACS
[0760] HEK293F suspension cells transiently transfected with
different anti-hCD19 CARTs were harvested 2 days after the
transfection. Around 1e6 cells were placed into each well of a
V-shape 96 well plate (Greiner Bio-One, Germany) and washed three
times with 0.2 ml FACS buffer (1.times.PBS containing 4% bovine
serum albumin (BSA) (BSA fraction V, Roche Diagnostics,
Indianapolis, Ind.). Cells were resuspended in 0.2 ml of the FCAS
buffer with either 0.2 .mu.g of biotinylated protein L (GenScript,
Piscataway, N.J.) or 100 nM of hCD19(AA 1-291)-hIgG1 Fc (Generated
in NIBRI) and incubated at 4.degree. C. for 30 minutes. Cells were
then washed with 0.2 ml of FACS buffer three times, and incubated
with 1 .mu.l Streptavidin Alexa Fluor 488 (Life Technologies, Grand
Island, N.Y.) in 0.2 ml of FACS buffer for samples with protein L,
or 2 .mu.l of PE anti-human Fcx (Jackson ImmunoResearch
Laboratories, West Grove, Pa.) in 0.2 ml of FACS buffer for samples
with hCD19-hIgG1 Fc for 30 minutes at 4.degree. C. in the dark.
After washing with 0.2 ml of FACS buffer three times, cells were
analyzed on a LSRII (BD Biosciences, San Jose, Calif.) machine
using the FACSDiva software (BD Biosciences, San Jose, Calif.).
Immunofluorescence staining was analyzed as the relative log
fluorescence of live cells, and the percentage of the Alexa Fluor
488 positive or PE positive cells were measured.
Analysis of Potential PMTs Generated During the Humanization
Process
[0761] Of interest was that during humanization, position 62 in the
CDRH2 region prefers to be a serine residue rather than the alanine
present in the murine CDRH2 as described in Example 1. Whether the
PTM site generated during humanization process was actually a
"true" PTM site or merely a theoretical one was tested. Two IgG
variants were generated in which the asparagine at position 60
(known to be a glycosylation site) was mutated to serine, or
glutamine and designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2
(N60Q), respectively. These constructs were generated in order to
eliminate the potential post-translational modification site (PTM)
and test for retained activity.
Results
[0762] Anti-CD19 humanized scFvs and mouse scFv were expressed in
293F cells and purified through His tag. The expression and yield
of all humanized scFvs was much higher than the original mouse scFv
(data not shown).
[0763] To confirm identity and assess integrity, the scFV
constructs are analyzed with or without incubation with N-glycanase
F (PNGaseF) followed by both high-performance liquid chromatography
mass spectrometry (HPLC-MS) (See FIG. 3) and SDS-PAGE (data not
shown). PNGaseF is an enzyme specific for the removal of N-linked
glycan structures from the consensus sequence N--X-S/T/C where X is
any amino acid except proline. Briefly, the samples are diluted in
water to 0.1 .mu.g/L and either left untreated or incubated with
PNGaseF at a 1:2 (w/w) PNGaseF: scFV ratio for 3 hours at
37.degree. C.
[0764] SDS-PAGE analysis is performed using a NuPAGE 4-12% Bis-Tris
gel from Novex. Approximately 2 .mu.g scFV are loaded into each
lane and the electrophoresis is conducted at 200 V constant for 40
minutes. Following electrophoresis, the gel is stained using
PhastGel Blue R 250 stain (Amersham Pharmacia) and destained with
10% acetic acid, 30% methanol.
[0765] HPLC-MS analysis is performed on the Water's Acquity UPLC
system coupled to a Xevo-Tof mass spectrometer. Approximately 1
.mu.g of each sample is loaded onto a R 1/10 2.1 .times.100 mm 10
am POROS column (Applied Biosciences) set to 60.degree. C. at a
flow rate of 0.5 mL/min. Mobile phases are composed of 0.1% formic
acid (A) and 0.1% formic acid, 75% isopropanol, 25% acetonitrile
(B). Protein is eluted from the column with a reverse phase
gradient from 25%-90% B in 12 minutes. The acquisition is performed
using electrospray positive scan at the m/z range of 600-4000 Da
with a source cone voltage ramp 20-50V. The resulting spectra are
deconvoluted using MaxEnt1.
[0766] The glycosylation site was introduced during the process of
humanization. The non-PTM variants (VH: N60S or N60Q) were without
this additional form. The construct was the only one with a
consensus site of N-linked glycosylation in HC CDR2. From the
SDS-PAGE analysis, the untreated samples migrated as single bands
consistent with the approximate molecular weights of the sequences
for all constructs except 103101-WT (S/N) for which doublet is
observed. This construct is the only one with a consensus site of
N-linked glycosylation in H-CDR2. When treated with PNGaseF, the
higher molecular weight band of the doublet is no longer present
suggesting partial occupancy of the site. Similarly, the observed
molecular weights from the deconvoluted mass spectra are consistent
with those predicted from the amino acid sequences. However, while
the other constructs demonstrated a single primary molecular
species, 103101-WT (S/N) also had a population 1217 Daltons higher
than that predicted from the sequence which is no longer present
after treatment with PNGaseF. This is consistent with the presence
of a single predominant N-linked glycoform, likely oligomannose 5
based upon mass. The presence of the glycosylated form was
confirmed by the MS analysis as shown in FIG. 3.
[0767] The conformation stability was measured by Differential
Scanning Fluorimetry (DSF). As shown in FIG. 4, the Tm of mouse
scFv was 57.degree. C., while the human variants showed higher Tm
at around 70.degree. C. The Tm for all the humanized scFv is much
better than the murine scFv, clearly showing that all the humanized
scFv are more stable than the murine scFv. This stability will
likely translate to the CART19 construct, likely leading to
improved therapeutic properties.
[0768] The activity of the purified scFv was measure by binding to
hCD19 expression cells as well as by binding to hCD19 antigen using
SPR based detection method. Mouse cell line 300 was used to
determine the binding of scFvs. The EC.sub.50 of mouse scFv for
hCD19 was around 06-1.6 nM. The humanized variants showed EC.sub.50
of the same range in the low or sub nM EC.sub.50s range.
Example 3: CD19 CAR Constructs
[0769] ScFv to be used in the final CAR construct were derived from
the humanized IgG described in Example 1. The order in which the VL
and VH domains appear in the scFv was varied (i.e., VL-VH, or VH-VL
orientation), and where either three or four copies of the "G4S"
(SEQ ID NO: 18) subunit, in which each subunit comprises the
sequence GGGGS (SEQ ID NO:18) (e.g., (G4S).sub.3 (SEQ ID NO:107) or
(G4S).sub.4 (SEQ ID NO:106)), connect the variable domains to
create the entirety of the scFv domain, as shown in Table 2.
TABLE-US-00015 TABLE 2 Humanized CD19 scFv constructs showing VH
and VL orientation and linker length ("3G4S" is disclosed as SEQ ID
NO: 107 and "4G4S" is disclosed as SEQ ID NO: 106). construct ID
Length aa annotation Vh change mscFvCTL019 486 VL-VH, 3G4S 104879
491 VL-VH, 4G4S N/S 104880 491 VL-VH, 4G4S N/Q 104881 491 VH-VL,
4G4S N/S 104882 491 VH-VL, 4G4S N/Q 104875 486 VL-VH, 3G4S N/S
104876 486 VL-VH, 3G4S N/Q 104877 486 VH-VL, 3G4S N/S 104878 486
VH-VL, 3G4S N/Q 105974 491 VL-VH, 4G4S S/N 105975 491 VH-VL, 4G4S
S/N 105976 486 VL-VH, 3G4S S/N 105977 486 VH-VL, 3G4S S/N
[0770] The sequences of the humanized scFv fragments (SEQ ID NOS:
1-12) are provided below in Table 3. Full CAR constructs were
generated using SEQ ID NOs: 1-12 with additional sequences, SEQ ID
NOs: 13-17, shown below, to generate full CAR constructs with SEQ
ID NOs: 31-42.
TABLE-US-00016 leader (amino acid sequence) (SEQ ID NO: 13)
MALPVTALLLPLALLLHAARP leader (nucleic acid sequence) (SEQ ID NO:
54) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCA
TGCCGCTAGACCC CD8 hinge (amino acid sequence) (SEQ ID NO: 14)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 hinge (nucleic
acid sequence) (SEQ ID NO: 55)
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTC
GCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG
CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT CD8 transmembrane (amino acid
sequence) (SEQ ID NO: 15) IYIWAPLAGTCGVLLLSLVITLYC transmembrane
(nucleic acid sequence) (SEQ ID NO: 56)
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTC
ACTGGTTATCACCCTTTACTGC 4-1BB Intracellular domain (amino acid
sequence) (SEQ ID NO: 16)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB Intracellular
domain (nucleic acid sequence) (SEQ ID NO: 60)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG
ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG
AAGAAGAAGAAGGAGGATGTGAACTG CD3 zeta domain (amino acid sequence)
(SEQ ID NO: 17) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CD3
zeta (nucleic acid sequence) (SEQ ID NO: 101)
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA
AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC
TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CD3 zeta domain (amino acid
sequence; NCBI Reference Sequence NM_000734.3) (SEQ ID NO: 43)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CD3
zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3);
(SEQ ID NO: 44) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA
AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC
TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC IgG4 Hinge (amino acid
sequence) (SEQ ID NO: 102)
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ
EGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge (nucleotide sequence)
(SEQ ID NO: 103) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCT
GGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGA
TGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAG
GAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
CAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGG
TGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAA
TACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAAC
CATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGC
CCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTG
GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG
CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACG
GCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAG
GAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCA
CTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG
[0771] These clones all contained a Q/K residue change in the
signal domain of the co-stimulatory domain derived from 4-1BB.
TABLE-US-00017 TABLE 3 Humanized CD19 CAR Constructs Name SEQ ID
Sequence CAR 1 CAR1 scFv 1
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHT domain
SRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGT
KLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPD
YGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKL
SSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS 103101 61
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR1
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Soluble
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nt
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactactcttcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagccaccaccatcatcaccatcaccat 103101 73
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR1
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aa
gvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslk
lssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 104875 85
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 1-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactactcttcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcc
cagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgca
tacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggta
cttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcgg
aagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactca
agaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaac
tgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaac
cagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaa
gcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaag
agggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagatt
ggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggact
cagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104875
31 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR 1-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Full-aa
ntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs
gvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslk
lssvtaadtavyycakhyyyggsyamdywgqgtlvtvsstttpaprpptpaptias
qplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgr
kkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeaysei
gmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 2 CAR2 scFv 2
eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domain
giparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs
ggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkgle
wigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyg
gsyamdywgqgtlvtvss 103102 62
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR2-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Soluble
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nt
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactaccaatcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagccaccaccatcatcaccatcaccat 103102 74
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR2-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aa
gvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslk
lssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 104876 86
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 2-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactaccaatcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcc
cagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgca
tacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggta
cttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcgg
aagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactca
agaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaac
tgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaac
cagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaa
gcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaag
agggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagatt
ggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggact
cagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104876
32 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR 2-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Full-aa
ntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs
gvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsksqvslk
lssvtaadtavyycakhyyyggsyamdywgqgtlvtvsstttpaprpptpaptias
qplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgr
kkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeaysei
gmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 3 CAR3 scFv 3
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domain
tyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq
gtivtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskyl
nwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcq
qgntlpytfgqgtkleik 103104 63
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 3-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Soluble
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc scFv-nt
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtcc
ctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaa
atacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctacc
acacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcggg
accgactacactctgaccatctcatctctccagcccgaggacttcgccgtctactt
ctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgaga
tcaaacatcaccaccatcatcaccatcac 103104 75
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR 3-
wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv-aa
lspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsg
tdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 104877 87
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 3-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-nt
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtcc
ctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaa
atacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctacc
acacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcggg
accgactacactctgaccatctcatctctccagcccgaggacttcgccgtctactt
ctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgaga
tcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctct
cagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgca
tacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggta
cttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcgg
aagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactca
agaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaac
tgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaac
cagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaa
gcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaag
agggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagatt
ggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggact
cagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104877
33 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR 3-
wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Full-aa
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls
lspgeratlscrasgdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsg
tdytltisslqpedfavyfcqqgntlpytfgqgtkleiktttpaprpptpaptias
qplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgr
kkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeaysei
gmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR4 CAR4 scFv 4
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domain
tyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq
gtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskyl
nwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcq
qgntlpytfgqgtkleik 103106 64
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR4-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Soluble
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc scFv-nt
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtcc
ctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaa
atacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctacc
acacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcggg
accgactacactctgaccatctcatctctccagcccgaggacttcgccgtctactt
ctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgaga
tcaaacatcaccaccatcatcaccatcac 103106 76
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR4-
wirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv-aa
lspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsg
tdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 104878 88
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 4-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-nt
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtcc
ctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaa
atacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctacc
acacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcggg
accgactacactctgaccatctcatctctccagcccgaggacttcgccgtctactt
ctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgaga
tcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctct
cagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgca
tacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggta
cttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcgg
aagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactca
agaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaac
tgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaac
cagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaa
gcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaag
agggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagatt
ggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggact
cagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104878
34 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR 4-
wirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadta Full-aa
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls
lspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsg
tdytltisslqpedfavyfcqqgntlpytfgqgtkleiktttpaprpptpaptias
qplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgr
kkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeaysei
gmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 5 CAR5 scFv 5
eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domain
giparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs
ggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqpp
gkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycak
hyyyggsyamdywgqgtlvtvss 99789 65
atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR5-
tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Soluble
agagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nt
tatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcct
ccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccc
tcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcagggg
aatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcgg
aggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaag
tgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctg
acttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgcca
gcctccggggaagggtcttgaatggattggggtgatttggggatcagagactactt
actactcttcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaac
caagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattg
tgccaaacattactattacggagggtcttatgctatggactactggggacagggga
ccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789 77
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR5-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFV-aa
tctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 104879 89
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR5-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagcggcggaggcgggagccagg
tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactg
acttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagaca
gccaccggggaagggtctggaatggattggagtgatttggggctctgagactactt
actactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat
caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattg
cgctaagcattactattatggcgggagctacgcaatggattactggggacagggta
ctctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggct
cctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 104879 35
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR5-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Full-aa
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl
tctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvsstttpaprpptpa
ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitly
ckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapay
kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmae
ayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 6 CAR6 6
eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs scFv
giparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs domain
ggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqpp
gkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycak
hyyyggsyamdywgqgtlvtvss 99790 66
atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR6-
tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Soluble
agagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nt
tatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcct
ccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccc
tcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcagggg
aatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcgg
aggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaag
tgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctg
acttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgcca
gcctccggggaagggtcttgaatggattggggtgatttggggatcagagactactt
actaccagtcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaac
caagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattg
tgccaaacattactattacggagggtcttatgctatggactactggggacagggga
ccctggtgactgtctctagccatcaccatcaccaccatcatcac 99790 78
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR6-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFv-aa
tctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 104880 90
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR6-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagcggaggcggagggagccagg
tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactg
acttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagaca
gccaccggggaagggtctggaatggattggagtgatttggggctctgagactactt
actaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat
caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattg
cgctaagcattactattatggcgggagctacgcaatggattactggggacagggta
ctctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggct
cctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 104880 36
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasgdiskylnw CAR6-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Full-aa
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl
tctvsgvslpdygvswirqppgkglewigviwgsettyygsslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvsstttpaprpptpa
ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitly
ckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapay
kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmae
ayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 7 CAR7 scFv 7
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domain
tyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq
gtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqd
iskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfa
vyfcqqgntlpytfgqgtkleik 100796 67
atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CAR7-
caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Soluble
ctctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nt
tggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttc
tgaaaccacctactactcatcttccctgaagtccagggtgaccatcagcaaggata
attccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgcc
gtgtattactgcgccaagcactactattacggaggaagctacgctatggactattg
gggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggag
gatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtca
ccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccag
ccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctc
gcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagc
ggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaaga
tttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagg
gaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796 79
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR7-
wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqs scFv-aa
patlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfs
gsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 104881 91
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 7
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-nt
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagc
cctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatc
acaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggccccta
ggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagc
gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgagga
cttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagg
gcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgcc
ccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 104881 37
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR 7
wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Full-aa
vyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqs
patlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfs
gsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleiktttpaprpptpa
ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitly
ckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapay
kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmae
ayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 8 CAR8 scFv 8
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domain
tyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq
gtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqd
iskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfa
vyfcqqgntlpytfgqgtkleik 100798 68
atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CAR8-
caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Soluble
ctctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nt
tggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttc
tgaaaccacctactaccagtcttccctgaagtccagggtgaccatcagcaaggata
attccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgcc
gtgtattactgcgccaagcactactattacggaggaagctacgctatggactattg
gggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggag
gatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtca
ccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccag
ccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctc
gcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagc
ggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaaga
tttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagg
gaaccaagctcgaaatcaagcaccatcaccatcatcatcaccac 100798 80
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR8-
wirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqs scFv-aa
patlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfs
gsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 104882 92
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 8-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-nt
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccggaggcggtgggtcagaaatcgtgatgacccagagc
cctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatc
acaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggccccta
ggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagc
gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgagga
cttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagg
gcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgcc
ccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 104882 38
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR 8-
wirqppgkglewigviwgsettyygsslksrvtiskdnsknqvslklssvtaadta Full-aa
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqs
patlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfs
gsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleiktttpaprpptpa
ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitly
ckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapay
kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmae
ayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 9 CAR9 scFv 9
eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domain
giparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs
ggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqpp
gkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycak
hyyyggsyamdywgqgtlvtvss 99789 69
atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR9-
tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Soluble
agagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nt
tatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcct
ccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccc
tcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcagggg
aatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcgg
aggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaag
tgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctg
acttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgcca
gcctccggggaagggtcttgaatggattggggtgatttggggatcagagactactt
actacaattcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaac
caagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattg
tgccaaacattactattacggagggtcttatgctatggactactggggacagggga
ccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789 81
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR9-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFv-aa
tctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 105974 93
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 9-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccagg
tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactg
acttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagaca
gccaccggggaagggtctggaatggattggagtgatttggggctctgagactactt
actacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat
caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattg
cgctaagcattactattatggcgggagctacgcaatggattactggggacagggta
ctctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggct
cctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 105974 39
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR 9-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Full-aa
ntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl
tctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnskn
qvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvsstttpaprpptpa
ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitly
ckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapay
kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmae
ayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR10 CAR10 10
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset scFv
tyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq domain
gtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqd
iskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfa
vyfcqqgntlpytfgqgtkleik 100796 70
atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CAR10-
caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Soluble
ctctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nt
tggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttc
tgaaaccacctactacaactcttccctgaagtccagggtgaccatcagcaaggata
attccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgcc
gtgtattactgcgccaagcactactattacggaggaagctacgctatggactattg
gggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggag
gatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtca
ccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccag
ccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctc
gcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagc
ggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaaga
tttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagg
gaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796 82
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR10-
wirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqs scFv-aa
patlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfs
gsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 105975 94
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 10
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccagg
tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactg
acttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagaca
gccaccggggaagggtctggaatggattggagtgatttggggctctgagactactt
actacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat
caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattg
cgctaagcattactattatggcgggagctacgcaatggattactggggacagggta
ctctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggct
cctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 105975 40
MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNW CAR 10
YQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQG Full-aa
NTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL
TCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKN
QVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY
CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR11 CAR11 11
eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs scFv
giparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs domain
ggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkgle
wigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyg
gsyamdywgqgtlvtvss 103101 71
Atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR11-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Soluble
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nt
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactacaattcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagccaccaccatcatcaccatcaccat 103101 83
MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR11-
yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Soluble
ntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aa
gvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslk
lssvtaadtavyycakhyyyggsyamdywgqgtlvtvsshhhhhhhh 105976 95
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 11
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-nt
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagc
cctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatc
acaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggccccta
ggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagc
gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgagga
cttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagg
gcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgcc
ccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagc
tggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg
cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttac
tgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcc
tgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagg
aaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctac
aagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagta
cgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgca
gaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaa
gcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacgg
actgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgc
aggccctgccgcctcgg 105976 41
MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVS CAR 11
WIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTA Full-aa
VYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQS
PATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFS
GSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY
CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR12 CAR12 12
qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset scFv
tyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq domain
gtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskyl
nwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcq
qgntlpytfgqgtkleik 103104 72
atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR12-
tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Soluble
ctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc
scFv-nt tggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtag
cgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggata
actcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgcc
gtgtattactgtgccaagcattactactatggagggtcctacgccatggactactg
gggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcg
ggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtcc
ctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaa
atacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctacc
acacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcggg
accgactacactctgaccatctcatctctccagcccgaggacttcgccgtctactt
ctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgaga
tcaaacatcaccaccatcatcaccatcac 103104 84
MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR12-
wirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadta Soluble
vyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv-aa
lspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsg
tdytltisslqpedfavyfcqqgntlpytfgqgtkleikhhhhhhhh 105977 96
atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 12-
tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-nt
agcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg
tatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggct
ccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccc
tcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaaggg
aacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggagg
tggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaa
gcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagc
ggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaaggg
tctggaatggattggagtgatttggggctctgagactacttactacaactcatccc
tcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaa
ctgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattacta
ttatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgt
ccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcc
cagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgca
tacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggta
cttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcgg
aagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactca
agaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaac
tgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaac
cagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaa
gcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaag
agggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagatt
ggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggact
cagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 105977
42 MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNW CAR 12-
YQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQG Full-aa
NTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVS
GVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDMSKMQVSLK
LSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIAS
QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR
KKLLYIFKQPFMRPVQTTQEEDGCSCREPEEEEGGCELRVKFSRSADAPAYKQGQN
QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKMPQEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
TABLE-US-00018 TABLE 7 Murine CD19 CAR Constructs CTL019 CTL019- 97
atggccctgcccgtcaccgctctgctgctgccccttgctctgcttcttcatgcagc Soluble
aaggccggacatccagatgacccaaaccacctcatccctctctgcctctcttggag
scFv-Histag-
acagggtgaccatttcttgtcgcgccagccaggacatcagcaagtatctgaactgg nt
tatcagcagaagccggacggaaccgtgaagctcctgatctaccatacctctcgcct
gcatagcggcgtgccctcacgcttctctggaagcggatcaggaaccgattattctc
tcactatttcaaatcttgagcaggaagatattgccacctatttctgccagcagggt
aataccctgccctacaccttcggaggagggaccaagctcgaaatcaccggtggagg
aggcagcggcggtggagggtctggtggaggtggttctgaggtgaagctgcaagaat
caggccctggacttgtggccccttcacagtccctgagcgtgacttgcaccgtgtcc
ggagtctccctgcccgactacggagtgtcatggatcagacaacctccacggaaagg
actggaatggctcggtgtcatctggggtagcgaaactacttactacaattcagccc
tcaaaagcaggctgactattatcaaggacaacagcaagtcccaagtctttcttaag
atgaactcactccagactgacgacaccgcaatctactattgtgctaagcactacta
ctacggaggatcctacgctatggattactggggacaaggtacttccgtcactgtct
cttcacaccatcatcaccatcaccatcac CTL019- 98
MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnw Soluble
yqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqg
scFv-Histag-
ntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvs aa
gvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflk
mnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsshhhhhhhh CTL019 99
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgc Full-nt
caggccggacatccagatgacacagactacatcctccctgtctgcctctctgggag
acagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattgg
tatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagatt
acactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctc
tcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggt
aatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcgg
tggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagt
caggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctca
ggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaaggg
tctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctc
tcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaa
atgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattatta
ctacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtct
cctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcg
cagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca
cacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccggga
cttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcaga
aagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactca
agaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaac
tgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaac
cagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaa
gagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg
aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagatt
gggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtct
cagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctc gc CTL019
58 MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnw Full-aa
yqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqg
ntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvs
gvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflk
mnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsstttpaprpptpaptias
qplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgr
kkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn
qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeaysei
gmkgerrrgkghdglyqglstatkdtydalhmqalppr CTL019 59
diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhs scFv
gvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggs domain
ggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkgle
wlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyg
gsyamdywgqgtsvtvss
[0772] The sequences of humanized CDR sequences of the scFv domains
are shown in Table 4 for the heavy chain variable domains and in
Table 5 for the light chain variable domains. "ID" stands for the
respective SEQ ID NO for each CDR.
TABLE-US-00019 TABLE 4 Heavy Chain Variable Domain CDRs (Kabat)
Candidate FW HCDR1 ID HCDR2 ID HCDR3 ID murine_CART19 GVSLPDYGVS 19
VIWGSETTYYNSALKS 20 HYYYGGSYAMDY 24 humanized_CART19 a VH4
GVSLPDYGVS 19 VIWGSETTYY S LKS 21 HYYYGGSYAMDY 24 humanized_CART19
b VH4 GVSLPDYGVS 19 VIWGSETTYY S LKS 22 HYYYGGSYAMDY 24
humanized_CART19 c VH4 GVSLPDYGVS 19 VIWGSETTYYNS LKS 23
HYYYGGSYAMDY 24
TABLE-US-00020 TABLE 5 Light Chain Variable Domain CDRs Candidate
FW LCDR1 ID LCDR2 ID LCDR3 ID murine_CART19 RASQDISKYLN 25 HTSRLHS
26 QQGNTLPYT 27 humanized_CART19 a VK3 RASQDISKYLN 25 HTSRLHS 26
QQGNTLPYT 27 humanized_CART19 b VK3 RASQDISKYLN 25 HTSRLHS 26
QQGNTLPYT 27 humanized_CART19 c VK3 RASQDISKYLN 25 HTSRLHS 26
QQGNTLPYT 27
[0773] Table 6 is an identification key correlating the CD19
constructs numerical names to the specific orientation of the light
and heavy chains of the scFv, the number of linker units (i.e.,
(G4S).sub.3 (SEQ ID NO: 107) or (G4S).sub.4 (SEQ ID NO: 106)),
separating the heavy and light chains, and the distinguishing amino
acid sequences in the heavy chain CDR2.
TABLE-US-00021 TABLE 6 CD19 CAR designations. Clone Alt. Chain Site
of Heavy SEQ ID ID/CAR# Clone ID Orientation Linkers CDR2 mutation
NO 104875 C2136 L2H 3x YSSSL 28 (CAR1) 104876 C2137 L2H 3x YQSSL 29
(CAR2) 104877 C2138 H2L 3x YSSSL 28 (CAR3) 104878 C2139 H2L 3x
YQSSL 29 (CAR4) 104879 C2140 L2H 4x YSSSL 28 (CAR5) 104880 C2141
L2H 4x YQSSL 29 (CAR6) 104881 C2142 H2L 4x YSSSL 28 (CAR7) 104882
C2143 H2L 4x YQSSL 29 (CAR8) 105974 C2144 L2H 4x YNSSL 30 (CAR9)
105975 C2145 H2L 4x YNSSL 30 (CAR10) 105976 C2146 L2H 3x YNSSL 30
(CAR11) 105977 C2147 H2L 3x YNSSL 30 (CAR12) CTL019 muCART19 L2H 3x
YNSAL 57
[0774] The CAR scFv fragments were then cloned into lentiviral
vectors to create a full length CAR construct in a single coding
frame, and using the EF1 alpha promoter for expression (SEQ ID NO:
100).
TABLE-US-00022 EF1 alpha promoter (SEQ ID NO: 100)
CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTC
CCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAG
GTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTT
TTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAAC
GTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTG
TGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTT
GAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGG
GTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTC
GCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGC
GAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA
GCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGA
TAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTG
GGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCG
AGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA
AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCC
CGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAA
AGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG
GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCT
TTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCG
TCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGG
TTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGG
AGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTT
GCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGT
TCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA.
Analysis of the humanized CAR constructs was conducted as described
in Example 4.
Example 4: Analysis of Humanized CD19 Constructs in CART
[0775] To evaluate the feasibility of targeting CD19 via a CAR
technology, the single chain variable fragments for an anti-CD19
antibody is cloned into a lentiviral CAR expression vector with the
CD3zeta chain and the 4-1BB costimulatory molecule in four
different configurations and the optimal construct is selected
based on the quantity and quality of the effector T cell response
of CD19 CAR transduced T cells ("CART19" or "CART19 T cells") in
response to CD19+ targets. Effector T cell responses include, but
are not limited to, cellular expansion, proliferation, doubling,
cytokine production and target cell killing or cytolytic activity
(degranulation).
Materials and Methods
Generation of Redirected Humanized CART19 T Cells
[0776] The humanized CART19 lentiviral transfer vectors are used to
produce the genomic material packaged into the VSVg psuedotyped
lentiviral particles. Lentiviral transfer vector DNA is mixed with
the three packaging components of VSVg, gag/pol and rev in
combination with lipofectamine reagent to transfect them together
in to 293T cells. After 24 and 48 hr, the media is collected,
filtered and concentrated by ultracentrifugation. The resulting
viral preparation is stored at -80 C. The number of transducing
units is determined by titration on SupT1 cells. Redirected CART19
T cells are produced by activating fresh naive T cells by engaging
with CD3.times.28 beads for 24 hrs and then adding the appropriate
number of transducing units to obtain the desired percentage of
transduced T cells. These modified T cells are allowed to expand
until they become rested and come down in size at which point they
are cryopreserved for later analysis. The cell numbers and sizes
are measured using a coulter multisizer III. Before cryopreserving,
percentage of cells transduced (expressing the CART19 on the cell
surface) and their relative fluorescence intensity of that
expression are determined by flow cytometric analysis on an LSRII.
From the histogram plots, the relative expression levels of the
CARs can be examined by comparing percentage transduced with their
relative fluorescent intensity.
Evaluating Cytolytic Activity, Proliferation Capabilities and
Cytokine Secretion of Humanized CART19 Redirected T Cells.
[0777] To evaluate the functional abilities of humanized CAR19 T
cells to kill, proliferate and secrete cytokines, the cells are
thawed and allowed to recover overnight. In addition to the
humanized CART19, the murine CART19 was used for comparative
purposes while SS1-BBz was used as non-targeting expressed CAR for
background CAR/T cell effect. The "control" gold standard (GS)
CART19 was used in all assays to compare assay variation.
Importantly, the GS CART19 are cells produced in research grade
(i.e., not clinical grade) manufacturing conditions and include the
addition of IL-2 to the growth culture. This likely impacts the
overall viability and functionality of these cells and should not
be evaluated as a direct comparison to the research grade
production of the other transduced T cell populations. The T cell
killing was directed towards K562, a chronic myelogenous leukemia
cell line expressing or not expressing CD19 or Pt14, B cells
isolated from CLL patients. For this flow based cytotoxicity assay,
the target cells are stained with CSFE to quantitate their
presence. The target cells were stained for CD19 expression to
confirm similar target antigens levels. The cytolytic activities of
CAR19 T cells are measured at a titration of effector:target cell
ratios of 10:1, 3:1, 1:1, 0.3:1 and 0:1 where effectors were
defined as T cells expressing the anti-CD19 chimeric receptor.
Assays were initiated by mixing an appropriate number of T cells
with a constant number of targets cells. After 16 hrs, total volume
of each mixture was removed and each well washed combining
appropriately. The T cells were stained for CD2 and all cells
stained with live/dead marker 7AAD. After the final wash, the
pelleted cells were re-suspended in a specific volume with a
predetermined number of counting beads. Cell staining data was
collected by LSRII flow cytometry and analyzed with FloJo software
using beads to quantitate results.
[0778] For measuring cell proliferation and cytokine production of
humanized CAR19 T cells, cells are thawed and allowed to recover
overnight. In addition to the humanized CART19, the murine CART19
was used for comparative purposes while SS1-BBz was used as a
non-targeting expressed CAR for background CAR/T cell effect. The
"control" gold standard (GS) CART19 was used in all assays to
compare assay variation. The T cells were directed either towards
K562, a chronic myelogenous leukemia cell line expressing or not
expressing CD19 or Pt14, B cells isolated from CLL patients. In
addition, CD3.times.28 beads were used to evaluate the potential of
T cells to respond to the endogenous immunological signals. To
analyze proliferation, T cells were stained with CSFE. The
proliferation is the dilution of the CSFE stain reflecting the
separation of the parental markings now into two daughter cells.
The assay tests only an effector:target ratios of 1:1 and 1:0 where
effectors were defined as T cells expressing the anti-CD19 chimeric
receptor. The assay is done in duplicate and 24 hrs after mixing of
the cells, 50% of the media is removed/replaced for cytokine
analysis using the Luminex 10-plex panel of human cytokines
detection. After 5 days, T cells were stained for CAR expression,
phenotyped as either CD4 or CD8 cells and stained for live/dead
with 7AAD. After the final wash, the pelleted cells were
re-suspended in a specific volume with a predetermined number of BD
counting beads. Cell staining data was collected by LSRII flow
cytometry and analyzed with FloJo software using beads to
quantitate results. Total cell counts were determined by number of
cells counted relative to a specific number of beads multiplied by
the fraction of beads yet to be counted.
[0779] To evaluate the potential for the humanized CART19 cells to
function similarly to the currently successful murine CART19, we
wanted to assess in vitro their ability to kill targeted cells, to
proliferate in response to the targeted antigen and to show signs
of persistence. By packaging each of the humanized CART19
lentiviral constructs and titering them on SupT1 cells, we are able
to determine the amount of virus to normalize transductions to be
around 50%. This allows for more direct comparisons of activity
starting with similar average intergration sites per cell.
[0780] The therapeutic CAR19 T cells are generated by starting with
the blood from a normal apheresed donor whose naive T cells are
obtained by negative selection for T cells, CD4+ and CD8+
lymphocytes. These cells are activated by CD3.times.28 beads in 10%
RPMI at 37C, 5% CO.sub.2.
[0781] After 24 hrs, the T cells are blasting and the normalized
amount of virus is added. The T cells begin to divide into a
logarithmic growth pattern which is monitored by measuring the cell
counts per ml and cell size. As the T cells begin to rest down, the
logarithmic growth wanes and the cell size shrinks. The combination
of slowing growth rate and T cell size approaching .about.300 fl
determines the state for T cells to be cryopreserved or
restimulated.
