U.S. patent application number 16/728557 was filed with the patent office on 2020-07-09 for treatment of cancer using a cll-1 chimeric antigen receptor.
The applicant listed for this patent is Novartis AG The Trustees of the University of Pennsylvania. Invention is credited to Jennifer Brogdon, Hilmar Erhard Ebersbach, Saar Gill, David Jonathan Glass, Julia Jascur, Saad Kenderian, Joan Mannick, Michael C. Milone, Leon Murphy, Celeste Richardson, Reshma Singh, Lai Wei, Qilong Wu, Qiumei Yang, Jiquan Zhang.
Application Number | 20200215171 16/728557 |
Document ID | / |
Family ID | 53761606 |
Filed Date | 2020-07-09 |
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United States Patent
Application |
20200215171 |
Kind Code |
A1 |
Brogdon; Jennifer ; et
al. |
July 9, 2020 |
TREATMENT OF CANCER USING A CLL-1 CHIMERIC ANTIGEN RECEPTOR
Abstract
The invention provides compositions and methods for treating
diseases associated with expression of CLL-1. The invention also
relates to chimeric antigen receptor (CAR) specific to CLL-1,
vectors encoding the same, and recombinant cells comprising the
CLL-1 CAR. The invention also includes methods of administering a
genetically modified cell expressing a CAR that comprises a CLL-1
binding domain.
Inventors: |
Brogdon; Jennifer; (Sudbury,
MA) ; Ebersbach; Hilmar Erhard; (Basel, CH) ;
Gill; Saar; (Philadelphia, PA) ; Glass; David
Jonathan; (Cambridge, MA) ; Jascur; Julia;
(Basel, CH) ; Kenderian; Saad; (Philadelphia,
PA) ; Mannick; Joan; (Cambridge, MA) ; Milone;
Michael C.; (Cherry Hill, NJ) ; Murphy; Leon;
(Cambridge, MA) ; Richardson; Celeste; (Cambridge,
MA) ; Singh; Reshma; (Cambridge, MA) ; Wei;
Lai; (Shanghai, CN) ; Wu; Qilong; (Shanghai,
CN) ; Yang; Qiumei; (Shanghai, CN) ; Zhang;
Jiquan; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG
The Trustees of the University of Pennsylvania |
Basel
Philadelphia |
PA |
CH
US |
|
|
Family ID: |
53761606 |
Appl. No.: |
16/728557 |
Filed: |
December 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14805075 |
Jul 21, 2015 |
10568947 |
|
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16728557 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7068 20130101;
A61P 37/04 20180101; C07K 16/2851 20130101; A61K 39/001102
20180801; A61P 43/00 20180101; C07K 14/7051 20130101; A61K
2039/5156 20130101; C07K 2319/02 20130101; A61K 35/17 20130101;
C07K 14/70578 20130101; A61K 39/0011 20130101; A61K 45/06 20130101;
C07K 2317/622 20130101; A61P 19/08 20180101; A61P 35/02 20180101;
A61P 35/00 20180101; C07K 2319/03 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/725 20060101 C07K014/725; C07K 16/28 20060101
C07K016/28; A61K 35/17 20060101 A61K035/17; A61K 31/7068 20060101
A61K031/7068; A61K 45/06 20060101 A61K045/06; C07K 14/705 20060101
C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 2014 |
CN |
PCT/CN2014/082602 |
Nov 6, 2014 |
CN |
PCT/CN2014/090500 |
Claims
1-50. (canceled)
51. A method of treating a mammal having a disease associated with
expression of CLL-1, comprising administering to the mammal an
effective amount of a cell, comprising an isolated nucleic acid
molecule encoding a chimeric antigen receptor (CAR), wherein the
CAR comprises an anti-CLL-1 binding domain, a transmembrane domain,
and an intracellular signaling domain, and wherein said anti-CLL-1
binding domain comprises 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 any anti-CLL-1 heavy chain binding domain amino acid
sequences listed in Table 2, 5, 7, or 3.
52. The method of claim 51, wherein the disease associated with
CLL-1 expression is: (i) a cancer or malignancy, or a precancerous
condition chosen from one or more of a myelodysplasia, a
myelodysplastic syndrome or a preleukemia, or (ii) a non-cancer
related indication associated with expression of CLL-1.
53. The method of claim 51, wherein the disease is a hematologic
cancer.
54. The method of claim 51, wherein the disease is an acute
leukemia chosen from one or more of acute myeloid leukemia (AML);
acute lymphoblastic B-cell leukemia (B-cell acute lymphoid
leukemia, BALL), acute lymphoblastic T-cell leukemia (T-cell acute
lymphoid leukemia (TALL), B-cell prolymphocytic leukemia, chronic
lymphocytic leukemia, chronic myeloid leukemia (CML),
myelodysplastic syndrome, plasma cell myeloma, or a combination
thereof.
55. The method of claim 51, wherein the cell is administered in
combination with one or more of: (i) an agent that increases the
efficacy of the cell comprising the CAR nucleic acid or CAR
polypeptide; (ii) an agent that ameliorates one or more side
effects associated with administration of the cell comprising the
CAR nucleic acid or CAR polypeptide; or (iii) an agent that treats
the disease associated with the expression of CLL-1.
56-57. (canceled)
58. The method of claim 51, wherein the cell comprising an isolated
nucleic acid molecule encoding the CAR further expresses an
inhibitory molecule that comprises a first polypeptide that
comprises at least a portion of an inhibitory molecule, associated
with a second polypeptide that comprises a positive signal from an
intracellular signaling domain, wherein the inhibitory molecule
comprises first polypeptide that comprises at least a portion of
PD1 and a second polypeptide comprising a costimulatory domain and
primary signaling domain.
59. (canceled)
60. The method of claim 55, wherein the agent is an mTOR inhibitor
and the subject is administered a low, immune enhancing, dose of an
mTOR inhibitor.
61-64. (canceled)
65. The method of claim 51, wherein a chemotherapeutic agent is
administered prior to administration of the cell, and optionally,
wherein the chemotherapeutic agent increases CLL-1 expression on
the cancer cell or wherein the chemotherapeutic agent is
cytarabine.
66. The method of claim 51, wherein said anti-CLL-1 binding domain
further comprises a light chain complementary determining region 1
(LC CDR1), a light chain complementary determining region 2 (LC
CDR2), and a light chain complementary determining region 3 (LC
CDR3) of any anti-CLL-1 light chain binding domain amino acid
sequences listed in Table 2, 6, 8, or 4.
67. The method of claim 51, wherein the isolated nucleic acid
molecule which encodes a CAR comprising: (a) (i) the amino acid
sequence of any light chain variable region listed in Table 2; (ii)
an amino acid sequence having at least one, two, or three
modifications but not more than 30, 20, or 10 modifications of the
amino acid sequence of any of the light chain variable regions
provided in Table 2; or (iii) an amino acid sequence with 95-99%
identity to the amino acid sequence of any of the light chain
variable regions provided in Table 2; and/or (b) (i) the amino acid
sequence of any heavy chain variable region listed in Table 2; (ii)
an amino acid sequence having at least one, two or three
modifications but not more than 30, 20 or 10 modifications of the
amino acid sequence of any of the heavy chain variable regions
provided in Table 2: or (iii) an amino acid sequence with 95-99%
identity to the amino acid sequence of any of the heavy chain
variable regions provided in Table 2.
68. The method of claim 51, wherein the encoded anti-CLL-1 binding
domain comprises: (a) an amino acid sequence: (i) selected from a
group consisting of SEQ ID NO:47, 44, 48, 49, 50, 39, 40, 41, 42,
43, 45, 46, 51, 73, 70, 74, 75, 76, 65, 66, 67, 68, 69, 71, 72, 77,
195, 86, 83, 87, 88, 89, 78, 79, 80, 81, 82, 84, 85, 90, or 196;
(ii) having at least one, two or three modifications but not more
than 30, 20 or 10 modifications to any of SEQ ID NO: 47, 44, 48,
49, 50, 39, 40, 41, 42, 43, 45, 46, 51, 73, 70, 74, 75, 76, 65, 66,
67, 68, 69, 71, 72, 77, 195, 86, 83, 87, 88, 89, 78, 79, 80, 81,
82, 84, 85, 90, or 196; or (iii) with 95-99% identity to any of SEQ
ID NO: 47, 44, 48, 49, 50, 39, 40, 41, 42, 43, 45, 46, 51, 73, 70,
74, 75, 76, 65, 66, 67, 68, 69, 71, 72, 77, 195, 86, 83, 87, 88,
89, 78, 79, 80, 81, 82, 84, 85, 90, or 196; or (b) a nucleotide
sequence selected from a group consisting of SEQ ID NO: 60, 44, 61,
62, or 63, or a sequence with 95-99% identity thereof.
69. The method of claim 51, wherein the encoded CAR includes a
transmembrane domain that comprises: (i) 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; (ii) the encoded transmembrane domain comprises
the amino acid sequence of SEQ ID NO: 6, an amino acid sequence
comprises at least one, two, or three modifications but not more
than 20, 10, or 5 modifications of the amino acid sequence of SEQ
ID NO:6, or a sequence with 95-99% identity to the amino acid
sequence of SEQ ID NO:6; and/or (iii) the nucleic acid sequence
encoding the transmembrane domain comprises a sequence of SEQ ID
NO:17, or a sequence with 95-99% identity thereof.
70. The method of claim 51, wherein the encoded CLL-1 binding
domain is connected to the transmembrane domain by a hinge region,
wherein: (i) the encoded hinge region comprises the amino acid
sequence of SEQ ID NO:2, or a sequence with 95-99% identity
thereof; or (ii) the nucleic acid sequence encoding the hinge
region comprises the nucleotide sequence of SEQ ID NO: 13, or a
sequence with 95-99% identity thereof.
71. The method of claim 51, wherein the CAR further comprises a
costimulatory domain, wherein the costimulatory domain: (i) is a
functional signaling domain obtained from a protein selected from
the group consisting of a MHC class I molecule, TNF receptor
proteins, Immunoglobulin-like proteins, cytokine receptors,
integrins, signaling lymphocytic activation molecules (SLAM
proteins), activating NK cell receptors, BTLA, a Toll ligand
receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,
LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83; (ii) comprises the amino acid
sequence of SEQ ID NO:7, or an amino acid sequence having at least
one, two, or three modifications but not more than 20, 10, or 5
modifications of the amino acid sequence of SEQ ID NO:7, or a
sequence with 95-99% identity to the amino acid sequence of SEQ ID
NO:7; and/or (iii) is encoded by a nucleic acid sequence comprising
the nucleotide sequence of SEQ ID NO:18, or a sequence with 95-99%
identity thereof.
72. The method of claim 51, wherein the encoded intracellular
signaling domain: (i) comprises a functional signaling domain of
4-1BB and/or a functional signaling domain of CD3 zeta; (ii)
comprises the amino acid sequence of SEQ ID NO: 7 and/or the
sequence of SEQ ID NO:9 or SEQ ID NO:10; or an amino acid sequence
having at least one, two, or three modifications but not more than
20, 10, or 5 modifications of the amino acid sequence of SEQ ID
NO:7 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10;
or a sequence with 95-99% identity to the amino acid sequence of
SEQ ID NO:7 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID
NO:10; (iii) comprises the sequence of SEQ ID NO:7 and the sequence
of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising
the intracellular signaling domain are expressed in the same frame
and as a single polypeptide chain; and/or (iv) comprises the
sequence of SEQ ID NO:18, or a sequence with 95-99% identity
thereof, and/or the sequence of SEQ ID NO:20 or SEQ ID NO:21, or a
sequence with 95-99% identity thereof.
73. The method of claim 51, further comprising a leader sequence
which encodes the amino acid sequence of SEQ ID NO:1.
74. The method of claim 51, wherein the CAR comprises: (a) an amino
acid sequence: (i) of any of SEQ ID NOs:99, 96, 100, 101, 102, 91,
92, 93, 94, 95, 97, 98, 103, or 197; (ii) having at least one, two
or three modifications but not more than 30, 20 or 10 modifications
to any of SEQ ID NOs: 99, 96, 100, 101, 102, 91, 92, 93, 94, 95,
97, 98, 103, or 197; or (iii) with 95-99% identity to any of SEQ ID
NOs: 99, 96, 100, 101, 102, 91, 92, 93, 94, 95, 97, 98, 103, or
197; or (b) the nucleotide sequence of any of SEQ ID NOs: 112, 109,
113, 114, 115, 104, 105, 106, 107, 108, 110, 111, 116, or 198, or a
nucleotide sequence with 95-99% identity to any of SEQ ID NOs: 112,
109, 113, 114, 115, 104, 105, 106, 107, 108, 110, 111, 116, or 198.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 14/805,075, filed Jul. 21, 2015, which claims priority to PCT
Application No. PCT/CN2014/082602, filed Jul. 21, 2014, and PCT
Application No. PCT/CN2014/090500, filed Nov. 6, 2014. The entire
contents of each of these applications are incorporated herein by
reference.
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 Oct. 7, 2015, is named N2067-7044WO3_SL.txt and is 347,719 bytes
in size.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the use of immune
effector cells (e.g., T cells, NK cells) engineered to express a
Chimeric Antigen Receptor (CAR) to treat a disease associated with
expression of C-type lectin-like-1 (CLL-1).
BACKGROUND OF THE INVENTION
[0004] C-type lectin-like-1 (CLL-1) is also known as MICL, CLEC12A,
CLEC-1, Dendritic Cell-Associated Lectin 1, and DCAL-2. CLL-1 is a
glycoprotein receptor and member of the large family of C-type
lectin-like receptors involved in immune regulation. CLL-1 is
expressed in hematopoietic cells, primarily on innate immune cells
including monocytes, DCs, pDCs, and granulocytes (Cancer Res. 2004;
J Immunol 2009) and myeloid progenitor cells (Blood, 2007). CLL-1
is also found on acute myeloid leukemia (AML) blasts and leukemic
stem cells (e.g., CD34.sup.+/CD38.sup.-) (Zhao et al.,
Haematologica. 2010, 95(1):71-78.). CLL-1 expression may also be
relevant for other myeloid leukemias, such as acute myelomonocytic
leukemia, acute monocytic leukemia, acute promyelocytic leukemia,
chronic myeloid leukemia (CML), and myelodysplastic syndrome
(MDS).
SUMMARY OF THE INVENTION
[0005] In a first aspect, the invention features an isolated
nucleic acid molecule encoding a chimeric antigen receptor (CAR),
wherein the CAR comprises an antibody or antibody fragment which
includes a human anti-CLL-1 binding domain, 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 a human anti-CLL-1 binding
domain 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).
[0006] In embodiments, the CAR comprises a human anti-CLL-1 binding
domain, a transmembrane domain, and an intracellular signaling
domain, and wherein said anti-CLL-1 binding domain comprises 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 any CLL-1
heavy chain binding domain amino acid sequences listed in Table 8.
In embodiments, the human CLL-1 binding domain further comprises a
light chain complementary determining region 1 (LC CDR1), a light
chain complementary determining region 2 (LC CDR2), and a light
chain complementary determining region 3 (LC CDR3). In embodiments,
the human CLL-1 binding domain comprises a light chain
complementary determining region 1 (LC CDR1), a light chain
complementary determining region 2 (LC CDR2), and a light chain
complementary determining region 3 (LC CDR3) of any CLL-1 light
chain binding domain amino acid sequences listed in Table 8.
[0007] In some embodiments, the CAR comprises an antibody or
antibody fragment which includes a human CLL-1 binding domain, a
transmembrane domain, and an intracellular signaling domain
comprising a costimulatory domain and/or a primary signaling
domain, and wherein said CLL-1 binding domain comprises one or more
of 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 any CLL-1light
chain binding domain amino acid sequences listed in Table 8, and
one or more of 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 any
CLL-1heavy chain binding domain amino acid sequences listed in
Table 8.
[0008] In one embodiment, the encoded human anti-CLL-1 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 human anti-CLL-1
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 human
anti-CLL-1 binding domain described herein, e.g., a human
anti-CLL-1 binding domain comprising one or more, e.g., all three,
LC CDRs and one or more, e.g., all three, HC CDRs.
[0009] In one embodiment, the encoded human anti-CLL-1 binding
domain comprises a light chain variable region described herein
(e.g., in Table 8) and/or a heavy chain variable region described
herein (e.g., in Table 8). In one embodiment, the encoded human
anti-CLL-1 binding domain is a scFv comprising a light chain and a
heavy chain of an amino acid sequence of Table 8. In an embodiment,
the human anti-CLL-1 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, e.g., conservative substitutions) but not more than
30, 20 or 10 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of a light chain variable
region provided in Table 8, or a sequence with 95-99% identity with
an amino acid sequence of Table 8; and/or a heavy chain variable
region comprising an amino acid sequence having at least one, two
or three modifications (e.g., substitutions, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of a heavy chain variable region provided in Table 8, or a
sequence with 95-99% identity to an amino acid sequence of Table
8.
[0010] In other embodiments, the encoded CLL-1 binding domain
comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any CLL-1 heavy
chain binding domain amino acid sequences listed in Table 8. In
embodiments, the CLL-1 binding domain further comprises a LC CDR1,
a LC CDR2, and a LC CDR3. In embodiments, the CLL-1 binding domain
comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any CLL-1 light
chain binding domain amino acid sequences listed in Table 8.
[0011] In some embodiments, the encoded CLL-1 binding domain
comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any
CLL-1 light chain binding domain amino acid sequences listed in
Table 8, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of
any CLL-1 heavy chain binding domain amino acid sequences listed in
Table 8.
[0012] In one embodiment, the encoded human anti-CLL-1 binding
domain comprises an amino acid sequence selected from a group
consisting of SEQ ID NO:39-51, 65-77, 195, 78-90, or 196. In an
embodiment, the encoded CLL-1 binding domain (e.g., an scFv)
comprises an amino acid sequence having at least one, two or three
modifications (e.g., substitutions, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of SEQ ID NO: 39-51, 65-77, 195, 78-90, or 196, or a
sequence with 95-99% identity with an amino acid sequence of SEQ ID
NO: 39-51, 65-77, 195, 78-90, or 196. In another embodiment, the
encoded CLL-1 binding domain comprises a heavy chain variable
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 65-77, or 195, or a sequence with 95-99%
identity thereof. In another embodiment, the encoded CLL-1 binding
domain comprises a light chain variable region comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:
66-74, or 196, or a sequence with 95-99% identity thereof. In one
embodiment, the nucleic acid molecule comprises a nucleotide
sequence selected from the group consisting of SEQ ID NO: 52-64, or
a sequence with 95-99% identity thereof.
[0013] In one embodiment, the encoded humanized anti-CLL-1 binding
domain includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5,
or 6, preferably 3 or 4 (SEQ ID NO:26). 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.
[0014] In one embodiment, the encoded CAR includes a transmembrane
domain that 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 encoded transmembrane domain comprises the sequence
of SEQ ID NO: 6. In one embodiment, the encoded transmembrane
domain comprises an amino acid sequence comprising at least one,
two or three modifications but not more than 20, 10 or 5
modifications of the amino acid sequence of SEQ ID NO:6, or a
sequence with 95-99% identity to an amino acid sequence of SEQ ID
NO:6. In one embodiment, the nucleic acid sequence encoding the
transmembrane domain comprises the sequence of SEQ ID NO: 17, or a
sequence with 95-99% identity thereof.
[0015] In one embodiment, the encoded anti-CLL-1 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:2, or a sequence with 95-99% identity
thereof. In one embodiment, the nucleic acid sequence encoding the
hinge region comprises the sequence of SEQ ID NO: 13, or a sequence
with 95-99% identity thereof.
[0016] In one embodiment, the isolated nucleic acid molecule
further comprises a sequence encoding a costimulatory domain, e.g.,
a costimulatory domain described herein. In embodiments, the
intracellular signaling domain comprises a costimulatory domain. In
embodiments, the intracellular signaling domain comprises a primary
signaling domain. In embodiments, the intracellular signaling
domain comprises a costimulatory domain and a primary signaling
domain.
[0017] In one embodiment, the encoded costimulatory domain is a
functional signaling domain obtained from a protein selected from
the group consisting of a MHC class I molecule, TNF receptor
proteins, Immunoglobulin-like proteins, cytokine receptors,
integrins, signaling lymphocytic activation molecules (SLAM
proteins), activating NK cell receptors, BTLA, a Toll ligand
receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,
LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83. In embodiments, the encoded
costimulatory domain comprises 4-1BB, CD27, CD28, or ICOS.
[0018] In one embodiment, the encoded costimulatory domain of 4-1BB
comprises the amino acid sequence of SEQ ID NO:7. In one
embodiment, the encoded costimulatory domain comprises an amino
acid sequence having at least one, two or three modifications but
not more than 20, 10 or 5 modifications of the amino acid sequence
of SEQ ID NO:7, or a sequence with 95-99% identity to the amino
acid sequence of SEQ ID NO:7. In one embodiment, the nucleic acid
sequence encoding the costimulatory domain comprises the nucleotide
sequence of SEQ ID NO:18, or a sequence with 95-99% identity
thereof. In another embodiment, the encoded costimulatory domain of
CD28 comprises the amino acid sequence of SEQ ID NO:482. In one
embodiment, the encoded costimulatory domain comprises an amino
acid sequence having at least one, two or three modifications but
not more than 20, 10 or 5 modifications of an amino acid sequence
of SEQ ID NO:482, or a sequence with 95-99% identity to an amino
acid sequence of SEQ ID NO:482. In one embodiment, the nucleic acid
sequence encoding the costimulatory domain of CD28 comprises the
nucleotide sequence of SEQ ID NO:483, or a sequence with 95-99%
identity thereof. In another embodiment, the encoded costimulatory
domain of CD27 comprises the amino acid sequence of SEQ ID NO:8. In
one embodiment, the encoded costimulatory domain comprises an amino
acid sequence having at least one, two or three modifications but
not more than 20, 10 or 5 modifications of an amino acid sequence
of SEQ ID NO:8, or a sequence with 95-99% identity to an amino acid
sequence of SEQ ID NO:8. In one embodiment, the nucleic acid
sequence encoding the costimulatory domain of CD27 comprises the
nucleotide sequence of SEQ ID NO:19, or a sequence with 95-99%
identity thereof.
[0019] In another embodiment, the encoded costimulatory domain of
ICOS comprises the amino acid sequence of SEQ ID NO:484. In one
embodiment, the encoded costimulatory domain comprises an amino
acid sequence having at least one, two or three modifications but
not more than 20, 10 or 5 modifications of an amino acid sequence
of SEQ ID NO:484, or a sequence with 95-99% identity to an amino
acid sequence of SEQ ID NO:484. In one embodiment, the nucleic acid
sequence encoding the costimulatory domain of ICOS comprises the
nucleotide sequence of SEQ ID NO:485, or a sequence with 95-99%
identity thereof.
[0020] In embodiments, the encoded primary signaling domain
comprises a functional signaling domain of CD3 zeta. In
embodiments, the functional signaling domain of CD3 zeta comprises
the sequence of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10
(wild-type human CD3 zeta), or a sequence with 95-99% identity
thereof.
[0021] In one embodiment, the encoded intracellular signaling
domain comprises a functional signaling domain of 4-1BB and/or a
functional signaling domain of CD3 zeta. In one embodiment, the
encoded intracellular signaling domain of 4-1BB comprises the amino
acid sequence of SEQ ID NO: 7 and/or the CD3 zeta amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:7
and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a
sequence with 95-99% identity to an amino acid sequence of SEQ ID
NO:7 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
In one embodiment, the encoded intracellular signaling domain
comprises the sequence of SEQ ID NO:7 and the sequence of SEQ ID
NO:9 or SEQ ID NO:10, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain. In one embodiment, the nucleic acid
sequence encoding the intracellular signaling domain of 4-1BB
comprises the nucleotide sequence of SEQ ID NO:18, or a sequence
with 95-99% identity thereof, and/or a sequence of SEQ ID NO:20 or
SEQ ID NO:21, or the CD3 zeta nucleotide sequence with 95-99%
identity thereof.
[0022] In one embodiment, the encoded intracellular signaling
domain comprises a functional signaling domain of CD27 and/or a
functional signaling domain of CD3 zeta. In one embodiment, the
encoded intracellular signaling domain of CD27 comprises the amino
acid sequence of SEQ ID NO: 8 and/or the CD3 zeta amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:8
and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a
sequence with 95-99% identity to an amino acid sequence of SEQ ID
NO:8 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
In one embodiment, the encoded intracellular signaling domain
comprises the sequence of SEQ ID NO:8 and the sequence of SEQ ID
NO:9 or SEQ ID NO:10, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain. In one embodiment, the nucleic acid
sequence encoding the intracellular signaling domain of CD27
comprises the nucleotide sequence of SEQ ID NO:19, or a sequence
with 95-99% identity thereof, and/or the CD3 zeta nucleotide
sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence with 95-99%
identity thereof.
[0023] In one embodiment, the encoded intracellular signaling
domain comprises a functional signaling domain of CD28 and/or a
functional signaling domain of CD3 zeta. In one embodiment, the
encoded intracellular signaling domain of CD28 comprises the amino
acid sequence of SEQ ID NO: 482 and/or the CD3 zeta amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID
NO:482 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID
NO:10, or a sequence with 95-99% identity to an amino acid sequence
of SEQ ID NO:482 and/or an amino acid sequence of SEQ ID NO:9 or
SEQ ID NO:10. In one embodiment, the encoded intracellular
signaling domain comprises the sequence of SEQ ID NO:482 and the
sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences
comprising the intracellular signaling domain are expressed in the
same frame and as a single polypeptide chain. In one embodiment,
the nucleic acid sequence encoding the intracellular signaling
domain of CD28 comprises the nucleotide sequence of SEQ ID NO:483,
or a sequence with 95-99% identity thereof, and/or the CD3 zeta
nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence
with 95-99% identity thereof.
[0024] In one embodiment, the encoded intracellular signaling
domain comprises a functional signaling domain of ICOS and/or a
functional signaling domain of CD3 zeta. In one embodiment, the
encoded intracellular signaling domain of ICOS comprises the amino
acid sequence of SEQ ID NO: 484 and/or the CD3 zeta amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID
NO:484 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID
NO:10, or a sequence with 95-99% identity to an amino acid sequence
of SEQ ID NO:484 and/or an amino acid sequence of SEQ ID NO:9 or
SEQ ID NO:10. In one embodiment, the encoded intracellular
signaling domain comprises the sequence of SEQ ID NO:484 and the
sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences
comprising the intracellular signaling domain are expressed in the
same frame and as a single polypeptide chain. In one embodiment,
the nucleic acid sequence encoding the intracellular signaling
domain of ICOS comprises the nucleotide sequence of SEQ ID NO:485,
or a sequence with 95-99% identity thereof, and/or the CD3 zeta
nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence
with 95-99% identity thereof.
[0025] In one embodiment, the isolated 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: 1, or a sequence with 95-99% identity
to an amino acid sequence of SEQ ID NO:1.
[0026] In another aspect, the invention pertains to an isolated
nucleic acid molecule encoding a CAR construct comprising a leader
sequence, e.g., a leader sequence described herein, e.g., the amino
acid sequence of SEQ ID NO: 1, an anti-CLL-1 binding domain
described herein, e.g., human anti-CLL-1 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 human anti-CLL-1 binding domain described
in Table 8, or a sequence with 95-99% identify thereof, a hinge
region described herein, e.g., the amino acid sequence of SEQ ID
NO:2, a transmembrane domain described herein, e.g., having a
sequence of SEQ ID NO: 6, and an intracellular signaling domain,
e.g., an intracellular signaling domain described herein. In one
embodiment, the encoded intracellular signaling domain comprises a
costimulatory domain, e.g., a costimulatory domain described herein
(e.g., a 4-1BB costimulatory domain having the amino acid sequence
of SEQ ID NO:7, a CD28 costimulatory domain having the amino acid
sequence of SEQ ID NO: 482, or an ICOS costimulatory domain having
the amino acid sequence of SEQ ID NO: 484, or a CD27 costimulatory
domain having the amino acid sequence of SEQ ID NO:8), 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:9 or SEQ ID NO:10).
[0027] In one embodiment, the isolated nucleic acid molecule
encoding the CAR construct includes a human anti-CLL-1 binding
domain sequence encoded by the nucleic acid sequence of SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ
ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,
SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64, or a sequence with
95-99% identity thereto.
[0028] In one embodiment, the isolated nucleic acid molecule
comprises (e.g., consists of) a nucleic acid encoding a CAR amino
acid sequence of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ
ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:
98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,
SEQ ID NO:103, or SEQ ID NO: 197; or an amino acid sequence having
one, two or three modifications (e.g., substitutions, e.g.,
conservative substitutions) but not more than 30, 20, or 10
modifications of an amino acid sequence of SEQ ID NO: 91, SEQ ID
NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,
SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID
NO: 101, SEQ ID NO: 102, SEQ ID NO:103, or SEQ ID NO: 197; or an
amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99%
identity to an amino acid sequence of SEQ ID NO: 91, SEQ ID NO: 92,
SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:
101, SEQ ID NO: 102, SEQ ID NO:103 or SEQ ID NO:197.
[0029] In one embodiment, the isolated nucleic acid molecule
comprises (e.g., consists of) a nucleic acid sequence of SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,
SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, or SEQ ID
NO:198; or a nucleic acid sequence having 85%, 90%, 95%, 96%, 97%,
98% or 99% identity to a nucleic acid sequence of SEQ ID NO:104,
SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113,
SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, or SEQ ID NO:198.
[0030] In one aspect, the invention pertains to an isolated nucleic
acid molecule encoding an anti-CLL-1 binding domain, wherein the
anti-CLL-1 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 an anti-CLL-1
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 an anti-CLL-1
binding domain described herein, e.g., a human anti-CLL-1 binding
domain comprising one or more, e.g., all three, LC CDRs and one or
more, e.g., all three, HC CDRs.
[0031] In other embodiments, the CLL-1 binding domain comprises a
HC CDR1, a HC CDR2, and a HC CDR3 of any CLL-1 heavy chain binding
domain amino acid sequences listed in Table 8. In embodiments, the
CLL-1 binding domain further comprises a LC CDR1, a LC CDR2, and a
LC CDR3. In embodiments, the CLL-1 binding domain comprises a LC
CDR1, a LC CDR2, and a LC CDR3)\ of any CLL-1 light chain binding
domain amino acid sequences listed in Table 8.
[0032] In some embodiments, the CLL-1 binding domain comprises one,
two or all of LC CDR1, LC CDR2, and LC CDR3 of any CLL-1 light
chain binding domain amino acid sequences listed in Table 8, and
one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any CLL-1 heavy
chain binding domain amino acid sequences listed in Table 8.
[0033] In one embodiment, the encoded anti-CLL-1 binding domain
comprises a light chain variable region described herein (e.g., in
SEQ ID NO:78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or
196) and/or a heavy chain variable region described herein (e.g.,
in SEQ ID NO:65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or
195). In one embodiment, the encoded anti-CLL-1 binding domain is a
scFv comprising a light chain and a heavy chain of an amino acid
sequence of in SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, or 51. In an embodiment, the anti-CLL-1 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, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of a light chain variable region provided in SEQ ID NO:
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 196, or a
sequence with 95-99% identity with an amino acid sequence of SEQ ID
NO: 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 196;
and/or a heavy chain variable region comprising an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions, e.g., conservative substitutions) but not more than
30, 20 or 10 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of a heavy chain variable
region provided in SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, or 195, or a sequence with 95-99% identity to an
amino acid sequence in SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, or 195. In one embodiment, the anti-CLL-1
binding domain comprises a sequence selected from a group
consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID
NO:51, or a sequence with 95-99% identify thereof. In one
embodiment, the encoded anti-CLL-1 binding domain is a scFv, and a
light chain variable region comprising an amino acid sequence
described herein, e.g., in Table 8, is attached to a heavy chain
variable region comprising an amino acid sequence described herein,
e.g., in Table 8, via a linker, e.g., a linker described herein. In
one embodiment, the encoded anti-CLL-1 binding domain includes a
(Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4
(SEQ ID NO: 26). 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. In one embodiment, the isolated nucleic acid
sequence encoding the human anti-CLL-1 binding domain comprises a
sequence selected from a group consisting of SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ
ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:63, and SEQ ID NO:64, or a sequence with 95-99% identity
thereof.
[0034] In another aspect, the invention pertains to an isolated CAR
(e.g., a polypeptide) molecule encoded by the nucleic acid
molecule. In one embodiment, the isolated CAR molecule comprises a
sequence selected from the group consisting of SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:197 or a sequence with
95-99% identify thereof.
[0035] In another aspect, the invention pertains to an isolated
chimeric antigen receptor (CAR) molecule (e.g., polypeptide)
comprising an anti-CLL-1 binding domain (e.g., a human antibody or
antibody fragment that specifically binds to CLL-1), 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-CLL-1 binding domain described herein (e.g., a human antibody
or antibody fragment that specifically binds to CLL-1 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).
[0036] In one embodiment, the anti-CLL-1 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 an anti-CLL-1 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 an anti-CLL-1 binding domain described herein, e.g., a
human anti-CLL-1 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-CLL-1 binding domain comprises a light chain
variable region described herein (e.g., in Table 8) and/or a heavy
chain variable region described herein (e.g., in Table 8). In one
embodiment, the anti-CLL-1 binding domain is a scFv comprising a
light chain and a heavy chain of an amino acid sequence listed in
Table 8. In an embodiment, the anti-CLL-1 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, e.g., conservative substitutions) but not
more than 30, 20 or 10 modifications (e.g., substitutions, e.g.,
conservative substitutions) of an amino acid sequence of a light
chain variable region provided in Table 8, or a sequence with
95-99% identity with an amino acid sequence provided in Table 8;
and/or a heavy chain variable region comprising an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions, e.g., conservative substitutions) but not more than
30, 20 or 10 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of a heavy chain variable
region provided in Table 8, or a sequence with 95-99% identity to
an amino acid sequence provided in Table 8.
[0037] In other embodiments, the encoded CLL-1 binding domain
comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any CLL-1 heavy
chain binding domain amino acid sequences listed in Table 8. In
embodiments, the CLL-1 binding domain further comprises a LC CDR1,
a LC CDR2, and a LC CDR3. In embodiments, the CLL-1 binding domain
comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any CLL-1 light
chain binding domain amino acid sequences listed in Table 8.
[0038] In some embodiments, the encoded CLL-1 binding domain
comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any
CLL-1 light chain binding domain amino acid sequences listed in
Table 8, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of
any CLL-1 heavy chain binding domain amino acid sequences listed in
Table 8.
[0039] In one embodiment, the anti-CLL-1 binding domain comprises a
sequence selected from a group consisting of SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 65-90, or SEQ ID NO:
195-196, or an amino acid sequence having at least one, two or
three modifications (e.g., substitutions, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) to any of the
aforesaid sequences; or a sequence with 95-99% identify thereof. In
one embodiment, the anti-CLL-1 binding domain is a scFv, and a
light chain variable region comprising an amino acid sequence
described herein, e.g., in Table 8, is attached to a heavy chain
variable region comprising an amino acid sequence described herein,
e.g., in Table 8, via a linker, e.g., a linker described herein. In
one embodiment, the anti-CLL-1 binding domain includes a
(Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4
(SEQ ID NO: 26). 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.
[0040] In one embodiment, the isolated CAR molecule comprises a
transmembrane domain of a protein selected from the group
consisting of the 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: 6. In one embodiment, the transmembrane domain comprises an
amino acid sequence having at least one, two or three modifications
(e.g., substitutions, e.g., conservative substitutions) but not
more than 20, 10 or 5 modifications (e.g., substitutions, e.g.,
conservative substitutions) of an amino acid sequence of SEQ ID NO:
6, or a sequence with 95-99% identity to an amino acid sequence of
SEQ ID NO: 6.
[0041] In one embodiment, the anti-CLL-1 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:2, or a sequence with 95-99% identity
thereof.
[0042] In embodiments, the intracellular signaling domain of the
isolated CAR molecule comprises a costimulatory domain. In
embodiments, the intracellular signaling domain of the isolated CAR
molecule comprises a primary signaling domain. In embodiments, the
intracellular signaling domain of the isolated CAR molecule
comprises a costimulatory domain and a primary signaling domain. In
one embodiment, the isolated 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 a MHC class I molecule, TNF
receptor proteins, Immunoglobulin-like proteins, cytokine
receptors, integrins, signaling lymphocytic activation molecules
(SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand
receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,
LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83. In one embodiment, the costimulatory
domain of 4-1BB comprises the amino acid sequence of SEQ ID NO:7.
In one embodiment, the costimulatory domain comprises an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions, e.g., conservative substitutions) but not more than
20, 10 or 5 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of SEQ ID NO:7, or a
sequence with 95-99% identity to an amino acid sequence of SEQ ID
NO:7. In another embodiment, the costimulatory domain of CD28
comprises the amino acid sequence of SEQ ID NO:482. In one
embodiment, the costimulatory domain comprises an amino acid
sequence having at least one, two or three modifications but not
more than 20, 10 or 5 modifications of an amino acid sequence of
SEQ ID NO:482, or a sequence with 95-99% identity to an amino acid
sequence of SEQ ID NO:482. In another embodiment, the costimulatory
domain of CD27 comprises the amino acid sequence of SEQ ID NO:8. In
one embodiment, the costimulatory domain comprises an amino acid
sequence having at least one, two or three modifications but not
more than 20, 10 or 5 modifications of an amino acid sequence of
SEQ ID NO:8, or a sequence with 95-99% identity to an amino acid
sequence of SEQ ID NO:8. In another embodiment, the costimulatory
domain of ICOS comprises the amino acid sequence of SEQ ID NO:484.
In one embodiment, the costimulatory domain comprises an amino acid
sequence having at least one, two or three modifications but not
more than 20, 10 or 5 modifications of an amino acid sequence of
SEQ ID NO:484, or a sequence with 95-99% identity to an amino acid
sequence of SEQ ID NO:484.
[0043] In embodiments, the primary signaling domain comprises a
functional signaling domain of CD3 zeta. In embodiments, the
functional signaling domain of CD3 zeta comprises the amino acid
sequence of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild
type human CD3 zeta), or a sequence with 95-99% identity
thereof.
[0044] 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:
7 and/or the sequence of SEQ ID NO:9 or SEQ ID NO:10. In one
embodiment, the intracellular signaling domain comprises an amino
acid sequence having at least one, two or three modifications
(e.g., substitutions, e.g., conservative substitutions) but not
more than 20, 10 or 5 modifications (e.g., substitutions, eg.,
conservative substitutions) of an amino acid sequence of SEQ ID NO:
7 and/or the sequence of SEQ ID NO:9 or SEQ ID NO:10, or a sequence
with 95-99% identity to an amino acid sequence of SEQ ID NO: 7
and/or the sequence of SEQ ID NO:9 or SEQ ID NO:10. In one
embodiment, the intracellular signaling domain comprises the
sequence of SEQ ID NO: 7 and/or the sequence of SEQ ID NO:9 or SEQ
ID NO:10, wherein the sequences comprising the intracellular
signaling domain are expressed in the same frame and as a single
polypeptide chain.
[0045] 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 of CD27 comprises the amino acid sequence of SEQ
ID NO: 8 and/or the CD3 zeta amino acid sequence of SEQ ID NO:9 or
SEQ ID NO:10. In one embodiment, the intracellular signaling domain
comprises an amino acid sequence having at least one, two or three
modifications but not more than 20, 10 or 5 modifications of an
amino acid sequence of SEQ ID NO:8 and/or an amino acid sequence of
SEQ ID NO:9 or SEQ ID NO:10, or a sequence with 95-99% identity to
an amino acid sequence of SEQ ID NO:8 and/or an amino acid sequence
of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises the sequence of SEQ ID
NO:8 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the
sequences comprising the intracellular signaling domain are
expressed in the same frame and as a single polypeptide chain.
[0046] In one embodiment, the intracellular signaling domain
comprises a functional signaling domain of CD28 and/or a functional
signaling domain of CD3 zeta. In one embodiment, the encoded
intracellular signaling domain of CD28 comprises the amino acid
sequence of SEQ ID NO: 482 and/or the CD3 zeta amino acid sequence
of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:
482 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10,
or a sequence with 95-99% identity to an amino acid sequence of SEQ
ID NO: 482 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID
NO:10. In one embodiment, the intracellular signaling domain
comprises the sequence of SEQ ID NO: 482 and the sequence of SEQ ID
NO:9 or SEQ ID NO:10, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain.
[0047] In one embodiment, the intracellular signaling domain
comprises a functional signaling domain of ICOS and/or a functional
signaling domain of CD3 zeta. In one embodiment, the encoded
intracellular signaling domain of ICOS comprises the amino acid
sequence of SEQ ID NO: 484 and/or the CD3 zeta amino acid sequence
of SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the
intracellular signaling domain comprises an amino acid sequence
having at least one, two or three modifications but not more than
20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:
484 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10,
or a sequence with 95-99% identity to an amino acid sequence of SEQ
ID NO: 482 and/or an amino acid sequence of SEQ ID NO:9 or SEQ ID
NO:10. In one embodiment, the intracellular signaling domain
comprises the sequence of SEQ ID NO: 484 and the sequence of SEQ ID
NO:9 or SEQ ID NO:10, wherein the sequences comprising the
intracellular signaling domain are expressed in the same frame and
as a single polypeptide chain.
[0048] In one embodiment, the isolated 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: 1, or a sequence with 95-99% identity
to an amino acid sequence of SEQ ID NO:1.
[0049] In another aspect, the invention pertains to an isolated CAR
molecule comprising a leader sequence, e.g., a leader sequence
described herein, e.g., a leader sequence of SEQ ID NO: 1, or
having 95-99% identity thereof, an anti-CLL-1 binding domain
described herein, e.g., an anti-CLL-1 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., an anti-CLL-1 binding domain described in
Table 8, 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:2, 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: 6 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:7, 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:9 or SEQ ID
NO:10, or having 95-99% identity thereof. 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:7, 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:9 or SEQ ID NO:10.
[0050] In one embodiment, the isolated CAR molecule comprises
(e.g., consists of) an amino acid sequence of SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ
ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:197, or an amino acid
sequence having at least one, two, three, four, five, 10, 15, 20 or
30 modifications (e.g., substitutions, e.g., conservative
substitutions) but not more than 60, 50 or 40 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,
SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,
or SEQ ID NO:197, or an amino acid sequence having 85%, 90%, 95%,
96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID
NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ
ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,
SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:197.
[0051] In one aspect, the invention pertains to an anti-CLL-1
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-CLL-1
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 an anti-CLL-1
binding domain described herein, e.g., a human anti-CLL-1 binding
domain comprising one or more, e.g., all three, LC CDRs and one or
more, e.g., all three, HC CDRs.
[0052] In other embodiments, the encoded CLL-1 binding domain
comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any CLL-1 heavy
chain binding domain amino acid sequences listed in Table 8. In
embodiments, the CLL-1 binding domain further comprises a LC CDR1,
a LC CDR2, and a LC CDR3. In embodiments, the CLL-1 binding domain
comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any CLL-1 light
chain binding domain amino acid sequences listed in Table 8.
[0053] In some embodiments, the encoded CLL-1 binding domain
comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any
CLL-1 light chain binding domain amino acid sequences listed in
Table 8, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of
any CLL-1 heavy chain binding domain amino acid sequences listed in
Table 8.
[0054] In one embodiment, the anti-CLL-1 binding domain comprises a
light chain variable region described herein (e.g., in SEQ ID NO:
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 196) and/or
a heavy chain variable region described herein (e.g. in SEQ ID NO:
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 195). In one
embodiment, the anti-CLL-1 binding domain is a scFv comprising a
light chain and a heavy chain of an amino acid sequence of SEQ ID
NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51. In an
embodiment, the anti-CLL-1 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, e.g., conservative substitutions) but not more than
30, 20 or 10 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of a light chain variable
region provided, in SEQ ID NO: 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, or 196 or a sequence with 95-99% identity with an
amino acid sequence in SEQ ID NO: 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, or 196; and/or a heavy chain variable region
comprising an amino acid sequence having at least one, two or three
modifications (e.g., substitutions, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of a heavy chain variable region provided in SEQ ID NO:
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 195, or a
sequence with 95-99% identity to an amino acid sequence in SEQ ID
NO: 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 195. In
one embodiment, the anti-CLL-1 binding domain comprises a sequence
selected from a group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, or SEQ ID NO:51 or a sequence with 95-99% identify thereof.
In one embodiment, the anti-CLL-1 binding domain is a scFv, and a
light chain variable region comprising an amino acid sequence
described herein, e.g., in Table 2, is attached to a heavy chain
variable region comprising an amino acid sequence described herein,
e.g., in Table 8, via a linker, e.g., a linker described herein. In
one embodiment, the anti-CLL-1 binding domain includes a
(Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4
(SEQ ID NO: 26). 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.
[0055] In another aspect, the invention pertains to a vector
comprising a nucleic acid molecule described herein, e.g., a
nucleic acid molecule encoding a CAR described herein. 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.
[0056] 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: 11. In another
embodiment, the promoter is a PGK promoter, e.g., a truncated PGK
promoter as described herein.
[0057] 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 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:312). In one embodiment, the nucleic acid sequence
in the 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 vector further comprises promoter, e.g., a T2A
promoter.
[0058] In another aspect, the invention pertains to a cell
comprising a vector described herein. In one embodiment, the cell
is a cell described herein, e.g., an immune effector cell, e.g., a
human T cell, e.g., a human T cell described herein; or a human NK
cell, e.g., a human NK cell described herein. In one embodiment,
the human T cell is a CD8+ T cell.
[0059] In another embodiment, 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. Examples of inhibitory molecules include PD1, PD-L1,
PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, 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,
PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, and 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).
[0060] In another aspect, the invention pertains to a method of
making a cell comprising transducing a cell described herein, e.g.,
an immune effector cell described herein, e.g., a T cell or a NK
cell described herein, with a vector of comprising a nucleic acid
encoding a CAR, e.g., a CAR described herein.
[0061] The present invention also provides a method of generating a
population of RNA-engineered cells, e.g., cells described herein,
e.g., immune effector cells, e.g., T cells or NK cells, transiently
expressing exogenous RNA. The method comprises introducing an in
vitro transcribed RNA or synthetic RNA into a cell, where the RNA
comprises a nucleic acid encoding a CAR molecule described
herein.
[0062] In another aspect, the invention pertains to a method of
providing an anti-tumor immunity in a mammal comprising
administering to the mammal an effective amount of a cell
expressing a CAR molecule, e.g., a cell expressing a CAR molecule
described herein. In one embodiment, the cell is an autologous
immune effector cell, e.g., T cell. In one embodiment, the cell is
an allogeneic immune effector cell, e.g., T cell. In one
embodiment, the mammal is a human, e.g., a patient with a
hematologic cancer. In another aspect, the invention pertains to a
method of treating a mammal having a disease associated with
expression of CLL-1 (e.g., a proliferative disease, a precancerous
condition, and a noncancer related indication associated with the
expression of CLL-1) comprising administering to the mammal an
effective amount of the cells expressing a CAR molecule, e.g., a
CAR molecule described herein. In one embodiment, the mammal is a
human, e.g., a patient with a hematologic cancer.
[0063] In one embodiment, the disease is a disease described
herein. In one embodiment, the disease associated with CLL-1
expression is selected from a hematologic cancer such as acute
leukemias including but not limited to acute myeloid leukemia
(AML); myelodysplastic syndrome; myeloproliferative neoplasms;
chronic myeloid leukemia (CML); Blastic plasmacytoid dendritic cell
neoplasm; and to disease associated with CLL-1 expression
including, but not limited to atypical and/or non-classical
cancers, malignancies, precancerous conditions or proliferative
diseases expressing CLL-1; and combinations thereof. In one
embodiment, the disease associated with CLL-1 expression is a
hematologic cancer selected from the group consisting of one or
more acute leukemias including but not limited to acute myelogenous
leukemia (or acute myeloid leukemia, AML); chronic myelogenous
leukemia (or chronic myeloid leukemia, CML): acute lymphoid
leukemia (or acute lymphoblastic leukemia, ALL); chronic lymphoid
leukemia (or chronic lymphocytic leukemia, CLL) and myelodysplastic
syndrome, B-cell acute lymphoid leukemia ("BALL", or acute
lymphoblastic B-cell leukemia), T-cell acute lymphoid leukemia
("TALL", or acute lymphoblastic T-cell leukemia), 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 B cell prolymphocytic
leukemia, blastic plasmacytoid dendritic cell neoplasm, lymphomas
including but not limited to multiple myeloma; non-Hodgkin's
lymphoma; Burkitt's lymphoma; small cell-follicular lymphoma; and
large cell-follicular lymphomaBurkitt'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, Marginal zone lymphoma, multiple myeloma, myelodysplasia
and myelodysplastic syndrome, Hodgkin's lymphoma, plasmablastic
lymphoma, plasmacytoid dendritic cell neoplasm, plasma cell
myeloma, Waldenstrom macroglobulinemia, 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 CLL-1 expression including, but not
limited to atypical and/or non-classical cancers, malignancies,
precancerous conditions or proliferative diseases expressing CLL-1;
and combinations thereof.
[0064] In another aspect, the invention pertains to a method of
conditioning a subject prior to cell transplantation comprising
administering to the subject an effective amount of the cell of
comprising a CAR molecule disclosed herein. In one embodiment, the
cell transplantation is a stem cell transplantation. The stem cell
transplantation is a hematopoietic stem cell stransplantation or a
bone marrow transplantation. In one embodiment, the cell
transplantation is allogeneic or autologous.
[0065] In one embodiment, the conditioning a subject prior to cell
transplantation comprises reducing the number of CLL-1-expressing
cells in a subject. The CLL-1-expressing cells in the subject are
CLL-1-expressing normal cells or CLL-1-expressing cancer cells, and
in some cases, the condition in the subject will reduce both
CLL-1-expressing normal and cancer cells prior to a cell
transplantation.
[0066] In one embodiment, the cells expressing a CAR molecule,
e.g., a CAR molecule described herein, are administered in
combination with an agent that increases the efficacy of a cell
expressing a CAR molecule, e.g., an agent described herein.
[0067] 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.
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.
[0068] In an embodiment this approach can be used to optimize the
performance of CAR cells 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 of a CLL-1 CAR expressing cell is improved. In other
embodiments, cells, e.g., T cells, which have, or will be that
expresses 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.
[0069] 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 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.
[0070] In an embodiment, the invention provides an mTOR inhibitor
for use in the treatment of a subject, wherein said mTOR inhibitor
enhances an immune response of said subject, and wherein said
subject has received, is receiving or is about to receive an immune
effector cell that expresses a CLL-1 CAR as described herein.
[0071] In an embodiment, the cell, e.g., T 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.
[0072] In one embodiment, the cells expressing a CAR molecule,
e.g., a CAR molecule described herein, are 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.
[0073] In one embodiment, the cells expressing a CAR molecule,
e.g., a CAR molecule described herein, are administered in
combination with an agent that treats the disease associated with
CLL-1, e.g., an agent described herein. In another embodiment, the
cells expressing a CAR molecule, e.g., a CAR molecule described
herein, are administered in combination with a chemotherapeutic
agent, e.g., a chemotherapeutic agent described herein. In an
embodiment, the chemotherapeutic agent is administered prior to
administration of the cell expressing a CAR molecule, e.g., a CAR
molecule described herein. For example, in chemotherapeutic
regimens where more than one administration of the chemotherapeutic
agent is desired, the chemotherapeutic regimen is initiated or
completed prior to administration of a cell expressing a CAR
molecule, e.g., a CAR molecule described herein. In embodiments,
the chemotherapeutic agent is administered at least 5 days, 10
days, 15 days, 30 days prior to administration of the cell
expressing the CAR molecule. In embodiments, the chemotherapeutic
agent is a chemotherapeutic agent that increases CLL-1 expression
on the cancer cells, e.g., the tumor cells, e.g., as compared to
CLL-1 expression on normal or non-cancer cells. For example, the
chemotherapeutic agent is cytarabine (Ara-C). In embodiments, the
combination of chemotherapy and a cell expressing a CAR molecule
described herein is useful for treating a hematological cancer,
e.g., a leukemia, e.g., AML, or a minimal residual disease (MRD) of
a hematological cancer described herein.
[0074] In another aspect, the invention pertains to the isolated
nucleic acid molecule encoding a CAR of the invention, the isolated
polypeptide molecule of a CAR of the invention, the vector
comprising a CAR of the invention, and the cell comprising a CAR of
the invention for use as a medicament, e.g., as described herein.
In another aspect, the invention pertains to a the isolated nucleic
acid molecule encoding a CAR of the invention, the isolated
polypeptide molecule of a CAR of the invention, the vector
comprising a CAR of the invention, and the cell comprising a CAR of
the invention for use in the treatment of a disease expressing
CLL-1, e.g., a disease expressing CLL-1 as described herein.
[0075] Additional features and embodiments of the aforesaid
compositions and methods include one or more of the following:
[0076] In certain embodiments, the CLL-1 CAR molecule (e.g., a
CLL-1 CAR nucleic acid or a CLL-1 CAR polypeptide as described
herein), or the CLL-1 binding domain as described herein, includes
one, two or three CDRs from the heavy chain variable region (e.g.,
HC CDR1, HC CDR2 and/or HC CDR3), provided in Table 1; and/or one,
two or three CDRs from the light chain variable region (e.g., LC
CDR1, LC CDR2 and/or LC CDR3) of CLL-1 CAR-1, CLL-1 CAR-2, CLL-1
CAR-3, CLL-1 CAR-4, CLL-1 CAR-5, CLL-1 CAR-6, CLL-1 CAR-7, CLL-1
CAR-8, CLL-1 CAR-9, CLL-1 CAR-10, CLL-1 CAR-11, CLL-1 CAR-12, CLL-1
CAR-13, or 181268 provided in Table 2; or a sequence substantially
identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino
acid changes, e.g., substitutions (e.g., e.g., conservative
substitutions)) to any of the aforesaid sequences.
[0077] In certain embodiments, the CLL-1 CAR molecule (e.g., a
CLL-1 CAR nucleic acid or a CLL-1 CAR polypeptide as described
herein), or the CLL-1 binding domain as described herein, includes
one, two or three CDRs from the heavy chain variable region (e.g.,
HC CDR1, HC CDR2 and/or HC CDR3), provided in Table 3; and/or one,
two or three CDRs from the light chain variable region (e.g., LC
CDR1, LC CDR2 and/or LC CDR3) of CLL-1 CAR-1, CLL-1 CAR-2, CLL-1
CAR-3, CLL-1 CAR-4, CLL-1 CAR-5, CLL-1 CAR-6, CLL-1 CAR-7, CLL-1
CAR-8, CLL-1 CAR-9, CLL-1 CAR-10, CLL-1 CAR-11, CLL-1 CAR-12, CLL-1
CAR-13, or 181268 provided in Table 4; or a sequence substantially
identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino
acid changes, e.g., substitutions (e.g., e.g., conservative
substitutions)) to any of the aforesaid sequences.
[0078] In certain embodiments, the CLL-1 CAR molecule (e.g., a
CLL-1 CAR nucleic acid or a CLL-1 CAR polypeptide as described
herein), or the CLL-1 binding domain as described herein, includes
one, two or three CDRs from the heavy chain variable region (e.g.,
HC CDR1, HC CDR2 and/or HC CDR3), provided in Table 5; and/or one,
two or three CDRs from the light chain variable region (e.g., LC
CDR1, LC CDR2 and/or LC CDR3) of CLL-1 CAR-1, CLL-1 CAR-2, CLL-1
CAR-3, CLL-1 CAR-4, CLL-1 CAR-5, CLL-1 CAR-6, CLL-1 CAR-7, CLL-1
CAR-8, CLL-1 CAR-9, CLL-1 CAR-10, CLL-1 CAR-11, CLL-1 CAR-12, CLL-1
CAR-13, or 181268 provided in Table 6; or a sequence substantially
identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino
acid changes, e.g., substitutions (e.g., e.g., conservative
substitutions)) to any of the aforesaid sequences.
[0079] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0080] (i) a LC CDR1 of SEQ ID NO: 156, LC CDR2 of SEQ ID NO: 169
and LC CDR3 of SEQ ID NO: 182 of CLL-1 CAR-1;
[0081] (ii) a LC CDR1 of SEQ ID NO: 157, LC CDR2 of SEQ ID NO: 170
and LC CDR3 of SEQ ID NO: 183 of CLL-1 CAR-2;
[0082] (iii) a LC CDR1 of SEQ ID NO: 158, LC CDR2 of SEQ ID NO: 171
and LC CDR3 of SEQ ID NO: 184 of CLL-1 CAR-3;
[0083] (iv) a LC CDR1 of SEQ ID NO: 159, LC CDR2 of SEQ ID NO: 172
and LC CDR3 of SEQ ID NO: 185 of CLL-1 CAR-4;
[0084] (v) a LC CDR1 of SEQ ID NO: 160, LC CDR2 of SEQ ID NO: 173
and LC CDR3 of SEQ ID NO: 186 of CLL-1 CAR-5;
[0085] (vi) a LC CDR1 of SEQ ID NO: 161, LC CDR2 of SEQ ID NO: 174
and LC CDR3 of SEQ ID NO: 187 of CLL-1 CAR-6;
[0086] (vii) a LC CDR1 of SEQ ID NO: 162, LC CDR2 of SEQ ID NO: 175
and LC CDR3 of SEQ ID NO: 188 of CLL-1 CAR-7;
[0087] (viii) a LC CDR1 of SEQ ID NO: 163, LC CDR2 of SEQ ID NO:
176 and LC CDR3 of SEQ ID NO: 189 of CLL-1 CAR-8; or
[0088] (ix) a LC CDR1 of SEQ ID NO: 164, LC CDR2 of SEQ ID NO: 177
and LC CDR3 of SEQ ID NO: 190 of CLL-1 CAR-9;
[0089] (x) a LC CDR1 of SEQ ID NO: 165, LC CDR2 of SEQ ID NO: 178
and LC CDR3 of SEQ ID NO: 191 of CLL-1 CAR-10;
[0090] (xi) a LC CDR1 of SEQ ID NO: 166, LC CDR2 of SEQ ID NO: 179
and LC CDR3 of SEQ ID NO: 192 of CLL-1 CAR-11;
[0091] (xii) a LC CDR1 of SEQ ID NO: 167, LC CDR2 of SEQ ID NO: 180
and LC CDR3 of SEQ ID NO: 193 of CLL-1 CAR-12;
[0092] (xiii) a LC CDR1 of SEQ ID NO: 168, LC CDR2 of SEQ ID NO:
181 and LC CDR3 of SEQ ID NO: 194 of CLL-1 CAR-13;
[0093] (xiv) a LC CDR1 of SEQ ID NO: 202, LC CDR2 of SEQ ID NO: 203
and LC CDR3 of SEQ ID NO: 204 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0094] (i) a HC CDR1 of SEQ ID NO: 117, HC CDR2 of SEQ ID NO: 130
and HC CDR3 of SEQ ID NO: 143 of CLL-1 CAR-1;
[0095] (ii) a HC CDR1 of SEQ ID NO: 118, HC CDR2 of SEQ ID NO: 131
and HC CDR3 of SEQ ID NO: 144 of CLL-1 CAR-2;
[0096] (iii) a HC CDR1 of SEQ ID NO: 119, HC CDR2 of SEQ ID NO: 132
and HC CDR3 of SEQ ID NO: 145 of CLL-1 CAR-3;
[0097] (iv) a HC CDR1 of SEQ ID NO: 120, HC CDR2 of SEQ ID NO: 133
and HC CDR3 of SEQ ID NO: 146 of CLL-1 CAR-4;
[0098] (v) a HC CDR1 of SEQ ID NO: 121, HC CDR2 of SEQ ID NO: 134
and HC CDR3 of SEQ ID NO: 147 of CLL-1 CAR-5;
[0099] (vi) a HC CDR1 of SEQ ID NO: 122, HC CDR2 of SEQ ID NO: 135
and HC CDR3 of SEQ ID NO: 148 of CLL-1 CAR-6;
[0100] (vii) a HC CDR1 of SEQ ID NO: 123, HC CDR2 of SEQ ID NO: 136
and HC CDR3 of SEQ ID NO: 149 of CLL-1 CAR-7;
[0101] (viii) a HC CDR1 of SEQ ID NO: 124, HC CDR2 of SEQ ID NO:
137 and HC CDR3 of SEQ ID NO: 150 of CLL-1 CAR-8; or
[0102] (ix) a HC CDR1 of SEQ ID NO: 125, HC CDR2 of SEQ ID NO: 138
and HC CDR3 of SEQ ID NO: 151 of CLL-1 CAR-9;
[0103] (x) a HC CDR1 of SEQ ID NO: 126, HC CDR2 of SEQ ID NO: 139
and HC CDR3 of SEQ ID NO: 152 of CLL-1 CAR-10;
[0104] (xi) a HC CDR1 of SEQ ID NO: 127, HC CDR2 of SEQ ID NO: 140
and HC CDR3 of SEQ ID NO: 153 of CLL-1 CAR-11;
[0105] (xii) a HC CDR1 of SEQ ID NO: 128, HC CDR2 of SEQ ID NO: 141
and HC CDR3 of SEQ ID NO: 154 of CLL-1 CAR-12;
[0106] (xiii) a HC CDR1 of SEQ ID NO: 129, HC CDR2 of SEQ ID NO:
142 and HC CDR3 of SEQ ID NO: 155 of CLL-1 CAR-13;
[0107] (xiv) a HC CDR1 of SEQ ID NO: 199, HC CDR2 of SEQ ID NO: 200
and HC CDR3 of SEQ ID NO: 201 of 181286.
[0108] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) or a CLL-1
binding domain includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0109] (i) a LC CDR1 of SEQ ID NO: 356, LC CDR2 of SEQ ID NO: 370
and LC CDR3 of SEQ ID NO: 384 of CLL-1 CAR-1;
[0110] (ii) a LC CDR1 of SEQ ID NO: 357, LC CDR2 of SEQ ID NO: 371
and LC CDR3 of SEQ ID NO: 385 of CLL-1 CAR-2;
[0111] (iii) a LC CDR1 of SEQ ID NO: 358, LC CDR2 of SEQ ID NO: 372
and LC CDR3 of SEQ ID NO: 386 of CLL-1 CAR-3;
[0112] (iv) a LC CDR1 of SEQ ID NO: 359, LC CDR2 of SEQ ID NO: 373
and LC CDR3 of SEQ ID NO: 387 of CLL-1 CAR-4;
[0113] (v) a LC CDR1 of SEQ ID NO: 360, LC CDR2 of SEQ ID NO: 374
and LC CDR3 of SEQ ID NO: 388 of CLL-1 CAR-5;
[0114] (vi) a LC CDR1 of SEQ ID NO: 361, LC CDR2 of SEQ ID NO: 375
and LC CDR3 of SEQ ID NO: 389 of CLL-1 CAR-6;
[0115] (vii) a LC CDR1 of SEQ ID NO: 362, LC CDR2 of SEQ ID NO: 376
and LC CDR3 of SEQ ID NO: 390 of CLL-1 CAR-7;
[0116] (viii) a LC CDR1 of SEQ ID NO: 363, LC CDR2 of SEQ ID NO:
377 and LC CDR3 of SEQ ID NO: 391 of CLL-1 CAR-8; or
[0117] (ix) a LC CDR1 of SEQ ID NO: 364, LC CDR2 of SEQ ID NO: 378
and LC CDR3 of SEQ ID NO: 392 of CLL-1 CAR-9;
[0118] (x) a LC CDR1 of SEQ ID NO: 365, LC CDR2 of SEQ ID NO: 379
and LC CDR3 of SEQ ID NO: 393 of CLL-1 CAR-10;
[0119] (xi) a LC CDR1 of SEQ ID NO: 366, LC CDR2 of SEQ ID NO: 380
and LC CDR3 of SEQ ID NO: 394 of CLL-1 CAR-11;
[0120] (xii) a LC CDR1 of SEQ ID NO: 367, LC CDR2 of SEQ ID NO: 381
and LC CDR3 of SEQ ID NO: 395 of CLL-1 CAR-12;
[0121] (xiii) a LC CDR1 of SEQ ID NO: 368, LC CDR2 of SEQ ID NO:
382 and LC CDR3 of SEQ ID NO: 396 of CLL-1 CAR-13;
[0122] (xiv) a LC CDR1 of SEQ ID NO: 369, LC CDR2 of SEQ ID NO: 383
and LC CDR3 of SEQ ID NO: 397 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0123] (i) a HC CDR1 of SEQ ID NO: 314, HC CDR2 of SEQ ID NO: 328
and HC CDR3 of SEQ ID NO: 342 of CLL-1 CAR-1;
[0124] (ii) a HC CDR1 of SEQ ID NO: 315, HC CDR2 of SEQ ID NO: 329
and HC CDR3 of SEQ ID NO: 343 of CLL-1 CAR-2;
[0125] (iii) a HC CDR1 of SEQ ID NO: 316, HC CDR2 of SEQ ID NO: 330
and HC CDR3 of SEQ ID NO: 344 of CLL-1 CAR-3;
[0126] (iv) a HC CDR1 of SEQ ID NO: 317, HC CDR2 of SEQ ID NO: 331
and HC CDR3 of SEQ ID NO: 345 of CLL-1 CAR-4;
[0127] (v) a HC CDR1 of SEQ ID NO: 318, HC CDR2 of SEQ ID NO: 332
and HC CDR3 of SEQ ID NO: 346 of CLL-1 CAR-5;
[0128] (vi) a HC CDR1 of SEQ ID NO: 319, HC CDR2 of SEQ ID NO: 333
and HC CDR3 of SEQ ID NO: 347 of CLL-1 CAR-6;
[0129] (vii) a HC CDR1 of SEQ ID NO: 320, HC CDR2 of SEQ ID NO: 334
and HC CDR3 of SEQ ID NO: 348 of CLL-1 CAR-7;
[0130] (viii) a HC CDR1 of SEQ ID NO: 321, HC CDR2 of SEQ ID NO:
335 and HC CDR3 of SEQ ID NO: 349 of CLL-1 CAR-8; or
[0131] (ix) a HC CDR1 of SEQ ID NO: 322, HC CDR2 of SEQ ID NO: 336
and HC CDR3 of SEQ ID NO: 350 of CLL-1 CAR-9;
[0132] (x) a HC CDR1 of SEQ ID NO: 323, HC CDR2 of SEQ ID NO: 337
and HC CDR3 of SEQ ID NO: 351 of CLL-1 CAR-10;
[0133] (xi) a HC CDR1 of SEQ ID NO: 324, HC CDR2 of SEQ ID NO: 338
and HC CDR3 of SEQ ID NO: 352 of CLL-1 CAR-11;
[0134] (xii) a HC CDR1 of SEQ ID NO: 325, HC CDR2 of SEQ ID NO: 339
and HC CDR3 of SEQ ID NO: 353 of CLL-1 CAR-12;
[0135] (xiii) a HC CDR1 of SEQ ID NO: 326, HC CDR2 of SEQ ID NO:
340 and HC CDR3 of SEQ ID NO: 354 of CLL-1 CAR-13;
[0136] (xiv) a HC CDR1 of SEQ ID NO: 327, HC CDR2 of SEQ ID NO: 341
and HC CDR3 of SEQ ID NO: 355 of 181286.
