U.S. patent application number 16/730951 was filed with the patent office on 2020-11-26 for methods and compositions for chimeric antigen receptor targeting cancer cells.
The applicant listed for this patent is The General Hospital Corporation, The University of North Carolina at Chapel Hill. Invention is credited to Gianpietro Dotti, Hongwei Du, Cristina Ferrone, Soldano Ferrone, Xinhui Wang.
Application Number | 20200369743 16/730951 |
Document ID | / |
Family ID | 1000005016190 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200369743 |
Kind Code |
A1 |
Dotti; Gianpietro ; et
al. |
November 26, 2020 |
METHODS AND COMPOSITIONS FOR CHIMERIC ANTIGEN RECEPTOR TARGETING
CANCER CELLS
Abstract
The present invention provides a chimeric antigen receptor (CAR)
that recognizes B7-H3 (CD276), as well as methods of use in the
treatment of diseases and disorders.
Inventors: |
Dotti; Gianpietro; (Chapel
Hill, NC) ; Ferrone; Soldano; (Boston, MA) ;
Du; Hongwei; (Chapel Hill, NC) ; Wang; Xinhui;
(Boston, MA) ; Ferrone; Cristina; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill
The General Hospital Corporation |
Chapel Hill
Boston |
NC
MA |
US
US |
|
|
Family ID: |
1000005016190 |
Appl. No.: |
16/730951 |
Filed: |
December 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16262490 |
Jan 30, 2019 |
10519214 |
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16730951 |
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16012432 |
Jun 19, 2018 |
10233226 |
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16262490 |
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62523105 |
Jun 21, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57492 20130101;
G01N 33/574 20130101; C07K 14/70532 20130101; C07K 14/70596
20130101; A61K 35/17 20130101; C07K 14/70521 20130101; A61K 38/00
20130101; A61P 35/00 20180101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61P 35/00 20060101 A61P035/00; G01N 33/574 20060101
G01N033/574; A61K 35/17 20060101 A61K035/17 |
Claims
1. A chimeric antigen receptor (CAR) comprising: a) a signal
peptide; b) a light chain variable region comprising the amino acid
sequence: TABLE-US-00006 DIVMTQSHKFMSTSIGARVSITCKASQDVRTAVAWYQQKPGQ
SPKWYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVY
YCQQHYGTPPWTFGGGTKLEIK
c) a linker peptide; d) a heavy chain variable region comprising
the amino acid sequence: TABLE-US-00007
EVQLVESGGGLVKPGGSLKLSCEASRFTFSSYAMSWVRQTPE
KRLEWVAAISGGGRYTYYPDSMKGRFTISRDNAKNFLYLQMS
SLRSEDTAMYYCARHYDGYLDYWGQGTTLTVSS;
e) a CD8.alpha. hinge polypeptide; f) a CD8.alpha. transmembrane
domain; g) a 4-1BB costimulatory domain; and h) a CD3.zeta.
signaling domain.
2. A chimeric antigen receptor (CAR) comprising: a) a signal
peptide; b) a light chain variable region comprising the amino acid
sequence: TABLE-US-00008
DIVMTQSHKFMSTSIGARVSITCKASQDVRTAVAWYQQKPGQSP
KWYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQ HYGTPPWTFGGGTKLEIK
c) a linker peptide; d) a heavy chain variable region comprising
the amino acid sequence: TABLE-US-00009
EVQLVESGGGLVKPGGSLKLSCEASRFTFSSYAMSWVRQTPEKR
LEWVAAISGGGRYTYYPDSMKGRFTISRDNAKNFLYLQMSSLRS
EDTAMYYCARHYDGYLDYWGQGTTLTVSS;
e) a CD8.alpha. hinge polypeptide; f) a CD8.alpha. transmembrane
domain; g) a CD28 costimulatory domain; and h) a CD3.zeta.
signaling domain.
3. A cell that expresses the CAR of claim 1.
4. The cell of claim 3, wherein the cell is selected from the group
consisting of a .alpha..beta.T cell, a natural killer (NK) cell, a
cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer
T (NKT) cell, a Th17 cell, and a .gamma..delta.T cell.