[0782] There is a very similar trend of T cells resting down as
seen by size. The almost overlapping pattern between the humanized
CART cells with the current murine CART19 and UTD population
indicates no unusual effect of the humanized CAR19 on the normal T
cell expansion following activation. As a control, SS1-BBz is used
to define unwanted antigen independent CAR activity. The expansion
profile in total cell numbers shows the differences in the actual
numbers in the individual expansions are likely due mainly to
different starting number of cells. By normalizing starting T cell
numbers, a tight cluster is seen for all the CART19 cells. In
addition, the unwanted effect of antigen independent CAR activation
is detected in the line running lower and away from the group.
[0783] The level of surface expression for each of these CAR19
expressing cells was determined. The titered virus normalized for
transduction show comparable expression levels correlating with
transduction efficiency, percent cells transduced. Some CARs had
their titers extrapolated from earlier packagings, and though their
percentages transduced are lower, their MFI are also reduced as
expected. The results indicate that there is no detectable negative
effect of the humanized CAR19 on the cells ability to expand
normally when compared to the UTD and murine CAR19 T cells.
[0784] The ability of the humanized CART19 cells to selectively
discern a cell surface specific epitope expressed on cells and
destroy them is analyzed. Wild type K562 cells do not express CD19
but can be transduced to express CD19. Comparing these killing
curves, titrating the amount of effector cells shows that those
cells expressing CD19 are destroyed. Redirected T cells from the
same donor and modified with either humanized CART19 cells or
current clinical murine CART19 cells indicate no difference in
their ability to kill. The killing curves show that a very similar
killing capacity is found among humanized CART19 cells targeting
CD19+ CLL cells from patient 14. Interestingly, there is a decrease
in overall cytolytic activity, in particularly GS CART19,
suggesting these cells may possess specific inhibitory properties.
The similar level of CD19 expressed on the targets cells indicates
the expression level is not the reason for differences in cell
killing.
[0785] The necessary property of the humanized CART19 cells to
proliferate after seeing target cells is found in all constructs
after being stimulated by the control CD3.times.28 beads and the
CD19 expressing targets. Targeting Pt14 CLL cells appear to
indicate a slightly greater proliferation rate with scFvs with a
light to heavy chain orientation with no bias seen when having a
3.times. or 4.times.GGGGS linkage (SEQ ID NOS 107 and 106,
respectively). The proliferative results reflect the total number
of cells accumulated over the 5 days, indicating that the humanized
CART19s, 2146, 2144, 2136, 2141 and 2137 drive a more proliferative
signal to the T cells. Impressively, this was detected in the
humanized CART19 cells targeting Pt14 CLL cells.
[0786] Overall, the humanized CART19 constructs exhibit very
similar characteristics to the current murine CART19 in cytolytic
activity, proliferative response and cytokine secretion to antigen
specific targets. The potential of humanized CART19 cells, (2146,
2144, 2136, 2141 and 2137), to drive a more proliferative signal to
the T cells upon target activation would seem to be an extra
benefit of these new constructs to potentially enhance therapeutic
response.
Results
[0787] Using both degranulation and cytokine production assays, it
is demonstrated that the engineered CART19 T cells specifically
target CD19+ cells.
[0788] ND317 cells transduced with humanized CD19CAR constructs
(a.k.a. "huCART19") of the invention were analyzed. There was a
tight similarity in size of the T cells during their expansions
after CD3.times.28 activation and transduction with the humanized
CART19 candidates relative to the murine CART19 and unmodified
(UTD) T cells.
[0789] Experiments showed little difference in the number of T
cells that accumulated during their expansions after CD3.times.28
activation and transduction with the different humanized CART19
candidates relative to the murine CART19 and unmodified (UTD) T
cells.
[0790] Cell surface expressions of humanized CART19 are comparable
and their expression level very similar to murine CART19. The
overlay of histograms plotting the cell surface expression staining
pattern of each humanized CART19 transduced T cells and the mean
fluorescent intensity (MFI) calculated from these profiles
correlates well with the percentage of cells transduced.
[0791] Furthermore the humanized CART19 have similar specific
cytotoxic activities in targeting CD19 expressing target cells and
comparable to murine CART19. Plots from 16 hr-flow-based killing
assays using titrating Effector to Target (E:T) ratios with
effector humanized CART19 cells targeting CSFE labeled K562cc (FIG.
1A. non-expressing CD19 controls), K562.CD19 (FIG. 1B, K562 cells
transduced to express CD19) or Pt14 (FIG. 1C, B cells from CLL
patient). The cytolytic activities of all the humanized CART19
cells are similar and comparable to the murine CART19. The
differences in the cytolytic activity between different targets is
similar and comparable indicating the murine CART19's activity is
preserved in the humanized form of CART19.
[0792] Histogram overlays of CFSE marked humanized CART19 cells 6
days after being mixed with target cells show their proliferative
capacity (FIG. 5). The proliferative response delivered from the
CAR19 is a necessary response after engagement with and killing of
target cells to develop a positive clinical response. The dilution
of SS1-BBz CSFE staining, an indicator of dividing daughter cells
diluting out the parental cell's stain, is a result of unrested T
cells maintaining divisions in a targeting independent
mechanism.
[0793] The cell populations overall ability to proliferate is
evaluated with CD3.times.28 beads which mimics the endogenous
engagement of the TCR and the co-stimulator CD28. Data indicates
each cell population has a comparable proliferation potential. All
humanized and murine CART19 cells proliferate strongly and
comparably upon engagement with K562 cells expressing CD19.
Humanized CART19 cells also responded well to B cells obtained from
a CLL patient though some seem to respond slightly less. As shown
in FIGS. 2A and 2B, the humanized CART19 cells 2136, 2137, 2140,
2141, 2144 and 2146 can be seen to have a slightly more robust
proliferation as evidenced by the greater dilution of CSFE
staining. These constructs all have the same variable chain
orientation of light to heavy, indicating that this is the
orientation of choice. A closer look at the amino acid changes in
the heavy CDR2 site (Table 1) reveals that each of the three
variations YSSSL, YQSSL and YNSSL (SEQ ID NOS:28, 29 and 30,
respectively) are represented in the constructs that appeared to
have the more robust proliferations after seeing targets. In
addition, these observed constructs have both the G4S linker
containing 3 copies of the subunit (3G4S) (SEQ ID NO: 107) and the
G4S linker containing 4 copies of the subunit (4G4S) (SEQ ID NO:
106), indicating the linker size did not influence function.
[0794] From the proliferative expansions described above, the total
cell numbers after 5 days post tumor engagement is determined. The
cells show a decline in numbers than were initially seeded,
indicating activation is required to maintain survival. An
endogenous activation control is analyzed to show that the total
cell count at the end of 6 days was similar. Humanized CART19 cells
targeting K562 cells expressing CD19 show that the two murine
CART19 cells both end up with the higher cell numbers, with 2146
slightly above all the other constructs with similar values. Total
cell numbers were also analyzed 6 days after exposure to B cells
from Patient 14 (pt14), and interestingly shows that the previously
selected out humanized CART19 constructs 2146, 2144, 2136, 2141 and
2137, all of which have the light to heavy chain orientation and
represent the three amino acid variations YSSSL, YQSSL and YNSSL
(SEQ ID NOS: 28, 29 and 30, respectively), resulted in higher total
cell numbers, higher than the murine CART19s. This unexpected
differentiation between the various humanized anti-CD19CAR clones
may translate to better clinical efficacy of CART cells transduced
with these constructs.
[0795] Background levels of cytokine produced from humanized CART19
cells after exposure to the control K562 cells not expressing CD19
were analyzed. 24 hr supernatants were analyzed using a luminex
30-plex panel. The potential cytokine profile from stimulation of
the endogenous immune system with the CD3.times.28 beads indicate
each of the cell populations have a comparable cytokine
profile.
[0796] Data also shows that the humanized CART19 and murine CART19
produce similar cytokine profiles at similar levels when responding
to the same targets. The cytokine profile was lower but similar
when targeting the Pt14 target cells.
Example 5: Humanized CD19 CAR T Cell Treatment in an In Vivo ALL
Model
[0797] Primary human ALL cells can be grown in immune compromised
mice without having to culture them in vitro. These mice can be
used to test the efficacy of chimeric antigen receptor (CAR) T
cells in a model that represents the patient population that will
be found in the clinic. The model used here, HALLX5447, was
passaged twice in NOD.Cg-Prkdc.sup.scidIl2rg.sup.tm1WjlSzJ (NSG)
mice, prior to use in studies testing the efficacy of CAR T
cells.
[0798] Murine CD19 CAR T cells have previously been shown to target
and kill leukemia cells in an NSG mouse model of primary human ALL.
The CD19 scFv (single chain Fc variable fragment) has been
humanized and the present example compares the ability of T cells
expressing a humanized CD19 CAR (CAR 2) to eliminate ALL tumor
cells in vivo to that of the murine CD19 CAR T cells. Here, the
efficacy of these cells has been directly compared in mice with
established primary human ALL, as assayed by peripheral blood FACS
analysis of human CD19 cells. Following an implant of
1.5.times.10.sup.6 primary ALL cells intravenously, a disease
burden of 2.5-4% CD19 human cells in the blood was achieved by 2
weeks post-tumor implantation. This CD19 percentage is of total
cells in the blood of the mice. 100% of human cells in the mice
prior to treatment with CAR T cells are tumor cells. Percentages
above 2% CD19 human cells in the peripheral blood are considered to
be established human ALL disease in this model. The
leukemia-bearing mice were treated with the CAR T cells once the
leukemia is established in the mice, approximately two to three
weeks after tumor implantation. Mice in each group were treated
with 5.times.10.sup.6 total human T cells. The transduction
efficiencies of the donor human T cells with the CAR expressing
lentivirus were between 40-60%. Following treatment with the T
cells, mice were bled weekly for analysis of the percentage of CD19
human cells in the blood as a biomarker for disease
progression.
Materials and Methods:
[0799] Primary Human ALL Cells:
[0800] Primary cells were not cultured in vitro prior to
implantation. These cells were harvested from a patient with ALL
and then transferred into mice for establishment and expansion.
After the tumor cells were expanded in the mice, the bone marrow
and splenocytes were harvested and viably frozen in separate
batches for re-implantation. The cells were frozen in 90% FBS and
10% DMSO at a minimum concentration of 5.times.10.sup.6 cells per
milliliter. For re-implantation, the frozen ALL cells were thawed
and then injected intravenously in to NSG mice, in order to
generate mice with ALL that will be used to compare the anti-tumor
efficacy of the humanized CD19 CAR T cells and the murine CD19 CAR
T cells.
[0801] Mice:
[0802] 6 week old NSG (NOD.Cg-Prkdc.sup.scidIl2rg.sup.tm1Wjl/SzJ)
mice were received from the Jackson Laboratory (stock number
005557). Animals were allowed to acclimate to the Novartis NIBRI
animal facility for at least 3 days prior to experimentation.
Animals were handled in accordance with Novartis ACUC regulations
and guidelines.
[0803] Tumor Implantation:
[0804] In vivo serially passaged primary human ALL cells, model
HALLX5447, were thawed in a 37.degree. C. water bath. The cells
were then transferred to a 15 ml conical tube and washed twice with
cold sterile PBS. The primary ALL cells were then counted and
resuspended at a concentration of 15.times.10.sup.6 cells per
milliliter of PBS. The cells were placed on ice and immediately
(within one hour) implanted in the mice. The ALL cells were
injected intravenously via the tail vein in a 100 .mu.l volume, for
a total of 1.5.times.10.sup.6 cells per mouse.
[0805] CAR T Cell Dosing:
[0806] Mice were administered 5.times.10.sup.6 T cells 16 days
after tumor implantation. Cells were partially thawed in a 37
degree Celsius water bath and then completely thawed by the
addition of 1 ml of cold sterile PBS to the tube containing the
cells. The thawed cells were transferred to a 15 ml falcon tube and
adjusted to a final volume of 10 mls with PBS. The cells were
washed twice at 1000 rpm for 10 minutes each time and then counted
on a hemocytometer. T cells were then resuspended at a
concentration of 50.times.10.sup.6 cells per ml of cold PBS and
kept on ice until the mice were dosed. The mice were injected
intravenously via the tail vein with 100 .mu.l of the CAR T cells
for a dose of 5.times.10.sup.6 T cells per mouse. Five mice per
group were treated either with 100 .mu.l of PBS alone (PBS),
untransduced T cells (Mock), murine CD19 CAR T cells (muCTL019), or
humanized CD19 CAR T cells (huCTL019). The untransduced T cells,
muCTL019 T cells, and huCTL019 T cells were all prepared from the
same human donor in parallel.
[0807] Animal Monitoring:
[0808] The health status of the mice was monitored daily, including
twice weekly body weight measurements. The percent change in body
weight was calculated as
(BW.sub.current-BW.sub.initial)/(BW.sub.initial).times.100%. Tumor
burden was monitored weekly by peripheral blood FACS analysis. Mice
were bled weekly via the tail vein into EDTA coated tubes that were
kept on ice. 10-20 .mu.l of blood was plated from the tubes into 96
well plates on ice. Red blood cells were lysed with ACK red blood
cell lysis buffer (Life Technologies, catalog number A10492-01) and
then washed twice with cold PBS. The cells were incubated with an
Fc blocking mix of human and mouse Fc block (Miltenyi Biotec,
catalog numbers 130-059-901 and 130-092-575) for 30 minutes and
then incubated with an anti-human CD19 antibody for 30 minutes. The
cells were fixed with a 2% paraformaldehyde solution for 20
minutes, washed and stored in PBS+2% FBS overnight prior to
analysis on a BD Canto or Fortessa, followed by further analysis
using the FlowJo FACS analysis software. The cells were analyzed to
determine the percent of human CD19.sup.+ cells in the blood of the
human HALLX5447 ALL tumor-bearing NSG mice. CD19 percentages in the
blood are reported as the mean.+-.standard error of the mean
(SEM).
[0809] Percent treatment/control (T/C) values were calculated using
the following formula:
% T/C=100 .times..DELTA.T/.DELTA.C if .DELTA.T.gtoreq.0;
% Regression=100 .times..DELTA.T/T.sub.initial if
.DELTA.T<0;
where T=mean peripheral blood CD19 percentage of the drug-treated
group on the final day of the study; T.sub.initial=peripheral blood
CD19 percentage of the drug-treated group on initial day of dosing;
.DELTA.T=mean peripheral blood CD19 percentage of the drug-treated
group on the final day of the study-mean peripheral blood CD19
percentage of the drug treated group on the initial day of dosing;
C=mean peripheral blood CD19 percentage of the control group on the
final day of the study; and .DELTA.C=mean peripheral blood CD19
percentage of the control group on the final day of the study -mean
peripheral blood CD19 percentage of the control group on the
initial day of dosing.
[0810] T/C values in the range of 100% to 42% are interpreted to
have no or minimal anti-tumor activity; T/C values that are
.ltoreq.42% and >10% are interpreted to have anti-tumor activity
or tumor growth inhibition. T/C values .ltoreq.10% or regression
values .gtoreq.-10% are interpreted to be tumor stasis. Regression
values <-10% are reported as regression.
Results:
[0811] The anti-tumor activity of murine and humanized CD19 CAR T
cells were evaluated and directly compared in a primary model of
human ALL. Following tumor implantation on day 0, mice were
randomized into treatment groups and treated with 5.times.10.sup.6
T cells intravenously on day 16. ALL disease burden and animal
health were monitored until animals achieved endpoint. The mice in
all the groups were euthanized on day 65 post-tumor implantation
when disease burden in the control groups was above 80% human
CD19.sup.+ cells in the peripheral blood.
[0812] A clear difference in disease burden was seen between the
control groups and the groups treated with either the murine or the
humanized CD19 CAR T cells with P<0.01 from day 24 after tumor
implantation, and continuing to the end of the study at day 65. The
murine and human CD19 CAR T cells demonstrate a similar ability to
control human HALLX5447 ALL tumor cell growth in NSG mice. Both
groups showed a peak peripheral blood disease level of 12-15% human
CD19.sup.+ cells at day 21 post HALLX5447 implantation. 42 days
after tumor cell implantation, no human CD19.sup.+ cells were
detectable in the huCTL019 group, while the percentage of human
CD19.sup.+ cells in the muCTL019 group dropped to about 1%. Both
the murine and the humanized CD19 CAR T cells resulted in a
comparable ability to control the expansion of primary human ALL
cells in this model (P>0.05). The % T/C values for the mock
transduced T cell group was 94.40%, demonstrating that the mock
transduced T cells had no anti-tumor activity. The percent
regression of the muCTL019 group was -89.75% and the huCTL019 group
was -90.46%, demonstrating that both of these treatments were able
to cause a regression of the HALLX5447 tumor model. The peripheral
blood human CD19.sup.+ cell percentages as a measure of the disease
burden in these mice is shown in FIG. 7. The PBS treatment group,
which did not receive any T cells, demonstrated baseline primary
ALL tumor growth kinetics in intravenously implanted NSG mice. The
Mock treatment group received untransduced T cells that underwent
the same in vitro expansion process as the CAR T cells. These cells
serve as a T cell control to show the non-specific response of the
T cells in this tumor model. Both the PBS and Mock transduced T
cell treatment groups demonstrated continuous tumor progression
throughout the experiment. Both the murine and the humanized CD19
CAR T cells control the progression of disease within one week of
the 5.times.10.sup.6 T cell injections and demonstrate a similar
ability to sustain disease control over the course of this 65 day
study.
[0813] The anti-tumor activity of murine and humanized CD19 CAR
transduced T cells was assessed in an efficacy study in NSG mice
bearing a primary human ALL model, HALLX5447. This study
demonstrated that both the murine and humanized CD19 CAR T cells
(muCTL019 and huCTL019) are capable of mounting an anti-tumor
response in a primary model of human ALL. In addition, this
response, as assayed by peripheral blood disease burden is the same
for the muCTL019 and huCTL019 cells. Both the murine and humanized
CD19 CAR T cells control primary ALL growth within a week of the
mice being dosed with the T cells. Initially after treatment, the
disease burden continued to increase before decreasing to virtually
undetectable levels. One treatment with either the murine or
humanized CAR T cells resulted in a sustained anti-tumor response
over the course of the 65 day disease progression in control
treated mice. The humanized CD19 CAR T cells demonstrated a similar
ability to mount an efficacious anti-CD19 tumor response and
control ALL disease burden as was seen with the murine CD19 CAR T
cells.
Example 6: CD19 CAR T Cells for Use in Treating Multiple
Myeloma
[0814] Even with current regimens of chemotherapy, targeted
therapies, and autologous stem cell transplant, myeloma is
considered an incurable disease. The present example describes
treating multiple myeloma (MM) with autologous T cells directed to
CD19 with a chimeric antigen receptor
(lentivirus/CD19:4-1BB:CD3zeta; also known as "CART19" or CTL019).
This example demonstrates that CD19-directed CAR therapies have the
potential to establish deep, long-term durable remissions based on
targeting the myeloma stem cell and/or tumor cells that express
very low (undetectable by most methods) levels of CD19.
[0815] In treating a patient with an aggressive secondary plasma
cell leukemia, we found that CART19 administered two days after a
salvage autologous stem cell transplant resulted in rapid clearance
of plasma cell leukemia and a very good partial response in a
patient who had progressed through multiple lines of chemotherapy.
This patient was transfusion-dependent for months prior to the
treatment; at two months after the treatment, she has recovered her
blood counts (with normal-range platelet counts and white blood
cell counts) and has not required transfusions since she was
discharged from the hospital from her treatment.
[0816] Because myeloma cells do not naturally express CD19, the
finding that CART19 treatment induced a rapid and significant tumor
response in this tumor was surprising. Without wishing to be bound
by a particular theory, it was reasoned that CART19 could be used
to treat myeloma because: (1) while myeloma cells are traditionally
thought to be negative for CD19 expression by flow cytometry, there
are data indicating that myeloma cells may express very low levels
of CD19, such that expression is detectable by RNA but not by flow
cytometry or immunohistochemistry; and (2) the concept of targeting
the clonotypic B cell, which is thought to be the cancerous stem
cell that gives rise to multiple myeloma, and is particularly
resistant to chemotherapy. There is a clonal relationship between B
cells and myeloma tumor cells, but traditional myeloma therapy is
aimed at the malignant plasma cells rather than B cells. CART19 for
treating myeloma therefore targets a different cell population than
most myeloma therapies.
[0817] In our single patient experience, the patient had
circulating plasma cells, and we were able to test her tumor cells
for the expression of CD19. Approximately 1-2% of her tumor cells
expressed the CD19 antigen. (FIG. 8). Thus, it was reasoned that
CART19 may have a direct effect on a very small population of her
tumor cells; a very good partial response, though would not have
been predicted based on targeting only the very small population of
CD19+ tumor cells.
[0818] In this case, CART19 was administered following autologous
stem cell transplant rescue after high-dose melphalan. Although
this is a standard therapy in myeloma, it is not curative.
Furthermore, this patient had previously undergone tandem
autologous stem cell transplants and relapsed early (<6 months)
after transplant. Without wishing to be bound by a particular
theory, use of CART19 cells as described in the present example may
have a non-overlapping mechanism in the treatment of myeloma when
combined with a salvage autologous stem cell transplant.
[0819] A patient with refractory multiple myeloma was treated with
CTL019 after myeloablative chemotherapy and ASCT. Remission was
maintained despite loss of detectable CTL019 and reconstitution of
normal CD19-positive B cells, indicating that this response did not
require sustained CTL019 activity. Moreover, this patient's
response was realized even though the vast majority (99.95%) of the
neoplastic plasma cells were CD19-negative by both flow cytometry
and RT-PCR.
[0820] The absence of detectable CD19 expression in this patient's
dominant neoplastic plasma cell population suggests that the
clinically relevant target of CTL019 resided outside this dominant
CD19-negative population. Neoplastic plasma cells in multiple
myeloma patients exhibit genetic, immunophenotypic, and functional
heterogeneity. Particular subpopulations may be required for
survival of the clone through anti-myeloma therapy. In the patient
reported here, for example, the small CD19-expressing subset of
plasma cells might have been relatively melphalan-resistant but
sensitive to CTL019. This finding suggests that therapeutically
targeting a small subset of the clone can lead to durable clinical
benefit when coupled with conventional anti-myeloma therapy.
[0821] Alternatively, the clinically relevant target of CTL019 in
this patient may have resided outside the neoplastic plasma cell
population. For instance, the CTL019 may target a stem cell
population that is relatively small but gives rise to neoplastic
plasma cells. Multiple myeloma may therefore be a disease of
multiple late B-lineage cell types, not just terminally
differentiated plasma cells, such that therapies like CTL019 that
target B lymphocytes might be useful adjuncts to therapies that
directly target plasma cells.
[0822] Ten additional multiple myeloma patients will be treated
with CART19 in a Phase I trial, at least three patients have been
treated to date.
Dose Rationale and Risks/Benefits
[0823] We have chosen to use flat dosing via the intravenous route
of administration for this protocol. The primary objective of this
protocol was to test the safety and feasibility of administering
CART-19 cells to patients with multiple myeloma. The primary
toxicities that were anticipated are (I) cytokine release when the
CARs encounter their surrogate CD19 antigen on malignant or normal
B cells; (2) depletion of normal B cells, similar to rituximab
therapy; (3) steroid-responsive skin and gastrointestinal syndromes
resembling graft-versus-host disease as has been seen previously
when expanded/costimulated autologous T-cells have been coupled
with ASCT for MM. A theoretical concern was whether transformation
or uncontrolled proliferation of the CART-19 T cells might occur in
response to high levels of CD19. This was less a concern in this
application compared to another study of CLL patients, as the
burden of clonotypic B-cells in MM is expected to be far lower than
the burden of malignant B-cells in the refractory CLL patients
treated on that study.
Dose Rationale
[0824] With the first 3 patients, we have observed clinical
activity at doses ranging from 1.4.times.10.sup.7 to
1.1.times.10.sup.9 CART-19 cells. This observation demonstrates, at
least in the first 3 patients treated, that there is not an obvious
dose response relationship. A complete response was observed in
patients administered with two log fold difference in dose. Thus,
unlike standard drugs that are metabolized, CAR T cells can have a
wide dose response range. This is most likely because the CAR T
cells are able to proliferate extensively in the patients. We
therefore set a dose range of 1-5.times.10.sup.8 CART-19 cells for
infusion. In this single-patient study offered on a compassionate
use basis, the patient was offered up to 5.times.10.sup.8 CART19
cells, with no lower dose limit. For the ten patient trial,
patients will be offered 1-5.times.10.sup.7 CART-19 cells.
General Design
[0825] This was single patient-study offered on a compassionate use
basis; it was modeled after a Phase I study to determine if the
infusion of autologous T cells transduced to express CART-19 is
safe. The primary goals of the study were to determine the safety,
tolerability and engraftment potential of CART-19 T cells in
patients undergoing salvage ASCT after early relapse following
first ASCT. The protocol consists of an open label pilot study.
[0826] At entry subjects will undergo a bone marrow biopsy and
routine laboratory and imaging assessment of their MM. Eligible
subjects will undergo steady-state apheresis to obtain large
numbers of peripheral blood mononuclear cells (PBMC) for CART-19
manufacturing. The T cells will be purified from the PBMC,
transduced with TCR.zeta./4-1BB lentiviral vector, expanded in
vitro and then frozen for future administration. The number of
patients who have inadequate T cell collections, expansion or
manufacturing compared to the number of patients who have T cells
successfully manufactured will be recorded; feasibility of product
manufacturing is not expected to be problematic in this patient
population.
[0827] Subjects will generally have had adequate peripheral blood
stem cells remaining stored from the mobilization/collection
performed in preparation for their first ASCT to conduct two
additional ASCT. Those who do not will undergo a second
mobilization/collection procedure either before or after their
steady-state apheresis with a regimen according to the treating
physician's preference. Approximately two weeks after the initial
leukapheresis, subjects will be admitted to the hospital and
receive high-dose melphalan (day -2) followed by infusion of
autologous stem cells two days later (day 0), and all subjects will
receive infusion of CART-19 cells twelve to fourteen days later
(day +12-14). Up to 10 patients will be enrolled.
[0828] All subjects will have blood tests to assess safety, and
engraftment and persistence of the CART-19 cells at regular
intervals through week 4 of the study. At day +42 and day +100,
subjects will undergo bone marrow aspirates/biopsies to assess the
bone marrow plasma cell burden and trafficking of CART-19 cells to
the bone marrow. A formal response assessment will be made at day
100 according to International Myeloma Working Group (IMWG)
criteria136, and TTP will be monitored according to routine
clinical practice for patients with multiple myeloma. The main
efficacy outcome measured in this study will be a comparison of TTP
after a patient's initial ASCT to TTP after the ASCT on this
study.
[0829] As the primary endpoint of this study is safety and
feasibility of infusion of CART-19 cells with ASCT, the study will
employ an early stopping rule. Briefly, if less than 2 severe,
unexpected adverse events occur among the first five subjects
treated, the study will then accrue an additional five subjects
towards a target enrollment of 10. We will observe treated subjects
for 40 days after CART-19 infusion (i.e., through the first
official response assessment at day 42) before enrolling a
subsequent subject until five subjects have been enrolled and so
observed. For treatment of the second group of five patients, no
waiting period will be required between subjects.
[0830] Following the 6 months of intensive follow-up, subjects will
be evaluated at least quarterly for two years with a medical
history, physical examination, and blood tests. Following this
evaluation, subjects will enter a roll-over study for annual
follow-up by phone and questionnaire for up to additional thirteen
years to assess for the diagnosis of long-term health problems,
such as development of new malignancy.
Primary Study Endpoints
[0831] This pilot trial is designed to test the safety and
feasibility of the autologous T cells transduced with the CD19
TCR/4-1BB in patients undergoing salvage ASCT for MM following
early relapse after first ASCT.
Primary Safety and Feasibility Endpoints Include:
[0832] Occurrence of study-related adverse events, defined as NCJ
CTC 2: grade 3 signs/symptoms, laboratory toxicities and clinical
events that are possibly, likely or definitely related to study
treatment at any time from the infusion until week 24. This will
include infusional toxicity and any toxicity possibly related to
the CART-19 cells including but not limited to:
a. Fevers b. Rash c. Neutropenia, thrombocytopenia, anemia, marrow
aplasia d. Hepatic dysfunction e. Pulmonary infiltrates or other
pulmonary toxicity f. GVHD-like syndromes affecting
gastrointestinal tract or skin.
[0833] Feasibility to manufacture CART-19 cells from patient
apheresis products. The number of manufactured products that do not
meet release criteria for vector transduction efficiency, T cell
purity, viability, sterility and tumor contamination will be
determined.
[0834] The depth and duration of response following autologous stem
cell transplant with CART19 will be compared to the depth and
duration of response that each patient initially achieved following
standard autologous stem cell transplant.
Subject Selection and Withdrawal
Inclusion Criteria
[0835] Subjects must have undergone a prior ASCT for MM and have
progressed within 365 days of stem cell infusion. Subjects who have
undergone two prior ASCTs as part of a planned tandem ASCT
consolidation regimen are eligible. Progression will be defined
according to IMWG criteria for progressive disease or, for patients
who attained CR or sCR after initial ASCT, criteria for relapse
from CR (Durie et al. Leukemia 2006; 20(9):1467-1473). N.B.: There
is no requirement that patients must enroll within 365 days of
prior ASCT, and patients may be treated with other agents,
including experimental agents, following relapse/progression after
prior ASCT before enrollment on this study.
[0836] Subjects must have signed written, informed consent.
[0837] Subjects must have adequate vital organ function to receive
high-dose melphalan as defined by the following criteria, measured
within 12 weeks prior to the date of melphalan infusion: a. Serum
creatinine .ltoreq.2.5 or estimated creatinine clearance .gtoreq.30
ml/min and not dialysis-dependent. b. SGOT .ltoreq.3.times. the
upper limit of normal and total bilirubin .ltoreq.2.0 mg/dl (except
for patients in whom hyperbilirubinemia is attributed to Gilbert's
syndrome). c. Left ventricular ejection fraction (LVEF) .gtoreq.45%
or, if LVEF is <45%, a formal evaluation by a cardiologist
identifying no clinically significant cardiovascular function
impairment. LVEF assessment must have been performed within six
weeks of enrollment. d. Adequate pulmonary function with FEV1, FVC,
TLC, DLCO (after appropriate adjustment for lung volume and
hemoglobin concentration) .gtoreq.40% of predicted values.
Pulmonary function testing must have been performed within six
weeks of enrollment.
[0838] Subjects must have an ECOG performance status of 0-2, unless
a higher performance status is due solely to bone pain.
Exclusion Criteria
[0839] Subjects must not:
[0840] Have any active and uncontrolled infection.
[0841] Have active hepatitis B, hepatitis C, or HIV infection.
[0842] Any uncontrolled medical disorder that would preclude
participation as outlined.
Treatment Regimen
[0843] Therapy for Relapsed/Progressive Multiple Myeloma
[0844] Patients may receive, prior to enrollment, therapy for
relapsed/progressive multiple myeloma according to the preference
of their treating physicians. Therapy may continue upon
enrollment.
[0845] Patients must stop all therapy for two weeks prior to
apheresis and for two weeks prior to high-dose melphalan. If more
than two weeks are expected to lapse between apheresis and
high-dose melphalan, patients may resume therapy after apheresis at
the discretion of their treating physicians.
[0846] High-Dose Melphalan (Day -2)
[0847] Patients will be admitted to the hospital on day -3 or -2
and will undergo examination by the attending physician and routine
laboratory tests, which will include monitoring parameters for
tumor lysis syndrome, prior to commencement of the treatment
protocol. Blood for MM monitoring laboratory tests (SPEP,
quantitative immunoglobulins, and serum free light chain analysis),
will be drawn prior to initiation of therapy if such tests had not
been drawn within 7 days of admission.
[0848] High-dose therapy will consist of melphalan at a dose of 200
mg/m.sup.2 administered intravenously over approximately 20 minutes
on day -2. The dose of melphalan will be reduced to 140 mg/m.sup.2
for patients >70 years of age or for patients of any age whom,
at the discretion of the treating physician, may not tolerate a
dose of 200 mg/m.sup.2 All patients will receive standard
anti-emetic prophylaxis, which may include dexamethasone, and
standard antibiotic prophylaxis.
[0849] Stem-Cell Re-Infusion (Day 0)
[0850] Stem cell infusion will take place on day 0, at least 18
hours after the administration of the high-dose melphalan. Stem
cells will be infused intravenously over approximately 20-60
minutes following premedication according to standard institutional
practice. At least 2.times.10.sup.6 CD34+ progenitors/kg body
weight should be infused. In addition, at least 1.times.10.sup.6
CD34+ progenitors/kg body weight should be available as a back-up
stem-cell product to be infused in the event of delayed engraftment
or late graft failure. G-CSF should be administered SQ beginning on
day +5, dosed according to standard institutional practice. Other
supportive care measures such as transfusion support will be done
in accordance with standard institutional guidelines.
[0851] CART19 Cell Infusion (Day +12-14)
[0852] A single dose of CART-19 transduced T cells will be given
consisting of up to 5.times.10.sup.7 CART-19 cells. The minimal
acceptable dose for infusion of cells transduced with the CD19 TCR
4-1BB vector is 1.times.10.sup.7. CART-19 cells will be given as a
single dose by rapid i.v. infusion on day +12-14 after stem cell
infusion. If patient fails to meet any of the inclusion criteria
described herein in the 12-14 day window, the CART-19 infusion may
be delayed beyond day +12-14 until the criteria is satisfied.
[0853] Maintenance Lenalidomide
[0854] Subjects who received and tolerated maintenance lenalidomide
after their first ASCT will re-initiate lenalidomide maintenance
therapy at approximately day +100, assuming there are no
contraindications in the judgment of the treating physician. The
starting dose will be 10 mg daily unless prior experience dictates
an alternative starting dose for a particular patient. Maintenance
therapy will continue until disease progression or intolerance.