[0137] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0138] (i) a LC CDR1 of SEQ ID NO: 440, LC CDR2 of SEQ ID NO: 454
and LC CDR3 of SEQ ID NO: 468 of CLL-1 CAR-1;
[0139] (ii) a LC CDR1 of SEQ ID NO: 441, LC CDR2 of SEQ ID NO: 455
and LC CDR3 of SEQ ID NO: 469 of CLL-1 CAR-2;
[0140] (iii) a LC CDR1 of SEQ ID NO: 442, LC CDR2 of SEQ ID NO: 456
and LC CDR3 of SEQ ID NO: 470 of CLL-1 CAR-3;
[0141] (iv) a LC CDR1 of SEQ ID NO: 443, LC CDR2 of SEQ ID NO: 457
and LC CDR3 of SEQ ID NO: 471 of CLL-1 CAR-4;
[0142] (v) a LC CDR1 of SEQ ID NO: 444, LC CDR2 of SEQ ID NO: 458
and LC CDR3 of SEQ ID NO: 472 of CLL-1 CAR-5;
[0143] (vi) a LC CDR1 of SEQ ID NO: 445, LC CDR2 of SEQ ID NO: 459
and LC CDR3 of SEQ ID NO: 473 of CLL-1 CAR-6;
[0144] (vii) a LC CDR1 of SEQ ID NO: 446, LC CDR2 of SEQ ID NO: 460
and LC CDR3 of SEQ ID NO: 474 of CLL-1 CAR-7;
[0145] (viii) a LC CDR1 of SEQ ID NO: 447, LC CDR2 of SEQ ID NO:
461 and LC CDR3 of SEQ ID NO: 475 of CLL-1 CAR-8; or
[0146] (ix) a LC CDR1 of SEQ ID NO: 448, LC CDR2 of SEQ ID NO: 462
and LC CDR3 of SEQ ID NO: 476 of CLL-1 CAR-9;
[0147] (x) a LC CDR1 of SEQ ID NO: 449, LC CDR2 of SEQ ID NO: 463
and LC CDR3 of SEQ ID NO: 477 of CLL-1 CAR-10;
[0148] (xi) a LC CDR1 of SEQ ID NO: 450, LC CDR2 of SEQ ID NO: 464
and LC CDR3 of SEQ ID NO: 478 of CLL-1 CAR-11;
[0149] (xii) a LC CDR1 of SEQ ID NO: 451, LC CDR2 of SEQ ID NO: 465
and LC CDR3 of SEQ ID NO: 479 of CLL-1 CAR-12;
[0150] (xiii) a LC CDR1 of SEQ ID NO: 452, LC CDR2 of SEQ ID NO:
466 and LC CDR3 of SEQ ID NO: 480 of CLL-1 CAR-13;
[0151] (xiv) a LC CDR1 of SEQ ID NO: 453, LC CDR2 of SEQ ID NO: 467
and LC CDR3 of SEQ ID NO: 481 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0152] (i) a HC CDR1 of SEQ ID NO: 398, HC CDR2 of SEQ ID NO: 412
and HC CDR3 of SEQ ID NO: 426 of CLL-1 CAR-1;
[0153] (ii) a HC CDR1 of SEQ ID NO: 399, HC CDR2 of SEQ ID NO: 413
and HC CDR3 of SEQ ID NO: 427 of CLL-1 CAR-2;
[0154] (iii) a HC CDR1 of SEQ ID NO: 400, HC CDR2 of SEQ ID NO: 414
and HC CDR3 of SEQ ID NO: 428 of CLL-1 CAR-3;
[0155] (iv) a HC CDR1 of SEQ ID NO: 401, HC CDR2 of SEQ ID NO: 415
and HC CDR3 of SEQ ID NO: 429 of CLL-1 CAR-4;
[0156] (v) a HC CDR1 of SEQ ID NO: 402, HC CDR2 of SEQ ID NO: 416
and HC CDR3 of SEQ ID NO: 430 of CLL-1 CAR-5;
[0157] (vi) a HC CDR1 of SEQ ID NO: 403, HC CDR2 of SEQ ID NO: 417
and HC CDR3 of SEQ ID NO: 431 of CLL-1 CAR-6;
[0158] (vii) a HC CDR1 of SEQ ID NO: 404, HC CDR2 of SEQ ID NO: 418
and HC CDR3 of SEQ ID NO: 432 of CLL-1 CAR-7;
[0159] (viii) a HC CDR1 of SEQ ID NO: 405, HC CDR2 of SEQ ID NO:
419 and HC CDR3 of SEQ ID NO: 433 of CLL-1 CAR-8; or
[0160] (ix) a HC CDR1 of SEQ ID NO: 406, HC CDR2 of SEQ ID NO: 420
and HC CDR3 of SEQ ID NO: 434 of CLL-1 CAR-9;
[0161] (x) a HC CDR1 of SEQ ID NO: 407, HC CDR2 of SEQ ID NO: 421
and HC CDR3 of SEQ ID NO: 435 of CLL-1 CAR-10;
[0162] (xi) a HC CDR1 of SEQ ID NO: 408, HC CDR2 of SEQ ID NO: 422
and HC CDR3 of SEQ ID NO: 436 of CLL-1 CAR-11;
[0163] (xii) a HC CDR1 of SEQ ID NO: 409, HC CDR2 of SEQ ID NO: 423
and HC CDR3 of SEQ ID NO: 437 of CLL-1 CAR-12;
[0164] (xiii) a HC CDR1 of SEQ ID NO: 410, HC CDR2 of SEQ ID NO:
424 and HC CDR3 of SEQ ID NO: 438 of CLL-1 CAR-13;
[0165] (xiv) a HC CDR1 of SEQ ID NO: 411, HC CDR2 of SEQ ID NO: 425
and HC CDR3 of SEQ ID NO: 439 of 181286.
[0166] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative
only and not intended to be limiting. 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. 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
[0167] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0168] FIG. 1, comprising FIGS. 1A 1B, and 1C, is a series of
images demonstrating luciferase levels in target-positive (PL21,
THP1, HL60, U937) or target-negative (K562) cell lines mixed with a
JNL cell line transduced with anti-CLL-1 CAR.
[0169] FIG. 2, comprising FIGS. 2A, 2B, and 2C, is a series of
images demonstrating CAR expression as evaluated by FACS in a JNL
cell line transduced with anti-CLL-1 CAR.
[0170] FIG. 3, comprising FIGS. 3A and 3B, is a series of images
demonstrating histogram plots of relative fluorescent intensity
from that FACS showed the percentage of transduced T cells. FIG. 3A
shows detection of CART expression in primary T cells using Protein
L. FIG. 3B shows detection of CART expression in primary T cells
using recombinant CLL-1 protein.
[0171] FIG. 4, comprising FIGS. 4A, 4B, and 4C, is a series of
images demonstrating anti-CLL-1 CART cell killing of luciferized
PL21 (FIG. 4A), HL60 (FIG. 4B) and U87 cells (FIG. 4C).
[0172] FIG. 5, comprising FIGS. 5A, 5B, and 5C, is a series of
images demonstrating cytokine production in CART-CLL-1 cells.
Untransduced T cells (UTD) were used as a non-specific control for
background T cell effects. TNF-alpha (FIG. 5A), IL-2 (FIG. 5B), and
interferon (IFN)-gamma (FIG. 5C) were measured.
[0173] FIG. 6 is an image demonstrating that CLL-1 is expressed in
most primary patient samples with AML (AML blasts were gated using
standard side scatter .sup.low CD45.sup.thm characteristics). CLL-1
was measured by flow cytometry using a commercially available
antibody (clone HIM3-4, eBioscience)
[0174] FIG. 7, comprising FIGS. 7A and 7B, is a series of images
demonstrating the transduction efficiency of T cells transduced
with CAR.
[0175] FIG. 8, comprising FIGS. 8A and 8B, is a series of images
demonstrating that CLL1-CART cells undergo specific degranulation
to CLL1+ cell lines and primary AML samples. CD107a degranulation
was measured by flow cytometry (FIG. 8A). CLL-1 CART cells
underwent specific degranulation to THP1 and primary AML samples
and not to the control cell line (FIG. 8B).
[0176] FIG. 9, comprising FIGS. 9A and 9B, is a series of images
demonstrating CLL1-CART cells produce TNF-.alpha. after incubation
with CLL1+ cell line and primary AML samples.
[0177] FIG. 10, comprising FIGS. 10A and 10B, is a series of images
demonstrating CLL1-CART cells produce IL-2 after incubation with
CLL1+ cell line and primary AML samples.
[0178] FIG. 11, comprising FIGS. 11A-11D, is a series of images
demonstrating CLL1-CART cells specifically kill the CLL-1+ cell
lines MOLM14 and THP-1 and primary AML samples. CLL1-CART cells
results in specific lysis of MOLM14 (FIG. 11D), THP-1 (FIG. 11A)
and the primary AML sample (FIG. 11B) and not to the control cell
line JEKO (FIG. 11C), at the indicated E:T ratios.
[0179] FIG. 12, comprising FIGS. 12A and 12B, is a series of images
demonstrating CLL1-CART cells proliferate in response to MOLM14,
THP-1 and primary AML samples.
[0180] FIG. 13 is an image illustrating a schematic diagram for
assaying hematopoietic stem cell yoxicity of CLL-1 CART cells using
autologous xenografts.
[0181] FIG. 14, comprising FIGS. 14A, 14B, and 14C, is a series of
images demonstrating that CLL-1 is expressed on different myeloid
lineage cells and B cells in humanized mice. A representative FACS
plots of the peripheral blood analysis of one mouse is shown (FIG.
14A). CLL-1 is expressed on monocytes (CD14+ cells), myeloid cells
(CD33+ and CD123+ cells), B cells (CD19+ cells), but not on
platelets (CD41+ cells) or T cells (CD3+ cells). A representative
histogram presentation is shown (FIG. 14B). A schematic plot
representation of peripheral blood analysis from 24 mice is shown
(FIG. 14C).
[0182] FIG. 15, comprising FIGS. 15A, 15B, 15C, and 15D, is a
series of images demonstrating CLL-1 is expressed on different
myeloid lineage cells and B cells in humanized mice.
[0183] FIG. 16, comprising FIGS. 16A, 16B, 16C, and 16D, is a
series of images demonstrating that CLL-1 is expressed on different
myeloid progenitors and on hematopoietic stem cells in humanized
mice.
[0184] FIG. 17 is an image illustrating a schematic diagram for
assaying hematopoietic stem cell toxicity of CLL-1 CART cells using
a Humanized Immune System (HIS) xenografts.
[0185] FIG. 18, comprising FIGS. 18A and 18B, is a series of images
demonstrating bone marrow analysis 4 weeks post CLL-1 CAR T cell
infusions. Flow cytometry analysis was performed in the CD34+CD38-
component (hematopoietic stem cells) (FIG. 18A) and CD34+CD38+
component (Progenitor cells) (FIG. 18B).
[0186] FIG. 19, comprising FIGS. 19A, 19B, 19C, 19D, and 19E, is a
series of images demonstrating bone marrow analysis 4 weeks post T
cells.
[0187] FIG. 20 is an image demonstrating bone marrow analysis in
HIS mice 4 weeks post T cells. Hematopoietic stem cell toxicity of
CLL1-CART cells using HIS xenografts.
[0188] FIG. 21, comprising FIGS. 21A, 21B, 21C, 21D, and 21E, is a
series of images demonstrating bone marrow analysis in HIS mice 4
weeks post T cells. Representative plots of bone marrows from mice
treated with different CART cells are shown.
[0189] FIG. 22, comprising FIGS. 22A and 22B, is a series of
histogram plots showing the relative fluorescent intensity from
FACS analysis showing the percentage of transduced T cells.
[0190] FIG. 22A shows detection of CART expression in primary T
cells using Protein L. FIG. 22B shows detection of CART expression
in primary T cells using recombinant CLL-1 protein.
[0191] FIG. 23, comprising FIGS. 23A and 23B, are two graphs
showing the proliferation capacity of the CLL-1 CART cells when
cultured with target cells.
[0192] FIG. 24 is a graphic representation demonstrating AML
disease progression in the PL-21-luc xenograft model after
treatment with CLL-1 CAR T cells. Mean bioluminescence (+/-SEM) of
the tumor cells show disease burden in the whole animal is shown as
photons/second (p/s) of the ROI (region of interest), which is the
whole mouse.
[0193] FIG. 25, comprising FIGS. 25A and 25B, are two graphs
showing the quantification of CD4+(FIG. 25A) and CD8+(FIG. 25B)
CARP T cells in the peripheral blood of PL-21-luc tumor-bearing
mice.
[0194] FIG. 26, comprising FIGS. 26A, 26B, 26C, and 26D, are bar
graphs quantifying the CD4+ T cells (FIG. 26A), CD4.sup.+ CLL-1
CAR-expressing T cells (FIG. 26B), CD8+ T cells (FIG. 26C), and the
CD8.sup.+ CLL-1 CAR-expressing T cells (FIG. 26D) in the bone
marrow of the PL-21-luc tumor-bearing mice. Mean T cell number
(+/-SEM) per million bone marrow cells is shown. Significance is
calculated by one way ANOVA and is denoted as * P<0.05 and
**P<0.01.
[0195] FIG. 27, comprising FIGS. 27A, 27B, 27C, and 27D, are bar
graphs quantifying the CD4+ T cells (FIG. 27A), CD4.sup.+ CLL-1
CAR-expressing T cells (FIG. 27B), CD8+ T cells (FIG. 27C), and the
CD8.sup.+ CLL-1 CAR-expressing T cells (FIG. 27D) in the spleen of
the PL-21-luc tumor-bearing mice. Mean T cell number (+/-SEM) per
million splenocytes is shown. Significance is calculated by one way
ANOVA and is denoted as * P<0.05 and **P<0.01.
[0196] FIG. 28, comprising FIGS. 28A, 28B, 28C, and 28D, shows
treatment with induction chemotherapy followed by CLL1-CART cells
results in leukemic eradication in primary AML xenografts. FIG. 28A
is a schematic illustrating the experimental schema for the
combined therapy of chemotherapy and CLL-1-CART cells in primary
AML xenografts. FIG. 28B is a bar graph showing the the mean
fluorescence intensity (MFI) of CLL1 in leukemic cells (live
huCD45dim compartment). FIG. 28C is a graph showing the
quantification of peripheral blood leukemic blast count per 1 ul of
peripheral blood (mean+/-SD) at different time points post AML
injection as indicated. The arrows denote administration Ara-C(grey
arrows) and administration of T cells (untransduced or CLL-1
CAR-expressing T cells, black arrows). FIG. 28D is a graph showing
the survival of the AML xenografts.
[0197] FIG. 29, comprising FIGS. 29A, 29B, 29C, 29D, and 29E, shows
the various configurations on a single vector, e.g., where the U6
regulated shRNA is upstream or downstream of the EF1 alpha
regulated CAR encoding elements. In the exemplary constructs
depicted in FIGS. 29A and 29B, the transcription occurs through the
U6 and EF1 alpha promoters in the same direction. In the exemplary
constructs depicted in FIGS. 29C and 29D, the transcription occurs
through the U6 and EF1 alpha promoters in different directions. In
FIG. 29E, the shRNA (and corresponding U6 promoter) is on a first
vector, and the CAR (and corresponding EF1 alpha promoter) is on a
second vector.
[0198] FIG. 30 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.
[0199] FIG. 31 shows that the proliferation of CAR-expressing,
transduced T cells is enhanced by low doses of RAD001 in a cell
culture system. CARTs were co-cultured with Nalm-6 cells in the
presence of different concentrations of RAD001. The number of
CAR-positive CD3-positive T cells (black) and total T cells (gray)
was assessed after 4 days of co-culture.
[0200] FIG. 32 depicts tumor growth measurements of NALM6-luc cells
with daily RAD001 dosing at 0.3, 1, 3, and 10 mg/kg (mpk) or
vehicle dosing. Circles denote the vehicle; squares denote the 10
mg/kg dose of RAD001; triangles denote the 3 mg/kg dose of RAD001,
inverted triangles denote the 1 mg/kg dose of RAD001; and diamonds
denote the 0.3 mg/kg dose of RAD001.
[0201] FIG. 33, comprising FIGS. 33A and 33B, shows pharmacokinetic
curves showing the amount of RAD001 in the blood of NSG mice with
NALM6 tumors. FIG. 33A shows day 0 PK following the first dose of
RAD001. FIG. 33B shows Day 14 PK following the final RAD001 dose.
Diamonds denote the 10 mg/kg dose of RAD001; squares denote the 1
mg/kg dose of RAD001; triangles denote the 3 mg/kg dose of RAD001;
and x's denote the 10 mg/kg dose of RAD001.
[0202] FIG. 34, comprising FIGS. 34A and 34B, shows in vivo
proliferation of humanized CD19 CART cells with and without RAD001
dosing. Low doses of RAD001 (0.003 mg/kg) daily lead to an
enhancement in CAR T cell proliferation, above the normal level of
huCAR19 proliferation. FIG. 34A shows CD4.sup.+ CAR T cells; FIG.
34B shows CD8.sup.+ CAR T cells. Circles denote PBS; squares denote
huCTL019; triangles denote huCTL019 with 3 mg/kg RAD001; inverted
triangles denote huCTL019 with 0.3 mg/kg RAD001; diamonds denote
huCTL019 with 0.03 mg/kg RAD001; and circles denote huCTL019 with
0.003 mg/kg RAD001.
DETAILED DESCRIPTION
Definitions
[0203] 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.
[0204] 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.
[0205] 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.
[0206] The term "Chimeric Antigen Receptor" or alternatively a
"CAR" refers to a recombinant polypeptide construct comprising 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 as defined
below. In some embodiments, the domains in the CAR polypeptide
construct are in the same polypeptide chain, e.g., comprise a
chimeric fusion protein. In some embodiments, the domains in the
CAR polypeptide construct are not contiguous with each other, e.g.,
are in different polypeptide chains, e.g., as provided in an RCAR
as described herein
[0207] In one aspect, the stimulatory molecule of the CAR is the
zeta chain associated with the T cell receptor complex. In one
aspect, the cytoplasmic signaling domain comprises a primary
signaling domain (e.g., a primary signaling domain of CD3-zeta). 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 4-1BB (i.e., CD137), CD27,
ICOS, and/or CD28. In one aspect, the CAR comprises a chimeric
fusion protein comprising an extracellular antigen recognition
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 recognition
domain, a transmembrane domain and an intracellular signaling
domain comprising a functional signaling domain derived from a
co-stimulatory 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
recognition domain, a transmembrane domain and an intracellular
signaling domain comprising two functional signaling domains
derived from one or more co-stimulatory 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 recognition domain, a transmembrane domain
and an intracellular signaling domain comprising at least two
functional signaling domains derived from one or more
co-stimulatory 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
recognition domain, wherein the leader sequence is optionally
cleaved from the antigen recognition domain (e.g., aa scFv) during
cellular processing and localization of the CAR to the cellular
membrane.
[0208] A CAR that comprises an antigen binding domain (e.g., a
scFv, a single domain antibody, or TCR (e.g., a TCR alpha binding
domain or TCR beta binding domain)) that specifically binds a
specific tumor marker X, wherein X can be a tumor marker as
described herein, is also referred to as XCAR. For example, a CAR
that comprises an antigen binding domain that specifically binds
CLL-1 is referred to as CLL-1 CAR. The CAR can be expressed in any
cell, e.g., an immune effector cell as described herein (e.g., a T
cell or an NK cell).
[0209] 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.
[0210] As used herein, the term "CLL-1" refers to C-type
lectin-like molecule-1, which is an antigenic determinant
detectable on leukemia precursor cells and on normal immune cells.
C-type lectin-like-1 (CLL-1) is also known as MICL, CLEC12A,
CLEC-1, Dendritic Cell-Associated Lectin 1, and DCAL-2. 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 CLL-1 can be found as
UniProt/Swiss-Prot Accession No. Q5QGZ9 and the nucleotide sequence
encoding of the human CLL-1 can be found at Accession Nos. NM
001207010.1, NM 138337.5, NM 201623.3, and NM 201625.1. In one
embodiment, the antigen-binding portion of the CAR recognizes and
binds an epitope within the extracellular domain of the CLL-1
protein or a fragment thereof. In one embodiment, the CLL-1 protein
is expressed on a cancer cell.
[0211] 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.
[0212] The term "antibody fragment" refers to at least one portion
of an intact antibody, or recombinant variants thereof, and refers
to the antigen binding domain, e.g., an antigenic determining
variable region of an intact antibody, that is sufficient to confer
recognition and specific binding of the antibody fragment to a
target, such as an antigen. Examples of antibody fragments include,
but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv
antibody fragments, linear antibodies, single domain antibodies
such as sdAb (either VL or VH), camelid VHH domains, and
multi-specific molecules formed from antibody fragments such as a
bivalent fragment comprising two or more, e.g., two, Fab fragments
linked by a disulfide bridge at the hinge region, or two or more,
e.g., two, isolated CDR or other epitope binding fragments of an
antibody linked. An antibody 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). Antibody 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).
[0213] 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 via 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.
[0214] The term "complementarity determining region" or "CDR," as
used herein, refers to the sequences of amino acids within antibody
variable regions which confer antigen specificity and binding
affinity. For example, in general, there are three CDRs in each
heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and
three CDRs in each light chain variable region (LCDR1, LCDR2, and
LCDR3). 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. Under the Kabat
numbering scheme, in some embodiments, the CDR amino acid residues
in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1),
50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues
in the light chain variable domain (VL) are numbered 24-34 (LCDR1),
50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering
scheme, in some embodiments, the CDR amino acids in the VH are
numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the
CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52
(LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia
numbering scheme, in some embodiments, the CDRs correspond to the
amino acid residues that are part of a Kabat CDR, a Chothia CDR, or
both. For instance, in some embodiments, the CDRs correspond to
amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102
(HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino
acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a
VL, e.g., a mammalian VL, e.g., a human VL.
[0215] The portion of the CAR composition of the invention
comprising an antibody or antibody fragment thereof may exist in a
variety of forms, for example, where the antigen binding domain is
expressed as part of a polypeptide chain including, for example, a
single domain antibody fragment (sdAb), a single chain antibody
(scFv), e.g., a human 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, New York; 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.
[0216] As used herein, the term "binding domain" or "antibody
molecule" (also referred to herein as "anti-target (e.g., CLL-1)
binding domain") 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.
[0217] 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.
[0218] 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 (.kappa.) and
lambda (.lamda.) light chains refer to the two major antibody light
chain isotypes.
[0219] 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.
[0220] 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.
[0221] 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 the number of metastases, an increase in
life expectancy, decrease in tumor cell proliferation, decrease in
tumor cell survival, or amelioration of various physiological
symptoms associated with the cancerous condition. An "anti-tumor
effect" can also be manifested by the ability of the peptides,
polynucleotides, cells and antibodies of the invention in
prevention of the occurrence of tumor in the first place.
[0222] The term "autologous" refers to any material derived from
the same individual to whom it is later to be re-introduced into
the individual.
[0223] 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
[0224] The term "xenogeneic" refers to a graft derived from an
animal of a different species.
[0225] The term "apheresis" as used herein refers to the
art-recognized extracorporeal process by which the blood of a donor
or patient is removed from the donor or patient and passed through
an apparatus that separates out selected particular constituent(s)
and returns the remainder to the circulation of the donor or
patient, e.g., by retransfusion. Thus, in the context of "an
apheresis sample" refers to a sample obtained using apheresis.
[0226] The term "combination" refers to either a fixed combination
in one dosage unit form, or a combined administration where a
compound of the present invention and a combination partner (e.g.
another drug as explained below, also referred to as "therapeutic
agent" or "co-agent") may be administered independently at the same
time or separately within time intervals, especially where these
time intervals allow that the combination partners show a
cooperative, e.g. synergistic effect. The single components may be
packaged in a kit or separately. One or both of the components
(e.g., powders or liquids) may be reconstituted or diluted to a
desired dose prior to administration. The terms "co-administration"
or "combined administration" or the like as utilized herein are
meant to encompass administration of the selected combination
partner to a single subject in need thereof (e.g. a patient), and
are intended to include treatment regimens in which the agents are
not necessarily administered by the same route of administration or
at the same time. The term "pharmaceutical combination" as used
herein means a product that results from the mixing or combining of
more than one active ingredient and includes both fixed and
non-fixed combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. a compound of
the present invention and a combination partner, are both
administered to a patient simultaneously in the form of a single
entity or dosage. The term "non-fixed combination" means that the
active ingredients, e.g. a compound of the present invention and a
combination partner, are both administered to a patient as separate
entities either simultaneously, concurrently or sequentially with
no specific time limits, wherein such administration provides
therapeutically effective levels of the two compounds in the body
of the patient. The latter also applies to cocktail therapy, e.g.
the administration of three or more active ingredients
[0227] The term "cancer" refers to a disease characterized by the
rapid and 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.
[0228] "Derived from" as that term is used herein, indicates a
relationship between a first and a second molecule. It generally
refers to structural similarity between the first molecule and a
second molecule and does not connotate or include a process or
source limitation on a first molecule that is derived from a second
molecule. For example, in the case of an intracellular signaling
domain that is derived from a CD3zeta molecule, the intracellular
signaling domain retains sufficient CD3zeta structure such that is
has the required function, namely, the ability to generate a signal
under the appropriate conditions. It does not connotate or include
a limitation to a particular process of producing the intracellular
signaling domain, e.g., it does not mean that, to provide the
intracellular signaling domain, one must start with a CD3zeta
sequence and delete unwanted sequence, or impose mutations, to
arrive at the intracellular signaling domain.
[0229] The phrase "disease associated with expression of CLL-1"
includes, but is not limited to, a disease associated with a cell
which expresses CLL-1 or condition associated with a cell which
expresses CLL-1 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 a cell which expresses
CLL-1 (e.g., wild-type or mutant CLL-1). For the avoidance of
doubt, a disease associated with expression of CLL-1 may include a
condition associated with a cell which do not presently express
CLL-1, e.g., because CLL-1 expression has been downregulated, e.g.,
due to treatment with a molecule targeting CLL-1, e.g., a CLL-1
inhibitor described herein, but which at one time expressed CLL-1.
In one aspect, a cancer associated with expression of CLL-1 is a
hematological cancer. In one aspect, a hematological cancer
includes but is not limited to leukemia (such as acute myelogenous
leukemia, chronic myelogenous leukemia, acute lymphoid leukemia,
chronic lymphoid leukemia and myelodysplastic syndrome) and
malignant lymphoproliferative conditions, including lymphoma (such
as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma,
and small cell- and large cell-follicular lymphoma). Further
diseases associated with expression of CLL-1 expression include,
but not limited to, e.g., atypical and/or non-classical cancers,
malignancies, precancerous conditions or proliferative diseases
associated with expression of CLL-1. Non-cancer related indications
associated with expression of CLL-1 may also be included. In some
embodiments, the tumor antigen-expressing cell expresses, or at any
time expressed, mRNA encoding the tumor antigen. In an embodiment,
the tumor antigen-expressing cell produces 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 cell produced detectable
levels of a tumor antigen protein at one point, and subsequently
produced substantially no detectable tumor antigen protein.
[0230] The terms "conservative sequence modifications" or
"conservative substitutions" 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
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.
[0231] The term "stimulation," refers to a primary response induced
by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with
its cognate ligand thereby mediating a signal transduction event,
such as, but not limited to, signal transduction via the TCR/CD3
complex. Stimulation can mediate altered expression of certain
molecules, such as downregulation of TGF-.beta., and/or
reorganization of cytoskeletal structures, and the like.
[0232] The term "stimulatory molecule," refers to a molecule
expressed by a T cell that provides the primary cytoplasmic
signaling sequence(s) that regulate primary activation of the TCR
complex in a stimulatory way for at least some aspect of the T cell
signaling pathway. In one aspect, the primary signal 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 primary cytoplasmic signaling
sequence that is of particular use in the invention includes, but
is not limited to, those derived from TCR zeta, FcR gamma, FcR
beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b,
CD278 (also known as "ICOS"), FccRI, CD66d, 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:9, 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:10, or the equivalent residues from a non-human
species, e.g., mouse, rodent, monkey, ape and the like.
[0233] 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.
[0234] 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. In embodiments, the intracellular signal
domain transduces the effector function signal and directs the cell
to perform a specialized function. While 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.
[0235] 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.
[0236] 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, FcR gamma, FcR beta, CD3 gamma, CD3
delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as
"ICOS"), FccRI, CD66d, DAP10, and DAP12.
[0237] 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 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:9. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta
stimulatory domain" is the sequence provided as SEQ ID NO:10.
[0238] 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 required for an
efficient immune response. Costimulatory molecules include, but are
not limited to an a MHC class I molecule, TNF receptor proteins,
Immunoglobulin-like proteins, cytokine receptors, integrins,
signaling lymphocytic activation molecules (SLAM proteins),
activating NK cell receptors, BTLA, a Toll ligand receptor, OX40,
CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18),
4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR,
LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30,
NKp46, 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, 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 (SLAMF1, CD150, IPO-3),
BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp,
CD19a, and a ligand that specifically binds with CD83.
[0239] A costimulatory intracellular signaling domain refers to the
intracellular portion of a costimulatory molecule.
[0240] 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 thereof.
[0241] 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 Acc. No. 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:7 or the equivalent residues from a non-human
species, e.g., mouse, rodent, monkey, ape and the like.
[0242] "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.
[0243] "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.
[0244] 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.
[0245] 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.
[0246] 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).
[0247] 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.
[0248] The term "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0249] The term "exogenous" refers to any material introduced from
or produced outside an organism, cell, tissue or system.
[0250] The term "expression" refers to the transcription and/or
translation of a particular nucleotide sequence driven by a
promoter.
[0251] 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.
[0252] 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.
[0253] 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.
[0254] 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.
[0255] 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.
[0256] "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.
[0257] "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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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)).
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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 (SEQ ID NO: 38)., 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 In one embodiment, the flexible
polypeptide linkers include, but are not limited to, (Gly4
Ser).sub.4 (SEQ ID NO:27) or (Gly4 Ser).sub.3 (SEQ ID NO:28). In
another embodiment, the linkers include multiple repeats of
(Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29). Also included
within the scope of the invention are linkers described in
WO2012/138475, incorporated herein by reference.
[0270] 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.
[0271] 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.
[0272] 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: 30), 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.
[0273] 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.
[0274] 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.
[0275] As used herein, the terms "treat", "treatment" and
"treating" refer to the reduction or amelioration of the
progression, severity and/or duration of a proliferative disorder,
or the amelioration of one or more symptoms (preferably, one or
more discernible symptoms) of a proliferative disorder resulting
from the administration of one or more therapies (e.g., one or more
therapeutic agents such as a CAR of the invention). In specific
embodiments, the terms "treat", "treatment" and "treating" refer to
the amelioration of at least one measurable physical parameter of a
proliferative disorder, such as growth of a tumor, not necessarily
discernible by the patient. In other embodiments the terms "treat",
"treatment" and "treating"-refer to the inhibition of the
progression of a proliferative disorder, either physically by,
e.g., stabilization of a discernible symptom, physiologically by,
e.g., stabilization of a physical parameter, or both. In other
embodiments the terms "treat", "treatment" and "treating" refer to
the reduction or stabilization of tumor size or cancerous cell
count.
[0276] 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.
[0277] The term "subject" is intended to include living organisms
in which an immune response can be elicited (e.g., mammals,
human).
[0278] 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.
[0279] The term "therapeutic" as used herein means a treatment. A
therapeutic effect is obtained by reduction, suppression,
remission, or eradication of a disease state.
[0280] The term "prophylaxis" as used herein means the prevention
of or protective treatment for a disease or disease state.
[0281] 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's lymphoma, Hodgkins 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.
[0282] 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.
[0283] The term "specifically binds," refers to an antibody, or a
ligand, which recognizes and binds with a cognate binding partner
(e.g., a stimulatory and/or costimulatory molecule present on a T
cell) protein present in a sample, but which antibody or ligand
does not substantially recognize or bind other molecules in the
sample.
[0284] "Regulatable chimeric antigen receptor (RCAR)," as used
herein, 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 regulatable intracellular signal generation.
In some embodiments, an RCAR comprises at least an extracellular
antigen binding domain, a transmembrane 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
herein in the context of a CAR molecule. In some embodiments, the
set of polypeptides in the RCAR are not contiguous with each other,
e.g., are in different polypeptide chains. In some embodiments, the
RCAR includes 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 some embodiments, the RCAR is expressed in a
cell (e.g., an immune effector cell) as described herein, e.g., an
RCAR-expressing cell (also referred to herein as "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. The RCAR can provide the
RCAR-expressing 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 RCAR-expressing cell. In embodiments, an RCAR 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.
[0285] "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.
[0286] "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.
[0287] "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.
[0288] 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.
[0289] The term "low, immune enhancing, dose" when used in
conjunction 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:
[0290] an increase in the expression of one or more of the
following markers: CD62L.sup.high, CD127.sup.high, CD27.sup.+, and
BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0291] a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; and
[0292] 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;
[0293] wherein any of the changes described above occurs, e.g., at
least transiently, e.g., as compared to a non-treated subject.
[0294] "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 resistant during a treatment. A refractory cancer
is also called a resistant cancer.
[0295] "Relapsed" or a "relapse" as used herein refers to the
reappearance of a disease (e.g., cancer) or the signs and symptoms
of a disease such as cancer after a period of improvement or
responsiveness, e.g., after prior treatment of a therapy, e.g.,
cancer therapy. For example, the 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%.
[0296] 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.
[0297] Description
[0298] Provided herein are compositions of matter and methods of
use for the treatment of a disease such as cancer using CLL-1
chimeric antigen receptors (CAR).
[0299] 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 CLL-1 protein or a fragment
thereof. In one aspect, the invention provides a cell (e.g., an
immune effector cell, e.g., a T cell or a NK cell) engineered to
express a CAR, wherein the CAR T cell ("CART") exhibits an
antitumor property. In one aspect a cell is transformed with the
CAR and at least part of the CAR construct is expressed on the cell
surface. In some embodiments, the cell (e.g., an immune effector
cell, e.g., a T cell or a NK 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., an immune
effector cell, e.g., a T cell or a NK 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.
[0300] In one aspect, the human anti-CLL-1 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
efficacy, as the IgG antibody having the same heavy and light chain
variable regions. 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.
[0301] 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 herein as SEQ
ID NO: 91-103.
[0302] In one aspect, the anti-CLL-1 binding domain, e.g., human
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.
[0303] In one aspect, the human CLL-1 binding domain comprises the
scFv portion provided in SEQ ID NOs:39-51. In one embodiment, the
human anti-CLL-1 binding domain comprises the scFv portion provided
in SEQ ID NO:39. In one embodiment, the human anti-CLL-1 binding
domain comprises the scFv portion provided in SEQ ID NO:40. In one
embodiment, the human anti-CLL-1 binding domain comprises the scFv
portion provided in SEQ ID NO:41. In one embodiment, the human
anti-CLL-1 binding domain comprises the scFv portion provided in
SEQ ID NO:42. In one embodiment, the human anti-CLL-1 binding
domain comprises the scFv portion provided in SEQ ID NO:43. In one
embodiment, the human anti-CLL-1 binding domain comprises the scFv
portion provided in SEQ ID NO:44. In one embodiment, the human
anti-CLL-1 binding domain comprises the scFv portion provided in
SEQ ID NO:45. In one embodiment, the human anti-CLL-1 binding
domain comprises the scFv portion provided in SEQ ID NO:46. In one
embodiment, the human anti-CLL-1 binding domain comprises the scFv
portion provided in SEQ ID NO:47. In one embodiment, the human
anti-CLL-1 binding domain comprises the scFv portion provided in
SEQ ID NO:48. In one embodiment, the human anti-CLL-1 binding
domain comprises the scFv portion provided in SEQ ID NO:49. In one
embodiment, the human anti-CLL-1 binding domain comprises the scFv
portion provided in SEQ ID NO:50. In one embodiment, the human
anti-CLL-1 binding domain comprises the scFv portion provided in
SEQ ID NO:51. 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 antigen binding domain binds to CLL-1. In one aspect,
the CLL-1 CAR comprises a CAR selected from the sequence provided
in one or more of SEQ ID NOs: 91-103 or 197. In one aspect, the
CLL-1 CAR comprises the sequence provided in SEQ ID NO:91. In one
aspect, the CLL-1 CAR comprises the sequence provided in SEQ ID
NO:92. In one aspect, the CLL-1 CAR comprises the sequence provided
in SEQ ID NO:93. In one aspect, the CLL-1 CAR comprises the
sequence provided in SEQ ID NO:94. In one aspect, the CLL-1 CAR
comprises the sequence provided in SEQ ID NO:95. In one aspect, the
CLL-1 CAR comprises the sequence provided in SEQ ID NO:96. In one
aspect, the CLL-1 CAR comprises the sequence provided in SEQ ID
NO:97. In one aspect, the CLL-1 CAR comprises the sequence provided
in SEQ ID NO:98. In one aspect, the CLL-1 CAR comprises the
sequence provided in SEQ ID NO:99. In one aspect, the CLL-1 CAR
comprises the sequence provided in SEQ ID NO:100. In one aspect,
the CLL-1 CAR comprises the sequence provided in SEQ ID NO:101. In
one aspect, the CLL-1 CAR comprises the sequence provided in SEQ ID
NO:102. In one aspect, the CLL-1 CAR comprises the sequence
provided in SEQ ID NO:103. In one aspect, the CLL-1 CAR comprises
the sequence provided in SEQ ID NO:197. Furthermore, the present
invention provides CLL-1 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 CLL-1.
[0304] In one aspect, the CAR of the invention can be used to
eradicate CLL-1-expressing normal cells, thereby applicable for use
as a cellular conditioning therapy prior to cell transplantation.
In one aspect, the CLL-1-expressing normal cell is a
CLL-1-expressing normal stem cell and the cell transplantation is a
stem cell transplantation.
[0305] In one aspect, the invention provides a cell (e.g., an
immune effector cell, e.g., a T cell or a NK cell) engineered to
express a chimeric antigen receptor (CAR) of the present invention,
wherein the cell (e.g., CAR-expressing immune effector cell, e.g.,
CAR T cell, e.g., "CART") exhibits an antitumor property. A
preferred antigen is CLL-1. In one aspect, the antigen binding
domain of the CAR comprises a human anti-CLL-1 antibody fragment.
In one aspect, the antigen binding domain of the CAR comprises
human anti-CLL-1 antibody fragment comprising an scFv. In one
embodiment, the antigen binding domain of the CAR comprises a human
anti-CLL-1 scFv. Accordingly, the invention provides a CLL-1-CAR
that comprises an anti-CLL-1 binding domain and is engineered into
an immune effector cell, e.g., a T cell or a NK cell and methods of
their use for adoptive therapy. In one aspect, the CLL-1-CAR
comprises a human anti-CLL-1 binding domain.
[0306] In one aspect, the CLL-1-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 CLL-1-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.
[0307] Chimeric Antigen Receptor (CAR)
[0308] The present invention provides a CAR (e.g., a CAR
polypeptide) that comprises an anti-CLL-1 binding domain (e.g.,
human or humanized CLL-1 binding domain as described herein), a
transmembrane domain, and an intracellular signaling domain, and
wherein said anti-CLL-1 binding domain comprises 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 any anti-CLL-1
heavy chain binding domain amino acid sequences listed in Table 8.
The anti-CLL-1 binding domain of the CAR can further comprise a
light chain complementary determining region 1 (LC CDR1), a light
chain complementary determining region 2 (LC CDR2), and a light
chain complementary determining region 3 (LC CDR3) of any
anti-CLL-1 light chain binding domain amino acid sequences listed
in Table 8.
[0309] The present invention also provides nucleic acid molecules
encoding the CAR as described herein, e.g., encoding a CAR that
comprises an anti-CLL-1 binding domain (e.g., human or humanized
CLL-1 binding domain as described herein), a transmembrane domain,
and an intracellular signaling domain, and wherein said anti-CLL-1
binding domain comprises 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 any anti-CLL-1 heavy chain binding domain amino acid
sequences listed in Table 8.
[0310] In one embodiment, the encoded anti-CLL-1 binding domain of
the CAR can further comprise a light chain complementary
determining region 1 (LC CDR1), a light chain complementary
determining region 2 (LC CDR2), and a light chain complementary
determining region 3 (LC CDR3) of any anti-CLL-1 light chain
binding domain amino acid sequences listed in Table 8.
[0311] In specific aspects, a CAR construct of the invention
comprises a scFv domain selected from the group consisting of SEQ
ID NOs:39-51, wherein the scFv may be preceded by an optional
leader sequence such as provided in SEQ ID NO: 1, and followed by
an optional hinge sequence such as provided in SEQ ID NO:2 or SEQ
ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, a transmembrane region such
as provided in SEQ ID NO:6, an intracellular signalling domain that
includes SEQ ID NO:7 or SEQ ID NO:8 and a CD3 zeta sequence that
includes SEQ ID NO:9 or SEQ ID NO:10, e.g., 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: 39-51. 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: 39-51, and each of the domains of SEQ ID
NOs: 1, 2, and 6-9. In one aspect an exemplary CLL-1 CAR 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 CLL-1
CAR 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.
[0312] In some embodiments, full-length CAR sequences are also
provided herein as SEQ ID NOs: 91-103, as shown in Table 8.
[0313] An exemplary leader sequence is provided as SEQ ID NO: 1. An
exemplary hinge/spacer sequence is provided as SEQ ID NO:2 or SEQ
ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5. An exemplary transmembrane
domain sequence is provided as SEQ ID NO:6. An exemplary sequence
of the intracellular signaling domain of the 4-1BB protein is
provided as SEQ ID NO: 7. An exemplary sequence of the
intracellular signaling domain of CD27 is provided as SEQ ID NO:8.
An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 9 or
SEQ ID NO:10.
[0314] 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 a CLL-1 binding
domain, e.g., described herein, e.g., that is contiguous with and
in the same reading frame as a nucleic acid sequence encoding an
intracellular signaling domain. In one aspect, the CLL-1 binding
domain is selected from one or more of SEQ ID NOs:39-51. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:39. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:40. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:41. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:42. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:43. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:44. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:45. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:46. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:47. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:48. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:49. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:50. In one
embodiment, the CLL-1 binding domain comprises SEQ ID NO:51.
[0315] 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-CLL-1 binding domain selected from one or
more of SEQ ID NOs:39-51, 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:104-116, or
198. In one aspect the nucleic acid sequence of a CAR construct is
SEQ ID NO:104. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:105. In one aspect the nucleic acid sequence
of a CAR construct is SEQ ID NO:106. In one aspect the nucleic acid
sequence of a CAR construct is SEQ ID NO:107. In one aspect the
nucleic acid sequence of a CAR construct is SEQ ID NO:108. In one
aspect the nucleic acid sequence of a CAR construct is SEQ ID
NO:109. In one aspect the nucleic acid sequence of a CAR construct
is SEQ ID NO:110. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:111. In one aspect the nucleic acid sequence
of a CAR construct is SEQ ID NO:112. In one aspect the nucleic acid
sequence of a CAR construct is SEQ ID NO:113. In one aspect the
nucleic acid sequence of a CAR construct is SEQ ID NO:114. In one
aspect the nucleic acid sequence of a CAR construct is SEQ ID
NO:115. In one aspect the nucleic acid sequence of a CAR construct
is SEQ ID NO:116. In one aspect the nucleic acid sequence of a CAR
construct is SEQ ID NO:198. 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.
[0316] The present invention includes retroviral and lentiviral
vector constructs expressing a CAR that can be directly transduced
into a cell.
[0317] 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:35). 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.
[0318] Antigen Binding Domain
[0319] The CARs of the present invention comprise a target-specific
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.
[0320] 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.
[0321] In one aspect, the CAR of the present invention comprises a
binding domain that specifically binds CLL-1. In one aspect, the
antigen binding domain specifically binds human CLL-1.
[0322] 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 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. 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.
[0323] 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. Thus, in one aspect, the antigen
binding domain comprises a human antibody or an antibody
fragment.
[0324] Thus, in one aspect, the antigen binding domain comprises a
human antibody or an antibody fragment. In one embodiment, the
human anti-CLL-1 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 human
anti-CLL-1 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 human anti-CLL-1 binding domain described herein, e.g., a
human anti-CLL-1 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 human anti-CLL-1 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 human anti-CLL-1 binding domain described herein, e.g., the
human anti-CLL-1 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 human anti-CLL-1 binding
domain comprises a human light chain variable region described
herein (e.g., in Table 2) and/or a human heavy chain variable
region described herein (e.g., in Table 1). In one embodiment, the
human anti-CLL-1 binding domain comprises a human heavy chain
variable region described herein (e.g., in Table 1), e.g., at least
two human heavy chain variable regions described herein (e.g., in
Table 1). In one embodiment, the anti-CLL-1 binding domain is a
scFv comprising a light chain and a heavy chain of an amino acid
sequence of Table 2. In an embodiment, the anti-CLL-1 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, e.g., conservative
substitutions) but not more than 30, 20 or 10 modifications (e.g.,
substitutions, e.g., conservative substitutions) of an amino acid
sequence of a light chain variable region provided in Table 2, or a
sequence with 95-99% identity with an amino acid sequence of Table
2; and/or a heavy chain variable region comprising an amino acid
sequence having at least one, two or three modifications (e.g.,
substitutions, e.g., conservative substitutions) but not more than
30, 20 or 10 modifications (e.g., substitutions, e.g., conservative
substitutions) of an amino acid sequence of a heavy chain variable
region provided in Table 1, or a sequence with 95-99% identity to
an amino acid sequence of Table 1. In one embodiment, the human
anti-CLL-1 binding domain comprises a sequence selected from a
group consisting of SEQ ID NO:39-51, or a sequence with 95-99%
identity thereof. In one embodiment, the nucleic acid sequence
encoding the human anti-CLL-1 binding domain comprises a sequence
selected from a group consisting of SEQ ID NO:52-64, or a sequence
with 95-99% identity thereof. In one embodiment, the human
anti-CLL-1 binding domain is a scFv, and a light chain variable
region comprising an amino acid sequence described herein, e.g., in
Table 8, is attached to a heavy chain variable region comprising an
amino acid sequence described herein, e.g., in Table 8, via a
linker, e.g., a linker described herein. In one embodiment, the
human anti-CLL-1 binding domain includes a (Gly4-Ser)n linker,
wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:26).
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.
[0325] In one aspect, the antigen binding domain portion comprises
one or more sequence selected from SEQ ID NOs:39-41. In one aspect
the CAR is selected from one or more sequence selected from SEQ ID
NOs: 91-103, or 197.
[0326] In one aspect, the anti-CLL-1 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 CLL-1.
[0327] 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 CLL-1 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: 39-51. In one aspect,
the antigen binding domain comprises an amino acid sequence of an
scFv selected from SEQ ID NOs: 39-51. 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:1.
[0328] In one aspect, the human anti-CLL-1 binding domain is a
fragment, e.g., a single chain variable fragment (scFv). In one
aspect, the human anti-CLL-1 binding domain is a Fv, a Fab, a
(Fab').sub.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 CLL-1 protein or a fragment thereof with wild-type or
enhanced affinity.
[0329] In some instances a human scFv be derived from a display
library. A display library is a collection of entities; each entity
includes an accessible polypeptide component and a recoverable
component that encodes or identifies the polypeptide component. The
polypeptide component is varied so that different amino acid
sequences are represented. The polypeptide component can be of any
length, e.g. from three amino acids to over 300 amino acids. A
display library entity can include more than one polypeptide
component, for example, the two polypeptide chains of a Fab. In one
exemplary embodiment, a display library can be used to identify a
human CLL-1 binding domain. In a selection, the polypeptide
component of each member of the library is probed with CLL-1, or a
fragment there, and if the polypeptide component binds to CLL-1,
the display library member is identified, typically by retention on
a support.
[0330] Retained display library members are recovered from the
support and analyzed. The analysis can include amplification and a
subsequent selection under similar or dissimilar conditions. For
example, positive and negative selections can be alternated. The
analysis can also include determining the amino acid sequence of
the polypeptide component, i.e., the CLL-1 binding domain, and
purification of the polypeptide component for detailed
characterization.
[0331] A variety of formats can be used for display libraries.
Examples include the phage display. In phage display, the protein
component is typically covalently linked to a bacteriophage coat
protein. The linkage results from translation of a nucleic acid
encoding the protein component fused to the coat protein. The
linkage can include a flexible peptide linker, a protease site, or
an amino acid incorporated as a result of suppression of a stop
codon. Phage display is described, for example, in U.S. Pat. No.
5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO
91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO
92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem
274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;
Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al.
(2005) Nat Biotechnol. 23(3)344-8. Bacteriophage displaying the
protein component can be grown and harvested using standard phage
preparatory methods, e.g. PEG precipitation from growth media.
After selection of individual display phages, the nucleic acid
encoding the selected protein components can be isolated from cells
infected with the selected phages or from the phage themselves,
after amplification. Individual colonies or plaques can be picked,
the nucleic acid isolated and sequenced.
[0332] Other display formats include cell based display (see, e.g.,
WO 03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat.
No. 6,207,446), ribosome display (See, e.g., Mattheakis et al.
(1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000)
Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol.
328:404-30; and Schaffitzel et al. (1999) J Immunol Methods.
231(1-2):119-35), and E. coli periplasmic display (2005 Nov.
22;PMID: 16337958).
[0333] 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. WO2006/020258 and
WO2007/024715, is incorporated herein by reference.
[0334] An 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:25). In one embodiment, the linker can be
(Gly4Ser).sub.4 (SEQ ID NO:27) or (Gly4Ser).sub.3(SEQ ID NO:28).
Variation in the linker length may retain or enhance activity,
giving rise to superior efficacy in activity studies.
Exemplary Human CLL-1 CAR Constructs and Antigen Binding
Domains
[0335] Exemplary CLL-1 CAR constructs disclose herein comprise an
scFv (e.g., a human scFv as disclosed in Tables 8 herein,
optionally preceded with an optional leader sequence (e.g., SEQ ID
NO:1 and SEQ ID NO:12 for exemplary leader amino acid and
nucleotide sequences, respectively). The sequences of the human
scFv fragments (amino acid sequences of SEQ ID NOs:39-51, and
nucleotide sequences of SEQ ID NOs:52-64) are provided herein in
Table 8. The CLL-1 CAR construct can further include an optional
hinge domain, e.g., a CD8 hinge domain (e.g., including the amino
acid sequence of SEQ ID NO: 2 or encoded by a nucleic acid sequence
of SEQ ID NO:13); a transmembrane domain, e.g., a CD8 transmembrane
domain (e.g., including the amino acid sequence of SEQ ID NO: 6 or
encoded by the nucleotide sequence of SEQ ID NO: 17); an
intracellular domain, e.g., a 4-1BB intracellular domain (e.g.,
including the amino acid sequence of SEQ ID NO: 7 or encoded by the
nucleotide sequence of SEQ ID NO: 18; and a functional signaling
domain, e.g., a CD3 zeta domain (e.g., including amino acid
sequence of SEQ ID NO: 9 or 10, or encoded by the nucleotide
sequence of SEQ ID NO: 20 or 21). In certain embodiments, the
domains are contiguous with and in the same reading frame to form a
single fusion protein. In other embodiments, the domain are in
separate polypeptides, e.g., as in an RCAR molecule as described
herein.
[0336] In certain embodiments, the full length CLL-1 CAR molecule
includes the amino acid sequence of, or is encoded by the
nucleotide sequence of, CLL-1 CAR-1, CLL-1 CAR-2, CLL-1 CAR-3,
CLL-1 CAR-4, CLL-1 CAR-5, CLL-1 CAR-6, CL-L1 CAR-7, CLL-1 CAR-8,
CLL-1 CAR-9, CLL-1 CAR-10, CLL-1 CAR-11, CLL-1 CAR-12, CLL-1
CAR-13, 181268 provided in Table 8, or a sequence substantially
(e.g., 95-99%) identical thereto.
[0337] In certain embodiments, the CLL-1 CAR molecule, or the
anti-CLL-1 antigen binding domain, includes the scFv amino acid
sequence of CLL-1 CAR-1, CLL-1 CAR-2, CLL-1 CAR-3, CLL-1 CAR-4,
CLL-1 CAR-5, CLL-1 CAR-6, CL-L1 CAR-7, CLL-1 CAR-8, CLL-1 CAR-9,
CLL-1 CAR-10, CLL-1 CAR-11, CLL-1 CAR-12, CLL-1 CAR-13, 181268,
provided in Table 8; or includes the scFv amino acid sequence of,
or is encoded by the nucleotide sequence of, CLL-1 CAR-1, CLL-1
CAR-2, CLL-1 CAR-3, CLL-1 CAR-4, CLL-1 CAR-5, CLL-1 CAR-6, CL-L1
CAR-7, CLL-1 CAR-8, CLL-1 CAR-9, CLL-1 CAR-10, CLL-1 CAR-11, CLL-1
CAR-12, CLL-1 CAR-13, 181268, or a sequence substantially identical
(e.g., 95-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or
1 amino acid changes, e.g., substitutions (e.g., conservative
substitutions)) to any of the aforesaid sequences.
[0338] In certain embodiments, the CLL-1 CAR molecule, or the
anti-CLL-1 antigen binding domain, includes the heavy chain
variable region and/or the light chain variable region of CLL-1
CAR-1, CLL-1 CAR-2, CLL-1 CAR-3, CLL-1 CAR-4, CLL-1 CAR-5, CLL-1
CAR-6, CL-L1 CAR-7, CLL-1 CAR-8, CLL-1 CAR-9, CLL-1 CAR-10, CLL-1
CAR-11, CLL-1 CAR-12, CLL-1 CAR-13, 181268, provided in Table 8, or
a sequence substantially identical (e.g., 95-99% identical, or up
to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid changes, e.g.,
substitutions (e.g., conservative substitutions)) to any of the
aforesaid sequences.
[0339] In certain embodiments, the CLL-1 CAR molecule, or the
anti-CLL-1 antigen binding domain, includes one, two or three CDRs
from the heavy chain variable region (e.g., HCDR1, HCDR2 and/or
HCDR3), provided in Table 1; and/or one, two or three CDRs from the
light chain variable region (e.g., LCDR1, LCDR2 and/or LCDR3) of
CLL-1 CAR-1, CLL-1 CAR-2, CLL-1 CAR-3, CLL-1 CAR-4, CLL-1 CAR-5,
CLL-1 CAR-6, CL-L1 CAR-7, CLL-1 CAR-8, CLL-1 CAR-9, CLL-1 CAR-10,
CLL-1 CAR-11, CLL-1 CAR-12, CLL-1 CAR-13, 181268, provided in Table
2; or a sequence substantially identical (e.g., 95-99% identical,
or up to 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions
(e.g., conservative substitutions)) to any of the aforesaid
sequences.
[0340] In certain embodiments, the CLL-1 CAR molecule, or the
anti-CLL-1 antigen binding domain, includes one, two or three CDRs
from the heavy chain variable region (e.g., HCDR1, HCDR2 and/or
HCDR3), provided in Table 3; and/or one, two or three CDRs from the
light chain variable region (e.g., LCDR1, LCDR2 and/or LCDR3) of
CLL-1 CAR-1, CLL-1 CAR-2, CLL-1 CAR-3, CLL-1 CAR-4, CLL-1 CAR-5,
CLL-1 CAR-6, CL-L1 CAR-7, CLL-1 CAR-8, CLL-1 CAR-9, CLL-1 CAR-10,
CLL-1 CAR-11, CLL-1 CAR-12, CLL-1 CAR-13, 181268, provided in Table
4; or a sequence substantially identical (e.g., 95-99% identical,
or up to 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions
(e.g., conservative substitutions)) to any of the aforesaid
sequences.
[0341] In certain embodiments, the CLL-1 CAR molecule, or the
anti-CLL-1 antigen binding domain, includes one, two or three CDRs
from the heavy chain variable region (e.g., HCDR1, HCDR2 and/or
HCDR3), provided in Table 5; and/or one, two or three CDRs from the
light chain variable region (e.g., LCDR1, LCDR2 and/or LCDR3) of
CLL-1 CAR-1, CLL-1 CAR-2, CLL-1 CAR-3, CLL-1 CAR-4, CLL-1 CAR-5,
CLL-1 CAR-6, CL-L1 CAR-7, CLL-1 CAR-8, CLL-1 CAR-9, CLL-1 CAR-10,
CLL-1 CAR-11, CLL-1 CAR-12, CLL-1 CAR-13, 181268, provided in Table
6; or a sequence substantially identical (e.g., 95-99% identical,
or up to 5, 4, 3, 2, or 1 amino acid changes, e.g., substitutions
(e.g., conservative substitutions)) to any of the aforesaid
sequences.
[0342] The sequences of humanized CDR sequences of the scFv domains
are shown in Tables 1, 3, and 5 for the heavy chain variable
domains and in Tables 2, 4, and 6 for the light chain variable
domains. "ID" stands for the respective SEQ ID NO for each CDR.
[0343] The CDRs provided in Tables 1 and 2 are according to a
combination of the Kabat and Chothia numbering scheme.
TABLE-US-00001 TABLE 1 Heavy Chain Variable Domain CDRs Candi- date
HCDR1 ID HCDR2 ID HCDR3 ID CLL-1 ANTFSDHVMH 125 YIHAANGGTH 138
GGYNSDAFDI 151 CAR 9 YSQKFQD CLL-1 GGSFSGYYWS 122 EINHSGSTNY 135
GSGLVVYAIR 148 CAR 6 NPSLKS VGSGWFDY CLL-1 GFTFSSYSMN 126
YISSSSSTIY 139 DLSVRAIDAF 152 CAR 10 YADSVKG DI CLL-1 GFTFNSYGLH
127 LIEYDGSNKY 140 EGNEDLAFDI 153 CAR 11 YGDSVKG CLL-1 GFNVSSNYMT
128 VIYSGGATYY 141 DRLYCGNNCY 154 CAR 12 GDSVKG LYYYYGMDV CLL-1
GGTFSSYAIS 117 GIIPIFGTAN 130 DLEMATIMGG 143 CAR 1 YAQKFQ Y CLL-1
GFTFDDYAMH 118 LISGDGGSTY 131 VFDSYYMDV 144 CAR 2 YADSVKG CLL-1
GGSISSSSYY 119 SIYYSGSTYY 132 PGTYYDFLSG 145 CAR 3 WG NPSLKS YYPFY
CLL-1 GFTFSSYWMS 120 NINEDGSAKF 133 DLRSGRY 146 CAR 4 YVDSVKG CLL-1
GGPVRSGSHY 121 YIYYSGSTNY 134 GTATFDWNFP 147 CAR 5 WN NPSLEN FDS
CLL-1 GFTFSSYSMN 123 SISSSSSYIY 136 DPSSSGSYYM 149 CAR 7 YADSVKG
EDSYYYGMDV CLL-1 GFTFSSYEMN 124 YISSSGSTIY 137 EALGSSWE 150 CAR 8
YADSVKG CLL-1 GYPFTGYYIQ 129 WIDPNSGNTG 142 DSYGYYYGMD 155 CAR 13
YAQKFQG V 181268 GFTFSSYEMN 199 YISSSGSTIY 200 DPYSSSWHDA 201
YADSVKG FDI
TABLE-US-00002 TABLE 2 Light Chain Variable Domain CDRs Candi- date
LCDR1 ID LCDR2 ID LCDR3 ID CLL-1 RASQDISSWL 164 AASSLQS 177
QQSYSTPLT 190 CAR 9 A CLL-1 RASQSISSYL 161 AASSLQS 174 QQSYSTPPWT
187 CAR 6 N CLL-1 QASQDISNYL 165 DASNLET 178 QQAYSTPFT 191 CAR 10 N
CLL-1 QASQFIKKNL 166 DASSLQT 179 QQHDNLPLT 192 CAR 11 N CLL-1
RASQSISSYL 167 AASSLQS 180 QQSYSTPPLT 193 CAR 12 N CLL-1 TGTSSDVGGY
156 DVSNRPS 169 SSYTSSSTLD 182 CAR 1 NYVS VV CLL-1 RSSQSLVYTD 157
KVSNRDS 170 MQGTHWSFT 183 CAR 2 GNTYLN CLL-1 RASQGISSYL 158 AASTLQS
171 QQLNSYPYT 184 CAR 3 A CLL-1 RASQSISGSF 159 GASSRAT 172
QQYGSSPPT 185 CAR 4 LA CLL-1 RASQSISSYL 160 AASSLQS 173 QQSYSTPWT
186 CAR 5 N CLL-1 TGSSGSIASN 162 EDNQRPS 175 QSYDSSNQVV 188 CAR 7
YVQ CLL-1 QASQDISNYL 163 DASNLET 176 QQYDNLPLT 189 CAR 8 N CLL-1
RASQGISSAL 168 DASSLES 181 QQFNNYPLT 194 CAR 13 A 181268 RASQSVSSSY
202 GASSRAT 203 QQYGSSPLT 204 LA
TABLE-US-00003 TABLE 3 Heavy Chain Variable Domain CDRs according
to the Kabat numbering scheme (Kabat et al. (1991), "Sequences of
Proteins of Immunological Inter- est," 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, MD) Candi- date
HCDR1 ID HCDR2 ID HCDR3 ID 146259- DHVMH 322 YIHAANGGTH 336
GGYNSDAFDI 350 CLL-1- YSQKFQD CAR9 139119- GYYWS 319 EINHSGSTNY 333
GSGLVVYAIR 347 CLL-1- NPSLKS VGSGWFDY CAR6 146261- SYSMN 323
YISSSSSTIY 337 DLSVRAIDAF 351 CLL-1- YADSVKG DI CAR10 146262- SYGLH
324 LIEYDGSNKY 338 EGNEDLAFDI 352 CLL-1- YGDSVKG CAR11 146263-
SNYMT 325 VIYSGGATYY 339 DRLYCGNNCY 353 CLL-1- GDSVKG LYYYYGMDV
CAR12 139115- SYAIS 314 GIIPIFGTAN 328 DLEMATIMGG 342 CLL-1-
YAQKFQG Y CAR1 139116- DYAMH 315 LISGDGGSTY 329 VFDSYYMDV 343
CLL-1- YADSVKG CAR2 139118- SSSYYWG 316 SIYYSGSTYY 330 PGTYYDFLSG
344 CLL-1- NPSLKS YYPFY CAR3 139122- SYWMS 317 NINEDGSAKF 331
DLRSGRY 345 CLL-1- YVDSVKG CAR4 139117- SGSHYWN 318 YIYYSGSTNY 332
GTATFDWNFP 346 CLL-1- NPSLEN FDS CAR5 139120- SYSMN 320 SISSSSSYIY
334 DPSSSGSYYM 348 CLL-1- YADSVKG EDSYYYGMDV CAR7 139121- SYEMN 321
YISSSGSTIY 335 EALGSSWE 349 CLL-1- YADSVKG CAR8 146264- GYYIQ 326
WIDPNSGNTG 340 DSYGYYYGMD 354 CLL-1- YAQKFQG V CAR13 181268 SYEMN
327 YISSSGSTIY 341 DPYSSSWHDA 355 YADSVKG FDI
TABLE-US-00004 TABLE 4 Light Chain Variable Domain CDRs according
to the Kabat numbering scheme (Kabat et al. (1991), "Sequences of
Proteins of Immuno- logical Interest," 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, MD) Candi- date
LCDR1 ID LCDR2 ID LCDR3 ID 146259- RASQDISSWL 364 AASSLQS 378
QQSYSTPLT 392 CLL-1- A CAR9 139119- RASQSISSYL 361 AASSLQS 375
QQSYSTPPWT 389 CLL-1- N CAR6 146261- QASQDISNYL 365 DASNLET 379
QQAYSTPFT 393 CLL-1- N CAR10 146262- QASQFIKKNL 366 DASSLQT 380
QQHDNLPLT 394 CLL-1- N CAR11 146263- RASQSISSYL 367 AASSLQS 381
QQSYSTPPLT 395 CLL-1- N CAR12 139115- TGTSSDVGGY 356 DVSNRPS 370
SSYTSSSTLD 384 CLL-1- NYVS VV CAR1 139116- RSSQSLVYTD 357 KVSNRDS
371 MQGTHWSFT 385 CLL-1- GNTYLN CAR2 139118- RASQGISSYL 358 AASTLQS
372 QQLNSYPYT 386 CLL-1- A CAR3 139122- RASQSISGSF 359 GASSRAT 373
QQYGSSPPT 387 CLL-1- LA CAR4 139117- RASQSISSYL 360 AASSLQS 374
QQSYSTPWT 388 CLL-1- N CAR5 139120- TGSSGSIASN 362 EDNQRPS 376
QSYDSSNQVV 390 CLL-1- YVQ CAR7 139121- QASQDISNYL 363 DASNLET 377
QQYDNLPLT 391 CLL-1- N CAR8 146264- RASQGISSAL 368 DASSLES 382
QQFNNYPLT 396 CLL-1- A CAR13 181268 RASQSVSSSY 369 GASSRAT 383
QQYGSSPLT 397 LA
TABLE-US-00005 TABLE 5 Heavy Chain Variable Domain CDRs according
to the Chothia numbering scheme (Al-Lazikani et al., (1997) JMB
273, 927-948) Candidate HCDR1 ID HCDR2 ID HCDR3 ID 146259- ANTFSDH
406 HAANGG 420 GGYNSDAFDI 434 CLL-1- CAR9 139119- GGSFSGY 403 NHSGS
417 GSGLVVYAIR 431 CLL-1- VGSGWFDY CAR6 146261- GFTFSSY 407 SSSSST
421 DLSVRAIDAF 435 CLL-1- DI CAR10 146262- GFTFNSY 408 EYDGSN 422
EGNEDLAFDI 436 CLL-1- CAR11 146263- GFNVSSN 409 YSGGA 423
DRLYCGNNCY 437 CLL-1- LYYYYGMDV CAR12 139115- GGTFSSY 398 IPIFGT
412 DLEMATIMGG 426 CLL-1- Y CAR1 139116- GFTFDDY 399 SGDGGS 413
VFDSYYMDV 427 CLL-1- CAR2 139118- GGSISSSSY 400 YYSGS 414
PGTYYDFLSG 428 CLL-1- YYPFY CAR3 139122- GFTFSSY 401 NEDGSA 415
DLRSGRY 429 CLL-1- CAR4 139117- GGPVRSGSH 402 YYSGS 416 GTATFDWNFP
430 CLL-1- FDS CAR5 139120- GFTFSSY 404 SSSSSY 418 DPSSSGSYYM 432
CLL-1- EDSYYYGMDV CAR7 139121- GFTFSSY 405 SSSGST 419 EALGSSWE 433
CLL-1- CAR8 146264- GYPFTGY 410 DPNSGN 424 DSYGYYYGMD 438 CLL-1- V
CAR13 181268 GFTFSSY 411 SSSGST 425 DPYSSSWHDA 439 FDI
TABLE-US-00006 TABLE 6 Light Chain Variable Domain CDRs according
to the Chothia numbering scheme (Al-Lazikani et al., (1997) JMB
273, 927-948) Candi- date LCDR1 ID LCDR2 ID LCDR3 ID 146259-
SQDISSW 448 AAS 462 SYSTPL 476 CLL-1- CAR9 139119- SQSISSY 445 AAS
459 SYSTPPW 473 CLL-1- CAR6 146261- SQDISNY 449 DAS 463 AYSTPF 477
CLL-1- CAR10 146262- SQFIKKN 450 DAS 464 HDNLPL 478 CLL-1- CAR11
146263- SQSISSY 451 AAS 465 SYSTPPL 479 CLL-1- CAR12 139115-
TSSDVGGYNY 440 DVS 454 YTSSSTLDV 468 CLL-1- CAR1 139116-
SQSLVYTDGNTY 441 KVS 455 GTHWSF 469 CLL-1- CAR2 139118- SQGISSY 442
AAS 456 LNSYPY 470 CLL-1- CAR3 139122- SQSISGSF 443 GAS 457 YGSSPP
471 CLL-1- CAR4 139117- SQSISSY 444 AAS 458 SYSTPW 472 CLL-1- CAR5
139120- SSGSIASNY 446 EDN 460 YDSSNQV 474 CLL-1- CAR7 139121-
SQDISNY 447 DAS 461 YDNLPL 475 CLL-1- CAR8 146264- SQGISSA 452 DAS
466 FNNYPL 480 CLL-1- CAR13 181268 SQSVSSSY 453 GAS 467 YGSSPL
481
[0344] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0345] (i) a LC CDR1 of SEQ ID NO: 156, LC CDR2 of SEQ ID NO: 169
and LC CDR3 of SEQ ID NO: 182 of CLL-1 CAR-1;
[0346] (ii) a LC CDR1 of SEQ ID NO: 157, LC CDR2 of SEQ ID NO: 170
and LC CDR3 of SEQ ID NO: 183 of CLL-1 CAR-2;
[0347] (iii) a LC CDR1 of SEQ ID NO: 158, LC CDR2 of SEQ ID NO: 171
and LC CDR3 of SEQ ID NO: 184 of CLL-1 CAR-3;
[0348] (iv) a LC CDR1 of SEQ ID NO: 159, LC CDR2 of SEQ ID NO: 172
and LC CDR3 of SEQ ID NO: 185 of CLL-1 CAR-4;
[0349] (v) a LC CDR1 of SEQ ID NO: 160, LC CDR2 of SEQ ID NO: 173
and LC CDR3 of SEQ ID NO: 186 of CLL-1 CAR-5;
[0350] (vi) a LC CDR1 of SEQ ID NO: 161, LC CDR2 of SEQ ID NO: 174
and LC CDR3 of SEQ ID NO: 187 of CLL-1 CAR-6;
[0351] (vii) a LC CDR1 of SEQ ID NO: 162, LC CDR2 of SEQ ID NO: 175
and LC CDR3 of SEQ ID NO: 188 of CLL-1 CAR-7;
[0352] (viii) a LC CDR1 of SEQ ID NO: 163, LC CDR2 of SEQ ID NO:
176 and LC CDR3 of SEQ ID NO: 189 of CLL-1 CAR-8; or
[0353] (ix) a LC CDR1 of SEQ ID NO: 164, LC CDR2 of SEQ ID NO: 177
and LC CDR3 of SEQ ID NO: 190 of CLL-1 CAR-9;
[0354] (x) a LC CDR1 of SEQ ID NO: 165, LC CDR2 of SEQ ID NO: 178
and LC CDR3 of SEQ ID NO: 191 of CLL-1 CAR-10;
[0355] (xi) a LC CDR1 of SEQ ID NO: 166, LC CDR2 of SEQ ID NO: 179
and LC CDR3 of SEQ ID NO: 192 of CLL-1 CAR-11;
[0356] (xii) a LC CDR1 of SEQ ID NO: 167, LC CDR2 of SEQ ID NO: 180
and LC CDR3 of SEQ ID NO: 193 of CLL-1 CAR-12;
[0357] (xiii) a LC CDR1 of SEQ ID NO: 168, LC CDR2 of SEQ ID NO:
181 and LC CDR3 of SEQ ID NO: 194 of CLL-1 CAR-13;
[0358] (xiv) a LC CDR1 of SEQ ID NO: 202, LC CDR2 of SEQ ID NO: 203
and LC CDR3 of SEQ ID NO: 204 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0359] (i) a HC CDR1 of SEQ ID NO: 117, HC CDR2 of SEQ ID NO: 130
and HC CDR3 of SEQ ID NO: 143 of CLL-1 CAR-1;
[0360] (ii) a HC CDR1 of SEQ ID NO: 118, HC CDR2 of SEQ ID NO: 131
and HC CDR3 of SEQ ID NO: 144 of CLL-1 CAR-2;
[0361] (iii) a HC CDR1 of SEQ ID NO: 119, HC CDR2 of SEQ ID NO: 132
and HC CDR3 of SEQ ID NO: 145 of CLL-1 CAR-3;
[0362] (iv) a HC CDR1 of SEQ ID NO: 120, HC CDR2 of SEQ ID NO: 133
and HC CDR3 of SEQ ID NO: 146 of CLL-1 CAR-4;
[0363] (v) a HC CDR1 of SEQ ID NO: 121, HC CDR2 of SEQ ID NO: 134
and HC CDR3 of SEQ ID NO: 147 of CLL-1 CAR-5;
[0364] (vi) a HC CDR1 of SEQ ID NO: 122, HC CDR2 of SEQ ID NO: 135
and HC CDR3 of SEQ ID NO: 148 of CLL-1 CAR-6;
[0365] (vii) a HC CDR1 of SEQ ID NO: 123, HC CDR2 of SEQ ID NO: 136
and HC CDR3 of SEQ ID NO: 149 of CLL-1 CAR-7;
[0366] (viii) a HC CDR1 of SEQ ID NO: 124, HC CDR2 of SEQ ID NO:
137 and HC CDR3 of SEQ ID NO: 150 of CLL-1 CAR-8; or
[0367] (ix) a HC CDR1 of SEQ ID NO: 125, HC CDR2 of SEQ ID NO: 138
and HC CDR3 of SEQ ID NO: 151 of CLL-1 CAR-9;
[0368] (x) a HC CDR1 of SEQ ID NO: 126, HC CDR2 of SEQ ID NO: 139
and HC CDR3 of SEQ ID NO: 152 of CLL-1 CAR-10;
[0369] (xi) a HC CDR1 of SEQ ID NO: 127, HC CDR2 of SEQ ID NO: 140
and HC CDR3 of SEQ ID NO: 153 of CLL-1 CAR-11;
[0370] (xii) a HC CDR1 of SEQ ID NO: 128, HC CDR2 of SEQ ID NO: 141
and HC CDR3 of SEQ ID NO: 154 of CLL-1 CAR-12;
[0371] (xiii) a HC CDR1 of SEQ ID NO: 129, HC CDR2 of SEQ ID NO:
142 and HC CDR3 of SEQ ID NO: 155 of CLL-1 CAR-13;
[0372] (xiv) a HC CDR1 of SEQ ID NO: 199, HC CDR2 of SEQ ID NO: 200
and HC CDR3 of SEQ ID NO: 201 of 181286.