5. The cell of claim 4, wherein the cell is an autologous cell.
6. A composition comprising the cell of claim 4.
7. A polynucleotide encoding the CAR of claim 1.
8. A cell comprising the polynucleotide of claim 7.
9. The cell of claim 8, wherein the cell is selected from the group
consisting of a .alpha..beta.T cell, a natural killer (NK) cell, a
cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer
T (NKT) cell, a Th17 cell, and a .gamma..delta.T cell.
10. The cell of claim 9, wherein the cell is an autologous
cell.
11. A composition comprising the cell of claim 9.
12. A retroviral vector comprising the polynucleotide of claim
7.
13. A cell comprising the retroviral vector of claim 12.
14. The cell of claim 13, wherein the cell is selected from the
group consisting of a .alpha..delta.T cell, a natural killer (NK)
cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a
natural killer T (NKT) cell, a Th17 cell, and a .gamma..delta.T
cell.
15. The cell of claim 14, wherein the cell is an autologous
cell.
16. A composition comprising the cell of claim 14.
17. A cell that expresses the CAR of claim 2.
18. The cell of claim 17, wherein the cell is selected from the
group consisting of a .alpha..delta.T cell, a natural killer (NK)
cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a
natural killer T (NKT) cell, a Th17 cell, and a .gamma..delta.T
cell.
19. The cell of claim 18, wherein the cell is an autologous
cell.
20. A composition comprising the cell of claim 18.
21.-30. (canceled)
Description
STATEMENT OF PRIORITY
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/262,490, filed Jan. 30, 2019, (now
U.S. Pat. No. 10,519,214), which is a continuation application of
U.S. patent application Ser. No. 16/012,432, filed Jun. 19, 2018,
(now U.S. Pat. No. 10,233,226), which claims the benefit, under 35
U.S.C. .sctn. 119(e), of U.S. Provisional Application Ser. No.
62/523,105, filed Jun. 21, 2017, the entire contents of each of
which are incorporated by reference herein.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn. 1.821, entitled 5470-802CT_ST25.txt, 30,659 bytes in
size, generated on Jan. 29, 2019 and filed via EFS-Web, is provided
in lieu of a paper copy. This Sequence Listing is hereby
incorporated herein by reference into the specification for its
disclosures.
FIELD OF THE INVENTION
[0003] The present invention is directed to chimeric antigen
receptor (CAR) compositions and methods of their use in cancer
immunotherapy.
BACKGROUND OF THE INVENTION
[0004] B7-H3 (CD276) is a type I transmembrane protein and a member
of the B7 superfamily of ligands that has an inhibitory effect on
T-cells. B7-H3 is highly expressed in several human malignancies
and its expression correlates with poor survival. B7-H3 is of
interest as a target of chimeric antigen receptor (CAR)-redirected
T cells, since it is expressed in tumor cells, but has a restricted
distribution in normal tissues. In view of the broad tumor
expression of B7-H3, there is much interest in the applicability of
the B7-H3.CAR derived from particular monoclonal antibodies for the
treatment of many types of solid and liquid human tumors. This
invention describes compositions and methods for a chimeric antigen
receptor (CAR) that targets the B7-H3 (CD276) transmembrane
protein.
[0005] The present invention overcomes previous shortcomings in the
art by providing a chimeric antigen receptor (CAR) that targets the
B7-H3 (CD276) transmembrane protein and methods of its use in
treating cancer.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods and compositions for
the treatment of cancer, including treatment of cancer employing
immunotherapy. In particular cases, the immunotherapy includes T
lymphocytes engineered to target certain cancers.
[0007] Thus, in one embodiment, the present invention provides a
chimeric antigen receptor (CAR) comprising the amino acid
sequence:
TABLE-US-00001 (SEQ ID NO: 1)
MEFGLSWLFLVAILKGVQCDIVMTQSHKFMSTSIGARVSITC
KASQDVRTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGS
GSGTDFTFTISSVQAEDLAVYYCQQHYGTPPWTFGGGTKLEI
KGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCEASR
FTFSSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRF
TISRDNAKNFLYLQMSSLRSEDTAMYYCARHYDGYLDYWGQG
TTLTVSSTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR
LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR.