[0855] Preparation and Administration of Study Drug
[0856] The CART-19 T cells are prepared in the CVPF and are not
released from the CVPF until FDA approved release criteria for the
infused cells (e.g., cell dose, cell purity, sterility, average
copy number of vectors/cell, etc.) are met. Upon release, the cells
are taken to the bedside for administration.
[0857] Cell thawing. The frozen cells will be transported in dry
ice to the subject's bedside. The cells will be thawed at the
bedside using a water bath maintained at 36.degree. C. to
38.degree. C. The bag will be gently massaged until the cells have
just thawed. There should be no frozen clumps left in the
container. If the CART-19 cell product appears to have a damaged or
the bag to be leaking, or otherwise appears to be compromised, it
should not be infused and should be returned to the CVPF as
specified below.
[0858] Premedication. Side effects following T cell infusions
include transient fever, chills, and/or nausea; see Cruz et al. for
review (Cytotherapy 2010; 12(6):743-749). It is recommended that
the subject be pre-medicated with acetaminophen and diphenhydramine
hydrochloride prior to the infusion of CART-19 cells. These
medications may be repeated every six hours as needed. A course of
non-steroidal anti-inflammatory medication may be prescribed if the
patient continues to have fever not relieved by acetaminophen. It
is recommended that patients not receive systemic corticosteroids
such as hydrocortisone, prednisone, methylprednisolone or
dexamethasone at any time, except in the case of a life-threatening
emergency, since this may have an adverse effect on T cells.
[0859] Febrile reaction. In the unlikely event that the subject
develops sepsis or systemic bacteremia following CAR T cell
infusion, appropriate cultures and medical management should be
initiated. If a contaminated CART-19 T cell product is suspected,
the product can be retested for sterility using archived samples
that are stored in the CVPF.
[0860] Administration. The infusion will take place in an isolated
room in Rhoads, using precautions for immunosuppressed patients.
The transduced T cells will be administered by rapid intravenous
infusion at a flow rate of approximately 10 mL to 20 ml per minute
through an 18-gauge latex free Y-type blood set with a 3-way
stopcock. The duration of the infusion will be based on the total
volume to be infused and the recommended infusion rate. Each
infusion bag will have affixed to it a label containing the
following: "FOR AUTOLOGOUS USE ONLY." In addition the label will
have at least two unique identifiers such as the subject's
initials, birth date, and study number. Prior to the infusion, two
individuals will independently verify all this information in the
presence of the subject and so confirm that the information is
correctly matched to the participant.
[0861] Emergency medical equipment (i.e., emergency trolley) will
be available during the infusion in case the subject has an
allergic response, or severe hypotensive crisis, or any other
reaction to the infusion. Vital signs (temperature, respiration
rate, pulse, and blood pressure) will be taken before and after
infusion, then every 15 minutes for at least one hour and until
these signs are satisfactory and stable. The subject will be asked
not to leave until the physician considers it is safe for him or
her to do so.
Packaging
[0862] Infusion will be comprised of a single dose of
1-5.times.10.sup.7 CA T19-transduced cells, with a minimal
acceptable dose of 1.times.10.sup.7 CART-19 cells for infusion.
Each bag will contain an aliquot (volume dependent upon dose) of
cryomedia containing the following infusible grade reagents (%
v/v): 31.25% plasmalyte-A, 31.25% dextrose (5%), 0.45% NaCl, up to
7.5% DMSO, 1% dextran 40, 5% human serum albumin.
Apheresis
[0863] A large volume (12-15 liters or 4-6 blood volumes) apheresis
procedure is carried out at the apheresis center. PBMC are obtained
for CART-19 during this procedure. From a single leukapheresis, the
intention is to harvest at least 5.times.10.sup.9 white blood cells
to manufacture CART-19 T cells. Baseline blood leukocytes for FDA
look-back requirements and for research are also obtained and
cryopreserved. The cell product is expected to be ready for release
approximately 2-4 weeks later. Flow cytometry lymphocyte subset
quantitation, including CD19 and CD20 B cell determination.
Baseline assessment is made for human anti-VSV-G and anti-murine
antibody (HAMA). If a subject has previously had an adequate
apberesis collection banked according to current Good Manufacturing
Practices at the Clinical Cell and Vaccine Production Facility
these cells may be used as the source of cells for CART-19
manufacturing. Using a banked apheresis product would avert the
expense, time, and risk to the subject of undergoing an additional
apheresis collection.
Cytoreductive Chemotherapy
[0864] The lymphodepleting chemotherapy will be high-dose melphalan
as described herein.
CART-19 Infusion
[0865] Infusion will begin on day +12-14 after stem-cell
reinfusion.
[0866] On day +12-14 prior to the first infusion, patients will
have a CBC with differential, and assessment of CD3, CD4 and CD8
counts since chemotherapy is given in part to induce
lymphopenia.
[0867] The first dose will be administered using a single dose. The
cells are thawed at the patient's bedside. The thawed cells will be
given at as rapid an infusion rate as tolerated such that the
duration of the infusion will be approximately 10-15 minutes. In
order to facilitate mixing, the cells will be administered
simultaneously using a Y-adapter. Subjects will be infused and
premedicated as described herein. Subjects' vital signs will be
assessed and pulse oxymetry done prior to dosing, at the end of the
infusion, and every 15 minutes thereafter for 1 hour and until
these are stable and satisfactory. A blood sample for determination
of a baseline CART-19 level is obtained any time prior to the first
infusion and 20 minutes to 4 hours after each infusion (and sent to
TCSL).
[0868] Patients experiencing toxicities related to high-dose
melphalan will have their infusion schedule delayed until these
toxicities have resolved. The specific toxicities warranting delay
of T cell infusions include: 1) Pulmonary: Requirement for
supplemental oxygen to keep saturation greater than 95% or presence
of radiographic abnormalities on chest x-ray that are progressive;
2) Cardiac: New cardiac arrhythmia not controlled with medical
management 3) Hypotension requiring vasopressor support. 4) Active
Infection: Positive blood cultures for bacteria, fungus, or virus
within 48-hours of T cell infusion.
Management of Toxicity
[0869] Uncontrolled T cell proliferation. Toxicity associated with
allogeneic or autologous T cell infusions has been managed with a
course of pharmacologic immunosuppression. T body associated
toxicity has been reported to respond to systemic corticosteroids.
If uncontrolled T cell proliferation occurs (grade 3 or 4 toxicity
related to CART-19 cells), subjects may be treated with
corticosteroids. Subjects will be treated with pulse
methylprednisolone (2 mg/kg i.v. divided q8 hr.times.2 days),
followed by a rapid taper.
[0870] In addition, based on the observations of subjects treated
on another protocol, there is some concern for macrophage
activation syndrome (MAS), though the CD19+ tumor burden is
expected to be much lower in patients with myeloma than in patients
with CLL. Treatment and timing of treatment of this toxicity will
be at the discretion of the patient's physician and the study
investigator. Suggested management might include: if the subject
has a fever greater than 101.degree. F. that lasts more than 2
consecutive days and there is no evidence of infection (negative
blood cultures, CXR or other source), tocilizumab 4 mg/kg can be
considered. The addition of corticosteroids and anti-TNF therapy
can be considered at the physician's discretion.
[0871] B cell depletion. It is possible that B cell depletion and
hypogammaglobulinemia will occur. This is common with anti-CD20
directed therapies. In the event of clinically significant
hypogammaglobulinemia (i.e. systemic infections), subjects will be
given intravenous immunoglobulin (IVIG) by established clinical
dosing guidelines to restore normal levels of serum immunoglobulin
levels, as has been done with Rituximab.
[0872] Primary graft failure. Primary graft failure (i.e.,
non-engraftment) may be more common after second ASCT compared to
first ASCT. Eligibility criteria stipulate that sufficient stem
cells must be available for rescue reinfusion at the discretion of
the treating physician in the event of primary graft failure.
[0873] Results
[0874] Three treatment-refractory, advanced multiple myeloma
patients have now been treated with CTL019 in this ongoing trial.
Results for two of these patients show that both have had
substantial anti-tumor effects from the CTL019 therapy based on the
primary efficacy assessment at the three-month time-point. The
third patient has not yet reached the three-month time point. The
results for the two patients are described in more detail
below.
[0875] The first myeloma patient has completed her +100 day
response assessment and she had a very good response to the CART19
therapy. The following tests were performed with the following
results: [0876] SPEP/immunofixation: negative [0877] urine
immunofixation: faint unmeasurable kappa light chain band on her
immunofixation (also present at day 38, so not new) Otherwise, the
patient meet the criteria for stringent complete remission
including: [0878] serum free light chain ratio: normal [0879] bone
marrow biopsy: negative [0880] IgA immunophenotyping: IgA is below
the limit of detection
[0881] Other than the faint unmeasurable kappa light chain result
from urine immunofixation, the patient met all criteria for
"stringent complete remission". The summary of the plasma cell
immunophenotyping at 3 time points (day -2, day +38, day +103) is
shown in FIG. 39, and demonstrates that the patient's IgA is below
the limit of detection. The summary shows heavy myeloma burden at
day -2 and none detectable at day +38 and +103, which classifies
the patient as "MRD negative" by flow analysis. At day +103, the
summary shows recovery of normal, polyclonal, CD19+ plasma cells
and B cells. The patient had no symptoms of disease or therapy and
is functioning like a normal person.
[0882] The second patient treated has not yet reached the +100 day
time point. However, at this time point, she is doing well but it
is too early to determine the effect of the CTL019 infusion.
Example 7: Kinase Inhibitor/CAR19 T-Cell Combined Therapy for
Mantle Cell Lymphoma
[0883] Adoptive T-cell therapy holds considerable promise for the
treatment of lymphoid malignancies. Promising clinical responses in
small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL)
and acute lymphocytic leukemia (ALL), using adoptive transfer of
autologous T cells transduced with chimeric antigen receptors (CAR)
against the B-cell specific CD19 antigen (CAR19 T cells/CART19
cells) using CTL109. We have recently reported initial data on 3
patients with chemotherapy-refractive SLL/CLL enrolled in a phase I
trial to treat CD19-positive malignancies using CD19-specific CAR
(CAR19 T cells/CART19 cells). The approach used involved the
genetic modification of patient-derived bulk T cells using a
lentivirus to express a CD19-targeting CAR that contains signaling
domains derived from CD137 and TcRz. For this study cells were
expanded using our anti-CD3 and -CD28 bead expansion methodology,
and cells were infused early post lymphodepletion without cytokine
support (Kalos M, et al. (2011) Sci Transl Med. 3: 95ra73; and
Porter D L, et al. (2011) N Engl J Med. 365: 725-733). These early
results were extremely promising: (i) following a single course of
treatment 2/3 patients achieved complete remissions and remain
disease free now at 15+ months post treatment, while the third
patient, who was treated with corticosteroids soon after the T cell
infusion demonstrated a strong partial response. (ii) In these
patients we were able to recapitulate the elements thought to be
required for ultimate efficacy of adoptive T cell therapy-based
strategies, namely robust in vivo T cell expansion, disease
eradication, T cell contraction, and long-term functional
persistence. To date, 10 SLL/CLL patients have been treated with 2
remaining in the complete clinical and molecular remission, 5
experiencing partial remission, and 3 displaying lack of measurable
response. In another study, two patients with B cell ALL achieved
complete remission with 1 relapsing with leukemic cells lacking
CD19 expression.
[0884] Mantle cell lymphoma (MCL), both before and after large cell
transformation, will also likely to benefit from the CART19-based
adoptive therapy, in particular when combined with kinase
inhibitors such as those that directly affect MCL cells.
[0885] To further analyze the combination of CART19-based adoptive
therapy in combination with kinase inhibitors, high throughput
screens will be used to evaluate several inhibitors targeting the
kinases critical for MCL pathogenesis: CDK4/6, BTK, and mTOR in
combination with CART19 cells. The most promising combinations will
be evaluated in greater detail, both in vitro and in vivo, in MCL
xenotransplant mouse model, which ultimately may guide the
development of a clinical protocol to evaluate combination of small
molecule kinase inhibitor and the CART cell immunotherapy in MCL
patients.
[0886] In this study, preclinical studies will be performed to
determine potential clinical efficacy of this approach in the
various subtypes of MCL and to evaluate the ability to
therapeutically target MCL cells using CART19 cells either alone or
in will combination with small molecule inhibitors of selected
proteins from the kinase family expression and activity of which is
critical for survival and growth of MCL cells.
Research Plan
[0887] In a pre-clinical setting, the ability to therapeutically
target MCL cells, both cultured and primary-type cells, using
inhibitors of kinases with documented pathogenic relevance in MCL
and CART19 cells will be evaluated. A high throughput MTT assay
will be used to determine the effect of these agents to identify
potential optimal combinations, dosing and timing of the agent
application. The most promising 2-3 combinations will be evaluated
in the greater detail in regard to cell function,
phosphorylation-based cell signaling, and gene expression first in
vitro and later in vivo in the MCL xenotransplant model.
In-Vitro Studies to Characterize the Ability of Kinase
Inhibitor/CART-19 Cell Combinations to Effectively Target MCL
Cells
[0888] In this aim, detailed functional, phenotypic, biochemical,
and molecular assays listed above to study in-vitro the impact of
the small molecule kinase inhibitors on MCL cells as well as to
examine interactions between CART19 cells and MCL cells and the
impact of the inhibitors on these interactions will be
examined.
[0889] The benchmarks for accomplishing this aim will be to
generate a comprehensive data set to: [0890] i. document that
CART19 cells are activated by and lyse the cultured and primary MCL
cells; [0891] ii. demonstrate that the selected kinase inhibitors
enhance the ability of each other and/or CART19 cells to eliminate
MCL cells without negatively impacting CART19 cell function when
appropriately applied in regard to the dose and, for some, timing
of the inhibitor vs. CART19 cell administration; and [0892] iii.
establish a regimen for the schedule and dosing for the BTK
inhibitor to be used in the in vivo MCL xenotransplant experiments
using the NSG mice.
[0893] The goals of this study are, e.g., to evaluate whether
identify the optimal therapeutic combinations of small molecule
inhibitors targeting kinases critical for MCL pathobiology: CDK4,
BTK, and mTOR together with CART19 cells, monitor CART19 activity,
and characterize the functional, biochemical, and molecular effects
of the therapy on MCL.
[0894] These studies should to establish a rational schema for
schedule for the timing and dose of BTK treatment kinase inhibitor
in conjunction with CART19 therapy to be evaluated in-vivo in aim
2.
In-Vivo Studies to Evaluate the Ability of CART19 Cells to Target
Follicular Lymphoma, Alone and in Combination with BTK
Inhibitor
[0895] In this aim, we will test in animal models the ability of
the selected inhibitor/CART19 cell combination(s) to affect growth
of established and primary MCL cells.
[0896] The benchmarks for accomplishing this aim will be to
generate a data set to: [0897] i. demonstrate that the selected
inhibitor/CART19 cell combination markedly enhances the survival of
animals engrafted with MCL as compared to the controls (single and
mock agent treated animals); [0898] ii. establish a regimen for the
schedule and dosing for the selected kinase inhibitor/CART19
combination to be used as the basis for a future clinical
trial.
[0899] The goals of this study include evaluating the treatment and
dose schedule defined in aim 1 for the identified kinase
inhibitor/CART19 plus BTK inhibitor combination, and to test
whether BTK treatment synergizes with CART19 to target MCL in NSG
mice xenotransplanted with MCL cells, both cultured and
primary.
[0900] The following cell types, compounds, animals and
experimental methodologies will be used to accomplish the proposed
aims:
[0901] MCL Cells:
[0902] Four MCL cell lines (Jeko-1, Mino, SP-49, and SP-53) and
viably frozen samples from 15 primary MCL (12 typical and 3
blastoid). While the cell lines grow well spontaneously, the
primary cells will be cultured alone as well as in the presence of
conditioned medium collected from HS5 bone marrow stromal cells to
improve their viability.
[0903] CART19 Cells:
[0904] Primary human T cells engineered to express CAR19 will be
generated using lentivirus transduction and using the established
protocols ((Kalos M, et al. (2011) Sci Transl Med. 3: 95ra73; and
Porter D L, et al. (2011) N Engl J Med. 365: 725-733). Following a
single transduction event T cells typically express CAR19 at
frequencies exceeding 30%.
[0905] Our studies will use CART19 populations from five SLL/CLL
patients (50-100 vials/patient at with 1.times.10.sup.7 cells/vial
are already available). CART19 cells will be identified using an
anti-CAR19-specific idiotype antibody (STM). CART19 activity will
be controlled both in vitro and in vivo in NSG mice in the
standardized manner using CD19+ NALM-6, CD19-negative K562, and
CD19-transduced K562 cell lines. Although CART19 cell function is
not MHC restricted, CART19 cell from at least 5 MCL patients will
also be used.
[0906] Kinase Inhibitors.
[0907] Inhibitors of the following kinases will be tested: CDK4/6
(PD0332991), BTK (PCI-32765), mTORC1 (rapamycin), MNK
(4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine (Marzec M,
et al. PLoS One 6:e24849); and a novel compound from Eli Lilly
(Gupta M, et al. (2012) Blood 119:476-487); mTOR (OSI-027), and
dual PI3K/mTOR (PF-04691502).
[0908] The compounds will be evaluated first at the pre-determined
spectrum of effective doses, including the non-toxic concentrations
reached in patients' sera, to assure optimal kinase inhibition.
[0909] Animals:
[0910] The in-vivo experiments will be performed using
NOD-SCID-IL-2Rgc null (NSG) mice which are bred and available from
the Stem Cell and Xenograft Core using breeders obtained from
Jackson Laboratory (Bar Harbor). Mice will be housed in sterile
conditions using HEPAfiltered microisolators and fed with
irradiated food and acidified water.
[0911] Transplanted mice are treated with antibiotics (neomycin and
polymixin) for the duration of the experiment. Six to eight
week-old animals, equal mixes of males and females, will be
utilized for all studies in accordance with protocols approved by
the Institutional Animal Care and Use Committee.
[0912] We have used NSG animals in previous T cell adoptive
transfer studies specifically to evaluate differential activity of
CART19 cells (Witzig T E, et al. (2010) Hematology Am Soc Hematol
Educ Program. 2010:265-270, MTT assay). The high throughput MTT
assay to evaluate MCL cell growth will be performed first in
response to the kinase inhibitors applied either alone or in
various combinations. This assay is able to simultaneously
determine cell proliferation rate and viability, allowing efficient
evaluation of many possible combinations of small molecule
inhibitors in the presence or absence of CART19 cells. The key
aspects of this analysis will be to characterize the drug effect in
regard to potential synergistic, additive, or antagonistic effect.
In addition, the effect of the small molecule inhibitors on CART19
cells will be evaluated. While BTK inhibition should be B-cell
specific, mTOR and CDK4/6 inhibition will affect CART19 cells.
Establishing the proper timing of the drug application to minimize
their potential effect on CART19 cells will be one of the aims of
these experiments.
[0913] To perform the test, MCL cells will be seeded in 96-well
plates at 1.times.10.sup.4 cells/well, in triplicates, and exposed
to medium or kinase inhibitors in various combinations and various
concentrations of CART-19 cells. After 48 and 74 hrs, the relative
number of metabolically active cells will be determined by the use
of MTT reduction colorimetric assay (Promega).
[0914] The significance of difference between the mean values
(+/-S.D.) of the controls and different treatment conditions will
be evaluated using Student's t-test with the P value of <0.05
considered to be statistically significant.
[0915] Cell Proliferation and Apoptosis Assays:
[0916] The most promising drug combinations will be next evaluated
in the CFSE labeling and terminal dUTP nick-end labeling (tunel)
assays to determine both cytostatic and cytotoxic components of MCL
cell growth inhibition, respectively. In the former assay, MCL
cells will be labeled with CFSE addition of the BTK inhibitor
and/or unlabeled CART-19 cells. After 48 hrs, the cultured cells
will be the analyzed by FACS for the CFSE labeling pattern of the
MCL-type cells. The tunel assay will be done using the ApoAlert DNA
Fragmentation Assay Kit from BD Biosciences according to the
manufacturer's protocol.
[0917] In brief, MCL cells will be cultured with the inhibitors
and/or CART19 cells for 48 or 72 hours. After being washed, cells
will be stained with labeled anti-CD20 antibody and permeabilized,
washed, and incubated in TdT buffer for 1 hour at 37.degree. C. The
reaction will be stopped, the cells washed, resuspended, and
analyzed by flow cytometry using the CellQuest PRO software.
[0918] CART19 Functional Assays:
[0919] We will measure effector activity of CART19 cells against
MCL cell lines using CD107 degranulation, Intracellular Cytokine
Secretion (ICS) assays, proliferationcytolysis assays, and
multiplex cytokine detection assays (32). For degranulation and ICS
assays, effector (T cells) and Target (tumor cells) will be
co-incubated in the presence of anti-CD107 antibody for 4 hours at
E:T of 0.2:1 followed by staining for surface (CAR19, CD3, CD8,
CD4) and intracellular cytokine markers as per established
protocols. Cytolysis of MCL cells will be assessed using
flow-cytometry-based cytolysis assays. For proliferation assays,
effector cells will be pre-loaded with CFSE (Carboxyfluorescein
succinimidyl estercarboxy-fluoroscein-succinil esterase), mixed
with target cells at E:T of 0.2:1, co-incubated at 37.degree. C.
for 4 days, stained for surface markers (CAR19, CD3, CD8, CD4) and
analyzed for dilution of CFSE by flow-cytometry.
[0920] Multiplex Cytokine Assays.
[0921] We will measure production of cytokines by CART19 cells in
response to MCL targets using Luminex-based bead assays as
described in (STM32). For these analyses we will employ the
Invitrogen 30-plex kit that simultaneously measures IL-1.beta.,
IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12
(p40/p70), IL-13, IL-15, IL-17, TNF-.alpha., IFN-.alpha.,
IFN-.gamma., GM-CSF, MIP-1.alpha., MIP-1.beta., IP-10, MIG,
Eotaxin, RANTES, MCP-1, VEGF, G-CSF, EGF, FGF-basic, and HGF in
serum, plasma, or tissue culture supernatant.
[0922] Multiparametric Flow Cytometry Analysis of CART19T
Cells:
[0923] We will measure the modulation of surface markers associated
with functional activation and suppression on CART19 cells
following co-incubation with tumor cells using four color flow
cytometry and a custom BD LSR II equipped with 4 lasers (blue (488
nM), violet (405 nM), Green (532 nM), and Red (633 nM) available
through the University of Pennsylvania Abramson Cancer Center Flow
Cytometry Core. All flow cytometry data will be analyzed using
FlowJo software (TreeStar, San Carlos, Calif.). These analyses will
be performed essentially as described in (STM42), using a dump
channel to exclude dead cells and target cells (CD19+), and a CAR19
idiotype-specific reagent to detect CART19 cells (STM). We will
evaluate the following markers on CART19-positive and -negative
cells (CD3+/CD8+ and CD3+/CD4+) post co-incubation with tumor
cells, on either intact or permeabilized cells as needed. We have
established multi-parametric panels for these markers: [0924]
activation/effector function: CD25, CD154, CD134, CD137, CD69,
CD57, CD28, T-bet [0925] inhibition: CD152 (CTLA4), PD1, LAG3,
CD200 [0926] suppression (Treg) CD4+/CD25++/CD127-, Fox-P3+
[0927] Simultaneously, MCL cells identified by CD19 and CD5
staining, will be examined for expression of the immunosuppressive
proteins: CD174 (PD-L1) CD173 (PD-L2) and CD152.
[0928] Inhibitor Impact on Cell Signaling:
[0929] This part of the study will focus only on the selected
compounds; the ones that proved to be the most effective in the
functional assays (cell growth, proliferation, and apoptosis)
described above. The effect will be studied separately for each
drug and for the selected combinations and the studies will be
adjusted to the specific compounds. For example, while the mTORC1
and MNK inhibitor combination will evaluate mTORC1 signaling, in
particular the eIF-4E phosphorylation, BTK inhibition will focus on
the PI3K-AKT and MEK-ERK pathways, and CDK4/6 inhibition on Rb
phosphorylation. These studies will be performed by Western
blotting using phospho-specific antibodies as described (Marzec M,
et al. (2006) Blood. 108:1744-1750; Marzec M, et al. (2008) Blood
111: 2181-2189; Zhang Q, et al. (2011) Proc Natl Acad Sci USA 108:
11977-11982). In brief, the MCL cells will be lysed and the protein
extracts will be assayed using the Lowry method (Bio-Rad) and
loaded into the polyacrylamide gel. To examine protein
phosphorylation, the blotted membranes will incubated with the
phosphor-specific antibodies, for example the ones specific for
S6rp S235/236, eIF4E S209, 4E-BP1 T37/46, 4E-BP1 T70 (Cell
Signaling) to evaluate the mTORC1 and MNK activity and their
inhibition. Next, the membranes will be incubated with the
appropriate secondary, peroxidase-conjugated antibodies. The blots
will be developed using the ECL Plus System from Amersham.
[0930] Genome-Scale Gene Expression Analysis:
[0931] Inhibition of cell signaling typically leads to changes in
gene transcription. To determine the effects of the selected
inhibitor, or a few inhibitors on gene transcription in MCL, a
genome-scale gene expression analysis will be performed as done as
described in Marzec M, et al. (2008) Blood 111: 2181-2189; Zhang Q,
et al. (2011) Proc Natl Acad Sci USA 108: 11977-11982. In brief,
the cells will be treated in triplicate cultures with the selected
inhibitor or its diluent for 0, 4, and 8 hours. The total RNA will
be further purified to enrich for mRNA which will be reverse
transcribed, labeled and examined by hybridization to the
Affimetrix microchip against all known gene exons. The microarray
data will be normalized and summarized using RMA as implemented in
GeneSpring and MASS algorithm. The resulting p values will be
corrected for multiple testing using false discovery rate (FDR) by
the Benjamini-Hochberg step-up method. Differential expression
testing will be accomplished using a variety of tools including SAM
and PartekPro. The emerging genes of interest will then be
clustered based on expression patterns (GeneSpring or Spotfire),
and clusters will be analyzed for functional groups and pathways in
KEGG, Ingenuity Pathway Analysis, and Gene Ontology databases using
the NIH-David as the search tool. For the genes identified based on
the data, the independent expression conformation by the
quantitative RTPCR will be performed on a larger pool of samples
(at least 20) of various types of MCL (standard vs. blastoid and
SOX11-positive vs. SOX11-negative).
[0932] Whole-Exome DNA Sequence Analysis:
[0933] To better characterize the MCL cases in regard to their
pathogenesis and, to the extent possible, response to the proposed
here combination therapies, the sequence of exomic DNA will be
examined. Whole-exome capture and next generation sequencing of the
MCL and normal peripheral blood DNA samples will performed using
the NimbleGen Sequence Capture 2.1M Human Exome Array and the HiSeq
2000/1000 Illumina instrument.
[0934] Evaluation of the Treatment Effect in the Xenotransplanted
Tumors:
[0935] The NSG mice will carry the MCL tumors tumors (derived from
both MCL cell lines: Jeko and Mino and primary cells implanted as
either tissue fragments or, less preferably, cell suspensions). The
tumors will be propagated by subcutaneous implantation of the small
tumor fragments. The therapy will be initiated once the tumors
reach 0.2-0.3 cm in the diameter. The kinase inhibitor(s) will
administered by gavage at dose and timing preselected in vitro (for
example, we expect to apply BTK inhibitor simultaneously with
CART19 cells, given its B-cell specificity and expected lack of any
inhibitory effect on CART19 cells). CART-19 cells will be injected
into the tail vein of the tumor-bearing mice at a dose of
1.times.10.sup.7/animal, the kinase inhibitor(s) will administered
by gavage at dose and timing preselected in vitro. a dose we have
established to be sufficient to reproducibly eradicate malignant
cells and, at the same time, not to induce xeno-graft versus host
disease. A large master stock of CART19 cells (lx 10.sup.10) will
be generated and frozen to minimize variability associated with
effector cell differences. The primary measure from these
experiments will be survival, which we will assess using
Kaplan/Meier curves. As a secondary measure we will evaluate
differential expansion of CART19 cells in animals following T cell
infusion. This will be made possible by the fact that the infused T
cell product will be composed of CART19-positive and -negative
cells at a defined ratio. For these analyses, animals will be bled
weekly by tail vein bleed (25 microliters each time), followed by
red blood cell lysis and staining for human CD3, CD4, CD8, and
CART19. Preferential expansion of CART19 cells (at least a 2-fold
increase in the CART19+/CART19-ratio will be evidence for selective
MCL-driven CART19 cell expansion. To assess the treatment results,
volumes of the implanted subcutaneous tumors will be measured
determined as follows, according to the formula: volume=0.4ab2,
where a and b designate respectively long and short diameters of
the tumor. Tumor volumes differences between the treated and
untreated groups of mice will be statistically analyzed using a
standard t-test. Mice will be sacrificed at either the end-point of
the experiments (>30 days), or if tumors reach >1.2 cm in
diameter, or when any evidence of the animal distress noted. Tumor
volumes differences between the treated and untreated groups of
mice will be statistically analyzed using a standard t-test. The
tumors as well as the internal organs will be harvested, processed
and analyzed by histology and, for selected tissues, by
immuno-histochemistry using the battery of antibodies against B
cells (CD20, CD79a, Pax-5, CD10, BCL-6) and T cells (CD2, CD3, CD4,
CD5, CD7, CD8, TIA-1), and the proliferation marker Ki-67.
[0936] Statistical Analysis:
[0937] In the in vitro functional studies, the significance of
difference between the mean values (+/-S.D.) of the controls and
different treatment conditions will be evaluated using Student's
t-test with the P value of <0.05 considered to be statistically
significant. Based on our previous experiences, the differences
between the experimental mouse groups are expected to be large.
Thus, 10 NSG mice will be used for each treatment group, which will
ensure at least 90% power at 0.05 type 1 error level with a
two-sided two sample/-test, given the ratio between the difference
in treatment means and the standard deviation is at least 3, which
is expected. Data will be presented as mean.+-.SEM. Comparison
among groups will be made using the two sample t-test. A value of
p<0.05 is considered to be significant. For tumor-free survival
studies, groups of 10 mice will be used for survival comparison,
and the disease status (tumor vs. no tumor) and tumor-free time for
each mouse will be recorded. The Kaplan-Meier survival curve will
be plotted and the log-rank test will be performed to compare the
survival curves. The significance level is controlled at 0.05.
Example 8: Ibrutinib/CAR19 T-Cell Combined Therapy for Mantle Cell
Lymphoma
[0938] The experiments described in this example characterize
CART19 activity in combination with ibrutinib treatment for
treating mantle cell lymphoma in vitro and in vivo. Ibrutinib is a
small molecule inhibitor of BTK often used for treatment of some
hematological cancers. The in vitro experiments described herein
include assessment of proliferation, cytokine production, CD107a
degranulation, and cytotoxicity. Xenoplant mouse models were
utilized to investigate the efficacy and optimal dosage of CART19
with ibrutinib treatment in vivo. Although ibrutinib displays
considerable activity in MCL, about 30% of patients do not respond,
and among the responders, only 21% to about one-third experience
complete remission (Wang et al. NEJM 369.6(2013):507-16).
Achievement of a complete remission is associated with improved
progression-free survival. Furthermore, therapy can lead to drug
resistance with the duration of median response of 17.5 months. In
some settings, mutations in BTK binding sites or immediately
downstream have been observed after ibrutinib therapy, highlighting
a mechanism of drug resistance that may become increasingly
frequent. See, e.g., Woyach et al. NEJM. 370.24(2014):2286-94.
Also, blockade of BTK function leads to inhibition of B cell
receptor (BCR) signaling and is not directly cytotoxic. See, e.g.,
Ponader et al. Blood. 119.5(2012): 1182-89. Lack of cytotoxicity
and failure to eradicate malignant clones predispose to clonal
evolution under a selection pressure. Also, preliminary findings of
increased transformation to aggressive disease in patients treated
with ibrutinib for CLL are concerning. See, e.g., Byrd et al. NEJM.
369.1(2013):32-42; and Parikh et al. Blood.
123.11(2014):1647-57.
[0939] Infusion of autologous T cells transduced with chimeric
antigen receptors (CAR) against the B-cell specific CD19 antigen
(CTL019, CART19) leads to dramatic clinical responses in the
majority of patients with various B-cell neoplasms, foremost acute
lymphoblastic leukemia (ALL). See, e.g., Maude et al. NEJM.
371.16(2014):1507-17; and Ruella et al. Expert Opin. Biol. Ther.
(2015):1-6. The presence of lymph node masses or bulky disease may
lead to decreased T cell infiltration and consequent reduced
anti-tumor activity. Bulky lymphadenopathy does not appear to
impair the response to ibrutinib. Wang et al. NEJM.
369.6(2013):507-16. Also, ibrutinib has shown particular efficacy
in reducing tumor masses and mobilizing neoplastic B cells in the
peripheral blood.
Methods
Cell Lines and Primary Samples.
[0940] MCL cell lines were obtained from ATCC (Mino, Jeko-1, SP-49)
while MCL-RL was generated from a progressive pleural effusion of a
MCL patient. For in vitro experiments, cell lines were maintained
in culture with RPMI media supplemented with 10% fetal calf serum,
penicillin, and streptomycin. For some experiments, MCL-RL and
Jeko-1 cells were transduced with click beetle green
luciferase/eGFP and then sorted to obtain a >99% positive
population. The acute leukemia cell lines MOLM-14, K562 or NALM-6
and the T-ALL cell line JURKAT were used as controls. These cell
lines were originally obtained from the ATCC. De-identified primary
human MCL bone marrow (BM) and peripheral blood (PB) specimens were
obtained from the clinical practices of University of Pennsylvania.
For all functional studies, primary cells were thawed at least 12
hours before experiment and rested at 37.degree. C.
Generation of CAR Constructs and CAR T Cells.
[0941] The murine anti-CD19 Chimeric antigen receptor (containing a
CD8 hinge, 41BB costimulatory domain and CD3 zeta signaling domain)
was generated as previously described. See, e.g., Milone et al.
Molecular Therapy: the Journal of the American Society of Gene
Therapy. 17.8(2009):1453-64. Production of CAR-expressing T cells
was performed as previously described. See, e.g., Gill et al.