[0373] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) or a CLL-1
binding domain includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0374] (i) a LC CDR1 of SEQ ID NO: 356, LC CDR2 of SEQ ID NO: 370
and LC CDR3 of SEQ ID NO: 384 of CLL-1 CAR-1;
[0375] (ii) a LC CDR1 of SEQ ID NO: 357, LC CDR2 of SEQ ID NO: 371
and LC CDR3 of SEQ ID NO: 385 of CLL-1 CAR-2;
[0376] (iii) a LC CDR1 of SEQ ID NO: 358, LC CDR2 of SEQ ID NO: 372
and LC CDR3 of SEQ ID NO: 386 of CLL-1 CAR-3;
[0377] (iv) a LC CDR1 of SEQ ID NO: 359, LC CDR2 of SEQ ID NO: 373
and LC CDR3 of SEQ ID NO: 387 of CLL-1 CAR-4;
[0378] (v) a LC CDR1 of SEQ ID NO: 360, LC CDR2 of SEQ ID NO: 374
and LC CDR3 of SEQ ID NO: 388 of CLL-1 CAR-5;
[0379] (vi) a LC CDR1 of SEQ ID NO: 361, LC CDR2 of SEQ ID NO: 375
and LC CDR3 of SEQ ID NO: 389 of CLL-1 CAR-6;
[0380] (vii) a LC CDR1 of SEQ ID NO: 362, LC CDR2 of SEQ ID NO: 376
and LC CDR3 of SEQ ID NO: 390 of CLL-1 CAR-7;
[0381] (viii) a LC CDR1 of SEQ ID NO: 363, LC CDR2 of SEQ ID NO:
377 and LC CDR3 of SEQ ID NO: 391 of CLL-1 CAR-8; or
[0382] (ix) a LC CDR1 of SEQ ID NO: 364, LC CDR2 of SEQ ID NO: 378
and LC CDR3 of SEQ ID NO: 392 of CLL-1 CAR-9;
[0383] (x) a LC CDR1 of SEQ ID NO: 365, LC CDR2 of SEQ ID NO: 379
and LC CDR3 of SEQ ID NO: 393 of CLL-1 CAR-10;
[0384] (xi) a LC CDR1 of SEQ ID NO: 366, LC CDR2 of SEQ ID NO: 380
and LC CDR3 of SEQ ID NO: 394 of CLL-1 CAR-11;
[0385] (xii) a LC CDR1 of SEQ ID NO: 367, LC CDR2 of SEQ ID NO: 381
and LC CDR3 of SEQ ID NO: 395 of CLL-1 CAR-12;
[0386] (xiii) a LC CDR1 of SEQ ID NO: 368, LC CDR2 of SEQ ID NO:
382 and LC CDR3 of SEQ ID NO: 396 of CLL-1 CAR-13;
[0387] (xiv) a LC CDR1 of SEQ ID NO: 369, LC CDR2 of SEQ ID NO: 383
and LC CDR3 of SEQ ID NO: 397 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0388] (i) a HC CDR1 of SEQ ID NO: 314, HC CDR2 of SEQ ID NO: 328
and HC CDR3 of SEQ ID NO: 342 of CLL-1 CAR-1;
[0389] (ii) a HC CDR1 of SEQ ID NO: 315, HC CDR2 of SEQ ID NO: 329
and HC CDR3 of SEQ ID NO: 343 of CLL-1 CAR-2;
[0390] (iii) a HC CDR1 of SEQ ID NO: 316, HC CDR2 of SEQ ID NO: 330
and HC CDR3 of SEQ ID NO: 344 of CLL-1 CAR-3;
[0391] (iv) a HC CDR1 of SEQ ID NO: 317, HC CDR2 of SEQ ID NO: 331
and HC CDR3 of SEQ ID NO: 345 of CLL-1 CAR-4;
[0392] (v) a HC CDR1 of SEQ ID NO: 318, HC CDR2 of SEQ ID NO: 332
and HC CDR3 of SEQ ID NO: 346 of CLL-1 CAR-5;
[0393] (vi) a HC CDR1 of SEQ ID NO: 319, HC CDR2 of SEQ ID NO: 333
and HC CDR3 of SEQ ID NO: 347 of CLL-1 CAR-6;
[0394] (vii) a HC CDR1 of SEQ ID NO: 320, HC CDR2 of SEQ ID NO: 334
and HC CDR3 of SEQ ID NO: 348 of CLL-1 CAR-7;
[0395] (viii) a HC CDR1 of SEQ ID NO: 321, HC CDR2 of SEQ ID NO:
335 and HC CDR3 of SEQ ID NO: 349 of CLL-1 CAR-8; or
[0396] (ix) a HC CDR1 of SEQ ID NO: 322, HC CDR2 of SEQ ID NO: 336
and HC CDR3 of SEQ ID NO: 350 of CLL-1 CAR-9;
[0397] (x) a HC CDR1 of SEQ ID NO: 323, HC CDR2 of SEQ ID NO: 337
and HC CDR3 of SEQ ID NO: 351 of CLL-1 CAR-10;
[0398] (xi) a HC CDR1 of SEQ ID NO: 324, HC CDR2 of SEQ ID NO: 338
and HC CDR3 of SEQ ID NO: 352 of CLL-1 CAR-11;
[0399] (xii) a HC CDR1 of SEQ ID NO: 325, HC CDR2 of SEQ ID NO: 339
and HC CDR3 of SEQ ID NO: 353 of CLL-1 CAR-12;
[0400] (xiii) a HC CDR1 of SEQ ID NO: 326, HC CDR2 of SEQ ID NO:
340 and HC CDR3 of SEQ ID NO: 354 of CLL-1 CAR-13;
[0401] (xiv) a HC CDR1 of SEQ ID NO: 327, HC CDR2 of SEQ ID NO: 341
and HC CDR3 of SEQ ID NO: 355 of 181286.
[0402] In certain embodiments, the CAR molecule described herein
(e.g., the CAR nucleic acid or the CAR polypeptide) includes:
(1) one, two, or three light chain (LC) CDRs chosen from one of the
following:
[0403] (i) a LC CDR1 of SEQ ID NO: 440, LC CDR2 of SEQ ID NO: 454
and LC CDR3 of SEQ ID NO: 468 of CLL-1 CAR-1;
[0404] (ii) a LC CDR1 of SEQ ID NO: 441, LC CDR2 of SEQ ID NO: 455
and LC CDR3 of SEQ ID NO: 469 of CLL-1 CAR-2;
[0405] (iii) a LC CDR1 of SEQ ID NO: 442, LC CDR2 of SEQ ID NO: 456
and LC CDR3 of SEQ ID NO: 470 of CLL-1 CAR-3;
[0406] (iv) a LC CDR1 of SEQ ID NO: 443, LC CDR2 of SEQ ID NO: 457
and LC CDR3 of SEQ ID NO: 471 of CLL-1 CAR-4;
[0407] (v) a LC CDR1 of SEQ ID NO: 444, LC CDR2 of SEQ ID NO: 458
and LC CDR3 of SEQ ID NO: 472 of CLL-1 CAR-5;
[0408] (vi) a LC CDR1 of SEQ ID NO: 445, LC CDR2 of SEQ ID NO: 459
and LC CDR3 of SEQ ID NO: 473 of CLL-1 CAR-6;
[0409] (vii) a LC CDR1 of SEQ ID NO: 446, LC CDR2 of SEQ ID NO: 460
and LC CDR3 of SEQ ID NO: 474 of CLL-1 CAR-7;
[0410] (viii) a LC CDR1 of SEQ ID NO: 447, LC CDR2 of SEQ ID NO:
461 and LC CDR3 of SEQ ID NO: 475 of CLL-1 CAR-8; or
[0411] (ix) a LC CDR1 of SEQ ID NO: 448, LC CDR2 of SEQ ID NO: 462
and LC CDR3 of SEQ ID NO: 476 of CLL-1 CAR-9;
[0412] (x) a LC CDR1 of SEQ ID NO: 449, LC CDR2 of SEQ ID NO: 463
and LC CDR3 of SEQ ID NO: 477 of CLL-1 CAR-10;
[0413] (xi) a LC CDR1 of SEQ ID NO: 450, LC CDR2 of SEQ ID NO: 464
and LC CDR3 of SEQ ID NO: 478 of CLL-1 CAR-11;
[0414] (xii) a LC CDR1 of SEQ ID NO: 451, LC CDR2 of SEQ ID NO: 465
and LC CDR3 of SEQ ID NO: 479 of CLL-1 CAR-12;
[0415] (xiii) a LC CDR1 of SEQ ID NO: 452, LC CDR2 of SEQ ID NO:
466 and LC CDR3 of SEQ ID NO: 480 of CLL-1 CAR-13;
[0416] (xiv) a LC CDR1 of SEQ ID NO: 453, LC CDR2 of SEQ ID NO: 467
and LC CDR3 of SEQ ID NO: 481 of 181286; and/or
(2) one, two, or three heavy chain (HC) CDRs from one of the
following:
[0417] (i) a HC CDR1 of SEQ ID NO: 398, HC CDR2 of SEQ ID NO: 412
and HC CDR3 of SEQ ID NO: 426 of CLL-1 CAR-1;
[0418] (ii) a HC CDR1 of SEQ ID NO: 399, HC CDR2 of SEQ ID NO: 413
and HC CDR3 of SEQ ID NO: 427 of CLL-1 CAR-2;
[0419] (iii) a HC CDR1 of SEQ ID NO: 400, HC CDR2 of SEQ ID NO: 414
and HC CDR3 of SEQ ID NO: 428 of CLL-1 CAR-3;
[0420] (iv) a HC CDR1 of SEQ ID NO: 401, HC CDR2 of SEQ ID NO: 415
and HC CDR3 of SEQ ID NO: 429 of CLL-1 CAR-4;
[0421] (v) a HC CDR1 of SEQ ID NO: 402, HC CDR2 of SEQ ID NO: 416
and HC CDR3 of SEQ ID NO: 430 of CLL-1 CAR-5;
[0422] (vi) a HC CDR1 of SEQ ID NO: 403, HC CDR2 of SEQ ID NO: 417
and HC CDR3 of SEQ ID NO: 431 of CLL-1 CAR-6;
[0423] (vii) a HC CDR1 of SEQ ID NO: 404, HC CDR2 of SEQ ID NO: 418
and HC CDR3 of SEQ ID NO: 432 of CLL-1 CAR-7;
[0424] (viii) a HC CDR1 of SEQ ID NO: 405, HC CDR2 of SEQ ID NO:
419 and HC CDR3 of SEQ ID NO: 433 of CLL-1 CAR-8; or
[0425] (ix) a HC CDR1 of SEQ ID NO: 406, HC CDR2 of SEQ ID NO: 420
and HC CDR3 of SEQ ID NO: 434 of CLL-1 CAR-9;
[0426] (x) a HC CDR1 of SEQ ID NO: 407, HC CDR2 of SEQ ID NO: 421
and HC CDR3 of SEQ ID NO: 435 of CLL-1 CAR-10;
[0427] (xi) a HC CDR1 of SEQ ID NO: 408, HC CDR2 of SEQ ID NO: 422
and HC CDR3 of SEQ ID NO: 436 of CLL-1 CAR-11;
[0428] (xii) a HC CDR1 of SEQ ID NO: 409, HC CDR2 of SEQ ID NO: 423
and HC CDR3 of SEQ ID NO: 437 of CLL-1 CAR-12;
[0429] (xiii) a HC CDR1 of SEQ ID NO: 410, HC CDR2 of SEQ ID NO:
424 and HC CDR3 of SEQ ID NO: 438 of CLL-1 CAR-13;
[0430] (xiv) a HC CDR1 of SEQ ID NO: 411, HC CDR2 of SEQ ID NO: 425
and HC CDR3 of SEQ ID NO: 439 of 181286.
[0431] In embodiments, fully human anti-CLL-1 single chain variable
fragments are generated and cloned into lentiviral CAR expression
vectors with the intracellular CD3zeta domain and the intracellular
co-stimulatory domain of 4-1BBNames of exemplary fully human CLL-1
scFvs are depicted in Table 7.
TABLE-US-00007 TABLE 7 CAR-CLL-1 constructs Construct ID Nickname
139115 CLL-1 (or CLL-1 CAR-1) 139116 CLL-2 (or CLL-1 CAR-2) 139117
CLL-3 (or CLL-1 CAR-3) 139118 CLL-4 (or CLL-1 CAR-4) 139119 CLL-5
(or CLL-1 CAR-5) 139120 CLL-6 (or CLL-1 CAR-6) 139121 CLL-7 (or
CLL-1 CAR-7) 139122 CLL-8 (or CLL-1 CAR-8) 146259 CLL-9 (or CLL-1
CAR-9) 146261 CLL-10 (or CLL-1 CAR-10) 146262 CLL-11 (or CLL-1
CAR-11) 146263 CLL-12 (or CLL-1 CAR-12) 146264 CLL-13 (or CLL-1
CAR-13)
[0432] In embodiments, the order in which the VL and VH domains
appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation),
and where either three or four copies of the "G4S" (SEQ ID NO:25)
subunit, in which each subunit comprises the sequence GGGGS (SEQ ID
NO:25) (e.g., (G4S).sub.3 (SEQ ID NO:28) or (G4S).sub.4(SEQ ID
NO:27)), connect the variable domains to create the entirety of the
scFv domain, as shown in Table 8.
[0433] The amino acid and nucleic acid sequences of the CLL-1 scFv
domains and CLL-1 CAR molecules are provided in Table 8. The amino
acid sequences for the variable heavy chain and variable light
chain for each scFv is also provided in Table 8 It is noted that
the scFv fragments (SEQ ID NOs: 39-51) with a leader sequence
(e.g., the amino acid sequence of SEQ ID NO: 1 or the nucleotide
sequence of SEQ ID NO: 12) are also encompassed by the present
invention.
TABLE-US-00008 Leader (amino acid sequence) (SEQ ID NO: 1)
MALPVTALLLPLALLLHAARP Leader (nucleic acid sequence) (SEQ ID NO:
12) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAG
ACCC CD8 hinge (amino acid sequence) (SEQ ID NO: 2)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 hinge (nucleic
acid sequence) (SEQ ID NO: 13)
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCC
TGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG
GGCTGGACTTCGCCTGTGA CD8 transmembrane (amino acid sequence) (SEQ ID
NO: 6) IYIWAPLAGTCGVLLLSLVITLYC CD8 transmembrane (nucleic acid
sequence) (SEQ ID NO: 17)
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATC
ACCCTTTACTGC 4-1BB Intracellular domain (amino acid sequence) (SEQ
ID NO: 7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB
Intracellular domain (nucleic acid sequence) (SEQ ID NO: 18)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC
AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG
GATGTGAACTG CD28 Intracellular domain (amino acid sequence) (SEQ ID
NO: 482) (SEQ ID NO: 482) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 Intracellular domain (nucleotide sequence) (SEQ ID NO: 483)
(SEQ ID NO: 483)
AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCG
CCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT
CGCAGCCTATCGCTCC ICOS Intracellular domain (amino acid sequence)
(SEQ ID NO: 484) (SEQ ID NO: 484) T K K K Y S S S V H D P N G E Y M
F M R A V N T A K K S R L T D V T L ICOS Intracellular domain
(nucleotide sequence) (SEQ ID NO: 485) (SEQ ID NO: 485)
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGA
GAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA CD3 zeta domain
(amino acid sequence) (SEQ ID NO: 9)
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta
(nucleic acid sequence) (SEQ ID NO: 20)
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAG
CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC
GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC
TGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA
AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAG
CCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC CD3 zeta domain
(amino acid sequence; NCBI Reference Sequence NM_000734.3) (SEQ ID
NO: 10) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta
(nucleic acid sequence; NCBI Reference Sequence NM_000734.3); (SEQ
ID NO: 21) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG
AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT
TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG
AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC
ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGC IgG4 Hinge (amino acid sequence) (SEQ
ID NO: 36)
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge
(nucleotide sequence) (SEQ ID NO: 37)
GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACC
CAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCG
AGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTT
CAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAAC
GGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAA
ACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTA
GCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTA
CCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA
GACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCG
TGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGC
CCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG
[0434] In embodiments, these clones (e.g., in Table 8) all
contained a Q/K residue change in the signal domain of the
co-stimulatory domain derived from CD3zeta chain.
TABLE-US-00009 TABLE 8 Amino Acid and Nucleic Acid Sequences of the
anti-CLL-1 scFv domains and CLL-1 CAR molecules SEQ Name/ ID
Description NO: Sequence 146259 146259- aa 47
QVQLVQSGAEVKEPGASVKVSCKAPANTFSDHVMHWVRQAPGQRFEWMGY ScFv domain
IHAANGGTHYSQKFQDRVTITRDTSANTVYMDLSSLRSEDTAVYYCARGG CLL-1 CAR 9
YNSDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSV
SASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSR
FNGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK 146259- nt 60
CAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTCAAGGAACCCGGAGCCTC ScFv domain
CGTGAAAGTGTCCTGCAAAGCTCCTGCCAACACTTTCTCGGACCACGTGA CLL-1 CAR 9
TGCACTGGGTGCGCCAGGCGCCGGGCCAGCGCTTCGAATGGATGGGATAC
ATTCATGCCGCCAATGGCGGTACCCACTACTCCCAAAAGTTCCAGGATAG
AGTCACCATCACCCGGGACACCAGCGCCAACACCGTGTATATGGATCTGT
CCAGCCTGAGGTCCGAGGATACCGCCGTGTACTACTGCGCCCGGGGCGGA
TACAACTCAGACGCGTTCGACATTTGGGGACAGGGTACTATGGTCACCGT
GTCATCCGGGGGCGGTGGCAGCGGGGGCGGAGGCTCTGGCGGAGGCGGAT
CAGGGGGAGGAGGGTCCGACATCGTGATGACCCAGTCCCCGTCATCGGTG
TCCGCGTCCGTGGGAGACAGAGTGACCATCACGTGTCGCGCCAGCCAGGA
CATCTCCTCGTGGTTGGCATGGTACCAGCAGAAGCCTGGAAAGGCCCCGA
AGCTGCTCATCTACGCCGCCTCCTCCCTTCAATCGGGAGTGCCCTCGCGG
TTCAACGGAAGCGGAAGCGGGACAGATTTTACCCTGACTATTAGCTCGCT
GCAGCCCGAGGACTTCGCTACTTACTACTGCCAACAGAGCTACTCCACCC
CACTGACTTTCGGCGGGGGTACCAAGGTCGAGATCAAG 146259- aa 73
QVQLVQSGAEVKEPGASVKVSCKAPANTFSDHVMHWVRQAPGQRFEWMGY VH of ScFv
IHAANGGTHYSQKFQDRVTITRDTSANTVYMDLSSLRSEDTAVYYCARGG CLL-1 CAR 9
YNSDAFDIWGQGTMVTVSS 146259- aa 86
DIVMTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYA VL of ScFv
ASSLQSGVPSRFNGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG CLL-1 CAR 9
GTKVEIK 146259- aa 99
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKEPGASVKVSCKAPANTF Full CAR
SDHVMHWVRQAPGQRFEWMGYIHAANGGTHYSQKFQDRVTITRDTSANTV CLL-1 CAR 9
YMDLSSLRSEDTAVYYCARGGYNSDAFDIWGQGTMVTVSSGGGGSGGGGS
GGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKP
GKAPKLLIYAASSLQSGVPSRFNGSGSGTDFTLTISSLQPEDFATYYCQQ
SYSTPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 146259- nt 112
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTCAAGG CLL-1 CAR 9
AACCCGGAGCCTCCGTGAAAGTGTCCTGCAAAGCTCCTGCCAACACTTTC
TCGGACCACGTGATGCACTGGGTGCGCCAGGCGCCGGGCCAGCGCTTCGA
ATGGATGGGATACATTCATGCCGCCAATGGCGGTACCCACTACTCCCAAA
AGTTCCAGGATAGAGTCACCATCACCCGGGACACCAGCGCCAACACCGTG
TATATGGATCTGTCCAGCCTGAGGTCCGAGGATACCGCCGTGTACTACTG
CGCCCGGGGCGGATACAACTCAGACGCGTTCGACATTTGGGGACAGGGTA
CTATGGTCACCGTGTCATCCGGGGGCGGTGGCAGCGGGGGCGGAGGCTCT
GGCGGAGGCGGATCAGGGGGAGGAGGGTCCGACATCGTGATGACCCAGTC
CCCGTCATCGGTGTCCGCGTCCGTGGGAGACAGAGTGACCATCACGTGTC
GCGCCAGCCAGGACATCTCCTCGTGGTTGGCATGGTACCAGCAGAAGCCT
GGAAAGGCCCCGAAGCTGCTCATCTACGCCGCCTCCTCCCTTCAATCGGG
AGTGCCCTCGCGGTTCAACGGAAGCGGAAGCGGGACAGATTTTACCCTGA
CTATTAGCTCGCTGCAGCCCGAGGACTTCGCTACTTACTACTGCCAACAG
AGCTACTCCACCCCACTGACTTTCGGCGGGGGTACCAAGGTCGAGATCAA
GACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCT
CCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGG
GCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGC
CCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTC
TTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCC
TTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCG
GTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCC
GCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAAC
GAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAG
AGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG
AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGC
GAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACT
GTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACA
TGCAGGCCCTGCCGCCTCGG 139119 139119- aa 44
QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWVGE ScFv domain
INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSG CLL-1 CAR 6
LVVYAIRVGSGWFDYWGQGTLVTVSSGGGGSGGGDSGGGGSDIQMTQSPS
SLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLMYAASSLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPWTFGQGTKVDIK 139119- nt 57
CAAGTGCAACTTCAAGAATCAGGCGCAGGACTTCTCAAGCCATCCGAAAC ScFv domain
ACTCTCCCTCACTTGCGCGGTGTACGGGGGAAGCTTCTCGGGATACTACT CLL-1 CAR 6
GGTCCTGGATTAGGCAGCCTCCCGGCAAAGGCCTGGAATGGGTCGGGGAG
ATCAACCACTCCGGTTCAACCAACTACAACCCGTCGCTGAAGTCCCGCGT
GACCATTTCCGTGGACACCTCTAAGAATCAGTTCAGCCTGAAGCTCTCGT
CCGTGACCGCGGCGGACACCGCCGTCTACTACTGCGCTCGGGGATCAGGA
CTGGTGGTGTACGCCATCCGCGTGGGCTCGGGCTGGTTCGATTACTGGGG
CCAGGGAACCCTGGTCACTGTGTCGTCCGGCGGAGGAGGTTCGGGGGGCG
GAGACAGCGGTGGAGGGGGTAGCGACATCCAGATGACCCAGTCCCCGTCC
TCGCTGTCCGCCTCCGTGGGAGATAGAGTGACCATCACCTGTCGGGCATC
CCAGAGCATTTCCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGAAAGG
CCCCTAAGCTGTTGATGTACGCCGCCAGCAGCTTGCAGTCGGGCGTGCCG
AGCCGGTTTTCCGGTTCCGGCTCCGGGACTGACTTCACCCTGACTATCTC
ATCCCTGCAACCCGAGGACTTCGCCACTTATTACTGCCAGCAGTCCTACT
CAACCCCTCCCTGGACGTTCGGACAGGGCACCAAGGTCGATATCAAG 139119- aa 70
QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWVGE VH of ScFv
INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSG CLL-1 CAR 6
LVVYAIRVGSGWFDYWGQGTLVTVSS 139119- aa 83
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLMYA VL of ScFv
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPWTFG CLL-1 CAR 6
QGTKVDIK 139119- aa 96
MALPVTALLLPLALLLHAARPQVQLQESGAGLLKPSETLSLTCAVYGGSF Full CAR
SGYYWSWIRQPPGKGLEWVGEINHSGSTNYNPSLKSRVTISVDTSKNQFS CLL-1 CAR 6
LKLSSVTAADTAVYYCARGSGLVVYAIRVGSGWFDYWGQGTLVTVSSGGG
GSGGGDSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ
KPGKAPKLLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
QQSYSTPPWTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF
KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139119- nt 109
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTTCAAGAATCAGGCGCAGGACTTCTCA CLL-1 CAR 6
AGCCATCCGAAACACTCTCCCTCACTTGCGCGGTGTACGGGGGAAGCTTC
TCGGGATACTACTGGTCCTGGATTAGGCAGCCTCCCGGCAAAGGCCTGGA
ATGGGTCGGGGAGATCAACCACTCCGGTTCAACCAACTACAACCCGTCGC
TGAAGTCCCGCGTGACCATTTCCGTGGACACCTCTAAGAATCAGTTCAGC
CTGAAGCTCTCGTCCGTGACCGCGGCGGACACCGCCGTCTACTACTGCGC
TCGGGGATCAGGACTGGTGGTGTACGCCATCCGCGTGGGCTCGGGCTGGT
TCGATTACTGGGGCCAGGGAACCCTGGTCACTGTGTCGTCCGGCGGAGGA
GGTTCGGGGGGCGGAGACAGCGGTGGAGGGGGTAGCGACATCCAGATGAC
CCAGTCCCCGTCCTCGCTGTCCGCCTCCGTGGGAGATAGAGTGACCATCA
CCTGTCGGGCATCCCAGAGCATTTCCAGCTACCTGAACTGGTATCAGCAG
AAGCCCGGAAAGGCCCCTAAGCTGTTGATGTACGCCGCCAGCAGCTTGCA
GTCGGGCGTGCCGAGCCGGTTTTCCGGTTCCGGCTCCGGGACTGACTTCA
CCCTGACTATCTCATCCCTGCAACCCGAGGACTTCGCCACTTATTACTGC
CAGCAGTCCTACTCAACCCCTCCCTGGACGTTCGGACAGGGCACCAAGGT
CGATATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTA
CCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCA
GCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTA
CATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCG
TGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTT
AAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTG
TTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGA
AATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAG
CTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGA
CAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGA
ATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAA
GCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCA
CGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACG
CTCTTCACATGCAGGCCCTGCCGCCTCGG 146261 146261- aa 48
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSY ScFv domain
ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDL CLL-1 CAR 10
SVRAIDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPS
SLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVP
SRFSGSGSGTDFTFTISSLQPEDFATYYCQQAYSTPFTFGPGTKVEIK 146261- nt 61
CAAGTGCAACTTGTTCAATCCGGTGGAGGTCTTGTGCAGCCCGGAGGATC ScFv domain
ACTCAGACTGTCGTGCGCCGCCTCTGGGTTCACTTTCTCCTCATACTCGA CLL-1 CAR 10
TGAACTGGGTGCGCCAGGCGCCGGGAAAGGGCCTGGAATGGGTGTCATAC
ATCTCCTCCTCATCCTCCACCATCTACTACGCCGATTCCGTGAAGGGCCG
CTTCACTATTTCCCGGGACAACGCGAAAAACTCGCTCTATCTGCAAATGA
ACTCCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGGGACCTG
AGCGTGCGGGCTATTGATGCGTTCGACATCTGGGGACAGGGCACCATGGT
CACAGTGTCCAGCGGAGGCGGCGGCAGCGGTGGAGGAGGATCAGGGGGAG
GAGGTTCGGGGGGCGGTGGCTCCGATATCGTGCTGACCCAGAGCCCGTCG
AGCCTCTCCGCCTCCGTCGGCGACAGAGTGACCATCACGTGTCAGGCATC
CCAGGACATTAGCAACTACCTGAATTGGTACCAGCAGAAGCCTGGAAAGG
CACCCAAGTTGCTGATCTACGACGCCTCCAACCTGGAAACCGGAGTGCCA
TCCAGGTTCTCGGGCAGCGGCTCGGGAACCGACTTCACTTTTACTATCTC
CTCCCTGCAACCCGAGGATTTCGCGACCTACTACTGCCAGCAGGCCTACA
GCACCCCTTTCACCTTCGGGCCGGGAACTAAGGTCGAAATCAAG 146261- aa 74
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSY VH of ScFv
ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDL CLL-1 CAR 10
SVRAIDAFDIWGQGTMVTVSS 146261- aa 87
DIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD VL of ScFv
ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQAYSTPFTFGP CLL-1 CAR 10
GTKVEIK 146261- aa 100
MALPVTALLLPLALLLHAARPQVQLVQSGGGLVQPGGSLRLSCAASGFTF Full CAR
SSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSL CLL-1 CAR 10
YLQMNSLRAEDTAVYYCARDLSVRAIDAFDIWGQGTMVTVSSGGGGSGGG
GSGGGGSGGGGSDIVLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ
KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYC
QQAYSTPFTFGPGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAA
GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK
QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 146261- nt 113
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTTGTTCAATCCGGTGGAGGTCTTGTGC CLL-1 CAR 10
AGCCCGGAGGATCACTCAGACTGTCGTGCGCCGCCTCTGGGTTCACTTTC
TCCTCATACTCGATGAACTGGGTGCGCCAGGCGCCGGGAAAGGGCCTGGA
ATGGGTGTCATACATCTCCTCCTCATCCTCCACCATCTACTACGCCGATT
CCGTGAAGGGCCGCTTCACTATTTCCCGGGACAACGCGAAAAACTCGCTC
TATCTGCAAATGAACTCCCTGCGCGCCGAGGACACCGCCGTGTACTACTG
CGCCCGGGACCTGAGCGTGCGGGCTATTGATGCGTTCGACATCTGGGGAC
AGGGCACCATGGTCACAGTGTCCAGCGGAGGCGGCGGCAGCGGTGGAGGA
GGATCAGGGGGAGGAGGTTCGGGGGGCGGTGGCTCCGATATCGTGCTGAC
CCAGAGCCCGTCGAGCCTCTCCGCCTCCGTCGGCGACAGAGTGACCATCA
CGTGTCAGGCATCCCAGGACATTAGCAACTACCTGAATTGGTACCAGCAG
AAGCCTGGAAAGGCACCCAAGTTGCTGATCTACGACGCCTCCAACCTGGA
AACCGGAGTGCCATCCAGGTTCTCGGGCAGCGGCTCGGGAACCGACTTCA
CTTTTACTATCTCCTCCCTGCAACCCGAGGATTTCGCGACCTACTACTGC
CAGCAGGCCTACAGCACCCCTTTCACCTTCGGGCCGGGAACTAAGGTCGA
AATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCA
TCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCT
GGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACAT
TTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGA
TCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAG
CAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTC
ATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAAT
TCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTC
TACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAA
GCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATC
CCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCC
TATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGA
CGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTC
TTCACATGCAGGCCCTGCCGCCTCGG 146262 146262- aa 49
EVQLVQSGGGVVRSGRSLRLSCAASGFTFNSYGLHWVRQAPGKGLEWVAL ScFv domain
IEYDGSNKYYGDSVKGRFTISRDKSKSTLYLQMDNLRAEDTAVYYCAREG CLL-1 CAR 11
NEDLAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPSSL
SASVGDRVTITCQASQFIKKNLNWYQHKPGKAPKLLIYDASSLQTGVPSR
FSGNRSGTTFSFTISSLQPEDVATYYCQQHDNLPLTFGGGTKVEIK 146262- nt 62
GAAGTGCAATTGGTGCAATCAGGAGGAGGAGTGGTCAGATCTGGAAGAAG ScFv domain
CCTGAGACTGTCATGCGCGGCTTCGGGCTTTACCTTCAACTCCTACGGCC CLL-1 CAR 11
TCCACTGGGTGCGCCAGGCCCCCGGAAAAGGCCTCGAATGGGTCGCACTG
ATTGAGTACGACGGGTCCAACAAGTACTACGGAGATAGCGTGAAGGGCCG
CTTCACCATCTCACGGGACAAGTCCAAGTCCACCCTGTATCTGCAAATGG
ACAACCTGAGGGCCGAGGATACTGCCGTGTACTACTGCGCCCGCGAAGGA
AACGAAGATCTGGCCTTCGATATTTGGGGCCAGGGTACTCTTGTGACCGT
GTCGAGCGGAGGCGGAGGCTCCGGTGGAGGAGGATCGGGGGGTGGTGGTT
CCGGCGGCGGGGGGAGCGAAATCGTGCTGACCCAGTCGCCTTCCTCCCTC
TCCGCTTCCGTGGGGGACCGGGTCACTATTACGTGTCAGGCGTCCCAATT
CATCAAGAAGAATCTGAACTGGTACCAGCACAAGCCGGGAAAGGCCCCCA
AACTGCTCATCTACGACGCCAGCTCGCTGCAGACTGGCGTGCCTTCCCGG
TTTTCCGGGAACCGGTCGGGAACCACCTTCTCATTCACCATCAGCAGCCT
CCAGCCGGAGGACGTGGCGACCTACTACTGCCAGCAGCATGACAACCTTC
CACTGACTTTCGGCGGGGGCACCAAGGTCGAGATTAAG
146262- aa 75 EVQLVQSGGGVVRSGRSLRLSCAASGFTFNSYGLHWVRQAPGKGLEWVAL VH
of ScFv IEYDGSNKYYGDSVKGRFTISRDKSKSTLYLQMDNLRAEDTAVYYCAREG CLL-1
CAR 11 NEDLAFDIWGQGTLVTVSS 146262- aa 88
EIVLTQSPSSLSASVGDRVTITCQASQFIKKNLNWYQHKPGKAPKLLIYD VL of ScFv
ASSLQTGVPSRFSGNRSGTTFSFTISSLQPEDVATYYCQQHDNLPLTFGG CLL-1 CAR 11
GTKVEIK 146262- aa 101
MALPVTALLLPLALLLHAARPEVQLVQSGGGVVRSGRSLRLSCAASGFTF Full CAR
NSYGLHWVRQAPGKGLEWVALIEYDGSNKYYGDSVKGRFTISRDKSKSTL CLL-1 CAR 11
YLQMDNLRAEDTAVYYCAREGNEDLAFDIWGQGTLVTVSSGGGGSGGGGS
GGGGSGGGGSEIVLTQSPSSLSASVGDRVTITCQASQFIKKNLNWYQHKP
GKAPKLLIYDASSLQTGVPSRFSGNRSGTTFSFTISSLQPEDVATYYCQQ
HDNLPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 146262- nt 114
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAATTGGTGCAATCAGGAGGAGGAGTGGTCA CLL-1 CAR 11
GATCTGGAAGAAGCCTGAGACTGTCATGCGCGGCTTCGGGCTTTACCTTC
AACTCCTACGGCCTCCACTGGGTGCGCCAGGCCCCCGGAAAAGGCCTCGA
ATGGGTCGCACTGATTGAGTACGACGGGTCCAACAAGTACTACGGAGATA
GCGTGAAGGGCCGCTTCACCATCTCACGGGACAAGTCCAAGTCCACCCTG
TATCTGCAAATGGACAACCTGAGGGCCGAGGATACTGCCGTGTACTACTG
CGCCCGCGAAGGAAACGAAGATCTGGCCTTCGATATTTGGGGCCAGGGTA
CTCTTGTGACCGTGTCGAGCGGAGGCGGAGGCTCCGGTGGAGGAGGATCG
GGGGGTGGTGGTTCCGGCGGCGGGGGGAGCGAAATCGTGCTGACCCAGTC
GCCTTCCTCCCTCTCCGCTTCCGTGGGGGACCGGGTCACTATTACGTGTC
AGGCGTCCCAATTCATCAAGAAGAATCTGAACTGGTACCAGCACAAGCCG
GGAAAGGCCCCCAAACTGCTCATCTACGACGCCAGCTCGCTGCAGACTGG
CGTGCCTTCCCGGTTTTCCGGGAACCGGTCGGGAACCACCTTCTCATTCA
CCATCAGCAGCCTCCAGCCGGAGGACGTGGCGACCTACTACTGCCAGCAG
CATGACAACCTTCCACTGACTTTCGGCGGGGGCACCAAGGTCGAGATTAA
GACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCT
CCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGG
GCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGC
CCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTC
TTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCC
TTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCG
GTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCC
GCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAAC
GAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAG
AGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAG
AGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGC
GAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACT
GTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACA
TGCAGGCCCTGCCGCCTCGG 146263 146263- aa 50
QVQLVESGGGLVQPGGSLRLSCAASGFNVSSNYMTWVRQAPGKGLEWVSV ScFv domain
IYSGGATYYGDSVKGRFTVSRDNSKNTVYLQMNRLTAEDTAVYYCARDRL CLL-1 CAR 12
YCGNNCYLYYYYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQ
VTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASS
LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPLTFGQGT KVEIK 146263- nt
63 CAAGTGCAACTCGTGGAATCAGGCGGAGGACTCGTGCAACCCGGAGGTTC ScFv domain
CCTTAGACTGTCATGTGCCGCTTCCGGGTTCAATGTGTCCAGCAACTACA CLL-1 CAR 12
TGACCTGGGTCAGACAGGCGCCGGGAAAGGGACTTGAATGGGTGTCCGTG
ATCTACTCCGGTGGAGCAACATACTACGGAGACTCCGTGAAAGGCCGCTT
TACCGTGTCCCGCGATAACTCGAAGAACACCGTGTACTTGCAGATGAACA
GGCTGACTGCCGAGGACACCGCCGTGTATTATTGCGCCCGGGACAGGCTG
TACTGTGGAAACAACTGCTACCTGTACTACTACTACGGGATGGACGTGTG
GGGACAGGGCACTCTCGTCACTGTGTCATCCGGGGGGGGCGGTAGCGGTG
GCGGAGGGTCCGGCGGAGGAGGCTCAGGGGGAGGCGGAAGCGATATCCAG
GTCACCCAGTCTCCCTCCTCGCTGTCCGCCTCCGTGGGCGACCGCGTCAC
CATTACTTGCCGGGCGTCGCAGTCGATCAGCTCCTACCTGAACTGGTACC
AGCAGAAGCCTGGAAAGGCCCCGAAGCTGCTGATCTACGCGGCCTCGTCC
CTGCAAAGCGGCGTCCCGTCGCGGTTCAGCGGTTCCGGTTCGGGAACCGA
CTTCACCCTGACTATTTCCTCCCTGCAACCCGAGGATTTCGCCACTTACT
ACTGCCAGCAGTCCTACTCCACCCCACCTCTGACCTTCGGCCAAGGAACC AAGGTCGAAATCAAG
146263- aa 76 QVQLVESGGGLVQPGGSLRLSCAASGFNVSSNYMTWVRQAPGKGLEWVSV VH
of ScFv IYSGGATYYGDSVKGRFTVSRDNSKNTVYLQMNRLTAEDTAVYYCARDRL CLL-1
CAR 12 YCGNNCYLYYYYGMDVWGQGTLVTVSS 146263- aa 89
DIQVTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA VL of ScFv
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPLTFG CLL-1 CAR 12
QGTKVEIK 146263- aa 102
MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFNV Full CAR
SSNYMTWVRQAPGKGLEWVSVIYSGGATYYGDSVKGRFTVSRDNSKNTVY CLL-1 CAR 12
LQMNRLTAEDTAVYYCARDRLYCGNNCYLYYYYGMDVWGQGTLVTVSSGG
GGSGGGGSGGGGSGGGGSDIQVTQSPSSLSASVGDRVTITCRASQSISSY
LNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQSYSTPPLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRP
EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 146263- nt 115
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTCGTGGAATCAGGCGGAGGACTCGTGC CLL-1 CAR 12
AACCCGGAGGTTCCCTTAGACTGTCATGTGCCGCTTCCGGGTTCAATGTG
TCCAGCAACTACATGACCTGGGTCAGACAGGCGCCGGGAAAGGGACTTGA
ATGGGTGTCCGTGATCTACTCCGGTGGAGCAACATACTACGGAGACTCCG
TGAAAGGCCGCTTTACCGTGTCCCGCGATAACTCGAAGAACACCGTGTAC
TTGCAGATGAACAGGCTGACTGCCGAGGACACCGCCGTGTATTATTGCGC
CCGGGACAGGCTGTACTGTGGAAACAACTGCTACCTGTACTACTACTACG
GGATGGACGTGTGGGGACAGGGCACTCTCGTCACTGTGTCATCCGGGGGG
GGCGGTAGCGGTGGCGGAGGGTCCGGCGGAGGAGGCTCAGGGGGAGGCGG
AAGCGATATCCAGGTCACCCAGTCTCCCTCCTCGCTGTCCGCCTCCGTGG
GCGACCGCGTCACCATTACTTGCCGGGCGTCGCAGTCGATCAGCTCCTAC
CTGAACTGGTACCAGCAGAAGCCTGGAAAGGCCCCGAAGCTGCTGATCTA
CGCGGCCTCGTCCCTGCAAAGCGGCGTCCCGTCGCGGTTCAGCGGTTCCG
GTTCGGGAACCGACTTCACCCTGACTATTTCCTCCCTGCAACCCGAGGAT
TTCGCCACTTACTACTGCCAGCAGTCCTACTCCACCCCACCTCTGACCTT
CGGCCAAGGAACCAAGGTCGAAATCAAGACCACTACCCCAGCACCGAGGC
CACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCG
GAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGA
CTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGG
TCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAG
AAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTAC
TCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCG
GCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTAC
AAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA
GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCG
GGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAA
AAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACG
CAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCA
CCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 139115 139115- aa
39 EVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG ScFv domain
IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDL CLL-1 CAR 1
EMATIMGGYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPG
QSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLDVVFGGGTKLTVL 139115- nt 52
GAAGTGCAACTCCAACAGTCAGGCGCAGAAGTCAAGAAGCCCGGATCGTC ScFv domain
AGTGAAAGTGTCCTGCAAAGCCTCCGGCGGAACCTTCAGCTCCTACGCAA CLL-1 CAR 1
TCAGCTGGGTGCGGCAGGCGCCCGGACAGGGACTGGAGTGGATGGGCGGT
ATCATTCCGATCTTTGGCACCGCCAATTACGCCCAGAAGTTCCAGGGACG
CGTCACAATCACCGCCGACGAATCGACTTCCACCGCCTACATGGAGCTGT
CGTCCTTGAGGAGCGAAGATACCGCCGTGTACTACTGCGCTCGGGATCTG
GAGATGGCCACTATCATGGGGGGTTACTGGGGCCAGGGGACCCTGGTCAC
TGTGTCCTCGGGAGGAGGGGGATCAGGCGGCGGCGGTTCCGGGGGAGGAG
GAAGCCAGTCCGCGCTGACTCAGCCAGCTTCCGTGTCTGGTTCGCCGGGA
CAGTCCATCACTATTAGCTGTACCGGCACCAGCAGCGACGTGGGCGGCTA
CAACTATGTGTCATGGTACCAGCAGCACCCGGGGAAGGCGCCTAAGCTGA
TGATCTACGACGTGTCCAACCGCCCTAGCGGAGTGTCCAACAGATTCTCC
GGTTCGAAGTCAGGGAACACTGCCTCCCTCACGATTAGCGGGCTGCAAGC
CGAGGATGAAGCCGACTACTACTGCTCCTCCTATACCTCCTCCTCGACCC
TGGACGTGGTGTTCGGAGGAGGCACCAAGCTCACCGTCCTT 139115- aa 65
EVQLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG VH of ScFv
IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDL CLL-1 CAR 1
EMATIMGGYWGQGTLVTVSS 139115- aa 78
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI VL of ScFv
YDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLD CLL-1 CAR 1
VVFGGGTKLTVL 139115- aa 91
MALPVTALLLPLALLLHAARPEVQLQQSGAEVKKPGSSVKVSCKASGGTF Full CAR
SSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTA CLL-1 CAR 1
YMELSSLRSEDTAVYYCARDLEMATIMGGYWGQGTLVTVSSGGGGSGGGG
SGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK
APKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYT
SSSTLDVVFGGGTKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139115- nt 104
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAACTCCAACAGTCAGGCGCAGAAGTCAAGA CLL-1 CAR 1
AGCCCGGATCGTCAGTGAAAGTGTCCTGCAAAGCCTCCGGCGGAACCTTC
AGCTCCTACGCAATCAGCTGGGTGCGGCAGGCGCCCGGACAGGGACTGGA
GTGGATGGGCGGTATCATTCCGATCTTTGGCACCGCCAATTACGCCCAGA
AGTTCCAGGGACGCGTCACAATCACCGCCGACGAATCGACTTCCACCGCC
TACATGGAGCTGTCGTCCTTGAGGAGCGAAGATACCGCCGTGTACTACTG
CGCTCGGGATCTGGAGATGGCCACTATCATGGGGGGTTACTGGGGCCAGG
GGACCCTGGTCACTGTGTCCTCGGGAGGAGGGGGATCAGGCGGCGGCGGT
TCCGGGGGAGGAGGAAGCCAGTCCGCGCTGACTCAGCCAGCTTCCGTGTC
TGGTTCGCCGGGACAGTCCATCACTATTAGCTGTACCGGCACCAGCAGCG
ACGTGGGCGGCTACAACTATGTGTCATGGTACCAGCAGCACCCGGGGAAG
GCGCCTAAGCTGATGATCTACGACGTGTCCAACCGCCCTAGCGGAGTGTC
CAACAGATTCTCCGGTTCGAAGTCAGGGAACACTGCCTCCCTCACGATTA
GCGGGCTGCAAGCCGAGGATGAAGCCGACTACTACTGCTCCTCCTATACC
TCCTCCTCGACCCTGGACGTGGTGTTCGGAGGAGGCACCAAGCTCACCGT
CCTTACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCG
CCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGT
GGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTG
GGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCA
CTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAA
CCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATG
CCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCA
GCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTAC
AACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCG
GAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCC
AAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTAT
AGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGG
ACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTC
ACATGCAGGCCCTGCCGCCTCGG 139116 139116- aa 40
EVQLVESGGGVVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSL ScFv domain
ISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARVF CLL-1 CAR 2
DSYYMDVWGKGTTVTVSSGGGGSGGGGSGSGGSEIVLTQSPLSLPVTPGQ
PASISCRSSQSLVYTDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRF
SGSGSDTDFTLKISRVEAEDVGIYYCMQGTHWSFTFGQGTRLEIK 139116- nt 53
GAAGTGCAATTGGTGGAAAGCGGAGGAGGAGTGGTGCAACCTGGAGGAAG ScFv domain
CCTGAGACTGTCATGTGCCGCCTCGGGATTCACTTTCGATGACTACGCAA CLL-1 CAR 2
TGCACTGGGTCCGCCAGGCCCCCGGAAAGGGTCTGGAATGGGTGTCCCTC
ATCTCCGGCGATGGGGGTTCCACTTACTATGCGGATTCTGTGAAGGGCCG
CTTCACAATCTCCCGGGACAATTCCAAGAACACTCTGTACCTTCAAATGA
ACTCCCTGAGGGTGGAGGACACCGCTGTGTACTACTGCGCGAGAGTGTTT
GACTCGTACTATATGGACGTCTGGGGAAAGGGCACCACCGTGACCGTGTC
CAGCGGTGGCGGTGGATCGGGGGGCGGCGGCTCCGGGAGCGGAGGTTCCG
AGATTGTGCTGACTCAGTCGCCGTTGTCACTGCCTGTCACCCCCGGGCAG
CCGGCCTCCATTTCATGCCGGTCCAGCCAGTCCCTGGTCTACACCGATGG
GAACACTTACCTCAACTGGTTCCAGCAGCGCCCAGGACAGTCCCCGCGGA
GGCTGATCTACAAAGTGTCAAACCGGGACTCCGGCGTCCCCGATCGGTTC
TCGGGAAGCGGCAGCGACACCGACTTCACGCTGAAGATTTCCCGCGTGGA
AGCCGAGGACGTGGGCATCTACTACTGTATGCAGGGCACCCACTGGTCGT
TTACCTTCGGACAAGGAACTAGGCTCGAGATCAAG 139116- aa 66
EVQLVESGGGVVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSL VH of ScFv
ISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARVF CLL-1 CAR 2
DSYYMDVWGKGTTVTVSS 139116- aa 79
EIVLTQSPLSLPVTPGQPASISCRSSQSLVYTDGNTYLNWFQQRPGQSPR VL of ScFv
RLIYKVSNRDSGVPDRFSGSGSDTDFTLKISRVEAEDVGIYYCMQGTHWS CLL-1 CAR 2
FTFGQGTRLEIK 139116- aa 92
MALPVTALLLPLALLLHAARPEVQLVESGGGVVQPGGSLRLSCAASGFTF Full CAR
DDYAMHWVRQAPGKGLEWVSLISGDGGSTYYADSVKGRFTISRDNSKNTL CLL-1 CAR 2
YLQMNSLRVEDTAVYYCARVFDSYYMDVWGKGTTVTVSSGGGGSGGGGSG
SGGSEIVLTQSPLSLPVTPGQPASISCRSSQSLVYTDGNTYLNWFQQRPG
QSPRRLIYKVSNRDSGVPDRFSGSGSDTDFTLKISRVEAEDVGIYYCMQG
THWSFTFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE
LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139116- nt 105
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAATTGGTGGAAAGCGGAGGAGGAGTGGTGC CLL-1 CAR 2
AACCTGGAGGAAGCCTGAGACTGTCATGTGCCGCCTCGGGATTCACTTTC
GATGACTACGCAATGCACTGGGTCCGCCAGGCCCCCGGAAAGGGTCTGGA
ATGGGTGTCCCTCATCTCCGGCGATGGGGGTTCCACTTACTATGCGGATT
CTGTGAAGGGCCGCTTCACAATCTCCCGGGACAATTCCAAGAACACTCTG
TACCTTCAAATGAACTCCCTGAGGGTGGAGGACACCGCTGTGTACTACTG
CGCGAGAGTGTTTGACTCGTACTATATGGACGTCTGGGGAAAGGGCACCA
CCGTGACCGTGTCCAGCGGTGGCGGTGGATCGGGGGGCGGCGGCTCCGGG
AGCGGAGGTTCCGAGATTGTGCTGACTCAGTCGCCGTTGTCACTGCCTGT
CACCCCCGGGCAGCCGGCCTCCATTTCATGCCGGTCCAGCCAGTCCCTGG
TCTACACCGATGGGAACACTTACCTCAACTGGTTCCAGCAGCGCCCAGGA
CAGTCCCCGCGGAGGCTGATCTACAAAGTGTCAAACCGGGACTCCGGCGT
CCCCGATCGGTTCTCGGGAAGCGGCAGCGACACCGACTTCACGCTGAAGA
TTTCCCGCGTGGAAGCCGAGGACGTGGGCATCTACTACTGTATGCAGGGC
ACCCACTGGTCGTTTACCTTCGGACAAGGAACTAGGCTCGAGATCAAGAC
CACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCC
AGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCC
GTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCC
TCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTT
ACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTC
ATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTT
CCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCA
GCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAA
CTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGG
ACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGG
GCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAG
ATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTA
CCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGC
AGGCCCTGCCGCCTCGG 139118 139118- aa 41
QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWI ScFv domain
GSIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLKYVTAADTAVYYCATP CLL-1 CAR 3
GTYYDFLSGYYPFYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSS
LSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSYPYTFGQGTKLEIK 139118- nt 54
CAAGTGCAGCTTCAAGAAAGCGGTCCAGGACTCGTCAAGCCATCAGAAAC ScFv domain
TCTTTCCCTCACTTGTACCGTGTCGGGAGGCAGCATCTCCTCGAGCTCCT CLL-1 CAR 3
ACTACTGGGGTTGGATTAGACAGCCCCCGGGAAAGGGGTTGGAGTGGATC
GGTTCCATCTACTACTCCGGGTCGACCTACTACAACCCTTCCCTGAAATC
TCGGGTGTCCATCTCCGTCGACACCTCCAAGAACCAGTTCAGCCTGAAGC
TGAAATATGTGACCGCGGCCGATACTGCCGTGTACTATTGCGCCACCCCG
GGAACCTACTACGACTTCCTCTCGGGGTACTACCCGTTTTACTGGGGACA
GGGGACTCTCGTGACCGTGTCCTCGGGCGGCGGAGGTTCAGGCGGTGGCG
GATCGGGGGGAGGAGGCTCAGACATTGTGATGACCCAGAGCCCGTCCAGC
CTGAGCGCCTCCGTGGGCGATAGGGTCACGATTACTTGCCGGGCGTCCCA
GGGAATCTCAAGCTACCTGGCCTGGTACCAACAGAAGCCCGGAAAGGCAC
CCAAGTTGCTGATCTATGCCGCTAGCACTCTGCAGTCCGGGGTGCCTTCC
CGCTTCTCCGGCTCCGGCTCGGGCACCGACTTCACCCTGACCATTTCCTC
ACTGCAACCCGAGGACTTCGCCACTTACTACTGCCAGCAGCTGAACTCCT
ACCCTTACACATTCGGACAGGGAACCAAGCTGGAAATCAAG 139118- aa 67
QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWI VH of ScFv
GSIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLKYVTAADTAVYYCATP CLL-1 CAR 3
GTYYDFLSGYYPFYWGQGTLVTVSS 139118- aa 80
DIVMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYA VL of ScFv
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSYPYTFGQ CLL-1 CAR 3
GTKLEIK 139118- aa 93
MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGGSI Full CAR
SSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVSISVDTSKNQ CLL-1 CAR 3
FSLKLKYVTAADTAVYYCATPGTYYDFLSGYYPFYWGQGTLVTVSSGGGG
SGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQK
PGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
QLNSYPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139118- nt 106
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAGCTTCAAGAAAGCGGTCCAGGACTCGTCA CLL-1 CAR 3
AGCCATCAGAAACTCTTTCCCTCACTTGTACCGTGTCGGGAGGCAGCATC
TCCTCGAGCTCCTACTACTGGGGTTGGATTAGACAGCCCCCGGGAAAGGG
GTTGGAGTGGATCGGTTCCATCTACTACTCCGGGTCGACCTACTACAACC
CTTCCCTGAAATCTCGGGTGTCCATCTCCGTCGACACCTCCAAGAACCAG
TTCAGCCTGAAGCTGAAATATGTGACCGCGGCCGATACTGCCGTGTACTA
TTGCGCCACCCCGGGAACCTACTACGACTTCCTCTCGGGGTACTACCCGT
TTTACTGGGGACAGGGGACTCTCGTGACCGTGTCCTCGGGCGGCGGAGGT
TCAGGCGGTGGCGGATCGGGGGGAGGAGGCTCAGACATTGTGATGACCCA
GAGCCCGTCCAGCCTGAGCGCCTCCGTGGGCGATAGGGTCACGATTACTT
GCCGGGCGTCCCAGGGAATCTCAAGCTACCTGGCCTGGTACCAACAGAAG
CCCGGAAAGGCACCCAAGTTGCTGATCTATGCCGCTAGCACTCTGCAGTC
CGGGGTGCCTTCCCGCTTCTCCGGCTCCGGCTCGGGCACCGACTTCACCC
TGACCATTTCCTCACTGCAACCCGAGGACTTCGCCACTTACTACTGCCAG
CAGCTGAACTCCTACCCTTACACATTCGGACAGGGAACCAAGCTGGAAAT
CAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCG
CCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGT
GGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTG
GGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCA
CTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAA
CCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATG
CCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCA
GCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTAC
AACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCG
GAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCC
AAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGTGGCAGAAGCCTAT
AGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGG
ACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTC
ACATGCAGGCCCTGCCCCTCGG 139122 139122- aa 42
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVAN ScFv domain
INEDGSAKFYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARDL CLL-1 CAR 4
RSGRYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGGRA
TLSCRASQSISGSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGSSPPTFGLGTKLEIK 139122- nt 55
CAAGTGCAACTCGTGGAATCTGGTGGAGGACTCGTGCAACCCGGAGGATC ScFv domain
ATTGCGACTCTCGTGTGCGGCATCCGGCTTTACCTTTTCATCCTACTGGA CLL-1 CAR 4
TGTCCTGGGTCAGACAGGCCCCCGGGAAGGGACTGGAATGGGTCGCGAAC
ATCAACGAGGACGGCTCGGCCAAGTTCTACGTGGACTCCGTGAAGGGCCG
CTTCACGATCTCACGGGATAACGCCAAGAATTCCCTGTATCTGCAAATGA
ACAGCCTGAGGGCCGAGGACACTGCGGTGTACTTCTGCGCACGCGACCTG
AGGTCCGGGAGATACTGGGGACAGGGCACCCTCGTGACCGTGTCGAGCGG
AGGAGGGGGGTCGGGCGGCGGCGGTTCCGGTGGCGGCGGTAGCGAAATTG
TGTTGACCCAGTCCCCTGGAACCCTGAGCCTGTCACCTGGAGGACGCGCC
ACCCTGTCCTGCCGGGCCAGCCAGAGCATCTCAGGGTCCTTCCTGGCTTG
GTACCAGCAGAAGCCGGGACAGGCTCCGAGACTTCTGATCTACGGCGCCT
CCTCGCGGGCGACCGGAATCCCGGATCGGTTCTCCGGCTCGGGAAGCGGA
ACTGACTTACTCTTACCATTTCCCGCCTGGAGCCGGAAGATTTCGCCGT
GTACTACTGCCAGCAGTACGGGTCATCCCCTCCAACCTTCGGCCTGGGAA
CTAAGCTGGAAATCAAA 139122- aa 68
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVAN VH of ScFv
INEDGSAKFYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARDL CLL-1 CAR 4
RSGRYWGQGTLVTVSS 139122- aa 81
EIVLTQSPGTLSLSPGGRATLSCRASQSISGSFLAWYQQKPGQAPRLLIY VL of ScFv
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPTFG CLL-1 CAR 4
LGTKLEIK 139122- aa 94
MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGFTF Full CAR
SSYWMSWVRQAPGKGLEWVANINEDGSAKFYVDSVKGRFTISRDNAKNSL CLL-1 CAR 4
YLQMNSLRAEDTAVYFCARDLRSGRYWGQGTLVTVSSGGGGSGGGGSGGG
GSEIVLTQSPGTLSLSPGGRATLSCRASQSISGSFLAWYQQKPGQAPRLL
IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPT
FGLGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL
DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139122- nt 107
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTCGTGGAATCTGGTGGAGGACTCGTGC CLL-1 CAR 4
AACCCGGAGGATCATTGCGACTCTCGTGTGCGGCATCCGGCTTTACCTTT
TCATCCTACTGGATGTCCTGGGTCAGACAGGCCCCCGGGAAGGGACTGGA
ATGGGTCGCGAACATCAACGAGGACGGCTCGGCCAAGTTCTACGTGGACT
CCGTGAAGGGCCGCTTCACGATCTCACGGGATAACGCCAAGAATTCCCTG
TATCTGCAAATGAACAGCCTGAGGGCCGAGGACACTGCGGTGTACTTCTG
CGCACGCGACCTGAGGTCCGGGAGATACTGGGGACAGGGCACCCTCGTGA
CCGTGTCGAGCGGAGGAGGGGGGTCGGGCGGCGGCGGTTCCGGTGGCGGC
GGTAGCGAAATTGTGTTGACCCAGTCCCCTGGAACCCTGAGCCTGTCACC
TGGAGGACGCGCCACCCTGTCCTGCCGGGCCAGCCAGAGCATCTCAGGGT
CCTTCCTGGCTTGGTACCAGCAGAAGCCGGGACAGGCTCCGAGACTTCTG
ATCTACGGCGCCTCCTCGCGGGCGACCGGAATCCCGGATCGGTTCTCCGG
CTCGGGAAGCGGAACTGACTTCACTCTTACCATTTCCCGCCTGGAGCCGG
AAGATTTCGCCGTGTACTACTGCCAGCAGTACGGGTCATCCCCTCCAACC
TTCGGCCTGGGAACTAAGCTGGAAATCAAAACCACTACCCCAGCACCGAG
GCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTC
CGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTT
GACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGG
GGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGA
AGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACT
ACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGG
CGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCT
ACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGA
GAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGG
CGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCC
AAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAA
CGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGC
CACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 139117 139117- aa
43 EVQLQQSGPGLVRPSETLSLTCTVSGGPVRSGSHYWNWIRQPPGRGLEWI ScFv domain
GYIYYSGSTNYNPSLENRVTISIDTSNNHFSLKLSSVTAADTALYFCARG CLL-1 CARS
TATFDWNFPFDSWGQGTLVTVSSGGGGSGGGGSGSGGSDIQMTQSPSSLS
ASIGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKLEIK 139117- nt 56
GAAGTGCAACTCCAACAATCCGGTCCAGGACTCGTCAGACCCTCCGAAAC ScFv domain
TCTCTCGCTTACATGCACTGTGTCCGGCGGCCCTGTGCGGTCCGGCTCTC CLL-1 CAR 5
ATTACTGGAACTGGATTCGCCAGCCCCCGGGACGCGGACTGGAGTGGATC
GGCTACATCTATTACTCGGGGTCGACTAACTACAACCCGAGCCTGGAAAA
TAGAGTGACCATCTCAATCGACACGTCCAACAACCACTTCTCGCTGAAGT
TGTCCTCCGTGACTGCCGCCGATACTGCCCTGTACTTCTGTGCTCGCGGA
ACCGCCACCTTCGACTGGAACTTCCCTTTTGACTCATGGGGCCAGGGGAC
CCTTGTGACCGTGTCCAGCGGAGGAGGAGGCTCCGGTGGTGGCGGGAGCG
GTAGCGGCGGAAGCGACATCCAGATGACCCAGTCACCGTCCTCGCTGTCC
GCATCCATTGGGGATCGGGTCACTATTACTTGCCGGGCGTCCCAGTCCAT
CTCGTCCTACCTGAACTGGTATCAGCAGAAGCCAGGGAAAGCCCCCAAGC
TGCTGATCTACGCGGCCAGCAGCCTGCAGTCAGGAGTGCCTTCAAGGTTT
AGCGGCAGCGGATCGGGAACCGACTTCACCCTGACCATTTCCTCCCTCCA
ACCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCTACTCCACCCCGT
GGACCTTCGGACAGGGAACCAAGCTGGAGATCAAG 139117- aa 69
EVQLQQSGPGLVRPSETLSLTCTVSGGPVRSGSHYWNWIRQPPGRGLEWI VH of ScFv
GYIYYSGSTNYNPSLENRVTISIDTSNNHFSLKLSSVTAADTALYFCARG CLL-1 CAR 5
TATFDWNFPFDSWGQGTLVTVSS 139117- aa 82
DIQMTQSPSSLSASIGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA VL of ScFv
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQ CLL-1 CAR 5
GTKLEIK 139117- aa 95
MALPVTALLLPLALLLHAARPEVQLQQSGPGLVRPSETLSLTCTVSGGPV Full CAR
RSGSHYWNWIRQPPGRGLEWIGYIYYSGSTNYNPSLENRVTISIDTSNNH CLL-1 CAR 5
FSLKLSSVTAADTALYFCARGTATFDWNFPFDSWGQGTLVTVSSGGGGSG
GGGSGSGGSDIQMTQSPSSLSASIGDRVTITCRASQSISSYLNWYQQKPG
KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQS
YSTPWTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE
LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139117- nt 108
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAACTCCAACAATCCGGTCCAGGACTCGTCA CLL-1 CAR 5
GACCCTCCGAAACTCTCTCGCTTACATGCACTGTGTCCGGCGGCCCTGTG
CGGTCCGGCTCTCATTACTGGAACTGGATTCGCCAGCCCCCGGGACGCGG
ACTGGAGTGGATCGGCTACATCTATTACTCGGGGTCGACTAACTACAACC
CGAGCCTGGAAAATAGAGTGACCATCTCAATCGACACGTCCAACAACCAC
TTCTCGCTGAAGTTGTCCTCCGTGACTGCCGCCGATACTGCCCTGTACTT
CTGTGCTCGCGGAACCGCCACCTTCGACTGGAACTTCCCTTTTGACTCAT
GGGGCCAGGGGACCCTTGTGACCGTGTCCAGCGGAGGAGGAGGCTCCGGT
GGTGGCGGGAGCGGTAGCGGCGGAAGCGACATCCAGATGACCCAGTCACC
GTCCTCGCTGTCCGCATCCATTGGGGATCGGGTCACTATTACTTGCCGGG
CGTCCCAGTCCATCTCGTCCTACCTGAACTGGTATCAGCAGAAGCCAGGG
AAAGCCCCCAAGCTGCTGATCTACGCGGCCAGCAGCCTGCAGTCAGGAGT
GCCTTCAAGGTTTAGCGGCAGCGGATCGGGAACCGACTTCACCCTGACCA
TTTCCTCCCTCCAACCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCC
TACTCCACCCCGTGGACCTTCGGACAGGGAACCAAGCTGGAGATCAAGAC
CACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCC
AGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCC
GTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCC
TCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTT
ACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTC
ATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTT
CCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCA
GCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAA
CTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGG
ACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGG
GCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAG
ATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTA
CCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGC
AGGCCCTGCCGCCTCGG 139120 139120- aa 45
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS ScFv domain
ISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDP CLL-1 CAR 7
SSSGSYYMEDSYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSNFMLTQ
PHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRP
SGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNQVVFGGGT KLTVL 139120- nt
58 GAAGTGCAATTGGTGGAATCTGGAGGAGGACTTGTGAAACCTGGTGGAAG ScFv domain
CCTGAGACTTTCCTGTGCGGCCTCGGGATTCACTTTCTCCTCCTACTCCA CLL-1 CAR 7
TGAACTGGGTCAGACAGGCCCCTGGGAAGGGACTGGAATGGGTGTCATCC
ATCTCCTCCTCATCGTCGTACATCTACTACGCCGATAGCGTGAAGGGGCG
GTTCACCATTTCCCGGGACAACGCTAAGAACAGCCTCTATCTGCAAATGA
ATTCCCTCCGCGCCGAGGACACTGCCGTGTACTACTGCGCGAGGGACCCC
TCATCAAGCGGCAGCTACTACATGGAGGACTCGTATTACTACGGAATGGA
CGTCTGGGGCCAGGGAACCACTGTGACGGTGTCCTCCGGTGGAGGGGGCT
CCGGGGGCGGGGGATCTGGCGGAGGAGGCTCCAACTTCATGCTGACCCAG
CCGCACTCCGTGTCCGAAAGCCCCGGAAAGACCGTGACAATTTCCTGCAC
CGGGTCCTCCGGCTCGATCGCATCAAACTACGTGCAGTGGTACCAGCAGC
GCCCGGGCAGCGCCCCCACCACTGTCATCTACGAGGATAACCAGCGGCCG
TCGGGTGTCCCAGACCGGTTTTCCGGTTCGATCGATAGCAGCAGCAACAG
CGCCTCCCTGACCATTTCCGGCCTCAAGACCGAGGATGAGGCTGACTACT
ACTGCCAGTCGTATGACTCCTCGAACCAAGTGGTGTTCGGTGGCGGCACC AAGCTGACTGTGCTG
139120- aa 71 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS VH
of ScFv ISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDP CLL-1
CAR 7 SSSGSYYMEDSYYYGMDVWGQGTTVTVSS 139120- aa 84
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSAPTTVIY VL of ScFv
EDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNQV CLL-1 CAR 7
VFGGGTKLTVL 139120- aa 97
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTF Full CAR
SSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSL CLL-1 CAR 7
YLQMNSLRAEDTAVYYCARDPSSSGSYYMEDSYYYGMDVWGQGTTVTVSS
GGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQ
WYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTED
EADYYCQSYDSSNQVVFGGGTKLTVLTTTPAPRPPTPAPTIASQPLSLRP
EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139120- nt 110
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAATTGGTGGAATCTGGAGGAGGACTTGTGA CLL-1 CAR 7
AACCTGGTGGAAGCCTGAGACTTTCCTGTGCGGCCTCGGGATTCACTTTC
TCCTCCTACTCCATGAACTGGGTCAGACAGGCCCCTGGGAAGGGACTGGA
ATGGGTGTCATCCATCTCCTCCTCATCGTCGTACATCTACTACGCCGATA
GCGTGAAGGGGCGGTTCACCATTTCCCGGGACAACGCTAAGAACAGCCTC
TATCTGCAAATGAATTCCCTCCGCGCCGAGGACACTGCCGTGTACTACTG
CGCGAGGGACCCCTCATCAAGCGGCAGCTACTACATGGAGGACTCGTATT
ACTACGGAATGGACGTCTGGGGCCAGGGAACCACTGTGACGGTGTCCTCC
GGTGGAGGGGGCTCCGGGGGCGGGGGATCTGGCGGAGGAGGCTCCAACTT
CATGCTGACCCAGCCGCACTCCGTGTCCGAAAGCCCCGGAAAGACCGTGA
CAATTTCCTGCACCGGGTCCTCCGGCTCGATCGCATCAAACTACGTGCAG
TGGTACCAGCAGCGCCCGGGCAGCGCCCCCACCACTGTCATCTACGAGGA
TAACCAGCGGCCGTCGGGTGTCCCAGACCGGTTTTCCGGTTCGATCGATA
GCAGCAGCAACAGCGCCTCCCTGACCATTTCCGGCCTCAAGACCGAGGAT
GAGGCTGACTACTACTGCCAGTCGTATGACTCCTCGAACCAAGTGGTGTT
CGGTGGCGGCACCAAGCTGACTGTGCTGACCACTACCCCAGCACCGAGGC
CACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCG
GAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGA
CTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGG
TCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAG
AAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTAC
TCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCG
GCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTAC
AAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGA
GGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCG
GGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAA
AAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACG
CAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCA
CCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 139121 139121- aa
46 QVNLRESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSY ScFv domain
ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREA CLL-1 CAR 8
LGSSWEWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR
VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSG
TDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIK 139121- nt 59
CAAGTGAACCTGAGAGAAAGCGGCGGAGGACTTGTGCAACCTGGAGGAAG ScFv domain
CCTGAGACTGTCATGTGCCGCGTCCGGCTTCACCTTCTCGTCCTACGAGA CLL-1 CAR 8
TGAACTGGGTCCGCCAGGCACCGGGCAAAGGACTGGAATGGGTGTCCTAC
ATTTCCTCGTCCGGGTCCACCATCTATTACGCCGACTCCGTGAAGGGACG
GTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTCTACCTCCAAATGA
ACTCACTGAGGGCAGAGGACACTGCGGTCTACTACTGCGCCCGCGAAGCT
TTGGGTAGCTCCTGGGAGTGGGGCCAGGGAACCACTGTGACCGTGTCCTC
GGGTGGAGGGGGCTCCGGTGGCGGGGGTTCAGGGGGTGGCGGAAGCGATA
TCCAGATGACTCAGTCACCAAGCTCCCTGAGCGCCTCAGTGGGAGATCGG
GTCACAATCACGTGCCAGGCGTCCCAGGACATTTCTAACTACCTCAATTG
GTACCAGCAGAAGCCGGGGAAGGCCCCCAAGCTTCTGATCTACGATGCCT
CCAACCTGGAAACCGGCGTGCCCTCCCGCTTCTCGGGATCGGGCAGCGGC
ACTGACTTCACCTTTACCATCTCGTCCCTGCAACCTGAGGACATCGCCAC
CTATTACTGCCAGCAGTACGATAACCTCCCGCTGACTTTCGGAGGCGGAA
CTAAGCTGGAGATTAAG 139121- aa 72
QVNLRESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSY VH of ScFv
ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREA CLL-1 CAR 8
LGSSWEWGQGTTVTVSS 139121- aa 85
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD VL of ScFv
ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGG CLL-1 CAR 8
GTKLEIK 139121- aa 98
MALPVTALLLPLALLLHAARPQVNLRESGGGLVQPGGSLRLSCAASGFTF Full CAR
SSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSL CLL-1 CAR 8
YLQMNSLRAEDTAVYYCAREALGSSWEWGQGTTVTVSSGGGGSGGGGSGG
GGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLL
IYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLT
FGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL
DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139121- nt 111
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGAACCTGAGAGAAAGCGGCGGAGGACTTGTGC CLL-1 CAR 8
AACCTGGAGGAAGCCTGAGACTGTCATGTGCCGCGTCCGGCTTCACCTTC
TCGTCCTACGAGATGAACTGGGTCCGCCAGGCACCGGGCAAAGGACTGGA
ATGGGTGTCCTACATTTCCTCGTCCGGGTCCACCATCTATTACGCCGACT
CCGTGAAGGGACGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTC
TACCTCCAAATGAACTCACTGAGGGCAGAGGACACTGCGGTCTACTACTG
CGCCCGCGAAGCTTTGGGTAGCTCCTGGGAGTGGGGCCAGGGAACCACTG
TGACCGTGTCCTCGGGTGGAGGGGGCTCCGGTGGCGGGGGTTCAGGGGGT
GGCGGAAGCGATATCCAGATGACTCAGTCACCAAGCTCCCTGAGCGCCTC
AGTGGGAGACGGGTCACAATCACGTGCCAGGCGTCCCAGGACATTTCTA
ACTACCTCAATTGGTACCAGCAGAAGCCGGGGAAGGCCCCCAAGCTTCTG
ATCTACGATGCCTCCAACCTGGAAACCGGCGTGCCCTCCCGCTTCTCGGG
ATCGGGCAGCGGCACTGACTTCACCTTTACCATCTCGTCCCTGCAACCTG
AGGACATCGCCACCTATTACTGCCAGCAGTACGATAACCTCCCGCTGACT
TTCGGAGGCGGAACTAAGCTGGAGATTAAGACCACTACCCCAGCACCGAG
GCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTC
CGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTT
GACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGG
GGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGA
AGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACT
ACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGG
CGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCT
ACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGA
GAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGG
CGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCC
AAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAA
CGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGC
CACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG 146264 146264- aa
51 QVQLVQSGAEVKKSGASVKVSCKASGYPFTGYYIQWVRQAPGQGLEWMGW ScFv domain
IDPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCASDS CLL-1 CAR 13
YGYYYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSS
LSASVGDRVTFTCRASQGISSALAWYQQKPGKPPKLLIYDASSLESGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQFNNYPLTFGGGTKVEIK 146264- nt 64
CAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTGAAAAAGAGCGGAGCCTC ScFv domain
AGTGAAAGTGTCCTGCAAGGCCTCCGGTTACCCCTTCACTGGATACTACA CLL-1 CAR 13
TTCAGTGGGTCCGCCAAGCCCCGGGACAGGGTCTGGAGTGGATGGGGTGG
ATTGACCCTAACTCGGGAAATACGGGATACGCGCAGAAGTTCCAGGGCCG
CGTGACCATGACCAGGAACACCTCGATCAGCACCGCCTACATGGAACTGT
CCTCCCTGCGGTCGGAGGATACTGCCGTGTACTACTGCGCCTCCGATTCC
TATGGGTACTACTACGGAATGGACGTCTGGGGACAGGGCACCCTCGTGAC
CGTGTCCTCGGGAGGCGGAGGGAGCGGCGGGGGTGGATCGGGAGGAGGCG
GCTCCGGCGGCGGCGGTAGCGACATCCAGATGACCCAGTCACCATCAAGC
CTTAGCGCCTCCGTGGGCGACAGAGTGACATTCACTTGTCGGGCGTCCCA
GGGAATCTCCTCCGCTCTGGCTTGGTATCAGCAGAAGCCTGGGAAGCCTC
CGAAGCTGTTGATCTACGACGCGAGCAGCCTGGAATCAGGGGTGCCCTCC
CGGTTTTCCGGGTCCGGTTCTGGCACCGATTTCACCCTGACCATTTCGTC
CCTCCAACCCGAGGACTTCGCCACTTACTACTGCCAGCAGTTCAACAACT
ACCCGCTGACCTTCGGAGGAGGCACTAAGGTCGAGATCAAG 146264- aa 77
QVQLVQSGAEVKKSGASVKVSCKASGYPFTGYYIQWVRQAPGQGLEWMGW VH of ScFv
IDPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCASDS CLL-1 CAR 13
YGYYYGMDVWGQGTLVTVSS 146264- aa 90
DIQMTQSPSSLSASVGDRVTFTCRASQGISSALAWYQQKPGKPPKLLIYD VL of ScFv
ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNNYPLTFGG CLL-1 CAR 13
GTKVEIK 146264- aa 103
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKSGASVKVSCKASGYPF Full CAR
TGYYIQWVRQAPGQGLEWMGWIDPNSGNTGYAQKFQGRVTMTRNTSISTA CLL-1 CAR 13
YMELSSLRSEDTAVYYCASDSYGYYYGMDVWGQGTLVTVSSGGGGSGGGG
SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTFTCRASQGISSALAWYQQK
PGKPPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
QFNNYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 146264- nt 116
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCCAAGTGCAACTCGTCCAGTCCGGTGCAGAAGTGAAAA CLL-1 CAR 13
AGAGCGGAGCCTCAGTGAAAGTGTCCTGCAAGGCCTCCGGTTACCCCTTC
ACTGGATACTACATTCAGTGGGTCCGCCAAGCCCCGGGACAGGGTCTGGA
GTGGATGGGGTGGATTGACCCTAACTCGGGAAATACGGGATACGCGCAGA
AGTTCCAGGGCCGCGTGACCATGACCAGGAACACCTCGATCAGCACCGCC
TACATGGAACTGTCCTCCCTGCGGTCGGAGGATACTGCCGTGTACTACTG
CGCCTCCGATTCCTATGGGTACTACTACGGAATGGACGTCTGGGGACAGG
GCACCCTCGTGACCGTGTCCTCGGGAGGCGGAGGGAGCGGCGGGGGTGGA
TCGGGAGGAGGCGGCTCCGGCGGCGGCGGTAGCGACATCCAGATGACCCA
GTCACCATCAAGCCTTAGCGCCTCCGTGGGCGACAGAGTGACATTCACTT
GTCGGGCGTCCCAGGGAATCTCCTCCGCTCTGGCTTGGTATCAGCAGAAG
CCTGGGAAGCCTCCGAAGCTGTTGATCTACGACGCGAGCAGCCTGGAATC
AGGGGTGCCCTCCCGGTTTTCCGGGTCCGGTTCTGGCACCGATTTCACCC
TGACCATTTCGTCCCTCCAACCCGAGGACTTCGCCACTTACTACTGCCAG
CAGTTCAACAACTACCCGCTGACCTTCGGAGGAGGCACTAAGGTCGAGAT
CAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCG
CCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGT
GGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTG
GGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCA
CTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAA
CCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATG
CCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCA
GCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTAC
AACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCG
GAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCC
AAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTAT
AGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGG
ACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTC
ACATGCAGGCCCTGCCGCCTCGG 181268 181268- aa 195
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSY VH of ScFv
ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDP
YSSSWHDAFDIWGQGTMVTVSS 181268- aa 196
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY VL of ScFv
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFG GGTKVDIK 181268-
aa 197 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTF Full CAR
SSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSL
YLQMNSLRAEDTAVYYCARDPYSSSWHDAFDIWGQGTMVTVSSGGGGSGG
GGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG
QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPLTFGGGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE
LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 181268- nt 198
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCA Full CAR
CGCCGCTCGGCCCGAAGTGCAACTCGTGGAAAGCGGTGGAGGTCTTGTGC
AACCTGGAGGTTCCTTGCGCCTGTCATGTGCAGCTTCCGGCTTCACTTTC
TCCTCGTACGAGATGAATTGGGTGCGGCAGGCGCCTGGAAAGGGGCTGGA
ATGGGTGTCCTACATCTCAAGCTCCGGCTCGACCATCTACTACGCGGACA
GCGTGAAGGGGCGGTTCACGATTTCGAGGGACAACGCCAAGAACTCGCTC
TATCTGCAAATGAACTCCCTGAGAGCCGAGGACACCGCTGTGTATTACTG
CGCCCGGGACCCCTACTCCTCCTCATGGCACGACGCCTTTGATATCTGGG
GCCAGGGAACCATGGTCACCGTCAGCAGCGGGGGCGGAGGTTCCGGGGGA
GGGGGCTCCGGCGGAGGAGGCTCCGAGATTGTGTTGACTCAGAGCCCGGG
TACCCTGTCGCTGAGCCCCGGAGAGCGGGCCACCCTTTCATGCCGCGCCA
GCCAGTCCGTGTCCTCATCCTACCTCGCGTGGTACCAGCAGAAACCTGGC
CAGGCCCCGCGGCTGCTGATCTACGGCGCCTCCTCGCGCGCAACCGGAAT
CCCCGACCGGTTCTCCGGGTCTGGCAGCGGAACCGACTTCACTCTCACCA
TTTCGAGGCTGGAGCCGGAAGATTTCGCCGTGTACTACTGCCAGCAGTAC
GGCTCCTCGCCACTGACTTTCGGCGGAGGAACCAAGGTCGATATCAAGAC
CACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCC
AGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCC
GTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCC
TCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTT
ACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTC
ATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTT
CCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCA
GCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAA
CTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGG
ACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGG
GCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAG
ATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTA
CCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGC
AGGCCCTGCCGCCTCGG
[0435] In embodiments, 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: 11).