[0008] In another embodiment, the present invention provides a
chimeric antigen receptor (CAR) comprising the amino acid
sequence:
TABLE-US-00002 (SEQ ID NO: 2)
MEFGLSWLFLVAILKGVQCDIVMTQSHKFMSTSIGARVSITC
KASQDVRTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGS
GSGTDFTFTISSVQAEDLAVYYCQQHYGTPPWTFGGGTKLEI
KGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCEASR
FTFSSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRF
TISRDNAKNFLYLQMSSLRSEDTAMYYCARHYDGYLDYWGQG
TTLTVSSTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR
SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
QGLSTATKDTYDALHMQALPPR.
[0009] In a further embodiment, the present invention provides a
nucleic acid molecule encoding the CAR of this invention,
including, in some embodiments, the nucleotide sequence of SEQ ID
NO:3, which comprises a nucleotide sequence that encodes the amino
acid sequence of SEQ ID NO:1 and in some embodiments, the
nucleotide sequence of SEQ ID NO:4, which comprises a nucleotide
sequence that encodes the amino acid sequence of SEQ ID NO:2. The
present invention further provides vectors and cells comprising the
nucleic acid molecule of this invention. The present invention also
provides a nucleic acid molecule having the nucleotide sequence of
SEQ ID NO:5.
[0010] In a further embodiment, the present invention provides a
method of stimulating a T cell-mediated immune response to a B7-H3
expressing target cell population or tissue in a subject,
comprising administering to the subject an effective amount of the
nucleic acid molecule, vector and/or cell of this invention,
thereby stimulating a T cell-mediated immune response to the B7-H3
expressing target cell population or tissue in the subject.
[0011] In additional embodiments, the present invention provides a
method of providing an anti-tumor immunity in a subject, comprising
administering to the subject an effective amount of the nucleic
acid molecule, vector, and/or the cell of this invention, thereby
providing an anti-tumor immunity in the subject.
[0012] The present invention further provides a method of treating
a subject having a disease or disorder associated with elevated
expression of B7-H3 (CD276) by a cell of the subject, comprising
administering to the subject an effective amount of the nucleic
acid molecule, vector, and/or cell of this invention, thereby
treating the subject having the disease or disorder associated with
elevated expression of B7-H3 by the cell of the subject.
[0013] In an additional embodiment, the present invention provides
a method of generating a persisting population of genetically
engineered T cells in a subject (e.g., a subject diagnosed with
cancer), comprising administering to the subject a T cell
genetically engineered to express the CAR of this invention,
wherein the persisting population of genetically engineered T cells
persists in the subject following administration.
[0014] In a further embodiment, the present invention provides a
method of expanding a population of genetically engineered T cells
in a subject (e.g., a subject diagnosed with cancer), comprising
administering to the subject a T cell genetically engineered to
express a CAR of this invention, wherein the administered
genetically engineered T cell produces a population of progeny T
cells in the subject.
[0015] In an additional embodiment, the present invention provides
a method of treating cancer in a subject, comprising administering
to the subject an effective amount of the nucleic acid molecule,
vector and/or the cell of this invention, thereby treating cancer
in the subject.
[0016] The present invention also provides a method of targeting a
cancer cell and/or a cancer initiating cell (CIC) having a B7-H3
(CD276) antigen, comprising contacting the cancer cell and/or the
CIC with a cell comprising the CAR of this invention.
[0017] Also provided herein is a method of detecting cancer cells
and/or cancer initiating cells (CICs) in a cell sample, comprising:
a) contacting the cell sample with the CAR of this invention under
conditions whereby a binding complex can form; and b) detecting
formation of the binding complex, wherein detection of the binding
complex is indicative of cancer cells and/or CICs in the cell
sample.
[0018] Another embodiment of this invention is a method of
detecting cancer cells and/or cancer initiating cells (CICs) in a
subject, comprising: a) contacting a cell sample obtained from the
subject with the CAR of this invention under conditions whereby a
binding complex can form; and b) detecting formation of the binding
complex, wherein detection of the binding complex is indicative of
the presence of cancer cells and/or CICs in the subject.
[0019] Further embodiments of the invention provide related nucleic
acid molecules, recombinant expression vectors, host cells,
populations of cells, antibodies or antigen binding portions
thereof, antibody fragments and pharmaceutical compositions
relating to the CARs of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-C. B7-H3 is highly expressed in pancreatic cancer.
(1A) Immunohistochemistry of frozen tissues of normal human
pancreas and pancreatic ductal adenocarcinoma (PDAC). Staining was
performed using the anti-B7-H3 mAb 376.96, from which the B7-H3.CAR
was derived. The final concentration of the mAb was 1 .mu.g/mL.