Blood. 123.15(2014):2343-54. Normal donor CD4 and CD8 T cells or PB
mononuclear cells (PBMC) were obtained from the Human Immunology
Core of the University of Pennsylvania. T cells were plated at
1.times.10.sup.6/ml, with a CD4:CD8 ratio of 1:1 and expanded in
X-vivo 15 media (Lonza, 04-418Q), human serum AB 5% (Gemini,
100-512), penicillin/streptomycin (Gibco, 15070063) and Glutamax
(Gibco, 35050061) using anti-CD3/CD28 Dynabeads (Life Technologies,
11161D) added on the day 1 of culture and removed on day 6. T cells
were transduced with lentivirus on day 2. T cells were expanded in
culture for 8-15 days and harvested when the median cell volume was
below 300 fl. T cells were then cryopreserved in FBS 10% DMSO for
future experiments. Prior to all experiments, T cells were thawed
and rested overnight at 37.degree. C.
Ibrutinib.
[0942] Ibrutinib (PCI-32765) was purchased from MedKoo (#202171) or
Selleck Biochemicals (#S2680) as a powder or DMSO solution. For in
vitro experiments, ibrutinib was diluted to the concentrations of
10, 100 and 1000 nM. For in vivo experiments, ibrutinib powder was
dissolved in a 10% HP-beta-cyclodextrin solution (1.6 mg/ml) and
administered to mice in the drinking water.
Multiparametric Flow Cytometry Analysis.
[0943] Anti-human antibodies were purchased from Biolegend,
eBioscience, or Becton Dickinson. Cells were isolated from in vitro
culture or from animals, washed once in PBS supplemented with 2%
fetal calf serum, and stained for 15 minutes at room temperature.
For cell number quantitation, Countbright (Invitrogen) beads were
used according to the manufacturer's instructions. In all analyses,
the population of interest was gated based on forward vs. side
scatter characteristics followed by singlet gating, and live cells
were gated using Live Dead Aqua (Invitrogen). Time gating was
included for quality control. Surface expression of CAR19 was
detected as previously described. See, e.g., Kalos et al. Science
Translational Medicine. 3.95(2011):95ra73. Flow cytometry was
performed on a four-laser Fortessa-LSR cytometer (Becton-Dickinson)
and analyzed with FlowJo X 10.0.7r2 (Tree Star).
Degranulation Assay.
[0944] Degranulation assay was performed as previously described.
See, e.g., Kalos et al. Science Translational Medicine.
3.95(2011):95ra73. T cells were incubated with target cells at a
1:5 ratio in T cell media. Anti-CD107a-PECY7 (Biolegend), anti-CD28
(BD Biosciences), anti-CD49d (BD Biosciences) antibodies and
monensin (BD Biosciences) were added to the co-culture. After 4
hours, cells were harvested and stained for CAR expression, CD3,
CD8 and Live Dead aqua staining (Invitrogen). Cells were fixed and
permeabilized (Invitrogen Fix/Perm buffers) and intracellular
staining was then performed to detect multiple cytokines (IFN,
TNF.alpha., IL-2, GM-CSF, MIP1b).
Proliferation Assay.
[0945] T cells were washed and resuspended at 1.times.10.sup.7/ml
in 100 ul of PBS and stained with 100 ul of CFSE 2.5 uM
(Invitrogen) for 5 minutes at 37.degree. C. The reaction was then
quenched with cold media, and cells were washed three times.
Targets were irradiated at a dose of 100 Gy. T cells were incubated
at a 1:1 ratio with irradiated target cells for 120 hours, adding
media at 24 hours. Cells were then harvested, stained for CD3, CAR
and Live Dead aqua (Invitrogen), and Countbright beads (Invitrogen)
were added prior to flow cytometric analysis for absolute
quantification.
Cytotoxicity Assays.
[0946] Luciferase/eGFP+ NALM-6 or RL cells were used for
cytotoxicity assay as previously described. See, e.g., Gill et al.
Blood. 123.15(2014):2343-54. Targets were incubated at the
indicated ratios with effector T cells for 4 or 16 hours. Killing
was calculated by bioluminescence imaging on a Xenogen IVIS-200
Spectrum camera.
[0947] Cytokine measurements. Effector and target cells were
co-incubated at a 1:1 ratio in T cell media for 24 h. Supernatant
was harvested and analyzed by 30-plex Luminex array (Luminex Corp,
FLEXMAP 3D) according to the manufacturer's protocol (Invitrogen).
See, e.g., Kalos et al. Science Translational Medicine.
3.95(2011):95ra73.
In Vivo Experiments.
[0948] NOD-SCID-.gamma. chain-/- (NSG) mice originally obtained
from Jackson Laboratories were purchased from the Stem Cell and
Xenograft Core of the University of Pennsylvania. All experiments
were performed on protocols approved by the Institutional Animal
Care and Use Committee (IACUC). Schematics of the utilized
xenograft models are discussed herein. Cells (MCL cell lines or T
cells) were injected in 200 ul of PBS at the indicated
concentration into the tail veins of mice. Bioluminescent imaging
was performed using a Xenogen IVIS-200 Spectrum camera and analyzed
with LivingImage software v. 4.3.1 (Caliper LifeSciences). Animals
were euthanized at the end of the experiment or when they met
pre-specified endpoints according to the IACUC protocols.
Immunohistochemistry.
[0949] Immuno-histochemical (IHC) staining of formalin fixed
paraffin embedded tissues was performed on a Leica Bond-III
instrument using the Bond Polymer Refine Detection System.
Antibodies against CD3, CD4, CD8, Pax5 and CyclinD1 were used
undiluted. Heat-induced epitope retrieval was done for 20 minutes
with ER2 solution (Leica Microsystems AR9640). Images were
digitally acquired using the Aperio ScanScope.TM..
Statistical Analysis.
[0950] All statistics were performed using GraphPad Prism 6 for
windows, version 6.04.
Mantle Cell Lymphoma Cell Lines
[0951] Most mantle cell lymphoma (MCL) lines in existence have been
immortalized and propagated for many generations in vitro, thus
losing their dependence on B cell receptor signaling. Consequently,
they are poorly sensitive to ibrutinib. In vitro experiments were
performed to determine the sensitivity of MCL cell lines on
ibrutinib treatment. These cell lines were also used to assess the
efficacy of ibrutinib/CART19 combination treatment in experiments
discussed further in this example. MCL cells were harvested from
the pleural effusion of a patient with multiply relapsed MCL. Both
the original cells (RL.sup.primary) and a cell line derived from
them (RL) had a blastoid morphology, typical MCL immunophenotype
and were positive for the classical t(11;14) translocation by
fluorescence in-situ hybridization (FISH) (FIG. 52A, 52B, 52C).
[0952] RL and JEKO-1 cells were cultured with different doses of
ibrutinib (0.1 nM, 1 nM, 10 nM, 100 nM, 1 .mu.M, and 10 .mu.M) and
sensitivity to ibrutinib was determined by measuring the reduction
of bioluminescence (BLI). As shown in FIG. 31A, RL cells were
sensitive to ibrutinib treatment in a dose-dependent manner.
However, JEKO-1 cells were resistant to ibrutinib treatment (no
demonstration of reduced bioluminescence as a function of increased
ibrutinib dosage).
[0953] Sensitivity of RL, Jeko-1, and Mino cells to ibrutinib were
also assayed using a MTT assay. Exposure of RL to increasing
concentrations of ibrutinib in vitro led to a dose-dependent
inhibition of proliferation and of downstream mediator
phosphorylation, indicating an on-target effect of ibrutinib, with
an IC50 of 10 nM. In contrast, the established MCL cell lines
Jeko-1 and Mino were relatively resistant to ibrutinib, with IC50
results up to 10 .mu.M (FIG. 52D). To confirm RL as a viable model
for in vivo experiments, immunodeficient NOD-SCID-.gamma. chain
knockout (NSG) mice were engrafted with 1.times.10.sup.6
luciferase-expressing RL cells (FIG. 52E). Their tumor burden and
survival were assessed. After intravenous injection, MCL engrafted
in all mice and localized to the spleen and liver, followed by
dissemination to bone marrow, blood and lymph nodes (FIG. 52F).
Histology of affected spleen and liver is consistent with
parenchymal infiltration of MCL. (FIG. 52G).
[0954] These results showed that these different cell lines could
therefore be used to model both ibrutinib-sensitive and
ibrutinib-resistant MCL.
Mantle Cell Lymphoma Cells are Sensitive to Killing by CART19
[0955] Most preclinical work showing the efficacy of CART19 has
been done using B-ALL cell lines, which are not sensitive to
ibrutinib. Furthermore, the best clinical responses to date have
been reported in patients with B-ALL, whereas patients with
indolent B-cell malignancies have reportedly lower responses.
[0956] To show that MCL is sensitive to killing by CART19 in this
model, healthy donor T cells were transduced with an anti-CD19 CAR
construct that has been used in clinical trials. See, e.g., Porter,
NEJM 2011. A series of in vitro experiments were performed to show
that the ibrutinib-sensitive cell line RL and the
ibrutinib-resistant cell line Jeko-1 lead to equivalent CART-19
degranulation, cytokine production, killing and proliferation (FIG.
53A, 53B, 53C, 53D). In addition, peripheral blood or bone marrow
was obtained from two patients with MCL in leukemic phase,
permitting the expansion and transduction of autologous T cells
with anti-CD19 CAR. Autologous patient-derived CART19 cells were
reactive to MCL whereas the untransduced T cells were unreactive to
their respective autologous MCL (FIG. 53E, 53F). These results
indicate that MCL are sensitive to the effector functions of
CART19.
Assessment of Ibrutinib/CART19 Treatment In Vitro
[0957] An effect of ibrutinib on T cells was previously discounted
based on short-term activity assays, as described in Honigberg et
al. Proc. Natl. Acad. Sci. USA. 107(2010):13075-80. Later, an
analysis of the effect of ibrutinib on the T cell kinase ITK was
reported to support an immunomodulatory role of ibrutinib on CD4 T
cells by inhibiting Th2-type polarization. See Dubovsky et al.
Blood 122.15(2013):2539-49. Cytokine analysis of patients treated
with CART19 by several groups indicates that CART19 therapy is
associated with both Th1 (IL2, IFN.gamma., TNF), Th2 (IL-4, IL-5,
IL-10) and other cytokines (see, e.g., Kalos et al. Science
Translational Medicine 3.95(2011):95ra73). In this example, the
effect of CART19 function was evaluated with ibrutinib at, above,
and below the ibrutinib concentrations that would be expected in
patients (mean peak concentration in serum 100-150 ng/ml) See
Advani et al. J. Clin. Oncol. 2013; 31:88.
[0958] CART19 cells were found to contain ITK. Non-specific
stimulation of CART19 cells via the TCR in the presence of
ibrutinib led to a reduction in phosphorylated ITK (pITK-Y.sub.180)
as previously reported for CD4+ T cells. See Dubovsky et al. Blood
122.15(2013):2539-49. In contrast, specific stimulation of CART19
cells via the CAR did not lead to diminished ITK activation (FIG.
54A). This observation indicated that CART19 function would likely
not be adversely affected by exposure to ibrutinib.
[0959] In addition, the short- and long-term in vitro function of
CART19 cells in the presence of ibrutinib was determined. Ibrutinib
at clinically relevant concentrations did not impair CART19 cell
proliferation, degranulation or cytokine production; although at
supra-physiologic concentrations, there was inhibition of CART19
cell functions, likely representing non-specific toxicity (FIG.
54B, 54C, 10B).
[0960] In particular, PBMCs isolated from a normal healthy donor
was transduced with a lentiviral anti-CD19 CAR construct as
described above. The resulting CAR19-expressing T cells (CART19)
were cultured and passaged to determine proliferation and expansion
capacity. A 1:1 ratio of CD4:CD8 expressing T cells were cultured
culture with or without different concentrations of ibrutinib (10
nM, 100 nM, and 1000 nM ibrutinib). Ibrutinib was added at each
cell passage. The number of cells was counted at day 0, day 5, day
6, day 7, day 9, and day 10 (FIG. 9A). Cell volume was also
monitored (FIG. 9B).
[0961] CFSE-staining and flow cytometry analysis was also used to
assess proliferation of the CART19 cells in the presence of
ibrutinib after stimulation by tumor cell lines MOLM14, JEKO-1, and
RL. MOLM14 is an AML cell line, and JEKO-1 and RL are mantle cell
lymphoma cell lines. Specifically, the RL cells are a novel MCL
cell line derived from neoplastic B-cells obtained from a pleural
effusion of a relapsed MCL patient. CART19 cells and tumor cells
were mixed in a 1:1 ratio and proliferation was assessed over 5
days. Percentage of proliferating cells are designated in each
histogram in FIGS. 10A-1, 10A-2, 10A-3, and 10A-4. Quantification
of proliferation as shown in FIG. 10B shows that high doses of
ibrutinib might inhibit CART19 cell proliferation during co-culture
with MCL cell lines.
[0962] Degranulation of T cells indicates the activation of
cytolytic T cells and the ability to initiate antigen-specific
cytotoxicity. CD107a is a functional marker of degranulation of T
cells transiently expressed on the cell surface after T cell
stimulation. Flow cytometry analysis was used to quantify
CD107a-expressing CART19 T cells after stimulation with tumor cell
lines MOLM14, JEKO-1, and RL. CD107a-expressing cells were present
in Q2 (quadrant 2) of the cell profiles shown in FIGS. 11A-1,
11A-2, 11A-3, 11A-4, 11A-5, and 11A-6. Quantification of the
results obtained from the profiles of FIGS. 11A-1, 11A-2, 11A-3,
11A-4, 11A-5, and 11A-6 is shown in FIG. 11B. These results
indicate that co-culture of CART19 cells with either MCL-RL or
JEKO-1 led to massive CAR-specific CD107a degranulation.
[0963] Cytokine production by CART19 T cells in the presence of
ibrutinib was also quantified after stimulation by different tumor
cell lines. IL-2, TNF-.alpha., and IFN-.gamma. production was
assessed by flow cytometry. Cells producing the cytokines are
present in quadrant 2 of the profiles shown in FIGS. 12-1, 12-1,
12-3, 12-4, 12-5, and 12-6, FIGS. 13-1, 13-2, 13-3, 13-4, 13-5, and
13-6, and FIGS. 14-1, 14-2, 14-3, 14-4, 14-5, and 14-6. CART19 T
cells stimulated by JEKO-1 and RL showed an increase in cytokine
expression. Also, increasing concentrations of ibrutinib treatment
did not affect the percentage of the CART19 cytokine-producing
cells.
[0964] Cytokine secretion from CART19 cells after stimulation by
tumor cells and in the presence of varying concentration of
ibrutinib was analyzed by 30-plex LUMINEX assay. Cytokines secreted
by T.sub.H1 cells, such as IL-2, IFN-.gamma., and TNF-.alpha., was
assayed. Cytokines secreted by T.sub.H2 cells, including IL-4,
IL-5, IL-6, IL-10, IL-13, IL-15, IL-17, MIP1a, GM-CSF, MIP-1b,
CP-1, IL-1Ra, IL-7, IP-10, IL-1b, VEGF, G-CSF, EGF, HGF, IFNa,
IL-12, RANTES, Eotaxin, IL-2R, MIG, and IL-8, was assayed. As shown
in FIGS. 15-1 through 15-10, T.sub.H1 and T.sub.H2 cytokines were
secreted by the CART19 T cells.
[0965] Also, experiments using two different techniques indicated
that there were no differences in Th1/Th2 polarization between
ibrutinib-exposed and ibrutinib-unexposed CART19 cells (FIGS. 54D-1
and 54D-2).
[0966] Killing of MCL cells by CART19 cells was augmented in the
presence of ibrutinib, suggesting an at least additive effect from
the combination (FIG. 54E). However, intrinsic cytotoxic function
of CART19 cells was not augmented in the presence of ibrutinib.
(FIG. 54F).
[0967] Additional bioluminescence assays were performed to assess
CART19 cell killing of tumor cells. CART19 cells were plated with
tumor cells MOLM14, JEKO-1, and RL carrying a luciferase reporter
in varying ratios, such as 1:1; 1:0.5; 1:0.25; and 1:0 in a 96 well
plate, in duplicate. After 24 hours, the bioluminescence was
detected and quantified. Results indicated that bioluminescence in
MOLM14 samples did not decrease after incubation with CART19 cells.
However, for JEKO-1 and RL cells, the bioluminescence decreased in
the presence of CART19 cells, indicating that CART19 cells mediated
JEKO-1 and RL cell killing (FIGS. 16A, 16B, 16C, 16D, 16E, and
16F). There was a further decrease in bioluminescence when treated
with ibrutinib, indicating that the combination of ibrutinib and
CART19 caused increased JEKO-1 and RL cell killing than with CART19
treatment alone. Consistent with these results, calculation of
total cells after each treatment showed that CART19 treatment of
JEKO-1 and RL cells caused reduction in cell number and a further
reduction when treated with CART19 and ibrutinib (FIGS. 17B and
17C), suggesting that the combination therapy was not only
efficacious for killing MCL cells, but led to efficient killing of
MCL cell lines.
Assessment of Ibrutinib/CART19 Treatment In Vivo
[0968] In these experiments, mouse models of mantle cell lymphoma
were used to assess CART19 and ibrutinib combination therapy in
vivo.
[0969] Schematics such as those shown in FIGS. 20, 55, and 5A were
used in this example. FIG. 20 shows a schematic of testing
CART19/ibrutinib combination therapy in the in vivo mouse models of
MCL. 1.times.10.sup.6 cells from RL (MCL-3), JEKO-1 (MCL-4), and
NALM6 (MCL-5) cell lines are injected in NSG mice (20 mice for each
experiment). After 1 week to allow engraftment, treatment is
initiated at Day 0, in which the treatment is: 0.5-1.times.10.sup.6
CART19 cells (via injection), 25 mg/kg/day ibrutinib (oral gavage),
or CART19+ ibrutinib treatment. At Day 7, Day 14, Day 21, and Day
28, the bioluminescence is imaged to monitor tumor size. Mice that
are receiving ibrutinib treatment are continuously treated with
ibrutinib. Survival of the mice is also monitored. FIGS. 55 and 56
show additional schematics of testing CART19/ibrutinib combination
therapy in the in vivo mouse models of MCL. 2.times.106 MCL-RL
cells are injected into NSG mice. After a week to allow engraftment
(engraftment confirmed by bioluminescence imaging), treatment is
initiated at Day 7 (after MCL-RL cell injection), in which
treatment is: vehicle control, CART19 cells (2.times.10.sup.6
cells), and/or ibrutinib (125 mg/kg/day). At days 14, 21, 28, and
35 (after injection of MCL-RL cells), the bioluminescence is imaged
to monitor tumor size.
[0970] The effect of ibrutinib treatment alone on an in vivo mouse
model of MCL was examined. RL cells transfected with the
GFP/luciferase gene were intravenously injected into
immunodeficient NSG mice, resulting in 100% MCL engraftment in
liver and spleen, with eventual spread into lymph nodes and bone
marrow. Mice were treated with varying doses of ibrutinib, 25
mg/kg/day and 250 mg/kg/day. Mean bioluminescence, representing
tumor growth, was assessed at various timepoints. As shown in FIG.
32, RL-derived tumors demonstrated dose-related sensitivity.
Additional experiments titrating ibrutinib doses in RL
cell-containing mice were performed and are shown in FIG. 57. A
higher dose led to a better antitumor activity without increasing
toxicity, in line with the higher dose of ibrutinib used in the
clinic for MCL (Wang et al. NEJM 369.6(2013):507-16).
[0971] CART19 dose finding was also performed. Two MCL cell lines,
RL (MCL-1) and JEKO-1 (MCL-2), carrying a GFP-luciferase reporter
were injected into NSG mice at day 0. CART19 T cells were injected
at day 7 at varying dosages, for example at 0.5.times.e6 cells,
1.times.e6 cells, or 2.times.e6 cells. Mice were monitored, for
example, for 100 days. At various timepoints, the mice were
monitored for tumor size (e.g., bioluminescence imaging) (FIGS. 18A
and 18B, and FIG. 19A), and for overall survival (e.g.,
Kaplan-Meier survival curve) (FIG. 18C and FIG. 19B). Different
doses of CART19 cells showing a dose-dependent anti-tumor efficacy,
with 2.times.10.sup.6 CART19cells/mouse being the most effective
dose (FIG. 19A).
[0972] These studies provided an opportunity to conduct a
head-to-head comparison of the two of therapies for MCL. As shown
in FIG. 58, long-term survival was achieved only in mice treated
with CART-19 cells. There was no difference in anti-tumor effect
when comparing untransduced T cells plus ibrutinib with ibrutinib
alone. Therefore, in all subsequent experiments, the control groups
were vehicle and ibrutinib alone (FIG. 59).
[0973] The addition of CART19 to ibrutinib was also tested, as
detailed in the schematic in FIG. 56. Evaluation of the effect of
ibrutinib on tumor burden indicated modestly delayed tumor growth
at early time points. In contrast, CART19 cell therapy led to a
clear decrease in tumor burden for several weeks. In mice receiving
CART19 cells alone, this was followed by an indolent relapse
beginning at Day 40, whereas mice that were treated with CART19
cells as well as ibrutinib had no detectable disease until Day 80
(FIG. 60). Histopathology of organs harvested at the end of the
experiment showed persistence of disease in all control and
ibrutinib treated mice with foci of tumor necrosis in the
ibrutinib-treated. Most of the mice treated with CART19 alone
showed indolent relapse at long term that was accompanied by the
persistence of CART19 cells, while mice treated with
CART19-ibrutinib showed clearance of the tumor and disappearance of
CART19 from involved organs (data not shown).
Mechanism of Combined Effect of CART19 and Ibrutinib
[0974] The in vitro experiments herein indicated that ibrutinib
neither impaired nor clearly augmented short-term CART19 effector
functions. The in vivo studies showed that ibrutinib monotherapy
had a modest anti-tumor effect. The results indicated that
ibrutinib could significantly enhance the anti-tumor function of
CART19 cells (FIG. 60). Therefore, experiments were performed to
determine the mechanism for this effect.
[0975] Inhibition of ITK has been shown to inhibit Th2 polarization
and skew towards a Th1 phenotype (Dubovsky et al. Blood
122.15(2013):2539-49). In mice treated with CART19 cells and
ibrutinib, an increase in Th1 cells when compared with CART19 cell
monotherapy was not observed using this assay (FIG. 61A, 61B).
However, exposure of mice to ibrutinib led to an increase in
peripheral CART19 cells. There was no difference in the
proliferation marker Ki67 between the treatment group and a control
group (FIG. 61C), so this assay did not detect a difference in
proliferation. Similarly, there was no difference in the
anti-apoptotic marker Bcl2 or the apoptosis marker phosphotidyl
serine, suggesting that the difference in CART cell numbers was not
related to an impairment of apoptosis (FIG. 61D). As ibrutinib has
been associated with peripheral lymphocytosis in patients,
experiments were done to determine whether this was also found in
mice treated with ibrutinib alone. One week after beginning
ibrutinib, there were more circulating MCL cells and fewer
nodal/organ MCL cells in ibrutinib-treated mice. Interestingly,
this increase was observed in both ibrutinib-sensitive and
-resistant in vivo model, as it was also observed in NSG mice
engrafted with the acute leukemia cell line NALM-6 (data not
shown).
[0976] In order to understand the role of ibrutinib in T cell
expansion in vivo we engrafted NSG mice with MCL-RL WT cells and
treated them with luciferase-positive T cells. Both CTL019 and
CTL019-ibrutinib treated mice showed intense T cell expansion
compared to UTD or UTD-ibrutinib (data not shown). We then
investigated the frequency of different T cells subsets in vivo and
did not see differences in PB T cells 1 week after T cells
infusion. (data not shown). Since CXCR4 is involved in
ibrutinib-driven B cell mobilization in humans, we checked the
expression of CXCR4 in vivo in PB T cells of mice treated with
CTL019 or CTL019-ibrutinib: CXCR4 level were similar in the 2
groups (data not shown). Lastly, expression of
inhibitory/costimulatory receptor on PB of T cells of mice treated
with CART19 and CART19-ibrutinib was analized. No difference in
expression of TIM3, LAG3, CD137 or CTLA4 was evident, however a
trend to a reduced PD-1 expression was noted in mice treated with
CTL019 and UTD combined with ibrutinib.
Conclusions
[0977] Therapies for B-cell malignancies include small molecule
inhibitors of BCR signaling and CD19-directed T cell based
therapies. In the setting of relapsed MCL, the BTK inhibitor
ibrutinib is now approved by the FDA and engenders high initial
response rates. Unfortunately, these responses tend to be transient
and require higher drug doses than those used for CLL. CART-19
leads to durable responses in patients with high-risk B-ALL, and it
may be efficacious in other B-cell malignancies as well.
Preliminary data suggest that the responses of mature B-cell
malignancies to CART-19 may be lower than those of B-ALL, but the
mechanism of this disparity has not yet been ascertained. This
example investigated the impact of adding ibrutinib to CART19 in
the treatment of MCL.
[0978] Different MCL cell lines with variable sensitivities to
ibrutinib (IC50 ranging from 10 nM to 10 pM) were used for in vitro
experiments. These different cell lines were used to model both
ibrutinib-sensitive and ibrutinib-resistant MCL. At all but the
highest doses of ibrutinib, CART19 cell function was unimpaired,
with intact T cell expansion kinetics, tumor recognition and
killing, and cytokine production. Furthermore, the results did not
reveal a T helper polarization upon ibrutinib exposure. This
finding may be due to a combination of factors, including the use
of a mixed culture of CD4 and CD8 cells, in contrast to the model
of CD4-only experimentation performed by Dubovksy et al. Blood
122.15(2013):2539-49. Both ibrutinib-sensitive and
ibrutinib-resistant cell lines strongly activated CART19 cells and
induced killing, cytokine production and proliferation. Combination
of CART19 and ibrutinib in vitro led to at least additive tumor
killing. The results in this example show a superiority of CART19
over ibrutinib when each was used as monotherapy at clinically
relevant doses and schedules of administration (single dose for
CART19, continuous administration for ibrutinib).
[0979] A systemic xenograft MCL model was also generated in this
example, using the MCL-RL cell line generated in a laboratory.
Treatment of these mice with different doses of allogeneic CAR19 T
cells led to a dose dependent anti-tumor effect. A similar dose
response to CART-19 was also observed in the ibrutinib resistant
JEKO-1 cell line. MCL-RL was treated in vivo with different doses
(e.g., 0, 25 and 125 mg/kg/day) of Ibrutinib, leading to a median
overall survival respectively of 70, 81 and 100 days (p<0.001).
A direct in vivo comparison of the ibrutinib 125 mg/kg and CART19
showed a significantly improved tumor control for CART19 treated
mice. Also, MCL-RL engrafted mice were treated with vehicle,
ibrutinib, CART19 or the combination of CART19 and ibrutinib
(iCART19). At clinically relevant doses, monotherapy of MCL with
CART19 was superior over monotherapy with ibrutinib, and the
combination of ibrutinib with CART19 led to an augmented anti-tumor
effect. In particular, the iCART19 combination in vivo led to
initially higher circulating levels of CART19 cells, followed by
deep tumor responses, and relapses were significantly delayed when
ibrutinib was added to CART19. The iCART19 combination resulted in
an improved tumor control with 80% of mice reaching complete
remission and long-term disease-free survival. Mechanistically,
mice treated with ibrutinib had higher numbers of circulating
CART19 cells without changes in Th1/Th2 or memory phenotype. Thus,
the results herein show that ibrutinib can be combined with CART19
in a rational manner and suggest that the properties of each of
these therapies may compensate for deficiencies of the other, thus
leading to enhanced long-term anti-tumor effect. The experiments
and results of combining BCR signaling inhibition with anti-CD19
directed T cell therapy pave the way to rational combinations of
non-crossresistant therapies for B cell malignancies.
[0980] The kinetics of tumor response and relapse suggest that
ibrutinib serves either to deepen the initial response achieved by
CTL019 alone, or to enhance the long-term immunosurveillance
capacity of CTL019 cells.
Example 9: Ibrutinib/CAR19 T-Cell Combined Therapy for Chronic
Lymphocytic Leukemia
[0981] Ibrutinib is also utilized for treatment of chronic
lymphocytic leukemia (CLL). Ibrutinib has not demonstrated
cytotoxic effects on T cells or NK cells. However, in CLL cells,
ibrutinib promotes programmed cell death and inhibits tumor cell
migration and adhesion. In this example, experiments were performed
to examine: 1) the effect of ibrutinib on CART19 production from
patients undergoing ibrutinib therapy; and 2) the optimal timing of
treatment with ibrutinib in combination with CART19 for optimal in
vivo function.
Effect of Ibrutinib on CART19 Production and Function
[0982] Normal donor PBMCs were obtained using apheresis as
described herein, for example in Example 6. The PBMCs were
incubated with 5 .mu.M of ibrutinib for 30 minutes or were left
untreated (for control), and then the cells were washed twice. The
cells were then transduced with lentiviral constructs containing
CAR19 to generate CART19 T cells using the methods described
herein, for example, in Example 4. The number of CART19 T cells
generated from ibrutinib-treated PBMCs FACs analysis was performed
to determine the number of CART19 T cells generated from
ibrutinib-treated PBMCs compared to untreated PBMCs. As shown in
FIG. 21, 15% of the transduced ibrutinib-treated PBMCs expressed
CAR19 and 12% of the transduced untreated PBMCs expressed CAR19.
These results demonstrate that ibrutinib treatment does not affect
lentiviral transduction efficiency or CART19 T cell production.
Therefore, CART19 T cells can be manufactured from CLL patients
undergoing ibrutinib treatment.
[0983] Further in vitro analysis was performed to determine the
effect of ibrutinib on CART19 T cell proliferation, CART19
cytotoxicity, and the ratio of T.sub.H1:T.sub.H2 cytokine
production.
[0984] For measuring cell proliferation, cells were stained with
CFSE for detection of proliferating cells and analyzed by FACS, as
described in Example 4. CART19 T cells were incubated with varying
concentrations of ibrutinib (0.1 .mu.M, 0.5 .mu.M, 1 .mu.M, and 5
.mu.M), and were either stimulated with CD3/CD28 beads or were left
unstimulated. In FIG. 22, histograms were overlayed to compare the
unstimulated to the CD3/CD28 stimulated CART19 T cells at each
concentration of ibrutinib. For each concentration of ibrutinib,
CD3/CD28-stimulated T cell proliferation was observed, thereby
demonstrating that ibrutinib treatment did not affect CART19 cell
proliferation.
[0985] The effect of ibrutinib on cytotoxicity of CART19 T cells
was also assessed, using methods described in Example 4.
Untransduced and CAR19-transduced T cells were treated with media,
DMSO, or 1.mu.M ibrutinib. A flow-based killing assay was performed
using titrating effector to target (E;T) ratios with effector
CART19 cells (media, DMSO, or ibrutinib-treated) to determined
specific cytotoxic activity against CD19-expressing target cells.
As shown in FIG. 23, CART19 T cells treated with ibrutinib
demonstrated the same percentage of specific cytotoxic activity
against CD19-expressing target cells as controls (CART19 T cells
treated with media or DMSO). Thus, ibrutinib treatment does not
affect CART19 cytotoxicity.
[0986] Ibrutinib treatment can limit T.sub.H2 activation, and
therefore may promote T.sub.H1 selective pressure in T cells and
skew T.sub.H1/T.sub.H2 cytokines in human CLL patients. Assays were
performed to measure T.sub.H1 and T.sub.H2 cytokine production in
the presence or absence of ibrutinib or DMSO (control). As shown in
FIG. 24, ibrutinib does not promote skewing of T.sub.H1 and
T.sub.H2 cytokines in CART19 cells.
Efficacy of Ibrutinib/CART19 Combination Treatment In Vivo
[0987] CART19 function was assessed in an in vivo mouse model.
Nalm/6 cells (human acute lymphoblastic leukemia cell line) were
implanted into NSG mice, and the mice were monitored daily for at
least 50 days. The Nalm/6 tumor model produces tumors that are not
sensitive to ibrutinib, and therefore allow analysis of CART19
function in vivo and efficacy in reducing tumor volume/treating
cancer. Starting on day 7, mice were administered either DMSO
(control) or ibrutinib daily by oral gavage. CART19 was
administered on day 7 or day 9, or mice were left untreated
(control). At days 4, 11, 18, 25 and 32, the number of Nalm/6 cells
circulating in the mice were measured, e.g., by peripheral blood
FACS analysis, to determine the efficacy of the CART19 for clearing
Nalm/6 cells in the presence or absence of ibrutinib. For example,
the cells were stained with anti-human CD19 antibody to determine
the percent of human CD19+(Nalm/6) cells in the blood of the
tumor-bearing NSG mice.
[0988] As shown in FIG. 25A, mice that did not receive CART19
injections exhibited an increase in tumor Nalm/6 cells. However,
mice that received CART19 injections in combination with daily
administration of DMSO showed successful clearing (reduction) of
Nalm/6 cells. Mice that received CART19 injections in combination
with daily administration of ibrutinib also showed successful
clearing of the Nalm/6 cells with the same kinetics and efficacy as
the mice that received DMSO treatment. Therefore, these results
demonstrate that ibrutinib treatment does not impair CART19
function in clearing Nalm/6 cells from this tumor model.
[0989] Health status of the mice were monitored for at least 50
days after injection of the Nalm/6 cells. The Kaplan-Meier survival
curve of FIG. 25B shows that none of the mice that were untreated
(did not receive CART19 T cells) and received either DMSO or
ibrutinib survived greater than 30 days after injection of the
Nalm/6 cells. However, treatment with CART19 T cells, with DMSO or
with ibrutinib increased the survival of the mice. Thus, these
results taken together with the tumor burden results from FIGS.
13-1, 13-2, 13-3, 13-4, 13-5, and 13-6 demonstrate that ibrutinib
treatment does not impair CART19 function in clearing tumor cells
from the in vivo NSG-Nalm/6 tumor model.