TABLE-US-00010 EF1 alpha promoter
CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC
CCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGT
GGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTC
CCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC
TTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTT
CCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTA
CTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAA
GTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTG
CTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGT
GGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA
ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAA
ATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCG
GCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGC
GAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTG
CTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAA
GGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCG
GCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGG
CGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCG
CTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGT
TCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATG
CGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTT
GGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTT
GGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTC AGGTGTCGTGA
Gly/Ser (SEQ ID NO: 25) GGGGS Gly/Ser (SEQ ID NO: 26): This
sequence may encompass 1-6 "Gly Gly Gly Gly Ser" repeating units
GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS Gly/Ser (SEQ ID NO: 27) GGGGSGGGGS
GGGGSGGGGS Gly/Ser (SEQ ID NO: 28) GGGGSGGGGS GGGGS Gly/Ser (SEQ ID
NO: 29) GGGS PolyA: (A).sub.5000 (SEQ ID NO: 30) This sequence may
encompass 50-5000 adenines. PolyA: (T).sub.100 (SEQ ID NO: 31)
PolyA: (T).sub.5000 (SEQ ID NO: 32) This sequence may encompass
50-5000 thymines. PolyA: (A).sub.5000 (SEQ ID NO: 33) This sequence
may encompass 100-5000 adenines. PolyA: (A).sub.400 (SEQ ID NO: 34)
PolyA: (A).sub.2000 (SEQ ID NO: 35) Gly/Ser (SEQ ID NO: 38): This
sequence may encompass 1-10 "Gly Gly Gly Ser" repeating units
GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS
[0436] In one embodiment, the CLL-1 CAR may comprise one or more,
e.g., one, two, or three, CDRs of the heavy chain variable domain
and/or one or more, e.g., one, two, or three, CDRs of the light
chain variable domain, or the variable heavy chain (VH) or the
variable light chain (VL) of of the anti-CLL-1 (CLEC12A) antibody
disclosed in PCT Publication WO2014/051433, the entire contents of
which are hereby incorporated by reference.
[0437] The CAR scFv fragments can be 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: 11).
[0438] The CAR construct can include a Gly/Ser linker having one or
more of the following sequences: GGGGS (SEQ ID NO:25); encompassing
1-6 "Gly Gly Gly Gly Ser" repeating units, e.g., GGGGSGGGGS
GGGGSGGGGS GGGGSGGGGS (SEQ ID NO:26); GGGGSGGGGS GGGGSGGGGS (SEQ ID
NO:27); GGGGSGGGGS GGGGS (SEQ ID NO:28); GGGS (SEQ ID NO:29); or
encompassing 1-10 "Gly Gly Gly Ser" repeating units, e.g.,
GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (SEQ ID NO:38). In
embodiments, the CAR construct include a poly A sequence, e.g., a
sequence encompassing 50-5000 or 100-5000 adenines (e.g., SEQ ID
NO:30, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35), or a sequence
encompassing 50-5000 thymines (e.g., SEQ ID NO:31, SEQ ID NO:32).
Alternatively, the CAR construct can include, for example, a linker
including the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 486)
[0439] Bispecific CARs
[0440] 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.
[0441] In certain embodiments, the antibody molecule is a
multi-specific (e.g., a bispecific or a trispecific) antibody
molecule. Protocols for generating bispecific or heterodimeric
antibody molecules are known in the art; including but not limited
to, for example, the "knob in a hole" approach described in, e.g.,
U.S. Pat. No. 5,731,168; the electrostatic steering Fc pairing as
described in, e.g., WO 09/089004, WO 06/106905 and WO 2010/129304;
Strand Exchange Engineered Domains (SEED) heterodimer formation as
described in, e.g., WO 07/110205; Fab arm exchange as described in,
e.g., WO 08/119353, WO 2011/131746, and WO 2013/060867; double
antibody conjugate, e.g., by antibody cross-linking to generate a
bi-specific structure using a heterobifunctional reagent having an
amine-reactive group and a sulfhydryl reactive group as described
in, e.g., U.S. Pat. No. 4,433,059; bispecific antibody determinants
generated by recombining half antibodies (heavy-light chain pairs
or Fabs) from different antibodies through cycle of reduction and
oxidation of disulfide bonds between the two heavy chains, as
described in, e.g., U.S. Pat. No. 4,444,878; trifunctional
antibodies, e.g., three Fab' fragments cross-linked through
sulfhdryl reactive groups, as described in, e.g., U.S. Pat. No.
5,273,743; biosynthetic binding proteins, e.g., pair of scFvs
cross-linked through C-terminal tails preferably through disulfide
or amine-reactive chemical cross-linking, as described in, e.g.,
U.S. Pat. No. 5,534,254; bifunctional antibodies, e.g., Fab
fragments with different binding specificities dimerized through
leucine zippers (e.g., c-fos and c-jun) that have replaced the
constant domain, as described in, e.g., U.S. Pat. No. 5,582,996;
bispecific and oligospecific mono- and oligovalent receptors, e.g.,
VH-CH1 regions of two antibodies (two Fab fragments) linked through
a polypeptide spacer between the CH1 region of one antibody and the
VH region of the other antibody typically with associated light
chains, as described in, e.g., U.S. Pat. No. 5,591,828; bispecific
DNA-antibody conjugates, e.g., crosslinking of antibodies or Fab
fragments through a double stranded piece of DNA, as described in,
e.g., U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., an
expression construct containing two scFvs with a hydrophilic
helical peptide linker between them and a full constant region, as
described in, e.g., U.S. Pat. No. 5,637,481; multivalent and
multispecific binding proteins, e.g., dimer of polypeptides having
first domain with binding region of Ig heavy chain variable region,
and second domain with binding region of Ig light chain variable
region, generally termed diabodies (higher order structures are
also encompassed creating for bispecifc, trispecific, or
tetraspecific molecules, as described in, e.g., U.S. Pat. No.
5,837,242; minibody constructs with linked VL and VH chains further
connected with peptide spacers to an antibody hinge region and CH3
region, which can be dimerized to form bispecific/multivalent
molecules, as described in, e.g., U.S. Pat. No. 5,837,821; VH and
VL domains linked with a short peptide linker (e.g., 5 or 10 amino
acids) or no linker at all in either orientation, which can form
dimers to form bispecific diabodies; trimers and tetramers, as
described in, e.g., U.S. Pat. No. 5,844,094; String of VH domains
(or VL domains in family members) connected by peptide linkages
with crosslinkable groups at the C-terminus further associated with
VL domains to form a series of FVs (or scFvs), as described in,
e.g., U.S. Pat. No. 5,864,019; and single chain binding
polypeptides with both a VH and a VL domain linked through a
peptide linker are combined into multivalent structures through
non-covalent or chemical crosslinking to form, e.g., homobivalent,
heterobivalent, trivalent, and tetravalent structures using both
scFV or diabody type format, as described in, e.g., U.S. Pat. No.
5,869,620. Additional exemplary multispecific and bispecific
molecules and methods of making the same are found, for example, in
U.S. Pat. Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830,
6,005,079, 6,239,259, 6,294,353, U.S. Pat. Nos. 6,333,396,
6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441,
7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181,
US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1,
US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1,
US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1,
US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1,
US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1,
US2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1,
US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1,
US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1,
US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1,
US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1,
US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1,
US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2,
WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2,
WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1,
WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1,
WO9637621A2, WO9964460A1. The contents of the above-referenced
applications are incorporated herein by reference in their
entireties.
[0442] Within each antibody or antibody fragment (e.g., scFv) of a
bispecific antibody molecule, the VH can be upstream or downstream
of the VL. In some embodiments, the upstream antibody or antibody
fragment (e.g., scFv) is arranged with its VH (VH.sub.1) upstream
of its VL (VL.sub.1) and the downstream antibody or antibody
fragment (e.g., scFv) is arranged with its VL (VL.sub.2) upstream
of its VH (VH.sub.2), such that the overall bispecific antibody
molecule has the arrangement VH.sub.1-VL.sub.1-VL.sub.2-VH.sub.2.
In other embodiments, the upstream antibody or antibody fragment
(e.g., scFv) is arranged with its VL (VL.sub.1) upstream of its VH
(VH.sub.1) and the downstream antibody or antibody fragment (e.g.,
scFv) is arranged with its VH (VH.sub.2) upstream of its VL
(VL.sub.2), such that the overall bispecific antibody molecule has
the arrangement VL.sub.1-VH.sub.1-VH.sub.2-VL.sub.2. Optionally, a
linker is disposed between the two antibodies or antibody fragments
(e.g., scFvs), e.g., between VL.sub.1 and VL.sub.2 if the construct
is arranged as VH.sub.1-VL.sub.1-VL.sub.2-VH.sub.2, or between
VH.sub.1 and VH.sub.2 if the construct is arranged as
VL.sub.1-VH.sub.1-VH.sub.2-VL.sub.2. The linker may be a linker as
described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1, 2, 3,
4, 5, or 6, preferably 4 (SEQ ID NO: 64). In general, the linker
between the two scFvs should be long enough to avoid mispairing
between the domains of the two scFvs. Optionally, a linker is
disposed between the VL and VH of the first scFv. Optionally, a
linker is disposed between the VL and VH of the second scFv. In
constructs that have multiple linkers, any two or more of the
linkers can be the same or different. Accordingly, in some
embodiments, a bispecific CAR comprises VLs, VHs, and optionally
one or more linkers in an arrangement as described herein.
[0443] In one aspect, the bispecific antibody molecule is
characterized by a first immunoglobulin variable domain sequence,
e.g., a scFv, which has binding specificity for CLL-1, e.g.,
comprises a scFv as described herein, e.g., as described in Table
8, or comprises the light chain CDRs and/or heavy chain CDRs from a
CLL-1 scFv described herein, and a second immunoglobulin variable
domain sequence that has binding specificity for a second epitope
on a different antigen. In some aspects the second immunoglobulin
variable domain sequence has binding specificity for an antigen
expressed on AML cells, e.g., an antigen other than CLL-1. For
example, the second immunoglobulin variable domain sequence has
binding specificity for CD123. As another example, the second
immunoglobulin variable domain sequence has binding specificity for
CD33. As another example, the second immunoglobulin variable domain
sequence has binding specificity for CD34. As another example, the
second immunoglobulin variable domain sequence has binding
specificity for FLT3. For example, the second immunoglobulin
variable domain sequence has binding specificity for folate
receptor beta. In some aspects, the second immunoglobulin variable
domain sequence has binding specificity for an antigen expressed on
B-cells, for example, CD19, CD20, CD22 or ROR1.
[0444] Chimeric TCR
[0445] In one aspect, the CLL-1 antibodies and antibody fragments
of the present invention (for example, those disclosed in Tables 8)
can be grafted to one or more constant domain of a T cell receptor
("TCR") chain, for example, a TCR alpha or TCR beta chain, to
create an chimeric TCR that binds specificity to CLL-1. Without
being bound by theory, it is believed that chimeric TCRs will
signal through the TCR complex upon antigen binding. For example, a
CLL-1 scFv as disclosed herein, can be grafted to the constant
domain, e.g., at least a portion of the extracellular constant
domain, the transmembrane domain and the cytoplasmic domain, of a
TCR chain, for example, the TCR alpha chain and/or the TCR beta
chain. As another example, a CLL-1 antibody fragment, for example a
VL domain as described herein, can be grafted to the constant
domain of a TCR alpha chain, and a CLL-1 antibody fragment, for
example a VH domain as described herein, can be grafted to the
constant domain of a TCR beta chain (or alternatively, a VL domain
may be grafted to the constant domain of the TCR beta chain and a
VH domain may be grafted to a TCR alpha chain). As another example,
the CDRs of a CLL-1 antibody or antibody fragment, e.g., the CDRs
of a CLL-1 antibody or antibody fragment as described in Tables 1,
2, 3, 4, 5, or 6 may be grafted into a TCR alpha and/or beta chain
to create a chimeric TCR that binds specifically to CLL-1. For
example, the LCDRs disclosed herein may be grafted into the
variable domain of a TCR alpha chain and the HCDRs disclosed herein
may be grafted to the variable domain of a TCR beta chain, or vice
versa. Such chimeric TCRs may be produced by methods known in the
art (For example, Willemsen R A et al, Gene Therapy 2000; 7:
1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggen
et al, Gene Ther. 2012 April; 19(4):365-74).
[0446] Transmembrane Domain
[0447] 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 is used. 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 CART cell surface. 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 CART.
[0448] 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 (e.g., CD8 alpha, CD8 beta), 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 a costimulatory molecule, e.g., a MHC
class I molecule, TNF receptor proteins, Immunoglobulin-like
proteins, cytokine receptors, integrins, signaling lymphocytic
activation molecules (SLAM proteins), activating NK cell receptors,
BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30,
CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS,
ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,
SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83.
[0449] 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, or a CD8a hinge. In
one embodiment, the hinge or spacer comprises (e.g., consists of)
the amino acid sequence of SEQ ID NO:2. In one aspect, the
transmembrane domain comprises (e.g., consists of) a transmembrane
domain of SEQ ID NO: 6.
[0450] 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:3). In some embodiments, the hinge or spacer comprises a hinge
encoded by a nucleotide sequence of
TABLE-US-00011 (SEQ ID NO: 14)
GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTG
GGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATG
ATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAG
GACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAAC
GCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTG
TCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAG
TGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGC
AAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGC
CAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAG
AACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTC
CTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTC
TTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG
AGCCTGAGCCTGTCCCTGGGCAAGATG.
[0451] 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
PVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPG
STTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:4).
In some embodiments, the hinge or spacer comprises a hinge encoded
by a nucleotide sequence of
TABLE-US-00012 (SEQ ID NO: 15)
AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAG
CCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACG
CGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAA
GAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAG
CCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGA
GATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCC
CATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAA
GGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTC
ACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTA
AATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCC
GCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCC
CCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCC
AACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGC
TTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCC
TGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATAC
ACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGG
AGTCTGGAGGTTTCCTACGTGACTGACCATT.
[0452] 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.
[0453] 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:5). In some embodiments, the
linker is encoded by a nucleotide sequence of
TABLE-US-00013 (SEQ ID NO: 16) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC.
[0454] In one aspect, the hinge or spacer comprises a KIR2DS2
hinge.
Cytoplasmic Domain
[0455] The cytoplasmic domain or region of a CAR of the present
invention 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.
[0456] 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.
[0457] 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).
[0458] 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.
[0459] Examples of ITAM containing primary intracellular signaling
domains that are of particular use in the invention include those
of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3
epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"),
FccRI, DAP10, DAP12, and CD66d. In one embodiment, a CAR of the
invention comprises an intracellular signaling domain, e.g., a
primary signaling domain of CD3-zeta.
[0460] 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.
[0461] 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.
[0462] The intracellular signalling domain of the CAR can comprise
the primary signalling domain, e.g., 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 primary signalling domain, e.g., 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 a MHC
class I molecule, TNF receptor proteins, Immunoglobulin-like
proteins, cytokine receptors, integrins, signaling lymphocytic
activation molecules (SLAM proteins), activating NK cell receptors,
BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30,
CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS,
ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,
SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, 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). 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.
[0463] 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.
[0464] 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: 7. In
one aspect, the signaling domain of CD3-zeta is a signaling domain
of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human
CD3 zeta).
[0465] 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
TABLE-US-00014 (SEQ ID NO: 8)
QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.
In one aspect, the signaling domain of CD27 is encoded by a nucleic
acid sequence of
TABLE-US-00015 (SEQ ID NO: 19)
AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCC
CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC
GACTTCGCAGCCTATCGCTCC.
[0466] In one aspect, the intracellular is designed to comprise the
signaling domain of CD3-zeta and the signaling domain of CD28. In
one aspect, the signaling domain of CD28 comprises an amino acid
sequence of SEQ ID NO: 482. In one aspect, the signaling domain of
CD28 is encoded by a nucleic acid sequence of SEQ ID NO: 483.
[0467] In one aspect, the intracellular is designed to comprise the
signaling domain of CD3-zeta and the signaling domain of ICOS. In
one aspect, the signaling domain of CD28 comprises an amino acid
sequence of SEQ ID NO: 484. In one aspect, the signaling domain of
ICOS is encoded by a nucleic acid sequence of SEQ ID NO: 485.
[0468] 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 (CLL-1)
or a different target (e.g., CD123, CD33, CD34, FLT3, or folate
receptor beta). In one embodiment, the second CAR includes an
antigen binding domain to a target expressed on acute myeloid
leukemia cells, such as, e.g., CD123, CD33, CD34, FLT3, or folate
receptor beta. In one embodiment, the CAR-expressing cell comprises
a first CAR that specifically binds a first antigen and includes an
intracellular signaling domain having a costimulatory signaling
domain but not a primary signaling domain, and a second CAR that
specifically binds a second, different, antigen and includes an
intracellular signaling domain having a primary signaling domain
but not a costimulatory signaling domain. While not wishing to be
bound by theory, placement of a co stimulatory signaling domain,
e.g., 4-1BB, CD28, CD27, ICOS, or OX-40, onto the first CAR, and
the primary signaling domain, e.g., CD3 zeta, on the second CAR can
limit the CAR activity to cells where both targets are expressed.
In one embodiment, the CAR expressing cell comprises a first CLL-1
CAR that includes a CLL-1 binding domain, a transmembrane domain
and a costimulatory domain and a second CAR that specifically binds
an antigen other than CLL-1 (e.g., an antigen expressed on AML
cells, e.g., CD123, CD33, CD34, FLT3, or folate receptor beta) and
includes an antigen binding domain, a transmembrane domain and a
primary signaling domain. In another embodiment, the CAR expressing
cell comprises a first CLL-1 CAR that includes a CLL-1 binding
domain, a transmembrane domain and a primary signaling domain and a
second CAR that specifically binds an antigen other than CLL-1
(e.g., an antigen expressed on AML cells, e.g., CD123, CD33, CD34,
FLT3, or folate receptor beta) and includes an antigen binding
domain to the antigen, a transmembrane domain and a costimulatory
signaling domain.
[0469] In one embodiment, the CAR-expressing cell comprises a CLL-1
CAR described herein and an inhibitory CAR. In one embodiment, the
inhibitory CAR comprises an antigen binding domain that binds an
antigen found on normal cells but not cancer cells, e.g., normal
cells that also express CLL. In one embodiment, the inhibitory CAR
comprises the antigen binding domain, a transmembrane domain and an
intracellular domain of an inhibitory molecule. For example, the
intracellular domain of the inhibitory CAR can be an intracellular
domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or
CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and
TGFR beta.
[0470] 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.
[0471] In some embodiments, the antigen binding domain comprises a
single domain antigen binding (SDAB) molecules include molecules
whose complementary determining regions are part of a single domain
polypeptide. Examples include, but are not limited to, heavy chain
variable domains, binding molecules naturally devoid of light
chains, single domains derived from conventional 4-chain
antibodies, engineered domains and single domain scaffolds other
than those derived from antibodies. SDAB molecules may be any of
the art, or any future single domain molecules. SDAB molecules may
be derived from any species including, but not limited to mouse,
human, camel, llama, lamprey, fish, shark, goat, rabbit, and
bovine. This term also includes naturally occurring single domain
antibody molecules from species other than Camelidae and
sharks.
[0472] In one aspect, an SDAB molecule can be derived from a
variable region of the immunoglobulin found in fish, such as, for
example, that which is derived from the immunoglobulin isotype
known as Novel Antigen Receptor (NAR) found in the serum of shark.
Methods of producing single domain molecules derived from a
variable region of NAR ("IgNARs") are described in WO 03/014161 and
Streltsov (2005) Protein Sci. 14:2901-2909.
[0473] According to another aspect, an SDAB molecule is a naturally
occurring single domain antigen binding molecule known as heavy
chain devoid of light chains. Such single domain molecules are
disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993)
Nature 363:446-448, for example. For clarity reasons, this variable
domain derived from a heavy chain molecule naturally devoid of
light chain is known herein as a VHH or nanobody to distinguish it
from the conventional VH of four chain immunoglobulins. Such a VHH
molecule can be derived from Camelidae species, for example in
camel, llama, dromedary, alpaca and guanaco. Other species besides
Camelidae may produce heavy chain molecules naturally devoid of
light chain; such VHHs are within the scope of the invention.
[0474] The SDAB molecules can be recombinant, CDR-grafted,
humanized, camelized, de-immunized and/or in vitro generated (e.g.,
selected by phage display).
[0475] It has also been discovered, that cells having a plurality
of chimeric membrane embedded receptors comprising an antigen
binding domain that interactions between the antigen binding domain
of the receptors can be undesirable, e.g., because it inhibits the
ability of one or more of the antigen binding domains to bind its
cognate antigen. Accordingly, disclosed herein are cells having a
first and a second non-naturally occurring chimeric membrane
embedded receptor comprising antigen binding domains that minimize
such interactions. Also disclosed herein are nucleic acids encoding
a first and a second non-naturally occurring chimeric membrane
embedded receptor comprising a antigen binding domains that
minimize such interactions, as well as methods of making and using
such cells and nucleic acids. In an embodiment the antigen binding
domain of one of said first said second non-naturally occurring
chimeric membrane embedded receptor, comprises an scFv, and the
other comprises a single VH domain, e.g., a camelid, shark, or
lamprey single VH domain, or a single VH domain derived from a
human or mouse sequence.
[0476] In some embodiments, the claimed invention comprises a first
and second CAR, wherein the antigen binding domain of one of said
first CAR said second CAR does not comprise a variable light domain
and a variable heavy domain. In some embodiments, the antigen
binding domain of one of said first CAR said second CAR is an scFv,
and the other is not an scFv. In some embodiments, the antigen
binding domain of one of said first CAR said second CAR comprises a
single VH domain, e.g., a camelid, shark, or lamprey single VH
domain, or a single VH domain derived from a human or mouse
sequence. In some embodiments, the antigen binding domain of one of
said first CAR said second CAR comprises a nanobody. In some
embodiments, the antigen binding domain of one of said first CAR
said second CAR comprises a camelid VHH domain.
[0477] In some embodiments, the antigen binding domain of one of
said first CAR said second CAR comprises an scFv, and the other
comprises a single VH domain, e.g., a camelid, shark, or lamprey
single VH domain, or a single VH domain derived from a human or
mouse sequence. In some embodiments, the antigen binding domain of
one of said first CAR said second CAR comprises an scFv, and the
other comprises a nanobody. In some embodiments, the antigen
binding domain of one of the first CAR or the second CAR comprises
an scFv, and the other comprises a camelid VHH domain.
[0478] In some embodiments, when present on the surface of a cell,
binding of the antigen binding domain of said first CAR to its
cognate antigen is not substantially reduced by the presence of
said second CAR. In some embodiments, binding of the antigen
binding domain of said first CAR to its cognate antigen in the
presence of said second CAR is 85%, 90%, 95%, 96%, 97%, 98% or 99%
of binding of the antigen binding domain of said first CAR to its
cognate antigen in the absence of said second CAR.
[0479] In some embodiments, when present on the surface of a cell,
the antigen binding domains of said first CAR said second CAR,
associate with one another less than if both were scFv antigen
binding domains. In some embodiments, the antigen binding domains
of said first CAR said second CAR, associate with one another 85%,
90%, 95%, 96%, 97%, 98% or 99% less than if both were scFv antigen
binding domains.
[0480] 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, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or
CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, 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, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3
and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,
CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270),
KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and 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., 4-1BB, CD27, ICOS, 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). In embodiments, the CAR-expressing cell described herein
comprises a switch costimulatory receptor, e.g., as described in WO
2013/019615, which is incorporated herein by reference in its
entirety. 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.
[0481] 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 CLL-1 CAR described herein, improves the
persistence of the CAR-expressing cell, e.g., T cell or NK cell. In
one embodiment, the CAR is a PD1 CAR comprising the extracellular
domain of PD1 indicated as underlined in SEQ ID NO: 24. In one
embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID
NO:24.
TABLE-US-00016 (SEQ ID NO: 24)
Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdna
tftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlp
ngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevp
tahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaagg
avhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpf
mrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynel
nlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseig
mkgerrrgkghdglyqglstatkdtydalhmqalppr.
[0482] In one embodiment, the PD1 CAR comprises the amino acid
sequence provided below (SEQ ID NO:22).
TABLE-US-00017 (SEQ ID NO: 22)
pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrms
psnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtyl
cgaislapkaqikeslraelrvterraeptahpspsprpagqfqtlvtttp
aprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagt
cgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeee
ggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemg
gkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstat
kdtydalhmqalppr.
[0483] 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: 23
TABLE-US-00018 (SEQ ID NO: 23)
atggccctccctgtcactgccctgcttctccccctcgcactcctgctccac
gccgctagaccacccggatggtttctggactctccggatcgcccgtggaat
cccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcg
accttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactgg
taccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaa
gatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccg
aatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactcc
gggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaa
gagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgcca
actgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctg
gtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcg
agccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggt
gctgtgcatacccggggattggacttcgcatgcgacatctacatttgggct
cctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctg
tactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttc
atgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttc
cccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagc
gccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactg
aacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgg
gaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctg
tataacgagctgcagaaggacaagatggccgaggcctactccgaaattggg
atgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaagga
ctgtccaccgccaccaaggacacatacgatgccctgcacatgcaggccctt ccccctcgc.
[0484] In another aspect, the present invention provides a
population of CAR-expressing cells, e.g., CART cells or
CAR-expressing NK 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 (e.g., CART cells or CAR-expressing NK cells)
can include a first cell expressing a CAR having an anti-CLL-1
binding domain described herein, and a second cell expressing a CAR
having a different anti-CLL-1 binding domain, e.g., an anti-CLL-1
binding domain described herein that differs from the anti-CLL-1
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-CLL-1 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 CLL-1
(e.g., CD123, CD33, CD34, FLT3, or folate receptor beta). 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, e.g., a costimulatory signaling
domain.
[0485] 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-CLL-1 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, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, 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,
PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, and 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., 4-1BB, CD27 ICOS, 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).
[0486] In one aspect, the present invention provides methods
comprising administering a population of CAR-expressing cells
(e.g., CART cells or CAR-expressing NK cells), e.g., a mixture of
cells expressing different CARs, in combination with another agent,
e.g., a kinase inhibitor, such as a kinase inhibitor described
herein. In another aspect, the present invention provides methods
comprising administering a population of cells wherein at least one
cell in the population expresses a CAR having an anti-cancer
associated antigen binding domain as described herein, and a second
cell expressing another agent, e.g., an agent which enhances the
activity of a CAR-expressing cell, in combination with another
agent, e.g., a kinase inhibitor, such as a kinase inhibitor
described herein.
[0487] Natural Killer Cell Receptor (NKR) CARs
[0488] In an embodiment, the CAR molecule described herein
comprises one or more components of a natural killer cell receptor
(NKR), thereby forming an NKR-CAR. The NKR component can be a
transmembrane domain, a hinge domain, or a cytoplasmic domain from
any of the following natural killer cell receptors: killer cell
immunoglobulin-like receptor (KIR), e.g., KIR2DL1, KIR2DL2/L3,
KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4,
DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1;
natural cyotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46;
signaling lymphocyte activation molecule (SLAM) family of immune
cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME,
and CD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49
receptors, e.g., LY49A, LY49C. The NKR-CAR molecules described
herein may interact with an adaptor molecule or intracellular
signaling domain, e.g., DAP12. Exemplary configurations and
sequences of CAR molecules comprising NKR components are described
in International Publication No. WO2014/145252, the contents of
which are hereby incorporated by reference.
Strategies for Regulating Chimeric Antigen Receptors
[0489] There are many ways CAR activities can be regulated. 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. For example, inducing apoptosis using, e.g., a caspase
fused to a dimerization domain (see, e.g., Di 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 another
example, CAR-expressing cells can also express an inducible
Caspase-9 (iCaspase-9) molecule that, upon administration of a
dimerizer drug (e.g., rimiducid (also called AP1903 (Bellicum
Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the
Caspase-9 and apoptosis of the cells. The iCaspase-9 molecule
contains a chemical inducer of dimerization (CID) binding domain
that mediates dimerization in the presence of a CID. This results
in inducible and selective depletion of CAR-expressing cells. In
some cases, the iCaspase-9 molecule is encoded by a nucleic acid
molecule separate from the CAR-encoding vector(s). In some cases,
the iCaspase-9 molecule is encoded by the same nucleic acid
molecule as the CAR-encoding vector. The iCaspase-9 can provide a
safety switch to avoid any toxicity of CAR-expressing cells. See,
e.g., Song et al. Cancer Gene Ther. 2008; 15(10):667-75; Clinical
Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med.
2011; 365:1673-83.
[0490] Alternative strategies for regulating the CAR therapy of the
instant invention include utilizing small molecules or antibodies
that deactivate or turn off CAR activity, e.g., by deleting
CAR-expressing cells, e.g., by inducing antibody dependent
cell-mediated cytotoxicity (ADCC). For example, CAR-expressing
cells described herein may also express an antigen that is
recognized by molecules capable of inducing cell death, e.g., ADCC
or compliment-induced cell death. For example, CAR expressing cells
described herein may also express a receptor capable of being
targeted by an antibody or antibody fragment. Examples of such
receptors include EpCAM, VEGFR, integrins (e.g., integrins
.alpha.v.beta.3, .alpha.4, .alpha.I3/4.beta.3, .alpha.4.beta.7,
.alpha.5.beta.1, .alpha.v.beta.3, .alpha.v), members of the TNF
receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor,
interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA,
CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4,
CD5, CD11, CD11 a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22,
CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44,
CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4,
CD154/CD40L, CD195/CCR5, CD319/SLAMF7, and EGFR, and truncated
versions thereof (e.g., versions preserving one or more
extracellular epitopes but lacking one or more regions within the
cytoplasmic domain). For example, CAR-expressing cells described
herein may also express a truncated epidermal growth factor
receptor (EGFR) which lacks signaling capacity but retains the
epitope that is recognized by molecules capable of inducing ADCC,
e.g., cetuximab (ERBITUX.RTM.), such that administration of
cetuximab induces ADCC and subsequent depletion of the
CAR-expressing cells (see, e.g., WO2011/056894, and Jonnalagadda et
al., Gene Ther. 2013; 20(8)853-860). Another strategy includes
expressing a highly compact marker/suicide gene that combines
target epitopes from both CD32 and CD20 antigens in the
CAR-expressing cells described herein, which binds rituximab,
resulting in selective depletion of the CAR-expressing cells, e.g.,
by ADCC (see, e.g., Philip et al., Blood. 2014; 124(8)1277-1287).
Other methods for depleting CAR-expressing cells described herein
include administration of CAMPATH, a monoclonal anti-CD52 antibody
that selectively binds and targets mature lymphocytes, e.g.,
CAR-expressing cells, for destruction, e.g., by inducing ADCC. In
other embodiments, the CAR-expressing cell can be selectively
targeted using a CAR ligand, e.g., an anti-idiotypic antibody. In
some embodiments, the anti-idiotypic antibody can cause effector
cell activity, e.g, ADCC or ADC activities, thereby reducing the
number of CAR-expressing cells. In other embodiments, the CAR
ligand, e.g., the anti-idiotypic antibody, can be coupled to an
agent that induces cell killing, e.g., a toxin, thereby reducing
the number of CAR-expressing cells. Alternatively, the CAR
molecules themselves can be configured such that the activity can
be regulated, e.g., turned on and off, as described below.
[0491] In some embodiments, 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. Additional description and
exemplary configurations of such regulatable CARs are provided
herein and in International Publiciation No. WO 2015/090229, hereby
incorporated by reference in its entirety.
[0492] 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
specifically binds a tumor antigen described herein, 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).
[0493] 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.
[0494] 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.
[0495] 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.
[0496] 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 4-1BB,
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:
4-1BB-CD27; 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB;
OX40-CD28; CD28-OX40; CD28-4-1BB; or 4-1BB-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.
[0497] 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.
[0498] 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.
[0499] 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
4-1BB, 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.
[0500] In any of the RCAR configurations described here, the first
and second switch domains comprise a FKBP-FRB based switch as
described herein.
[0501] 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.
[0502] 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.
[0503] 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.
[0504] Dimerization Switches
[0505] 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.
[0506] 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.)
[0507] In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based
switch can use a dimerization molecule, e.g., rapamycin or a
rapamycin analog.
[0508] The amino acid sequence of FKBP is as follows:
TABLE-US-00019 (SEQ ID NO: 205) 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
[0509] 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: 205, which is:
TABLE-US-00020 (SEQ ID NO: 206) 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
[0510] The amino acid sequence of FRB is as follows:
TABLE-US-00021 (SEQ ID NO: 207) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV
LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR
ISK
[0511] "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: 54 or 55; 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: 56. In an embodiment, a RCAR described herein comprises one
switch domain comprises amino acid residues disclosed in SEQ ID NO:
205 (or SEQ ID NO: 206), and one switch domain comprises amino acid
residues disclosed in SEQ ID NO: 207.
[0512] 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 (E20321), e.g., SEQ
ID NO: 208, or leucine (E2032L), e.g., SEQ ID NO: 209. 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: 210. 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: 211. In
an embodiment, a mutant FRB comprises an E20321 and a T2098L
mutation, e.g., SEQ ID NO: 212. In an embodiment, a mutant FRB
comprises an E2032L and a T2098L mutation, e.g., SEQ ID NO:
213.
TABLE-US-00022 TABLE 9 Exemplary mutant FRB having increased
affinity for a dimerization molecule. SEQ FRB ID mutant Amino Acid
Sequence NO: E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLH 208 mutant
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLTQAWDLYYHVFRRISKTS
E2032L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLH 209 mutant
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLTQAWDLYYHVFRRISKTS
T2098L ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLH 210 mutant
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLLQAWDLYYHVFRRISKTS
E2032, ILWHEMWHEGLXEASRLYFGERNVKGMFEVLEPLH 211 T2098
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK mutant
SGNVKDLXQAWDLYYHVFRRISKTS E2032I,
ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLH 212 T2098L
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK mutant
SGNVKDLLQAWDLYYHVFRRISKTS E2032L,
ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLH 213 T2098L
AMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK mutant
SGNVKDLLQAWDLYYHVFRRISKTS
[0513] 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.
[0514] Dimerization Molecule
[0515] 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.
[0516] 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 "Combination with a low
dose mTOR inhibitor".
Split CAR
[0517] 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, incorporated herein
by reference. 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. In embodiments
the first antigen binding domain recognizes CLL-1, e.g., comprises
an antigen binding domain described herein, and the second antigen
binding domain recognizes an antigen expressed on acute myeloid
leukemia cells, e.g., CD123, CD33, CD34, FLT3, or folate receptor
beta. In embodiments the first antigen binding domain recognizes
CLL-1, e.g., comprises an antigen binding domain described herein,
and the second antigen binding domain recognizes an antigen
expressed on B-cells, e.g., CD19, CD20, CD22 or ROR1.
[0518] Stability and Mutations
[0519] The stability of a CLL-1 binding domain, e.g., scFv
molecules (e.g., soluble scFv) can be evaluated in reference to the
biophysical properties (e.g., thermal stability) of a conventional
control scFv molecule or a full length antibody. In one embodiment,
the human scFv has a thermal stability that is greater than about
0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about
1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4,
about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about
7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees,
about 11 degrees, about 12 degrees, about 13 degrees, about 14
degrees, or about 15 degrees Celsius than a control binding
molecule (e.g. a conventional scFv molecule) in the described
assays.
[0520] The improved thermal stability of the anti-CLL-1 binding
domain, e.g., scFv is subsequently conferred to the entire CLL-1
CAR construct, leading to improved therapeutic properties of the
CLL-1 CAR construct. The thermal stability of the anti-CLL-1
binding domain, e.g., scFv can be improved by at least about
2.degree. C. or 3.degree. C. as compared to a conventional
antibody. In one embodiment, the anti-CLL-1 binding domain, e.g.,
scFv has a 1.degree. C. improved thermal stability as compared to a
conventional antibody. In another embodiment, the anti-CLL-1
binding domain, e.g., scFv has a 2.degree. C. improved thermal
stability as compared to a conventional antibody. In another
embodiment, the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15.degree. C. improved thermal stability as compared to a
conventional antibody. Comparisons can be made, for example,
between the scFv molecules disclosed herein and full length
antibodies. Thermal stability can be measured using methods known
in the art. For example, in one embodiment, Tm can be measured.
Methods for measuring Tm and other methods of determining protein
stability are described in more detail below.
[0521] Mutations in scFv alter the stability of the scFv and
improve the overall stability of the scFv and the CART CLL-1
construct. Stability of the human scFv is determined using
measurements such as Tm, temperature denaturation and temperature
aggregation.
[0522] The binding capacity of the mutant scFvs can be determined
using assays described in the Examples.
[0523] In one embodiment, the anti-CLL-1 binding domain, e.g., scFv
comprises at least one mutation such that the mutated scFv confers
improved stability to the CLL-1 CAR construct. In another
embodiment, the anti-CLL-1 binding domain, e.g., scFv comprises at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the
humanization process such that the mutated scFv confers improved
stability to the CLL-1 CAR construct.
[0524] Methods of Evaluating Protein Stability
[0525] The stability of an antigen binding domain may be assessed
using, e.g., the methods described below. Such methods allow for
the determination of multiple thermal unfolding transitions where
the least stable domain either unfolds first or limits the overall
stability threshold of a multidomain unit that unfolds
cooperatively (e.g., a multidomain protein which exhibits a single
unfolding transition). The least stable domain can be identified in
a number of additional ways. Mutagenesis can be performed to probe
which domain limits the overall stability. Additionally, protease
resistance of a multidomain protein can be performed under
conditions where the least stable domain is known to be
intrinsically unfolded via DSC or other spectroscopic methods
(Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al.
(2009) J. Mol. Biol. 393: 672-692). Once the least stable domain is
identified, the sequence encoding this domain (or a portion
thereof) may be employed as a test sequence in the methods.
[0526] a) Thermal Stability
[0527] The thermal stability of the compositions may be analyzed
using a number of non-limiting biophysical or biochemical
techniques known in the art. In certain embodiments, thermal
stability is evaluated by analytical spectroscopy.
[0528] An exemplary analytical spectroscopy method is Differential
Scanning calorimetry (DSC). DSC employs a calorimeter which is
sensitive to the heat absorbances that accompany the unfolding of
most proteins or protein domains (see, e.g. Sanchez-Ruiz, et al.,
Biochemistry, 27: 1648-52, 1988). To determine the thermal
stability of a protein, a sample of the protein is inserted into
the calorimeter and the temperature is raised until the Fab or scFv
unfolds. The temperature at which the protein unfolds is indicative
of overall protein stability.
[0529] Another exemplary analytical spectroscopy method is Circular
Dichroism (CD) spectroscopy. CD spectrometry measures the optical
activity of a composition as a function of increasing temperature.
Circular dichroism (CD) spectroscopy measures differences in the
absorption of left-handed polarized light versus right-handed
polarized light which arise due to structural asymmetry. A
disordered or unfolded structure results in a CD spectrum very
different from that of an ordered or folded structure. The CD
spectrum reflects the sensitivity of the proteins to the denaturing
effects of increasing temperature and is therefore indicative of a
protein's thermal stability (see van Mierlo and Steemsma, J.
Biotechnol., 79(3):281-98, 2000).
[0530] Another exemplary analytical spectroscopy method for
measuring thermal stability is Fluorescence Emission Spectroscopy
(see van Mierlo and Steemsma, supra). Yet another exemplary
analytical spectroscopy method for measuring thermal stability is
Nuclear Magnetic Resonance (NMR) spectroscopy (see, e.g. van Mierlo
and Steemsma, supra).
[0531] The thermal stability of a composition can be measured
biochemically. An exemplary biochemical method for assessing
thermal stability is a thermal challenge assay. In a "thermal
challenge assay", a composition is subjected to a range of elevated
temperatures for a set period of time. For example, in one
embodiment, test scFv molecules or molecules comprising scFv
molecules are subject to a range of increasing temperatures, e.g.,
for 1-1.5 hours. The activity of the protein is then assayed by a
relevant biochemical assay. For example, if the protein is a
binding protein (e.g. an scFv or scFv-containing polypeptide) the
binding activity of the binding protein may be determined by a
functional or quantitative ELISA.
[0532] Such an assay may be done in a high-throughput format and
those disclosed in the Examples using E. coli and high throughput
screening. A library of anti-CLL-1 binding domain, e.g., scFv
variants may be created using methods known in the art. Anti-CLL-1
binding domain, e.g., scFv expression may be induced and the
anti-CLL-1 binding domain, e.g., scFv may be subjected to thermal
challenge. The challenged test samples may be assayed for binding
and those anti-CLL-1 binding domain, e.g., scFvs which are stable
may be scaled up and further characterized.
[0533] Thermal stability is evaluated by measuring the melting
temperature (Tm) of a composition using any of the above techniques
(e.g. analytical spectroscopy techniques). The melting temperature
is the temperature at the midpoint of a thermal transition curve
wherein 50% of molecules of a composition are in a folded state
(See e.g., Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In one
embodiment, Tm values for an anti-CLL-1 binding domain, e.g., scFv
are about 40.degree. C., 41.degree. C., 42.degree. C., 43.degree.
C., 44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.,
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., 66.degree. C., 67.degree. C.,
68.degree. C., 69.degree. C., 70.degree. C., 71.degree. C.,
72.degree. C., 73.degree. C., 74.degree. C., 75.degree. C.,
76.degree. C., 77.degree. C., 78.degree. C., 79.degree. C.,
80.degree. C., 81.degree. C., 82.degree. C., 83.degree. C.,
84.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C. In one embodiment, Tm values for an IgG is about
40.degree. C., 41.degree. C., 42.degree. C., 43.degree. C.,
44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.,
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., 66.degree. C., 67.degree. C.,
68.degree. C., 69.degree. C., 70.degree. C., 71.degree. C.,
72.degree. C., 73.degree. C., 74.degree. C., 75.degree. C.,
76.degree. C., 77.degree. C., 78.degree. C., 79.degree. C.,
80.degree. C., 81.degree. C., 82.degree. C., 83.degree. C.,
84.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C. In one embodiment, Tm values for an multivalent
antibody is about 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C., 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., 66.degree. C.,
67.degree. C., 68.degree. C., 69.degree. C., 70.degree. C.,
71.degree. C., 72.degree. C., 73.degree. C., 74.degree. C.,
75.degree. C., 76.degree. C., 77.degree. C., 78.degree. C.,
79.degree. C., 80.degree. C., 81.degree. C., 82.degree. C.,
83.degree. C., 84.degree. C., 85.degree. C., 86.degree. C.,
87.degree. C., 88.degree. C., 89.degree. C., 90.degree. C.,
91.degree. C., 92.degree. C., 93.degree. C., 94.degree. C.,
95.degree. C., 96.degree. C., 97.degree. C., 98.degree. C.,
99.degree. C., 100.degree. C.
[0534] Thermal stability is also evaluated by measuring the
specific heat or heat capacity (Cp) of a composition using an
analytical calorimetric technique (e.g. DSC). The specific heat of
a composition is the energy (e.g. in kcal/mol) is required to rise
by 1.degree. C., the temperature of 1 mol of water. As large Cp is
a hallmark of a denatured or inactive protein composition. The
change in heat capacity (.DELTA.Cp) of a composition is measured by
determining the specific heat of a composition before and after its
thermal transition. Thermal stability may also be evaluated by
measuring or determining other parameters of thermodynamic
stability including Gibbs free energy of unfolding (.DELTA.G),
enthalpy of unfolding (.DELTA.H), or entropy of unfolding
(.DELTA.S). One or more of the above biochemical assays (e.g. a
thermal challenge assay) are used to determine the temperature
(i.e. the Tc value) at which 50% of the composition retains its
activity (e.g. binding activity).
[0535] In addition, mutations to the anti-CLL-1 binding domain,
e.g., scFv alter the thermal stability of the anti-CLL-1 binding
domain, e.g., scFv compared with the unmutated anti-CLL-1 binding
domain, e.g., scFv. In one embodiment, the anti-CLL-1 binding
domain, e.g., scFv comprises a single mutation that confers thermal
stability to the anti-CLL-1 binding domain, e.g., scFv. In another
embodiment, the anti-CLL-1 binding domain, e.g., scFv comprises
multiple mutations that confer thermal stability to the anti-CLL-1
binding domain, e.g., scFv. In one embodiment, the multiple
mutations in the anti-CLL-1 binding domain, e.g., scFv have an
additive effect on thermal stability of the anti-CLL-1 binding
domain, e.g., scFv.
[0536] b) % Aggregation
[0537] The stability of a composition can be determined by
measuring its propensity to aggregate. Aggregation can be measured
by a number of non-limiting biochemical or biophysical techniques.
For example, the aggregation of a composition may be evaluated
using chromatography, e.g. Size-Exclusion Chromatography (SEC). SEC
separates molecules on the basis of size. A column is filled with
semi-solid beads of a polymeric gel that will admit ions and small
molecules into their interior but not large ones. When a protein
composition is applied to the top of the column, the compact folded
proteins (i.e. non-aggregated proteins) are distributed through a
larger volume of solvent than is available to the large protein
aggregates. Consequently, the large aggregates move more rapidly
through the column, and in this way the mixture can be separated or
fractionated into its components. Each fraction can be separately
quantified (e.g. by light scattering) as it elutes from the gel.
Accordingly, the % aggregation of a composition can be determined
by comparing the concentration of a fraction with the total
concentration of protein applied to the gel. Stable compositions
elute from the column as essentially a single fraction and appear
as essentially a single peak in the elution profile or
chromatogram.
[0538] c) Binding Affinity
[0539] The stability of a composition can be assessed by
determining its target binding affinity. A wide variety of methods
for determining binding affinity are known in the art. An exemplary
method for determining binding affinity employs surface plasmon
resonance. Surface plasmon resonance is an optical phenomenon that
allows for the analysis of real-time biospecific interactions by
detection of alterations in protein concentrations within a
biosensor matrix, for example using the BIAcore system (Pharmacia
Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further
descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.
51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson,
B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et
al. (1991) Anal. Biochem. 198:268-277.
[0540] In one aspect, the antigen binding domain of the CAR
comprises an amino acid sequence that is homologous to an antigen
binding domain amino acid sequence described herein, and the
antigen binding domain retains the desired functional properties of
the anti-CLL-1 antibody fragments described herein. In one specific
aspect, the CAR composition of the invention comprises an antibody
fragment. In a further aspect, that antibody fragment comprises an
scFv.
[0541] In various aspects, the antigen binding domain of the CAR is
engineered by modifying one or more amino acids within one or both
variable regions (e.g., VH and/or VL), for example within one or
more CDR regions and/or within one or more framework regions. In
one specific aspect, the CAR composition of the invention comprises
an antibody fragment. In a further aspect, that antibody fragment
comprises an scFv.
[0542] It will be understood by one of ordinary skill in the art
that the antibody or antibody fragment of the invention may further
be modified such that they vary in amino acid sequence (e.g., from
wild-type), but not in desired activity. For example, additional
nucleotide substitutions leading to amino acid substitutions at
"non-essential" amino acid residues may be made to the protein For
example, a nonessential amino acid residue in a molecule may be
replaced with another amino acid residue from the same side chain
family. In another embodiment, a string of amino acids can be
replaced with a structurally similar string that differs in order
and/or composition of side chain family members, e.g., a
conservative substitution, in which an amino acid residue is
replaced with an amino acid residue having a similar side chain,
may be made.
[0543] 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).
[0544] 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.
[0545] 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).
[0546] 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.
[0547] 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.
[0548] 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-CLL-1 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-CLL-1 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.
[0549] RNA Transfection
[0550] 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:35). RNA so produced can efficiently transfect different
kinds of cells. In one aspect, the template includes sequences for
the CAR.
[0551] In one aspect the anti-CLL-1 CAR is encoded by a messenger
RNA (mRNA). In one aspect the mRNA encoding the anti-CLL-1 CAR is
introduced into a T cell for production of a CART cell.
[0552] 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.
[0553] 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.
[0554] 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.
[0555] 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.
[0556] 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.
[0557] 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.
[0558] 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.
[0559] 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.
[0560] 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.
[0561] 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).
[0562] 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.
[0563] 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: 31) (size can be 50-5000 T (SEQ
ID NO: 32)), 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:
33).