Scale bars are 100 .mu.m. Human PDAC tumor cell lines (1B) and
primary pancreatic tumor cell lines derived from PDX (1C) were also
stained with anti-B7-H3 mAb 376.96, and the expression of the
antigen was assessed by flow cytometry.
[0021] FIG. 2. Limited expression of B7-H3 in normal human tissues.
Immunohistochemistry of frozen microarrays of normal human tissues.
Staining was performed using the anti-B7-H3 mAb 376.96. The final
concentration of the Ab was 1 .mu.g/mL. Representative
photomicrographs are shown. Black boxes indicate zoomed in cutout.
Data represent at least three sections per tissue. Scale bars are
200 .mu.m.
[0022] FIGS. 3A-G. B7-H3.CAR-T cells recognize human and mouse
B7-H3. (3A) Schematic structure of the retroviral vector SFG
encoding the B7-H3.CARs including either CD28 or 4-1BB
co-stimulatory domains. (3B) Representative expression of the
B7-H3.CARs in transduced human T cells. CAR expression was detected
using an anti-mouse-FAB antibody and analyzed by flow cytometry.
(3C) The human lymphoma tumor cell line Raji was engineered to
express either the two human isoforms of B7-H3 (2Ig-hB7-H3 or
4Ig-hB7-H3) or the mouse isoform (mB7-H3) via retrovirus gene
transfer. Single clones were then selected. The anti-B7-H3 mAb
376.96 recognizes both human and mouse B7-H3, as assessed by flow
cytometry. (3D) Wild type (WT) Raji cells and Raji cells modified
to express either human or mouse B7-H3 were co-cultured with either
control T cells or B7-H3.CAR-Ts (1:1 ratio). By day 5, tumor cells
(CD19+) and B7-H3.CAR-Ts (CD3+) were enumerated by flow cytometry.
(3E) Statistics of the tumor cell frequency by day 5 of co-culture
(n=4). (3F) IFN.gamma. and (3G) IL2 released by control T cells and
B7-H3.CAR-T cells after 24 hours of co-cultured with Raji cells as
measured by ELISA (n=4).
[0023] FIGS. 4A-F. B7-H3.CAR-Ts target PDAC cell lines in vitro.
(4A) Six PDAC cell lines were co-cultured with control (NT) or
B7-H3.CAR-Ts at the T cell to PDAC ratio of 1:5 or 1:10. PDAC tumor
cell lines were labeled with green fluorescent protein (GFP). By
day 7, PDAC (GFP+) and B7-H3.CAR-T cells (CD3+) were enumerated by
flow cytometry. (4B) Statistics of the tumor cell frequency for T
cell to PDAC ratio 1:5, and (4C) T cell to PDAC ratio 1:10 (n=4).
(4D) IFN.gamma. and (4E) IL2 released by control and B7-H3.CAR-Ts
after 24 hours co-cultured with PDAC as measured by ELISA (n=4).
(4F) CFSE-labeled B7-H3.CAR-Ts were co-cultured with PDAC for 5
days at 1:1 ratio. Proliferation of CAR-T cells was measured by
CFSE dilution by flow cytometry. CFSE-labeled B7-H3.CAR-T cells
alone were used as control, which is shown as filled gray peak.
[0024] FIGS. 5A-F. B7-H3.CAR-T cells target primary PDAC cell lines
derived from PDX. (5A) Three primary PDAC cell lines derived from
PDX were co-cultured with control T cells (NT) or B7-H3.CAR-T cells
at the T cell to PDAC ratio 1:5. PDAC tumor cell lines were labeled
with GFP. By day 7, tumor cells (GFP+) and B7-H3.CAR-T cells (CD3+)
were enumerated by flow cytometry. (5B) Statistics of the tumor
cell frequency after 7 days co-culture with either control or
B7-H3.CAR-T cells for T cell to PDAC ratio 1:5 (n=6), and (5C) for
T cell to PDAC ratio 1:10 (n=4). (5D) IFN.gamma. and (5E) IL2
released by either T cells or B7-H3.CAR-Ts after 24 hours of
co-cultured with PDAC as measured by ELISA (n=4). (5F) CFSE-labeled
B7-H3.CAR-T cells were co-cultured with PDAC for 5 days at 1:1
ratio. Proliferation of B7-H3.CAR-T cells was measured by CFSE
dilution and analyzed by flow cytometry. CFSE-labeled B7-H3.CAR-T
cells alone were used as control, which is shown as filled gray
peak.