Optimal Timing of Ibrutinib/CART19 Combination Treatment in CLL
Patients
[0990] The optimal time during ibrutinib treatment of CLL for
administration of CART19 was assessed using samples from CLL
patients who were undergoing ibrutinib treatment for one year. PBMC
samples from 9 CLL patients (Patient 111330026, Patient 111330030,
Patient 111330039, Patient 111330056, Patient 111330073, Patient
111330074, Patient 111330081, Patient 111330086, and Patient
111330111) were isolated at different cycles of the ibrutinib
treatment and were used to manufacture CART19 T cells. The PBMC
samples were collected before ibrutinib treatment to establish a
baseline, and then collected during ibrutinib treatment at cycle 2,
day 1, and cycle 12, day 1. Several different parameters of CART19
manufacturing were assessed, such as transduction, proliferation,
cyotoxicity, and cytokine production. Other evaluations can include
ex vivo immunophenotyping, such as assessment of memory, inhibitor
molecules, and exhaustion.
[0991] FIG. 26 shows the results from flow cytometry analysis from
CAR19 transduction of T cells from Patient 111330030, which was
representative of the results obtained from the other 8 patients
after CAR transduction. PBMCs were collected from the patients at
the indicated times (baseline, e.g., before treatment; cycle 2 at
day 1; and cycle 12 at day 1) and were transduced with lentiviral
vectors containing CAR19, using methods as described, for example,
in Example 4. The transduced PBMCs were then stained for annexin,
CD3, CD4, and GAM (to detect CAR), and then analyzed by FACS
analysis. The boxed region in the graphs in FIGS. 14-1, 14-2, 14-3,
14-4, 14-5, and 14-6 shows the percentage of cells that were GAM
positive, and successfully transduced to CART19 T cells. The lower
three graphs show that CAR19 was successfully transduced in about
23% of cells at baseline, 28% of cells at cycle 2, day 1, and 44%
of cells at cycle 12, day 1.
[0992] Next, the proliferation rate (or population doublings) of
the CART19 cells or the untransduced cells (control) was assessed
at each time point (baseline, cycle 2 at day 1, and cycle 12 at day
1) for 12 days. FIG. 27-1 through 27-3 shows the graphic
representations of the population doublings over 12 days for three
patients, Patient 111330039 (#39), Patient 111330026 (#26), and
Patient 111330030 (#30). These results indicate that with regard to
proliferation rate, PBMCs isolated between or at cycle 2, day 1 and
cycle 12, day 1 are preferred for CAR transduction.
Potential Mechanisms for Ibrutinib Treatment Affecting CART19
Function
[0993] Various mechanisms of ibrutinib treatment that may affect
CART19 function was assessed from CLL patient samples.
[0994] Analysis of CD19-expressing (CD19+) cells was assessed by
FACS analysis. PBMCs from a CLL patient undergoing ibrutinib
treatment were isolated at baseline (before ibrutinib treatment),
at cycle 2, day 1, and at cycle 12, day 1, and were subsequently
stained for CD19. FACS analysis showed that ibrutinib causes a
decrease in CD19-expressing cells (FIG. 28A, FIG. 28B, and FIG.
28C). Thus, these results indicate that ibrutinib treatment induces
lymphocytosis.
[0995] Additional analysis was performed to examine CD200
expression on tumor cells over time during ibrutinib treatment. The
immune-suppressive molecule CD200 is up-regulated on primary B cell
CLL tumor cells. CD200 binds to its receptor, CD200R, which is
expressed on cells of the monocyte/macrophage lineage and on T
lymphocytes. Interaction of CD200 with its receptor delivers an
inhibitory signal to the macrophage lineage altering cytokine
profiles from T.sub.H1 to T.sub.H2 and results in the induction of
regulatory T cells. Samples from Patients 111330030, 111330026, and
111330039 at baseline (screen), cycle 2, day 1, and cycle 12, day 1
were stained for annexin, CD19 (to sort for CD19-expressing tumor
cells), and CD200 and analyzed by FACS. The histograms detecting
CD200 expression on tumor cells from each timepoint was overlaid
for each patient (FIGS. 29A, 29B, and 29C). Generally, CD200
expression on tumor cells decreased over time during ibrutinib
treatment.
[0996] The frequency of PD1-expressing T cells during ibrutinib
treatment was also assessed. Samples from patients were obtained at
baseline, cycle 2, day 1, and cycle 12, day 1 and stained with
annexin, CD3, CD8, and PD1. Cells that were negative for annexin
and positive for CD3 were analyzed for CD8 and PD1 expression. The
cells that express CD8 and PD1 are designated by the box in FIGS.
30A, 30B, and 30C. Comparison of FACS profiles of cells at baseline
(FIG. 30A), cycle 2, day 1 (FIG. 30B), and cycle 12, day 1 (FIG.
30C), indicates that ibrutinib treatment decreases the frequency of
PD1-expressing cells over time.
[0997] The data obtained from the experiments described above
indicate that the optimal time for administering CART19 therapy to
CLL patients receiving ibrutinib is between cycle 2 and cycle 12,
or at cycle 12.
Example 10: CAR19 T Cell Therapy for Hodgkin Lymphoma
[0998] CAR19 T cell therapy can also be used to treat Hodgkin
lymphoma (HL). Hodgkin lymphoma is characterized by the presence of
malignant Hodgkin Reed-Sternberg (HRS) cells that are derived from
clonal germinal center B cells. There are several factors that
indicate the therapeutic efficacy of CAR19 T cell therapy for HL.
CD19 staining of HL tumors shows CD19-expressing (CD19.sup.+) cells
within the tumor and tumor microenvironment (FIG. 33). A study has
shown that a clonal B cell population (CD20+CD27+ ALDH.sup.+) that
expresses CD19 is responsible for the generation and maintenance of
Hodgkin lymphoma cell lines, and also circulates in the blood of
most HL patients (Jones et al., Blood, 2009, 113(23):5920-5926).
This clonal B cell population has also been suggested to give rise
to or contribute to the generation of the malignant HRS cells.
Thus, CART19 therapy would deplete this B cell population that
contributes to tumorigenesis or maintenance of tumor cells. Another
study showed that B cell depletion retards solid tumor growth in
multiple murine models (Kim et al., J Immunotherapy, 2008,
31(5):446-57). In support of the idea that depletion of B cells in
the HL tumormicroenvironment results in some anti-tumor effect,
current therapies, such as rituxan, are being clinically tested for
targeting and depletion of tumoral B cells in HL (Younes et al.,
Blood, 2012, 119(18):4123-8). De novo carcinogenesis related to
chronic inflammation has also been shown to be B-cell dependent (de
Visser, et al., Cancer Cell, 2005, 7(5):411-23). The results from
these studies indicate that targeting of the B cell population,
particularly in the HL tumor microenvironment, would be useful for
treating HL, by reducing or inhibiting disease progression or tumor
growth.
[0999] In addition, normal CD19-expressing B cells also infiltrate
the tumor microenvironment in HL. Previous studies with CART19
therapy in CLL and ALL (e.g., described in Examples 4 and 5) show
that CART19 exposure to CD19+ targets leads to cytokine production
and macrophage production. Thus, modulation of the HL tumor
microenvironment from a pro-tumor microenvironment to an anti-tumor
microenvironment can be achieved by infusing CART19 to interact
with normal CD19+B cells present in the HL. For example, CART19
exposure to CD19-expressing targets causes cytokine production,
e.g., inflammatory cytokines, that promote anti-tumor activity
through the expansion of cytotoxic T cells, activation of
macrophages, and recruitment of other immune effector cells with
various functions that inhibit tumor growth, such as leukocytes,
macrophages, and antigen-presenting cells. Because the target
CD19+B cells may not be malignant (e.g., normally circulating B
cells), a transient rather than protracted CART19 effect may be
preferred for modulation of the tumor microenvironment.
[1000] A study to examine the therapeutic efficacy of CART19
therapy in HL patients can be performed as described below (FIG.
34). The study will also assess the safety and tolerability of
CART19 in HL subjects, and determine the effect of CART19 cells on
the HL tumor microenvironment.
[1001] 8 patients with classical HL are treated in this study.
Patients are of all ages, though separate protocols for drug
delivery can be established for pediatric and adult patients.
Patients in this study have no available potentially curative
treatment options (such as autologous (ASCT) or allogeneic stem
cell transplantation), or are not suitable for such curative
treatment options. For example, patients can be any of the
following: PET+ after salvage chemotherapy, PET+ after treatment
with brentuximab, or PET+ after ASCT with or without prior
brentuximab exposure. The patients will have a limited prognosis
(several months to less than or equal to 2 year expected survival)
with currently available therapies. And finally, the patients will
not have received anti-CD20 antibody therapy. Patients are excluded
due to lack of feasibility, e.g., if the patient has insufficient
numbers of T cells for 6 infusions of CART19.
[1002] An mRNA CAR19 is produced by in vitro transcription. The
CAR19 mRNA is electroporated into donor T cells, and the resulting
cells are expanded and stimulated by incubation with CD3/CD28
beads. Dosages containing 1.times.10.sup.8-5.times.10.sup.8
RNA-electroporated CAR19 T cells are delivered to the patient three
times a week for two weeks (e.g., at day 0, 2, 4, 7, 9 and 11). The
overall response rate will be assessed by clinical, CT, and PET
scanning at 1 month after treatment. Response and survival will be
monitored monthly for the first 6 months, then every 3 months until
2 years after the first CART19 infusion (day 0). Monitoring
techniques include biopsy of the tumor or lymph node (e.g., for
immunohistochemical analysis and/or RNA for gene expression
profiling) and PET scanning before and after CART19 treatment. For
example, the effect of the CART19 cells on the HL tumor
microenvironment are analyzed by comparing the results of gene
expression profiling performed on accessible lymph node biopsies
from selected patients before treatment and approximately one week
after treatment (or the appropriate time after treatment to allow
for alteration of cellular phenotype). To assess the safety and
tolerability of CART19 treatment, the frequency and severity of
adverse events are reported, including the frequency of cytokine
release syndrome (CRS) and macrophage activation syndrome
(MAS).
[1003] Chemotherapy may be administered concurrently with CART19
treatment. The first dose of CART19 can be preceded by
lymphodepleting chemotherapy, e.g., cytoxan.
Example 11: Non-Responder Subset of CLL Patients Exhibit Increased
Expression of Immune Checkpoint Inhibitor Molecules
[1004] In this study, CART19 cells from clinical manufacture from
34 CLL patients were assessed for expression of immune checkpoint
inhibitor molecules, such as PD-1, LAG3, and TIM3. The response of
this cohort to CART19 was known and hence a correlation between
response and biomarker expression patterns could be assessed.
[1005] Manufactured CART19 cells from CLL patients with different
responses to CART therapy were analyzed by flow cytometry to
determine the expression of CAR and the immune checkpoint inhibitor
molecules PD-1, LAG3, and TIM3. The CART19 cells were from: healthy
donors (HD) (n=2); CLL patients that responded to CART therapy (CR)
(n=5); CLL patients that partially responded to CART therapy (PR)
(n=8); CLL patients that did not respond to CART therapy (NR)
(n=21). Cells were stained with fluorescently labeled antibodies
that specifically recognize CD3, CD4, CD8, CD27, CD45RO, the CAR19
molecule, and immune checkpoint molecules PD-1, LAG3, and TIM3,
according to standard methods for flow cytometry analysis known in
the art. Expression of each marker, e.g., CD4+, CD8+, etc., was
determined by flow cytometry analysis software, and subpopulations
(e.g., CD4+ T cells, CD8+ T cells, or CAR19-expressing T cells)
were further analyzed for the expression of immune checkpoint
molecules PD-1, LAG3, and TIM3.
[1006] An example of the flow cytometry profiles analysis used to
determine surface marker expression is shown in FIGS. 35A and 35B.
T cells expressing CD4 were determined using flow cytometry, and
were further analyzed for CAR19 and PD-1 expression, such that the
x-axis of the profiles indicate CAR19 expression (the top left (Q5)
and bottom left (Q8) quadrants show the CAR19-negative CD4+ cells,
while the top right (Q6) and bottom right (Q7) quadrants show the
CAR19-expressing CD4+ cells) and the y-axis shows PD-1 expression
(the bottom left (Q8) and right (Q7) quadrants show the PD-1
negative CD4+ cells and the top left (Q5) and right (Q6) quadrants
show the PD-1-expressing CD4+ cells). In the CD4+ population from a
CART responder, 44.7% of the CD4+ cells overall expressed PD-1, and
about 22.3% of the CAR19-expressing cells were PD-1 positive, while
27.2% of CAR19-expressing cells were PD-1 negative (FIG. 35A). In
contrast, in the CD4+ population from a non-responder, there was a
significant decrease in CAR19-expressing cells overall (about 15.3%
compared to the 49.5% in CR), with 14.7% of the CAR19-expressing
cells being PD-1 positive while only 0.64% were PD-1 negative (FIG.
35B). Comparison between the profiles in FIG. 35A and FIG. 35B
shows that a much higher percentage of the CD4+ cells from a
non-responder express PD-1 (about 92.9%) compared to the CART
responder (about 44.7%).
[1007] Using the methods and analysis described above, the
percentage of PD-1 expressing (PD-1+) cells of the CD4+ population
and the CD8+ population was determined for each patient in each
response group. Non-responders were shown to have a greater
percentage of PD-1+ cells in both the CD4+(FIG. 35C) and CD8+(FIG.
35D) populations compared to those that responded to CAR therapy
(CR); the increase of average PD-1 percentage was statistically
significant for both CD4+ and CD8+ populations. Partial responders
(PR) exhibited higher percentages of PD-1+ cells than responders
(CR) in both CD4+(FIG. 35C) and CD8+(FIG. 35D) populations.
[1008] Next, the percentage of PD-1 expressing (PD-1+) cells of the
CAR19-expressing CD4+ population and the CAR19-expressing CD8+
population was determined for each patient in each response group.
Similar analysis was performed as above, with the additional step
of analyzing the CD4+ and CD8+ cells for CAR19-expression, and
after identification of the CAR19-expressing cells, determining the
percentage of cells with PD-1 expression from the populations of
CAR19-expressing cells. A similar trend as that observed in the
CD4+ and CD8+ overall populations was observed for the CAR19
expressing CD4+ and CD8+ populations: non-responders were shown to
have a greater percentage of PD-1+ cells in both the CD4+(FIG. 36A)
and CD8+(FIG. 36B) populations compared to those that responded to
CAR therapy (CR); the increase of average PD-1 percentage was
statistically significant for both CD4+ and CD8+ populations.
Partial responders (PR) exhibited higher percentages of PD-1+ cells
than responders (CR) in both CD4+(FIG. 36A) and CD8+(FIG. 36B)
populations.
[1009] Further analysis was performed to determine the distribution
of cells expressing PD-1, LAG3, and TIM3 from patients with
different responses to CAR therapy. Representative cell profile
analysis for PD-1, LAG3, and TIM3expression in the CD4+ population
is shown in FIG. 37. The cell populations were first analyzed for
CD4+ and CD8+ expression. The CD4+ population (or CD8+ population,
not shown) was then analyzed for PD-1 and CAR19 expression (FIG.
37, left profiles). As described previously, non-responders (NR)
had a significantly increased percentage of cells that were PD-1+
overall compared to CART responders (CR) (about 92.9% PD-1 positive
for NR compared to 44.7% PD-1 positive for CR). Moreover, in
non-responders, CAR19-expressing cells were mostly PD-1 positive
(14.7% PD-1 positive and CAR+ compared to 0.64% PD-1 negative and
CAR+). Then the populations were analyzed for PD-1 and LAG3
co-expression (FIG. 37, middle profiles). Cells that expressed both
PD-1 and LAG3 are shown in the top right quadrant (Q2).
Non-responders had a significantly increased percentage of cells
that expressed both immune checkpoint inhibitors, PD-1 and LAG3,
compared to CART responders (67.3% compared to 7.31%). PD-1
expression was also analyzed with TIM3 expression. In FIG. 37,
right profiles, the box indicates the cells that express both PD-1
and TIM3. Similar to the results obtained with PD-1 and LAG3, the
non-responders had a significantly higher percentage of cells that
expressed both immune checkpoint inhibitors, PD-1 and TIM3,
compared to CART responders (83.3% compared to 28.5%). The
percentage of PD-1 expressing cells (PD1+), PD-1 and
LAG3-expressing cells (PD1+ LAG3+), and PD-1 and TIM3-expressing
cells (PD1+TIM3+) was determined for each patient in each response
group using the flow cytometry analysis as described above.
Non-responders were shown to have an increased percentage of PD1+
LAG3+ cells (FIG. 38A) and PD1+TIM3+ cells (FIG. 38B) compared to
CART responders that was statistically significant for both cell
populations. Partial responders also showed an increased percentage
of both cell populations compared to CART responders, with the
averages being decreased compared to the non-responders.
[1010] These results indicate that patients that do not respond to
CAR therapy exhibit increased expression of immune checkpoint
inhibitors (e.g., PD-1, LAG3, and TIM3) compared to patients that
respond or partially respond to CAR therapy. Thus, these results
show that agents that inhibit or decrease expression of immune
checkpoint inhibitors, e.g., PD-1, LAG3, or TIM3, may be useful for
administration to patients receiving CAR therapy to prevent immune
suppression through immune checkpoint pathways (e.g., mediated by
PD-1, LAG3, or TIM3), thereby increasing the efficacy of the
CAR-expressing cells.
Example 12: Effects of mTOR Inhibition on Immunosenescence in the
Elderly
[1011] One of the pathways most clearly linked to aging is the mTOR
pathway. The mTOR inhibitor rapamycin has been shown to extend
lifespan in mice and improve a variety of aging-related conditions
in old mice (Harrison, D E et al. (2009) Nature 460:392-395;
Wilkinson J E et al. (2012) Aging Cell 11:675-682; and Flynn, J M
et al. (2013) Aging Cell 12:851-862). Thus, these findings indicate
that mTOR inhibitors may have beneficial effects on aging and
aging-related conditions in humans.
[1012] An age-related phenotype that can be studied in a short
clinical trial timeframe is immunosenescence. Immunosenescence is
the decline in immune function that occurs in the elderly, leading
to an increased susceptibility to infection and a decreased
response to vaccination, including influenza vaccination. The
decline in immune function with age is due to an accumulation of
immune defects, including a decrease in the ability of
hematopoietic stem cells (HSCs) to generate naive lymphocytes, and
an increase in the numbers of exhausted PD-1 positive lymphocytes
that have defective responses to antigenic stimulation (Boraschi, D
et al. (2013) Sci. Transl. Med. 5:185ps8; Lages, C S et al. (2010)
Aging Cell 9:785-798; and Shimatani, K et al., (2009) Proc. Natl.
Acad. Sci. USA 106:15807-15812). Studies in elderly mice showed
that 6 weeks of treatment with the mTOR inhibitor rapamycin
rejuvenated HSC function leading to increased production of naive
lymphocytes, improved response to influenza vaccination, and
extended lifespan (Chen, C et al. (2009) Sci. Signal. 2:ra75).
[1013] To assess the effects of mTOR inhibition on human
aging-related phenotypes and whether the mTOR inhibitor RAD001
ameliorates immunosenescence, the response to influenza vaccine in
elderly volunteers receiving RAD001 or placebo was evaluated. The
findings presented herein suggest that RAD001 enhanced the response
to influenza vaccine in elderly volunteers at doses that were well
tolerated. RAD001 also reduced the percentage of programmed death
(PD)-1 positive CD4 and CD8 T lymphocytes that accumulate with age.
These results show that mTOR inhibition has beneficial effects on
immunosenescence in elderly volunteers.
[1014] As described herein, a 6 week treatment with the mTOR
inhibitor RAD001, an analog of rapamycin, improved the response to
influenza vaccination in elderly human volunteers.
Methods
Study Population
[1015] Elderly volunteers >=65 years of age without unstable
underlying medical diseases were enrolled at 9 sites in New Zealand
and Australia. Exclusion criteria at screening included hemoglobin
<9.0 g/dL, white blood cell count <3,500/mm.sup.3, neutrophil
count <2,000/mm.sup.3, or platelet count <125,000/mm.sup.3,
uncontrolled diabetes, unstable ischemic heart disease, clinically
significant underlying pulmonary disease, history of an
immunodeficiency or receiving immunosuppressive therapy, history of
coagulopathy or medical condition requiring long-term
anticoagulation, estimated glomerular filtration rate <30
ml/min, presence of severe uncontrolled hypercholesterolemia
(>350 mg/dL, 9.1 mmol/L) or hypertriglyceridemia (>500 mg/dL,
5.6 mmol/L).
[1016] Baseline demographics between the treatment arms were
similar (Table 7). Of the 218 subjects enrolled, 211 completed the
study. Seven subjects withdrew from the study. Five subjects
withdrew due to adverse events (AEs), one subject withdrew consent,
and one subject left the study as a result of a protocol
violation.
TABLE-US-00023 TABLE 8 Demographic and Baseline characteristics of
the Study Patients RAD001 RAD001 RAD001 0.5 mg 5 mg 20 mg Placebo
daily weekly weekly pooled Total Population N = 53 N = 53 N = 53 N
= 59 N = 218 Age (Years) Mean (SD) 70.8 (5.0) 72.0 (5.3) 71.4 (5.2)
71.1 (5.1) 71.3 (5.2) Gender Male - n 34 (64%) 27 (51%) 32 (60%) 31
(53%) 124 (57%) (%) BMI* Mean (SD) 27.4 (4.2) 28.8 (5.0) 28.0 (4.1)
28.0 (4.2) 28.0 (4.4) (kg/m2) Race - n (%) Caucasian 48 (91%) 50
(94%) 46 (87%) 54 (92%) 198 (91%) Other 5 (9%) 3 (6%) 7 (13%) 5
(8%) 20 (9%) *The body-mass index is weight in kilograms divided by
the square of the height in meters Study Design and Conduct
[1017] From December 2011 to April 2012, 218 elderly volunteers
were enrolled in a randomized, observer-blind, placebo-controlled
trial. The subjects were randomized to treatment arms using a
validated automated randomization system with a ratio of RAD001 to
placebo of 5:2 in each treatment arm. The treatment arms were:
[1018] RAD001 0.5 mg daily or placebo
[1019] RAD001 5 mg weekly or placebo
[1020] RAD001 20 mg weekly or placebo
[1021] The trial was observer-blind because the placebo in the
RAD001 0.5 mg daily and 20 mg weekly cohorts differed slightly from
the RAD001 tablets in those cohorts. The study personnel evaluating
the subjects did not see the study medication and therefore were
fully blinded. The treatment duration for all cohorts was 6 weeks
during which time subjects underwent safety evaluations in the
clinic every 2 weeks. After subjects had been dosed for 4 weeks,
RAD001 steady state levels were measured pre-dose and at one hour
post dose. After completing the 6 week course of study drug,
subjects were given a 2 week drug free break to reverse any
possible RAD001-induced immunosuppression, and then were given a
2012 seasonal influenza vaccination (Agrippal.RTM., Novartis
Vaccines and Diagnostics, Siena, Italy) containing the strains H1N1
A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008.
Four weeks after influenza vaccination, subjects had serum
collected for influenza titer measurements. Antibody titers to the
3 influenza vaccine strains as well as to 2 heterologous strains
(A/H1N1 strain A/New Jersy/8/76 and A/H3N2 strain
A/Victoria/361/11) were measured by standard hemagglutination
inhibition assay (Kendal, A P et al. (1982) Concepts and procedures
for laboratory-based influenza surveillance. Atlanta: Centers for
Disease Control and Prevention B17-B35). Levels of IgG and IgM
specific for the A/H1N1/California/07/2009 were measured in serum
samples taken before and 4 weeks after influenza vaccination as
described previously (Spensieri, F. et al. (2013) Proc. Natl. Acad.
Sci. USA 110:14330-14335). Results were expressed as fluorescence
intensity.
[1022] All subjects provided written informed consent. The study
was conducted in accordance with the principals of Good Clinical
Practice and was approved by the appropriate ethics committees and
regulatory agencies.
Safety
[1023] Adverse event assessment and blood collection for
hematologic and biochemical safety assessments were performed
during study visits. Adverse event information was also collected
in diaries that subjects filled out at home during the 6 weeks they
were on study drug. Data on all adverse events were collected from
the time of informed consent until 30 days after the last study
visit. Events were classified by the investigators as mild,
moderate or severe.
Statistical Analysis
[1024] The primary analysis of geometric mean titer ratios was done
using a normal Bayesian regression model with non-informative
priors. This model was fitted to each antibody titer on the log
scale. The primary outcome in each model was the Day 84
measurement. The Day 63 measurement was included in the outcome
vector. The model fitted using SAS 9.2 proc mixed with the prior
statement. The covariance structure of the matrix was considered as
unstructured (option type=UN). A flat prior was used. For the
secondary analysis of seroconversion rates, logistic regression was
used.
[1025] The intention to treat population was defined as all
subjects who received at least one full dose of study drug and who
had no major protocol deviations impacting efficacy data. 199 out
of the total of 218 subjects enrolled in the study were in the
intention to treat population.
Immunophenotyping
[1026] Peripheral blood mononuclear cells were isolated from whole
blood collected at 3 time points: baseline; after 6 weeks of study
drug treatment; and at the end of study when subjects had been off
study drug for 6 weeks and 4 weeks after influenza vaccination.
Seventy-six PBMC subsets were analyzed by flow cytometry using
8-color immunophenotyping panels at the Human Immune Monitoring
Center at Stanford University, CA, USA as described previously
(Maecker, H T et al. (2012) Nat Rev Immunol. 12:191-200).
Seventy-six PBMC subsets were analyzed by flow cytometry using
8-color lyophilized immunophenotyping panels (BD Lyoplate, BD
Biosciences, San Diego, Calif.). PBMC samples with viability
>80% and yield of 2.times.10.sup.6 cells or greater were
included in the analysis.
[1027] Relative changes of the immunophenotypes from baseline to
Week 6 of study drug treatment and from baseline to the end of
study (Week 12) were calculated for each of the RAD001 dosing
cohorts. Student T test was conducted to examine if the relative
change of the immunophenotypes from baseline to the two blood
sampling time points was significantly different from zero,
respectively, within each dosing group after adjusting for placebo
effect. Missing data imputation in treatment effect analysis was
not conducted. Therefore if a patient has a missing phenotype data
at baseline, this patient was not be included in the analysis for
this phenotype. If a patient had a missing phenotype data at 6 or
12 weeks, then this patient did not contribute to the analysis of
this phenotype for the affected timepoint.
[1028] 608 tests in 76 phenotypes under 3 dosing groups were
conducted to compare the treatment effect against the placebo
effect. Stratified false discovery rate (FDR) control methodology
was implemented to control the occurrence of false positives
associated with multiple testing yet provide considerably better
power. The cell type group was taken as the stratification factor
and conducted FDR (q-value) calculation within each stratum
respectively. All null-hypotheses were rejected at 0.05
significance level with corresponding q-value .ltoreq.0.1. The
multiple testing adjustment strategy with rejecting at 0.05
significance level and corresponding q<0.1 ensured that less
than 10% of the findings are false.
[1029] In a second analysis, the immunophenotype changes between
pooled treatment and placebo groups, where all three RAD001 dosing
groups were combined. To determine which immunophenotype changes
differed between the treated and placebo groups, within-patient
cell count ratios for each measured phenotype were calculated
between baseline and Week 6 of study drug treatment and between
baseline and the end of study (Week 12). The ratios were log
transformed, and analyzed by analysis of covariance at each time
point in order to detect a difference between the pooled treatment
and placebo groups. 152 tests in 76 phenotypes were performed to
compare the pooled treatment effect against the placebo effect.
Stratified false discovery rate (FDR) control methodology was
implemented to control the occurrence of false positives associated
with multiple testing yet provide considerably better power
(Benjamini, Y. et al. (1995) J. Roy. Statist. 57:289-300; and Sun,
L. et al. (2006) Genet. Epidemiol. 30:519-530). The cell type group
was taken as the stratification factor and FDR (q-value)
calculation was conducted within each stratum respectively. All
null-hypotheses at 0.05 significance level and q-value less than
20% were rejected. This can be interpreted as rejecting only those
hypotheses with P values less than 0.05 and less than 20%
probability that the each observed significant result is due to
multiple testing.
Results
[1030] In general, RAD001 was well tolerated, particularly the 0.5
mg daily and 5 mg weekly dosing regimens. No deaths occurred during
the study. Three subjects experienced four serious adverse events
(SAEs) that were assessed as unrelated to RAD001. The 4 SAEs were
retinal hemorrhage of the left eye with subsequent blindness in a
subject with normal platelet counts who had completed a 6 week
course of 5 mg weekly RAD001 6 weeks previously; severe back pain
in a subject treated with placebo and severe gastroenteritis in a
subject treated with placebo. A list of treatment-related adverse
events (AEs) with an incidence >2% in any treatment group is
provided in Table 9. The most common RAD001-related AE was mouth
ulcer that, in the majority of cases, was of mild severity.
Overall, subjects who received RAD001 had a similar incidence of
severe AEs as those treated with placebo. Only one severe AE was
assessed as related to RAD001 mouth ulcers in a subject treated
with 20 mg weekly RAD001.
TABLE-US-00024 TABLE 9 Incidence of treatment-related AEs >2% in
any treatment group by preferred term RAD001 RAD001 RAD001 Placebo,
0.5 mg daily 5 mg weekly 20 mg weekly pooled Total N = 53 N = 53 N
= 53 N = 59 N = 218 n (%) n (%) n (%) n (%) n (%) Total AE(s) 35 46
109 21 211 Patients with AE(s) 22 (41.5%) 20 (37.7%) 27 (50.9%) 12
(20.3%) 81 (37.2%) Mouth ulceration 6 (11.3%) 2 (3.8%) 9 (17.0%) 3
(5.1%) 20 (9.2%) Headache 0 2 (3.8%) 9 (17.0%) 1 (1.7%) 12 (5.5%)
Blood cholesterol 2 (3.8%) 2 (3.8%) 2 (3.8%) 0 6 (2.8%) increased
Diarrhea 1 (1.9%) 4 (7.5%) 1 (1.9%) 0 6 (2.8%) Dyspepsia 0 3 (5.7%)
2 (3.8%) 1 (1.7%) 6 (2.8%) Fatigue 0 2 (3.8%) 4 (7.5%) 0 6 (2.8%)
Low density lipoprotein 2 (3.8%) 1 (1.9%) 2 (3.8%) 0 5 (2.3%)
increased Tongue ulceration 3 (5.7%) 1 (1.9%) 0 1 (1.7%) 5 (2.3%)
Insomnia 1 (1.9%) 2 (3.8%) 1 (1.9%) 0 4 (1.8%) Dry mouth 0 0 2
(3.8%) 1 (1.7%) 3 (1.4%) Neutropenia 0 0 3 (5.7%) 0 3 (1.4%) Oral
pain 0 2 (3.8%) 1 (1.9%) 0 3 (1.4%) Pruritus 0 2 (3.8%) 1 (1.9%) 0
3 (1.4%) Conjunctivitis 0 2 (3.8%) 0 0 2 (0.9%) Erythema 0 2 (3.8%)
0 0 2 (0.9%) Limb discomfort 0 2 (3.8%) 0 0 2 (0.9%) Mucosal
inflammation 0 0 2 (3.8%) 0 2 (0.9%) Paresthesia oral 2 (3.8%) 0 0
0 2 (0.9%) Stomatitis 0 0 2 (3.8%) 0 2 (0.9%) Thrombocytopenia 0 0
2 (3.8%) 0 2 (0.9%) Urinary tract infection 0 0 2 (3.8%) 0 2
(0.9%)
[1031] The ability of RAD001 to improve immune function in elderly
volunteers was evaluated by measuring the serologic response to the
2012 seasonal influenza vaccine. The hemagglutination inhibition
(HI) geometric mean titers (GMT) to each of the 3 influenza vaccine
strains at baseline and 4 weeks after influenza vaccination are
provided in Table 10. The primary analysis variable was the HI GMT
ratio (4 weeks post vaccination/baseline). The study was powered to
be able to demonstrate that in at least 2 out of 3 influenza
vaccine strains there was 1) a .gtoreq.1.2-fold GMT increase
relative to placebo; and 2) a posterior probability no lower than
80% that the placebo-corrected GMT ratio exceeded 1. This endpoint
was chosen because a 1.2-fold increase in the influenza GMT ratio
induced by the MF-59 vaccine adjuvant was associated with a
decrease in influenza illness (Iob, A et al. (2005) Epidemiol
Infect 133:687-693).
TABLE-US-00025 TABLE 10 HI GMTs for each influenza vaccine strain
at baseline and at 4 weeks after influenza vaccination Influenza
RAD001 RAD001 5 mg RAD001 Vaccine 0.5 mg daily weekly 20 mg weekly
Placebo Strain Time N = 50 N = 49 N = 49 N = 55 A/H1N1 GMT (CV %)
Baseline 102.8 (186.9) 84.2 (236.4) 90.1 (188.4) 103.2 (219.7) Week
4 190.2 (236.9) 198.73 (195.6) 129.7 (175.9) 169.4 (259.8) GMT
ratio 2.6 (302.5) 2.5 (214.3) 1.8 (201.5) 2.0 (132.7) (CV %) A/H3N2
GMT (CV %) Baseline 106.8 (168.2) 126.04 (162.6) 137.1 (211.5)
131.7 (162.3) Week 4 194.4 (129.1) 223.0 (118.8) 223.0 (163.6)
184.3 (153.2) GMT ratio 2.1 (152.6) 2.0 (189.2) 2.1 (277.3) 1.6
(153.6) (CV %) B GMT (CV %) Baseline 44.2 (96.6) 64.8 (87.3) 58.0
(156.0) 57.0 (112.6) Week 4 98.4 (94.8) 117.3 (99.9) 99.2 (124.1)
114.6 (136.7) GMT ratio 2.5 (111.2) 2.2 (112.8) 2.1 (126.5) 2.2
(109.2) (CV %) Baseline indicates 2 weeks prior to influenza
vaccination Week 4 indicates 4 weeks after influenza vaccination N
is number of subjects per cohort GMT is geometric mean titer GMT
ratio is the GMT at week 4 post vaccination/GMT at baseline CV %
indicates coefficient of variation
[1032] In the intent-to-treat (ITT) population, the low, immune
enhancing, dose RAD001 (0.5 mg daily or 5 mg weekly) cohorts but
not higher dose (20 mg weekly) cohort met the primary endpoint of
the study (FIG. 40A). This demonstrates that there is a distinct
immunomodulatory mechanism of RAD001 at the lower doses, and that
at the higher dose the known immunosuppressive effects of mTOR
inhibition may come into play. Furthermore, the results suggest a
trend toward improved immune function in the elderly after low,
immune enhancing, dose RAD001 treatment.