[0564] 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: 34) 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.
[0565] 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)).
[0566] 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.
[0567] 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).
[0568] Non-Viral Delivery Methods
[0569] 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.
[0570] 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.
[0571] 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.
[0572] 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.
[0573] 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
Tc 1/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.
[0574] 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.
[0575] 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.
[0576] 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).
[0577] 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.
[0578] Nucleic Acid Constructs Encoding a CAR
[0579] 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.
[0580] 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-CLL-1 binding domain (e.g.,
a human anti-CLL-1 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-CLL-1 binding
domain is an anti-CLL-1 binding domain described herein, e.g., an
anti-CLL-1 binding domain which comprises a sequence selected from
a group consisting of SEQ ID NO:39-51, or a sequence with 95-99%
identity thereof. In one embodiment, the transmembrane domain is
transmembrane domain of a protein described herein, e.g., 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: 6, or a sequence with 95-99% identity thereof. In one
embodiment, the anti-CLL-1 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:2
or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5, 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 described herein, e.g., selected from
the group consisting of a MHC class I molecule, TNF receptor
proteins, Immunoglobulin-like proteins, cytokine receptors,
integrins, signaling lymphocytic activation molecules (SLAM
proteins), activating NK cell receptors, BTLA, a Toll ligand
receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1,
LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83.
[0581] In one embodiment, the costimulatory domain comprises a
sequence of SEQ ID NO:7, 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: 7 or SEQ ID
NO:8, or a sequence with 95-99% identity thereof, and the sequence
of SEQ ID NO: 9 or SEQ ID NO:10, 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.
[0582] In another aspect, the invention pertains to an isolated
nucleic acid molecule encoding a CAR construct comprising a leader
sequence of SEQ ID NO: 1, a scFv domain having a sequence selected
from the group consisting of SEQ ID NO:39-51, (or a sequence with
95-99% identity thereof), a hinge region of SEQ ID NO:2 or SEQ ID
NO:3 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequence with 95-99%
identity thereof), a transmembrane domain having a sequence of SEQ
ID NO: 6 (or a sequence with 95-99% identity thereof), a 4-1BB
costimulatory domain having a sequence of SEQ ID NO:7 or a CD27
costimulatory domain having a sequence of SEQ ID NO:8 (or a
sequence with 95-99% identity thereof) or a CD28 costimulatory
domain having a sequence of SEQ ID NO:482 (or a sequence with
95-99% identity thereof) or a ICOS costimulatory domain having a
sequence of SEQ ID NO: 483 (or a sequence with 95-99% identity
thereof), and a CD3 zeta stimulatory domain having a sequence of
SEQ ID NO:9 or SEQ ID NO:10 (or a sequence with 95-99% identity
thereof).
[0583] 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:91-103, or a
sequence with 95-99% identity thereof.
[0584] In another aspect, the invention pertains to a nucleic acid
molecule encoding a chimeric antigen receptor (CAR) molecule that
comprises an anti-CLL-1 binding domain, a transmembrane domain, and
an intracellular signaling domain comprising a stimulatory domain,
and wherein said anti-CLL-1 binding domain comprises a sequence
selected from the group consisting of SEQ ID NO:39-51, or a
sequence with 95-99% identity thereof.
[0585] 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 a MHC
class I molecule, TNF receptor proteins, Immunoglobulin-like
proteins, cytokine receptors, integrins, signaling lymphocytic
activation molecules (SLAM proteins), activating NK cell receptors,
BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30,
CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS,
ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,
SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83. In one embodiment, the 4-1BB
costimulatory domain comprises an amino acid sequence of SEQ ID
NO:7. In one embodiment, the CD27 costimulatory domain comprises an
amino acid sequence of SEQ ID NO:8. In one embodiment, the CD28
costimulatory domain comprises an amino acid sequence of SEQ ID
NO:482. In one embodiment, the ICOS costimulatory domain comprises
an amino acid sequence of SEQ ID NO:484.
[0586] 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, CD154, a MHC class I
molecule, TNF receptor proteins, Immunoglobulin-like proteins,
cytokine receptors, integrins, signaling lymphocytic activation
molecules (SLAM proteins), activating NK cell receptors, BTLA, a
Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS,
ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, 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, 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 (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that
specifically binds with CD83. In one embodiment, the transmembrane
domain comprises a sequence of SEQ ID NO:6. 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: 7 and the sequence of SEQ ID NO: 9, 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-CLL-1 binding domain is connected to the
transmembrane domain by a hinge region. In one embodiment, the
hinge region comprises SEQ ID NO:2. In one embodiment, the hinge
region comprises SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5.
[0587] In another aspect, the invention pertains to an encoded CAR
molecule comprising a leader sequence of SEQ ID NO: 1, a scFv
domain having a sequence selected from the group consisting of SEQ
ID NO:59-51, or a sequence with 95-99% identity thereof, a hinge
region of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5,
a transmembrane domain having a sequence of SEQ ID NO: 6, a 4-1BB
costimulatory domain having a sequence of SEQ ID NO:7 or a CD27
costimulatory domain having a sequence of SEQ ID NO:8, and a CD3
zeta stimulatory domain having a sequence of SEQ ID NO:9 or SEQ ID
NO:10. In one embodiment, the encoded CAR molecule comprises a
sequence selected from a group consisting of SEQ ID NO:91-103, or a
sequence with 95-99% identity thereof.
[0588] 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.
[0589] 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.
[0590] 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.
[0591] 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.
[0592] 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.
[0593] 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.
[0594] 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).
[0595] 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.
[0596] 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.
[0597] 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:11.
[0598] 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.
[0599] Another example of a promoter is the phosphoglycerate kinase
(PGK) promoter. In embodiments, a truncated PGK promoter (e.g., a
PGK promoter with one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or
400, nucleotide deletions when compared to the wild-type PGK
promoter sequence) may be desired. The nucleotide sequences of
exemplary PGK promoters are provided below.
TABLE-US-00023 WT PGK Promoter (SEQ ID NO: 487)
ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCAC
GCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG
GTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGAC
GAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCA
GCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCAC
TCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTT
GGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCC
CATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCT
CTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGC
GCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTGGGG CACCATAAGCT
Exemplary truncated PGK Promoters: PGK100: (SEQ ID NO: 488)
ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCAC
GCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG
GTGTGATGGCGGGGTG PGK200: (SEQ ID NO: 489)
ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCAC
GCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG
GTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGAC
GAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCA
GCCGCGCGACGGTAACG PGK300: (SEQ ID NO: 490)
ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCAC
GCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG
GTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGAC
GAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCA
GCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCAC
TCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTT
GGAAGGGCTGAATCCCCG PGK400: (SEQ ID NO: 491)
ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCAC
GCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGG
GTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGAC
GAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCA
GCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCAC
TCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTT
GGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCC
CATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCT
CTGGGTCCCAGCCG
[0600] 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).
[0601] 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.
[0602] 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.
[0603] In one embodiment, the vector can further comprise a nucleic
acid encoding a second CAR. In one embodiment, the second CAR
includes an antigen binding domain to a target expressed on acute
myeloid leukemia cells, such as, e.g., CD123, CD33, CD34, FLT3, or
folate receptor beta. In one embodiment, the vector comprises a
nucleic acid sequence encoding a first CAR that specifically binds
a first antigen and includes an intracellular signaling domain
having a costimulatory signaling domain but not a primary signaling
domain, and a nucleic acid encoding a second CAR that specifically
binds a second, different, antigen and includes an intracellular
signaling domain having a primary signaling domain but not a
costimulatory signaling domain. In one embodiment, the vector
comprises a nucleic acid encoding a first CLL-1 CAR that includes a
CLL-1 binding domain, a transmembrane domain and a costimulatory
domain and a nucleic acid encoding a second CAR that specifically
binds an antigen other than CLL-1 (e.g., an antigen expressed on
AML cells, e.g., CD123, CD33, CD34, FLT3, or folate receptor beta)
and includes an antigen binding domain, a transmembrane domain and
a primary signaling domain. In another embodiment, the vector
comprises a nucleic acid encoding a first CLL-1 CAR that includes a
CLL-1 binding domain, a transmembrane domain and a primary
signaling domain and a nucleic acid encoding a second CAR that
specifically binds an antigen other than CLL-1 (e.g., an antigen
expressed on AML cells, e.g., CD123, CD33, CD34, FLT3, or folate
receptor beta) and includes an antigen binding domain to the
antigen, a transmembrane domain and a costimulatory signaling
domain.
[0604] In one embodiment, the vector comprises a nucleic acid
encoding a CLL-1 CAR described herein and a nucleic acid encoding
an inhibitory CAR. In one embodiment, the inhibitory CAR comprises
an antigen binding domain that binds an antigen found on normal
cells but not cancer cells, e.g., normal cells that also express
CLL. In one embodiment, the inhibitory CAR comprises the antigen
binding domain, a transmembrane domain and an intracellular domain
of an inhibitory molecule. For example, the intracellular domain of
the inhibitory CAR can be an intracellular domain of PD1, PD-L1,
PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.
[0605] In embodiments, the vector may comprise two or more nucleic
acid sequences encoding a CAR, e.g., a CLL-1 CAR described herein
and a second CAR, e.g., an inhibitory CAR or a CAR that
specifically binds to an antigen other than CLL-1 (e.g., an antigen
expressed on AML cells, e.g., CD123, CD33, CD34, FLT3, or folate
receptor beta). In such embodiments, the two or more nucleic acid
sequences encoding the CAR are encoded by a single nucleic molecule
in the same frame and as a single polypeptide chain. In this
aspect, the two or more CARs, can, e.g., be separated by one or
more peptide cleavage sites. (e.g., an auto-cleavage site or a
substrate for an intracellular protease). Examples of peptide
cleavage sites include the following, wherein the GSG residues are
optional:
TABLE-US-00024 T2A: (SEQ ID NO: 492) (GSG) E G R G S L L T C G D V
E E N P G P P2A: (SEQ ID NO: 493) (GSG) A T N F S L L K Q A G D V E
E N P G P E2A: (SEQ ID NO: 494) (GSG) Q C T N Y A L L K L A G D V E
S N P G P F2A: (SEQ ID NO: 495) (GSG) V K Q T L N F D L L K L A G D
V E S N P G P
[0606] 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.
[0607] 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
[0608] 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.
[0609] 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.
[0610] 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.
[0611] 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.
[0612] 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.
[0613] 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., an immune effector cell,
e.g., a T cell or NK 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.
[0614] Sources of Cells
[0615] Prior to expansion and genetic modification, a source of
cells (e.g., immune effector cells, e.g., T cells or NK cells) is
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, 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.
[0616] In certain aspects of the present invention, any number of
immune effector cell (e.g., T cell or NK cell) lines available in
the art, may be used. In certain aspects of the present invention,
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 to place the cells in an appropriate buffer
or media for subsequent processing steps. In one aspect of the
invention, the cells are washed with phosphate buffered saline
(PBS). In an alternative aspect, the wash solution lacks calcium
and may lack magnesium or may lack many if not all divalent
cations. 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.
[0617] It is recognized that the methods of the application can
utilize culture media conditions comprising 5% or less, for example
2%, human AB serum, and employ known culture media conditions and
compositions, for example those described in Smith et al., "Ex vivo
expansion of human T cells for adoptive immunotherapy using the
novel Xeno-free CTS Immune Cell Serum Replacement" Clinical &
Translational Immunology (2015) 4, e31;
doi:10.1038/cti.2014.31.
[0618] 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. A specific
subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+,
and CD45RO+ T cells, can be further isolated by positive or
negative selection techniques. For example, in one aspect, T cells
are 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 aspect, the time period is about 30
minutes. In a further aspect, the time period ranges from 30
minutes to 36 hours or longer and all integer values there between.
In a further aspect, the time period is at least 1, 2, 3, 4, 5, or
6 hours. In yet another preferred aspect, the time period is 10 to
24 hours. In one aspect, the incubation time period is 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. The skilled artisan would recognize that
multiple rounds of selection can also be used in the context of
this invention. In certain aspects, it may be desirable to perform
the selection procedure and use the "unselected" cells in the
activation and expansion process. "Unselected" cells can also be
subjected to further rounds of selection.
[0619] Enrichment of a T cell population by negative selection can
be accomplished 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
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8. In certain aspects, it may be desirable to enrich for or
positively select for regulatory T cells which typically express
CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain
aspects, T regulatory cells are depleted by anti-C25 conjugated
beads or other similar method of selection.
[0620] 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.
[0621] 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.
[0622] 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.
[0623] 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).
[0624] 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.
[0625] 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.
[0626] 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.
[0627] 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.
[0628] 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.
[0629] 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.
[0630] 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.
[0631] 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.
[0632] 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 PD1, PD-L1, PD-L2,
CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),
LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3
(CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC
class I, MHC class II, GAL9, adenosine, and TGFR beta. 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.
[0633] In one embodiment, a T cell population can be selected that
expresses one or more of IFN-.gamma., TNF.alpha., IL-17A, IL-2,
IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or
other appropriate molecules, e.g., other cytokines. Methods for
screening for cell expression can be determined, e.g., by the
methods described in PCT Publication No.: WO 2013/126712.
[0634] 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 2 billion
cells/ml is used. In one aspect, a concentration of 1 billion
cells/ml is used. In a further 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, 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.
[0635] 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.10e6/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.
[0636] 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.
[0637] 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
1.degree. 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.
[0638] 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.
[0639] 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 immune effector cells, e.g., T cells or NK cells, isolated
and frozen for later use in cell therapy, e.g., T cell therapy, for
any number of diseases or conditions that would benefit from cell
therapy, e.g., T 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 immune effector cells, e.g., T cells or NK
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.
[0640] 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.
[0641] 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.
[0642] 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.
[0643] 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.
[0644] 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.
[0645] 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.
[0646] 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).
[0647] Allogeneic CAR
[0648] 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.
[0649] 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 (e.g., engineered such that it does not
express (or exhibits reduced expression) of TCR alpha, TCR beta,
TCR gamma, TCR delta, TCR epsilon, or TCR zeta) 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.
[0650] 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. In some aspects, downregulation of HLA may be
accomplished by reducing or eliminating expression of beta-2
microglobulin (B2M).
[0651] 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.
[0652] 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).
[0653] In some embodiments, the allogeneic cell can be a cell which
does not expresses or expresses at low levels an inhibitory
molecule, e.g., a cell engineered 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, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, 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.
[0654] siRNA and shRNA to Inhibit TCR or HLA
[0655] 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, and/or an inhibitory molecule described
herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g.,
CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT,
LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM
(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,
adenosine, and TGFR beta), in a cell.
[0656] Expression of siRNA and shRNAs in T cells can be achieved
using any conventional expression system, e.g., such as a
lentiviral expression system.
[0657] 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.
[0658] CRISPR to Inhibit TCR or HLA
[0659] "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, and/or an inhibitory molecule
described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM
(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAGS, VISTA, BTLA,
TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1),
HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,
GAL9, adenosine, and TGFR beta).
[0660] 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.
[0661] 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.
[0662] 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.
[0663] 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.
[0664] 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 Cas1 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.
[0665] 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.
[0666] 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.
[0667] TALEN to Inhibit TCR and/or HLA
[0668] "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, and/or an inhibitory molecule described herein
(e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or
CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and
TGFR beta).
[0669] 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.
[0670] 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.
[0671] 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.
[0672] 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.
[0673] 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.
[0674] 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.
[0675] Zinc Finger Nuclease to Inhibit HLA and/or TCR
[0676] "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, and/or an inhibitory molecule described herein (e.g., PD1,
PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or
CEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,
B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR,
MHC class I, MHC class II, GAL9, adenosine, and TGFR beta).
[0677] 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.
[0678] 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.
[0679] 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.
[0680] 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.
[0681] 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; Guo et al.
(2010) J. Mol. Biol. 400: 96; U.S. Patent Publication 2011/0158957;
and U.S. Patent Publication 2012/0060230.
[0682] Telomerase Expression
[0683] 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.
[0684] In one aspect, the disclosure features a method of making a
population of immune effector cells (e.g., T cells, 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.
[0685] 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.
[0686] 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-00025 (SEQ ID NO: 214)
MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALV
AQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFA
LLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLL
ARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWN
HSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGS
WAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAG
PPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGA
RRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVL
LKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPW
QVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTW
KMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFF
YVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREA
RPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFS
VLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVT
GAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVST
LTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHH
AVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLV
DDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTA
FVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRN
MRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLP
FHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQ
WLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPA LPSDFKTILD
[0687] In an embodiment, the hTERT has a sequence at least 80%,
85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 214. In an embodiment, the hTERT has a sequence of SEQ
ID NO: 214. 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.
[0688] 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-00026 (SEQ ID NO: 215) 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
[0689] 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: 215. In an embodiment, the
hTERT is encoded by a nucleic acid of SEQ ID NO: 215.
[0690] Activation and Expansion of T Cells
[0691] 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.
[0692] Generally, the T cells of the invention 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).
[0693] 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.
[0694] 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 of the
present invention, 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 of the invention, 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.
[0695] 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.
[0696] In further aspects of the present invention, 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.
[0697] 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.
[0698] 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
CLL-1 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 CLL-1 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 CLL-1
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 CLL-1 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.
[0699] In one aspect of the present invention, the mixture may be
cultured for several hours (about 3 hours) to about 14 days or any
hourly integer value in between. In one aspect, the mixture may be
cultured for 21 days. In one aspect of the invention the beads and
the T cells are cultured together for about eight days. In one
aspect, the beads and T cells are cultured together for 2-3 days.
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).
[0700] 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).
[0701] 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.
[0702] 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.
[0703] 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.
[0704] 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.
[0705] 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.
[0706] Once a CLL-1 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 CLL-1 CAR are described in
further detail below
[0707] 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+ and CD8+ 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-t cytoplasmic domain and the
endogenous TCR-t chain are detected by western blotting using an
antibody to the TCR-t chain. The same T cell subsets are used for
SDS-PAGE analysis under non-reducing conditions to permit
evaluation of covalent dimer formation.
[0708] In vitro expansion of CARP 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 aAPCs 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 can be re-stimulated with CLL-1 expressing
cells.
[0709] Sustained CARP 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 III 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.
[0710] Animal models can also be used to measure a CART activity.
For example, xenograft model using human CLL-1-specific CARP T
cells to treat a primary human AML in immunodeficient mice can be
used. Very briefly, after establishment of the tumors, mice are
randomized as to treatment groups. CLL-1 CART cells are injected
into the immunodeficient mice, e.g., intravenously. Animals are
assessed for cancer cellsat weekly intervals. Peripheral blood
CLL-1-expressing AML cell counts are measured in mice that are
injected with CLL-1 CART cells or mock-transduced T cells. Survival
curves for the groups are compared using the log-rank test.
[0711] Cytotoxicity can be assessed by a standard 51Cr-release
assay. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464
(2009). Briefly, target cells are loaded with 51Cr (as NaCrO4, 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.
[0712] 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 CARP
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 CLL-1 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 CARP PBLs) can be generated.
[0713] 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 CLL-1 CAR constructs of the invention.
[0714] Alternatively, or in combination to the methods disclosed
herein, methods and compositions for one or more of: detection
and/or quantification of CAR-expressing cells (e.g., in vitro or in
vivo (e.g., clinical monitoring)); immune cell expansion and/or
activation; and/or CAR-specific selection, that involve the use of
a CAR ligand, are disclosed. In one exemplary embodiment, the CAR
ligand is an antibody that binds to the CAR molecule, e.g., binds
to the extracellular antigen binding domain of CAR (e.g., an
antibody that binds to the antigen binding domain, e.g., an
anti-idiotypic antibody; or an antibody that binds to a constant
region of the extracellular binding domain). In other embodiments,
the CAR ligand is a CAR antigen molecule (e.g., a CAR antigen
molecule as described herein).
[0715] In one aspect, a method for detecting and/or quantifying
CAR-expressing cells is disclosed. For example, the CAR ligand can
be used to detect and/or quantify CAR-expressing cells in vitro or
in vivo (e.g., clinical monitoring of CAR-expressing cells in a
patient, or dosing a patient). The method includes:
[0716] providing the CAR ligand (optionally, a labelled CAR ligand,
e.g., a CAR ligand that includes a tag, a bead, a radioactive or
fluorescent label);
[0717] acquiring the CAR-expressing cell (e.g., acquiring a sample
containing CAR-expressing cells, such as a manufacturing sample or
a clinical sample);
[0718] contacting the CAR-expressing cell with the CAR ligand under
conditions where binding occurs, thereby detecting the level (e.g.,
amount) of the CAR-expressing cells present. Binding of the
CAR-expressing cell with the CAR ligand can be detected using
standard techniques such as FACS, ELISA and the like.
[0719] In another aspect, a method of expanding and/or activating
cells (e.g., immune effector cells) is disclosed. The method
includes:
[0720] providing a CAR-expressing cell (e.g., a first
CAR-expressing cell or a transiently expressing CAR cell);
[0721] contacting said CAR-expressing cell with a CAR ligand, e.g.,
a CAR ligand as described herein), under conditions where immune
cell expansion and/or proliferation occurs, thereby producing the
activated and/or expanded cell population.
[0722] In certain embodiments, the CAR ligand is present on (e.g.,
is immobilized or attached to a substrate, e.g., a non-naturally
occurring substrate). In some embodiments, the substrate is a
non-cellular substrate. The non-cellular substrate can be a solid
support chosen from, e.g., a plate (e.g., a microtiter plate), a
membrane (e.g., a nitrocellulose membrane), a matrix, a chip or a
bead. In embodiments, the CAR ligand is present in the substrate
(e.g., on the substrate surface). The CAR ligand can be
immobilized, attached, or associated covalently or non-covalently
(e.g., cross-linked) to the substrate. In one embodiment, the CAR
ligand is attached (e.g., covalently attached) to a bead. In the
aforesaid embodiments, the immune cell population can be expanded
in vitro or ex vivo. The method can further include culturing the
population of immune cells in the presence of the ligand of the CAR
molecule, e.g., using any of the methods described herein.
[0723] In other embodiments, the method of expanding and/or
activating the cells further comprises addition of a second
stimulatory molecule, e.g., CD28. For example, the CAR ligand and
the second stimulatory molecule can be immobilized to a substrate,
e.g., one or more beads, thereby providing increased cell expansion
and/or activation.
[0724] In yet another aspect, a method for selecting or enriching
for a CAR expressing cell is provided. The method includes
contacting the CAR expressing cell with a CAR ligand as described
herein; and selecting the cell on the basis of binding of the CAR
ligand.
[0725] In yet other embodiments, a method for depleting, reducing
and/or killing a CAR expressing cell is provided. The method
includes contacting the CAR expressing cell with a CAR ligand as
described herein; and targeting the cell on the basis of binding of
the CAR ligand, thereby reducing the number, and/or killing, the
CAR-expressing cell. In one embodiment, the CAR ligand is coupled
to a toxic agent (e.g., a toxin or a cell ablative drug). In
another embodiment, the anti-idiotypic antibody can cause effector
cell activity, e.g., ADCC or ADC activities.
[0726] Exemplary anti-CAR antibodies that can be used in the
methods disclosed herein are described, e.g., in WO 2014/190273 and
by Jena et al., "Chimeric Antigen Receptor (CAR)-Specific
Monoclonal Antibody to Detect CD19-Specific T cells in Clinical
Trials", PLOS March 2013 8:3 e57838, the contents of which are
incorporated by reference. In one embodiment, the anti-idiotypic
antibody molecule recognizes an anti-CD19 antibody molecule, e.g.,
an anti-CD19 scFv. For instance, the anti-idiotypic antibody
molecule can compete for binding with the CD19-specific CAR mAb
clone no. 136.20.1 described in Jena et al., PLOS March 2013 8:3
e57838; may have the same CDRs (e.g., one or more of, e.g., all of,
VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3, using the
Kabat definition, the Chothia definition, or a combination of the
Kabat and Chothia definitions) as the CD19-specific CAR mAb clone
no. 136.20.1; may have one or more (e.g., 2) variable regions as
the CD19-specific CAR mAb clone no. 136.20.1, or may comprise the
CD19-specific CAR mAb clone no. 136.20.1. In some embodiments, the
anti-idiotypic antibody was made according to a method described in
Jena et al. In another embodiment, the anti-idiotypic antibody
molecule is an anti-idiotypic antibody molecule described in WO
2014/190273. In some embodiments, the anti-idiotypic antibody
molecule has the same CDRs (e.g., one or more of, e.g., all of, VH
CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2, and VL CDR3) as an
antibody molecule of WO 2014/190273 such as 136.20.1; may have one
or more (e.g., 2) variable regions of an antibody molecule of WO
2014/190273, or may comprise an antibody molecule of WO 2014/190273
such as 136.20.1. In other embodiments, the anti-CAR antibody binds
to a constant region of the extracellular binding domain of the CAR
molecule, e.g., as described in WO 2014/190273. In some
embodiments, the anti-CAR antibody binds to a constant region of
the extracellular binding domain of the CAR molecule, e.g., a heavy
chain constant region (e.g., a CH2--CH3 hinge region) or light
chain constant region. For instance, in some embodiments the
anti-CAR antibody competes for binding with the 2D3 monoclonal
antibody described in WO 2014/190273, has the same CDRs (e.g., one
or more of, e.g., all of, VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL
CDR2, and VL CDR3) as 2D3, or has one or more (e.g., 2) variable
regions of 2D3, or comprises 2D3 as described in WO
2014/190273.
[0727] In some aspects and embodiments, the compositions and
methods herein are optimized for a specific subset of T cells,
e.g., as described in U.S. Ser. No. 62/031,699 filed Jul. 31, 2014,
the contents of which are incorporated herein by reference in their
entirety. In some embodiments, the optimized subsets of T cells
display an enhanced persistence compared to a control T cell, e.g.,
a T cell of a different type (e.g., CD8.sup.+ or CD4.sup.+)
expressing the same construct.
[0728] In some embodiments, a CD4.sup.+ T cell comprises a CAR
described herein, which CAR comprises an intracellular signaling
domain suitable for (e.g., optimized for, e.g., leading to enhanced
persistence in) a CD4.sup.+ T cell, e.g., an ICOS domain. In some
embodiments, a CD8.sup.+ T cell comprises a CAR described herein,
which CAR comprises an intracellular signaling domain suitable for
(e.g., optimized for, e.g., leading to enhanced persistence of) a
CD8.sup.+ T cell, e.g., a 4-1BB domain, a CD28 domain, or another
costimulatory domain other than an ICOS domain. In some
embodiments, the CAR described herein comprises an antigen binding
domain described herein, e.g., a CAR comprising an antigen binding
domain that specifically binds CLL-1, e.g., a CAR of Table 8.
[0729] In an aspect, described herein is a method of treating a
subject, e.g., a subject having cancer. The method includes
administering to said subject, an effective amount of:
[0730] 1) a CD4.sup.+ T cell comprising a CAR (the
CAR.sup.CD4+)
[0731] comprising:
[0732] an antigen binding domain, e.g., an antigen binding domain
described herein, e.g., an antigen binding domain that specifically
binds CLL-1, e.g., an antigen-binding domain of Table 8;
[0733] a transmembrane domain; and
[0734] an intracellular signaling domain, e.g., a first
costimulatory domain, e.g., an ICOS domain; and
[0735] 2) a CD8.sup.+ T cell comprising a CAR (the CAR.sup.CD8+)
comprising:
[0736] an antigen binding domain, e.g., an antigen binding domain
described herein, e.g., an antigen binding domain that specifically
binds CLL-1, e.g., an antigen-binding domain of Table 8;
[0737] a transmembrane domain; and
[0738] an intracellular signaling domain, e.g., a second co
stimulatory domain, e.g., a 4-1BB domain, a CD28 domain, or another
costimulatory domain other than an ICOS domain;
[0739] wherein the CAR.sup.CD4+ and the CAR.sup.CD8+ differ from
one another.
[0740] Optionally, the method further includes administering:
[0741] 3) a second CD8.sup.+ T cell comprising a CAR (the second
CAR.sup.CD8+) comprising:
[0742] an antigen binding domain, e.g., an antigen binding domain
described herein, e.g., an antigen binding domain that specifically
binds CLL-1, e.g., an antigen-binding domain of Table 8;
[0743] a transmembrane domain; and
an intracellular signaling domain, wherein the second CAR.sup.CD8+
comprises an intracellular signaling domain, e.g., a costimulatory
signaling domain, not present on the CAR.sup.CD8+, and, optionally,
does not comprise an ICOS signaling domain.
Therapeutic Application
[0744] CLL-1 Associated Diseases and/or Disorders
[0745] The present invention provides, among other things,
compositions and methods for treating cancer. In one aspect, the
cancer is a hematologic cancer including but is not limited to
leukemia (such as acute myelogenous leukemia (AML), chronic
myelogenous leukemia (CML), acute lymphoid leukemia, chronic
lymphoid leukemia, acute lymphoblastic B-cell leukemia (B-cell
acute lymphoid leukemia, BALL), acute lymphoblastic T-cell leukemia
(T-cell acute lymphoid leukemia (TALL), B-cell prolymphocytic
leukemia, plasma cell myeloma, and myelodysplastic syndrome) and
malignant lymphoproliferative conditions, including lymphoma (such
as multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma,
and small cell- and large cell-follicular lymphoma).
Therapeutic Applications
[0746] In one aspect, the invention provides methods for treating a
disease associated with CLL-1 expression. In one aspect, the
invention provides methods for treating a disease wherein part of
the tumor is negative for CLL-1 and part of the tumor is positive
for CLL-1. For example, the CAR of the invention is useful for
treating subjects that have undergone treatment for a disease
associated with elevated expression of CLL-1, wherein the subject
that has undergone treatment for elevated levels of CLL-1 exhibits
a disease associated with elevated levels of CLL-1. In embodiments,
the CAR of the invention is useful for treating subjects that have
undergone treatment for a disease associated with expression of
CLL-1, wherein the subject that has undergone treatment related to
expression of CLL-1 exhibits a disease associated with expression
of CLL-1.
[0747] In one aspect, the invention pertains to a vector comprising
CLL-1 CAR operably linked to promoter for expression in mammalian
immune effector cells, e.g., T cells or NK cells. In one aspect,
the invention provides a recombinant immune effector cells, e.g., T
cells or NK cells expressing the CLL-1 CAR for use in treating
CLL-1-expressing tumors, wherein the recombinant immune effector
cells, e.g., T cells or NK cells expressing the CLL-1 CAR is termed
a CLL-1 CAR-expressing cell (e.g., CLL-1 CART or CLL-1
CAR-expressing NK cell). In one aspect, the CLL-1 CAR-expressing
cell (e.g., CLL-1 CART or CLL-1 CAR-expressing NK cell).of the
invention is capable of contacting a tumor cell with at least one
CLL-1 CAR of the invention expressed on its surface such that the
CLL-1 CAR-expressing cell (e.g., CLL-1 CART or CLL-1 CAR-expressing
NK cell) targets the tumor cell and growth of the tumor is
inhibited.
[0748] In one aspect, the invention pertains to a method of
inhibiting growth of a CLL-1-expressing tumor cell, comprising
contacting the tumor cell with a CLL-1 CAR-expressing cell (e.g.,
CLL-1 CART or CLL-1 CAR-expressing NK cell) cell of the present
invention such that the CLL-1 CAR-expressing cell (e.g., CLL-1 CART
or CLL-1 CAR-expressing NK cell) is activated in response to the
antigen and targets the cancer cell, wherein the growth of the
tumor is inhibited.
[0749] In one aspect, the invention pertains to a method of
treating cancer in a subject. The method comprises administering to
the subject a CLL-1 CAR-expressing cell (e.g., CLL-1 CART or CLL-1
CAR-expressing NK cell) of the present invention such that the
cancer is treated in the subject. An example of a cancer that is
treatable by the CLL-1 CAR-expressing cell (e.g., CLL-1 CART or
CLL-1 CAR-expressing NK cell) of the invention is a cancer
associated with expression of CLL-1. In one aspect, the cancer
associated with expression of CLL-1 is a hematological cancer. In
one aspect, a hematologic cancer including but is not limited to
leukemia (such as acute myelogenous leukemia, chronic myelogenous
leukemia, acute lymphoid leukemia, chronic lymphoid leukemia and
myelodysplastic syndrome) and malignant lymphoproliferative
conditions, including lymphoma (such as multiple myeloma,
non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell- and
large cell-follicular lymphoma). In other embodiments, a
hematologic cancer can include minimal residual disease, MRD, e.g.,
of a leukemia, e.g., of AML or MDS.
[0750] The invention includes a type of cellular therapy where
immune effector cell, e.g., T cells or NK cells, are genetically
modified to express a chimeric antigen receptor (CAR) and the CLL-1
CAR-expressing cell (e.g., CLL-1 CART or CLL-1 CAR-expressing NK
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 cells (e.g., T cells or NK cells) are able
to replicate in vivo resulting in long-term persistence that can
lead to sustained tumor control. In various aspects, the immune
effector cells (e.g., T cells or NK 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 immune
effector cell (e.g., T cell or NK cell) to the patient.
[0751] The invention also includes a type of cellular therapy where
immune effector cells (e.g., T cells or NK cells) are modified,
e.g., by in vitro transcribed RNA, to transiently express a
chimeric antigen receptor (CAR) and the CLL-1 CAR expressing cell
(e.g., CLL-1 CAR T cell or CLL-1 CAR-expressing NK 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 immune
effector cells (e.g., T cells or NK cells) administered to the
patient, is present for less than one month, e.g., three weeks, two
weeks, one week, after administration of the immune effector cells
(e.g., T cells or NK cells) to the patient.
[0752] Without wishing to be bound by any particular theory, the
anti-tumor immunity response elicited by the CAR-modified immune
effector cells (e.g., T cells or NK 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 immune
effector cells (e.g., T cells or NK cells) exhibit specific
proinflammatory cytokine secretion and potent cytolytic activity in
response to human cancer cells expressing the CLL-1, resist soluble
CLL-1 inhibition, mediate bystander killing and mediate regression
of an established human tumor. For example, antigen-less tumor
cells within a heterogeneous field of CLL-1-expressing tumor may be
susceptible to indirect destruction by CLL-1-redirected immune
effector cells (e.g., T cells or NK cells) that has previously
reacted against adjacent antigen-positive cancer cells.
[0753] In one aspect, the fully-human CAR-modified immune effector
cells (e.g., T cells or NK 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.
[0754] 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.
[0755] 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.
[0756] 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 comprises: (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.
[0757] In addition to using a cell-based vaccine in terms of ex
vivo immunization, the present invention also provides compositions
and methods for in vivo immunization to elicit an immune response
directed against an antigen in a patient.
[0758] 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-modified immune effector cells (e.g., T cells or NK cells)
of the invention are used in the treatment of diseases, disorders
and conditions associated with expression of CLL-1. In certain
aspects, the cells of the invention are used in the treatment of
patients at risk for developing diseases, disorders and conditions
associated with expression of CLL-1. Thus, the present invention
provides methods for the treatment or prevention of diseases,
disorders and conditions associated with expression of CLL-1
comprising administering to a subject in need thereof, a
therapeutically effective amount of the CAR-modified immune
effector cells (e.g., T cells or NK cells) of the invention. In one
aspect the CAR-expressing cells, e.g., CART cells or CAR-expressing
NK cells) of the inventions may be used to treat a proliferative
disease such as a cancer or malignancy or is a precancerous
condition such as a myelodysplasia, a myelodysplastic syndrome or a
preleukemiahyperproliferative disorder, hyperplasia or a dysplasia,
which is characterized by abnormal growth of cells.
[0759] In one aspect, the CAR-expressing cells (e.g., CART cells or
CAR-expressing NK cells) of the invention are used to treat a
cancer, wherein the cancer is a hematological cancer. Hematological
cancer conditions are the types of cancer such as leukemia and
malignant lymphoproliferative conditions that affect blood, bone
marrow and the lymphatic system.
[0760] In one aspect, the compositions and CAR-expressing cells
(e.g., CART cells or CAR-expressing NK cells) of the present
invention are particularly useful for treating myeloid leukemias,
AML and its subtypes, chronic myeloid leukemia (CML), and
myelodysplastic syndrome (MDS).
[0761] 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.
[0762] Lymphoma is a group of blood cell tumors that develop from
lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and
Hodgkin lymphoma.
[0763] In AML, malignant transformation and uncontrolled
proliferation of an abnormally differentiated, long-lived myeloid
progenitor cell results in high circulating numbers of immature
blood forms and replacement of normal marrow by malignant cells.
Symptoms include fatigue, pallor, easy bruising and bleeding,
fever, and infection; symptoms of leukemic infiltration are present
in only about 5% of patients (often as skin manifestations).
Examination of peripheral blood smear and bone marrow is
diagnostic. Existing treatment includes induction chemotherapy to
achieve remission and post-remission chemotherapy (with or without
stem cell transplantation) to avoid relapse.
[0764] AML has a number of subtypes that are distinguished from
each other by morphology, immunophenotype, and cytochemistry. Five
classes are described, based on predominant cell type, including
myeloid, myeloid-monocytic, monocytic, erythroid, and
megakaryocytic.
[0765] Remission induction rates range from 50 to 85%. Long-term
disease-free survival reportedly occurs in 20 to 40% of patients
and increases to 40 to 50% in younger patients treated with stem
cell transplantation.
[0766] Prognostic factors help determine treatment protocol and
intensity; patients with strongly negative prognostic features are
usually given more intense forms of therapy, because the potential
benefits are thought to justify the increased treatment toxicity.
The most important prognostic factor is the leukemia cell
karyotype; favorable karyotypes include t(15;17), t(8;21), and
inv16 (p13;q22). Negative factors include increasing age, a
preceding myelodysplastic phase, secondary leukemia, high WBC
count, and absence of Auer rods.
[0767] Initial therapy attempts to induce remission and differs
most from ALL in that AML responds to fewer drugs. The basic
induction regimen includes cytarabine by continuous IV infusion or
high doses for 5 to 7 days; daunorubicin or idarubicin is given IV
for 3 days during this time. Some regimens include 6-thioguanine,
etoposide, vincristine, and prednisone, but their contribution is
unclear. Treatment usually results in significant myelosuppression,
with infection or bleeding; there is significant latency before
marrow recovery. During this time, meticulous preventive and
supportive care is vital.
[0768] Chronic myelogenous (or myeloid) leukemia (CML) is also
known as chronic granulocytic leukemia, and is characterized as a
cancer of the white blood cells. Common treatment regimens for CML
include Bcr-Abl tyrosine kinase inhibitors, imatinib
(Gleevec.RTM.), dasatinib and nilotinib. Bcr-Abl tyrosine kinase
inhibitors are specifically useful for CML patients with the
Philadelphia chromosome translocation.
[0769] Myelodysplastic syndromes (MDS) are hematological medical
conditions characterized by disorderly and ineffective
hematopoiesis, or blood production. Thus, the number and quality of
blood-forming cells decline irreversibly. Some patients with MDS
can develop severe anemia, while others are asymptomatic.
[0770] The classification scheme for MDS is known in the art, with
criteria designating the ratio or frequency of particular blood
cell types, e.g., myeloblasts, monocytes, and red cell precursors.
MDS includes refractory anemia, refractory anemia with ring
sideroblasts, refractory anemia with excess blasts, refractory
anemia with excess blasts in transformation, chronic myelomonocytic
leukemia (CMML).
[0771] Treatment for MDS vary with the severity of the symptoms.
Aggressive forms of treatment for patients experiencing severe
symptoms include bone marrow transplants and supportive care with
blood product support (e.g., blood transfusions) and hematopoietic
growth factors (e.g., erythropoietin). Other agents are frequently
used to treat MDS: 5-azacytidine, decitabine, and lenalidomide. In
some cases, iron chelators deferoxamine (Desferal.RTM.) and
deferasirox (Exjade.RTM.) may also be administered.
[0772] In another embodiment, the CAR-expressing cells (e.g., CART
cells or CAR-expressing NK cells) of the present invention are used
to treat cancers or leukemias with leukemia stem cells. For
example, the leukemia stem cells are CD34.sup.+/CD38.sup.- leukemia
cells.
[0773] The present invention provides, among other things,
compositions and methods for treating cancer. In one aspect, the
cancer is a hematologic cancer including but is not limited to
leukemia (such as acute myelogenous leukemia, chronic myelogenous
leukemia, acute lymphoid leukemia, chronic lymphoid leukemia and
myelodysplastic syndrome) and malignant lymphoproliferative
conditions, including lymphoma (such as multiple myeloma,
non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell- and
large cell-follicular lymphoma).
[0774] In one aspect, the CAR-expressing cells (e.g., CART cells or
CAR-expressing NK cells) of the invention may be used to treat
other cancers and malignancies such as, but not limited to, e.g.,
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 lymphocytic leukemia (CLL); additional
hematologic cancers or hematologic conditions including, but 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, Marginal zone lymphoma, multiple myeloma, myelodysplasia
and myelodysplastic syndrome, non-Hodgkin's 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. The CAR-modified
immune effector cells (e.g., T cells or NK cells) of the present
invention 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.
[0775] The present invention also provides methods for inhibiting
the proliferation or reducing a CLL-1-expressing cell population,
the methods comprising contacting a population of cells comprising
a CLL-1-expressing cell with an CLL-1 CAR-expressing cell (e.g.,
CLL-1 CART cell or CLL-1 CAR-expressing NK cell) of the invention
that binds to the CLL-1-expressing cell. In a specific aspect, the
present invention provides methods for inhibiting the proliferation
or reducing the population of cancer cells expressing CLL-1, the
methods comprising contacting the CLL-1-expressing cancer cell
population with a CLL-1 CAR-expressing cell (e.g., CLL-1 CART cell
or CLL-1 CAR-expressing NK cell) of the invention that binds to the
CLL-1-expressing cell. In one aspect, the present invention
provides methods for inhibiting the proliferation or reducing the
population of cancer cells expressing CLL-1, the methods comprising
contacting the CLL-1-expressing cancer cell population with a CLL-1
CAR-expressing cell (e.g., CLL-1 CART cell or CLL-1 CAR-expressing
NK cell) of the invention that binds to the CLL-1-expressing cell.
In certain aspects, the CLL-1 CAR-expressing cell (e.g., CLL-1 CART
cell or CLL-1 CAR-expressing NK cell) cell of the invention 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 myeloid leukemia or
another cancer associated with CLL-1-expressing cells relative to a
negative control. In one aspect, the subject is a human.
[0776] The present invention also provides methods for preventing,
treating and/or managing a disease associated with CLL-1-expressing
cells (e.g., a hematologic cancer or atypical cancer expressing
CLL-1), the methods comprising administering to a subject in need a
CLL-1 CAR-expressing cell (e.g., CLL-1 CART cell or CLL-1
CAR-expressing NK cell) of the invention that binds to the
CLL-1-expressing cell. In one aspect, the subject is a human.
Non-limiting examples of disorders associated with CLL-1-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 CLL-1).
[0777] The present invention also provides methods for preventing,
treating and/or managing a disease associated with CLL-1-expressing
cells, the methods comprising administering to a subject in need an
a CLL-1 CAR-expressing cell (e.g., CLL-1 CART cell or CLL-1
CAR-expressing NK cell) of the invention that binds to the
CLL-1-expressing cell. In one aspect, the subject is a human.
[0778] The present invention provides methods for preventing
relapse of cancer associated with CLL-1-expressing cells, the
methods comprising administering to a subject in need thereof a
CLL-1 CAR-expressing cell (e.g., CLL-1 CART cell or CLL-1
CAR-expressing NK cell) of the invention that binds to the
CLL-1-expressing cell. In one aspect, the methods comprise
administering to the subject in need thereof an effective amount of
a CLL-1 CAR-expressing cell (e.g., CLL-1 CART cell or CLL-1
CAR-expressing NK cell) described herein that binds to the
CLL-1-expressing cell in combination with an effective amount of
another therapy.
[0779] Combination Therapies
[0780] A CAR-expressing cell described herein may be used in
combination with other known agents and therapies. 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.
[0781] A CAR-expressing cell described herein and 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.
[0782] 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 the other treatment,
concurrently with the treatment, post-treatment, or during
remission of the disorder.
[0783] When administered in combination, the CAR therapy and the
additional agent (e.g., second or 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., second 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., second 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.
[0784] In further aspects, a CAR-expressing cell described herein
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.
[0785] In certain instances, compounds of the present invention are
combined with other therapeutic agents, such as other anti-cancer
agents, anti-allergic agents, anti-nausea agents (or anti-emetics),
pain relievers, cytoprotective agents, and combinations
thereof.
[0786] In one embodiment, a CAR-expressing cell described herein
can be used in combination with a 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)), an mTOR inhibitor, 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).
[0787] 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.).
[0788] Treatment with a combination of a chemotherapeutic agent and
a cell expressing a CLL-1 CAR molecule described herein can be used
to treat a hematologic cancer described herein, e.g., AML. In
embodiments, the combination of a chemotherapeutic agent and a
CLL-1 CAR-expressing cell is useful for targeting, e.g., killing,
cancer stem cells, e.g., leukemic stem cells, e.g., in subjects
with AML. In embodiments, the combination of a chemotherapeutic
agent and a CLL-1 CAR-expressing cell is useful for treating
minimal residual disease (MRD). MRD refers to the small number of
cancer cells that remain in a subject during treatment, e.g.,
chemotherapy, or after treatment. MRD is often a major cause for
relapse. The present invention provides a method for treating
cancer, e.g., MRD, comprising administering a chemotherapeutic
agent in combination with a CLL-1 CAR-expressing cell, e.g., as
described herein.
[0789] In an embodiment, the chemotherapeutic agent is administered
prior to administration of the cell expressing a CAR molecule,
e.g., a CAR molecule described herein. In chemotherapeutic regimens
where more than one administration of the chemotherapeutic agent is
desired, the chemotherapeutic regimen is initiated or completed
prior to administration of a cell expressing a CAR molecule, e.g.,
a CAR molecule described herein. In embodiments, the
chemotherapeutic agent is administered at least 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11
days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, or 30
days prior to administration of the cell expressing the CAR
molecule. In embodiments, the chemotherapeutic regimen is initiated
or completed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,
14 days, 15 days, 20 days, 25 days, or 30 days prior to
administration of the cell expressing the CAR molecule. In
embodiments, the chemotherapeutic agent is a chemotherapeutic agent
that increases CLL-1 expression on the cancer cells, e.g., the
tumor cells, e.g., as compared to CLL-1 expression on normal or
non-cancer cells. CLL-1 expression can be determined, for example,
by immunohistochemical staining or flow cytometry analysis. For
example, the chemotherapeutic agent is cytarabine (Ara-C).
[0790] Anti-cancer agents of particular interest for combinations
with the compounds of the present invention include:
antimetabolites; drugs that inhibit either the calcium dependent
phosphatase calcineurin or the p70S6 kinase FK506) or inhibit the
p70S6 kinase; alkylating agents; mTOR inhibitors; immunomodulators;
anthracyclines; vinca alkaloids; proteosome inhibitors; GITR
agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase
inhibitor; a BTK kinase inhibitor; a MKN kinase inhibitor; a DGK
kinase inhibitor; or an oncolytic virus.
[0791] Exemplary antimetabolites include, without limitation, folic
acid antagonists (also referred to herein as antifolates),
pyrimidine analogs, purine analogs and adenosine deaminase
inhibitors): methotrexate (Rheumatrex.RTM., Trexall.RTM.),
5-fluorouracil (Adrucil.RTM., Efudex.RTM., Fluoroplex.RTM.),
floxuridine (FUDF.RTM.), cytarabine (Cytosar-U.RTM., Tarabine PFS),
6-mercaptopurine (Puri-Nethol.RTM.)), 6-thioguanine (Thioguanine
Tabloid.RTM.), fludarabine phosphate (Fludara.RTM.), pentostatin
(Nipent.RTM.), pemetrexed (Alimta.RTM.), raltitrexed
(Tomudex.RTM.), cladribine (Leustatin.RTM.), clofarabine
(Clofarex.RTM., Clolar.RTM.), mercaptopurine (Puri-Nethol.RTM.),
capecitabine (Xeloda.RTM.), nelarabine (Arranon.RTM.), azacitidine
(Vidaza.RTM.) and gemcitabine (Gemzar.RTM.). Preferred
antimetabolites include, e.g., 5-fluorouracil (Adrucil.RTM.,
Efudex.RTM., Fluoroplex.RTM.), floxuridine (FUDF.RTM.),
capecitabine (Xeloda.RTM.), pemetrexed (Alimta.RTM.), raltitrexed
(Tomudex.RTM.) and gemcitabine (Gemzar.RTM.).
[0792] 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.).
[0793] In embodiments, a CAR-expressing cell described herein 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/m2 (e.g.,
400-450, 450-500, 500-550, or 550-600 mg/m.sup.2), e.g.,
intravenously.
[0794] In embodiments, a CAR-expressing cell described herein 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/m2 (e.g., 70-80, 80-90, 90-100, or 100-110 mg/m2), e.g.,
intravenously. In embodiments, the rituximab is administered at a
dosage of about 400-600 mg/m2 (e.g., 400-450, 450-500, 500-550, or
550-600 mg/m.sup.2), e.g., intravenously.
[0795] In embodiments, a CAR-expressing cell described herein 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.
[0796] In embodiments, a CAR-expressing cell described herein 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.
[0797] In embodiments, a CAR-expressing cell described herein 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.
[0798] Exemplary mTOR inhibitors include, e.g., temsirolimus;
ridaforolimus (formally known as deferolimus, (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-15,17,21,23,2-
9,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0.s-
up.4,
9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexy-
l dimethylphosphinate, also known as AP23573 and MK8669, and
described in PCT Publication No. WO 03/064383); everolimus
(Afinitor.RTM. or RAD001); rapamycin (AY22989, Sirolimus.RTM.);
simapimod (CAS 164301-51-3); emsirolimus,
(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, CAS
1013101-36-4); and
N.sup.2[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholi-
nium-4-yl]methoxy]butyl]-L-arginylglycyl-L-.alpha.-aspartylL-serine-(SEQ
ID NO: 313), inner salt (SF1126, CAS 936487-67-1), and XL765.
[0799] Exemplary immunomodulators include, e.g., afutuzumab
(available from Roche.RTM.); pegfilgrastim (Neulasta.RTM.);
lenalidomide (CC-5013, Revlimid.RTM.); thalidomide (Thalomid.RTM.),
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).
[0800] 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.
[0801] 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.).
[0802] 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-[(1S)-2-[(-
2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide
(ONX-0912).
[0803] In embodiments, a CAR-expressing cell described herein 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's
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.
[0804] In embodiments, a CAR-expressing cell described herein 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's 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/m2
(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.
[0805] 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.
[0806] 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/2010/103705s53111bl.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.
[0807] 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).
[0808] 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/2009/1253261bl.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.
[0809] 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).
[0810] 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.
[0811] 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.
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/2013/125486s0001bl.pdf.
[0812] 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.
[0813] 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.
[0814] 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.
[0815] 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.
[0816] 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-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) is shown
below.
##STR00001##
[0817] 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.
[0818] In some embodiments, a CAR-expressing cell described herein
is administered in combination with an oncolytic virus. In
embodiments, oncolytic viruses are capable of selectively
replicating in and triggering the death of or slowing the growth of
a cancer cell. In some cases, oncolytic viruses have no effect or a
minimal effect on non-cancer cells. An oncolytic virus includes but
is not limited to an oncolytic adenovirus, oncolytic Herpes Simplex
Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic
vaccinia virus, oncolytic Sinbis virus, oncolytic influenza virus,
or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic
Newcastle Disease Virus (NDV), oncolytic measles virus, or
oncolytic vesicular stomatitis virus (VSV)).
[0819] In some embodiments, the oncolytic virus is a virus, e.g.,
recombinant oncolytic virus, described in US2010/0178684 A1, which
is incorporated herein by reference in its entirety. In some
embodiments, a recombinant oncolytic virus comprises a nucleic acid
sequence (e.g., heterologous nucleic acid sequence) encoding an
inhibitor of an immune or inflammatory response, e.g., as described
in US2010/0178684 A1, incorporated herein by reference in its
entirety. In embodiments, the recombinant oncolytic virus, e.g.,
oncolytic NDV, comprises a pro-apoptotic protein (e.g., apoptin), a
cytokine (e.g., GM-CSF, interferon-gamma, interleukin-2 (IL-2),
tumor necrosis factor-alpha), an immunoglobulin (e.g., an antibody
against ED-B firbonectin), tumor associated antigen, a bispecific
adapter protein (e.g., bispecific antibody or antibody fragment
directed against NDV HN protein and a T cell co-stimulatory
receptor, such as CD3 or CD28; or fusion protein between human IL-2
and single chain antibody directed against NDV HN protein). See,
e.g., Zamarin et al. Future Microbiol. 7.3(2012):347-67,
incorporated herein by reference in its entirety. In some
embodiments, the oncolytic virus is a chimeric oncolytic NDV
described in U.S. Pat. No. 8,591,881 B2, US 2012/0122185 A1, or US
2014/0271677 A1, each of which is incorporated herein by reference
in their entireties.
[0820] In some embodiments, the oncolytic virus comprises a
conditionally replicative adenovirus (CRAd), which is designed to
replicate exclusively in cancer cells. See, e.g., Alemany et al.
Nature Biotechnol. 18(2000):723-27. In some embodiments, an
oncolytic adenovirus comprises one described in Table 1 on page 725
of Alemany et al., incorporated herein by reference in its
entirety.
[0821] Exemplary oncolytic viruses include but are not limited to
the following:
[0822] Group B Oncolytic Adenovirus (ColoAd1) (PsiOxus Therapeutics
Ltd.) (see, e.g., Clinical Trial Identifier: NCT02053220);
[0823] ONCOS-102 (previously called CGTG-102), which is an
adenovirus comprising granulocyte-macrophage colony stimulating
factor (GM-CSF) (Oncos Therapeutics) (see, e.g., Clinical Trial
Identifier: NCT01598129);
[0824] VCN-01, which is a genetically modified oncolytic human
adenovirus encoding human PH20 hyaluronidase (VCN Biosciences,
S.L.) (see, e.g., Clinical Trial Identifiers: NCT02045602 and
NCT02045589);
[0825] Conditionally Replicative Adenovirus ICOVIR-5, which is a
virus derived from wild-type human adenovirus serotype 5 (Had5)
that has been modified to selectively replicate in cancer cells
with a deregulated retinoblastoma/E2F pathway (Institut Catala
d'Oncologia) (see, e.g., Clinical Trial Identifier:
NCT01864759);
[0826] Celyvir, which comprises bone marrow-derived autologous
mesenchymal stem cells (MSCs) infected with ICOVIR5, an oncolytic
adenovirus (Hospital Infantil Universitario Nino Jesus, Madrid,
Spain/Ramon Alemany) (see, e.g., Clinical Trial Identifier:
NCT01844661);
[0827] CG0070, which is a conditionally replicating oncolytic
serotype 5 adenovirus (Ad5) in which human E2F-1 promoter drives
expression of the essential E1a viral genes, thereby restricting
viral replication and cytotoxicity to Rb pathway-defective tumor
cells (Cold Genesys, Inc.) (see, e.g., Clinical Trial Identifier:
NCT02143804); or DNX-2401 (formerly named Delta-24-RGD), which is
an adenovirus that has been engineered to replicate selectively in
retinoblastoma (Rb)-pathway deficient cells and to infect cells
that express certain RGD-binding integrins more efficiently
(Clinica Universidad de Navarra, Universidad de Navarra/DNAtrix,
Inc.) (see, e.g., Clinical Trial Identifier: NCT01956734).
[0828] In some embodiments, an oncolytic virus described herein is
administering by injection, e.g., subcutaneous, intra-arterial,
intravenous, intramuscular, intrathecal, or intraperitoneal
injection. In embodiments, an oncolytic virus described herein is
administered intratumorally, transdermally, transmucosally, orally,
intranasally, or via pulmonary administration.
[0829] In an embodiment, cells expressing a CAR described herein
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.
[0830] In one embodiment, a CAR expressing cell described herein is
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 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. 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.
[0831] In one embodiment, a CAR expressing cell described herein is
administered to a subject in combination with an mTOR inhibitor,
e.g., an mTOR inhibitor described herein, e.g., a rapalog such as
everolimus. In one embodiment, the mTOR inhibitor is administered
prior to the CAR-expressing cell. For example, in one embodiment,
the mTOR inhibitor can be administered prior to apheresis of the
cells.
[0832] In one embodiment, a CAR expressing cell 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.
[0833] In one embodiment, a CAR expressing cell described herein is
administered to a subject in combination with a protein tyrosine
phosphatase inhibitor, e.g., a protein tyrosine phosphatase
inhibitor described herein. In one embodiment, the protein tyrosine
phosphatase inhibitor is an SHP-1 inhibitor, e.g., an SHP-1
inhibitor described herein, such as, e.g., sodium stibogluconate.
In one embodiment, the protein tyrosine phosphatase inhibitor is an
SHP-2 inhibitor, e.g., an SHP-2 inhibitor described herein.
[0834] In one embodiment, a CAR-expressing cell described herein
can be used in combination with a kinase inhibitor. 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-ylamino)-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 MNKla, MNK1b, MNK2a and/or MNK2b
inhibitor. In one embodiment, the kinase inhibitor is a dual
PI3K/mTOR inhibitor described herein, such as, e.g., PF-04695102.
In one embodiment, the kinase inhibitor is a DGK inhibitor, e.g., a
DGK inhibitor described herein, such as, e.g., DGKinh1 (D5919) or
DGKinh2 (D5794). 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).
[0835] 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.
[0836] In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with a cyclin-dependent
kinase (CDK) 4 or 6 inhibitor, e.g., a CDK4 inhibitor or a CDK6
inhibitor described herein. In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with a
CDK4/6 inhibitor (e.g., an inhibitor that targets both CDK4 and
CDK6), e.g., a CDK4/6 inhibitor described herein. In an embodiment,
the subject has MCL. MCL is an aggressive cancer that is poorly
responsive to currently available therapies, i.e., essentially
incurable. In many cases of MCL, cyclin D1 (a regulator of CDK4/6)
is expressed (e.g., due to chromosomal translocation involving
immunoglobulin and Cyclin D1 genes) in MCL cells. Thus, without
being bound by theory, it is thought that MCL cells are highly
sensitive to CDK4/6 inhibition with high specificity (i.e., minimal
effect on normal immune cells). CDK4/6 inhibitors alone have had
some efficacy in treating MCL, but have only achieved partial
remission with a high relapse rate. An exemplary CDK4/6 inhibitor
is LEE011 (also called ribociclib), the structure of which is shown
below.
##STR00002##
[0837] 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.
[0838] 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.
[0839] In one embodiment, the kinase inhibitor is a BTK inhibitor,
e.g., ibrutinib (PCI-32765). In embodiments, a CAR-expressing cell
described herein is administered to a subject in combination with a
BTK inhibitor (e.g., ibrutinib). In embodiments, a CAR-expressing
cell described herein is administered to a subject in combination
with ibrutinib (also called PCI-32765). 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.
##STR00003##
[0840] In embodiments, the subject has CLL, mantle cell lymphoma
(MCL), or small lymphocytic lymphoma (SLL). 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 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 has
refractory CLL or SLL. In other embodiments, the subject has
follicular lymphoma, e.g., relapse or refractory follicular
lymphoma. In some embodiments, ibrutinib is administered at a
dosage of about 300-600 mg/day (e.g., about 300-350, 350-400,
400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420
mg/day or about 560 mg/day), e.g., orally. In embodiments, 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. In some embodiments,
ibrutinib is administered in combination with rituximab. See, e.g.,
Burger et al. (2013) Ibrutinib In Combination With Rituximab (iR)
Is Well Tolerated and Induces a High Rate Of Durable Remissions In
Patients With High-Risk Chronic Lymphocytic Leukemia (CLL): New,
Updated Results Of a Phase II Trial In 40 Patients, Abstract 675
presented at 55.sup.th ASH Annual Meeting and Exposition, New
Orleans, LA 7-10 December Without being bound by theory, it is
thought that the addition of ibrutinib enhances the T cell
proliferative response and may shift T cells from a T-helper-2
(Th2) to T-helper-1 (Th1) phenotype. Th1 and Th2 are phenotypes of
helper T cells, with Th1 versus Th2 directing different immune
response pathways. A Th1 phenotype is associated with
proinflammatory responses, e.g., for killing cells, such as
intracellular pathogens/viruses or cancerous cells, or perpetuating
autoimmune responses. A Th2 phenotype is associated with eosinophil
accumulation and anti-inflammatory responses.
[0841] In some embodiments of the methods, uses, and compositions
herein, the BTK inhibitor is a BTK inhibitor described in
International Application WO/2015/079417, which is herein
incorporated by reference in its entirety. For instance, in some
embodiments, the BTK inhibitor is a compound of formula (I) or a
pharmaceutically acceptable salt thereof;
##STR00004##
[0842] wherein,
[0843] R1 is hydrogen, C1-C6 alkyl optionally substituted by
hydroxy;
[0844] R2 is hydrogen or halogen;
[0845] R3 is hydrogen or halogen;
[0846] R4 is hydrogen;
[0847] R5 is hydrogen or halogen;
[0848] or R4 and R5 are attached to each other and stand for a
bond, --CH2-, --CH2--CH2-, --CH.dbd.CH--, --CH.dbd.CH--CH2-;
--CH2--CH.dbd.CH--; or --CH2--CH2--CH2-;
[0849] R6 and R7 stand independently from each other for H, C1-C6
alkyl optionally substituted by hydroxyl, C3-C6 cycloalkyl
optionally substituted by halogen or hydroxy, or halogen;
[0850] R8, R9, R, R', R10 and R11 independently from each other
stand for H, or C1-C6 alkyl optionally substituted by C1-C6 alkoxy;
or any two of R8, R9, R, R', R10 and R11 together with the carbon
atom to which they are bound may form a 3-6 membered saturated
carbocyclic ring;
[0851] R12 is hydrogen or C1-C6 alkyl optionally substituted by
halogen or C1-C6 alkoxy; or R12 and any one of R8, R9, R, R', R10
or R11 together with the atoms to which they are bound may form a
4, 5, 6 or 7 membered azacyclic ring, which ring may optionally be
substituted by halogen, cyano, hydroxyl, C1-C6 alkyl or C1-C6
alkoxy;
[0852] n is 0 or 1; and
[0853] R13 is C2-C6 alkenyl optionally substituted by C1-C6 alkyl,
C1-C6 alkoxy or N,N-di-C1-C6 alkyl amino; C2-C6 alkynyl optionally
substituted by C1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylenyl
oxide optionally substituted by C1-C6 alkyl.
[0854] In some embodiments, the BTK inhibitor of Formula I is
chosen from:
N-(3-(5-((1-Acryloylazetidin-3-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2--
methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(E)-N-(3-(6-Amino-5-((1-(but-2-enoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5--
fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-((1-propioloylazetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluoro--
2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-((1-(but-2-ynoyl)azetidin-3-yl)oxy)pyrimidin-4-yl)-5-fluo-
ro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(5-((1-Acryloylpiperidin-4-yl)oxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-
-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(2-(N-methylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2--
methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(E)-N-(3-(6-Amino-5-(2-(N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fl-
uoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(2-(N-methylpropiolamido)ethoxy)pyrimidin-4-yl)-5-fluoro--
2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(E)-N-(3-(6-Amino-5-(2-(4-methoxy-N-methylbut-2-enamido)ethoxy)pyrimidin--
4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)ethoxy)pyrimidin-4-yl)-5-fluoro-
-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(2-((4-Amino-6-(3-(4-cyclopropyl-2-fluorobenzamido)-5-fluoro-2-methylph-
enyl)pyrimidin-5-yl)oxy)ethyl)-N-methyloxirane-2-carboxamide;
N-(2-((4-Amino-6-(3-(6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)phe-
nyl)pyrimidin-5-yl)oxy)ethyl)-N-methylacrylamide;
N-(3-(5-(2-Acrylamidoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphen-
yl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(2-(N-ethylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-m-
ethylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(2-(N-(2-fluoroethyl)acrylamido)ethoxy)pyrimidin-4-yl)-5--
fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(5-((1-Acrylamidocyclopropyl)methoxy)-6-aminopyrimidin-4-yl)-5-fluor-
o-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(5-(2-Acrylamidopropoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-meth-
ylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(6-Amino-5-(2-(but-2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-
-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(6-Amino-5-(2-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-flu-
oro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(6-Amino-5-(2-(N-methylbut-2-ynamido)propoxy)pyrimidin-4-yl)-5--
fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(3-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-
-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)--
5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(6-Amino-5-((1-(but-2-ynoyl)pyrrolidin-2-yl)methoxy)pyrimidin-4-
-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)-2-(3-(5-((1-Acryloylpyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-
-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H-
)-one;
N-(2-((4-Amino-6-(3-(6-cyclopropyl-1-oxo-3,4-dihydroisoquinolin-2(1-
H)-yl)-5-fluoro-2-(hydroxymethyl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N-methyl-
acrylamide;
N-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyr-
imidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methox-
y)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide-
;
2-(3-(5-(((2S,4R)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopy-
rimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroi-
soquinolin-1(2H)-one;
N-(3-(5-(((2S,4S)-1-Acryloyl-4-methoxypyrrolidin-2-yl)methoxy)-6-aminopyr-
imidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(((2S,4S)-1-(but-2-ynoyl)-4-methoxypyrrolidin-2-yl)methox-
y)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide-
;
N-(3-(5-(((2S,4R)-1-Acryloyl-4-fluoropyrrolidin-2-yl)methoxy)-6-aminopyr-
imidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(6-Amino-5-(((2S,4R)-1-(but-2-ynoyl)-4-fluoropyrrolidin-2-yl)methoxy-
)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5--
fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)--N-(3-(6-Amino-5-((1-propioloylazetidin-2-yl)methoxy)pyrimidin-4-yl)--
5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(S)-2-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-f-
luoro-2-(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)--
one;
(R)--N-(3-(5-((1-Acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl-
)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
(R)--N-(3-(5-((1-Acryloylpiperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-
-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(5-(((2R,3S)-1-Acryloyl-3-methoxypyrrolidin-2-yl)methoxy)-6-aminopyr-
imidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
N-(3-(5-(((2S,4R)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrim-
idin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide;
or
N-(3-(5-(((2S,4S)-1-Acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6-aminopyrim-
idin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide.
[0855] Unless otherwise provided, the chemical terms used above in
describing the BTK inhibitor of Formula I are used according to
their meanings as set out in International Application
WO/2015/079417, which is herein incorporated by reference in its
entirety
[0856] 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-15,17,21,23,2-
9,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0.s-
up.4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl
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-(SEQ
ID NO:313), inner salt (SF1126); and XL765.
[0857] 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.
[0858] 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.
[0859] In embodiments, a CAR-expressing cell described herein 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.
##STR00005##
[0860] Duvelisib (also called IPI-145; Infinity Pharmaceuticals and
Abbvie) 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.
##STR00006##
[0861] 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.
[0862] 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).
[0863] In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with an anaplastic
lymphoma kinase (ALK) inhibitor. Exemplary ALK kinases include but
are not limited to crizotinib (Pfizer), ceritinib (Novartis),
alectinib (Chugai), brigatinib (also called AP26113; Ariad),
entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (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.
[0864] 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.
[0865] 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.
[0866] In embodiments, a CAR-expressing cell described herein 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)
[0867] In embodiments, a CAR-expressing cell described herein 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.
##STR00007##
[0868] In embodiments, a CAR-expressing cell described herein is
administered to a subject in combination with a CD19 CART cell
(e.g., CTL019, e.g., as described in WO2012/079000, incorporated
herein by reference). In embodiments, the subject has acute myeloid
leukemia (AML), e.g., a CD19 positive AML or a CD19 negative AML.