[1033] In a subgroup analysis, the subset of subjects with low
baseline influenza titers (.ltoreq.1:40) experienced a greater
RAD001-associated increase in titers than did the ITT population
(FIG. 40B). These data show that RAD001 is particularly effective
at enhancing the influenza vaccine response of subjects who did not
have protective (>1:40) titers at baseline, and therefore were
at highest risk of influenza illness.
[1034] Scatter plots of RAD001 concentration versus increase in
titer to each influenza vaccine strain show an inverse
exposure/response relationship (FIG. 41). Modeling and simulation
based on mTOR mediated phosphorylation of S6 kinase (S6K) predicts
that the 20 mg weekly dosing regimen inhibits mTOR-mediated S6K
activity almost completely, the 5 mg weekly dosing regimen inhibits
S6K activity by over 50%, and the 0.5 mg daily dosing regiment
inhibits S6K phosphorylation by approximately 38% during the dosing
interval (Tanaka, C et al. (2008) J. Clin. Oncol 26:1596-1602).
Thus, partial mTOR inhibition, e.g., mTOR-mediated S6K
phosphorylation, with low, immune enhancing, dose RAD001 may be as,
if not more effective, than near complete mTOR inhibition with high
dose RAD001 at enhancing the immune response of the elderly.
[1035] Rates of seroconversion 4 weeks after influenza vaccination
were also evaluated. Seroconversion was defined as the change from
a negative pre-vaccination titer (i.e., HI titer <1:10) to
post-vaccination HI titer .gtoreq.1:40 or at least 4-fold increase
from a non-negative (.gtoreq.1:10) pre-vaccination HI titer. In the
intention-to-treat population, seroconversion rates for the H3N2
and B strains were increased in the RAD001 as compared to the
placebo cohorts although the increases did not meet statistical
significance (Table 11). In the subpopulation of subjects with
baseline influenza titers <=1:40, RAD001 treatment also
increased the rates of seroconversion to the H3N2 and B strains,
and these results reached statistical significance for the B strain
in the 0.5 mg daily dosing cohort. These data further show that
RAD001 enhanced the serologic response to influenza vaccination in
the elderly.
TABLE-US-00026 TABLE 11 Percent of subjects with seroconversion to
influenza 4 weeks after vaccination Placebo 0.5 mg 5 mg 20 mg N =
54 N = 48 N = 49 N = 48 Intention to Treat Population H1N1 24 27 27
17 H3N2 17 27 24 25 B 17 27 22 19 Subjects with Baseline Titers
<= 40 H1N1 40 42 45 36 H3N2 42 64 53 71 B 16 40* 33 28 *Odds
ratio for seroconversion between RAD001 and Placebo significantly
different than 1 (two-sided p-value < 0.05 obtained by logistic
regression with treatment as fixed effect)
[1036] Current seasonal influenza vaccines often provide inadequate
protection against continuously emerging strains of influenza that
present as variants of previously circulating viruses. However,
mice vaccinated against influenza in the presence of the mTOR
inhibitor rapamycin, as compared to placebo, developed a broader
serologic response to influenza. The broader serologic response
included antibodies to conserved epitopes expressed by multiple
subtypes of influenza that provided protection against infection
with heterologous strains of influenza not contained in the vaccine
(Keating, R et al. (2013) Nat Immunology 14:2166-2178). To
determine if RAD001 broadened the serologic response to influenza
in the elderly volunteers, HI titers to 2 heterologous strains of
influenza not contained in the influenza vaccine (A/H1N1 strain
A/New Jersey/8/76 and A/H3N2 strain A/Victoria/361/11) were
measured. The increase in the HI GMT ratios for the heterologous
strains was higher in the RAD001 as compared to placebo cohorts
(FIG. 42). In addition, seroconversion rates for the heterologous
strains were higher in the RAD001 as compared to placebo cohorts.
The increase in seroconversion rates in the 5 and 20 mg weekly
RAD001 dosing cohorts was statistically significant for the H3N2
heterologous strain (Table 12). The H3N2 seroconversion rate for
the pooled RAD001 cohorts was 39% versus 20% for the placebo cohort
(p=0.007). The results presented herein suggest that mTOR
inhibition broadens the serologic response of elderly volunteers to
influenza vaccination, and increases antibody titers to
heterologous strains of influenza not contained in the seasonal
influenza vaccine.
[1037] Broadened serologic response to heterologous strains of
influenza in mice treated with rapamycin has been associated with
an inhibition of class switching in B cells and an increase in
anti-influenza IgM levels (Keating, R. et al. (2013) Nat Immunol
14:2166-2178). However, inhibition of class switching may not be
involved in the broadened serologic response in humans treated with
RAD001 because the post-vaccination anti-influenza IgM and IgG
levels did not differ between RAD001 and placebo treated cohorts
(FIG. 43).
TABLE-US-00027 TABLE 12 Percentage of subjects who seroconvert to
heterologous strains of influenza 4 weeks after seasonal influenza
vaccination Placebo, RAD001 RAD001 RAD001 pooled 0.5 mg daily 5 mg
weekly 20 mg weekly A/H1N1 strain: 7% 17% 16% 8% A/NewJersey/8/76
A/H3N2 strain: 20% 38% 39%* 40% * A/Victoria/361/11 * Odds ratio
for seroconversion between RAD001 and Placebo significantly
different than 1 (two-sided p-value < 0.05 obtained by logistic
regression with treatment as fixed effect)
[1038] To address the mechanism by which RAD001 enhanced immune
function in elderly volunteers, immunophenotyping was performed on
PBMC samples obtained from subjects at baseline, after 6 weeks of
study drug treatment and 4 weeks after influenza vaccination (6
weeks after study drug discontinuation). Although the percentage of
most PBMC subsets did not differ between the RAD001 and placebo
cohorts, the percentage of PD-1 positive CD4 and CD8 cells was
lower in the RAD001 as compared to placebo cohorts (FIG. 44). PD-1
positive CD4 and CD8 cells accumulate with age and have defective
responses to antigen stimulation because PD-1 inhibits T cell
receptor-induced T cell proliferation, cytokine production and
cytolytic function (Lages, C S et al. (2010) Aging Cell 9:785-798).
There was an increase in percentage of PD-1 positive T cells over
time in the placebo cohort. At week 12 (4 weeks post-vaccination)
this increase may have been due to influenza vaccination since
influenza virus has been shown to increase PD-1 positive T cells
(Erikson, J J et al. (2012) JCI 122:2967-2982). However the
percentage of CD4 PD-1 positive T cells decreased from baseline at
week 6 and 12 in all RAD001 cohorts (FIG. 44A). The percentage of
CD8 PD-1 positive cells also decreased from baseline at both week 6
and 12 in the two lower dose RAD001 cohorts (FIG. 44B). The
percentage of PD-1 negative CD4 T cells was evaluated and increased
in the RAD001 cohorts as compared to the placebo cohorts (FIG.
44C).
[1039] Under more stringent statistical analysis, where the results
from the RAD001 cohorts were pooled and adjusted for differences in
baseline PD-1 expression, there was a statistically significant
decrease of 30.2% in PD-1 positive CD4 T cells at week 6 in the
pooled RAD cohort (n=84) compared to placebo cohort (n=25) with
p=0.03 (q=0.13) (FIG. 45A). The decrease in PD-1 positive CD4 T
cells at week 12 in the pooled RAD as compared to the placebo
cohort is 32.7% with p=0.05 (q=0.19). FIG. 45B shows a
statistically significant decrease of 37.4% in PD-1 positive CD8 T
cells at week 6 in the pooled RAD001 cohort (n=84) compared to
placebo cohort (n=25) with p=0.008 (q=0.07). The decrease in PD-1
positive CD8 T cells at week 12 in the pooled RAD001 as compared to
the placebo cohort is 41.4% with p=0.066 (q=0.21). Thus, the
results from FIGS. 44 and 45 together suggest that the
RAD001-associated decrease in the percentage of PD-1 positive CD4
and CD8 T cells may contribute to enhanced immune function.
Conclusion
[1040] In conclusion, the data presented herein show that the mTOR
inhibitor RAD001 ameliorates the age-related decline in
immunological function of the human elderly as assessed by response
to influenza vaccination, and that this amelioration is obtained
with an acceptable risk/benefit balance. In a study of elderly
mice, 6 weeks treatment with the mTOR inhibitor rapamycin not only
enhanced the response to influenza vaccination but also extended
lifespan, suggesting that amelioration of immunosenescence may be a
marker of a more broad effect on aging-related phenotypes.
[1041] Since RAD001 dosing was discontinued 2 weeks prior to
vaccination, the immune enhancing effects of RAD001 may be mediated
by changes in a relevant cell population that persists after
discontinuation of drug treatment. The results presented herein
show that RAD001 decreased the percentage of exhausted PD-1
positive CD4 and CD8 T cells as compared to placebo. PD-1
expression is induced by TCR signaling and remains high in the
setting of persistent antigen stimulation including chronic viral
infection. While not wishing to be bound by theory, is possible
that RAD001 reduced chronic immune activation in elderly volunteers
and thereby led to a decrease in PD-1 expression. RAD001 may also
directly inhibit PD-1 expression as has been reported for the
immunophilin cyclosporine A (Oestreich, K J et al. (2008) J
Immunol. 181:4832-4839). A RAD001-induced reduction in the
percentage of PD-1 positive T cells is likely to improve the
quality of T cell responses. This is consistent with previous
studies showing that mTOR inhibition improved the quality of memory
CD8 T cell response to vaccination in mice and primates (Araki, K
et al. (2009) Nature 460:108-112). In aged mice, mTOR inhibition
has also been shown to increase the number of hematopoietic stem
cells, leading to increased production of naive lymphocytes (Chen,
C et al. (2009) Sci Signal 2:ra75). Although significant
differences in the percentages of naive lymphocytes in the RAD001
versus placebo cohorts were not detected in this example, this
possible mechanism may be further investigated.
[1042] The mechanism by which RAD001 broadened the serologic
response to heterologous strains of influenza may be further
investigated. Rapamycin has also been shown to inhibit class
switching in B cells after influenza vaccination. As a result, a
unique repertoire of anti-influenza antibodies was generated that
promoted cross-strain protection against lethal infection with
influenza virus subtypes not contained in the influenza vaccine
(Keating, R et al. (2013) Nat Immunol. 14:2166-2178). The results
described herein did not show that RAD001 altered B cell class
switching in the elderly subjects who had discontinued RAD001 2
weeks prior to influenza vaccination. Although the underlying
mechanism requires further elucidation, the increased serologic
response to heterologous influenza strains described herein may
confer enhanced protection to influenza illness in years when there
is a poor match between the seasonal vaccine and circulating
strains of influenza in the community.
[1043] The effect of RAD001 on influenza antibody titers was
comparable to the effect of the MF59 vaccine adjuvant that is
approved to enhance the response of the elderly to influenza
vaccination (Podda, A (2001) Vaccine 19:2673-2680). Therefore,
RAD001-driven enhancement of the antibody response to influenza
vaccination may translate into clinical benefit as demonstrated
with MF59-adjuvanted influenza vaccine in the elderly (Iob, A et
al. (2005) Epidemiol Infect. 133:687-693). However, RAD001 is also
used to suppress the immune response of organ transplant patients.
These seemingly paradoxical findings raise the possibility that the
immunomodulatory effects of mTOR inhibitors may be dose and/or
antigen-dependent (Ferrer, I R et al. (2010) J Immunol.
185:2004-2008). A trend toward an inverse RAD001
exposure/vaccination response relationship was seen herein. It is
possible that complete mTOR inhibition suppresses immune function
through the normal cyclophilin-rapamycin mechanism, whereas partial
mTOR inhibition, at least in the elderly, enhances immune function
due to a distinct aging-related phenotype inhibition. Of interest,
mTOR activity is increased in a variety of tissues including
hematopoietic stem cells in aging animal models (Chen C. et al.
(2009) Sci Signal 2:ra75 and Barns, M. et al. (2014) Int J Biochem
Cell Biol. 53:174-185). Thus, turning down mTOR activity to levels
seen in young tissue, as opposed to more complete suppression of
mTOR activity, may be of clinical benefit in aging indications.
[1044] The safety profile of mTOR inhibitors such as RAD001 in the
treatment of aging-related indications has been of concern. The
toxicity of RAD001 at doses used in oncology or organ transplant
indications includes rates of stomatitis, diarrhea, nausea,
cytopenias, hyperlipidemia, and hyperglycemia that would be
unacceptable for many aging-related indications. However, these AEs
are related to the trough levels of RAD001 in blood. Therefore the
RAD001 dosing regimens used in this study were chosen to minimize
trough levels. The average RAD001 trough levels of the 0.5 mg
daily, 5 mg weekly and 20 mg weekly dosing cohorts were 0.9 ng/ml,
below 0.3 ng/ml (the lower limit of quantification), and 0.7 ng/ml,
respectively. These trough levels are significantly lower than the
trough levels associated with dosing regimens used in organ
transplant and cancer patients. In addition, the limited 6 week
course of treatment decreased the risk of adverse events. These
findings suggest that the dosing regimens used in this study may
have an acceptable risk/benefit for some conditions of the elderly.
Nonetheless, significant numbers of subjects in the experiments
described herein developed mouth ulcers even when dosed as low as
0.5 mg daily. Therefore the safety profile of low, immune
enhancing, dose RAD001 warrants further study. Development of mTOR
inhibitors with cleaner safety profiles than currently available
rapalogs may provide better therapeutic options in the future for
aging-associated conditions.
Example 13: Enhancement of Immune Response to Vaccine in Elderly
Subjects
[1045] Immune function declines in the elderly, leading to an
increase incidence of infection and a decreased response to
vaccination. As a first step in determining if mTOR inhibition has
anti-aging effects in humans, a randomized placebo-controlled trial
was conducted to determine if the mTOR inhibitor RAD001 reverses
the aging-related decline in immune function as assessed by
response to vaccination in elderly volunteers. In all cases,
appropriate patent consents were obtained and the study was
approved by national health authorities.
[1046] The following 3 dosing regimens of RAD001 were used in the
study:
20 mg weekly (trough level: 0.7 ng/ml) 5 mg weekly (trough level
was below detection limits) 0.5 mg daily (trough level: 0.9
ng/ml)
[1047] These dosing regimens were chosen because they have lower
trough levels than the doses of RAD001 approved for transplant and
oncology indications. Trough level is the lowest level of a drug in
the body. The trough level of RAD001 associated with the 10 mg
daily oncology dosing regimen is approximately 20 ng/ml. The trough
level associated with the 0.75-1.5 mg bid transplant dosing regimen
is approximately 3 ng/ml. In contrast, the trough level associated
with the dosing regimens used in our immunization study were 3-20
fold lower.
[1048] Since RAD001-related AEs are associated with trough levels,
the 3 dosing regimens were predicted to have adequate safety for
normal volunteers. In addition, the 3 doses were predicted to give
a range of mTOR inhibition. P70 S6 Kinase (P70 S6K) is a downstream
target that is phosphorylated by mTOR. Levels of P70 S6K
phosphorylation serve as a measure of mTOR activity. Based on
modeling and simulation of P70 S6K phosphorylation data obtained in
preclinical and clinical studies of RAD001, 20 mg weekly was
predicted to almost fully inhibit mTOR activity for a full week,
whereas 5 mg weekly and 0.5 mg daily were predicted to partially
inhibit mTOR activity.
[1049] Elderly volunteers >=65 years of age were randomized to
one of the 3 RAD001 treatment groups (50 subjects per arm) or
placebo (20 subjects per arm). Subjects were treated with study
drug for 6 weeks, given a 2 week break, and then received influenza
(Aggrippal, Novartis) and pneumoccal (Pneumovax 23, Merck),
vaccinations. Response to influenza vaccination was assessed by
measuring the geometric mean titers (GMTs) by hemagglutination
inhibition assay to the 3 influenza strains (H1N1, H3N2 and B
influenza subtypes) in the influenza vaccine 4 weeks after
vaccination. The primary endpoints of the study were (1) safety and
tolerability and (2) a 1.2 fold increase in influenza titers as
compared to placebo in 2/3 of the influenza vaccine strains 4 weeks
after vaccination. This endpoint was chosen because a 1.2 fold
increase in influenza titers is associated with a decrease in
influenza illness post vaccination, and therefore is clinically
relevant. The 5 mg weekly and 0.5 mg daily doses were well
tolerated and unlike the 20 mg weekly dose, met the GMT primary
endpoint (FIG. 40A). Not only did RAD001 improve the response to
influenza vaccination, it also improved the response to
pneumococcal vaccination as compared to placebo in elderly
volunteers. The pneumococcal vaccine contains antigens from 23
pneumococcal serotypes. Antibody titers to 7 of the serotypes were
measured in our subjects. Antibody titers to 6/7 serotypes were
increased in all 3 RAD cohorts compared to placebo.
[1050] The combined influenza and pneumococcal titer data suggest
that partial (less than 80-100%) mTOR inhibition is more effective
at reversing the aging-related decline in immune function than more
complete mTOR inhibition.
Example 14: Low Dose mTOR Inhibition Increases Energy and
Exercise
[1051] In preclinical models, mTOR inhibition with the rapalog
rapamycin increases spontaneous physical activity in old mice
(Wilkinson et al. Rapamycin slows aging in mice. (2012) Aging Cell;
11:675-82). Of interest, subjects in the 0.5 mg daily dosing cohort
described in Example 13 also reported increased energy and exercise
ability as compared to placebo in questionnaires administered one
year after dosing (FIG. 46). These data suggest that partial mTOR
inhibition with rapalogs may have beneficial effects on
aging-related morbidity beyond just immune function.
Example 15: P70 S6 Kinase Inhibition with RAD001
[1052] Modeling and simulation were performed to predict daily and
weekly dose ranges of RAD001 that are predicted to partially
inhibit mTOR activity. As noted above, P70 S6K is phosphorylated by
mTOR and is the downstream target of mTOR that is most closely
linked to aging because knockout of P70 S6K increases lifespan.
Therefore modeling was done of doses of RAD001 that partially
inhibit P70 S6K activity. Weekly dosing in the range of >=0.1 mg
and <20 mg are predicted to achieve partial inhibition of P70
S6K activity (FIG. 47).
[1053] For daily dosing, concentrations of RAD001 from 30 pM to 4
nM partially inhibited P70 S6K activity in cell lines (Table 13).
These serum concentrations are predicted to be achieved with doses
of RAD001 >=0.005 mg to <1.5 mg daily.
TABLE-US-00028 TABLE 13 Percent inhibition of P70 S6K activity in
HeLa cells in vitro RAD001 6 32 160 800 4 20 concentration 0 pM pM
pM pM nM nM % P70 S6K 0 0 18 16 62 90 95 inhibition
Conclusion
[1054] Methods of treating aging-related morbidity, or generally
enhancing an immune response, with doses of mTOR inhibitors that
only partially inhibit P70 S6K. The efficacy of partial mTOR
inhibition with low doses of RAD001 in aging indications is an
unexpected finding. RAD001 dose ranges between >=0.1 mg to
<20 mg weekly and >=0.005 mg to <1.5 mg daily will achieve
partial mTOR inhibition and therefore are expected to have efficacy
in aging-related morbidity or in the enhancement of the immune
response.
Example 16: Exogenous IL-7 Enhances the Function of CAR T Cells
[1055] After adoptive transfer of CAR T cells, some patients
experience limited persistence of the CAR T cells, which can result
in suboptimal levels of anti-tumor activity. In this example, the
effects of administration of exogenous human IL-7 is assessed in
mouse xenograft models where an initial suboptimal response to CAR
T cells has been observed.
[1056] Expression of the IL-7 receptor CD127 was first assessed in
different cancer cell lines and in CAR-expressing cells. Two mantle
cell lymphoma cell lines (RL and Jeko-1) and one B-ALL cell line
(Nalm-6) were analyzed by flow cytometry for CD127 expression. As
shown in FIG. 48A, out of the three cancer cell lines tested, RL
was shown to have the highest expression of CD127, followed by
Jeko-1 and Nalm-6. CART19 cells were infused into NSG mice and
CD127 expression was assessed on the circulating CART19 cells by
flow cytometry. As shown in FIG. 48B, CD127 is uniformly expressed
on all circulating CART19 cells.
[1057] Next, the effect of exogenous IL-7 treatment on anti-tumor
activity of CART19 cells was assessed in a lymphoma animal model.
NSG mice were engrafted with a luciferase-expressing mantle cell
line (RL luc) on Day 0 (D0), followed by treatment of CART19 cells
on Day 6. The NSG mice were divided into groups, where one group
received no CART19 cells, a second group received
0.5.times.10.sup.6 CART19 cells, a third group received
1.times.10.sup.6 CART19 cells, and a fourth group received
2.times.10.sup.6 CART19 cells. Tumor size was monitored by
measuring the mean bioluminescence of the engrafted tumors over
more than 80 days. Only mice receiving 2.times.10.sup.6 CART19
cells demonstrated rejection of the tumor and inhibition of tumor
growth (FIG. 49A). Mice from the two groups receiving
0.5.times.10.sup.6 CART19 cells or 1.times.10.sup.6 CART19 cells
were shown to s a suboptimal anti-tumor response. Mice from these
two groups were then randomized, where three mice (mouse #3827 and
#3829 which received 0.5.times.10.sup.6 CART19 cells, and mouse
#3815 which received 1.times.10.sup.6 CART19 cells) received
exogenous recombinant human IL-7 at a dosage of 200 ng/mouse by
intraperitoneal injection three times weekly starting at Day 85,
and two mice did not. The tumor burden of mice receiving exogenous
IL-7 from Day 85-125, as detected by mean bioluminescence, is shown
in FIG. 49B. All mice receiving IL-7 showed a dramatic response of
1-3 log reduction in tumor burden. Mice that originally received a
higher dose of CART19 cells (mouse #3815 which received
1.times.10.sup.6 CART19 cells) showed a more profound response.
When comparing the tumor burden of mice that received IL-7
treatment to control, before and after IL-7 treatment, tumor
reduction in tumor burden was only seen in the mice that had
received IL-7 treatment (FIG. 49C).
[1058] T cell dynamics following IL-7 treatment in the lymphoma
animal model was also examined. Human CART19 cells were not
detectable in the blood prior to IL-7 treatment. Upon treatment of
IL-7, there was rapid, but variable increase in the numbers of T
cells in the treated mice (FIG. 50A). The extent of T cell
expansion observed in mice receiving the IL-7 also correlated with
tumor response. The mouse with the highest number of T cells
detected in the blood at peak expansion during IL-7 treatment
(mouse #3815) had the most robust reduction in tumor burden (see
FIG. 49B). Moreover, the time of peak expansion correlated with the
T cell dose injected as baseline. The number/level CD3-expressing
cells in the blood were also measured before and after IL-7
treatment. In control mice, very few CD3-expressing cells were
detected, while IL-7-treated mice showed a significant increase in
CD3+ cells after IL-7 treatment (FIG. 50B).
[1059] Together, the results in this example demonstrate that
exogenous IL-7 treatment increases T cell proliferation and
anti-tumor activity in vivo, indicating that use of IL-7 in
patients with suboptimal results after CAR therapy can improve
anti-tumor response in these patients.
EQUIVALENTS
[1060] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific aspects, it is apparent
that other aspects and variations of this invention may be devised
by others skilled in the art without departing from the true spirit
and scope of the invention. The appended claims are intended to be
construed to include all such aspects and equivalent variations.
Sequence CWU 1
1
1321242PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Glu Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr His Thr Ser Arg
Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105
110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu
115 120 125Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu
Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val
Ser Trp Ile Arg145 150 155 160Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Ser Ser
Ser Leu Lys Ser Arg Val Thr Ile Ser 180 185 190Lys Asp Asn Ser Lys
Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr 195 200 205Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly
Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val225 230
235 240Ser Ser2242PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 2Glu Ile Val Met Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr His Thr
Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly
Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln
Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr
Leu Ser Leu Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp
Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Gly Lys Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr
Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser 180 185 190Lys Asp
Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr 195 200
205Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val225 230 235 240Ser Ser3242PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 3Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser
Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr
Tyr Ser Ser Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn
Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala 130 135
140Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
Ala145 150 155 160Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly 165 170 175Gln Ala Pro Arg Leu Leu Ile Tyr His Thr
Ser Arg Leu His Ser Gly 180 185 190Ile Pro Ala Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Leu 195 200 205Thr Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln 210 215 220Gln Gly Asn Thr
Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu225 230 235 240Ile
Lys4242PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 4Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly
Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys 50 55 60Ser Arg Val Thr
Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser
Pro Ala 130 135 140Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala145 150 155 160Ser Gln Asp Ile Ser Lys Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly 165 170 175Gln Ala Pro Arg Leu Leu Ile
Tyr His Thr Ser Arg Leu His Ser Gly 180 185 190Ile Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 195 200 205Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln 210 215 220Gln
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu225 230
235 240Ile Lys5247PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 5Glu Ile Val Met Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr His Thr
Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly
Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln 115 120 125Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu Thr 130 135 140Leu Ser Leu Thr Cys Thr Val Ser Gly
Val Ser Leu Pro Asp Tyr Gly145 150 155 160Val Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 165 170 175Val Ile Trp Gly
Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser 180 185 190Arg Val
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 195 200
205Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys
210 215 220His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
Gln Gly225 230 235 240Thr Leu Val Thr Val Ser Ser
2456247PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 6Glu Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr His Thr Ser Arg
Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105
110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu Thr 130 135 140Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu
Pro Asp Tyr Gly145 150 155 160Val Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile Gly 165 170 175Val Ile Trp Gly Ser Glu Thr
Thr Tyr Tyr Gln Ser Ser Leu Lys Ser 180 185 190Arg Val Thr Ile Ser
Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 195 200 205Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys 210 215 220His
Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly225 230
235 240Thr Leu Val Thr Val Ser Ser 2457247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 7Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser
Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr
Tyr Ser Ser Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn
Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met 130 135
140Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala
Thr145 150 155 160Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr His Thr Ser 180 185 190Arg Leu His Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Val Tyr Phe Cys
Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln225 230 235 240Gly
Thr Lys Leu Glu Ile Lys 2458247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 8Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser
Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr
Tyr Gln Ser Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn
Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met 130 135
140Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala
Thr145 150 155 160Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr His Thr Ser 180 185 190Arg Leu His Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Val Tyr Phe Cys
Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln225 230 235 240Gly
Thr Lys Leu Glu Ile Lys 2459247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 9Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp
Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr 130 135
140Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
Gly145 150 155 160Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile Gly 165 170 175Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
Asn Ser Ser Leu Lys Ser 180 185 190Arg Val Thr Ile Ser Lys Asp Asn
Ser Lys Asn Gln Val Ser Leu Lys 195 200 205Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys 210 215 220His Tyr Tyr Tyr
Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly225 230 235 240Thr
Leu Val Thr Val Ser Ser 24510247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 10Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser
Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr
Tyr Asn Ser Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn
Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120
125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met
130 135 140Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg
Ala Thr145 150 155 160Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys
Tyr Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile Tyr His Thr Ser 180 185 190Arg Leu His Ser Gly Ile Pro
Ala Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Val Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln225 230 235
240Gly Thr Lys Leu Glu Ile Lys 24511242PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 11Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp
Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 130 135
140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg145 150 155 160Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val
Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys
Ser Arg Val Thr Ile Ser 180 185 190Lys Asp Asn Ser Lys Asn Gln Val
Ser Leu Lys Leu Ser Ser Val Thr 195 200 205Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val225 230 235 240Ser
Ser12242PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 12Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met
Thr Gln Ser Pro Ala 130 135 140Thr Leu Ser Leu Ser Pro Gly Glu Arg
Ala Thr Leu Ser Cys Arg Ala145 150 155 160Ser Gln Asp Ile Ser Lys
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 165 170 175Gln Ala Pro Arg
Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 180 185 190Ile Pro
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 195 200
205Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln
210 215 220Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu225 230 235 240Ile Lys1321PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 13Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu1 5 10 15His Ala Ala Arg Pro 201445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 14Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
Thr Ile Ala1 5 10 15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro Ala Ala Gly 20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
Cys Asp 35 40 451524PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 15Ile Tyr Ile Trp Ala Pro
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr
Leu Tyr Cys 201642PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 16Lys Arg Gly Arg Lys
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu
Glu Glu Gly Gly Cys Glu Leu 35 4017112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 17Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110185PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Gly Gly Gly Gly Ser1 51910PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 19Gly Val Ser Leu Pro Asp Tyr Gly Val Ser1 5
102016PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Val Ile Trp Gly Ser Glu Thr Thr Tyr
Tyr Asn Ser Ala Leu Lys Ser1 5 10 152116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu
Lys Ser1 5 10 152216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 22Val Ile Trp Gly Ser Glu
Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser1 5 10 152316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 23Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu
Lys Ser1 5 10 152412PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 24His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp Tyr1 5 102511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn1 5
10267PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 26His Thr Ser Arg Leu His Ser1
5279PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 27Gln Gln Gly Asn Thr Leu Pro Tyr Thr1
5285PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 28Tyr Ser Ser Ser Leu1
5295PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 29Tyr Gln Ser Ser Leu1
5305PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 30Tyr Asn Ser Ser Leu1
531486PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 31Met Ala Leu Pro Val Thr Ala Leu
Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile
Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser Pro Gly Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg Leu Leu
Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75 80Ala Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser
Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105
110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gln 130 135 140Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu Thr145 150 155 160Leu Ser Leu Thr Cys Thr Val Ser Gly
Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile Gly 180 185 190Val Ile Trp Gly Ser
Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser 195 200 205Arg Val Thr
Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 210 215 220Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys225 230
235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
Gly 245 250 255Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro
Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala Ala Gly Gly Ala
Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys Asp Ile Tyr Ile
Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315 320Val Leu Leu Leu
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335Lys Lys
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345
350Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala 370 375 380Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
Glu Leu Asn Leu385 390 395 400Gly Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp 405 410 415Pro Glu Met Gly Gly Lys Pro
Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met465 470
475 480Gln Ala Leu Pro Pro Arg 48532486PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 32Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu 20 25 30Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Ile Pro65 70 75 80Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135
140Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
Thr145 150 155 160Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu
Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile Gly 180 185 190Val Ile Trp Gly Ser Glu Thr Thr
Tyr Tyr Gln Ser Ser Leu Lys Ser 195 200 205Arg Val Thr Ile Ser Lys
Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 210 215 220Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys225 230 235 240His
Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250
255Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
Pro Glu 275 280 285Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
Arg Gly Leu Asp 290 295 300Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
Leu Ala Gly Thr Cys Gly305 310 315 320Val Leu Leu Leu Ser Leu Val
Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335Lys Lys Leu Leu Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365Glu
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375
380Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
Leu385 390 395 400Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
Arg Gly Arg Asp 405 410 415Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
Asn Pro Gln Glu Gly Leu 420 425 430Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445Gly Met Lys Gly Glu Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460Gln Gly Leu Ser
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met465 470 475 480Gln
Ala Leu Pro Pro Arg 48533486PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 33Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu 20 25 30Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Val 35 40 45Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr65 70 75 80Ser Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys 85 90 95Asn Gln Val Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135
140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val
Met145 150 155 160Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
Glu Arg Ala Thr 165 170 175Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser
Lys Tyr Leu Asn Trp Tyr
180 185 190Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His
Thr Ser 195 200 205Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly 210 215 220Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala225 230 235 240Val Tyr Phe Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gln 245 250 255Gly Thr Lys Leu Glu
Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270Thr Pro Ala
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285Ala
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295
300Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
Gly305 310 315 320Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
Lys Arg Gly Arg 325 330 335Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met Arg Pro Val Gln 340 345 350Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365Glu Gly Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380Pro Ala Tyr Lys
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu385 390 395 400Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410
415Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
Glu Ile 435 440 445Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly Leu Tyr 450 455 460Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu His Met465 470 475 480Gln Ala Leu Pro Pro Arg
48534486PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 34Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser
Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Ile Val Met145 150 155 160Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr 165 170 175Leu Ser Cys Arg
Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr 180 185 190Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser 195 200
205Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
210 215 220Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala225 230 235 240Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
Tyr Thr Phe Gly Gln 245 250 255Gly Thr Lys Leu Glu Ile Lys Thr Thr
Thr Pro Ala Pro Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala
Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315
320Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
Val Gln 340 345 350Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
Pro Glu Glu Glu 355 360 365Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg Ser Ala Asp Ala 370 375 380Pro Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu385 390 395 400Gly Arg Arg Glu Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440
445Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His Met465 470 475 480Gln Ala Leu Pro Pro Arg 48535491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 35Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu 20 25 30Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Ile Pro65 70 75 80Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135
140Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val145 150 155 160Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr Ser 195 200 205Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys Asn 210 215 220Gln Val Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val225 230 235 240Tyr
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp 245 250
255Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
Pro Leu 275 280 285Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
Gly Ala Val His 290 295 300Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Tyr Ile Trp Ala Pro Leu305 310 315 320Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335Cys Lys Arg Gly Arg
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350Met Arg Pro
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365Phe
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375
380Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
Tyr385 390 395 400Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys 405 410 415Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro Arg Arg Lys Asn 420 425 430Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu 435 440 445Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460His Asp Gly Leu
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr465 470 475 480Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 49036491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 36Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu 20 25 30Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Ile Pro65 70 75 80Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135
140Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val145 150 155 160Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr Gln 195 200 205Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys Asn 210 215 220Gln Val Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val225 230 235 240Tyr
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp 245 250
255Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro
260 265 270Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
Pro Leu 275 280 285Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
Gly Ala Val His 290 295 300Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Tyr Ile Trp Ala Pro Leu305 310 315 320Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335Cys Lys Arg Gly Arg
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350Met Arg Pro
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365Phe
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375
380Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
Tyr385 390 395 400Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys 405 410 415Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro Arg Arg Lys Asn 420 425 430Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu 435 440 445Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460His Asp Gly Leu
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr465 470 475 480Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 49037491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 37Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu 20 25 30Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Val 35 40 45Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr65 70 75 80Ser Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys 85 90 95Asn Gln Val Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135
140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly145 150 155 160Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 195 200 205Ile Tyr His Thr Ser Arg
Leu His Ser Gly Ile Pro Ala Arg Phe Ser 210 215 220Gly Ser Gly Ser
Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln225 230 235 240Pro
Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 245 250
255Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro
260 265 270Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
Pro Leu 275 280 285Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
Gly Ala Val His 290 295 300Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Tyr Ile Trp Ala Pro Leu305 310 315 320Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335Cys Lys Arg Gly Arg
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350Met Arg Pro
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365Phe
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375
380Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
Tyr385 390 395 400Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys 405 410 415Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro Arg Arg Lys Asn 420 425 430Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu 435 440 445Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460His Asp Gly Leu
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr465 470 475 480Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 49038491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 38Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu 20 25 30Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Val 35 40 45Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr65 70 75 80Gln Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys 85 90 95Asn Gln Val Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly145 150 155 160Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 195 200 205Ile Tyr His Thr Ser
Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser 210 215 220Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln225 230 235
240Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
245 250 255Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr
Thr Pro 260 265 270Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
Ser Gln Pro Leu 275 280 285Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly Gly Ala Val His 290 295 300Thr Arg Gly Leu Asp Phe Ala Cys
Asp Ile Tyr Ile Trp Ala Pro Leu305 310 315 320Ala Gly Thr Cys Gly
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335Cys Lys Arg
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350Met
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360
365Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
370 375 380Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
Leu Tyr385 390 395 400Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys 405 410 415Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn 420 425 430Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr465 470 475
480Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485
49039491PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 39Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val145 150 155 160Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn 195 200
205Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn
210 215 220Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val225 230 235 240Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Thr Thr Thr Pro 260 265 270Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300Thr Arg Gly
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu305 310 315
320Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe 340 345 350Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg 355 360 365Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg Val Lys Phe Ser 370 375 380Arg Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr385 390 395 400Asn Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415Arg Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430Pro
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440
445Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
Thr Tyr465 470 475 480Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 49040491PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 40Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val145 150 155 160Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn 195 200
205Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn
210 215 220Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val225 230 235 240Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Thr Thr Thr Pro 260 265 270Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300Thr Arg Gly
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu305 310 315
320Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe 340 345 350Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg 355 360 365Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg Val Lys Phe Ser 370 375 380Arg Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr385 390 395 400Asn Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415Arg Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430Pro
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440
445Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
Thr Tyr465 470 475 480Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 49041491PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 41Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser
Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly145 150 155 160Ser Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 195 200
205Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser
210 215 220Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln225 230 235 240Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro 245 250 255Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Thr Thr Thr Pro 260 265 270Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300Thr Arg Gly
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu305 310 315
320Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe 340 345 350Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg 355 360 365Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg Val Lys Phe Ser 370 375 380Arg Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr385 390 395 400Asn Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415Arg Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430Pro
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440
445Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
Thr Tyr465 470 475 480Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 49042486PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 42Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln 130 135 140Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu Thr145 150 155 160Leu Ser Leu Thr Cys Thr
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 180 185 190Val Ile
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser 195 200
205Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys
210 215 220Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Lys225 230 235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
Tyr Trp Gly Gln Gly 245 250 255Thr Leu Val Thr Val Ser Ser Thr Thr
Thr Pro Ala Pro Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala
Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315
320Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
Val Gln 340 345 350Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
Pro Glu Glu Glu 355 360 365Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg Ser Ala Asp Ala 370 375 380Pro Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu385 390 395 400Gly Arg Arg Glu Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440
445Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His Met465 470 475 480Gln Ala Leu Pro Pro Arg 48543112PRTHomo
sapiens 43Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 11044336DNAHomo
sapiens 44agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca
gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa
gagacgtggc 120cgggaccctg agatgggggg aaagccgaga aggaagaacc
ctcaggaagg cctgtacaat 180gaactgcaga aagataagat ggcggaggcc
tacagtgaga ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga
tggcctttac cagggtctca gtacagccac caaggacacc 300tacgacgccc
ttcacatgca ggccctgccc cctcgc 33645230PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys Met225 23046690DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 46gagagcaagt
acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60agcgtgttcc
tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag
120gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt
caactggtac 180gtggacggcg tggaggtgca caacgccaag accaagcccc
gggaggagca gttcaatagc 240acctaccggg tggtgtccgt gctgaccgtg
ctgcaccagg actggctgaa cggcaaggaa 300tacaagtgta aggtgtccaa
caagggcctg cccagcagca tcgagaaaac catcagcaag 360gccaagggcc
agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg
420accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag
cgacatcgcc 480gtggagtggg agagcaacgg ccagcccgag aacaactaca
agaccacccc ccctgtgctg 540gacagcgacg gcagcttctt cctgtacagc
cggctgaccg tggacaagag ccggtggcag 600gagggcaacg tctttagctg
ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660aagagcctga
gcctgtccct gggcaagatg 69047282PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 47Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val
Pro Thr Ala1 5 10 15Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr
Thr Ala Pro Ala 20 25 30Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu
Lys Lys Lys Glu Lys 35 40 45Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr
Lys Thr Pro Glu Cys Pro 50 55 60Ser His Thr Gln Pro Leu Gly Val Tyr
Leu Leu Thr Pro Ala Val Gln65 70 75 80Asp Leu Trp Leu Arg Asp Lys
Ala Thr Phe Thr Cys Phe Val Val Gly 85 90 95Ser Asp Leu Lys Asp Ala
His Leu Thr Trp Glu Val Ala Gly Lys Val 100 105 110Pro Thr Gly Gly
Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly 115 120 125Ser Gln
Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn 130 135
140Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro
Pro145 150 155 160Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln
Ala Pro Val Lys 165 170 175Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp
Pro Pro Glu Ala Ala Ser 180 185 190Trp Leu Leu Cys Glu Val Ser Gly
Phe Ser Pro Pro Asn Ile Leu Leu 195 200 205Met Trp Leu Glu Asp Gln
Arg Glu Val Asn Thr Ser Gly Phe Ala Pro 210 215 220Ala Arg Pro Pro
Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser225 230 235 240Val
Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr 245 250
255Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg
260 265 270Ser Leu Glu Val Ser Tyr Val Thr Asp His 275
28048847DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 48aggtggcccg
aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca 60gaaggcagcc
tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc
120ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga
gaccaagacc 180cctgaatgtc catcccatac ccagccgctg ggcgtctatc
tcttgactcc cgcagtacag 240gacttgtggc ttagagataa ggccaccttt
acatgtttcg tcgtgggctc tgacctgaag 300gatgcccatt tgacttggga
ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360ttgctggagc
gccattccaa tggctctcag agccagcact caagactcac ccttccgaga
420tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag
cctgccccca 480cagcgtctga tggcccttag agagccagcc gcccaggcac
cagttaagct tagcctgaat 540ctgctcgcca gtagtgatcc cccagaggcc
gccagctggc tcttatgcga agtgtccggc 600tttagcccgc ccaacatctt
gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660ggcttcgctc
cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt
720gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg
tgttgtgtcc 780catgaagata gcaggaccct gctaaatgct tctaggagtc
tggaggtttc ctacgtgact 840gaccatt 8474910PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
105030PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 50Gly Gly Thr Gly Gly Cys Gly Gly
Ala Gly Gly Thr Thr Cys Thr Gly1 5 10 15Gly Ala Gly Gly Thr Gly Gly
Ala Gly Gly Thr Thr Cys Cys 20 25 305148PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 51Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro
Val Glu Pro1 5 10 15Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu
Glu Gly Ser Thr 20 25 30Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu
Pro Ala Cys Ser Pro 35 40 4552123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 52aggagtaaga ggagcaggct cctgcacagt gactacatga
acatgactcc ccgccgcccc 60gggcccaccc gcaagcatta ccagccctat gccccaccac
gcgacttcgc agcctatcgc 120tcc 1235330PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"misc_feature(1)..(30)/note="This sequence may encompass
1-6 repeating "Gly Gly Gly Gly Ser" repeating units" 53Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
305463DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 54atggccctgc ctgtgacagc
cctgctgctg cctctggctc tgctgctgca tgccgctaga 60ccc
6355135DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 55accacgacgc cagcgccgcg
accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc cagaggcgtg
ccggccagcg gcggggggcg cagtgcacac gagggggctg 120gacttcgcct gtgat
1355672DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 56atctacatct gggcgccctt
ggccgggact tgtggggtcc ttctcctgtc actggttatc 60accctttact gc
72575PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 57Tyr Asn Ser Ala Leu1
558486PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 58Met Ala Leu Pro Val Thr Ala Leu
Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Ile
Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu
Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75 80Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser
Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105
110Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu 130 135 140Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
Pro Ser Gln Ser145 150 155 160Leu Ser Val Thr Cys Thr Val Ser Gly
Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile Arg Gln Pro
Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190Val Ile Trp Gly Ser
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205Arg Leu Thr
Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220Met
Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys225 230
235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
Gly 245 250 255Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro
Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala Ala Gly Gly Ala
Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys Asp Ile Tyr Ile
Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315 320Val Leu Leu Leu
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335Lys Lys
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345
350Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala 370 375 380Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
Glu Leu Asn Leu385 390 395 400Gly Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp 405 410 415Pro Glu Met Gly Gly Lys Pro
Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met465 470
475 480Gln Ala Leu Pro Pro Arg 48559242PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 59Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp
Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly
Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135
140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val
Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile
Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser
Ser60126DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 60aaacggggca
gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag
aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg
12661813DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 61atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc
atcagaaact 480ctttcactga cttgtactgt gagcggagtg tctctccccg
attacggggt gtcttggatc 540agacagccac cggggaaggg tctggaatgg
attggagtga tttggggctc tgagactact 600tactactctt catccctcaa
gtcacgcgtc accatctcaa aggacaactc taagaatcag 660gtgtcactga
aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag
720cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac
tctggtcacc 780gtgtccagcc accaccatca tcaccatcac cat
81362813DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 62atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc
atcagaaact 480ctttcactga cttgtactgt gagcggagtg tctctccccg
attacggggt gtcttggatc 540agacagccac cggggaaggg tctggaatgg
attggagtga tttggggctc tgagactact 600tactaccaat catccctcaa
gtcacgcgtc accatctcaa aggacaactc taagaatcag 660gtgtcactga
aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag
720cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac
tctggtcacc 780gtgtccagcc accaccatca tcaccatcac cat
81363813DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 63atggctctgc
ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc 60ccacaagtcc
agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc
120ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg
gattagacag 180cctcccggaa agggactgga gtggatcgga gtgatttggg
gtagcgaaac cacttactat 240tcatcttccc tgaagtcacg ggtcaccatt
tcaaaggata actcaaagaa
tcaagtgagc 300ctcaagctct catcagtcac cgccgctgac accgccgtgt
attactgtgc caagcattac 360tactatggag ggtcctacgc catggactac
tggggccagg gaactctggt cactgtgtca 420tctggtggag gaggtagcgg
aggaggcggg agcggtggag gtggctccga aatcgtgatg 480acccagagcc
ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg
540gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg
acaggcccct 600aggcttctta tctaccacac ctctcgcctg catagcggga
ttcccgcacg ctttagcggg 660tctggaagcg ggaccgacta cactctgacc
atctcatctc tccagcccga ggacttcgcc 720gtctacttct gccagcaggg
taacaccctg ccgtacacct tcggccaggg caccaagctt 780gagatcaaac
atcaccacca tcatcaccat cac 81364813DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 64atggctctgc ccgtgaccgc actcctcctg ccactggctc
tgctgcttca cgccgctcgc 60ccacaagtcc agcttcaaga atcagggcct ggtctggtga
agccatctga gactctgtcc 120ctcacttgca ccgtgagcgg agtgtccctc
ccagactacg gagtgagctg gattagacag 180cctcccggaa agggactgga
gtggatcgga gtgatttggg gtagcgaaac cacttactat 240caatcttccc
tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc
300ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc
caagcattac 360tactatggag ggtcctacgc catggactac tggggccagg
gaactctggt cactgtgtca 420tctggtggag gaggtagcgg aggaggcggg
agcggtggag gtggctccga aatcgtgatg 480acccagagcc ctgcaaccct
gtccctttct cccggggaac gggctaccct ttcttgtcgg 540gcatcacaag
atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct
600aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg
ctttagcggg 660tctggaagcg ggaccgacta cactctgacc atctcatctc
tccagcccga ggacttcgcc 720gtctacttct gccagcaggg taacaccctg
ccgtacacct tcggccaggg caccaagctt 780gagatcaaac atcaccacca
tcatcaccat cac 81365828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 65atggccctcc cagtgaccgc tctgctgctg cctctcgcac
ttcttctcca tgccgctcgg 60cctgagatcg tcatgaccca aagccccgct accctgtccc
tgtcacccgg cgagagggca 120accctttcat gcagggccag ccaggacatt
tctaagtacc tcaactggta tcagcagaag 180ccagggcagg ctcctcgcct
gctgatctac cacaccagcc gcctccacag cggtatcccc 240gccagatttt
ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag
300cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta
caccttcggt 360caaggtacca agctggaaat caagggaggc ggaggatcag
gcggtggcgg aagcggagga 420ggtggctccg gaggaggagg ttcccaagtg
cagcttcaag aatcaggacc cggacttgtg 480aagccatcag aaaccctctc
cctgacttgt accgtgtccg gtgtgagcct ccccgactac 540ggagtctctt
ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg
600ggatcagaga ctacttacta ctcttcatca cttaagtcac gggtcaccat
cagcaaagat 660aatagcaaga accaagtgtc acttaagctg tcatctgtga
ccgccgctga caccgccgtg 720tactattgtg ccaaacatta ctattacgga
gggtcttatg ctatggacta ctggggacag 780gggaccctgg tgactgtctc
tagccatcac catcaccacc atcatcac 82866828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 66atggccctcc cagtgaccgc tctgctgctg cctctcgcac
ttcttctcca tgccgctcgg 60cctgagatcg tcatgaccca aagccccgct accctgtccc
tgtcacccgg cgagagggca 120accctttcat gcagggccag ccaggacatt
tctaagtacc tcaactggta tcagcagaag 180ccagggcagg ctcctcgcct
gctgatctac cacaccagcc gcctccacag cggtatcccc 240gccagatttt
ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag
300cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta
caccttcggt 360caaggtacca agctggaaat caagggaggc ggaggatcag
gcggtggcgg aagcggagga 420ggtggctccg gaggaggagg ttcccaagtg
cagcttcaag aatcaggacc cggacttgtg 480aagccatcag aaaccctctc
cctgacttgt accgtgtccg gtgtgagcct ccccgactac 540ggagtctctt
ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg
600ggatcagaga ctacttacta ccagtcatca cttaagtcac gggtcaccat
cagcaaagat 660aatagcaaga accaagtgtc acttaagctg tcatctgtga
ccgccgctga caccgccgtg 720tactattgtg ccaaacatta ctattacgga
gggtcttatg ctatggacta ctggggacag 780gggaccctgg tgactgtctc
tagccatcac catcaccacc atcatcac 82867828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 67atggcactgc ctgtcactgc cctcctgctg cctctggccc
tccttctgca tgccgccagg 60ccccaagtcc agctgcaaga gtcaggaccc ggactggtga
agccgtctga gactctctca 120ctgacttgta ccgtcagcgg cgtgtccctc
cccgactacg gagtgtcatg gatccgccaa 180cctcccggga aagggcttga
atggattggt gtcatctggg gttctgaaac cacctactac 240tcatcttccc
tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc
300cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc
caagcactac 360tattacggag gaagctacgc tatggactat tggggacagg
gcactctcgt gactgtgagc 420agcggcggtg gagggtctgg aggtggagga
tccggtggtg gtgggtcagg cggaggaggg 480agcgagattg tgatgactca
gtcaccagcc accctttctc tttcacccgg cgagagagca 540accctgagct
gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa
600ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc
tggtatcccc 660gctcggttta gcggatcagg atctggtacc gactacactc
tgaccatttc cagcctgcag 720ccagaagatt tcgcagtgta tttctgccag
cagggcaata cccttcctta caccttcggt 780cagggaacca agctcgaaat
caagcaccat caccatcatc accaccat 82868828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 68atggcactgc ctgtcactgc cctcctgctg cctctggccc
tccttctgca tgccgccagg 60ccccaagtcc agctgcaaga gtcaggaccc ggactggtga
agccgtctga gactctctca 120ctgacttgta ccgtcagcgg cgtgtccctc
cccgactacg gagtgtcatg gatccgccaa 180cctcccggga aagggcttga
atggattggt gtcatctggg gttctgaaac cacctactac 240cagtcttccc
tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc
300cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc
caagcactac 360tattacggag gaagctacgc tatggactat tggggacagg
gcactctcgt gactgtgagc 420agcggcggtg gagggtctgg aggtggagga
tccggtggtg gtgggtcagg cggaggaggg 480agcgagattg tgatgactca
gtcaccagcc accctttctc tttcacccgg cgagagagca 540accctgagct
gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa
600ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc
tggtatcccc 660gctcggttta gcggatcagg atctggtacc gactacactc
tgaccatttc cagcctgcag 720ccagaagatt tcgcagtgta tttctgccag
cagggcaata cccttcctta caccttcggt 780cagggaacca agctcgaaat
caagcaccat caccatcatc atcaccac 82869828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 69atggccctcc cagtgaccgc tctgctgctg cctctcgcac
ttcttctcca tgccgctcgg 60cctgagatcg tcatgaccca aagccccgct accctgtccc
tgtcacccgg cgagagggca 120accctttcat gcagggccag ccaggacatt
tctaagtacc tcaactggta tcagcagaag 180ccagggcagg ctcctcgcct
gctgatctac cacaccagcc gcctccacag cggtatcccc 240gccagatttt
ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag
300cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta
caccttcggt 360caaggtacca agctggaaat caagggaggc ggaggatcag
gcggtggcgg aagcggagga 420ggtggctccg gaggaggagg ttcccaagtg
cagcttcaag aatcaggacc cggacttgtg 480aagccatcag aaaccctctc
cctgacttgt accgtgtccg gtgtgagcct ccccgactac 540ggagtctctt
ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg
600ggatcagaga ctacttacta caattcatca cttaagtcac gggtcaccat
cagcaaagat 660aatagcaaga accaagtgtc acttaagctg tcatctgtga
ccgccgctga caccgccgtg 720tactattgtg ccaaacatta ctattacgga
gggtcttatg ctatggacta ctggggacag 780gggaccctgg tgactgtctc
tagccatcac catcaccacc atcatcac 82870828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 70atggcactgc ctgtcactgc cctcctgctg cctctggccc
tccttctgca tgccgccagg 60ccccaagtcc agctgcaaga gtcaggaccc ggactggtga
agccgtctga gactctctca 120ctgacttgta ccgtcagcgg cgtgtccctc
cccgactacg gagtgtcatg gatccgccaa 180cctcccggga aagggcttga
atggattggt gtcatctggg gttctgaaac cacctactac 240aactcttccc
tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc
300cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc
caagcactac 360tattacggag gaagctacgc tatggactat tggggacagg
gcactctcgt gactgtgagc 420agcggcggtg gagggtctgg aggtggagga
tccggtggtg gtgggtcagg cggaggaggg 480agcgagattg tgatgactca
gtcaccagcc accctttctc tttcacccgg cgagagagca 540accctgagct
gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa
600ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc
tggtatcccc 660gctcggttta gcggatcagg atctggtacc gactacactc
tgaccatttc cagcctgcag 720ccagaagatt tcgcagtgta tttctgccag
cagggcaata cccttcctta caccttcggt 780cagggaacca agctcgaaat
caagcaccat caccatcatc accaccat 82871813DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 71atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60cccgaaattg tgatgaccca gtcacccgcc actcttagcc
tttcacccgg tgagcgcgca 120accctgtctt gcagagcctc ccaagacatc
tcaaaatacc ttaattggta tcaacagaag 180cccggacagg ctcctcgcct
tctgatctac cacaccagcc ggctccattc tggaatccct 240gccaggttca
gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag
300ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta
cacctttgga 360cagggcacca agctcgagat taaaggtgga ggtggcagcg
gaggaggtgg gtccggcggt 420ggaggaagcc aggtccaact ccaagaaagc
ggaccgggtc ttgtgaagcc atcagaaact 480ctttcactga cttgtactgt
gagcggagtg tctctccccg attacggggt gtcttggatc 540agacagccac
cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact
600tactacaatt catccctcaa gtcacgcgtc accatctcaa aggacaactc
taagaatcag 660gtgtcactga aactgtcatc tgtgaccgca gccgacaccg
ccgtgtacta ttgcgctaag 720cattactatt atggcgggag ctacgcaatg
gattactggg gacagggtac tctggtcacc 780gtgtccagcc accaccatca
tcaccatcac cat 81372813DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 72atggctctgc ccgtgaccgc actcctcctg ccactggctc
tgctgcttca cgccgctcgc 60ccacaagtcc agcttcaaga atcagggcct ggtctggtga
agccatctga gactctgtcc 120ctcacttgca ccgtgagcgg agtgtccctc
ccagactacg gagtgagctg gattagacag 180cctcccggaa agggactgga
gtggatcgga gtgatttggg gtagcgaaac cacttactat 240aactcttccc
tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc
300ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc
caagcattac 360tactatggag ggtcctacgc catggactac tggggccagg
gaactctggt cactgtgtca 420tctggtggag gaggtagcgg aggaggcggg
agcggtggag gtggctccga aatcgtgatg 480acccagagcc ctgcaaccct
gtccctttct cccggggaac gggctaccct ttcttgtcgg 540gcatcacaag
atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct
600aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg
ctttagcggg 660tctggaagcg ggaccgacta cactctgacc atctcatctc
tccagcccga ggacttcgcc 720gtctacttct gccagcaggg taacaccctg
ccgtacacct tcggccaggg caccaagctt 780gagatcaaac atcaccacca
tcatcaccat cac 81373271PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 73Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser
Pro Ala Thr Leu 20 25 30Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala 50 55 60Pro Arg Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Ile Pro65 70 75 80Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu
Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135
140Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
Thr145 150 155 160Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu
Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile Gly 180 185 190Val Ile Trp Gly Ser Glu Thr Thr
Tyr Tyr Ser Ser Ser Leu Lys Ser 195 200 205Arg Val Thr Ile Ser Lys
Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 210 215 220Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys225 230 235 240His
Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250
255Thr Leu Val Thr Val Ser Ser His His His His His His His His 260
265 27074271PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 74Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln 130 135 140Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu Thr145 150 155 160Leu Ser Leu Thr Cys Thr
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 180 185 190Val Ile
Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser 195 200
205Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys
210 215 220Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Lys225 230 235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
Tyr Trp Gly Gln Gly 245 250 255Thr Leu Val Thr Val Ser Ser His His
His His His His His His 260 265 27075271PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 75Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu 20 25 30Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Val 35 40 45Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr65 70 75 80Ser Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys 85 90 95Asn Gln Val Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135
140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val
Met145 150 155 160Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
Glu Arg Ala Thr 165 170 175Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser
Lys Tyr Leu Asn Trp Tyr 180 185 190Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile Tyr His Thr Ser 195 200 205Arg Leu His Ser Gly Ile
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220Thr Asp Tyr Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala225 230 235 240Val
Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 245 250
255Gly Thr Lys Leu Glu Ile Lys His His His His His His His His 260
265 27076271PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 76Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr
Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met145 150 155 160Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr 165 170
175Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr
180 185 190Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His
Thr Ser 195 200 205Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly 210 215 220Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala225 230 235 240Val Tyr Phe Cys Gln Gln Gly
Asn Thr Leu Pro Tyr Thr Phe Gly Gln 245 250 255Gly Thr Lys Leu Glu
Ile Lys His His His His His His His His 260 265
27077276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 77Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val145 150 155 160Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser 195 200
205Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn
210 215 220Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val225 230 235 240Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser His His His His 260 265 270His His His His
27578276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 78Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val145 150 155 160Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln 195 200
205Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn
210 215 220Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val225 230 235 240Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser His His His His 260 265 270His His His His
27579276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 79Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser
Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly145 150 155 160Ser Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 195 200
205Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser
210 215 220Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln225 230 235 240Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro 245 250 255Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys His His His His 260 265 270His His His His
27580276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 80Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser
Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly145 150 155 160Ser Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 195 200
205Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser
210 215 220Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln225 230 235 240Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro 245 250 255Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys His His His His 260 265 270His His His His
27581276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 81Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val145 150 155 160Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175Leu Pro Asp Tyr
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190Leu Glu
Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn 195 200
205Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn
210 215 220Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val225 230 235 240Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
Ser Tyr Ala Met Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser His His His His 260 265 270His His His His
27582276PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 82Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val 35 40 45Ser Leu
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr65 70 75
80Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys
85 90 95Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser
Tyr Ala Met 115 120 125Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly145 150 155 160Ser Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 165 170 175Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys 180 185 190Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 195 200
205Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser
210 215 220Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln225 230 235 240Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly Asn Thr Leu Pro 245 250 255Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys His His His His 260 265 270His His His His
27583271PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 83Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30Ser Leu Ser
Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60Pro
Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro65 70 75
80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln 130 135 140Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu Thr145 150 155 160Leu Ser Leu Thr Cys Thr
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 180 185 190Val Ile
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser 195 200
205Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys
210 215 220Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Lys225 230 235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
Tyr Trp Gly Gln Gly 245 250 255Thr Leu Val Thr Val Ser Ser His His
His His His His His His 260 265 27084271PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 84Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu 20 25 30Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Val 35 40 45Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile
Arg Gln Pro Pro Gly Lys 50 55 60Gly Leu Glu Trp Ile Gly Val Ile Trp
Gly Ser Glu Thr Thr Tyr Tyr65 70 75 80Asn Ser Ser Leu Lys Ser Arg
Val Thr Ile Ser Lys Asp Asn Ser Lys 85 90 95Asn Gln Val Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110Val Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 115 120 125Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135
140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val
Met145 150 155 160Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
Glu Arg Ala Thr 165 170 175Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser
Lys Tyr Leu Asn Trp Tyr 180 185 190Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile Tyr His Thr Ser 195 200 205Arg Leu His Ser Gly Ile
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220Thr Asp Tyr Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala225 230 235 240Val
Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln 245 250
255Gly Thr Lys Leu Glu Ile Lys His His His His His His His His 260
265 270851458DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 85atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60cccgaaattg tgatgaccca gtcacccgcc actcttagcc
tttcacccgg tgagcgcgca 120accctgtctt gcagagcctc ccaagacatc
tcaaaatacc ttaattggta tcaacagaag 180cccggacagg ctcctcgcct
tctgatctac cacaccagcc ggctccattc tggaatccct 240gccaggttca
gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag
300ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta
cacctttgga 360cagggcacca agctcgagat taaaggtgga ggtggcagcg
gaggaggtgg gtccggcggt 420ggaggaagcc aggtccaact ccaagaaagc
ggaccgggtc ttgtgaagcc atcagaaact 480ctttcactga cttgtactgt
gagcggagtg tctctccccg attacggggt gtcttggatc 540agacagccac
cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact
600tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc
taagaatcag 660gtgtcactga aactgtcatc tgtgaccgca gccgacaccg
ccgtgtacta ttgcgctaag 720cattactatt atggcgggag ctacgcaatg
gattactggg gacagggtac tctggtcacc 780gtgtccagca ccactacccc
agcaccgagg ccacccaccc cggctcctac catcgcctcc 840cagcctctgt
ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc
900cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg
tacttgcggg 960gtcctgctgc tttcactcgt gatcactctt tactgtaagc
gcggtcggaa gaagctgctg 1020tacatcttta agcaaccctt catgaggcct
gtgcagacta ctcaagagga ggacggctgt 1080tcatgccggt tcccagagga
ggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140agcgcagatg
ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt
1200ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc
agaaatgggc 1260gggaagccgc gcagaaagaa tccccaagag ggcctgtaca
acgagctcca aaaggataag 1320atggcagaag cctatagcga gattggtatg
aaaggggaac gcagaagagg caaaggccac 1380gacggactgt accagggact
cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440caggccctgc cgcctcgg
1458861458DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 86atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc
atcagaaact 480ctttcactga cttgtactgt gagcggagtg tctctccccg
attacggggt gtcttggatc 540agacagccac cggggaaggg tctggaatgg
attggagtga tttggggctc tgagactact 600tactaccaat catccctcaa
gtcacgcgtc accatctcaa aggacaactc taagaatcag 660gtgtcactga
aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag
720cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac
tctggtcacc 780gtgtccagca ccactacccc agcaccgagg ccacccaccc
cggctcctac catcgcctcc 840cagcctctgt ccctgcgtcc ggaggcatgt
agacccgcag ctggtggggc cgtgcatacc 900cggggtcttg acttcgcctg
cgatatctac atttgggccc ctctggctgg tacttgcggg 960gtcctgctgc
tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg
1020tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga
ggacggctgt 1080tcatgccggt tcccagagga ggaggaaggc ggctgcgaac
tgcgcgtgaa attcagccgc 1140agcgcagatg ctccagccta caagcagggg
cagaaccagc tctacaacga actcaatctt 1200ggtcggagag aggagtacga
cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260gggaagccgc
gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag
1320atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg
caaaggccac 1380gacggactgt accagggact cagcaccgcc accaaggaca
cctatgacgc tcttcacatg 1440caggccctgc cgcctcgg
1458871458DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 87atggctctgc
ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc 60ccacaagtcc
agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc
120ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg
gattagacag 180cctcccggaa agggactgga gtggatcgga gtgatttggg
gtagcgaaac cacttactat 240tcatcttccc tgaagtcacg ggtcaccatt
tcaaaggata actcaaagaa tcaagtgagc 300ctcaagctct catcagtcac
cgccgctgac accgccgtgt attactgtgc caagcattac 360tactatggag
ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca
420tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga
aatcgtgatg 480acccagagcc ctgcaaccct gtccctttct cccggggaac
gggctaccct ttcttgtcgg 540gcatcacaag atatctcaaa atacctcaat
tggtatcaac agaagccggg acaggcccct 600aggcttctta tctaccacac
ctctcgcctg catagcggga ttcccgcacg ctttagcggg 660tctggaagcg
ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc
720gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg
caccaagctt 780gagatcaaaa ccactactcc cgctccaagg ccacccaccc
ctgccccgac catcgcctct 840cagccgcttt ccctgcgtcc ggaggcatgt
agacccgcag ctggtggggc cgtgcatacc 900cggggtcttg acttcgcctg
cgatatctac atttgggccc ctctggctgg tacttgcggg 960gtcctgctgc
tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg
1020tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga
ggacggctgt 1080tcatgccggt tcccagagga ggaggaaggc ggctgcgaac
tgcgcgtgaa attcagccgc 1140agcgcagatg ctccagccta caagcagggg
cagaaccagc tctacaacga actcaatctt 1200ggtcggagag aggagtacga
cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260gggaagccgc
gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag
1320atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg
caaaggccac 1380gacggactgt accagggact cagcaccgcc accaaggaca
cctatgacgc tcttcacatg 1440caggccctgc cgcctcgg
1458881458DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 88atggctctgc
ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc 60ccacaagtcc
agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc
120ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg
gattagacag 180cctcccggaa agggactgga gtggatcgga gtgatttggg
gtagcgaaac cacttactat 240caatcttccc tgaagtcacg ggtcaccatt
tcaaaggata actcaaagaa tcaagtgagc 300ctcaagctct catcagtcac
cgccgctgac accgccgtgt attactgtgc caagcattac 360tactatggag
ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca
420tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga
aatcgtgatg 480acccagagcc ctgcaaccct gtccctttct cccggggaac
gggctaccct ttcttgtcgg 540gcatcacaag atatctcaaa atacctcaat
tggtatcaac agaagccggg acaggcccct 600aggcttctta tctaccacac
ctctcgcctg catagcggga ttcccgcacg ctttagcggg 660tctggaagcg
ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc
720gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg
caccaagctt 780gagatcaaaa ccactactcc cgctccaagg ccacccaccc
ctgccccgac catcgcctct 840cagccgcttt ccctgcgtcc ggaggcatgt
agacccgcag ctggtggggc cgtgcatacc 900cggggtcttg acttcgcctg
cgatatctac atttgggccc ctctggctgg tacttgcggg 960gtcctgctgc
tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg
1020tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga
ggacggctgt 1080tcatgccggt tcccagagga ggaggaaggc ggctgcgaac
tgcgcgtgaa attcagccgc 1140agcgcagatg ctccagccta caagcagggg
cagaaccagc tctacaacga actcaatctt 1200ggtcggagag aggagtacga
cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260gggaagccgc
gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag
1320atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg
caaaggccac 1380gacggactgt accagggact cagcaccgcc accaaggaca
cctatgacgc tcttcacatg 1440caggccctgc cgcctcgg
1458891473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 89atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcg gcggaggcgg gagccaggtc caactccaag aaagcggacc
gggtcttgtg 480aagccatcag aaactctttc actgacttgt actgtgagcg
gagtgtctct ccccgattac 540ggggtgtctt ggatcagaca gccaccgggg
aagggtctgg aatggattgg agtgatttgg 600ggctctgaga ctacttacta
ctcttcatcc ctcaagtcac gcgtcaccat ctcaaaggac 660aactctaaga
atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg
720tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta
ctggggacag 780ggtactctgg tcaccgtgtc cagcaccact accccagcac
cgaggccacc caccccggct 840cctaccatcg cctcccagcc tctgtccctg
cgtccggagg catgtagacc cgcagctggt 900ggggccgtgc atacccgggg
tcttgacttc gcctgcgata tctacatttg ggcccctctg 960gctggtactt
gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt
1020cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca
gactactcaa 1080gaggaggacg gctgttcatg ccggttccca gaggaggagg
aaggcggctg cgaactgcgc 1140gtgaaattca gccgcagcgc agatgctcca
gcctacaagc aggggcagaa ccagctctac 1200aacgaactca atcttggtcg
gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1260gacccagaaa
tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag
1320ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg
ggaacgcaga 1380agaggcaaag gccacgacgg actgtaccag ggactcagca
ccgccaccaa ggacacctat 1440gacgctcttc acatgcaggc cctgccgcct cgg
1473901473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 90atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcg gaggcggagg gagccaggtc caactccaag aaagcggacc
gggtcttgtg 480aagccatcag aaactctttc actgacttgt actgtgagcg
gagtgtctct ccccgattac 540ggggtgtctt ggatcagaca gccaccgggg
aagggtctgg aatggattgg agtgatttgg 600ggctctgaga ctacttacta
ccaatcatcc ctcaagtcac gcgtcaccat ctcaaaggac 660aactctaaga
atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg
720tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta
ctggggacag 780ggtactctgg tcaccgtgtc cagcaccact accccagcac
cgaggccacc caccccggct 840cctaccatcg cctcccagcc tctgtccctg
cgtccggagg catgtagacc cgcagctggt 900ggggccgtgc atacccgggg
tcttgacttc gcctgcgata tctacatttg ggcccctctg 960gctggtactt
gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt
1020cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca
gactactcaa 1080gaggaggacg gctgttcatg ccggttccca gaggaggagg
aaggcggctg cgaactgcgc 1140gtgaaattca gccgcagcgc agatgctcca
gcctacaagc aggggcagaa ccagctctac 1200aacgaactca atcttggtcg
gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1260gacccagaaa
tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag
1320ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg
ggaacgcaga 1380agaggcaaag gccacgacgg actgtaccag ggactcagca
ccgccaccaa ggacacctat 1440gacgctcttc acatgcaggc cctgccgcct cgg
1473911473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 91atggctctgc
ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc 60ccacaagtcc
agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc
120ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg
gattagacag 180cctcccggaa agggactgga gtggatcgga gtgatttggg
gtagcgaaac cacttactat 240tcatcttccc tgaagtcacg ggtcaccatt
tcaaaggata actcaaagaa tcaagtgagc 300ctcaagctct catcagtcac
cgccgctgac accgccgtgt attactgtgc caagcattac 360tactatggag
ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca
420tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg
aggtggcgga 480agcgaaatcg tgatgaccca gagccctgca accctgtccc
tttctcccgg ggaacgggct 540accctttctt gtcgggcatc acaagatatc
tcaaaatacc tcaattggta tcaacagaag 600ccgggacagg cccctaggct
tcttatctac cacacctctc gcctgcatag cgggattccc 660gcacgcttta
gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag
720cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta
caccttcggc 780cagggcacca agcttgagat caaaaccact actcccgctc
caaggccacc cacccctgcc 840ccgaccatcg cctctcagcc gctttccctg
cgtccggagg catgtagacc cgcagctggt 900ggggccgtgc atacccgggg
tcttgacttc gcctgcgata tctacatttg ggcccctctg 960gctggtactt
gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt
1020cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca
gactactcaa 1080gaggaggacg gctgttcatg ccggttccca gaggaggagg
aaggcggctg cgaactgcgc 1140gtgaaattca gccgcagcgc agatgctcca
gcctacaagc aggggcagaa ccagctctac 1200aacgaactca atcttggtcg
gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1260gacccagaaa
tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag
1320ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg
ggaacgcaga 1380agaggcaaag gccacgacgg actgtaccag ggactcagca
ccgccaccaa ggacacctat 1440gacgctcttc acatgcaggc cctgccgcct cgg
1473921473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 92atggctctgc
ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc 60ccacaagtcc
agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc
120ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg
gattagacag 180cctcccggaa agggactgga gtggatcgga gtgatttggg
gtagcgaaac cacttactat 240caatcttccc tgaagtcacg ggtcaccatt
tcaaaggata actcaaagaa tcaagtgagc 300ctcaagctct catcagtcac
cgccgctgac accgccgtgt attactgtgc caagcattac 360tactatggag
ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca
420tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg
aggcggtggg 480tcagaaatcg tgatgaccca gagccctgca accctgtccc
tttctcccgg ggaacgggct 540accctttctt gtcgggcatc acaagatatc
tcaaaatacc tcaattggta tcaacagaag 600ccgggacagg cccctaggct
tcttatctac cacacctctc gcctgcatag cgggattccc 660gcacgcttta
gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag
720cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta
caccttcggc 780cagggcacca agcttgagat caaaaccact actcccgctc
caaggccacc cacccctgcc 840ccgaccatcg cctctcagcc gctttccctg
cgtccggagg catgtagacc cgcagctggt 900ggggccgtgc atacccgggg
tcttgacttc gcctgcgata tctacatttg ggcccctctg 960gctggtactt
gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt
1020cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca
gactactcaa 1080gaggaggacg gctgttcatg ccggttccca gaggaggagg
aaggcggctg cgaactgcgc 1140gtgaaattca gccgcagcgc agatgctcca
gcctacaagc aggggcagaa ccagctctac 1200aacgaactca atcttggtcg
gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1260gacccagaaa
tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag
1320ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg
ggaacgcaga 1380agaggcaaag gccacgacgg actgtaccag ggactcagca
ccgccaccaa ggacacctat 1440gacgctcttc acatgcaggc cctgccgcct cgg
1473931473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 93atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc
gggtcttgtg 480aagccatcag aaactctttc actgacttgt actgtgagcg
gagtgtctct ccccgattac 540ggggtgtctt ggatcagaca gccaccgggg
aagggtctgg aatggattgg agtgatttgg 600ggctctgaga ctacttacta
caactcatcc ctcaagtcac gcgtcaccat ctcaaaggac 660aactctaaga
atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg
720tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta
ctggggacag 780ggtactctgg tcaccgtgtc cagcaccact accccagcac
cgaggccacc caccccggct 840cctaccatcg cctcccagcc tctgtccctg
cgtccggagg catgtagacc cgcagctggt 900ggggccgtgc atacccgggg
tcttgacttc gcctgcgata tctacatttg ggcccctctg 960gctggtactt
gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt
1020cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca
gactactcaa 1080gaggaggacg gctgttcatg ccggttccca gaggaggagg
aaggcggctg cgaactgcgc 1140gtgaaattca gccgcagcgc agatgctcca
gcctacaagc aggggcagaa ccagctctac 1200aacgaactca atcttggtcg
gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1260gacccagaaa
tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag
1320ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg
ggaacgcaga 1380agaggcaaag gccacgacgg actgtaccag ggactcagca
ccgccaccaa ggacacctat 1440gacgctcttc acatgcaggc cctgccgcct cgg
1473941473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 94atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaaattg
tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca
120accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta
tcaacagaag 180cccggacagg ctcctcgcct tctgatctac cacaccagcc
ggctccattc tggaatccct 240gccaggttca gcggtagcgg atctgggacc
gactacaccc tcactatcag ctcactgcag 300ccagaggact tcgctgtcta
tttctgtcag caagggaaca ccctgcccta cacctttgga 360cagggcacca
agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt
420ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc
gggtcttgtg 480aagccatcag aaactctttc actgacttgt actgtgagcg
gagtgtctct ccccgattac 540ggggtgtctt ggatcagaca gccaccgggg
aagggtctgg aatggattgg
agtgatttgg 600ggctctgaga ctacttacta caactcatcc ctcaagtcac
gcgtcaccat ctcaaaggac 660aactctaaga atcaggtgtc actgaaactg
tcatctgtga ccgcagccga caccgccgtg 720tactattgcg ctaagcatta
ctattatggc gggagctacg caatggatta ctggggacag 780ggtactctgg
tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct
840cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc
cgcagctggt 900ggggccgtgc atacccgggg tcttgacttc gcctgcgata
tctacatttg ggcccctctg 960gctggtactt gcggggtcct gctgctttca
ctcgtgatca ctctttactg taagcgcggt 1020cggaagaagc tgctgtacat
ctttaagcaa cccttcatga ggcctgtgca gactactcaa 1080gaggaggacg
gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc
1140gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa
ccagctctac 1200aacgaactca atcttggtcg gagagaggag tacgacgtgc
tggacaagcg gagaggacgg 1260gacccagaaa tgggcgggaa gccgcgcaga
aagaatcccc aagagggcct gtacaacgag 1320ctccaaaagg ataagatggc
agaagcctat agcgagattg gtatgaaagg ggaacgcaga 1380agaggcaaag
gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat
1440gacgctcttc acatgcaggc cctgccgcct cgg 1473951473DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 95atggctctgc ccgtgaccgc actcctcctg ccactggctc
tgctgcttca cgccgctcgc 60ccacaagtcc agcttcaaga atcagggcct ggtctggtga
agccatctga gactctgtcc 120ctcacttgca ccgtgagcgg agtgtccctc
ccagactacg gagtgagctg gattagacag 180cctcccggaa agggactgga
gtggatcgga gtgatttggg gtagcgaaac cacttactat 240aactcttccc
tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc
300ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc
caagcattac 360tactatggag ggtcctacgc catggactac tggggccagg
gaactctggt cactgtgtca 420tctggtggag gaggtagcgg aggaggcggg
agcggtggag gtggctccgg aggtggcgga 480agcgaaatcg tgatgaccca
gagccctgca accctgtccc tttctcccgg ggaacgggct 540accctttctt
gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag
600ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag
cgggattccc 660gcacgcttta gcgggtctgg aagcgggacc gactacactc
tgaccatctc atctctccag 720cccgaggact tcgccgtcta cttctgccag
cagggtaaca ccctgccgta caccttcggc 780cagggcacca agcttgagat
caaaaccact actcccgctc caaggccacc cacccctgcc 840ccgaccatcg
cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt
900ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg
ggcccctctg 960gctggtactt gcggggtcct gctgctttca ctcgtgatca
ctctttactg taagcgcggt 1020cggaagaagc tgctgtacat ctttaagcaa
cccttcatga ggcctgtgca gactactcaa 1080gaggaggacg gctgttcatg
ccggttccca gaggaggagg aaggcggctg cgaactgcgc 1140gtgaaattca
gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac
1200aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg
gagaggacgg 1260gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc
aagagggcct gtacaacgag 1320ctccaaaagg ataagatggc agaagcctat
agcgagattg gtatgaaagg ggaacgcaga 1380agaggcaaag gccacgacgg
actgtaccag ggactcagca ccgccaccaa ggacacctat 1440gacgctcttc
acatgcaggc cctgccgcct cgg 1473961458DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 96atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60cccgaaattg tgatgaccca gtcacccgcc actcttagcc
tttcacccgg tgagcgcgca 120accctgtctt gcagagcctc ccaagacatc
tcaaaatacc ttaattggta tcaacagaag 180cccggacagg ctcctcgcct
tctgatctac cacaccagcc ggctccattc tggaatccct 240gccaggttca
gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag
300ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta
cacctttgga 360cagggcacca agctcgagat taaaggtgga ggtggcagcg
gaggaggtgg gtccggcggt 420ggaggaagcc aggtccaact ccaagaaagc
ggaccgggtc ttgtgaagcc atcagaaact 480ctttcactga cttgtactgt
gagcggagtg tctctccccg attacggggt gtcttggatc 540agacagccac
cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact
600tactacaact catccctcaa gtcacgcgtc accatctcaa aggacaactc
taagaatcag 660gtgtcactga aactgtcatc tgtgaccgca gccgacaccg
ccgtgtacta ttgcgctaag 720cattactatt atggcgggag ctacgcaatg
gattactggg gacagggtac tctggtcacc 780gtgtccagca ccactacccc
agcaccgagg ccacccaccc cggctcctac catcgcctcc 840cagcctctgt
ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc
900cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg
tacttgcggg 960gtcctgctgc tttcactcgt gatcactctt tactgtaagc
gcggtcggaa gaagctgctg 1020tacatcttta agcaaccctt catgaggcct
gtgcagacta ctcaagagga ggacggctgt 1080tcatgccggt tcccagagga
ggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140agcgcagatg
ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt
1200ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc
agaaatgggc 1260gggaagccgc gcagaaagaa tccccaagag ggcctgtaca
acgagctcca aaaggataag 1320atggcagaag cctatagcga gattggtatg
aaaggggaac gcagaagagg caaaggccac 1380gacggactgt accagggact
cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440caggccctgc cgcctcgg
145897813DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 97atggccctgc
ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60ccggacatcc
agatgaccca aaccacctca tccctctctg cctctcttgg agacagggtg
120accatttctt gtcgcgccag ccaggacatc agcaagtatc tgaactggta
tcagcagaag 180ccggacggaa ccgtgaagct cctgatctac catacctctc
gcctgcatag cggcgtgccc 240tcacgcttct ctggaagcgg atcaggaacc
gattattctc tcactatttc aaatcttgag 300caggaagata ttgccaccta
tttctgccag cagggtaata ccctgcccta caccttcgga 360ggagggacca
agctcgaaat caccggtgga ggaggcagcg gcggtggagg gtctggtgga
420ggtggttctg aggtgaagct gcaagaatca ggccctggac ttgtggcccc
ttcacagtcc 480ctgagcgtga cttgcaccgt gtccggagtc tccctgcccg
actacggagt gtcatggatc 540agacaacctc cacggaaagg actggaatgg
ctcggtgtca tctggggtag cgaaactact 600tactacaatt cagccctcaa
aagcaggctg actattatca aggacaacag caagtcccaa 660gtctttctta
agatgaactc actccagact gacgacaccg caatctacta ttgtgctaag
720cactactact acggaggatc ctacgctatg gattactggg gacaaggtac
ttccgtcact 780gtctcttcac accatcatca ccatcaccat cac
81398271PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 98Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30Ser Ala Ser
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60Val
Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75
80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Thr 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Glu 130 135 140Val Lys Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln Ser145 150 155 160Leu Ser Val Thr Cys Thr
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175Val Ser Trp Ile
Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190Val Ile
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200
205Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
210 215 220Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys
Ala Lys225 230 235 240His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
Tyr Trp Gly Gln Gly 245 250 255Thr Ser Val Thr Val Ser Ser His His
His His His His His His 260 265 270991458DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 99atggccttac cagtgaccgc cttgctcctg ccgctggcct
tgctgctcca cgccgccagg 60ccggacatcc agatgacaca gactacatcc tccctgtctg
cctctctggg agacagagtc 120accatcagtt gcagggcaag tcaggacatt
agtaaatatt taaattggta tcagcagaaa 180ccagatggaa ctgttaaact
cctgatctac catacatcaa gattacactc aggagtccca 240tcaaggttca
gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag
300caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta
cacgttcgga 360ggggggacca agctggagat cacaggtggc ggtggctcgg
gcggtggtgg gtcgggtggc 420ggcggatctg aggtgaaact gcaggagtca
ggacctggcc tggtggcgcc ctcacagagc 480ctgtccgtca catgcactgt
ctcaggggtc tcattacccg actatggtgt aagctggatt 540cgccagcctc
cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca
600tactataatt cagctctcaa atccagactg accatcatca aggacaactc
caagagccaa 660gttttcttaa aaatgaacag tctgcaaact gatgacacag
ccatttacta ctgtgccaaa 720cattattact acggtggtag ctatgctatg
gactactggg gccaaggaac ctcagtcacc 780gtctcctcaa ccacgacgcc
agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840cagcccctgt
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg
900agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg
gacttgtggg 960gtccttctcc tgtcactggt tatcaccctt tactgcaaac
ggggcagaaa gaaactcctg 1020tatatattca aacaaccatt tatgagacca
gtacaaacta ctcaagagga agatggctgt 1080agctgccgat ttccagaaga
agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140agcgcagacg
cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta
1200ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc
tgagatgggg 1260ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca
atgaactgca gaaagataag 1320atggcggagg cctacagtga gattgggatg
aaaggcgagc gccggagggg caaggggcac 1380gatggccttt accagggtct
cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440caggccctgc cccctcgc
14581001184DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 100cgtgaggctc
cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60tggggggagg
ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
120aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
ccgtatataa 180gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
tgccgccaga acacaggtaa 240gtgccgtgtg tggttcccgc gggcctggcc
tctttacggg ttatggccct tgcgtgcctt 300gaattacttc cacctggctg
cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360ggtgggagag
ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
420cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
gcctgtctcg 480ctgctttcga taagtctcta gccatttaaa atttttgatg
acctgctgcg acgctttttt 540tctggcaaga tagtcttgta aatgcgggcc
aagatctgca cactggtatt tcggtttttg 600gggccgcggg cggcgacggg
gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660tgcgagcgcg
gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
720tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
gcccggtcgg 780caccagttgc gtgagcggaa agatggccgc ttcccggccc
tgctgcaggg agctcaaaat 840ggaggacgcg gcgctcggga gagcgggcgg
gtgagtcacc cacacaaagg aaaagggcct 900ttccgtcctc agccgtcgct
tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960tcgattagtt
ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
1020cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
tggcacttga 1080tgtaattctc cttggaattt gccctttttg agtttggatc
ttggttcatt ctcaagcctc 1140agacagtggt tcaaagtttt tttcttccat
ttcaggtgtc gtga 1184101336DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 101agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca
agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg
ttttggacaa gagacgtggc 120cgggaccctg agatgggggg aaagccgaga
aggaagaacc ctcaggaagg cctgtacaat 180gaactgcaga aagataagat
ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240cggaggggca
aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc
300tacgacgccc ttcacatgca ggccctgccc cctcgc 336102230PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 102Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe1 5 10 15Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 20 25 30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala145 150 155 160Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 165 170 175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser 195 200 205Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly
Lys Met225 230103690DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 103gagagcaagt
acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60agcgtgttcc
tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag
120gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt
caactggtac 180gtggacggcg tggaggtgca caacgccaag accaagcccc
gggaggagca gttcaatagc 240acctaccggg tggtgtccgt gctgaccgtg
ctgcaccagg actggctgaa cggcaaggaa 300tacaagtgta aggtgtccaa
caagggcctg cccagcagca tcgagaaaac catcagcaag 360gccaagggcc
agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg
420accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag
cgacatcgcc 480gtggagtggg agagcaacgg ccagcccgag aacaactaca
agaccacccc ccctgtgctg 540gacagcgacg gcagcttctt cctgtacagc
cggctgaccg tggacaagag ccggtggcag 600gagggcaacg tctttagctg
ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660aagagcctga
gcctgtccct gggcaagatg 690104150DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 104aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
15010540PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polypeptide"misc_feature(1)..(40)/note="This sequence may encompass
1-10 repeating "Gly Gly Gly Ser" repeating units" 105Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30Gly
Gly Gly Ser Gly Gly Gly Ser 35 4010620PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 106Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly1 5 10 15Gly Gly Gly Ser 2010715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 107Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser1 5 10 151084PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 108Gly Gly Gly
Ser11095000DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 50-5000 nucleotides" 109aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1980aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2280aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2340aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2580aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2880aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2940aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3000aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3480aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3780aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4680aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4980aaaaaaaaaa aaaaaaaaaa 5000110100DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 110tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 60tttttttttt tttttttttt tttttttttt tttttttttt
1001115000DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 50-5000 nucleotides" 111tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 60tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 120tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 180tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
240tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 300tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 360tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 420tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 480tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
540tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 600tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 660tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 720tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 780tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
840tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 900tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 960tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 1020tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 1080tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
1140tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 1200tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 1260tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 1320tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 1380tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
1440tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 1500tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 1560tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 1620tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 1680tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
1740tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 1800tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 1860tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 1920tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 1980tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
2040tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 2100tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 2160tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 2220tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 2280tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
2340tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 2400tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 2460tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 2520tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 2580tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
2640tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 2700tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 2760tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 2820tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 2880tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
2940tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 3000tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 3060tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 3120tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 3180tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
3240tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 3300tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 3360tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 3420tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 3480tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
3540tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 3600tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 3660tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 3720tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 3780tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
3840tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 3900tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 3960tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 4020tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 4080tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
4140tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 4200tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 4260tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 4320tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 4380tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
4440tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 4500tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 4560tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 4620tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 4680tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
4740tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt 4800tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 4860tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 4920tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt tttttttttt 4980tttttttttt
tttttttttt 50001125000DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 100-5000 nucleotides" 112aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2100aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2340aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2760aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2940aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3000aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3060aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
3840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
4740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 4860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980aaaaaaaaaa
aaaaaaaaaa 5000113400DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(400)/note="This sequence may
encompass 100-400 nucleotides" 113aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 400114120PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 114Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65
70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120115120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 115Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120116120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 116Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120117107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 117Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr His
Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1051182000DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(2000)/note="This sequence may
encompass 50-2000 nucleotides" 118aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 300aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 360aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 600aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 660aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
840aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 900aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1440aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa
aaaaaaaaaa 2000119373PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 119Pro Gly Trp Phe
Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr1 5 10 15Phe Ser Pro
Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30Thr Cys
Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45Arg
Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55
60Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu65
70 75 80Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg
Asn 85 90 95Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro
Lys Ala 100 105 110Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
Thr Glu Arg Arg 115 120 125Ala Glu Val Pro Thr Ala His Pro Ser Pro
Ser Pro Arg Pro Ala Gly 130 135 140Gln Phe Gln Thr Leu Val Thr Thr
Thr Pro Ala Pro Arg Pro Pro Thr145 150 155 160Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 165 170 175Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 180 185 190Ala
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 195 200
205Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
210 215 220Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
Gln Thr225 230 235 240Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
Pro Glu Glu Glu Glu 245 250 255Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg Ser Ala Asp Ala Pro 260 265 270Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly 275 280 285Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 290 295 300Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr305 310 315
320Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
325 330 335Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
Tyr Gln 340 345 350Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln 355 360 365Ala Leu Pro Pro Arg
3701201182DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 120atggccctcc
ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga 60ccacccggat
ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg
120gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt
ctccaacacc 180tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt
caaaccagac cgacaagctc 240gccgcgtttc cggaagatcg gtcgcaaccg
ggacaggatt gtcggttccg cgtgactcaa 300ctgccgaatg gcagagactt
ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg 360acctacctgt
gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg
420gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc
atccccatcg 480cctcggcctg cggggcagtt tcagaccctg gtcacgacca
ctccggcgcc gcgcccaccg 540actccggccc caactatcgc gagccagccc
ctgtcgctga ggccggaagc atgccgccct 600gccgccggag gtgctgtgca
tacccgggga ttggacttcg catgcgacat ctacatttgg 660gctcctctcg
ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc
720aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag
gcccgtgcaa 780accacccagg aggaggacgg ttgctcctgc cggttccccg
aagaggaaga aggaggttgc 840gagctgcgcg tgaagttctc ccggagcgcc
gacgcccccg cctataagca gggccagaac 900cagctgtaca acgaactgaa
cctgggacgg cgggaagagt acgatgtgct ggacaagcgg 960cgcggccggg
accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg
1020tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg
gatgaaggga 1080gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag
gactgtccac cgccaccaag 1140gacacatacg atgccctgca catgcaggcc
cttccccctc gc 1182121394PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 121Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser
Pro Asp Arg Pro 20 25 30Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu
Val Val Thr Glu Gly 35 40 45Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser
Asn Thr Ser Glu Ser Phe 50 55 60Val Leu Asn Trp Tyr Arg Met Ser Pro
Ser Asn Gln Thr Asp Lys Leu65 70 75 80Ala Ala Phe Pro Glu Asp Arg
Ser Gln Pro Gly Gln Asp Cys Arg Phe 85 90 95Arg Val Thr Gln Leu Pro
Asn Gly Arg Asp Phe His Met Ser Val Val 100 105 110Arg Ala Arg Arg
Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser 115 120 125Leu Ala
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg 130 135
140Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro
Ser145 150 155 160Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr
Thr Thr Pro Ala 165 170 175Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser 180 185 190Leu Arg Pro Glu Ala Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr 195 200 205Arg Gly Leu Asp Phe Ala
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 210 215 220Gly Thr Cys Gly
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys225 230 235 240Lys
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 245 250
255Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
260 265 270Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg 275 280 285Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn 290 295 300Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg305 310 315 320Arg Gly Arg Asp Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro 325 330 335Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 340 345 350Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 355 360 365Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 370 375
380Ala Leu His Met Gln Ala Leu Pro Pro Arg385
390122132PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 122Asp Val Pro Asp Tyr
Ala Ser Leu Gly Gly Pro Ser Ser Pro Lys Lys1 5 10 15Lys Arg Lys Val
Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly 20 25 30Asp Gly Arg
Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr 35 40 45Thr Gly
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg 50 55 60Asn
Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly65 70 75
80Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
85 90 95Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly
Ile 100 105 110Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu
Leu Lys Leu 115 120 125Glu Thr Ser Tyr 130123108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 123Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
Phe Pro Lys1 5 10 15Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met
Leu Glu Asp Gly 20 25 30Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
Pro Phe Lys Phe Met 35 40 45Leu Gly Lys Gln Glu Val Ile Arg Gly Trp
Glu Glu Gly Val Ala Gln 50 55 60Met Ser Val Gly Gln Arg Ala Lys Leu
Thr Ile Ser Pro Asp Tyr Ala65 70 75 80Tyr Gly Ala Thr Gly His Pro
Gly Ile Ile Pro Pro His Ala Thr Leu 85 90 95Val Phe Asp Val Glu Leu
Leu Lys Leu Glu Thr Ser 100 10512493PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 124Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Thr Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys 85 9012595PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 125Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Thr Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Thr Ser 85 90 9512695PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 126Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg
Asp Leu Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser
Gly Asn Val Lys Asp Leu Thr Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr
His Val Phe Arg Arg Ile Ser Lys Thr Ser 85 90 9512795PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 127Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Leu Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Thr Ser 85 90 9512895PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"MOD_RES(12)..(12)Any amino acidMOD_RES(78)..(78)Any
amino acid 128Ile Leu Trp His Glu Met Trp His Glu Gly Leu Xaa Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Xaa Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Thr Ser 85 90 9512995PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 129Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Leu Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Thr Ser 85 90 9513095PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 130Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu
Ala Ser Arg1 5 10 15Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
Glu Val Leu Glu 20 25 30Pro Leu His Ala Met Met Glu Arg Gly Pro Gln
Thr Leu Lys Glu Thr 35 40 45Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu
Met Glu Ala Gln Glu Trp 50 55 60Cys Arg Lys Tyr Met Lys Ser Gly Asn
Val Lys Asp Leu Leu Gln Ala65 70 75 80Trp Asp Leu Tyr Tyr His Val
Phe Arg Arg Ile Ser Lys Thr Ser 85 90 951311132PRTHomo sapiens
131Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser1
5 10 15His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu
Gly 20 25 30Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala
Phe Arg 35 40 45Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp
Ala Arg Pro 50 55 60Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys
Leu Lys Glu Leu65 70 75 80Val Ala Arg Val Leu Gln Arg Leu Cys Glu
Arg Gly Ala Lys Asn Val 85 90 95Leu Ala Phe Gly Phe Ala Leu Leu Asp
Gly Ala Arg Gly Gly Pro Pro 100 105 110Glu Ala Phe Thr Thr Ser Val
Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125Asp Ala Leu Arg Gly
Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140Gly Asp Asp
Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val145 150 155
160Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr
165 170 175Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala
Ser Gly 180 185 190Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn
His Ser Val Arg 195 200 205Glu Ala Gly Val Pro Leu Gly Leu Pro Ala
Pro Gly Ala Arg Arg Arg 210 215 220Gly Gly Ser Ala Ser Arg Ser Leu
Pro Leu Pro Lys Arg Pro Arg Arg225 230 235 240Gly Ala Ala Pro Glu
Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255Ala His Pro
Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270Val
Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280
285Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His
290 295 300Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp
Thr Pro305 310 315 320Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe
Leu Tyr Ser Ser Gly 325 330 335Asp Lys Glu Gln Leu Arg Pro Ser Phe
Leu Leu Ser Ser Leu Arg Pro 340 345 350Ser Leu Thr Gly Ala Arg Arg
Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365Arg Pro Trp Met Pro
Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380Arg Tyr Trp
Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His385 390 395
400Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg
405 410 415Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys
Pro Gln 420 425 430Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp
Pro Arg Arg Leu 435 440 445Val Gln Leu Leu Arg Gln His Ser Ser Pro
Trp Gln Val Tyr Gly Phe 450 455 460Val Arg Ala Cys Leu Arg Arg Leu
Val Pro Pro Gly Leu Trp Gly Ser465 470 475 480Arg His Asn Glu Arg
Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495Leu Gly Lys
His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510Ser
Val Arg Gly Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520
525Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe
530 535 540Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg
Ser Phe545 550 555 560Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn
Arg Leu Phe Phe Tyr 565 570 575Arg Lys Ser Val Trp Ser Lys Leu Gln
Ser Ile Gly Ile Arg Gln His 580 585 590Leu Lys Arg Val Gln Leu Arg
Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605His Arg Glu Ala Arg
Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620Pro Lys Pro
Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val625 630 635
640Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser
645 650 655Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala
Arg Arg 660 665 670Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp
Asp Ile His Arg 675 680 685Ala Trp Arg Thr Phe Val Leu Arg Val Arg
Ala Gln Asp Pro Pro Pro 690 695 700Glu Leu Tyr Phe Val Lys Val Asp
Val Thr Gly Ala Tyr Asp Thr Ile705 710 715 720Pro Gln Asp Arg Leu
Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735Asn Thr Tyr
Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750Gly
His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760
765Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser
770 775 780Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu
Asn Glu785 790 795 800Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg
Phe Met Cys His His 805 810 815Ala Val Arg Ile Arg Gly Lys Ser Tyr
Val Gln Cys Gln Gly Ile Pro 820 825 830Gln Gly Ser Ile Leu Ser Thr
Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845Met Glu Asn Lys Leu
Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860Arg Leu Val
Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala865 870 875
880Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys
885 890 895Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu
Asp Glu 900 905 910Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala
His Gly Leu Phe 915 920 925Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg
Thr Leu Glu Val Gln Ser 930 935 940Asp Tyr Ser Ser Tyr Ala Arg Thr
Ser Ile Arg Ala Ser Leu Thr Phe945 950 955 960Asn Arg Gly Phe Lys
Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975Val Leu Arg
Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990Ser
Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995
1000 1005Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His
Gln 1010 1015 1020Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val
Ile Ser Asp 1025 1030 1035Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys
Ala Lys Asn Ala Gly 1040 1045 1050Met Ser Leu Gly Ala Lys Gly Ala
Ala Gly Pro Leu Pro Ser Glu 1055 1060 1065Ala Val Gln Trp Leu Cys
His Gln Ala Phe Leu Leu Lys Leu Thr 1070 1075 1080Arg His Arg Val
Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr 1085 1090 1095Ala Gln
Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr 1100 1105
1110Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys
1115 1120 1125Thr Ile Leu Asp 11301324027DNAHomo sapiens
132caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc
cccgcgatgc 60cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac
cgcgaggtgc 120tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg
ctggcggctg gtgcagcgcg 180gggacccggc ggctttccgc gcgctggtgg
cccagtgcct ggtgtgcgtg ccctgggacg 240cacggccgcc ccccgccgcc
ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 300cccgagtgct
gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg
360cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc
gtgcgcagct 420acctgcccaa cacggtgacc gacgcactgc gggggagcgg
ggcgtggggg ctgctgttgc 480gccgcgtggg cgacgacgtg ctggttcacc
tgctggcacg ctgcgcgctc tttgtgctgg 540tggctcccag ctgcgcctac
caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 600ctcaggcccg
gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg
660cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc
ccgggtgcga 720ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc
caagaggccc aggcgtggcg 780ctgcccctga gccggagcgg acgcccgttg
ggcaggggtc ctgggcccac ccgggcagga 840cgcgtggacc gagtgaccgt
ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 900ccacctcttt
ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc
960agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac
acgccttgtc 1020ccccggtgta cgccgagacc aagcacttcc tctactcctc
aggcgacaag gagcagctgc 1080ggccctcctt cctactcagc tctctgaggc
ccagcctgac tggcgctcgg aggctcgtgg 1140agaccatctt tctgggttcc
aggccctgga tgccagggac tccccgcagg ttgccccgcc 1200tgccccagcg
ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc
1260agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg
gtcaccccag 1320cagccggtgt ctgtgcccgg gagaagcccc agggctctgt
ggcggccccc gaggaggagg 1380acacagaccc ccgtcgcctg gtgcagctgc
tccgccagca cagcagcccc tggcaggtgt 1440acggcttcgt gcgggcctgc
ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1500acaacgaacg
ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca
1560agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct
tggctgcgca 1620ggagcccagg ggttggctgt gttccggccg cagagcaccg
tctgcgtgag gagatcctgg 1680ccaagttcct gcactggctg atgagtgtgt
acgtcgtcga gctgctcagg tctttctttt 1740atgtcacgga gaccacgttt
caaaagaaca ggctcttttt ctaccggaag agtgtctgga 1800gcaagttgca
aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt
1860cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg
tccagactcc 1920gcttcatccc caagcctgac gggctgcggc cgattgtgaa
catggactac gtcgtgggag 1980ccagaacgtt ccgcagagaa aagagggccg
agcgtctcac ctcgagggtg aaggcactgt 2040tcagcgtgct caactacgag
cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2100gcctggacga
tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc
2160cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac
accatccccc 2220aggacaggct cacggaggtc atcgccagca tcatcaaacc
ccagaacacg tactgcgtgc 2280gtcggtatgc cgtggtccag aaggccgccc
atgggcacgt ccgcaaggcc ttcaagagcc 2340acgtctctac cttgacagac
ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2400agaccagccc
gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca
2460gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg
cgcatcaggg 2520gcaagtccta cgtccagtgc caggggatcc cgcagggctc
catcctctcc acgctgctct 2580gcagcctgtg ctacggcgac atggagaaca
agctgtttgc ggggattcgg cgggacgggc 2640tgctcctgcg tttggtggat
gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2700ccttcctcag
gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga
2760agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct
tttgttcaga 2820tgccggccca cggcctattc ccctggtgcg gcctgctgct
ggatacccgg accctggagg 2880tgcagagcga ctactccagc tatgcccgga
cctccatcag agccagtctc accttcaacc 2940gcggcttcaa ggctgggagg
aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3000gtcacagcct
gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct
3060acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag
ctcccatttc 3120atcagcaagt ttggaagaac cccacatttt tcctgcgcgt
catctctgac acggcctccc 3180tctgctactc catcctgaaa gccaagaacg
cagggatgtc gctgggggcc aagggcgccg 3240ccggccctct gccctccgag
gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3300tgactcgaca
ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc
3360agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca
gccaacccgg 3420cactgccctc agacttcaag accatcctgg actgatggcc
acccgcccac agccaggccg 3480agagcagaca ccagcagccc tgtcacgccg
ggctctacgt cccagggagg gaggggcggc 3540ccacacccag gcccgcaccg
ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3600gcatgtccgg
ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc
3660tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg
gctccacccc 3720agggccagct tttcctcacc aggagcccgg cttccactcc
ccacatagga atagtccatc 3780cccagattcg ccattgttca cccctcgccc
tgccctcctt tgccttccac ccccaccatc 3840caggtggaga ccctgagaag
gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3900gccctgtaca
caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg
3960ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga
aaaaaaaaaa 4020aaaaaaa 4027
* * * * *
References