In embodiments, the subject has a CD19+ lymphoma, e.g., a CD19+
Non-Hodgkin's Lymphoma (NHL), a CD19+FL, or a CD19+ DLBCL. In
embodiments, the subject has a relapsed or refractory CD19+
lymphoma. In embodiments, a lymphodepleting chemotherapy is
administered to the subject prior to, concurrently with, or after
administration (e.g., infusion) of CD19 CART cells. In an example,
the lymphodepleting chemotherapy is administered to the subject
prior to administration of CD19 CART cells. For example, the
lymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4
days) prior to CD19 CART cell infusion. In embodiments, multiple
doses of CD19 CART cells are administered, e.g., as described
herein. For example, a single dose comprises about 5.times.10.sup.8
CD19 CART 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, e.g., a non-CD19 CAR-expressing cell. In embodiments, a
CD19 CART is administered to the subject prior to, concurrently
with, or after administration (e.g., infusion) of a non-CD19
CAR-expressing cell, e.g., a non-CD19 CAR-expressing cell described
herein.
[0869] In some embodiments, a CAR-expressing cell described herein
is administered to a subject in combination with a CD19
CAR-expressing cell, e.g., CTL019, e.g., as described in
WO2012/079000, incorporated herein by reference, for treatment of a
disease associated with the expression of CLL-1, e.g., a cancer
described herein. Without being bound by theory, it is believed
that administering a CD19 CAR-expressing cell in combination with a
CAR-expressing cell improves the efficacy of a CAR-expressing cell
described herein by targeting early lineage cancer cells, e.g.,
cancer stem cells, modulating the immune response, depleting
regulatory B cells, and/or improving the tumor microenvironment.
For example, a CD19 CAR-expressing cell targets cancer cells that
express early lineage markers, e.g., cancer stem cells and
CD19-expressing cells, while the CAR-expressing cell described
herein targets cancer cells that express later lineage markers,
e.g., CLL-1. This preconditioning approach can improve the efficacy
of the CAR-expressing cell described herein. In such embodiments,
the CD19 CAR-expressing cell is administered prior to, concurrently
with, or after administration (e.g., infusion) of a CAR-expressing
cell described herein.
[0870] In embodiments, a CAR-expressing cell described herein also
expresses a CAR targeting CD19, e.g., a CD19 CAR. In an embodiment,
the cell expressing a CAR described herein and a CD19 CAR is
administered to a subject for treatment of a cancer described
herein, e.g., AML. In an embodiment, the configurations of one or
both of the CAR molecules comprise a primary intracellular
signaling domain and a costimulatory signaling domain. In another
embodiment, the configurations of one or both of the CAR molecules
comprise a primary intracellular signaling domain and two or more,
e.g., 2, 3, 4, or 5 or more, costimulatory signaling domains. In
such embodiments, the CAR molecule described herein and the CD19
CAR may have the same or a different primary intracellular
signaling domain, the same or different costimulatory signaling
domains, or the same number or a different number of costimulatory
signaling domains. Alternatively, the CAR described herein and the
CD19 CAR are configured as a split CAR, in which one of the CAR
molecules comprises an antigen binding domain and a costimulatory
domain (e.g., 4-1BB), while the other CAR molecule comprises an
antigen binding domain and a primary intracellular signaling domain
(e.g., CD3 zeta).
[0871] In some embodiments, a CAR-expressing cell described herein
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.
[0872] In embodiments, a subject having a disease described herein,
e.g., a hematological disorder, e.g., AML or MDS, is administered a
CAR-expressing cell described herein in combination with an agent,
e.g., cytotoxic or chemotherapy agent, a biologic therapy (e.g.,
antibody, e.g., monoclonal antibody, or cellular therapy), or an
inhibitor (e.g., kinase inhibitor). In embodiments, the subject is
administered a CAR-expressing cell described herein in combination
with a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals),
cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals),
sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or
mitoxantrone. CPX-351 is a liposomal formulation comprising
cytarabine and daunorubicin at a 5:1 molar ratio. In embodiments,
the subject is administered a CAR-expressing cell described herein
in combination with a hypomethylating agent, e.g., a DNA
methyltransferase inhibitor, e.g., azacitidine or decitabine. In
embodiments, the subject is administered a CAR-expressing cell
described herein in combination with a biologic therapy, e.g., an
antibody or cellular therapy, e.g., 225Ac-lintuzumab (Actimab-A;
Actinium Pharmaceuticals), IPH2102 (Innate Pharma/Bristol Myers
Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin
(Mylotarg; Pfizer). SGN-CD33A is an antibody-drug conjugate (ADC)
comprising a pyrrolobenzodiazepine dimer that is attached to an
anti-CD33 antibody. Actimab-A is an anti-CD33 antibody (lintuzumab)
labeled with actinium. IPH2102 is a monoclonal antibody that
targets killer immunoglobulin-like receptors (KIRs). In
embodiments, the subject is administered a CAR-expressing cell
described herein in combination a FLT3 inhibitor, e.g., sorafenib
(Bayer), midostaurin (Novartis), quizartinib (Daiichi Sankyo),
crenolanib (Arog Pharmaceuticals), PLX3397 (Daiichi Sankyo),
AKN-028 (Akinion Pharmaceuticals), or ASP2215 (Astellas). In
embodiments, the subject is administered a CAR-expressing cell
described herein in combination with an isocitrate dehydrogenase
(IDH) inhibitor, e.g., AG-221 (Celgene/Agios) or AG-120
(Agios/Celgene). In embodiments, the subject is administered a
CAR-expressing cell described herein in combination with a cell
cycle regulator, e.g., inhibitor of polo-like kinase 1 (Plk1),
e.g., volasertib (Boehringer Ingelheim); or an inhibitor of
cyclin-dependent kinase 9 (Cdk9), e.g., alvocidib (Tolero
Pharmaceuticals/Sanofi Aventis). In embodiments, the subject is
administered a CAR-expressing cell described herein in combination
with a B cell receptor signaling network inhibitor, e.g., an
inihibitor of B-cell lymphoma 2 (Bcl-2), e.g., venetoclax
(Abbvie/Roche); or an inhibitor of Bruton's tyrosine kinase (Btk),
e.g., ibrutinib (Pharmacyclics/Johnson & Johnson Janssen
Pharmaceutical). In embodiments, the subject is administered a
CAR-expressing cell described herein in combination with an
inhibitor of M1 aminopeptidase, e.g., tosedostat (CTI
BioPharmaNernalis); an inhibitor of histone deacetylase (HDAC),
e.g., pracinostat (MEI Pharma); a multi-kinase inhibitor, e.g.,
rigosertib (Onconova Therapeutics/Baxter/SymBio); or a peptidic
CXCR4 inverse agonist, e.g., BL-8040 (BioLineRx). In embodiments,
the subject is administered a CLL-1-targeting CAR-expressing cell
in combination with a CAR-expressing cell that specifically binds
an antigen other than CLL-1, e.g., CLL, BCMA, CD123, CD19, FLT-3,
or folate receptor beta.
[0873] In another embodiment, the subjects receive an infusion of
the CLL-1 expressing cell compositions of the present invention
prior to transplantation, e.g., allogeneic stem cell transplant, of
cells. In a preferred embodiment, CLL-1 expressing cells
transiently express CLL-1 CAR, e.g., by electroporation of an mRNA
CLL-1 CAR, whereby the expression of the CLL-1 is terminated prior
to infusion of donor stem cells to avoid engraftment failure.
[0874] Some patients may experience allergic reactions to the
compounds of the present invention and/or other anti-cancer
agent(s) during or after administration; therefore, anti-allergic
agents are often administered to minimize the risk of an allergic
reaction. Suitable anti-allergic agents include corticosteroids,
such as dexamethasone (e.g., Decadron.RTM.), beclomethasone (e.g.,
Beclovent.RTM.), hydrocortisone (also known as cortisone,
hydrocortisone sodium succinate, hydrocortisone sodium phosphate,
and sold under the tradenames Ala-Cort.RTM., hydrocortisone
phosphate, Solu-Cortef.RTM., Hydrocort Acetate.RTM. and
Lanacort.RTM.), prednisolone (sold under the tradenames
Delta-Cortel.RTM., Orapred.RTM., Pediapred.RTM. and Prelone.RTM.),
prednisone (sold under the tradenames Deltasone.RTM., Liquid
Red.RTM., Meticorten.RTM. and Orasone.RTM.), methylprednisolone
(also known as 6-methylprednisolone, methylprednisolone acetate,
methylprednisolone sodium succinate, sold under the tradenames
Duralone.RTM., Medralone.RTM., Medrol.RTM., M-Prednisol.RTM. and
Solu-Medrol.RTM.); antihistamines, such as diphenhydramine (e.g.,
Benadryl.RTM.), hydroxyzine, and cyproheptadine; and
bronchodilators, such as the beta-adrenergic receptor agonists,
albuterol (e.g., Proventil.RTM.), and terbutaline
(Brethine.RTM.).
[0875] Some patients may experience nausea during and after
administration of the compound of the present invention and/or
other anti-cancer agent(s); therefore, anti-emetics are used in
preventing nausea (upper stomach) and vomiting. Suitable
anti-emetics include aprepitant (Emend.RTM.), ondansetron
(Zofran.RTM.), granisetron HCl (Kytril.RTM.), lorazepam
(Ativan.RTM., dexamethasone (Decadron.RTM.), prochlorperazine
(Compazine.RTM.), casopitant (Rezonic.RTM. and Zunrisa.RTM.), and
combinations thereof.
[0876] Medication to alleviate the pain experienced during the
treatment period is often prescribed to make the patient more
comfortable. Common over-the-counter analgesics, such Tylenol.RTM.,
are often used. However, opioid analgesic drugs such as
hydrocodone/paracetamol or hydrocodone/acetaminophen (e.g.,
Vicodin.RTM.), morphine (e.g., Astramorph.RTM. or Avinza.RTM.),
oxycodone (e.g., OxyContin.RTM. or Percocet.RTM.), oxymorphone
hydrochloride (Opana.RTM.), and fentanyl (e.g., Duragesic.RTM.) are
also useful for moderate or severe pain.
[0877] In an effort to protect normal cells from treatment toxicity
and to limit organ toxicities, cytoprotective agents (such as
neuroprotectants, free-radical scavengers, cardioprotectors,
anthracycline extravasation neutralizers, nutrients and the like)
may be used as an adjunct therapy. Suitable cytoprotective agents
include Amifostine (Ethyol.RTM.), glutamine, dimesna
(Tavocept.RTM.), mesna (Mesnex.RTM.), dexrazoxane (Zinecard.RTM. or
Totect.RTM.), xaliproden (Xaprila.RTM.), and leucovorin (also known
as calcium leucovorin, citrovorum factor and folinic acid).
[0878] The structure of the active compounds identified by code
numbers, generic or trade names may be taken from the actual
edition of the standard compendium "The Merck Index" or from
databases, e.g. Patents International (e.g. IMS World
Publications).
[0879] The above-mentioned compounds, which can be used in
combination with a compound of the present invention, can be
prepared and administered as described in the art, such as in the
documents cited above.
[0880] In one embodiment, the present invention provides
pharmaceutical compositions comprising at least one compound of the
present invention (e.g., a compound of the present invention) or a
pharmaceutically acceptable salt thereof together with a
pharmaceutically acceptable carrier suitable for administration to
a human or animal subject, either alone or together with other
anti-cancer agents.
[0881] In one embodiment, the present invention provides methods of
treating human or animal subjects suffering from a cellular
proliferative disease, such as cancer. The present invention
provides methods of treating a human or animal subject in need of
such treatment, comprising administering to the subject a
therapeutically effective amount of a compound of the present
invention (e.g., a compound of the present invention) or a
pharmaceutically acceptable salt thereof, either alone or in
combination with other anti-cancer agents.
[0882] In particular, compositions will either be formulated
together as a combination therapeutic or administered
separately.
[0883] In combination therapy, the compound of the present
invention and other anti-cancer agent(s) may be administered either
simultaneously, concurrently or sequentially with no specific time
limits, wherein such administration provides therapeutically
effective levels of the two compounds in the body of the
patient.
[0884] In a preferred embodiment, the compound of the present
invention and the other anti-cancer agent(s) is generally
administered sequentially in any order by infusion or orally. The
dosing regimen may vary depending upon the stage of the disease,
physical fitness of the patient, safety profiles of the individual
drugs, and tolerance of the individual drugs, as well as other
criteria well-known to the attending physician and medical
practitioner(s) administering the combination. The compound of the
present invention and other anti-cancer agent(s) may be
administered within minutes of each other, hours, days, or even
weeks apart depending upon the particular cycle being used for
treatment. In addition, the cycle could include administration of
one drug more often than the other during the treatment cycle and
at different doses per administration of the drug.
[0885] In another aspect of the present invention, kits that
include one or more compound of the present invention and a
combination partner as disclosed herein are provided.
Representative kits include (a) a compound of the present invention
or a pharmaceutically acceptable salt thereof, (b) at least one
combination partner, e.g., as indicated above, whereby such kit may
comprise a package insert or other labeling including directions
for administration.
[0886] A compound of the present invention may also be used to
advantage in combination with known therapeutic processes, for
example, the administration of hormones or especially radiation. A
compound of the present invention may in particular be used as a
radiosensitizer, especially for the treatment of tumors which
exhibit poor sensitivity to radiotherapy.
[0887] 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. CRS may include clinical constitutional signs and
symptoms such as fever, fatigue, anorexia, myalgias, arthalgias,
nausea, vomiting, and headache. CRS may include clinical skin signs
and symptoms such as rash. CRS may include clinical
gastrointestinal signs and symsptoms such as nausea, vomiting and
diarrhea. CRS may include clinical respiratory signs and symptoms
such as tachypnea and hypoxemia. CRS may include clinical
cardiovascular signs and symptoms such as tachycardia, widened
pulse pressure, hypotension, increased cardac output (early) and
potentially diminished cardiac output (late). CRS may include
clinical coagulation signs and symptoms such as elevated d-dimer,
hypofibrinogenemia with or without bleeding. CRS may include
clinical renal signs and symptoms such as azotemia. CRS may include
clinical hepatic signs and symptoms such as transaminitis and
hyperbilirubinemia. CRS may include clinical neurologic signs and
symptoms such as headache, mental status changes, confusion,
delirium, word finding difficulty or frank aphasia, hallucinations,
tremor, dymetria, altered gait, and seizures. Accordingly, the
methods described herein can comprise administering a
CAR-expressing cell described herein to a subject and further
administering one or more agents 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 and IL-6. In an
embodiment, the factor elevated in the subject is one or more of
IL-1, GM-CSF, IL-10, IL-8, IL-5 and fraktalkine. Therefore, an
agent administered to treat this side effect can be an agent that
neutralizes one or more of these soluble factors. In one
embodiment, the agent that neutralizes one or more of these soluble
forms is an antibody or antibody thereof.
[0888] 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 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 antibody
molecule is tocilizumab. An example of an IL-1R based inhibitor is
anakinra.
[0889] 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, e.g., the agent is a checkpoint inhibitor.
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, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or
CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, 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, 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.
[0890] In an embodiment, a nucleic acid molecule that encodes a
dsRNA molecule that inhibits expression of the molecule that
modulates or regulates, e.g., inhibits, T-cell function is operably
linked to a promoter, e.g., a H1- or a U6-derived promoter such
that the dsRNA molecule that inhibits expression of the molecule
that modulates or regulates, e.g., inhibits, T-cell function is
expressed, e.g., is expressed within a CAR-expressing cell. See
e.g., Tiscornia G., "Development of Lentiviral Vectors Expressing
siRNA," Chapter 3, in Gene Transfer: Delivery and Expression of DNA
and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R, et al.
(2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat.
Biotechnol. 19: 497-500. In an embodiment the nucleic acid molecule
that encodes a dsRNA molecule that inhibits expression of the
molecule that modulates or regulates, e.g., inhibits, T-cell
function is present on the same vector, e.g., a lentiviral vector,
that comprises a nucleic acid molecule that encodes a component,
e.g., all of the components, of the CAR. In such an embodiment, the
nucleic acid molecule that encodes a dsRNA molecule that inhibits
expression of the molecule that modulates or regulates, e.g.,
inhibits, T-cell function is located on the vector, e.g., the
lentiviral vector, 5'- or 3'- to the nucleic acid that encodes a
component, e.g., all of the components, of the CAR. The nucleic
acid molecule that encodes a dsRNA molecule that inhibits
expression of the molecule that modulates or regulates, e.g.,
inhibits, T-cell function can be transcribed in the same or
different direction as the nucleic acid that encodes a component,
e.g., all of the components, of the CAR. In an embodiment the
nucleic acid molecule that encodes a dsRNA molecule that inhibits
expression of the molecule that modulates or regulates, e.g.,
inhibits, T-cell function is present on a vector other than the
vector that comprises a nucleic acid molecule that encodes a
component, e.g., all of the components, of the CAR. In an
embodiment, the nucleic acid molecule that encodes a dsRNA molecule
that inhibits expression of the molecule that modulates or
regulates, e.g., inhibits, T-cell function it transiently expressed
within a CAR-expressing cell. In an embodiment, the nucleic acid
molecule that encodes a dsRNA molecule that inhibits expression of
the molecule that modulates or regulates, e.g., inhibits, T-cell
function is stably integrated into the genome of a CAR-expressing
cell. FIGS. 29A-29E depicts examples of vectors for expressing a
component, e.g., all of the components, of the CAR with a dsRNA
molecule that inhibits expression of the molecule that modulates or
regulates, e.g., inhibits, T-cell function.
[0891] Examples of dsRNA molecules useful for inhibiting expression
of a molecule that modulates or regulates, e.g., inhibits, T-cell
function, wherein the molecule that modulates or regulates, e.g.,
inhibits, T-cell function is PD-1 are provided below.
[0892] Provided in Table 10 below are the names of PDCD1 (PD1) RNAi
agents (derived from their position in the mouse PDCD1 gene
sequence NM_008798.2), along with the SEQ ID NOs: 216-263
representing the DNA sequence. Both sense (S) and antisense (AS)
sequences are presented as 19mer and 21mer sequences are in this
table. Also note that the position (PoS, e.g., 176) is derived from
the position number in the mouse PDCD1 gene sequence NM_008798.2.
SEQ ID NOs are indicated in groups of 12 that correspond with
"sense 19" SEQ ID NOs: 216-227; "sense 21" SEQ ID NOs: 228-239;
"asense 21" SEQ ID NOs: 240-251; "asense 19" SEQ ID NOs:
252-263.
TABLE-US-00027 TABLE 10 Mouse PDCD1 (PD1) shRNA sequences Position
on NM_008 Target 798.2 region Sense19 Sense21 Asense21 Asense19 176
CDS GGAGGTCCCT CTGGAGGTCC TAGAAGGTGA TAGAAGGTGA CACCTTCTA
CTCACCTTCT GGGACCTCCA GGGACCTCC (SEQ ID NO: A G (SEQ ID NO: 216)
(SEQ ID NO: (SEQ ID NO: 252) 228) 240) 260 CDS CGGAGGATCT
GTCGGAGGAT TTCAGCATAA TTCAGCATAA TATGCTGAA CTTATGCTGA GATCCTCCGA
GATCCTCCG (SEQ ID NO: A C (SEQ ID NO: 217) (SEQ ID NO: (SEQ ID NO:
253) 229) 241) 359 CDS CCCGCTTCCA TGCCCGCTTC TGTATGATCT TGTATGATCT
GATCATACA CAGATCATAC GGAAGCGGGC GGAAGCGGG (SEQ ID NO: A A (SEQ ID
NO: 218) (SEQ ID NO: (SEQ ID NO: 254) 230) 242) 528 CDS GGAGACCTCA
CTGGAGACCT ATATCTTGTTG ATATCTTGTT ACAAGATAT CAACAAGATA AGGTCTCCAG
GAGGTCTCC (SEQ ID NO: T (SEQ ID NO: (SEQ ID NO: 219) (SEQ ID NO:
243) 255) 231) 581 CDS AAGGCATGGT TCAAGGCATG ATACCAATGA ATACCAATGA
CATTGGTAT GTCATTGGTA CCATGCCTTG CCATGCCTT (SEQ ID NO: T A (SEQ ID
NO: 220) (SEQ ID NO: (SEQ ID NO: 256) 232) 244) 584 CDS GCATGGTCAT
AGGCATGGTC ATGATACCAA ATGATACCAA TGGTATCAT ATTGGTATCA TGACCATGCC
TGACCATGC (SEQ ID NO: T T (SEQ ID NO: 221) (SEQ ID NO: (SEQ ID NO:
257) 233) 245) 588 CDS GGTCATTGGT ATGGTCATTG ATGGTCATTG ATGGTCATTG
ATCATGAGT GTATCATGAG GTATCATGAG GTATCATGA (SEQ ID NO: T T (SEQ ID
NO: 222) (SEQ ID NO: (SEQ ID NO: 258) 234) 246) 609 CDS CCTAGTGGGT
GCCCTAGTGG GCCCTAGTGG GCCCTAGTGG ATCCCTGTA GTATCCCTGT GTATCCCTGT
GTATCCCTG (SEQ ID NO: A A (SEQ ID NO: 223) (SEQ ID NO: (SEQ ID NO:
259) 235) 247) 919 CDS GAGGATGGAC ATGAGGATGG ATGAGGATGG ATGAGGATGG
ATTGTTCTT ACATTGTTCTT ACATTGTTCTT ACATTGTTC (SEQ ID NO: (SEQ ID NO:
(SEQ ID NO: (SEQ ID NO: 224) 236) 248) 260) 1021 3'UTR GCATGCAGGC
GAGCATGCAG GAGCATGCAG GAGCATGCAG TACAGTTCA GCTACAGTTC GCTACAGTTC
GCTACAGTT (SEQ ID NO: A A (SEQ ID NO: 225) (SEQ ID NO: (SEQ ID NO:
261) 237) 249) 1097 3'UTR CCAGCACATG TTCCAGCACA TTCCAGCACA
TTCCAGCACA CACTGTTGA TGCACTGTTG TGCACTGTTG TGCACTGTT (SEQ ID NO: A
A (SEQ ID NO: 226) (SEQ ID NO: (SEQ ID NO: 262) 238) 250) 1101
3'UTR CACATGCACT AGCACATGCA AGCACATGCA AGCACATGCA GTTGAGTGA
CTGTTGAGTG CTGTTGAGTG CTGTTGAGT (SEQ ID NO: A A (SEQ ID NO: 227)
(SEQ ID NO: (SEQ ID NO: 263) 239) 251)
[0893] Provided in Table 11 below are the names of PDCD1 (PD1) RNAi
agents (derived from their position in the human PDCD1 gene
sequence, along with the SEQ ID NOs. 264-311 representing the DNA
sequence. Both sense (S) and antisense (AS) sequences are presented
as 19mer and 21mer sequences. SEQ ID NOs are indicated in groups of
12 that correspond with "sense 19" SEQ ID NOs: 264-275; "sense 21"
SEQ ID NOs: 276-287; "asense 21" SEQ ID NOs: 288-299; "asense 19"
SEQ ID NOs: 300-311.
TABLE-US-00028 TABLE 11 Human PDCD1 (PD1) shRNA sequences Position
on NM_005 Target 018.2 region Sense19 Asense19 Sense21 Asense21 145
CDS GGCCAGGATG TCTAAGAACC GCGGCCAGGA TCTAAGAACC GTTCTTAGA ATCCTGGCC
TGGTTCTTAG ATCCTGGCCG (SEQ ID NO: (SEQ ID NO: A C 264) 276) (SEQ ID
NO: (SEQ ID NO: 288) 300) 271 CDS GCTTCGTGCT TACCAGTTTA GAGCTTCGTG
TACCAGTTTA AAACTGGTA GCACGAAGC CTAAACTGGT GCACGAAGCT (SEQ ID NO:
(SEQ ID NO: A C 265) 277) (SEQ ID NO: (SEQ ID NO: 289) 301) 393 CDS
GGGCGTGACT TCATGTGGAA ACGGGCGTGA TCATGTGGAA TCCACATGA GTCACGCCC
CTTCCACATG GTCACGCCCG (SEQ ID NO: (SEQ ID NO: A T 266) 278) (SEQ ID
NO: (SEQ ID NO: 290) 302) 1497 3'UTR CAGGCCTAGA TGAAACTTCT
TGCAGGCCTA TGAAACTTCT GAAGTTTCA CTAGGCCTG GAGAAGTTTC CTAGGCCTGC
(SEQ ID NO: (SEQ ID NO: A A 267) 279) (SEQ ID NO: (SEQ ID NO: 291)
303) 1863 3'UTR CTTGGAACCC TTCAGGAATG TCCTTGGAAC TTCAGGAATG
ATTCCTGAA GGTTCCAAG CCATTCCTGA GGTTCCAAGG (SEQ ID NO: (SEQ ID NO: A
A 268) 280) (SEQ ID NO: (SEQ ID NO: 292) 304) 1866 3'UTR GGAACCCATT
AATTTCAGGA TTGGAACCCA AATTTCAGGA CCTGAAATT ATGGGTTCC TTCCTGAAAT
ATGGGTTCCA (SEQ ID NO: (SEQ ID NO: T A 269) 281) (SEQ ID NO: (SEQ
ID NO: 293) 305) 1867 3'UTR GAACCCATTC TAATTTCAGG TGGAACCCAT
TAATTTCAGG CTGAAATTA AATGGGTTC TCCTGAAATT AATGGGTTCC (SEQ ID NO:
(SEQ ID NO: A A 270) 282) (SEQ ID NO: (SEQ ID NO: 294) 306) 1868
3'UTR AACCCATTCC ATAATTTCAG GGAACCCATT ATAATTTCAG TGAAATTAT
GAATGGGTT CCTGAAATTA GAATGGGTTC (SEQ ID NO: (SEQ ID NO: T C 271)
283) (SEQ ID NO: (SEQ ID 295) NO:307) 1869 3'UTR ACCCATTCCT
AATAATTTCA GAACCCATTC AATAATTTCA GAAATTATT GGAATGGGT CTGAAATTAT
GGAATGGGTT (SEQ ID NO: (SEQ ID NO: T C 272) 284) (SEQ ID NO: (SEQ
ID NO: 296) 308) 1870 3'UTR CCCATTCCTG AAATAATTTC AACCCATTCC
AAATAATTTC AAATTATTT AGGAATGGG TGAAATTATT AGGAATGGGT (SEQ ID NO:
(SEQ ID NO: T T 273) 285) (SEQ ID NO: (SEQ ID NO: 297) 309) 2079
3'UTR CTGTGGTTCT TAATATAATA CCCTGTGGTT TAATATAATA ATTATATTA
GAACCACAG CTATTATATT GAACCACAGG (SEQ ID NO: (SEQ ID NO: A G 274)
286) (SEQ ID NO: (SEQ ID NO: 298) 310) 2109 3'UTR AAATATGAGA
TTAGCATGCT TTAAATATGA TTAGCATGCT GCATGCTAA CTCATATTT GAGCATGCTA
CTCATATTTA (SEQ ID NO: (SEQ ID NO: A A 275) 287) (SEQ ID NO: (SEQ
ID NO: 299) 311)
[0894] 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
embodiments, the agent that enhances the activity of a
CAR-expressing cell, e.g., inhibitor of an inhibitory molecule, is
administered in combination with an allogeneic CAR, e.g., an
allogeneic CAR described herein (e.g., described in the Allogeneic
CAR section herein).
[0895] PD-1 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 PD-1, PD-L1 and PD-L2 have
been shown to downregulate T cell activation upon binding to PD-1
(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 PD-1 with PD-L1.
Antibodies, antibody fragments, and other inhibitors of PD-1, PD-L1
and PD-L2 are available in the art and may be used combination with
a cars of the present invention 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 PD-1. Nivolumab (clone 5C4) and other human
monoclonal antibodies that specifically bind to PD-1 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
PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal
antibodies are disclosed in WO2009/101611. Pembrolizumab (formerly
known as lambrolizumab, and also referred to as MK03475; Merck) is
a humanized IgG4 monoclonal antibody that binds to PD-1.
Pembrolizumab and other humanized anti-PD-1 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-L1. 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 PD-1 and
B7-H1. Other anti-PD-1 antibodies include AMP 514 (Amplimmune),
among others, e.g., anti-PD-1 antibodies disclosed in U.S. Pat. No.
8,609,089, US 2010028330, and/or US 20120114649.
[0896] 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.
[0897] 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.
[0898] 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.
[0899] 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, BMS-986016
(Bristol-Myers Squib) is a monoclonal antibody that targets LAG-3.
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.
[0900] 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 antracellular 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-CLL-1 CAR.
[0901] In one embodiment, the agent which enhances activity of a
CAR-expressing cell described herein is miR-17-92.
[0902] 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.
[0903] In embodiments, the cytokine is administered in combination
with CAR-expressing T cells. The cytokine can be administered
simultaneously or concurrently with the CAR-expressing T cells,
e.g., administered on the same day. The cytokine may be prepared in
the same pharmaceutical composition as the CAR-expressing T 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 T 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 T 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 T 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 CAR-expressing T cells is IL-7, IL-15, or
IL-21.
[0904] In other embodiments, the cytokine is administered a period
of time after administration of 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 CAR-expressing cell 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 CAR-expressing cell therapy
can be administered a cytokine. Administration of the cytokine to
the subject that has sub-optimal response to the CAR-expressing
cell therapy improves CAR-expressing cell efficacy or anti-cancer
activity. In a preferred embodiment, the cytokine administered
after administration of CAR-expressing cells is IL-7.
Combination with a Low, Immune Enhancing, Dose of an mTOR
Inhibitor
[0905] Methods described herein use low, immune enhancing, doses of
mTOR inhibitors, e.g., allosteric mTOR inhibitors, including
rapalogs such as RAD001. Administration of a low, immune enhancing,
dose of an mTOR inhibitor (e.g., a dose that is insufficient to
completely suppress the immune system, but sufficient to improve
immune function) can optimize the performance of immune effector
cells, e.g., T cells or CAR-expressing cells, in the subject.
Methods for measuring mTOR inhibition, dosages, treatment regimens,
and suitable pharmaceutical compositions are described in U.S.
Patent Application No. 2015/01240036, hereby incorporated by
reference.
[0906] In an embodiment, administration of a low, immune enhancing,
dose of an mTOR inhibitor can result in one or more of the
following: [0907] i) a decrease in the number of PD-1 positive
immune effector cells; [0908] ii) an increase in the number of PD-1
negative immune effector cells; [0909] iii) an increase in the
ratio of PD-1 negative immune effector cells/PD-1 positive immune
effector cells; [0910] iv) an increase in the number of naive T
cells; [0911] v) an increase in the expression of one or more of
the following markers: CD62L.sup.high, CD127.sup.high, CD27.sup.+,
and BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0912] vi) a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; or [0913] vii) 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
foregoing, e.g., i), ii), iii), iv), v), vi), or vii), occurs e.g.,
at least transiently, e.g., as compared to a non-treated
subject.
[0914] In another embodiment, administration of a low, immune
enhancing, dose of an mTOR inhibitor results in increased or
prolonged proliferation or persistence of CAR-expressing cells,
e.g., in culture or in a subject, e.g., as compared to non-treated
CAR-expressing cells or a non-treated subject. In embodiments,
increased proliferation is associated with in an increase in the
number of CAR-expressing cells. Methods for measuring increased or
prolonged proliferation are described in Examples 8 and 9. In
another embodiment, administration of a low, immune enhancing, dose
of an mTOR inhibitor results in increased killing of cancer cells
by CAR-expressing cells, e.g., in culture or in a subject, e.g., as
compared to non-treated CAR-expressing cells or a non-treated
subject. In embodiments, increased killing of cancer cells is
associated with in a decrease in tumor volume. Methods for
measuring increased killing of cancer cells are described in
Example 2.
[0915] 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,
e.g., an allosteric mTOR inhibitor, e.g., RAD001, or a catalytic
mTOR inhibitor. For example, administration of the low, immune
enhancing, dose of the mTOR inhibitor can be initiated prior to
administration of a CAR-expressing cell described herein; completed
prior to administration of a CAR-expressing cell described herein;
initiated at the same time as administration of a CAR-expressing
cell described herein; overlapping with administration of a
CAR-expressing cell described herein; or continuing after
administration of a CAR-expressing cell described herein.
[0916] Alternatively or in addition, administration of a low,
immune enhancing, dose of an mTOR inhibitor can optimize immune
effector cells to be engineered to express a CAR molecule described
herein. In such embodiments, 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 or
completed prior to harvest of immune effector cells, e.g., T cells
or NK cells, to be engineered to express a CAR molecule described
herein, from a subject.
[0917] In another embodiment, immune effector cells, e.g., T cells
or NK cells, to be engineered to express a CAR molecule described
herein, e.g., after harvest from a subject, or CAR-expressing
immune effector cells, e.g., T cells or NK cells, e.g., prior to
administration to a subject, can be cultured in the presence of a
low, immune enhancing, dose of an mTOR inhibitor.
[0918] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in an immediate release dosage form, 0.1 to
20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs of RAD001, or a
bioequivalent dose thereof. In an embodiment, administering to the
subject a low, immune enhancing, dose of an mTOR inhibitor
comprises administering, e.g., once per week, e.g., in a sustained
release dosage form, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or
about 15 mgs of RAD001, or a bioequivalent dose thereof.
[0919] 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%, at least 70 but no more
than 90%, 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%, at least 60 but no
more than 80%, 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%, at least 50 but no more than 70%, 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%, at least 40 but no more than 60%, 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%, at least 40 but no more than 50%, 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%, at least 35 but no more than 40%,
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%.
[0920] The extent of mTOR inhibition can be conveyed as, or
corresponds to, 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 various methods, such as measuring
P70 S6 kinase activity by the Boulay assay, as described in U.S.
Patent Application No. 2015/01240036, hereby incorporated by
reference, or as described in U.S. Pat. No. 7,727,950, hereby
incorporated by reference; measuring the level of phosphorylated S6
by western blot; or evaluating a change in the ratio of PD1
negative immune effector cells to PD1 positive immune effector
cells.
[0921] 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. 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. In an
embodiment, an mTOR inhibitor is a catalytic inhibitor.
[0922] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygroscopicus having the structure shown in Formula
A.
##STR00008##
[0923] 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.
[0924] 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.
[0925] 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.
[0926] 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.
[0927] 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 mexthoxy 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.
[0928] Rapamycin analogs from WO95/16691 include, but are not
limited to, 16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,
16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,
16-demthoxy-16-(propargyl)oxy-rapamycin,
16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,
16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,
16-demthoxy-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.
[0929] 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.
[0930] Another suitable rapamycin analog is umirolimus as described
in US2005/0101624 the contents of which are incorporated by
reference.
[0931] 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-12-1(1R)-2-{[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methy-
lethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tri-
cyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone-
, as described in U.S. Pat. No. 5,665,772 and WO94/09010, the
contents of each are incorporated by reference.
[0932] 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 inhibtors
include zotarolimus (AB T578) and umirolimus.
[0933] 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.
[0934] 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).
[0935] 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).
[0936] mTOR inhibitors useful according to the present invention
also include prodrugs, derivatives, pharmaceutically acceptable
salts, or analogs thereof of any of the foregoing.
[0937] 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.
Methods and Biomarkers for Evaluating CAR-Effectiveness or Sample
Suitability
[0938] In another aspect, the invention features a method of
evaluating or monitoring the effectiveness of a CAR-expressing cell
therapy (e.g., a CLL-1 CAR therapy), in a subject (e.g., a subject
having a cancer, e.g., a hematological cancer), or the suitability
of a sample (e.g., an apheresis sample) for a CAR therapy (e.g., a
CLL-1 CAR therapy). The method includes acquiring a value of
effectiveness to the CAR therapy, or sample suitability, wherein
said value is indicative of the effectiveness or suitability of the
CAR-expressing cell therapy.
[0939] In embodiments, the value of effectiveness to the CAR
therapy, or sample suitability, comprises a measure of one, two,
three, four, five, six or more (all) of the following:
[0940] (i) the level or activity of one, two, three, or more (e.g.,
all) of resting T.sub.EFF cells, resting T.sub.REG cells, younger T
cells (e.g., younger CD4 or CD8 cells, or gamma/delta T cells), or
early memory T cells, or a combination thereof, in a sample (e.g.,
an apheresis sample or a manufactured CAR-expressing cell product
sample);
[0941] (ii) the level or activity of one, two, three, or more
(e.g., all) of activated T.sub.EFF cells, activated T.sub.REG
cells, older T cells (e.g., older CD4 or CD8 cells), or late memory
T cells, or a combination thereof, in a sample (e.g., an apheresis
sample or a manufactured CAR-expressing cell product sample);
[0942] (iii) the level or activity of an immune cell exhaustion
marker, e.g., one, two or more immune checkpoint inhibitors (e.g.,
PD-1, PD-L1, TIM-3 and/or LAG-3) in a sample (e.g., an apheresis
sample or a manufactured CAR-expressing cell product sample). In
one embodiment, an immune cell has an exhausted phenotype, e.g.,
co-expresses at least two exhaustion markers, e.g., co-expresses
PD-1 and TIM-3. In other embodiments, an immune cell has an
exhausted phenotype, e.g., co-expresses at least two exhaustion
markers, e.g., co-expresses PD-1 and LAG-3;
[0943] (iv) the level or activity of CD27 and/or CD45RO- (e.g.,
CD27+CD45RO-) immune effector cells, e.g., in a CD4+ or a CD8+ T
cell population, in a sample (e.g., an apheresis sample or a
manufactured CAR-expressing cell product sample);
[0944] (v) the level or activity of one, two, three, four, five,
ten, twenty or more of the biomarkers chosen from CCL20, IL-17a
and/or IL-6, PD-1, PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,
KLRG1;
[0945] (vi) a cytokine level or activity (e.g., quality of cytokine
reportoire) in a CAR-expressing cell product sample, e.g.,
CLL-1-expressing cell product sample; or
[0946] (vii) a transduction efficiency of a CAR-expressing cell in
a manufactured CAR-expressing cell product sample.
[0947] In some embodiments of any of the methods disclosed herein,
the CAR-expressing cell therapy comprises a plurality (e.g., a
population) of CAR-expressing immune effector cells, e.g., a
plurality (e.g., a population) of T cells or NK cells, or a
combination thereof. In one embodiment, the CAR-expressing cell
therapy is a CLL-1 CAR therapy.
[0948] In some embodiments of any of the methods disclosed herein,
the measure of one or more of (i)-(vii) is obtained from an
apheresis sample acquired from the subject. The apheresis sample
can be evaluated prior to infusion or re-infusion.
[0949] In some embodiments of any of the methods disclosed herein,
the measure of one or more of (i)-(vii) is obtained from a
manufactured CAR-expressing cell product sample, e.g., CLL-1 CAR-
expressing cell product sample. The manufactured CAR-expressing
cell product can be evaluated prior to infusion or re-infusion.
[0950] In some embodiments of any of the methods disclosed herein,
the subject is evaluated prior to receiving, during, or after
receiving, the CAR-expressing cell therapy.
[0951] In some embodiments of any of the methods disclosed herein,
the measure of one or more of (i)-(vii) evaluates a profile for one
or more of gene expression, flow cytometry or protein
expression.
[0952] In some embodiments of any of the methods disclosed herein,
the method further comprises identifying the subject as a
responder, a non-responder, a relapser or a non-relapser, based on
a measure of one or more of (i)-(vii).
[0953] In some embodiments of any of the methods disclosed herein,
a responder (e.g., a complete responder) has, or is identified as
having, a greater level or activity of one, two, or more (all) of
GZMK, PPF1BP2, or naive T cells as compared to a non-responder.
[0954] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, a greater level or
activity of one, two, three, four, five, six, seven, or more (e.g.,
all) of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T
cells, or regulatory T cells, as compared to a responder.
[0955] In an embodiment, a relapser is a patient having, or who is
identified as having, an increased level of expression of one or
more of (e.g., 2, 3, 4, or all of) the following genes, compared to
non relapsers: MIR199A1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1
and/or a decreased levels of expression of one or more of (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) the following genes,
compared to non relapsers: PPIAL4D, TTTY10, TXLNG2P, MIR4650-1,
KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1, and
EIF1AY.
[0956] In some embodiments of any of the methods disclosed herein,
a complete responder has, or is identified as having, a greater,
e.g., a statistically significant greater, percentage of CD8+ T
cells compared to a reference value, e.g., a non-responder
percentage of CD8+ T cells.
[0957] In some embodiments of any of the methods disclosed herein,
a complete responder has, or is identified as having, a greater
percentage of CD27+CD45RO- immune effector cells, e.g., in the CD8+
population, compared to a reference value, e.g., a non-responder
number of CD27+CD45RO- immune effector cells.
[0958] In some embodiments of any of the methods disclosed herein,
a complete responder or a partial responder has, or is identified
as having, a greater, e.g., a statistically significant greater,
percentage of CD4+ T cells compared to a reference value, e.g., a
non-responder percentage of CD4+ T cells.
[0959] In some embodiments of any of the methods disclosed herein,
a complete responder has, or is identified as having, a greater
percentage of one, two, three, or more (e.g., all) of resting
T.sub.EFF cells, resting T.sub.REG cells, younger T cells (e.g.,
younger CD4 or CD8 cells, or gamma/delta T cells), or early memory
T cells, or a combination thereof, compared to a reference value,
e.g., a non-responder number of resting T.sub.EFF cells, resting
T.sub.REG cells, younger T cells (e.g., younger CD4 or CD8 cells),
or early memory T cells.
[0960] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, a greater
percentage of one, two, three, or more (e.g., all) of activated
T.sub.EFF cells, activated T.sub.REG cells, older T cells (e.g.,
older CD4 or CD8 cells), or late memory T cells, or a combination
thereof, compared to a reference value, e.g., a responder number of
activated T.sub.EFF cells, activated T.sub.REG cells, older T cells
(e.g., older CD4 or CD8 cells), or late memory T cells.
[0961] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, a greater
percentage of an immune cell exhaustion marker, e.g., one, two or
more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3 and/or
LAG-3). In one embodiment, a non-responder has, or is identified as
having, a greater percentage of PD-1, PD-L1, or LAG-3 expressing
immune effector cells (e.g., CD4+ T cells and/or CD8+ T cells)
(e.g., CAR-expressing CD4+ cells and/or CD8+ T cells) compared to
the percentage of PD-1 or LAG-3 expressing immune effector cells
from a responder.
[0962] In one embodiment, a non-responder has, or is identified as
having, a greater percentage of immune cells having an exhausted
phenotype, e.g., immune cells that co-express at least two
exhaustion markers, e.g., co-expresses PD-1, PD-L1 and/or TIM-3. In
other embodiments, a non-responder has, or is identified as having,
a greater percentage of immune cells having an exhausted phenotype,
e.g., immune cells that co-express at least two exhaustion markers,
e.g., co-expresses PD-1 and LAG-3.
[0963] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, a greater
percentage of PD-1/PD-L1+/LAG-3+ cells in the CAR-expressing cell
population (e.g., a CLL-1 CAR+ cell population) compared to a
responder (e.g., a complete responder) to the CAR-expressing cell
therapy.
[0964] In some embodiments of any of the methods disclosed herein,
a partial responder has, or is identified as having, a higher
percentages of PD-1/PD-L1+/LAG-3+ cells, than a responder, in the
CAR-expressing cell population (e.g., a CLL-1 CAR+ cell
population).
[0965] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, an exhausted
phenotype of PD1/PD-L1+ CAR+ and co-expression of LAG3 in the
CAR-expressing cell population (e.g., a CLL-1 CAR+ cell
population).
[0966] In some embodiments of any of the methods disclosed herein,
a non-responder has, or is identified as having, a greater
percentage of PD-1/PD-L1+/TIM-3+ cells in the CAR-expressing cell
population (e.g., a CLL-1 CAR+ cell population) compared to the
responder (e.g., a complete responder).
[0967] In some embodiments of any of the methods disclosed herein,
a partial responders has, or is identified as having, a higher
percentage of PD-1/PD-L1+/TIM-3+ cells, than responders, in the
CAR-expressing cell population (e.g., a CLL-1 CAR+ cell
population).
[0968] In some embodiments of any of the methods disclosed herein,
the presence of CD8+CD27+CD45RO- T cells in an apheresis sample is
a positive predictor of the subject response to a CAR-expressing
cell therapy (e.g., a CLL-1 CAR therapy).
[0969] In some embodiments of any of the methods disclosed herein,
a high percentage of PD1+ CAR+ and LAG3+ or TIM3+ T cells in an
apheresis sample is a poor prognostic predictor of the subject
response to a CAR-expressing cell therapy (e.g., a CLL-1 CAR
therapy).
[0970] In some embodiments of any of the methods disclosed herein,
the responder (e.g., the complete or partial responder) has one,
two, three or more (or all) of the following profile:
[0971] (i) has a greater number of CD27+ immune effector cells
compared to a reference value, e.g., a non-responder number of
CD27+ immune effector cells;
[0972] (ii) (i) has a greater number of CD8+ T cells compared to a
reference value, e.g., a non-responder number of CD8+ T cells;
[0973] (iii) has a lower number of immune cells expressing one or
more checkpoint inhibitors, e.g., a checkpoint inhibitor chosen
from PD-1, PD-L1, LAG-3, TIM-3, or KLRG-1, or a combination,
compared to a reference value, e.g., a non-responder number of
cells expressing one or more checkpoint inhibitors; or
[0974] (iv) has a greater number of one, two, three, four or more
(all) of resting T.sub.EFF cells, resting T.sub.REG cells, naive
CD4 cells, unstimulated memory cells or early memory T cells, or a
combination thereof, compared to a reference value, e.g., a
non-responder number of resting T.sub.EFF cells, resting T.sub.REG
cells, naive CD4 cells, unstimulated memory cells or early memory T
cells.
[0975] In some embodiments of any of the methods disclosed herein,
the cytokine level or activity of (vi) is chosen from one, two,
three, four, five, six, seven, eight, or more (or all) of cytokine
CCL20/MIP3a, IL17A, IL6, GM-CSF, IFN.gamma., IL10, IL13, IL2, IL21,
IL4, IL5, IL9 or TNF.alpha., or a combination thereof. The cytokine
can be chosen from one, two, three, four or more (all) of IL-17a,
CCL20, IL2, IL6, or TNF.alpha.. In one embodiment, an increased
level or activity of a cytokine is chosen from one or both of
IL-17a and CCL20, is indicative of increased responsiveness or
decreased relapse.
[0976] In some embodiments of any of the methods disclosed herein,
a transduction efficiency of 15% or higher in (vii) is indicative
of increased responsiveness or decreased relapse.
[0977] In some embodiments of any of the methods disclosed herein,
a transduction efficiency of less than 15% in (vii) is indicative
of decreased responsiveness or increased relapse.
[0978] In embodiments, the responder, a non-responder, a relapser
or a non-relapser identified by the methods herein can be further
evaluated according to clinical criteria. For example, a complete
responder has, or is identified as, 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 NCCN Guidelines.RTM., or 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), as
described herein. A partial responder has, or is identified as, 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 NCCN Guidelines.RTM.,
or Cheson criteria as described herein. A non-responder has, or is
identified as, 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 NCCN Guidelines.RTM., or Cheson criteria as
described herein.
[0979] Alternatively, or in combination with the methods disclosed
herein, responsive to said value, performing one, two, three four
or more of:
[0980] administering e.g., to a responder or a non-relapser, a
CAR-expressing cell therapy;
[0981] administered an altered dosing of a CAR-expressing cell
therapy;
[0982] altering the schedule or time course of a CAR-expressing
cell therapy;
[0983] administering, e.g., to a non-responder or a partial
responder, an additional agent in combination with a CAR-expressing
cell therapy, e.g., a checkpoint inhibitor, e.g., a checkpoint
inhibitor described herein;
[0984] administering to a non-responder or partial responder a
therapy that increases the number of younger T cells in the subject
prior to treatment with a CAR-expressing cell therapy;
[0985] modifying a manufacturing process of a CAR-expressing cell
therapy, e.g., enriching for younger T cells prior to introducing a
nucleic acid encoding a CAR, or increasing the transduction
efficiency, e.g., for a subject identified as a non-responder or a
partial responder;
[0986] administering an alternative therapy, e.g., for a
non-responder or partial responder or relapser; or
[0987] if the subject is, or is identified as, a non-responder or a
relapser, decreasing the T.sub.REG cell population and/or T.sub.REG
gene signature, e.g., by one or more of CD25 depletion,
administration of cyclophosphamide, anti-GITR antibody, or a
combination thereof.
[0988] In certain embodiments, the subject is pre-treated with an
anti-GITR antibody. In certain embodiment, the subject is treated
with an anti-GITR antibody prior to infusion or re-infusion.
[0989] Biopolymer Delivery Methods
[0990] In some embodiments, one or more CAR-expressing cells as
disclosed herein 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.
[0991] 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 .alpha.-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.
[0992] 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.
[0993] Pharmaceutical Compositions and Treatments
[0994] 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.
[0995] 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.
[0996] 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.
[0997] 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).
[0998] 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 10 cc to 400 cc. In certain
aspects, T cells are activated from blood draws of 20 cc, 30 cc, 40
cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
[0999] 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 CAR-expressing cell
(e.g., T cell or NK cell) compositions of the present invention are
administered by i.v. injection. The compositions of CAR-expressing
cells (e.g., T cell or NK cell) may be injected directly into a
tumor, lymph node, or site of infection. 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., immune effector cells (e.g., T cells
or NK cells). These immune effector cells (e.g., T cells or NK
cells) 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-expressing cell (e.g., CAR T
cell or CAR-expressing NK 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-expressing cell (e.g., CAR T cell or CAR-expressing NK cell) of
the present invention. In an additional aspect, expanded cells are
administered before or following surgery.
[1000] 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).
[1001] In one embodiment, the CAR is introduced into immune
effector cells (e.g., T cells or NK cells), e.g., using in vitro
transcription, and the subject (e.g., human) receives an initial
administration of CAR-expressing immune effector cells (e.g., T
cells, NK cells) cells of the invention, and one or more subsequent
administrations of the CAR-expressing immune effector cells (e.g.,
T cells, NK cells) 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-expressing immune effector cells (e.g., T
cells, NK cells) of the invention are administered to the subject
(e.g., human) per week, e.g., 2, 3, or 4 administrations of the
CAR-expressing immune effector cells (e.g., T cells, NK 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-expressing immune effector cells (e.g., T cells, NK
cells) 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-expressing immune effector cells (e.g., T cells, NK cells)
administrations, and then one or more additional administration of
the CAR-expressing immune effector cells (e.g., T cells, NK
cells)(e.g., more than one administration of the CAR-expressing
immune effector cells (e.g., T cells, NK cells) per week) is
administered to the subject. In another embodiment, the subject
(e.g., human subject) receives more than one cycle of
CAR-expressing immune effector cells (e.g., T cells, NK 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-expressing immune effector cells
(e.g., T cells, NK cells) are administered every other day for 3
administrations per week. In one embodiment, the CAR-expressing
immune effector cells (e.g., T cells, NK cells) of the invention
are administered for at least two, three, four, five, six, seven,
eight or more weeks.
[1002] In one aspect, CLL-1 CAR-expressing cells, e.g., CLL-1 CARTs
or CLL-1 CAR-expressing NK cells) are generated using lentiviral
viral vectors, such as lentivirus. CAR-expressing cells, e.g.,
CLL-1 CARTs or CAR expressing NK cells, generated that way will
have stable CAR expression.
[1003] In one aspect, CAR-expressing cells, e.g., CARTs or
CAR-expressing NK cells, are generated using a viral vector such as
a gammaretroviral vector, e.g., a gammaretroviral vector described
herein. CAR-expressing cells, e.g., CARTs or CAR-expressing NK
cells, generated using these vectors can have stable CAR
expression.
[1004] In one aspect, CAR-expressing cells, e.g., CARTs or
CAR-expressing NK cells, 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.
[1005] A potential issue that can arise in patients being treated
using transiently expressing CAR-expressing cells, e.g., CARTs or
CAR-expressing NK cells, (particularly with murine scFv bearing
CARTs) is anaphylaxis after multiple treatments.
[1006] 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.
[1007] 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
[1008] 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.
[1009] 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: Generating CAR Constructs
[1010] Fully human anti-CD33 single chain variable fragments (scFv)
were generated and cloned into a lentiviral expression vector with
the intracellular CD3zeta chain and the intracellular
co-stimulatory domain of 4-1BB and given the names depicted in
Table 7 (which is shown in the Detailed Description).
[1011] 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:25) subunit, in which
each subunit comprises the sequence GGGGS (SEQ ID NO:25) (e.g.,
(G45).sub.3 (SEQ ID NO:28) or (G4S).sub.4(SEQ ID NO:27)), connect
the variable domains to create the entirety of the scFv domain, as
shown in Table 8.
[1012] The sequences of the human scFv fragments (SEQ ID NOS:
39-51) are provided herein in Table 8 (in the Detailed
Description). These clones all contained a Q/K residue change in
the signal domain of the co-stimulatory domain derived from CD3zeta
chain. 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:
11).
[1013] Sequences of CAR constructs and their domain sequences are
listed in the Detailed Description. Analysis of the human CAR
constructs was conducted as described in Examples 2-5.
Example 2: Analysis and In Vitro Activity of Human scFv Bearing
CARTs
[1014] Anti-CLL-1 CAR constructs were evaluated for activity using
a Jurkat cell line containing the luciferase reporter driven by the
NFAT promoter (termed JNL cells). CAR activity is measured as
activation of this NFAT-driven reporter. Lentiviral supernatants
containing the CART constructs were added to JNL cells for
transduction. 4-6 days after transduction, JNL cells were either
evaluated for CAR expression by FACS as described below (FIGS. 2A,
2B, and 2C) or mixed with target-positive (PL21, THP1, HL60, U937)
or target-negative (K562) cell lines at the indicated effector
(JNL) to target cell line (E:T) ratio to trigger activation. After
20 hours of co-incubation, luciferase signal was measured using the
Bright-Glo.TM. Luciferase Assay on the EnVision instrument (FIGS.
1A, 1B, and 2C).
[1015] Optimal anti-CLL-1 CAR constructs are selected based on the
quantity and quality of the effector T cell responses of CLL-1 CAR
transduced T cells ("CART-CLL-1" or "CART-CLL-1 T cells") in
response to CLL-1 expressing ("CLL-1+") 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).
Generation of CART-CLL-1
[1016] The human scFv encoding lentiviral transfer vectors were
used to produce the genomic material packaged into the VSVg
pseudotyped lentiviral particles. Lentiviral transfer vector DNA
was mixed with the three packaging components of VSVg, gag/pol and
rev in combination with lipofectamine reagent to transfect them
together in to Lenti-X 293T cells (Clontech).
[1017] After 30 hours, the media was collected, filtered and stored
at -80C. The therapeutic CART-CLL-1 were 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 were activated by CD3.times.28 beads
(Dynabeads.RTM. Human T-Expander CD3/CD28, Invitrogen) at a ratio
of 1:3 in RPMI 1640, 10% heat-inactivated fetal calf serum (FCS), 2
mM L-glutamine, lx Penicillin/Streptomycin, 100 .mu.M non-essential
amino acids, 1 mM NaPyruvate, 10 mM Hepes, and 55 .mu.M
2-mercaptoethanol at 37.degree. C., 5% CO.sub.2. T cells were
cultured at 1.times.10.sup.6 T cells in 0.5 mL medium per well of a
24-well plate. After 24 hours, the T cells are blasting and 0.5 mL
of viral supernatant was added. The T cells began to divide in a
logarithmic growth pattern, which was monitored by measuring the
cell counts per mL, and T cells were diluted in fresh medium every
two days. As the T cells began to rest down after approximately 10
days, the logarithmic growth waned. The combination of slowing
growth rate and T cell size approaching .about.300 fl determines
the state for T cells to be cryopreserved for later analysis.
[1018] Before cryopreserving, percentage of cells transduced
(expressing the anti-CLL-1 CAR on the cell surface) and their
relative fluorescence intensity of expression were determined by
flow cytometric analysis on a BD LSRFortessa or BD-FACSCanto using
either Protein L (FIG. 3A and 22A) or biotinylated recombinant
human CLL-1 protein as detection reagents (FIGS. 3B and 22B).
Histogram plots of relative fluorescent intensity from that FACS
showed the percentage of transduced T cells. Transduction result in
a range of CART positive cells from 10-50%.
Evaluating Cytolytic Activity and Cytokine Secretion of CART-CLL-1
Redirected T Cells.
[1019] To evaluate the functional abilities of CART-CLL-1 T cells
to kill and secrete cytokines, the cells were thawed and allowed to
recover overnight.
[1020] T cell killing was directed towards CLL-1-expressing PL21
(FIG. 4A) and HL-60 (FIG. 4B) acute myelogenous leukemia cell lines
stably expressing luciferase. Non-CLL-1 expressing U87 cells were
used as a control (FIG. 4C) and untransduced T cells were used to
determine non-specific background killing levels. The cytolytic
activities of CART-CLL-1 were measured as a titration of
effector:target cell ratios of 10:1 and 3-fold downward dilutions
of T cells where effectors were defined as T cells expressing the
anti-CLL-1 chimeric receptor. Assays were initiated by mixing an
appropriate number of T cells with a constant number of targets
cells. After 20 hours luciferase signal was measured using the
Bright-Glo.TM. Luciferase Assay on the EnVision instrument.
[1021] Comparing these killing curves, titrating the amount of
effector cells shows that those cells expressing CLL-1 were
destroyed. T cells from the same donor that were transduced with
either human scFv bearing CAR-CLL-1 cells were able to kill
selectively CLL-1+ targets. Interestingly, not all CART-CLL-1 cells
were active. CART cells containing clone 13 were inactive in this
assay even in the presence of target-expressing cells.
[1022] For measuring cytokine production of CART-CLL-1 cells, cells
were thawed and allowed to recover overnight. Untransduced T cells
(UTD) were used as a non-specific control for background T cell
effects. The T cells were directed towards HL-60, PL21, or U87
cells. The assay tested an effector:target ratio of 1:1 or 10:1 as
noted where effectors were defined as T cells expressing the
anti-CLL-1 CAR. The assay was run 24 hours after mixing of the
cells, when the media is removed for analysis of cytokines
TNF-alpha (FIG. 5A), IL-2 (FIG. 5B), and INF-gamma (FIG. 5C) using
the CBA-Flex kit for human cytokine detection.
[1023] When CART-CLL-1 T cells were cultured with cancer cells
endogenously expressing CLL-1, all CLL-1-CARTs except CLL-1-13
produced cytokines in response to target-expressing cells. The
difference in reactivity of the various CLL-1-CART clones toward
low CLL-1-expressing target cells may translate to better clinical
efficacy of CART cells transduced with these constructs.
Evaluating Proliferative Capacity of CART-CLL-1
[1024] CART-CLL1 T cells were tested for their ability to
proliferate in response to exposure to antigen on target cells.
Multiple CLL-1 CAR constructs were tested, CLL-6, CLL-9, CLL-10,
CLL-11, CLL-12, and CLL-13. Target cells included U937, PL-21,
HL60, and Molm13 cells. On the day of assay (Day 0), target cells
were counted and transferred to a 50 ml tube in 6 mL of T cell
media at 3e6 cells/ml. Target cells were irradiated on ice at
10,000 rad. After irradiation, target cells were washed twice in T
cell media, counted, and resuspended to 5e5 cells/ml in T cell
media on ice.
[1025] Frozen transduced T cells were thawed, washed in 10 mL
complete T cell media, spun at 300 g for 10 min, and resuspended
gently in 3 mL of complete T cell media at RT. T-cells were then
counted in a cellometer and resuspended to 2.5e6/mL in 10 mL of
media. In a 96 well U-bottom plate, 25,000 irradiated target cells
and 25,000 transduced CAR T cells (1:1 ratio) were combined in
duplicate wells. In a separate well, 75,000 Anti-CD3/CD28 beads
were added in 100 .mu.l of medium to 25,000 transduced T cells to
create a 1:3 cells-to-beads ratio as positive control; in another
well, 100 .mu.l of medium was added to 25,000 transduced T cells
alone as media-only control. Cells were incubated for 4 days at
37.degree. C., 5% CO.sub.2.
[1026] On day 4, cells were harvested and duplicates were combined
by pipetting and transferring into the same well on the U-bottom
plate for staining for FACS of CD4, CD8, and CAR using protein L or
recombinant human CLL1 protein. After staining, cells were
resuspended in 120p1 MACS+0.5% BSA buffer and 20p1/well countbright
beads were added to each well. Proliferation was measured as the
number of FACS positive cells detected in the period of time used
to count 2500 beads.
[1027] As shown in FIGS. 23A and 23B, cells expressing CLL-1 CAR
constructs CLL-6, CLL-9, CLL-10, CLL-11, and CLL-12 proliferated in
the presence of different target cells.
Example 3: CLL-1 Toxicity Studies
[1028] CLL-1 was measured by flow cytometry using a commercially
available antibody (clone HIM3-4, eBioscience). The results herein
demonstrate that CLL-1 was expressed in most primary patient
samples with AML (AML blasts were gated using standard side scatter
low CD45dim characteristics) (FIG. 6).
[1029] T cells from two different donors were transduced with CLL-1
(FIG. 6) and resulted in transduction efficiency of 35-45%. T cells
were first stimulated with CD3/CD28 Dynabeads (Invitrogen) and
maintained in serum free T cell media, along with IL-2 support. T
cell were then transduced with CLL-1 (FIG. 6) CAR using a
lentiviral vector the following day and were expanded in media for
about 10 days. T cells were then frozen when the median cell volume
approached 300 fl. CAR expression on T cells was detected by flow
cytometry using a biotinylated CLL-1 protein (Sino Biological) with
secondary staining using streptavidin. The results presented herein
demonstrate transduction efficiency of T cells transduced with CAR
(FIGS. 7A and 7B).
[1030] CART123, CLL1-CART cells and untransduced T cells were
incubated with the CD123+/CLL1+ cell line THP-1, two primary AML
samples that are CLL1+/CD123+ and a control ALL cell line NALM6 for
4 hours. CD107a degranulation was measured by flow cytometry (8A).
CLL-1 CART cells underwent specific degranulation to THP1 and
primary AML samples and not to the control cell line (FIG. 8B). The
results presented herein demonstrate that CLL1-CART cells underwent
specific degranulation to CLL1+ cell lines and primary AML
samples.
[1031] CART123, CLL1-CART cells and untransduced T cells were
incubated with the CD123+/CLL1+ cell line THP-1, two primary AML
samples that are CLL1+/CD123+ and a control ALL cell line NALM6 for
4 hours. Cells were then harvested and intracytoplasmic TNF.alpha.
were measured by flow cytometry. More CLL-1 CART cells produced
TNF-.alpha. after incubation specifically with THP1 and primary AML
samples and not to the control cell line. The results presented
herein demonstrate that CLL1-CART cells produced TNF-.alpha. after
incubation with CLL1+ cell line and primary AML samples (FIGS. 9A
and 9B).
[1032] CART123, CLL1-CART cells and untransduced T cells were
incubated with the CD123+/CLL1+ cell line THP-1, two primary AML
samples that are CLL1+/CD123+ and a control ALL cell line NALM6 for
4 hours. Cells were then harvested and intracytoplasmic IL-2 were
measured by flow cytometry. More CLL-1 CART cells produced IL-2
after incubation specifically with THP1 and primary AML samples and
not to the control cell line. The results presented herein
demonstrate that CLL1-CART cells produced IL-2 after incubation
with CLL1+ cell line and primary AML samples (FIGS. 10A and
10B).
[1033] CART123, CLL1-CART cells and untransduced T cells were
incubated with the CD123+/CLL1+ cell lines THP-1 and MOLM14, a
primary AML sample that is CLL1+/CD123+ and a control mantle cell
lymphoma cell line JEKO for 24 hours. Cells were then harvested and
7-AAD and counting beads were added. Killing was then measured
using a flow cytometry based assay after CFSE-labeling of the tumor
cells (e.g. Cao et al, Cytometry Part A 2010;7&A:534-545) or by
incubating CART cells with luciferase-expressing target cells at
various effector-to-target ratios for up to 20 hours, followed by
optical imaging for photons emitted by the target cells. In this
latter assay, number of live target cells correlates positively
with the number of photons emitted. CLL1-CART cells results in
specific lysis of MOLM14 (FIG. 11D), THP-1 (FIG. 11A) and the
primary AML sample (FIG. 11B) and not to the control cell line JEKO
(FIG. 11C), at the indicated E:T ratios. The results presented
herein demonstrate that CLL1-CART cells specifically killed the
CLL-1+ cell lines MOLM14 and THP-1 and primary AML samples (FIG.
11A-11D).
[1034] Proliferation of CART123, CART33 and CLL1-CART cells was
measured in response to MOLM14, THP-1, and two primary AML samples.
T cells were labeled with CFSE and incubated with targets for 120
hour at an effector:target ratio of 1:1. CLL1-CART cells underwent
specific proliferation in response to MOLM14, THP1 and primary AML
samples. Un-proliferated T cells retained a single bright peak of
CFSE expression (by green fluorescence in the FITC channel),
whereas proliferating CART cells had more than one CFSE peak and
expression that was lower than baseline. The results presented
herein demonstrate that CLL1-CART cells proliferated in response to
MOLM14, THP-1 and primary AML samples (FIGS. 12A and 12B).
[1035] FIG. 13 presents a schematic diagram for assaying
hematopoietic stem cell cyoxicity of CLL-1 CART cells using
autologous xenografts. NSGS (NOD-SCID-gamma mice that are
transgenic for IL-3, GM-CSF, stem cell factor) mice received
busulfan i.p. followed by T cell depleted bone marrow from a normal
donor the following day. Engraftment was confirmed by flow
cytometric analysis of peripheral blood after 4 weeks and defined
as circulating CD45 positive cells of >1%. Mice were then
treated with autologous T cells by intravenous tail vain injection.
T cells were derived from the same donor and were transduced with
CART33, CLL1-CAR or UTD. A forth group received no treatment. Mice
were then followed with retro-orbital bleeding on day 7, day 14 and
day 21.
[1036] CLL-1 expression was measured on different peripheral blood
cells from humanized xenografts. Mice were bled through the
retro-orbital vein after being anesthetized, using standard
techniques. A standard volume of 50-60 ul blood was then lysed in 1
ml of ACK lysis buffer. The blood was then stained using
fluorescently-labelled antibodies and the expression of CLL-1 on
different peripheral blood cells was detected using flow cytometry.
This analysis was performed at baseline, after engraftment with T
cell depleted normal donor bone marrow and prior to any treatments.
A representative FACS plots of the peripheral blood analysis of one
mouse is shown (FIG. 14A). CLL-1 is expressed on monocytes (CD14+
cells), myeloid cells (CD33+ and CD123+ cells), B cells (CD19+
cells), but not on platelets (CD41+ cells) or T cells (CD3+ cells).
A representative histogram presentation is shown (FIG. 14B). A
shcematic plot representation of peripheral blood analysis from 24
mice is shown (FIG. 14C). The results presented herein demonstrate
that CLL-1 was expressed on different myeloid lineage cells and B
cells in humanized mice.
[1037] Hematopoietic stem cell toxicity of CLL1-CART cells was
determined using an autologous model. Representative plot of
peripheral blood analysis by flow cytometry. Treatment with CART33
or CLL1-CART cells resulted in significant reduction in Myloid
cells (CD123+,CD33+, CLL1+), and in CD14+ monocytes. The results
presented herein demonstrate that CLL-1 was expressed on different
myeloid lineage cells and B cells in humanized mice.
[1038] CLL-1 expression on different bone marrow progenitor cells
from humanized xenografts was assayed. After 4 weeks of treatment
with T cells, mice were euthanized and bone marrow was harvested
and analyzed. Bone marrow was harvested by flushing of the femur
bones. Bone marrow samples from the control untreated animals were
used as a reference to analyze expression of CLL-1 on different
progenitors. The samples were then stained using
fluorescently-labelled antibodies and the expression of CLL-1 on
different progenitor cells was detected using flow cytometry. CLL-1
was expressed on the CD34+CD38-hematopoietic stem cells, CD123
bright, CD123 dim and CD33 positive cells. Gated on CD45.sup.dim,
LIN.sup.-, live cells. The results presented herein demonstrate
that CLL-1 was expressed on different myeloid progenitors and on
hematopoietic stem cells in humanized mice (FIG. 16A-16D).
[1039] FIG. 17 illustrates a schematic diagram for assaying
hematopoietic stem cell toxicity of CLL-1 CART cells using a
Humanized Immune System (HIS) xenografts. HIS mice were bled
retro-orbitally 6-8 weeks after injection of CD34+ fetal liver, to
confirm engraftment of human cells and then treated with either
CLL1-CARTs, CART123, CART33-CD8 hinge, CART33-IgG4 hinge,
untransduced T cells or with no treatment. Mice were then followed
by serial weekly retro-orbital bleedings. Mice were then euthanized
on day 28 and organs were harvested and analyzed.
[1040] Bone marrow was analyzed 4 weeks post T cell infusion.
Hematopoietic stem cell toxicity of CLL1-CART cells was measured
using HIS xenografts. Mice were euthanized 4 weeks after T cell
infusion, femur bones were harvested and flushed for bone marrow.
The samples were then stained using fluorescently-labelled
antibodies. Schematic plots of all mice treated with different CART
cells. Treatment with CLL1-CART cells resulted in significant
reduction in the CD34+CD38-component (hematopoietic stem cells)
(FIG. 18A) and CD34+CD38+ component (Progenitor cells) (FIG. 18B).
Representative plots of bone marrows from mice treated with
different CART cells are shown. Gated on live CD45dim LIN- cells
(FIG. 19A-19E).
[1041] Bone marrow was analyzed in HIS mice 4 weeks post T cell
infusion. Hematopoietic stem cell toxicity of CLL1-CART cells using
HIS xenografts. Mice were euthanized 4 weeks after T cell infusion,
femur bones were harvested and flushed for bone marrow. The samples
were then stained using fluorescently-labelled antibodies. A
schematic plots is shown of all mice treated with different CART
cells (FIG. 20). Treatment with CLL1-CART cells did not result in
significant reduction in the CD123 bright population. The samples
were then stained using fluorescently-labelled antibodies.
Representative plots of bone marrows from mice treated with
different CART cells are shown (FIG. 21).
Example 4: Evaluation of CLL-1 CART Cells In Vivo
[1042] PL-21 is a human acute myeloid leukemia cell line isolated
from the peripheral blood of a 24 year old male patient with
refractory acute promyelocytic leukemia, and can be grown as a
xenograft in immune compromised mice. The xenograft mimics disease
in the bone marrow as seen in humans, establishing a model with
which to test the efficacy of therapies on AMLs in the bone. These
mice can be used to test the efficacy of chimeric antigen receptor
(CAR) T cells specific for cellular markers found on acute myeloid
(or promyelocytic) leukemia cells, such as CLL-1 (a C-type
lectin-type molecule). PL-21 cells were tagged with a firefly
luciferase reporter gene and used in an orthotopic model of acute
myeloid leukemia (AML) in NOD.Cg-Prkdc.sup.scidll2rg.sup.tm1Wjl/SzJ
(NSG) mice to test the efficacy of CAR T cells specific for
CLL-1.
[1043] CLL-1 expression was tested on PL-21 cells and these cells
were used in in vitro assays to look at the ability of
CLL-1-specific CAR T cells to recognize and respond to the target.
In vivo PL-21 cells grow when implanted intravenously via the tail
vein and growth is limited primarily to the bone marrow. One week
after the tumor cells are implanted, the disease shifts fully to
the bones and begins to grow at an exponential rate. Left
untreated, mice will start to display clinical symptoms and hind
limb paralysis 4-6 weeks after tumor implantation. The study
described in this example investigates whether any of the CLL-1
specific scFv clones from the in vitro screen show activity against
tumors in this in vivo xenograft model.
[1044] The following materials and methods were used in the
experiments described herein.
Materials and Methods:
[1045] PL-21 Cell Line:
[1046] The PL-21 human AML cell line was developed from the
peripheral blood of a patient with acute promyelocytic leukemia.
The cells were then tagged with firefly luciferase. These
suspension cells grow in RPMI supplemented with 10% heat
inactivated fetal bovine serum.
[1047] Mice:
[1048] 6 week old NSG (NOD.Cg-Prkdc.sup.scidll2rg.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.
[1049] Tumor Implantation:
[1050] PL-21-luc cells were grown and expanded in vitro in RPMI
supplemented with 10% heat inactivated fetal bovine serum. The
cells were then transferred to a 50 ml conical tube and washed
twice with cold sterile PBS. The PL-21-luc cells were then counted
and resuspended at a concentration of 10.times.10.sup.6 cells per
milliliter of PBS. The cells were placed on ice and immediately
(within one hour) implanted in mice. PL-21-luc cells were injected
intravenously via the tail vein in a 100 .mu.l volume, for a total
of 1.times.10.sup.6 cells per mouse.
[1051] CAR T Cell Dosing:
[1052] Mice were administered 5.times.10.sup.6 CAR' T cells 8 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. The CAR T cells were normalized for CAR
transduction so that each group has the same percentage of CAR' T
cells. The 5.times.10.sup.6 CAR' T cells were then resuspended at a
concentration of 50.times.10.sup.6 CAR' T 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 CAR' T cells per mouse.
[1053] Eight mice per group were treated either with 100 .mu.l of
PBS alone (PBS), CD19 control CAR T cells (CD19), CLL-1-6 (clone 6)
CAR T cells, CLL-1-9 (clone 9) CAR T cells, CLL-1-10 (clone 10)
CART cells, CLL-1-11 (clone 11) CART cells, and CLL-1-12 (clone 12)
CAR T cells. The T cells were all prepared from the same human
donor in parallel.
[1054] Animal Monitoring:
[1055] 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 twice weekly by bioluminescent imaging. Mice
were intraperitoneally injected with D-luciferin 10 minutes prior
to anesthetizing and imaging the mice with a Xenogen. Disease
burden was calculated by calculating the bioluminescence of the
tumor cells (photons/second).
[1056] 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;
[1057] where T=mean bioluminescence of the drug-treated group on
the final day of the study; T.sub.initial==mean bioluminescence of
the drug-treated group on initial day of dosing; .DELTA.T==mean
bioluminescence of the drug-treated group on the final day of the
study -=mean bioluminescence of the drug treated group on the
initial day of dosing; C==mean bioluminescence of the control group
on the final day of the study; and .DELTA.C==mean bioluminescence
of the control group on the final day of the study -=mean
bioluminescence of the control group on the initial day of
dosing.
[1058] 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.
[1059] Peripheral Blood FACS Analysis:
[1060] T cells in the peripheral blood of the mice were also
monitored. Mice were bled weekly via the tail vein into EDTA coated
tubes that were kept on ice. 10 .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 anti-mouse CD11b, anti-human
CD45, anti-human CD4, anti-human CD8, and CLL-1-Fc or Protein L
antibodies, followed by a secondary. 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 Fortessa, followed
by further analysis using the FlowJo FACS analysis software. The
cells were analyzed to determine the number of CAR.sup.+CD4.sup.+
and CD8.sup.+ T cells per milliliter of blood in the PL-21-luc
tumor-bearing NSG mice. T cell numbers in the blood are reported as
the mean+standard error of the mean (SEM).
Results:
[1061] The anti-tumor activity of a panel of CLL-1 specific CAR T
cells (clone 6, 9 10, 11, and 12) were evaluated and directly
compared in the PL-21 model of human AML. Following tumor
implantation on day 0, mice were randomized into treatment groups
and treated with 5.times.10.sup.6 CARP T cells intravenously on day
8. AML disease burden and animal health were monitored until
animals achieved endpoint. The mice in the control PBS and CD19
groups along with the CLL-1-11 and CLL-1-12 groups were euthanized
on day 18 post-CART cell dosing (26 days post-tumor implantation)
when disease burden in the control groups was nearing maximum
luminescence via imaging. The mice in the remaining CLL-1 CAR T
cell treated groups (CLL-1-6, CLL-1-9, and CLL-1-10) were
euthanized on day 25 post-CAR T cell dosing (33 days post-tumor
implantation) to enable comparison of all of the groups terminal
samples.
[1062] A delay in disease progression was observed between the
control groups and the CLL-1-6, CLL-1-9, and CLL-1-10 treated
groups and slowing in tumor growth at late time points with the
CLL-1-11 and CLL-1-12 CAR T cell treated groups. Calculated at the
last time point with all the groups represented (day 18 post CAR T
cell dose), all of the CLL-1 CAR T cells groups were significantly
different from the control groups. Compared to the PBS treated
mice, the CD19 CAR T cell treated group did not demonstrate
significance with a P value of 0.496. All of the CLL-1 CAR T cell
treated groups compared to PBS had a P<0.01 (clone 6 P=0.0008;
clone 9 P=0.0006; clone 10 P=0.0006; clone 11 P=0.0013; clone 12
P=0.0109). The three CLL-1 clones that showed an initial delay in
tumor progression, clones 6, 9, and 10, showed stasis in tumor
growth on day 18 post CAR T cell dose. The other two CLL-1 clones,
clones 11 and 12, showed anti-tumor activity by this time point,
although at the earlier time points they did not show any delay in
disease progression. Calculated on day 18 post-T cell dose, the
percent delta T/C values for each treatment group compared to the
PBS control group were as follows: 98.38% for CD19, 3.15% for
CLL-1-6, 2.04% for CLL-1-9, 4.92% for CLL-1-10, 13.42% for
CLL-1-11, and 25.19% for CLL-1-12. Based on these values, the CD19
CAR T cell treated group showed no activity, the CLL-1-6, -9, and
-10 CAR T cell treated groups showed stasis in tumor growth, and
the CLL-1-11, and -12 CAR T cell treated groups demonstrated
anti-tumor activity.
[1063] The bioluminescence imaging results of this study are shown
in FIG. 24. The PBS treatment group, which did not receive any T
cells, demonstrated baseline PL-21 tumor growth kinetics in
intravenously implanted NSG mice. The CD19 treatment group received
control CD19 CAR T cells, not specific for PL-21 cells, which
underwent the same in vitro expansion process as the CAR T cells.
These cells served as a T cell control to show the non-specific
response of the T cells in this tumor model. Both the PBS and the
CD19 CAR T cell treatment groups demonstrated continuous tumor
progression throughout the experiment.
[1064] In addition to monitoring the disease burden via
bioluminescence, the CARP T cell numbers in each group was also
monitored via peripheral blood FACS analysis. The FACS results of
this study are shown in FIGS. 25A and 25B. The groups that showed
the greatest anti-tumor activity and a delay in tumor growth after
CAR T cell treatment showed an increase in both CD4.sup.+CAR.sup.+
(FIG. 25A) and CD8.sup.+CAR.sup.+ T cells (FIG. 25B) in the
peripheral blood at late time points. Limited numbers of cells were
observed in the peripheral blood at early time point. The AML was
primarily in the bone marrow and the CAR T cells may also be in the
bone marrow at these time points. However, this group showed no
anti-tumor activity. At the final time point, there was an
expanding population of CAR T cells in the peripheral blood for of
the CLL-1-6, CLL-1-9, and CLL-1-10 groups.
[1065] At the end of the study, spleen and bone marrow cells were
harvested from the mice and analyzed to determine if the CAR T
cells persist. As seen in FIGS. 26A-26D, CD4 and CD8 CAR+ T cells
were seen in appreciable numbers in the CLL-1-9 and CLL-1-10
treated groups. An expansion of T cells was also seen in some of
the other groups, in particular the CLL-1-12 CART cell treated
group. However, although large numbers of both CD4+ and CD8+ T
cells were seen in the bone marrow of this group, the cells are not
CAR+, indicating a non-specific expansion of T cells in these mice.
CLL-1-6, CLL-1-9, and CLL-10, showed the greatest anti-tumor effect
and delay in tumor growth and this corresponded to the terminal
bone marrow CAR+ T cell numbers. Of note, the CLL-1-6 CAR was not
well detected, so the CAR+ numbers in this group may be
underestimated. A slight persistence of both CD4 and CD8 T cells in
the CLL-1-11 group and larger numbers of these cells in the
CLL-1-12 group may correlate to a non-specific late stage activity
on the tumors resulting in the slight dip in tumor burden seen at
the late time points in FIG. 24.
[1066] At the termination of the study, the phenotype of the cells
in the spleen was also determined. Similar to what was observed in
the bone marrow samples of these mice, the only groups with
appreciable numbers of CD4 and CD8 CARP T cells in the spleen were
the CLL-1-9 and CLL-1-10 groups, as shown in FIGS. 27A-27D. These
were also the only groups that had large numbers of CD4.sup.+ and
CD8.sup.+ T cells in the spleen. The CLL-1-11 and CLL-12 groups did
not show the non-CAR accumulation of T cells in the spleen that was
observed in the bone marrow samples. Due to the large variation in
CAR+ T cells detected in the spleen samples of the CLL-1-10 mice,
this group did not correlate as significantly different when
compared to the control groups.
Discussion:
[1067] The anti-tumor activity of novel CLL-1 CAR transduced T
cells was assessed in an efficacy study in NSG mice bearing a
xenograft model of human AML. These studies showed that the
PL-21-luc model recapitulates human AML in the NSG mouse and is
capable of being targeted by CLL-1 CAR T cells (FIG. 24). This
study demonstrated that the three of the CLL-1 CARs (CLL-1-6,
CLL-1-9, and CLL-1-10) were capable of mounting an anti-tumor
response in a xenograft model of AML (FIG. 24). Minimal numbers of
CAR T cells were detected in the blood for all of the groups,
however at late time points, CAR T cells were seen in increasing
numbers in the CLL-1-6, CLL-1-9, and CLL-1-10 groups (FIGS. 25A and
25B). The groups treated with these three CAR T cells showed a
delay in tumor growth, and in particular, the CLL-1-9 and CLL-1-10
groups showed significant numbers of CAR T cells in the terminal
bone marrow and spleen samples at the end of the study (FIGS.
26A-26D and 27A-27D). The CLL-1-6 group also showed an initial and
prolonged delay in tumor growth, but the terminal CAR T cell
numbers could not accurately be calculated due to difficulties in
detecting the expression of the scFv on the surface of the T cells.
Taken together, the CLL-1-9 and CLL-1-10 groups showed a delay in
tumor growth, an increase in CD4.sup.+CAR.sup.+ T cells and
CD8.sup.+CAR.sup.+ T cells in the peripheral blood, along with
significant numbers of CD4.sup.+CAR.sup.+ T cells and
CD8.sup.+CAR.sup.+ T cells in the terminal bone marrow and spleen
samples. Only the CLL-1-9 group had significantly different amounts
of CD4.sup.+CAR.sup.+ and CD8.sup.+CAR.sup.+ T cells as compared to
the control groups in both the bone marrow and spleen.
Example 5: Chemotherapy and CLL1-CAR Combined Therapy
[1068] The effect of CLL-1 CAR therapy combined with chemotherapy
was examined using an in vivo AML mouse model. Treatment with
induction chemotherapy followed by CLL1-CART cells (clone 6) result
in leukemic eradication in primary AML xenografts.
[1069] NSG mice that are additionally transgenic for stem cell
factor, GM-CSF and IL-3 (NSG-S) were injected with primary AML
blasts (3.times.10.sup.6 via tail vain injection, day 0). After 4-6
weeks, peripheral blood (PB) was collected to confirm engraftment,
which was defined as the presence of >1% circulating leukemic
cells (live human CD45dim cells). AML xenografts were then treated
with cytarabine (Ara-C, 60 mg/kg by intra-peritoneal injection,
daily between days 35-39). Peripheral blood counts were monitored
and then these xenografts were randomized to receive CLL1 directed
CART cells or control untransduced T cells (1.times.10.sup.5 I.V.)
on day 55. Following that, serial retro-orbital bleedings were
performed and absolute leukemic blast count was calculated as a
measure of disease burden and the xenografts were followed up for
survival.
[1070] FIG. 33B comprises representative plots of the mean
fluorescence intensity (MFI) of CLL1 in leukemic cells (live
huCD45dim compartment), and demonstrates that treatment with
chemotherapy results in upregulation of CLL-1 antigen in the
residual AML. There was a significant increase in CLL1-MFI in AML
xenografts two weeks after treatment with cytarabine
chemotherapy.
[1071] Mice that were treated with induction chemotherapy followed
by untransduced T cells did not show reduction of peripheral blood
leukemic blast count. In contrast, mice treated with induction
chemotherapy followed by CLL-1-CARTs resulted in significant
reduction of the peripheral blood leukemic blast count and
eradication of leukemia in AML xenografts, as shown in FIG. 33C.
Plots are representative of the peripheral blood absolute leukemic
blast count per 1 ul of peripheral blood (mean+/-SD) at different
time points post AML injection as indicated. Analysis of overall
survival also showed that induction chemotherapy followed by
CLL1-CARTs, but not by untransduced T cells results in a
significant advantage in overall survival in AML xenografts (FIG.
33D).
Example 6: Low Dose RAD001 Stimulates CART Proliferation in a Cell
Culture Model
[1072] The effect of low doses of RAD001 on CAR T cell
proliferation in vitro was evaluated by co-culturing
CART-expressing cells with target cells in the presence of
different concentrations of RAD001.
Materials and Methods
[1073] Generation of CAR-Transduced T Cells
[1074] A humanized, anti-human CD19 CAR (huCART19) lentiviral
transfer vector was used to produce the genomic material packaged
into VSVg pseudotyped lentiviral particles. The amino acid and
nucleotide sequence of the humanized anti-human CD19 CAR (huCART19)
is CAR 1, ID 104875 described in PCT publication, WO2014/153270,
filed Mar. 15, 2014, and is designated SEQ ID NOs. 85 and 31
therein.
[1075] Lentiviral transfer vector DNA is mixed with the three
packaging components VSVg env, gag/pol and rev in combination with
lipofectamine reagent to transfect Lenti-X 293T cells. Medium is
changed after 24 h and 30 h thereafter, the virus-containing media
is collected, filtered and stored at -80.degree. C. CARTs are
generated by transduction of fresh or frozen naive T cells obtained
by negative magnetic selection of healthy donor blood or leukopak.
T cells are activated by incubation with anti-CD3/anti-CD28 beads
for 24 h, after which viral supernatant or concentrated virus
(MOI=2 or 10, respectively) is added to the cultures. The modified
T cells are allowed to expand for about 10 days. The percentage of
cells transduced (expressing the CARs on the cell surface) and the
level of CAR expression (relative fluorescence intensity, Geo Mean)
are determined by flow cytometric analysis between days 7 and 9.
The combination of slowing growth rate and T cell size approaching
.about.350 fL determines the state for T cells to be cryopreserved
for later analysis.
[1076] Evaluating Proliferation of CARTs
[1077] To evaluate the functionality of CARTs, the T cells are
thawed and counted, and viability is assessed by Cellometer. The
number of CAR-positive cells in each culture is normalized using
non-transduced T cells (UTD). The impact of RAD001 on CARTs was
tested in titrations with RAD001, starting at 50 nM. The target
cell line used in all co-culture experiments is Nalm-6, a human
pre-B cell acute lymphoblastic leukemia (ALL) cell line expressing
CD19 and transduced to express luciferase.
[1078] For measuring the proliferation of CARTs, T cells are
cultured with target cells at a ratio of 1:1. The assay is run for
4 days, when cells are stained for CD3, CD4, CD8 and CAR
expression. The number of T cells is assessed by flow cytometry
using counting beads as reference.
Results
[1079] The proliferative capacity of CART cells was tested in a 4
day co-culture assay. The number of CAR-positive CD3-positive T
cells (dark bars) and total CD3-positive T cells (light bars) was
assessed after culturing the CAR-transduced and non-transduced T
cells with Nalm-6 (FIG. 31). huCART19 cells expanded when cultured
in the presence of less than 0.016 nM of RAD001, and to a lesser
extent at higher concentrations of the compound. Importantly, both
at 0.0032 and 0.016 nM RAD001 the proliferation was higher than
observed without the addition of RAD001. The non-transduced T cells
(UTD) did not show detectable expansion.
Example 7: Low Dose RAD001 Stimulates CART Expansion In Vivo
[1080] This example evaluates the ability of huCAR19 cells to
proliferate in vivo with different concentrations of RAD001.
Materials and Methods:
[1081] NALM6-Luc Cells:
[1082] The NALM6 human acute lymphoblastic leukemia (ALL) cell line
was developed from the peripheral blood of a patient with relapsed
ALL. The cells were then tagged with firefly luciferase. These
suspension cells grow in RPMI supplemented with 10% heat
inactivated fetal bovine serum.
[1083] Mice:
[1084] 6 week old NSG (NOD.Cg-Prkdc.sup.scidll2rg.sup.tm1Wjl/SzJ)
mice were received from the Jackson Laboratory (stock number
005557).
[1085] Tumor Implantation:
[1086] NALM6-luc cells were grown and expanded in vitro in RPMI
supplemented with 10% heat inactivated fetal bovine serum. The
cells were then transferred to a 15 ml conical tube and washed
twice with cold sterile PBS. NALM6-luc cells were then counted and
resuspended at a concentration of 10.times.10.sup.6 cells per
milliliter of PBS. The cells were placed on ice and immediately
(within one hour) implanted in the mice. NALM6-luc cells were
injected intravenously via the tail vein in a 100 .mu.l volume, for
a total of 1.times.10.sup.6 cells per mouse.
[1087] CAR T cell dosing: Mice were administered 5.times.10.sup.6
CAR T cells 7 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 CAR T 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 CAR T cells per mouse. Eight mice
per group were treated either with 100 .mu.l of PBS alone (PBS), or
humanized CD19 CAR T cells.
[1088] RAD001 Dosing:
[1089] A concentrated micro-emulsion of 50 mg equal to 1 mg RAD001
was formulated and then resuspended in D5W (dextrose 5% in water)
at the time of dosing. Mice were orally dosed daily (via oral
gavage) with 200 .mu.l of the desired doses of RAD001.
[1090] PK Analysis:
[1091] Mice were dosed daily with RAD001 starting 7 days post tumor
implantation. Dosing groups were as follows: 0.3 mg/kg, 1 mg/kg, 3
mg/kg, and 10 mg/kg. Mice were bled on days 0 and 14 following the
first and last dose of RAD001, at the following time points for PK
analysis: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8
hours, 12 hours, and 24 hours.
Results:
[1092] The expansion and pharmacokinetics of RAD001 was tested in
NSG mice with NALM6-luc tumors. Daily oral dosing of RAD001 alone
did not have an impact on the growth of NALM6-luc tumors (FIG. 32).
The pharmacokinetic analysis of RAD001 shows that it is fairly
stable in the blood of tumor bearing mice (FIGS. 33A and 33B). Both
the day 0 and day 14 PK analyses show that the RAD001
concentrations in the blood is above 10 nm even 24 hours after
dosing at the lowest dose tested (0.3 mg/kg).
[1093] Based on these doses, huCAR19 CAR T cells were dosed with
and without RAD001 to determine the proliferative ability of these
cells. The highest dose used was 3 mg/kg based on the levels of
RAD001 in the blood 24 hours after dosing. As the concentration of
RAD001 was above 10 nM 24 hours after the final dose of RAD001,
several lower doses of RAD001 were used in the in vivo study with
CAR T cells. The CAR T cells were dosed IV one day prior to the
start of the daily oral RAD001 dosing. Mice were monitored via FACS
for T cell expansion.
[1094] The lowest doses of RAD001 show an enhanced proliferation of
the CAR T cells (FIG. 34). This enhanced proliferation is more
evident and prolonged with the CD4.sup.+CAR T cells than the
CD8.sup.+CAR T cells. However, with the CD8.sup.+CAR T cells,
enhanced proliferation can be seen at early time points following
the CAR T cell dose.
EQUIVALENTS
[1095] 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
495121PRTArtificial Sequencesource/note="Description of Artificial
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Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro
20245PRTArtificial Sequencesource/note="Description of Artificial
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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 453230PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Arg Glu Glu Gln Phe Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val
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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
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Phe Leu Tyr Ser Arg Leu 180 185 190Thr Val Asp Lys Ser Arg Trp Gln
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85 90 95Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys
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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
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Thr Asp His 275 280510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
10624PRTArtificial Sequencesource/note="Description of Artificial
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Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr Leu Tyr Cys
20742PRTArtificial Sequencesource/note="Description of Artificial
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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 40848PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 8Gln 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 459112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 9Arg 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
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sapiens 10Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
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Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110111184DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 11cgtgaggctc 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
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gttggaagtg 360ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
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tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
600gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
aggcggggcc 660tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
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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 11841263DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 12atggccctgc ctgtgacagc cctgctgctg cctctggctc
tgctgctgca tgccgctaga 60ccc 6313135DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 13accacgacgc cagcgccgcg accaccaaca ccggcgccca
ccatcgcgtc gcagcccctg 60tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg
cagtgcacac gagggggctg 120gacttcgcct gtgat 13514690DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 14gagagcaagt 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
69015847DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 15aggtggcccg
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 8471630DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 16ggtggcggag gttctggagg tggaggttcc
301772DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 17atctacatct gggcgccctt
ggccgggact tgtggggtcc ttctcctgtc actggttatc 60accctttact gc
7218126DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 18aaacggggca gaaagaaact
cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg
ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg
12619123DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 19aggagtaaga
ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60gggcccaccc
gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120tcc
12320336DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 20agagtgaagt
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 33621336DNAHomo sapiens 21agagtgaagt 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
33622373PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 22Pro 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
370231182DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 23atggccctcc
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
118224394PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 24Met 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 390255PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 25Gly Gly Gly Gly Ser1
52630PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide"misc_feature(1)..(30)/note="This
sequence may encompass 1-6 'Gly Gly Gly Gly Ser' repeating units,
wherein some positions may be absent"source/note="See specification
as filed for detailed description of substitutions and preferred
embodiments" 26Gly 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 302720PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 27Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
202815PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 28Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 15294PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 29Gly Gly Gly Ser1305000DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 50-5,000 nucleotides, wherein some positions may be
absent"source/note="See specification as filed for detailed
description of substitutions and preferred embodiments"
30aaaaaaaaaa 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 500031100DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 31tttttttttt tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt 60tttttttttt tttttttttt tttttttttt tttttttttt
100325000DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 50-5,000 nucleotides, wherein some positions may be
absent" 32tttttttttt 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
5000335000DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(5000)/note="This sequence may
encompass 100-5,000 nucleotides, wherein some positions
may be absent" 33aaaaaaaaaa 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
500034400DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(400)/note="This sequence may
encompass 100-400 nucleotides, wherein some positions may be
absent"source/note="See specification as filed for detailed
description of substitutions and preferred embodiments"
34aaaaaaaaaa 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 400352000DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide"misc_feature(1)..(2000)/note="This sequence may
encompass 50-2,000 nucleotides, wherein some positions may be
absent" 35aaaaaaaaaa 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
200036230PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 36Glu 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 23037690DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 37gagagcaagt 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 6903840PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"misc_feature(1)..(40)/note="This sequence may encompass
1-10 'Gly Gly Gly Ser' repeating units, wherein some positions may
be absent"source/note="See specification as filed for detailed
description of substitutions and preferred embodiments" 38Gly 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 4039247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 39Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala
Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Glu Met
Ala Thr Ile Met Gly Gly 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 Gln Ser Ala Leu Thr Gln Pro Ala Ser 130 135
140Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly
Thr145 150 155 160Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp
Tyr Gln Gln His 165 170 175Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr
Asp Val Ser Asn Arg Pro 180 185 190Ser Gly Val Ser Asn Arg Phe Ser
Gly Ser Lys Ser Gly Asn Thr Ala 195 200 205Ser Leu Thr Ile Ser Gly
Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr 210 215 220Cys Ser Ser Tyr
Thr Ser Ser Ser Thr Leu Asp Val Val Phe Gly Gly225 230 235 240Gly
Thr Lys Leu Thr Val Leu 24540245PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Leu Ile Ser Gly Asp Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Phe Asp Ser
Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr 100 105 110Thr Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Ser
Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu 130 135
140Pro Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln145 150 155 160Ser Leu Val Tyr Thr Asp Gly Asn Thr Tyr Leu Asn
Trp Phe Gln Gln 165 170 175Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile
Tyr Lys Val Ser Asn Arg 180 185 190Asp Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Asp Thr Asp 195 200 205Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Val Gly Ile Tyr 210 215 220Tyr Cys Met Gln
Gly Thr His Trp Ser Phe Thr Phe Gly Gln Gly Thr225 230 235 240Arg
Leu Glu Ile Lys 24541247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 41Gln 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 Gly Ser
Ile Ser Ser Ser 20 25 30Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser
Thr Tyr Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Ser Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe65 70 75 80Ser Leu Lys Leu Lys Tyr Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Thr Pro Gly Thr
Tyr Tyr Asp Phe Leu Ser Gly Tyr Tyr Pro 100 105 110Phe Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met 130 135
140Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr145 150
155 160Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp
Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Ala Ala Ser 180 185 190Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Gln Gln Leu
Asn Ser Tyr Pro Tyr Thr Phe Gly Gln225 230 235 240Gly Thr Lys Leu
Glu Ile Lys 24542239PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 42Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn
Ile Asn Glu Asp Gly Ser Ala Lys Phe Tyr Val Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95Ala Arg Asp Leu Arg Ser Gly Arg Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 115 120 125Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu 130 135 140Ser Pro Gly Gly Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Ile145 150 155 160Ser Gly Ser Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175Arg Leu Leu Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp 180 185 190Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200
205Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
210 215 220Ser Ser Pro Pro Thr Phe Gly Leu Gly Thr Lys Leu Glu Ile
Lys225 230 23543245PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 43Glu Val Gln Leu Gln
Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Pro Val Arg Ser Gly 20 25 30Ser His Tyr
Trp Asn Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu Glu 35 40 45Trp Ile
Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60Leu
Glu Asn Arg Val Thr Ile Ser Ile Asp Thr Ser Asn Asn His Phe65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Phe
85 90 95Cys Ala Arg Gly Thr Ala Thr Phe Asp Trp Asn Phe Pro Phe Asp
Ser 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Asp
Ile Gln Met Thr Gln 130 135 140Ser Pro Ser Ser Leu Ser Ala Ser Ile
Gly Asp Arg Val Thr Ile Thr145 150 155 160Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu 180 185 190Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200
205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
210 215 220Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp Thr Phe Gly Gln
Gly Thr225 230 235 240Lys Leu Glu Ile Lys 24544249PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 44Gln Val Gln Leu Gln Glu Ser Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ser Gly Leu Val
Val Tyr Ala Ile Arg Val Gly Ser Gly Trp 100 105 110Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125Gly Gly
Ser Gly Gly Gly Asp Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135
140Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val145 150 155 160Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr Leu Asn Trp 165 170 175Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Met Tyr Ala Ala 180 185 190Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Pro Pro Trp Thr Phe225 230 235 240Gly
Gln Gly Thr Lys Val Asp Ile Lys 24545255PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Ser Ser
Ser Gly Ser Tyr Tyr Met Glu Asp Ser Tyr 100 105 110Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135
140Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly
Lys145 150 155 160Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser
Ile Ala Ser Asn 165 170 175Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly
Ser Ala Pro Thr Thr Val 180 185 190Ile Tyr Glu Asp Asn Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 195 200 205Gly Ser Ile Asp Ser Ser
Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 210 215 220Leu Lys Thr Glu
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser225 230 235 240Ser
Asn Gln Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 245 250
25546239PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 46Gln Val Asn Leu Arg
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Glu Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr
Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Leu Gly Ser Ser Trp Glu Trp Gly Gln Gly Thr
Thr 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 115 120 125Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser 130 135 140Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Asp145 150 155 160Ile Ser Asn Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 165 170 175Lys Leu Leu Ile
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser 180 185 190Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 195 200
205Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp
210 215 220Asn Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys225 230 23547246PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 47Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Pro Ala Asn Thr Phe Ser Asp His 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met 35 40 45Gly Tyr
Ile His Ala Ala Asn Gly Gly Thr His Tyr Ser Gln Lys Phe 50 55 60Gln
Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Asn Thr Val Tyr65 70 75
80Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asn Ser Asp Ala Phe Asp Ile Trp Gly Gln
Gly 100 105 110Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Val Met Thr 130 135 140Gln Ser Pro Ser Ser Val Ser Ala Ser
Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln 165 170 175Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 180 185 190Leu Gln
Ser Gly Val Pro Ser Arg Phe Asn Gly Ser Gly Ser Gly Thr 195 200
205Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly
Gly Gly225 230 235 240Thr Lys Val Glu Ile Lys 24548248PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 48Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Ser Val
Arg Ala Ile Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 130 135
140Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val145 150 155 160Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr Leu Asn Trp 165 170 175Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr Asp Ala 180 185 190Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220Ala Thr Tyr Tyr
Cys Gln Gln Ala Tyr Ser Thr Pro Phe Thr Phe Gly225 230 235 240Pro
Gly Thr Lys Val Glu Ile Lys 24549246PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 49Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg
Ser Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asn Ser Tyr 20 25 30Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ala Leu Ile Glu Tyr Asp Gly Ser Asn Lys
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Lys Ser Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met Asp Asn Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Asn Glu
Asp Leu Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr 130 135
140Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile145 150 155 160Thr Cys Gln Ala Ser Gln Phe Ile Lys Lys Asn Leu
Asn Trp Tyr Gln 165 170 175His Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Asp Ala Ser Ser 180 185 190Leu Gln Thr Gly Val Pro Ser Arg
Phe Ser Gly Asn Arg Ser Gly Thr 195 200 205Thr Phe Ser Phe Thr Ile
Ser Ser Leu Gln Pro Glu Asp Val Ala Thr 210 215 220Tyr Tyr Cys Gln
Gln His Asp Asn Leu Pro Leu Thr Phe Gly Gly Gly225 230 235 240Thr
Lys Val Glu Ile Lys 24550255PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 50Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Val Ser Ser Asn 20 25 30Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Ser Gly Gly Ala Thr Tyr
Tyr Gly Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Val Ser Arg Asp Asn
Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Arg Leu Thr Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Asp Arg Leu Tyr Cys
Gly Asn Asn Cys Tyr Leu Tyr Tyr Tyr Tyr 100 105 110Gly Met Asp Val
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135
140Gly Gly Ser Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser
Ala145 150 155 160Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile 165 170 175Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 180 185 190Leu Leu Ile Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg 195 200 205Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 210 215 220Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser225 230 235 240Thr
Pro Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 245 250
25551247PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 51Gln Val Gln Leu Val
Gln Ser Gly
Ala Glu Val Lys Lys Ser Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Pro Phe Thr Gly Tyr 20 25 30Tyr Ile Gln Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ser Asp Ser Tyr Gly Tyr Tyr Tyr Gly Met Asp Val 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 Asp Ile
Gln Met 130 135 140Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arg Val Thr145 150 155 160Phe Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Ala Leu Ala Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Pro
Pro Lys Leu Leu Ile Tyr Asp Ala Ser 180 185 190Ser Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr
Tyr Tyr Cys Gln Gln Phe Asn Asn Tyr Pro Leu Thr Phe Gly Gly225 230
235 240Gly Thr Lys Val Glu Ile Lys 24552741DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 52gaagtgcaac tccaacagtc aggcgcagaa gtcaagaagc
ccggatcgtc agtgaaagtg 60tcctgcaaag cctccggcgg aaccttcagc tcctacgcaa
tcagctgggt gcggcaggcg 120cccggacagg gactggagtg gatgggcggt
atcattccga tctttggcac cgccaattac 180gcccagaagt tccagggacg
cgtcacaatc accgccgacg aatcgacttc caccgcctac 240atggagctgt
cgtccttgag gagcgaagat accgccgtgt actactgcgc tcgggatctg
300gagatggcca ctatcatggg gggttactgg ggccagggga ccctggtcac
tgtgtcctcg 360ggaggagggg gatcaggcgg cggcggttcc gggggaggag
gaagccagtc cgcgctgact 420cagccagctt ccgtgtctgg ttcgccggga
cagtccatca ctattagctg taccggcacc 480agcagcgacg tgggcggcta
caactatgtg tcatggtacc agcagcaccc ggggaaggcg 540cctaagctga
tgatctacga cgtgtccaac cgccctagcg gagtgtccaa cagattctcc
600ggttcgaagt cagggaacac tgcctccctc acgattagcg ggctgcaagc
cgaggatgaa 660gccgactact actgctcctc ctatacctcc tcctcgaccc
tggacgtggt gttcggagga 720ggcaccaagc tcaccgtcct t
74153735DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 53gaagtgcaat
tggtggaaag cggaggagga gtggtgcaac ctggaggaag cctgagactg 60tcatgtgccg
cctcgggatt cactttcgat gactacgcaa tgcactgggt ccgccaggcc
120cccggaaagg gtctggaatg ggtgtccctc atctccggcg atgggggttc
cacttactat 180gcggattctg tgaagggccg cttcacaatc tcccgggaca
attccaagaa cactctgtac 240cttcaaatga actccctgag ggtggaggac
accgctgtgt actactgcgc gagagtgttt 300gactcgtact atatggacgt
ctggggaaag ggcaccaccg tgaccgtgtc cagcggtggc 360ggtggatcgg
ggggcggcgg ctccgggagc ggaggttccg agattgtgct gactcagtcg
420ccgttgtcac tgcctgtcac ccccgggcag ccggcctcca tttcatgccg
gtccagccag 480tccctggtct acaccgatgg gaacacttac ctcaactggt
tccagcagcg cccaggacag 540tccccgcgga ggctgatcta caaagtgtca
aaccgggact ccggcgtccc cgatcggttc 600tcgggaagcg gcagcgacac
cgacttcacg ctgaagattt cccgcgtgga agccgaggac 660gtgggcatct
actactgtat gcagggcacc cactggtcgt ttaccttcgg acaaggaact
720aggctcgaga tcaag 73554741DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 54caagtgcagc ttcaagaaag cggtccagga ctcgtcaagc
catcagaaac tctttccctc 60acttgtaccg tgtcgggagg cagcatctcc tcgagctcct
actactgggg ttggattaga 120cagcccccgg gaaaggggtt ggagtggatc
ggttccatct actactccgg gtcgacctac 180tacaaccctt ccctgaaatc
tcgggtgtcc atctccgtcg acacctccaa gaaccagttc 240agcctgaagc
tgaaatatgt gaccgcggcc gatactgccg tgtactattg cgccaccccg
300ggaacctact acgacttcct ctcggggtac tacccgtttt actggggaca
ggggactctc 360gtgaccgtgt cctcgggcgg cggaggttca ggcggtggcg
gatcgggggg aggaggctca 420gacattgtga tgacccagag cccgtccagc
ctgagcgcct ccgtgggcga tagggtcacg 480attacttgcc gggcgtccca
gggaatctca agctacctgg cctggtacca acagaagccc 540ggaaaggcac
ccaagttgct gatctatgcc gctagcactc tgcagtccgg ggtgccttcc
600cgcttctccg gctccggctc gggcaccgac ttcaccctga ccatttcctc
actgcaaccc 660gaggacttcg ccacttacta ctgccagcag ctgaactcct
acccttacac attcggacag 720ggaaccaagc tggaaatcaa g
74155717DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 55caagtgcaac
tcgtggaatc tggtggagga ctcgtgcaac ccggaggatc attgcgactc 60tcgtgtgcgg
catccggctt taccttttca tcctactgga tgtcctgggt cagacaggcc
120cccgggaagg gactggaatg ggtcgcgaac atcaacgagg acggctcggc
caagttctac 180gtggactccg tgaagggccg cttcacgatc tcacgggata
acgccaagaa ttccctgtat 240ctgcaaatga acagcctgag ggccgaggac
actgcggtgt acttctgcgc acgcgacctg 300aggtccggga gatactgggg
acagggcacc ctcgtgaccg tgtcgagcgg aggagggggg 360tcgggcggcg
gcggttccgg tggcggcggt agcgaaattg tgttgaccca gtcccctgga
420accctgagcc tgtcacctgg aggacgcgcc accctgtcct gccgggccag
ccagagcatc 480tcagggtcct tcctggcttg gtaccagcag aagccgggac
aggctccgag acttctgatc 540tacggcgcct cctcgcgggc gaccggaatc
ccggatcggt tctccggctc gggaagcgga 600actgacttca ctcttaccat
ttcccgcctg gagccggaag atttcgccgt gtactactgc 660cagcagtacg
ggtcatcccc tccaaccttc ggcctgggaa ctaagctgga aatcaaa
71756735DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 56gaagtgcaac
tccaacaatc cggtccagga ctcgtcagac cctccgaaac tctctcgctt 60acatgcactg
tgtccggcgg ccctgtgcgg tccggctctc attactggaa ctggattcgc
120cagcccccgg gacgcggact ggagtggatc ggctacatct attactcggg
gtcgactaac 180tacaacccga gcctggaaaa tagagtgacc atctcaatcg
acacgtccaa caaccacttc 240tcgctgaagt tgtcctccgt gactgccgcc
gatactgccc tgtacttctg tgctcgcgga 300accgccacct tcgactggaa
cttccctttt gactcatggg gccaggggac ccttgtgacc 360gtgtccagcg
gaggaggagg ctccggtggt ggcgggagcg gtagcggcgg aagcgacatc
420cagatgaccc agtcaccgtc ctcgctgtcc gcatccattg gggatcgggt
cactattact 480tgccgggcgt cccagtccat ctcgtcctac ctgaactggt
atcagcagaa gccagggaaa 540gcccccaagc tgctgatcta cgcggccagc
agcctgcagt caggagtgcc ttcaaggttt 600agcggcagcg gatcgggaac
cgacttcacc ctgaccattt cctccctcca acccgaggat 660ttcgccacct
actactgcca gcagtcctac tccaccccgt ggaccttcgg acagggaacc
720aagctggaga tcaag 73557747DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 57caagtgcaac ttcaagaatc aggcgcagga cttctcaagc
catccgaaac actctccctc 60acttgcgcgg tgtacggggg aagcttctcg ggatactact
ggtcctggat taggcagcct 120cccggcaaag gcctggaatg ggtcggggag
atcaaccact ccggttcaac caactacaac 180ccgtcgctga agtcccgcgt
gaccatttcc gtggacacct ctaagaatca gttcagcctg 240aagctctcgt
ccgtgaccgc ggcggacacc gccgtctact actgcgctcg gggatcagga
300ctggtggtgt acgccatccg cgtgggctcg ggctggttcg attactgggg
ccagggaacc 360ctggtcactg tgtcgtccgg cggaggaggt tcggggggcg
gagacagcgg tggagggggt 420agcgacatcc agatgaccca gtccccgtcc
tcgctgtccg cctccgtggg agatagagtg 480accatcacct gtcgggcatc
ccagagcatt tccagctacc tgaactggta tcagcagaag 540cccggaaagg
cccctaagct gttgatgtac gccgccagca gcttgcagtc gggcgtgccg
600agccggtttt ccggttccgg ctccgggact gacttcaccc tgactatctc
atccctgcaa 660cccgaggact tcgccactta ttactgccag cagtcctact
caacccctcc ctggacgttc 720ggacagggca ccaaggtcga tatcaag
74758765DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 58gaagtgcaat
tggtggaatc tggaggagga cttgtgaaac ctggtggaag cctgagactt 60tcctgtgcgg
cctcgggatt cactttctcc tcctactcca tgaactgggt cagacaggcc
120cctgggaagg gactggaatg ggtgtcatcc atctcctcct catcgtcgta
catctactac 180gccgatagcg tgaaggggcg gttcaccatt tcccgggaca
acgctaagaa cagcctctat 240ctgcaaatga attccctccg cgccgaggac
actgccgtgt actactgcgc gagggacccc 300tcatcaagcg gcagctacta
catggaggac tcgtattact acggaatgga cgtctggggc 360cagggaacca
ctgtgacggt gtcctccggt ggagggggct ccgggggcgg gggatctggc
420ggaggaggct ccaacttcat gctgacccag ccgcactccg tgtccgaaag
ccccggaaag 480accgtgacaa tttcctgcac cgggtcctcc ggctcgatcg
catcaaacta cgtgcagtgg 540taccagcagc gcccgggcag cgcccccacc
actgtcatct acgaggataa ccagcggccg 600tcgggtgtcc cagaccggtt
ttccggttcg atcgatagca gcagcaacag cgcctccctg 660accatttccg
gcctcaagac cgaggatgag gctgactact actgccagtc gtatgactcc
720tcgaaccaag tggtgttcgg tggcggcacc aagctgactg tgctg
76559717DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 59caagtgaacc
tgagagaaag cggcggagga cttgtgcaac ctggaggaag cctgagactg 60tcatgtgccg
cgtccggctt caccttctcg tcctacgaga tgaactgggt ccgccaggca
120ccgggcaaag gactggaatg ggtgtcctac atttcctcgt ccgggtccac
catctattac 180gccgactccg tgaagggacg gttcaccatc tcccgggaca
acgccaagaa ctccctctac 240ctccaaatga actcactgag ggcagaggac
actgcggtct actactgcgc ccgcgaagct 300ttgggtagct cctgggagtg
gggccaggga accactgtga ccgtgtcctc gggtggaggg 360ggctccggtg
gcgggggttc agggggtggc ggaagcgata tccagatgac tcagtcacca
420agctccctga gcgcctcagt gggagatcgg gtcacaatca cgtgccaggc
gtcccaggac 480atttctaact acctcaattg gtaccagcag aagccgggga
aggcccccaa gcttctgatc 540tacgatgcct ccaacctgga aaccggcgtg
ccctcccgct tctcgggatc gggcagcggc 600actgacttca cctttaccat
ctcgtccctg caacctgagg acatcgccac ctattactgc 660cagcagtacg
ataacctccc gctgactttc ggaggcggaa ctaagctgga gattaag
71760738DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 60caagtgcaac
tcgtccagtc cggtgcagaa gtcaaggaac ccggagcctc cgtgaaagtg 60tcctgcaaag
ctcctgccaa cactttctcg gaccacgtga tgcactgggt gcgccaggcg
120ccgggccagc gcttcgaatg gatgggatac attcatgccg ccaatggcgg
tacccactac 180tcccaaaagt tccaggatag agtcaccatc acccgggaca
ccagcgccaa caccgtgtat 240atggatctgt ccagcctgag gtccgaggat
accgccgtgt actactgcgc ccggggcgga 300tacaactcag acgcgttcga
catttgggga cagggtacta tggtcaccgt gtcatccggg 360ggcggtggca
gcgggggcgg aggctctggc ggaggcggat cagggggagg agggtccgac
420atcgtgatga cccagtcccc gtcatcggtg tccgcgtccg tgggagacag
agtgaccatc 480acgtgtcgcg ccagccagga catctcctcg tggttggcat
ggtaccagca gaagcctgga 540aaggccccga agctgctcat ctacgccgcc
tcctcccttc aatcgggagt gccctcgcgg 600ttcaacggaa gcggaagcgg
gacagatttt accctgacta ttagctcgct gcagcccgag 660gacttcgcta
cttactactg ccaacagagc tactccaccc cactgacttt cggcgggggt
720accaaggtcg agatcaag 73861744DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 61caagtgcaac ttgttcaatc cggtggaggt cttgtgcagc
ccggaggatc actcagactg 60tcgtgcgccg cctctgggtt cactttctcc tcatactcga
tgaactgggt gcgccaggcg 120ccgggaaagg gcctggaatg ggtgtcatac
atctcctcct catcctccac catctactac 180gccgattccg tgaagggccg
cttcactatt tcccgggaca acgcgaaaaa ctcgctctat 240ctgcaaatga
actccctgcg cgccgaggac accgccgtgt actactgcgc ccgggacctg
300agcgtgcggg ctattgatgc gttcgacatc tggggacagg gcaccatggt
cacagtgtcc 360agcggaggcg gcggcagcgg tggaggagga tcagggggag
gaggttcggg gggcggtggc 420tccgatatcg tgctgaccca gagcccgtcg
agcctctccg cctccgtcgg cgacagagtg 480accatcacgt gtcaggcatc
ccaggacatt agcaactacc tgaattggta ccagcagaag 540cctggaaagg
cacccaagtt gctgatctac gacgcctcca acctggaaac cggagtgcca
600tccaggttct cgggcagcgg ctcgggaacc gacttcactt ttactatctc
ctccctgcaa 660cccgaggatt tcgcgaccta ctactgccag caggcctaca
gcaccccttt caccttcggg 720ccgggaacta aggtcgaaat caag
74462738DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 62gaagtgcaat
tggtgcaatc aggaggagga gtggtcagat ctggaagaag cctgagactg 60tcatgcgcgg
cttcgggctt taccttcaac tcctacggcc tccactgggt gcgccaggcc
120cccggaaaag gcctcgaatg ggtcgcactg attgagtacg acgggtccaa
caagtactac 180ggagatagcg tgaagggccg cttcaccatc tcacgggaca
agtccaagtc caccctgtat 240ctgcaaatgg acaacctgag ggccgaggat
actgccgtgt actactgcgc ccgcgaagga 300aacgaagatc tggccttcga
tatttggggc cagggtactc ttgtgaccgt gtcgagcgga 360ggcggaggct
ccggtggagg aggatcgggg ggtggtggtt ccggcggcgg ggggagcgaa
420atcgtgctga cccagtcgcc ttcctccctc tccgcttccg tgggggaccg
ggtcactatt 480acgtgtcagg cgtcccaatt catcaagaag aatctgaact
ggtaccagca caagccggga 540aaggccccca aactgctcat ctacgacgcc
agctcgctgc agactggcgt gccttcccgg 600ttttccggga accggtcggg
aaccaccttc tcattcacca tcagcagcct ccagccggag 660gacgtggcga
cctactactg ccagcagcat gacaaccttc cactgacttt cggcgggggc
720accaaggtcg agattaag 73863765DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 63caagtgcaac tcgtggaatc aggcggagga ctcgtgcaac
ccggaggttc ccttagactg 60tcatgtgccg cttccgggtt caatgtgtcc agcaactaca
tgacctgggt cagacaggcg 120ccgggaaagg gacttgaatg ggtgtccgtg
atctactccg gtggagcaac atactacgga 180gactccgtga aaggccgctt
taccgtgtcc cgcgataact cgaagaacac cgtgtacttg 240cagatgaaca
ggctgactgc cgaggacacc gccgtgtatt attgcgcccg ggacaggctg
300tactgtggaa acaactgcta cctgtactac tactacggga tggacgtgtg
gggacagggc 360actctcgtca ctgtgtcatc cggggggggc ggtagcggtg
gcggagggtc cggcggagga 420ggctcagggg gaggcggaag cgatatccag
gtcacccagt ctccctcctc gctgtccgcc 480tccgtgggcg accgcgtcac
cattacttgc cgggcgtcgc agtcgatcag ctcctacctg 540aactggtacc
agcagaagcc tggaaaggcc ccgaagctgc tgatctacgc ggcctcgtcc
600ctgcaaagcg gcgtcccgtc gcggttcagc ggttccggtt cgggaaccga
cttcaccctg 660actatttcct ccctgcaacc cgaggatttc gccacttact
actgccagca gtcctactcc 720accccacctc tgaccttcgg ccaaggaacc
aaggtcgaaa tcaag 76564741DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 64caagtgcaac tcgtccagtc cggtgcagaa gtgaaaaaga
gcggagcctc agtgaaagtg 60tcctgcaagg cctccggtta ccccttcact ggatactaca
ttcagtgggt ccgccaagcc 120ccgggacagg gtctggagtg gatggggtgg
attgacccta actcgggaaa tacgggatac 180gcgcagaagt tccagggccg
cgtgaccatg accaggaaca cctcgatcag caccgcctac 240atggaactgt
cctccctgcg gtcggaggat actgccgtgt actactgcgc ctccgattcc
300tatgggtact actacggaat ggacgtctgg ggacagggca ccctcgtgac
cgtgtcctcg 360ggaggcggag ggagcggcgg gggtggatcg ggaggaggcg
gctccggcgg cggcggtagc 420gacatccaga tgacccagtc accatcaagc
cttagcgcct ccgtgggcga cagagtgaca 480ttcacttgtc gggcgtccca
gggaatctcc tccgctctgg cttggtatca gcagaagcct 540gggaagcctc
cgaagctgtt gatctacgac gcgagcagcc tggaatcagg ggtgccctcc
600cggttttccg ggtccggttc tggcaccgat ttcaccctga ccatttcgtc
cctccaaccc 660gaggacttcg ccacttacta ctgccagcag ttcaacaact
acccgctgac cttcggagga 720ggcactaagg tcgagatcaa g
74165120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 65Glu Val Gln Leu Gln
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Glu Met Ala Thr Ile Met Gly Gly Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12066118PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 66Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Leu
Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Phe Asp Ser Tyr Tyr Met Asp Val Trp Gly Lys Gly
Thr 100 105 110Thr Val Thr Val Ser Ser 11567125PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 67Gln 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 Gly Ser
Ile Ser Ser Ser 20 25 30Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser
Thr Tyr Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Ser Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe65 70 75 80Ser Leu Lys Leu Lys Tyr Val
Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95Cys Ala Thr Pro Gly Thr Tyr Tyr Asp Phe Leu Ser Gly
Tyr Tyr Pro 100 105 110Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 12568116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 68Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Asn Glu Asp Gly Ser Ala Lys
Phe Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Asp Leu Arg Ser
Gly Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11569123PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 69Glu Val Gln Leu Gln
Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Pro Val Arg Ser Gly 20 25 30Ser His Tyr
Trp Asn Trp Ile Arg Gln Pro Pro Gly Arg Gly Leu Glu 35 40 45Trp Ile
Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60Leu
Glu Asn Arg Val Thr Ile Ser Ile Asp Thr Ser Asn Asn His Phe65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Phe
85 90 95Cys Ala Arg Gly Thr Ala Thr Phe Asp Trp Asn Phe Pro Phe Asp
Ser 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12070126PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 70Gln Val Gln Leu Gln
Glu Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu
Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Ser Gly Leu Val Val Tyr Ala Ile Arg Val Gly Ser Gly
Trp 100 105 110Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 12571129PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 71Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Pro Ser Ser Ser Gly Ser Tyr Tyr Met Glu Asp Ser
Tyr 100 105 110Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 125Ser72117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 72Gln Val Asn Leu Arg Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ala Leu Gly
Ser Ser Trp Glu Trp Gly Gln Gly Thr Thr 100 105 110Val Thr Val Ser
Ser 11573119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 73Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Pro Ala Asn Thr Phe Ser Asp His 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met 35 40 45Gly Tyr
Ile His Ala Ala Asn Gly Gly Thr His Tyr Ser Gln Lys Phe 50 55 60Gln
Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Asn Thr Val Tyr65 70 75
80Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asn Ser Asp Ala Phe Asp Ile Trp Gly Gln
Gly 100 105 110Thr Met Val Thr Val Ser Ser 11574121PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 74Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Ser Val
Arg Ala Ile Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met
Val Thr Val Ser Ser 115 12075119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 75Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg
Ser Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asn Ser Tyr 20 25 30Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ala Leu Ile Glu Tyr Asp Gly Ser Asn Lys
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Lys Ser Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met Asp Asn Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Asn Glu
Asp Leu Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Leu Val Thr
Val Ser Ser 11576127PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 76Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Val Ser Ser Asn 20 25 30Tyr Met Thr
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val
Ile Tyr Ser Gly Gly Ala Thr Tyr Tyr Gly Asp Ser Val Lys 50 55 60Gly
Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75
80Gln Met Asn Arg Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Asp Arg Leu Tyr Cys Gly Asn Asn Cys Tyr Leu Tyr Tyr Tyr
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 12577120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 77Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Ser Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro
Phe Thr Gly Tyr 20 25 30Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Asn Ser Gly Asn Thr
Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asn
Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Asp Ser Tyr Gly
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Leu Val
Thr Val Ser Ser 115 12078112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 78Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly
Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95Ser Thr Leu Asp Val Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
11079112PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 79Glu Ile Val Leu Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Thr 20 25 30Asp Gly Asn
Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg
Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Asp Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Gly
85 90 95Thr His Trp Ser Phe Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105 11080107PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 80Asp Ile Val Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10581108PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 81Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Gly Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Gly Ser 20 25 30Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Pro Thr Phe Gly Leu Gly Thr Lys Leu Glu Ile Lys 100
10582107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 82Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10583108PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 83Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro
85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100
10584111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 84Asn Phe Met Leu Thr
Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile
Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30Tyr Val Gln
Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45Ile Tyr
Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly
Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly65 70 75
80Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
85 90 95Ser Asn Gln Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11085107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 85Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10586107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 86Asp Ile Val Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Asn Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 10587107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 87Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Tyr Ser Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr
Lys Val Glu Ile Lys 100 10588107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 88Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Phe
Ile Lys Lys Asn 20 25 30Leu Asn Trp Tyr Gln His Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Gln Thr Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Asn Arg Ser Gly Thr Thr Phe Ser Phe
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Gln His Asp Asn Leu Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 10589108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 89Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95Leu Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10590107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 90Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro
Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Phe Asn Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 10591491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 91Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser
Gly Ala Glu Val 20 25 30Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly 35 40 45Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln 50 55 60Gly Leu Glu Trp Met Gly Gly Ile Ile
Pro Ile Phe Gly Thr Ala Asn65 70 75 80Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser 85 90 95Thr Ser Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110Ala Val Tyr Tyr
Cys Ala Arg Asp Leu Glu Met Ala Thr Ile Met Gly 115 120 125Gly 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 Gln Ser Ala
Leu145 150 155 160Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
Ser Ile Thr Ile 165 170 175Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr Asn Tyr Val Ser 180 185 190Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu Met Ile Tyr Asp 195 200 205Val Ser Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe Ser Gly Ser Lys 210 215 220Ser Gly Asn Thr
Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp225 230 235 240Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Asp 245 250
255Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 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 49092489PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 92Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val 20 25 30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe 35 40 45Thr Phe Asp Asp Tyr Ala Met His Trp Val
Arg Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ser Leu Ile Ser
Gly Asp Gly Gly Ser Thr Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Val Glu Asp Thr 100 105 110Ala Val Tyr Tyr
Cys Ala Arg Val Phe Asp Ser Tyr Tyr Met Asp Val 115 120 125Trp Gly
Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135
140Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Glu Ile Val Leu Thr
Gln145 150 155 160Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Gln Pro
Ala Ser Ile Ser 165 170 175Cys Arg Ser Ser Gln Ser Leu Val Tyr Thr
Asp Gly Asn Thr Tyr Leu 180 185 190Asn Trp Phe Gln Gln Arg Pro Gly
Gln Ser Pro Arg Arg Leu Ile Tyr 195 200 205Lys Val Ser Asn Arg Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 210 215 220Gly Ser Asp Thr
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu225 230 235 240Asp
Val Gly Ile Tyr Tyr Cys Met Gln Gly Thr His Trp Ser Phe Thr 245 250
255Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro
260 265 270Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
Ser Leu 275 280 285Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
Val His Thr Arg 290 295 300Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
Trp Ala Pro Leu Ala Gly305 310 315 320Thr Cys Gly Val Leu Leu Leu
Ser Leu Val Ile Thr Leu Tyr Cys Lys 325 330 335Arg Gly Arg Lys Lys
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 340 345 350Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 355 360 365Glu
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 370 375
380Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
Glu385 390 395 400Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg 405 410 415Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
Arg Arg Lys Asn Pro Gln 420 425 430Glu Gly Leu Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445Ser Glu Ile Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460Gly Leu Tyr Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala465 470 475 480Leu
His Met Gln Ala Leu Pro Pro Arg 48593491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 93Met 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 Gly 35 40 45Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly
Trp Ile Arg Gln Pro Pro 50 55 60Gly Lys Gly Leu Glu Trp Ile Gly Ser
Ile Tyr Tyr Ser Gly Ser Thr65 70 75 80Tyr Tyr Asn Pro Ser Leu Lys
Ser Arg Val Ser Ile Ser Val Asp Thr 85 90 95Ser Lys Asn Gln Phe Ser
Leu Lys Leu Lys Tyr Val Thr Ala Ala Asp 100 105 110Thr Ala Val Tyr
Tyr Cys Ala Thr Pro Gly Thr Tyr Tyr Asp Phe Leu 115 120 125Ser Gly
Tyr Tyr Pro Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 130 135
140Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly145 150 155 160Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val 165 170 175Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser 180 185 190Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu 195 200 205Ile Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 210 215 220Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln225 230 235 240Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr 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 49094483PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 94Met 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 Val Glu Ser
Gly Gly Gly Leu 20 25 30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe 35 40 45Thr Phe Ser Ser Tyr Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ala Asn Ile Asn
Glu Asp Gly Ser Ala Lys Phe65 70 75 80Tyr Val Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95Lys Asn Ser Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110Ala Val Tyr Phe
Cys Ala Arg Asp Leu Arg Ser Gly Arg Tyr Trp Gly 115 120 125Gln Gly
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135
140Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser
Pro145 150 155 160Gly Thr Leu Ser Leu Ser Pro Gly Gly Arg Ala Thr
Leu Ser Cys Arg 165 170 175Ala Ser Gln Ser Ile Ser Gly Ser Phe Leu
Ala Trp Tyr Gln Gln Lys 180 185 190Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr Gly Ala Ser Ser Arg Ala 195 200 205Thr Gly Ile Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 210 215 220Thr Leu Thr Ile
Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr225 230 235 240Cys
Gln Gln Tyr Gly Ser Ser Pro Pro Thr Phe Gly Leu Gly Thr Lys 245 250
255Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
260 265 270Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
Cys Arg 275 280 285Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
Asp Phe Ala Cys 290 295 300Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly Val Leu Leu305 310 315 320Leu Ser Leu Val Ile Thr Leu
Tyr Cys Lys Arg Gly Arg Lys Lys Leu 325 330 335Leu Tyr Ile Phe Lys
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 340 345 350Glu Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 355 360 365Cys
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 370 375
380Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
Arg385 390 395
400Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
405 410 415Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu 420 425 430Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys 435 440 445Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly Leu Tyr Gln Gly Leu 450 455 460Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala Leu His Met Gln Ala Leu465 470 475 480Pro Pro
Arg95489PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 95Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30Val Arg Pro
Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 35 40 45Pro Val
Arg Ser Gly Ser His Tyr Trp Asn Trp Ile Arg Gln Pro Pro 50 55 60Gly
Arg Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr65 70 75
80Asn Tyr Asn Pro Ser Leu Glu Asn Arg Val Thr Ile Ser Ile Asp Thr
85 90 95Ser Asn Asn His Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp 100 105 110Thr Ala Leu Tyr Phe Cys Ala Arg Gly Thr Ala Thr Phe
Asp Trp Asn 115 120 125Phe Pro Phe Asp Ser Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Ser Gly Gly Ser Asp145 150 155 160Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Ile Gly Asp 165 170 175Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu 180 185 190Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 195 200
205Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
210 215 220Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu225 230 235 240Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Pro Trp Thr 245 250 255Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Thr Thr Thr Pro Ala Pro 260 265 270Arg Pro Pro Thr Pro Ala Pro
Thr Ile Ala Ser Gln Pro Leu Ser Leu 275 280 285Arg Pro Glu Ala Cys
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 290 295 300Gly Leu Asp
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly305 310 315
320Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
325 330 335Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met Arg 340 345 350Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro 355 360 365Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser 370 375 380Ala Asp Ala Pro Ala Tyr Lys Gln
Gly Gln Asn Gln Leu Tyr Asn Glu385 390 395 400Leu Asn Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415Gly Arg Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430Glu
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440
445Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala465 470 475 480Leu His Met Gln Ala Leu Pro Pro Arg
48596493PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 96Met 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 Ala Gly Leu 20 25 30Leu Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly 35 40 45Ser Phe
Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr65 70 75
80Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
85 90 95Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Arg Gly Ser Gly Leu Val Val Tyr
Ala Ile Arg 115 120 125Val Gly Ser Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 130 135 140Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Asp Ser Gly Gly Gly145 150 155 160Gly Ser Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 165 170 175Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser 180 185 190Ser Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200
205Leu Met Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
210 215 220Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu225 230 235 240Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ser Tyr Ser Thr 245 250 255Pro Pro Trp Thr Phe Gly Gln Gly Thr
Lys Val Asp Ile Lys Thr Thr 260 265 270Thr Pro Ala Pro Arg Pro Pro
Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285Pro Leu Ser Leu Arg
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300Val His Thr
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala305 310 315
320Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln 340 345 350Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser 355 360 365Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu Leu Arg Val Lys 370 375 380Phe Ser Arg Ser Ala Asp Ala Pro
Ala Tyr Lys Gln Gly Gln Asn Gln385 390 395 400Leu Tyr Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415Asp Lys Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430Lys
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440
445Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp465 470 475 480Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
Pro Arg 485 49097499PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 97Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30Val Lys Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe
Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr65 70 75
80Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Pro Ser Ser Ser Gly
Ser Tyr Tyr 115 120 125Met Glu Asp Ser Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr 130 135 140Thr Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser145 150 155 160Gly Gly Gly Gly Ser Asn
Phe Met Leu Thr Gln Pro His Ser Val Ser 165 170 175Glu Ser Pro Gly
Lys Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly 180 185 190Ser Ile
Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser 195 200
205Ala Pro Thr Thr Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val
210 215 220Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser
Ala Ser225 230 235 240Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu
Ala Asp Tyr Tyr Cys 245 250 255Gln Ser Tyr Asp Ser Ser Asn Gln Val
Val Phe Gly Gly Gly Thr Lys 260 265 270Leu Thr Val Leu Thr Thr Thr
Pro Ala Pro Arg Pro Pro Thr Pro Ala 275 280 285Pro Thr Ile Ala Ser
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 290 295 300Pro Ala Ala
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys305 310 315
320Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
325 330 335Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
Lys Leu 340 345 350Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
Gln Thr Thr Gln 355 360 365Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu Gly Gly 370 375 380Cys Glu Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr385 390 395 400Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 405 410 415Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 420 425 430Gly
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 435 440
445Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
450 455 460Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
Gly Leu465 470 475 480Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His Met Gln Ala Leu 485 490 495Pro Pro Arg98483PRTArtificial
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 Gln Val Asn Leu Arg Glu Ser
Gly Gly Gly Leu 20 25 30Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe 35 40 45Thr Phe Ser Ser Tyr Glu Met Asn Trp Val
Arg Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ser Tyr Ile Ser
Ser Ser Gly Ser Thr Ile Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95Lys Asn Ser Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110Ala Val Tyr Tyr
Cys Ala Arg Glu Ala Leu Gly Ser Ser Trp Glu Trp 115 120 125Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135
140Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser145 150 155 160Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys 165 170 175Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu
Asn Trp Tyr Gln Gln Lys 180 185 190Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr Asp Ala Ser Asn Leu Glu 195 200 205Thr Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 210 215 220Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr225 230 235 240Cys
Gln Gln Tyr Asp Asn Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys 245 250
255Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
260 265 270Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
Cys Arg 275 280 285Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
Asp Phe Ala Cys 290 295 300Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly Val Leu Leu305 310 315 320Leu Ser Leu Val Ile Thr Leu
Tyr Cys Lys Arg Gly Arg Lys Lys Leu 325 330 335Leu Tyr Ile Phe Lys
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 340 345 350Glu Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 355 360 365Cys
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 370 375
380Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
Arg385 390 395 400Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
Asp Pro Glu Met 405 410 415Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
Glu Gly Leu Tyr Asn Glu 420 425 430Leu Gln Lys Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys 435 440 445Gly Glu Arg Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 450 455 460Ser Thr Ala Thr
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu465 470 475 480Pro
Pro Arg99490PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 99Met 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 Val Gln Ser Gly Ala Glu Val 20 25 30Lys Glu Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Pro Ala Asn 35 40 45Thr Phe
Ser Asp His Val Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60Arg
Phe Glu Trp Met Gly Tyr Ile His Ala Ala Asn Gly Gly Thr His65 70 75
80Tyr Ser Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Arg Asp Thr Ser
85 90 95Ala Asn Thr Val Tyr Met Asp Leu Ser Ser Leu Arg Ser Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Asn Ser Asp
Ala Phe Asp 115 120 125Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser145 150 155 160Asp Ile Val Met Thr Gln
Ser Pro Ser Ser Val Ser Ala Ser Val Gly 165 170 175Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp 180 185 190Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200
205Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Asn Gly
210 215 220Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro225 230 235 240Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Ser Thr Pro Leu 245 250 255Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Thr Thr Thr Pro Ala 260 265 270Pro Arg Pro Pro Thr Pro Ala
Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285Leu Arg Pro Glu Ala
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300Arg Gly Leu
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala305 310 315
320Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met 340 345 350Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg Phe 355 360 365Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg Val Lys Phe Ser Arg 370 375 380Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr Asn385 390 395 400Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425
430Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 450 455 460Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp465 470 475 480Ala Leu His Met Gln Ala Leu Pro Pro
Arg 485 490100492PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 100Met 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 Val Gln Ser Gly Gly Gly Leu 20 25 30Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe
Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr65 70 75
80Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Leu Ser Val Arg Ala
Ile Asp Ala 115 120 125Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Asp Ile Val Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 165 170 175Val Gly Asp Arg
Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser 180 185 190Asn Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 195 200
205Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe
210 215 220Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu225 230 235 240Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ala Tyr Ser Thr 245 250 255Pro Phe Thr Phe Gly Pro Gly Thr Lys
Val Glu Ile Lys Thr Thr Thr 260 265 270Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300His Thr Arg
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro305 310 315
320Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
Gln Pro 340 345 350Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys 355 360 365Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu Arg Val Lys Phe 370 375 380Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Lys Gln Gly Gln Asn Gln Leu385 390 395 400Tyr Asn Glu Leu Asn
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415Lys Arg Arg
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440
445Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr465 470 475 480Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
Arg 485 490101490PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 101Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val 20 25 30Val Arg Ser
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe
Asn Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Ala Leu Ile Glu Tyr Asp Gly Ser Asn Lys Tyr65 70 75
80Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser
85 90 95Lys Ser Thr Leu Tyr Leu Gln Met Asp Asn Leu Arg Ala Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Glu Gly Asn Glu Asp Leu
Ala Phe Asp 115 120 125Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser145 150 155 160Glu Ile Val Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Phe Ile Lys Lys Asn 180 185 190Leu Asn
Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200
205Tyr Asp Ala Ser Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly
210 215 220Asn Arg Ser Gly Thr Thr Phe Ser Phe Thr Ile Ser Ser Leu
Gln Pro225 230 235 240Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His
Asp Asn Leu Pro Leu 245 250 255Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Thr Thr Thr Pro Ala 260 265 270Pro Arg Pro Pro Thr Pro Ala
Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285Leu Arg Pro Glu Ala
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300Arg Gly Leu
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala305 310 315
320Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met 340 345 350Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe 355 360 365Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg 370 375 380Ser Ala Asp Ala Pro Ala Tyr Lys
Gln Gly Gln Asn Gln Leu Tyr Asn385 390 395 400Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425 430Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440
445Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp465 470 475 480Ala Leu His Met Gln Ala Leu Pro Pro Arg 485
490102499PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 102Met 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 Val Glu Ser Gly Gly Gly Leu 20 25 30Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Asn Val
Ser Ser Asn Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ala Thr Tyr Tyr65 70 75
80Gly Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys
85 90 95Asn Thr Val Tyr Leu Gln Met Asn Arg Leu Thr Ala Glu Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Arg Asp Arg Leu Tyr Cys Gly Asn
Asn Cys Tyr 115 120 125Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Leu Val 130 135 140Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly145 150 155 160Gly Gly Ser Gly Gly Gly
Gly Ser Asp Ile Gln Val Thr Gln Ser Pro 165 170 175Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 180 185 190Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 195 200
205Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
210 215 220Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr225 230 235 240Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys 245 250 255Gln Gln Ser Tyr Ser Thr Pro Pro Leu
Thr Phe Gly Gln Gly Thr Lys 260 265 270Val Glu Ile Lys Thr Thr Thr
Pro Ala Pro Arg Pro Pro Thr Pro Ala 275 280 285Pro Thr Ile Ala Ser
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 290 295 300Pro Ala Ala
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys305 310 315
320Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
325 330 335Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
Lys Leu 340 345 350Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
Gln Thr Thr Gln 355 360 365Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu Gly Gly 370 375 380Cys Glu Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr385 390 395 400Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 405 410 415Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 420 425 430Gly
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 435 440
445Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
450 455 460Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
Gly Leu465 470 475 480Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His Met Gln Ala Leu 485 490 495Pro Pro Arg103491PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 103Met 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 Val Gln Ser
Gly Ala Glu Val 20 25 30Lys Lys Ser Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr 35 40 45Pro Phe Thr Gly Tyr Tyr Ile Gln Trp Val
Arg Gln Ala Pro Gly Gln 50 55 60Gly Leu Glu Trp Met Gly Trp Ile Asp
Pro Asn Ser Gly Asn Thr Gly65 70 75 80Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Met Thr Arg Asn Thr Ser 85 90 95Ile Ser Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110Ala Val Tyr Tyr
Cys Ala Ser Asp Ser Tyr Gly Tyr Tyr Tyr Gly Met 115 120 125Asp Val
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 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val 165 170 175Gly Asp Arg Val Thr Phe Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser 180 185 190Ala Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Pro Pro Lys Leu Leu 195 200 205Ile Tyr Asp Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 210 215 220Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln225 230 235 240Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Asn Tyr Pro 245 250
255Leu Thr Phe Gly Gly Gly Thr Lys Val 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 4901041473DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 104atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60cccgaagtgc aactccaaca gtcaggcgca gaagtcaaga
agcccggatc gtcagtgaaa 120gtgtcctgca aagcctccgg cggaaccttc
agctcctacg caatcagctg ggtgcggcag 180gcgcccggac agggactgga
gtggatgggc ggtatcattc cgatctttgg caccgccaat 240tacgcccaga
agttccaggg acgcgtcaca atcaccgccg acgaatcgac ttccaccgcc
300tacatggagc tgtcgtcctt gaggagcgaa gataccgccg tgtactactg
cgctcgggat 360ctggagatgg ccactatcat ggggggttac tggggccagg
ggaccctggt cactgtgtcc 420tcgggaggag ggggatcagg cggcggcggt
tccgggggag gaggaagcca gtccgcgctg 480actcagccag cttccgtgtc
tggttcgccg ggacagtcca tcactattag ctgtaccggc 540accagcagcg
acgtgggcgg ctacaactat gtgtcatggt accagcagca cccggggaag
600gcgcctaagc tgatgatcta cgacgtgtcc aaccgcccta gcggagtgtc
caacagattc 660tccggttcga agtcagggaa cactgcctcc ctcacgatta
gcgggctgca agccgaggat 720gaagccgact actactgctc ctcctatacc
tcctcctcga ccctggacgt ggtgttcgga 780ggaggcacca agctcaccgt
ccttaccact 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 14731051467DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 105atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60cccgaagtgc aattggtgga aagcggagga ggagtggtgc
aacctggagg aagcctgaga 120ctgtcatgtg ccgcctcggg attcactttc
gatgactacg caatgcactg ggtccgccag 180gcccccggaa
agggtctgga atgggtgtcc ctcatctccg gcgatggggg ttccacttac
240tatgcggatt ctgtgaaggg ccgcttcaca atctcccggg acaattccaa
gaacactctg 300taccttcaaa tgaactccct gagggtggag gacaccgctg
tgtactactg cgcgagagtg 360tttgactcgt actatatgga cgtctgggga
aagggcacca ccgtgaccgt gtccagcggt 420ggcggtggat cggggggcgg
cggctccggg agcggaggtt ccgagattgt gctgactcag 480tcgccgttgt
cactgcctgt cacccccggg cagccggcct ccatttcatg ccggtccagc
540cagtccctgg tctacaccga tgggaacact tacctcaact ggttccagca
gcgcccagga 600cagtccccgc ggaggctgat ctacaaagtg tcaaaccggg
actccggcgt ccccgatcgg 660ttctcgggaa gcggcagcga caccgacttc
acgctgaaga tttcccgcgt ggaagccgag 720gacgtgggca tctactactg
tatgcagggc acccactggt cgtttacctt cggacaagga 780actaggctcg
agatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc
840atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc
tggtggggcc 900gtgcataccc ggggtcttga cttcgcctgc gatatctaca
tttgggcccc tctggctggt 960acttgcgggg tcctgctgct ttcactcgtg
atcactcttt actgtaagcg cggtcggaag 1020aagctgctgt acatctttaa
gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080gacggctgtt
catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa
1140ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct
ctacaacgaa 1200ctcaatcttg gtcggagaga ggagtacgac gtgctggaca
agcggagagg acgggaccca 1260gaaatgggcg ggaagccgcg cagaaagaat
ccccaagagg gcctgtacaa cgagctccaa 1320aaggataaga tggcagaagc
ctatagcgag attggtatga aaggggaacg cagaagaggc 1380aaaggccacg
acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct
1440cttcacatgc aggccctgcc gcctcgg 14671061473DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 106atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60ccccaagtgc agcttcaaga aagcggtcca ggactcgtca
agccatcaga aactctttcc 120ctcacttgta ccgtgtcggg aggcagcatc
tcctcgagct cctactactg gggttggatt 180agacagcccc cgggaaaggg
gttggagtgg atcggttcca tctactactc cgggtcgacc 240tactacaacc
cttccctgaa atctcgggtg tccatctccg tcgacacctc caagaaccag
300ttcagcctga agctgaaata tgtgaccgcg gccgatactg ccgtgtacta
ttgcgccacc 360ccgggaacct actacgactt cctctcgggg tactacccgt
tttactgggg acaggggact 420ctcgtgaccg tgtcctcggg cggcggaggt
tcaggcggtg gcggatcggg gggaggaggc 480tcagacattg tgatgaccca
gagcccgtcc agcctgagcg cctccgtggg cgatagggtc 540acgattactt
gccgggcgtc ccagggaatc tcaagctacc tggcctggta ccaacagaag
600cccggaaagg cacccaagtt gctgatctat gccgctagca ctctgcagtc
cggggtgcct 660tcccgcttct ccggctccgg ctcgggcacc gacttcaccc
tgaccatttc ctcactgcaa 720cccgaggact tcgccactta ctactgccag
cagctgaact cctaccctta cacattcgga 780cagggaacca agctggaaat
caagaccact 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 14731071449DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 107atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60ccccaagtgc aactcgtgga atctggtgga ggactcgtgc
aacccggagg atcattgcga 120ctctcgtgtg cggcatccgg ctttaccttt
tcatcctact ggatgtcctg ggtcagacag 180gcccccggga agggactgga
atgggtcgcg aacatcaacg aggacggctc ggccaagttc 240tacgtggact
ccgtgaaggg ccgcttcacg atctcacggg ataacgccaa gaattccctg
300tatctgcaaa tgaacagcct gagggccgag gacactgcgg tgtacttctg
cgcacgcgac 360ctgaggtccg ggagatactg gggacagggc accctcgtga
ccgtgtcgag cggaggaggg 420gggtcgggcg gcggcggttc cggtggcggc
ggtagcgaaa ttgtgttgac ccagtcccct 480ggaaccctga gcctgtcacc
tggaggacgc gccaccctgt cctgccgggc cagccagagc 540atctcagggt
ccttcctggc ttggtaccag cagaagccgg gacaggctcc gagacttctg
600atctacggcg cctcctcgcg ggcgaccgga atcccggatc ggttctccgg
ctcgggaagc 660ggaactgact tcactcttac catttcccgc ctggagccgg
aagatttcgc cgtgtactac 720tgccagcagt acgggtcatc ccctccaacc
ttcggcctgg gaactaagct ggaaatcaaa 780accactaccc cagcaccgag
gccacccacc ccggctccta ccatcgcctc ccagcctctg 840tccctgcgtc
cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt
900gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg
ggtcctgctg 960ctttcactcg tgatcactct ttactgtaag cgcggtcgga
agaagctgct gtacatcttt 1020aagcaaccct tcatgaggcc tgtgcagact
actcaagagg aggacggctg ttcatgccgg 1080ttcccagagg aggaggaagg
cggctgcgaa ctgcgcgtga aattcagccg cagcgcagat 1140gctccagcct
acaagcaggg gcagaaccag ctctacaacg aactcaatct tggtcggaga
1200gaggagtacg acgtgctgga caagcggaga ggacgggacc cagaaatggg
cgggaagccg 1260cgcagaaaga atccccaaga gggcctgtac aacgagctcc
aaaaggataa gatggcagaa 1320gcctatagcg agattggtat gaaaggggaa
cgcagaagag gcaaaggcca cgacggactg 1380taccagggac tcagcaccgc
caccaaggac acctatgacg ctcttcacat gcaggccctg 1440ccgcctcgg
14491081467DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 108atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaagtgc
aactccaaca atccggtcca ggactcgtca gaccctccga aactctctcg
120cttacatgca ctgtgtccgg cggccctgtg cggtccggct ctcattactg
gaactggatt 180cgccagcccc cgggacgcgg actggagtgg atcggctaca
tctattactc ggggtcgact 240aactacaacc cgagcctgga aaatagagtg
accatctcaa tcgacacgtc caacaaccac 300ttctcgctga agttgtcctc
cgtgactgcc gccgatactg ccctgtactt ctgtgctcgc 360ggaaccgcca
ccttcgactg gaacttccct tttgactcat ggggccaggg gacccttgtg
420accgtgtcca gcggaggagg aggctccggt ggtggcggga gcggtagcgg
cggaagcgac 480atccagatga cccagtcacc gtcctcgctg tccgcatcca
ttggggatcg ggtcactatt 540acttgccggg cgtcccagtc catctcgtcc
tacctgaact ggtatcagca gaagccaggg 600aaagccccca agctgctgat
ctacgcggcc agcagcctgc agtcaggagt gccttcaagg 660tttagcggca
gcggatcggg aaccgacttc accctgacca tttcctccct ccaacccgag
720gatttcgcca cctactactg ccagcagtcc tactccaccc cgtggacctt
cggacaggga 780accaagctgg agatcaagac cactacccca gcaccgaggc
cacccacccc ggctcctacc 840atcgcctccc agcctctgtc cctgcgtccg
gaggcatgta gacccgcagc tggtggggcc 900gtgcataccc ggggtcttga
cttcgcctgc gatatctaca tttgggcccc tctggctggt 960acttgcgggg
tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag
1020aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac
tcaagaggag 1080gacggctgtt catgccggtt cccagaggag gaggaaggcg
gctgcgaact gcgcgtgaaa 1140ttcagccgca gcgcagatgc tccagcctac
aagcaggggc agaaccagct ctacaacgaa 1200ctcaatcttg gtcggagaga
ggagtacgac gtgctggaca agcggagagg acgggaccca 1260gaaatgggcg
ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa
1320aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg
cagaagaggc 1380aaaggccacg acggactgta ccagggactc agcaccgcca
ccaaggacac ctatgacgct 1440cttcacatgc aggccctgcc gcctcgg
14671091479DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 109atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60ccccaagtgc
aacttcaaga atcaggcgca ggacttctca agccatccga aacactctcc
120ctcacttgcg cggtgtacgg gggaagcttc tcgggatact actggtcctg
gattaggcag 180cctcccggca aaggcctgga atgggtcggg gagatcaacc
actccggttc aaccaactac 240aacccgtcgc tgaagtcccg cgtgaccatt
tccgtggaca cctctaagaa tcagttcagc 300ctgaagctct cgtccgtgac
cgcggcggac accgccgtct actactgcgc tcggggatca 360ggactggtgg
tgtacgccat ccgcgtgggc tcgggctggt tcgattactg gggccaggga
420accctggtca ctgtgtcgtc cggcggagga ggttcggggg gcggagacag
cggtggaggg 480ggtagcgaca tccagatgac ccagtccccg tcctcgctgt
ccgcctccgt gggagataga 540gtgaccatca cctgtcgggc atcccagagc
atttccagct acctgaactg gtatcagcag 600aagcccggaa aggcccctaa
gctgttgatg tacgccgcca gcagcttgca gtcgggcgtg 660ccgagccggt
tttccggttc cggctccggg actgacttca ccctgactat ctcatccctg
720caacccgagg acttcgccac ttattactgc cagcagtcct actcaacccc
tccctggacg 780ttcggacagg gcaccaaggt cgatatcaag accactaccc
cagcaccgag gccacccacc 840ccggctccta ccatcgcctc ccagcctctg
tccctgcgtc cggaggcatg tagacccgca 900gctggtgggg ccgtgcatac
ccggggtctt gacttcgcct gcgatatcta catttgggcc 960cctctggctg
gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag
1020cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc
tgtgcagact 1080actcaagagg aggacggctg ttcatgccgg ttcccagagg
aggaggaagg cggctgcgaa 1140ctgcgcgtga aattcagccg cagcgcagat
gctccagcct acaagcaggg gcagaaccag 1200ctctacaacg aactcaatct
tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260ggacgggacc
cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac
1320aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat
gaaaggggaa 1380cgcagaagag gcaaaggcca cgacggactg taccagggac
tcagcaccgc caccaaggac 1440acctatgacg ctcttcacat gcaggccctg
ccgcctcgg 14791101497DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 110atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaagtgc
aattggtgga atctggagga ggacttgtga aacctggtgg aagcctgaga
120ctttcctgtg cggcctcggg attcactttc tcctcctact ccatgaactg
ggtcagacag 180gcccctggga agggactgga atgggtgtca tccatctcct
cctcatcgtc gtacatctac 240tacgccgata gcgtgaaggg gcggttcacc
atttcccggg acaacgctaa gaacagcctc 300tatctgcaaa tgaattccct
ccgcgccgag gacactgccg tgtactactg cgcgagggac 360ccctcatcaa
gcggcagcta ctacatggag gactcgtatt actacggaat ggacgtctgg
420ggccagggaa ccactgtgac ggtgtcctcc ggtggagggg gctccggggg
cgggggatct 480ggcggaggag gctccaactt catgctgacc cagccgcact
ccgtgtccga aagccccgga 540aagaccgtga caatttcctg caccgggtcc
tccggctcga tcgcatcaaa ctacgtgcag 600tggtaccagc agcgcccggg
cagcgccccc accactgtca tctacgagga taaccagcgg 660ccgtcgggtg
tcccagaccg gttttccggt tcgatcgata gcagcagcaa cagcgcctcc
720ctgaccattt ccggcctcaa gaccgaggat gaggctgact actactgcca
gtcgtatgac 780tcctcgaacc aagtggtgtt cggtggcggc accaagctga
ctgtgctgac cactacccca 840gcaccgaggc cacccacccc ggctcctacc
atcgcctccc agcctctgtc cctgcgtccg 900gaggcatgta gacccgcagc
tggtggggcc gtgcataccc ggggtcttga cttcgcctgc 960gatatctaca
tttgggcccc tctggctggt acttgcgggg tcctgctgct ttcactcgtg
1020atcactcttt actgtaagcg cggtcggaag aagctgctgt acatctttaa
gcaacccttc 1080atgaggcctg tgcagactac tcaagaggag gacggctgtt
catgccggtt cccagaggag 1140gaggaaggcg gctgcgaact gcgcgtgaaa
ttcagccgca gcgcagatgc tccagcctac 1200aagcaggggc agaaccagct
ctacaacgaa ctcaatcttg gtcggagaga ggagtacgac 1260gtgctggaca
agcggagagg acgggaccca gaaatgggcg ggaagccgcg cagaaagaat
1320ccccaagagg gcctgtacaa cgagctccaa aaggataaga tggcagaagc
ctatagcgag 1380attggtatga aaggggaacg cagaagaggc aaaggccacg
acggactgta ccagggactc 1440agcaccgcca ccaaggacac ctatgacgct
cttcacatgc aggccctgcc gcctcgg 14971111449DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 111atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60ccccaagtga acctgagaga aagcggcgga ggacttgtgc
aacctggagg aagcctgaga 120ctgtcatgtg ccgcgtccgg cttcaccttc
tcgtcctacg agatgaactg ggtccgccag 180gcaccgggca aaggactgga
atgggtgtcc tacatttcct cgtccgggtc caccatctat 240tacgccgact
ccgtgaaggg acggttcacc atctcccggg acaacgccaa gaactccctc
300tacctccaaa tgaactcact gagggcagag gacactgcgg tctactactg
cgcccgcgaa 360gctttgggta gctcctggga gtggggccag ggaaccactg
tgaccgtgtc ctcgggtgga 420gggggctccg gtggcggggg ttcagggggt
ggcggaagcg atatccagat gactcagtca 480ccaagctccc tgagcgcctc
agtgggagat cgggtcacaa tcacgtgcca ggcgtcccag 540gacatttcta
actacctcaa ttggtaccag cagaagccgg ggaaggcccc caagcttctg
600atctacgatg cctccaacct ggaaaccggc gtgccctccc gcttctcggg
atcgggcagc 660ggcactgact tcacctttac catctcgtcc ctgcaacctg
aggacatcgc cacctattac 720tgccagcagt acgataacct cccgctgact
ttcggaggcg gaactaagct ggagattaag 780accactaccc cagcaccgag
gccacccacc ccggctccta ccatcgcctc ccagcctctg 840tccctgcgtc
cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt
900gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg
ggtcctgctg 960ctttcactcg tgatcactct ttactgtaag cgcggtcgga
agaagctgct gtacatcttt 1020aagcaaccct tcatgaggcc tgtgcagact
actcaagagg aggacggctg ttcatgccgg 1080ttcccagagg aggaggaagg
cggctgcgaa ctgcgcgtga aattcagccg cagcgcagat 1140gctccagcct
acaagcaggg gcagaaccag ctctacaacg aactcaatct tggtcggaga
1200gaggagtacg acgtgctgga caagcggaga ggacgggacc cagaaatggg
cgggaagccg 1260cgcagaaaga atccccaaga gggcctgtac aacgagctcc
aaaaggataa gatggcagaa 1320gcctatagcg agattggtat gaaaggggaa
cgcagaagag gcaaaggcca cgacggactg 1380taccagggac tcagcaccgc
caccaaggac acctatgacg ctcttcacat gcaggccctg 1440ccgcctcgg
14491121470DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 112atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60ccccaagtgc
aactcgtcca gtccggtgca gaagtcaagg aacccggagc ctccgtgaaa
120gtgtcctgca aagctcctgc caacactttc tcggaccacg tgatgcactg
ggtgcgccag 180gcgccgggcc agcgcttcga atggatggga tacattcatg
ccgccaatgg cggtacccac 240tactcccaaa agttccagga tagagtcacc
atcacccggg acaccagcgc caacaccgtg 300tatatggatc tgtccagcct
gaggtccgag gataccgccg tgtactactg cgcccggggc 360ggatacaact
cagacgcgtt cgacatttgg ggacagggta ctatggtcac cgtgtcatcc
420gggggcggtg gcagcggggg cggaggctct ggcggaggcg gatcaggggg
aggagggtcc 480gacatcgtga tgacccagtc cccgtcatcg gtgtccgcgt
ccgtgggaga cagagtgacc 540atcacgtgtc gcgccagcca ggacatctcc
tcgtggttgg catggtacca gcagaagcct 600ggaaaggccc cgaagctgct
catctacgcc gcctcctccc ttcaatcggg agtgccctcg 660cggttcaacg
gaagcggaag cgggacagat tttaccctga ctattagctc gctgcagccc
720gaggacttcg ctacttacta ctgccaacag agctactcca ccccactgac
tttcggcggg 780ggtaccaagg tcgagatcaa gaccactacc ccagcaccga
ggccacccac cccggctcct 840accatcgcct cccagcctct gtccctgcgt
ccggaggcat gtagacccgc agctggtggg 900gccgtgcata cccggggtct
tgacttcgcc tgcgatatct acatttgggc ccctctggct 960ggtacttgcg
gggtcctgct gctttcactc gtgatcactc tttactgtaa gcgcggtcgg
1020aagaagctgc tgtacatctt taagcaaccc ttcatgaggc ctgtgcagac
tactcaagag 1080gaggacggct gttcatgccg gttcccagag gaggaggaag
gcggctgcga actgcgcgtg 1140aaattcagcc gcagcgcaga tgctccagcc
tacaagcagg ggcagaacca gctctacaac 1200gaactcaatc ttggtcggag
agaggagtac gacgtgctgg acaagcggag aggacgggac 1260ccagaaatgg
gcgggaagcc gcgcagaaag aatccccaag agggcctgta caacgagctc
1320caaaaggata agatggcaga agcctatagc gagattggta tgaaagggga
acgcagaaga 1380ggcaaaggcc acgacggact gtaccaggga ctcagcaccg
ccaccaagga cacctatgac 1440gctcttcaca tgcaggccct gccgcctcgg
14701131476DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 113atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60ccccaagtgc
aacttgttca atccggtgga ggtcttgtgc agcccggagg atcactcaga
120ctgtcgtgcg ccgcctctgg gttcactttc tcctcatact cgatgaactg
ggtgcgccag 180gcgccgggaa agggcctgga atgggtgtca tacatctcct
cctcatcctc caccatctac 240tacgccgatt ccgtgaaggg ccgcttcact
atttcccggg acaacgcgaa aaactcgctc 300tatctgcaaa tgaactccct
gcgcgccgag gacaccgccg tgtactactg cgcccgggac 360ctgagcgtgc
gggctattga tgcgttcgac atctggggac agggcaccat ggtcacagtg
420tccagcggag gcggcggcag cggtggagga ggatcagggg gaggaggttc
ggggggcggt 480ggctccgata tcgtgctgac ccagagcccg tcgagcctct
ccgcctccgt cggcgacaga 540gtgaccatca cgtgtcaggc atcccaggac
attagcaact acctgaattg gtaccagcag 600aagcctggaa aggcacccaa
gttgctgatc tacgacgcct ccaacctgga aaccggagtg 660ccatccaggt
tctcgggcag cggctcggga accgacttca cttttactat ctcctccctg
720caacccgagg atttcgcgac ctactactgc cagcaggcct acagcacccc
tttcaccttc 780gggccgggaa ctaaggtcga aatcaagacc actaccccag
caccgaggcc acccaccccg 840gctcctacca tcgcctccca gcctctgtcc
ctgcgtccgg aggcatgtag acccgcagct 900ggtggggccg tgcatacccg
gggtcttgac ttcgcctgcg atatctacat ttgggcccct 960ctggctggta
cttgcggggt cctgctgctt tcactcgtga tcactcttta ctgtaagcgc
1020ggtcggaaga agctgctgta catctttaag caacccttca tgaggcctgt
gcagactact 1080caagaggagg acggctgttc atgccggttc ccagaggagg
aggaaggcgg ctgcgaactg 1140cgcgtgaaat tcagccgcag cgcagatgct
ccagcctaca agcaggggca gaaccagctc 1200tacaacgaac tcaatcttgg
tcggagagag gagtacgacg tgctggacaa gcggagagga 1260cgggacccag
aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac
1320gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa
aggggaacgc 1380agaagaggca aaggccacga cggactgtac cagggactca
gcaccgccac caaggacacc 1440tatgacgctc ttcacatgca ggccctgccg cctcgg
14761141470DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 114atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaagtgc
aattggtgca atcaggagga ggagtggtca gatctggaag aagcctgaga
120ctgtcatgcg cggcttcggg ctttaccttc aactcctacg gcctccactg
ggtgcgccag 180gcccccggaa aaggcctcga atgggtcgca ctgattgagt
acgacgggtc caacaagtac 240tacggagata gcgtgaaggg ccgcttcacc
atctcacggg acaagtccaa gtccaccctg 300tatctgcaaa tggacaacct
gagggccgag gatactgccg tgtactactg cgcccgcgaa 360ggaaacgaag
atctggcctt cgatatttgg ggccagggta ctcttgtgac cgtgtcgagc
420ggaggcggag gctccggtgg aggaggatcg gggggtggtg gttccggcgg
cggggggagc 480gaaatcgtgc tgacccagtc gccttcctcc ctctccgctt
ccgtggggga ccgggtcact 540attacgtgtc aggcgtccca attcatcaag
aagaatctga actggtacca gcacaagccg 600ggaaaggccc ccaaactgct
catctacgac gccagctcgc tgcagactgg cgtgccttcc 660cggttttccg
ggaaccggtc gggaaccacc ttctcattca ccatcagcag cctccagccg
720gaggacgtgg cgacctacta ctgccagcag catgacaacc ttccactgac
tttcggcggg 780ggcaccaagg tcgagattaa gaccactacc ccagcaccga
ggccacccac cccggctcct 840accatcgcct
cccagcctct gtccctgcgt ccggaggcat gtagacccgc agctggtggg
900gccgtgcata cccggggtct tgacttcgcc tgcgatatct acatttgggc
ccctctggct 960ggtacttgcg gggtcctgct gctttcactc gtgatcactc
tttactgtaa gcgcggtcgg 1020aagaagctgc tgtacatctt taagcaaccc
ttcatgaggc ctgtgcagac tactcaagag 1080gaggacggct gttcatgccg
gttcccagag gaggaggaag gcggctgcga actgcgcgtg 1140aaattcagcc
gcagcgcaga tgctccagcc tacaagcagg ggcagaacca gctctacaac
1200gaactcaatc ttggtcggag agaggagtac gacgtgctgg acaagcggag
aggacgggac 1260ccagaaatgg gcgggaagcc gcgcagaaag aatccccaag
agggcctgta caacgagctc 1320caaaaggata agatggcaga agcctatagc
gagattggta tgaaagggga acgcagaaga 1380ggcaaaggcc acgacggact
gtaccaggga ctcagcaccg ccaccaagga cacctatgac 1440gctcttcaca
tgcaggccct gccgcctcgg 14701151497DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 115atggccctcc ctgtcaccgc cctgctgctt ccgctggctc
ttctgctcca cgccgctcgg 60ccccaagtgc aactcgtgga atcaggcgga ggactcgtgc
aacccggagg ttcccttaga 120ctgtcatgtg ccgcttccgg gttcaatgtg
tccagcaact acatgacctg ggtcagacag 180gcgccgggaa agggacttga
atgggtgtcc gtgatctact ccggtggagc aacatactac 240ggagactccg
tgaaaggccg ctttaccgtg tcccgcgata actcgaagaa caccgtgtac
300ttgcagatga acaggctgac tgccgaggac accgccgtgt attattgcgc
ccgggacagg 360ctgtactgtg gaaacaactg ctacctgtac tactactacg
ggatggacgt gtggggacag 420ggcactctcg tcactgtgtc atccgggggg
ggcggtagcg gtggcggagg gtccggcgga 480ggaggctcag ggggaggcgg
aagcgatatc caggtcaccc agtctccctc ctcgctgtcc 540gcctccgtgg
gcgaccgcgt caccattact tgccgggcgt cgcagtcgat cagctcctac
600ctgaactggt accagcagaa gcctggaaag gccccgaagc tgctgatcta
cgcggcctcg 660tccctgcaaa gcggcgtccc gtcgcggttc agcggttccg
gttcgggaac cgacttcacc 720ctgactattt cctccctgca acccgaggat
ttcgccactt actactgcca gcagtcctac 780tccaccccac ctctgacctt
cggccaagga accaaggtcg aaatcaagac cactacccca 840gcaccgaggc
cacccacccc ggctcctacc atcgcctccc agcctctgtc cctgcgtccg
900gaggcatgta gacccgcagc tggtggggcc gtgcataccc ggggtcttga
cttcgcctgc 960gatatctaca tttgggcccc tctggctggt acttgcgggg
tcctgctgct ttcactcgtg 1020atcactcttt actgtaagcg cggtcggaag
aagctgctgt acatctttaa gcaacccttc 1080atgaggcctg tgcagactac
tcaagaggag gacggctgtt catgccggtt cccagaggag 1140gaggaaggcg
gctgcgaact gcgcgtgaaa ttcagccgca gcgcagatgc tccagcctac
1200aagcaggggc agaaccagct ctacaacgaa ctcaatcttg gtcggagaga
ggagtacgac 1260gtgctggaca agcggagagg acgggaccca gaaatgggcg
ggaagccgcg cagaaagaat 1320ccccaagagg gcctgtacaa cgagctccaa
aaggataaga tggcagaagc ctatagcgag 1380attggtatga aaggggaacg
cagaagaggc aaaggccacg acggactgta ccagggactc 1440agcaccgcca
ccaaggacac ctatgacgct cttcacatgc aggccctgcc gcctcgg
14971161473DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 116atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60ccccaagtgc
aactcgtcca gtccggtgca gaagtgaaaa agagcggagc ctcagtgaaa
120gtgtcctgca aggcctccgg ttaccccttc actggatact acattcagtg
ggtccgccaa 180gccccgggac agggtctgga gtggatgggg tggattgacc
ctaactcggg aaatacggga 240tacgcgcaga agttccaggg ccgcgtgacc
atgaccagga acacctcgat cagcaccgcc 300tacatggaac tgtcctccct
gcggtcggag gatactgccg tgtactactg cgcctccgat 360tcctatgggt
actactacgg aatggacgtc tggggacagg gcaccctcgt gaccgtgtcc
420tcgggaggcg gagggagcgg cgggggtgga tcgggaggag gcggctccgg
cggcggcggt 480agcgacatcc agatgaccca gtcaccatca agccttagcg
cctccgtggg cgacagagtg 540acattcactt gtcgggcgtc ccagggaatc
tcctccgctc tggcttggta tcagcagaag 600cctgggaagc ctccgaagct
gttgatctac gacgcgagca gcctggaatc aggggtgccc 660tcccggtttt
ccgggtccgg ttctggcacc gatttcaccc tgaccatttc gtccctccaa
720cccgaggact tcgccactta ctactgccag cagttcaaca actacccgct
gaccttcgga 780ggaggcacta aggtcgagat caagaccact 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
147311710PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 117Gly Gly Thr Phe Ser Ser
Tyr Ala Ile Ser1 5 1011810PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 118Gly Phe Thr Phe Asp Asp Tyr Ala Met His1 5
1011912PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 119Gly Gly Ser Ile Ser Ser Ser Ser Tyr
Tyr Trp Gly1 5 1012010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 120Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser1 5
1012112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 121Gly Gly Pro Val Arg Ser Gly Ser His
Tyr Trp Asn1 5 1012210PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 122Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser1 5
1012310PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 123Gly Phe Thr Phe Ser Ser Tyr Ser Met
Asn1 5 1012410PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 124Gly Phe Thr Phe Ser Ser
Tyr Glu Met Asn1 5 1012510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 125Ala Asn Thr Phe Ser Asp His Val Met His1 5
1012610PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 126Gly Phe Thr Phe Ser Ser Tyr Ser Met
Asn1 5 1012710PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 127Gly Phe Thr Phe Asn Ser
Tyr Gly Leu His1 5 1012810PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 128Gly Phe Asn Val Ser Ser Asn Tyr Met Thr1 5
1012910PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 129Gly Tyr Pro Phe Thr Gly Tyr Tyr Ile
Gln1 5 1013016PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 130Gly Ile Ile Pro Ile Phe
Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10 1513117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 131Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly13216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 132Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1513317PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 133Asn Ile Asn Glu Asp
Gly Ser Ala Lys Phe Tyr Val Asp Ser Val Lys1 5 10
15Gly13416PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 134Tyr Ile Tyr Tyr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Glu Asn1 5 10 1513516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 135Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1513617PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 136Ser Ile Ser Ser Ser
Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly13717PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 137Tyr Ile Ser Ser Ser Gly
Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly13817PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 138Tyr Ile His Ala Ala Asn
Gly Gly Thr His Tyr Ser Gln Lys Phe Gln1 5 10
15Asp13917PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 139Tyr Ile Ser Ser Ser Ser
Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly14017PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 140Leu Ile Glu Tyr Asp Gly
Ser Asn Lys Tyr Tyr Gly Asp Ser Val Lys1 5 10
15Gly14116PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 141Val Ile Tyr Ser Gly Gly
Ala Thr Tyr Tyr Gly Asp Ser Val Lys Gly1 5 10 1514217PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 142Trp Ile Asp Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly14311PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 143Asp Leu Glu Met Ala Thr Ile Met Gly Gly Tyr1 5
101449PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 144Val Phe Asp Ser Tyr Tyr Met Asp Val1
514515PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 145Pro Gly Thr Tyr Tyr Asp Phe Leu Ser
Gly Tyr Tyr Pro Phe Tyr1 5 10 151467PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 146Asp Leu Arg Ser Gly Arg Tyr1 514713PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 147Gly Thr Ala Thr Phe Asp Trp Asn Phe Pro Phe Asp Ser1 5
1014818PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 148Gly Ser Gly Leu Val Val Tyr Ala Ile
Arg Val Gly Ser Gly Trp Phe1 5 10 15Asp Tyr14920PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 149Asp Pro Ser Ser Ser Gly Ser Tyr Tyr Met Glu Asp Ser Tyr
Tyr Tyr1 5 10 15Gly Met Asp Val 201508PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 150Glu Ala Leu Gly Ser Ser Trp Glu1 515110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 151Gly Gly Tyr Asn Ser Asp Ala Phe Asp Ile1 5
1015212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 152Asp Leu Ser Val Arg Ala Ile Asp Ala
Phe Asp Ile1 5 1015310PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 153Glu Gly Asn Glu Asp Leu Ala Phe Asp Ile1 5
1015419PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 154Asp Arg Leu Tyr Cys Gly Asn Asn Cys
Tyr Leu Tyr Tyr Tyr Tyr Gly1 5 10 15Met Asp Val15511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 155Asp Ser Tyr Gly Tyr Tyr Tyr Gly Met Asp Val1 5
1015614PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 156Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr Asn Tyr Val Ser1 5 1015716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 157Arg Ser Ser Gln Ser Leu Val Tyr Thr Asp Gly Asn Thr Tyr
Leu Asn1 5 10 1515811PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 158Arg Ala Ser Gln Gly
Ile Ser Ser Tyr Leu Ala1 5 1015912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 159Arg Ala Ser Gln Ser Ile Ser Gly Ser Phe Leu Ala1 5
1016011PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 160Arg Ala Ser Gln Ser Ile Ser Ser Tyr
Leu Asn1 5 1016111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 161Arg Ala Ser Gln Ser Ile
Ser Ser Tyr Leu Asn1 5 1016213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 162Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln1 5
1016311PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 163Gln Ala Ser Gln Asp Ile Ser Asn Tyr
Leu Asn1 5 1016411PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 164Arg Ala Ser Gln Asp Ile
Ser Ser Trp Leu Ala1 5 1016511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 165Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5
1016611PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 166Gln Ala Ser Gln Phe Ile Lys Lys Asn
Leu Asn1 5 1016711PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 167Arg Ala Ser Gln Ser Ile
Ser Ser Tyr Leu Asn1 5 1016811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 168Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala1 5
101697PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 169Asp Val Ser Asn Arg Pro Ser1
51707PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 170Lys Val Ser Asn Arg Asp Ser1
51717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 171Ala Ala Ser Thr Leu Gln Ser1
51727PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 172Gly Ala Ser Ser Arg Ala Thr1
51737PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 173Ala Ala Ser Ser Leu Gln Ser1
51747PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 174Ala Ala Ser Ser Leu Gln Ser1
51757PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 175Glu Asp Asn Gln Arg Pro Ser1
51767PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 176Asp Ala Ser Asn Leu Glu Thr1
51777PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 177Ala Ala Ser Ser Leu Gln Ser1
51787PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 178Asp Ala Ser Asn Leu Glu Thr1
51797PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 179Asp Ala Ser Ser Leu Gln Thr1
51807PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 180Ala Ala Ser Ser Leu Gln Ser1
51817PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 181Asp Ala Ser Ser Leu Glu Ser1
518212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 182Ser Ser Tyr Thr Ser Ser Ser Thr Leu
Asp Val Val1 5 101839PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 183Met Gln Gly Thr His
Trp Ser Phe Thr1 51849PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 184Gln Gln Leu Asn Ser Tyr Pro Tyr Thr1 51859PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 185Gln Gln Tyr Gly Ser Ser Pro Pro Thr1 51869PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 186Gln Gln Ser Tyr Ser Thr Pro Trp Thr1
518710PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 187Gln Gln Ser Tyr Ser Thr Pro Pro Trp
Thr1 5 1018810PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 188Gln Ser Tyr Asp Ser Ser
Asn Gln Val Val1 5 101899PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 189Gln Gln Tyr Asp Asn Leu Pro Leu Thr1 51909PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 190Gln Gln Ser Tyr Ser Thr Pro Leu Thr1 51919PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 191Gln Gln Ala Tyr Ser Thr Pro Phe Thr1 51929PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 192Gln Gln His Asp Asn Leu Pro Leu Thr1
519310PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 193Gln Gln Ser Tyr Ser Thr Pro Pro Leu
Thr1 5 101949PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 194Gln Gln Phe Asn Asn Tyr
Pro Leu Thr1 5195122PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 195Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Glu Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Asp Pro Tyr Ser Ser Ser Trp His Asp Ala Phe Asp
Ile Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120196108PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 196Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100
105197489PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 197Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe
Ser Ser Tyr Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly
Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr65 70 75
80Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Pro Tyr Ser Ser Ser
Trp His Asp 115 120 125Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Ile145 150 155 160Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 165 170 175Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu 180 185 190Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 195 200
205Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser
210 215 220Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
Pro Glu225 230 235 240Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Ser Ser Pro Leu Thr 245 250 255Phe Gly Gly Gly Thr Lys Val Asp Ile
Lys Thr Thr Thr Pro Ala Pro 260 265 270Arg Pro Pro Thr Pro Ala Pro
Thr Ile Ala Ser Gln Pro Leu Ser Leu 275 280 285Arg Pro Glu Ala Cys
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 290 295 300Gly Leu Asp
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly305 310 315
320Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
325 330 335Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met Arg 340 345 350Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro 355 360 365Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser 370 375 380Ala Asp Ala Pro Ala Tyr Lys Gln
Gly Gln Asn Gln Leu Tyr Asn Glu385 390 395 400Leu Asn Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415Gly Arg Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430Glu
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440
445Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala465 470 475 480Leu His Met Gln Ala Leu Pro Pro Arg
4851981467DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 198atggccctcc
ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60cccgaagtgc
aactcgtgga aagcggtgga ggtcttgtgc aacctggagg ttccttgcgc
120ctgtcatgtg cagcttccgg cttcactttc tcctcgtacg agatgaattg
ggtgcggcag 180gcgcctggaa aggggctgga atgggtgtcc tacatctcaa
gctccggctc gaccatctac 240tacgcggaca gcgtgaaggg gcggttcacg
atttcgaggg acaacgccaa gaactcgctc 300tatctgcaaa tgaactccct
gagagccgag gacaccgctg tgtattactg cgcccgggac 360ccctactcct
cctcatggca cgacgccttt gatatctggg gccagggaac catggtcacc
420gtcagcagcg ggggcggagg ttccggggga gggggctccg gcggaggagg
ctccgagatt 480gtgttgactc agagcccggg taccctgtcg ctgagccccg
gagagcgggc caccctttca 540tgccgcgcca gccagtccgt gtcctcatcc
tacctcgcgt ggtaccagca gaaacctggc 600caggccccgc ggctgctgat
ctacggcgcc tcctcgcgcg caaccggaat ccccgaccgg 660ttctccgggt
ctggcagcgg aaccgacttc actctcacca tttcgaggct ggagccggaa
720gatttcgccg tgtactactg ccagcagtac ggctcctcgc cactgacttt
cggcggagga 780accaaggtcg atatcaagac cactacccca gcaccgaggc
cacccacccc ggctcctacc 840atcgcctccc agcctctgtc cctgcgtccg
gaggcatgta gacccgcagc tggtggggcc 900gtgcataccc ggggtcttga
cttcgcctgc gatatctaca tttgggcccc tctggctggt 960acttgcgggg
tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag
1020aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac
tcaagaggag 1080gacggctgtt catgccggtt cccagaggag gaggaaggcg
gctgcgaact gcgcgtgaaa 1140ttcagccgca gcgcagatgc tccagcctac
aagcaggggc agaaccagct ctacaacgaa 1200ctcaatcttg gtcggagaga
ggagtacgac gtgctggaca agcggagagg acgggaccca 1260gaaatgggcg
ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa
1320aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg
cagaagaggc 1380aaaggccacg acggactgta ccagggactc agcaccgcca
ccaaggacac ctatgacgct 1440cttcacatgc aggccctgcc gcctcgg
146719910PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 199Gly Phe Thr Phe Ser Ser
Tyr Glu Met Asn1 5 1020017PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 200Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly20113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 201Asp Pro Tyr Ser Ser Ser Trp His Asp Ala Phe Asp Ile1 5
1020212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 202Arg Ala Ser Gln Ser Val Ser Ser Ser
Tyr Leu Ala1 5 102037PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 203Gly Ala Ser Ser Arg
Ala Thr1 52049PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 204Gln Gln Tyr Gly Ser Ser
Pro Leu Thr1 5205132PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 205Asp 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 130206108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 206Val 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 10520793PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 207Ile 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 9020895PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 208Ile 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 9520995PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 209Ile 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 9521095PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 210Ile 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 9521195PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide"MOD_RES(12)..(12)Any amino acidMOD_RES(78)..(78)Any
amino acid 211Ile 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 9521295PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 212Ile 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 9521395PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 213Ile 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 952141132PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 214Met 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 11302154027DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 215caggcagcgt 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 402721619DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 216ggaggtccct caccttcta 1921719DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 217cggaggatct tatgctgaa 1921819DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 218cccgcttcca gatcataca 1921919DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 219ggagacctca acaagatat 1922019DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 220aaggcatggt cattggtat 1922119DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 221gcatggtcat tggtatcat 1922219DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 222ggtcattggt atcatgagt 1922319DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 223cctagtgggt atccctgta 1922419DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 224gaggatggac attgttctt 1922519DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 225gcatgcaggc tacagttca 1922619DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 226ccagcacatg cactgttga 1922719DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 227cacatgcact gttgagtga 1922821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 228ctggaggtcc ctcaccttct a 2122921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 229gtcggaggat cttatgctga a 2123021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 230tgcccgcttc cagatcatac a 2123121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 231ctggagacct caacaagata t 2123221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 232tcaaggcatg gtcattggta t 2123321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 233aggcatggtc attggtatca t 2123421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 234atggtcattg gtatcatgag t 2123521DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 235gccctagtgg gtatccctgt a 2123621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 236atgaggatgg acattgttct t 2123721DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 237gagcatgcag gctacagttc a 2123821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 238ttccagcaca tgcactgttg a 2123921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 239agcacatgca ctgttgagtg a 2124021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 240tagaaggtga gggacctcca g 2124121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 241ttcagcataa gatcctccga c
2124221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 242tgtatgatct ggaagcgggc a
2124321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 243atatcttgtt gaggtctcca g
2124421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 244ataccaatga ccatgccttg a
2124521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 245atgataccaa tgaccatgcc t
2124621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 246atggtcattg gtatcatgag t
2124721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 247gccctagtgg gtatccctgt a
2124821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 248atgaggatgg acattgttct t
2124921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 249gagcatgcag gctacagttc a
2125021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 250ttccagcaca tgcactgttg a
2125121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 251agcacatgca ctgttgagtg a
2125219DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 252tagaaggtga gggacctcc
1925319DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 253ttcagcataa gatcctccg
1925419DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 254tgtatgatct ggaagcggg
1925519DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 255atatcttgtt gaggtctcc
1925619DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 256ataccaatga ccatgcctt
1925719DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 257atgataccaa tgaccatgc
1925819DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 258atggtcattg gtatcatga
1925919DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 259gccctagtgg gtatccctg
1926019DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 260atgaggatgg acattgttc
1926119DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 261gagcatgcag gctacagtt
1926219DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 262ttccagcaca tgcactgtt
1926319DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 263agcacatgca ctgttgagt
1926419DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 264ggccaggatg gttcttaga
1926519DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 265gcttcgtgct aaactggta
1926619DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 266gggcgtgact tccacatga
1926719DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 267caggcctaga gaagtttca
1926819DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 268cttggaaccc attcctgaa
1926919DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 269ggaacccatt cctgaaatt
1927019DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 270gaacccattc ctgaaatta
1927119DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 271aacccattcc tgaaattat
1927219DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 272acccattcct gaaattatt
1927319DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 273cccattcctg aaattattt
1927419DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 274ctgtggttct attatatta
1927519DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 275aaatatgaga gcatgctaa
1927619DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 276tctaagaacc atcctggcc
1927719DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 277taccagttta gcacgaagc
1927819DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 278tcatgtggaa gtcacgccc
1927919DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 279tgaaacttct ctaggcctg
1928019DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 280ttcaggaatg ggttccaag
1928119DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 281aatttcagga atgggttcc
1928219DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 282taatttcagg aatgggttc
1928319DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 283ataatttcag gaatgggtt
1928419DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 284aataatttca ggaatgggt
1928519DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 285aaataatttc aggaatggg
1928619DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 286taatataata gaaccacag
1928719DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 287ttagcatgct ctcatattt
1928821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 288gcggccagga tggttcttag a
2128921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 289gagcttcgtg ctaaactggt a
2129021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 290acgggcgtga cttccacatg a
2129121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 291tgcaggccta gagaagtttc a
2129221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 292tccttggaac ccattcctga a
2129321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 293ttggaaccca ttcctgaaat t
2129421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 294tggaacccat tcctgaaatt a
2129521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 295ggaacccatt cctgaaatta t
2129621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 296gaacccattc ctgaaattat t
2129721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 297aacccattcc tgaaattatt t
2129821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 298ccctgtggtt ctattatatt a
2129921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 299ttaaatatga gagcatgcta a
2130021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 300tctaagaacc atcctggccg c
2130121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 301taccagttta gcacgaagct c
2130221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 302tcatgtggaa gtcacgcccg t
2130321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 303tgaaacttct ctaggcctgc a
2130421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 304ttcaggaatg ggttccaagg a
2130521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 305aatttcagga atgggttcca a
2130621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 306taatttcagg aatgggttcc a
2130721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 307ataatttcag gaatgggttc c
2130821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 308aataatttca ggaatgggtt c
2130921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 309aaataatttc aggaatgggt t
2131021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 310taatataata gaaccacagg g
2131121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 311ttagcatgct ctcatattta a
21312150DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 312aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1503134PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 313Arg Gly Asp Ser13145PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 314Ser Tyr Ala Ile Ser1 53155PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 315Asp Tyr Ala Met His1 53167PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 316Ser Ser Ser Tyr Tyr Trp Gly1 53175PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 317Ser Tyr Trp Met Ser1 53187PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 318Ser Gly Ser His Tyr Trp Asn1 53195PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 319Gly Tyr Tyr Trp Ser1 53205PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 320Ser Tyr Ser Met Asn1 53215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 321Ser Tyr Glu Met Asn1 53225PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 322Asp His Val Met His1 53235PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 323Ser Tyr Ser Met Asn1 53245PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 324Ser Tyr Gly Leu His1 53255PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 325Ser Asn Tyr Met Thr1 53265PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 326Gly Tyr Tyr Ile Gln1 53275PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 327Ser Tyr Glu Met Asn1 532817PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 328Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly32917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 329Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly33016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 330Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1533117PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 331Asn Ile Asn Glu Asp
Gly Ser Ala Lys Phe Tyr Val Asp Ser Val Lys1 5 10
15Gly33216PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 332Tyr Ile Tyr Tyr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Glu Asn1 5 10 1533316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 333Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1533417PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 334Ser Ile Ser Ser Ser
Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly33517PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 335Tyr Ile Ser Ser Ser Gly
Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly33617PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 336Tyr Ile His Ala Ala Asn
Gly Gly Thr His Tyr Ser Gln Lys Phe Gln1 5
10 15Asp33717PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 337Tyr Ile Ser Ser Ser Ser
Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly33817PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 338Leu Ile Glu Tyr Asp Gly
Ser Asn Lys Tyr Tyr Gly Asp Ser Val Lys1 5 10
15Gly33916PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 339Val Ile Tyr Ser Gly Gly
Ala Thr Tyr Tyr Gly Asp Ser Val Lys Gly1 5 10 1534017PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 340Trp Ile Asp Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly34117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 341Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15Gly34211PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 342Asp Leu Glu Met Ala Thr Ile Met Gly Gly Tyr1 5
103439PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 343Val Phe Asp Ser Tyr Tyr Met Asp Val1
534415PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 344Pro Gly Thr Tyr Tyr Asp Phe Leu Ser
Gly Tyr Tyr Pro Phe Tyr1 5 10 153457PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 345Asp Leu Arg Ser Gly Arg Tyr1 534613PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 346Gly Thr Ala Thr Phe Asp Trp Asn Phe Pro Phe Asp Ser1 5
1034718PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 347Gly Ser Gly Leu Val Val Tyr Ala Ile
Arg Val Gly Ser Gly Trp Phe1 5 10 15Asp Tyr34820PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 348Asp Pro Ser Ser Ser Gly Ser Tyr Tyr Met Glu Asp Ser Tyr
Tyr Tyr1 5 10 15Gly Met Asp Val 203498PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 349Glu Ala Leu Gly Ser Ser Trp Glu1 535010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 350Gly Gly Tyr Asn Ser Asp Ala Phe Asp Ile1 5
1035112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 351Asp Leu Ser Val Arg Ala Ile Asp Ala
Phe Asp Ile1 5 1035210PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 352Glu Gly Asn Glu Asp Leu Ala Phe Asp Ile1 5
1035319PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 353Asp Arg Leu Tyr Cys Gly Asn Asn Cys
Tyr Leu Tyr Tyr Tyr Tyr Gly1 5 10 15Met Asp Val35411PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 354Asp Ser Tyr Gly Tyr Tyr Tyr Gly Met Asp Val1 5
1035513PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 355Asp Pro Tyr Ser Ser Ser Trp His Asp
Ala Phe Asp Ile1 5 1035614PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 356Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val
Ser1 5 1035716PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 357Arg Ser Ser Gln Ser Leu
Val Tyr Thr Asp Gly Asn Thr Tyr Leu Asn1 5 10 1535811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 358Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala1 5
1035912PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 359Arg Ala Ser Gln Ser Ile Ser Gly Ser
Phe Leu Ala1 5 1036011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 360Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5
1036111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 361Arg Ala Ser Gln Ser Ile Ser Ser Tyr
Leu Asn1 5 1036213PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 362Thr Gly Ser Ser Gly Ser
Ile Ala Ser Asn Tyr Val Gln1 5 1036311PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 363Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5
1036411PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 364Arg Ala Ser Gln Asp Ile Ser Ser Trp
Leu Ala1 5 1036511PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 365Gln Ala Ser Gln Asp Ile
Ser Asn Tyr Leu Asn1 5 1036611PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 366Gln Ala Ser Gln Phe Ile Lys Lys Asn Leu Asn1 5
1036711PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 367Arg Ala Ser Gln Ser Ile Ser Ser Tyr
Leu Asn1 5 1036811PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 368Arg Ala Ser Gln Gly Ile
Ser Ser Ala Leu Ala1 5 1036912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 369Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
103707PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 370Asp Val Ser Asn Arg Pro Ser1
53717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 371Lys Val Ser Asn Arg Asp Ser1
53727PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 372Ala Ala Ser Thr Leu Gln Ser1
53737PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 373Gly Ala Ser Ser Arg Ala Thr1
53747PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 374Ala Ala Ser Ser Leu Gln Ser1
53757PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 375Ala Ala Ser Ser Leu Gln Ser1
53767PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 376Glu Asp Asn Gln Arg Pro Ser1
53777PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 377Asp Ala Ser Asn Leu Glu Thr1
53787PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 378Ala Ala Ser Ser Leu Gln Ser1
53797PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 379Asp Ala Ser Asn Leu Glu Thr1
53807PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 380Asp Ala Ser Ser Leu Gln Thr1
53817PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 381Ala Ala Ser Ser Leu Gln Ser1
53827PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 382Asp Ala Ser Ser Leu Glu Ser1
53837PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 383Gly Ala Ser Ser Arg Ala Thr1
538412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 384Ser Ser Tyr Thr Ser Ser Ser Thr Leu
Asp Val Val1 5 103859PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 385Met Gln Gly Thr His
Trp Ser Phe Thr1 53869PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 386Gln Gln Leu Asn Ser Tyr Pro Tyr Thr1 53879PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 387Gln Gln Tyr Gly Ser Ser Pro Pro Thr1 53889PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 388Gln Gln Ser Tyr Ser Thr Pro Trp Thr1
538910PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 389Gln Gln Ser Tyr Ser Thr Pro Pro Trp
Thr1 5 1039010PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 390Gln Ser Tyr Asp Ser Ser
Asn Gln Val Val1 5 103919PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 391Gln Gln Tyr Asp Asn Leu Pro Leu Thr1 53929PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 392Gln Gln Ser Tyr Ser Thr Pro Leu Thr1 53939PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 393Gln Gln Ala Tyr Ser Thr Pro Phe Thr1 53949PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 394Gln Gln His Asp Asn Leu Pro Leu Thr1
539510PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 395Gln Gln Ser Tyr Ser Thr Pro Pro Leu
Thr1 5 103969PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 396Gln Gln Phe Asn Asn Tyr
Pro Leu Thr1 53979PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 397Gln Gln Tyr Gly Ser Ser
Pro Leu Thr1 53987PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 398Gly Gly Thr Phe Ser Ser
Tyr1 53997PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 399Gly Phe Thr Phe Asp Asp
Tyr1 54009PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 400Gly Gly Ser Ile Ser Ser
Ser Ser Tyr1 54017PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 401Gly Phe Thr Phe Ser Ser
Tyr1 54029PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 402Gly Gly Pro Val Arg Ser
Gly Ser His1 54037PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 403Gly Gly Ser Phe Ser Gly
Tyr1 54047PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 404Gly Phe Thr Phe Ser Ser
Tyr1 54057PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 405Gly Phe Thr Phe Ser Ser
Tyr1 54067PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 406Ala Asn Thr Phe Ser Asp
His1 54077PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 407Gly Phe Thr Phe Ser Ser
Tyr1 54087PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 408Gly Phe Thr Phe Asn Ser
Tyr1 54097PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 409Gly Phe Asn Val Ser Ser
Asn1 54107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 410Gly Tyr Pro Phe Thr Gly
Tyr1 54117PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 411Gly Phe Thr Phe Ser Ser
Tyr1 54126PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 412Ile Pro Ile Phe Gly Thr1
54136PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 413Ser Gly Asp Gly Gly Ser1
54145PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 414Tyr Tyr Ser Gly Ser1
54156PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 415Asn Glu Asp Gly Ser Ala1
54165PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 416Tyr Tyr Ser Gly Ser1
54175PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 417Asn His Ser Gly Ser1
54186PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 418Ser Ser Ser Ser Ser Tyr1
54196PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 419Ser Ser Ser Gly Ser Thr1
54206PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 420His Ala Ala Asn Gly Gly1
54216PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 421Ser Ser Ser Ser Ser Thr1
54226PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 422Glu Tyr Asp Gly Ser Asn1
54235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 423Tyr Ser Gly Gly Ala1
54246PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 424Asp Pro Asn Ser Gly Asn1
54256PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 425Ser Ser Ser Gly Ser Thr1
542611PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 426Asp Leu Glu Met Ala Thr Ile Met Gly
Gly Tyr1 5 104279PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 427Val Phe Asp Ser Tyr Tyr
Met Asp Val1 542815PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 428Pro Gly Thr Tyr Tyr
Asp Phe Leu Ser Gly Tyr Tyr Pro Phe Tyr1 5 10 154297PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 429Asp Leu Arg Ser Gly Arg Tyr1 543013PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 430Gly Thr Ala Thr Phe Asp Trp Asn Phe Pro Phe Asp Ser1 5
1043118PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 431Gly Ser Gly Leu Val Val Tyr Ala Ile
Arg Val Gly Ser Gly Trp Phe1 5 10 15Asp Tyr43220PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 432Asp Pro Ser Ser Ser Gly Ser Tyr Tyr Met Glu Asp Ser Tyr
Tyr Tyr1 5 10 15Gly Met Asp Val 204338PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 433Glu Ala Leu Gly Ser Ser Trp Glu1 543410PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 434Gly Gly Tyr Asn Ser Asp Ala Phe Asp Ile1 5
1043512PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 435Asp Leu Ser Val Arg Ala Ile Asp Ala
Phe Asp Ile1 5 1043610PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 436Glu Gly Asn Glu Asp Leu Ala Phe Asp Ile1 5
1043719PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 437Asp Arg Leu Tyr Cys Gly Asn Asn Cys
Tyr Leu Tyr Tyr Tyr Tyr Gly1 5 10 15Met Asp Val43811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 438Asp Ser Tyr Gly Tyr Tyr Tyr Gly Met Asp Val1 5
1043913PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 439Asp Pro Tyr Ser Ser Ser
Trp His Asp Ala Phe Asp Ile1 5 1044010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 440Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr1 5
1044112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 441Ser Gln Ser Leu Val Tyr Thr Asp Gly
Asn Thr Tyr1 5 104427PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 442Ser Gln Gly Ile Ser
Ser Tyr1 54438PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 443Ser Gln Ser Ile Ser Gly
Ser Phe1 54447PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 444Ser Gln Ser Ile Ser Ser
Tyr1 54457PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 445Ser Gln Ser Ile Ser Ser
Tyr1 54469PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 446Ser Ser Gly Ser Ile Ala
Ser Asn Tyr1 54477PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 447Ser Gln Asp Ile Ser Asn
Tyr1 54487PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 448Ser Gln Asp Ile Ser Ser
Trp1 54497PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 449Ser Gln Asp Ile Ser Asn
Tyr1 54507PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 450Ser Gln Phe Ile Lys Lys
Asn1 54517PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 451Ser Gln Ser Ile Ser Ser
Tyr1 54527PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 452Ser Gln Gly Ile Ser Ser
Ala1 54538PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 453Ser Gln Ser Val Ser Ser
Ser Tyr1 54543PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 454Asp Val
Ser14553PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 455Lys Val
Ser14563PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 456Ala Ala
Ser14573PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 457Gly Ala
Ser14583PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 458Ala Ala
Ser14593PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 459Ala Ala
Ser14603PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 460Glu Asp
Asn14613PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 461Asp Ala
Ser14623PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 462Ala Ala
Ser14633PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 463Asp Ala
Ser14643PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 464Asp Ala
Ser14653PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 465Ala Ala
Ser14663PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 466Asp Ala
Ser14673PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 467Gly Ala
Ser14689PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 468Tyr Thr Ser Ser Ser Thr
Leu Asp Val1 54696PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 469Gly Thr His Trp Ser Phe1
54706PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 470Leu Asn Ser Tyr Pro Tyr1
54716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 471Tyr Gly Ser Ser Pro Pro1
54726PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 472Ser Tyr Ser Thr Pro Trp1
54737PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 473Ser Tyr Ser Thr Pro Pro Trp1
54747PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 474Tyr Asp Ser Ser Asn Gln Val1
54756PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 475Tyr Asp Asn Leu Pro Leu1
54766PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 476Ser Tyr Ser Thr Pro Leu1
54776PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 477Ala Tyr Ser Thr Pro Phe1
54786PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 478His Asp Asn Leu Pro Leu1
54797PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 479Ser Tyr Ser Thr Pro Pro Leu1
54806PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 480Phe Asn Asn Tyr Pro Leu1
54816PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 481Tyr Gly Ser Ser Pro Leu1
548241PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 482Arg Ser Lys Arg Ser Arg Leu Leu
His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala Ala
Tyr Arg Ser 35 40483123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 483aggagtaaga ggagcaggct cctgcacagt gactacatga
acatgactcc ccgccgcccc 60gggcccaccc gcaagcatta ccagccctat gccccaccac
gcgacttcgc agcctatcgc 120tcc 12348435PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 484Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn
Gly Glu Tyr1 5 10 15Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser
Arg Leu Thr Asp 20 25 30Val Thr Leu 35485105DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 485acaaaaaaga agtattcatc cagtgtgcac gaccctaacg
gtgaatacat gttcatgaga 60gcagtgaaca cagccaaaaa atccagactc acagatgtga
cccta 10548618PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 486Gly Ser Thr Ser Gly Ser
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr1 5 10 15Lys
Gly487521DNAUnknownsource/note="Description of Unknown Wild-type
PGK promoter polynucleotide" 487acccctctct ccagccacta agccagttgc
tccctcggct gacggctgca cgcgaggcct 60ccgaacgtct tacgccttgt ggcgcgcccg
tccttgtccc gggtgtgatg gcggggtgtg 120gggcggaggg cgtggcgggg
aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180gataaccggt
gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga
240cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg
gcgcttggcg 300ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc
ggccccccgg gtgttcccat 360cgccgcttct aggcccactg cgacgcttgc
ctgcacttct tacacgctct gggtcccagc 420cgcggcgacg caaagggcct
tggtgcgggt ctcgtcggcg cagggacgcg tttgggtccc 480gacggaacct
tttccgcgtt ggggttgggg caccataagc t 521488118DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 488acccctctct ccagccacta agccagttgc tccctcggct
gacggctgca cgcgaggcct 60ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc
gggtgtgatg gcggggtg 118489221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 489acccctctct ccagccacta agccagttgc tccctcggct
gacggctgca cgcgaggcct 60ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc
gggtgtgatg gcggggtgtg 120gggcggaggg cgtggcgggg aagggccggc
gacgagagcc gcgcgggacg actcgtcggc 180gataaccggt gtcgggtagc
gccagccgcg cgacggtaac g 221490324DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 490acccctctct ccagccacta agccagttgc tccctcggct
gacggctgca cgcgaggcct 60ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc
gggtgtgatg gcggggtgtg 120gggcggaggg cgtggcgggg aagggccggc
gacgagagcc gcgcgggacg actcgtcggc 180gataaccggt gtcgggtagc
gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240cgctcccatg
atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg
300ttccttggaa gggctgaatc cccg 324491422DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 491acccctctct ccagccacta agccagttgc tccctcggct
gacggctgca cgcgaggcct 60ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc
gggtgtgatg gcggggtgtg 120gggcggaggg cgtggcgggg aagggccggc
gacgagagcc gcgcgggacg actcgtcggc 180gataaccggt gtcgggtagc
gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240cgctcccatg
atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg
300ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg
gtgttcccat 360cgccgcttct aggcccactg cgacgcttgc ctgcacttct
tacacgctct gggtcccagc 420cg 42249221PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"VARIANT(1)..(3)/replace="
"MISC_FEATURE(1)..(3)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 492Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr
Cys Gly Asp Val Glu1 5 10 15Glu Asn Pro Gly Pro
2049322PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide"VARIANT(1)..(3)/replace="
"MISC_FEATURE(1)..(3)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 493Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
Gln Ala Gly Asp Val1 5 10 15Glu Glu Asn Pro Gly Pro
2049423PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide"VARIANT(1)..(3)/replace="
"MISC_FEATURE(1)..(3)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 494Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu
Lys Leu Ala Gly Asp1 5 10 15Val Glu Ser Asn Pro Gly Pro
2049525PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide"VARIANT(1)..(3)/replace="
"MISC_FEATURE(1)..(3)/note="Variant residues given in the sequence
have no preference with respect to those in the annotations for
variant positions" 495Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala1 5 10 15Gly Asp Val Glu Ser Asn Pro Gly Pro 20
25
* * * * *
References