[0025] FIGS. 6A-J. B7-H3.CAR-Ts showed antitumor activity in
xenograft model. (6A) Schema of the orthotopic mouse models. FFluc
labeled Panc-1 (2.times.10.sup.5/mouse) or BxPC-3
(1.times.10.sup.5/mouse) human PDAC tumor cell lines were implanted
into the pancreas of NSG mice. By day 12, mice were infused with
either control CD19.CAR-T cells or B7-H3.CAR-T cells encoding
either CD28 or 4-1BB co-stimulatory domains (10.sup.7 cells/mouse
by intravenous (i.v.) route). Tumor growth was monitored by
luminescence imaging weekly after T cell infusion. (6B)
Bioluminescence of Panc-1 orthotopic model. (6C) Representative
ultrasound (US) measurement of Panc-1 by day 50 after tumor implant
in mice treated with CD19.CAR-T cells. (6D) Bioluminescence signal
measurements of Panc-1. (6E) Bioluminescence of BxPC-3 orthotopic
model. (6F) Bioluminescence signal measurement of BxPC-3. (6G)
Kaplan-Meier survival curve analysis of the BxPC-3 orthotopic
model. (6H) Schema of the metastatic model of Panc-1. (6I)
Bioluminescence of Panc-1 metastatic model. (6J) Bioluminescence
signal measurement of metastatic Panc-1.
[0026] FIG. 7. Plasmid map and nucleotide sequence of plasmid
encoding CD28 version of CAR (SEQ ID NO:3) with translation into
amino acid sequence (SEQ ID NO:1).
[0027] FIG. 8. Plasmid map and nucleotide sequence of plasmid
encoding 4-1-BB version of CAR (SEQ ID NO:4) with translation into
amino acid sequence (SEQ ID NO:2).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention is explained in greater detail below.
This description is not intended to be a detailed catalog of all
the different ways in which the invention may be implemented, or
all the features that may be added to the instant invention. For
example, features illustrated with respect to one embodiment may be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that
embodiment. In addition, numerous variations and additions to the
various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure which do not
depart from the instant invention. Hence, the following
specification is intended to illustrate some particular embodiments
of the invention, and not to exhaustively specify all permutations,
combinations and variations thereof.
[0029] Unless the context indicates otherwise, it is specifically
intended that the various features of the invention described
herein can be used in any combination. Moreover, the present
invention also contemplates that in some embodiments of the
invention, any feature or combination of features set forth herein
can be excluded or omitted.
[0030] In the following description, certain details are set forth
such as specific quantities, sizes, etc. so as to provide a
thorough understanding of the present embodiments disclosed herein.
However, it will be obvious to those skilled in the art that the
present disclosure may be practiced without such specific details.
In many cases, details concerning such considerations and the like
have been omitted inasmuch as such details are not necessary to
obtain a complete understanding of the present disclosure and are
within the skills of persons of ordinary skill in the relevant
art.
[0031] The present invention is based on the discovery of a
chimeric antigen receptor (CAR) that targets cancer cells and/or
cancer initiating cells (CICs) having a B7-H3 antigen. Accordingly,
the present invention provides a chimeric antigen receptor (CAR)
that targets cancer cells and/or CICs having a B7-H3 antigen,
wherein the CAR comprises, consists essentially of and/or consists
of the components described herein.
[0032] Thus, in one embodiment, the present invention provides a
chimeric antigen receptor (CAR) comprising, consisting essentially
of, or consisting of the amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 1)
MEFGLSWLFLVAILKGVQCDIVMTQSHKFMSTSIGARVSITC
KASQDVRTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGS
GSGTDFTFTISSVQAEDLAVYYCQQHYGTPPWTFGGGTKLEI
KGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCEASR
FTFSSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRF
TISRDNAKNFLYLQMSSLRSEDTAMYYCARHYDGYLDYWGQG
TTLTVSSTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR
LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR
(B7-H3.CAR including CD28 co-stimulatory domain).
[0033] In another embodiment, the present invention provides a
chimeric antigen receptor (CAR) comprising, consisting essentially
of, or consisting of the amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 2)
MEFGLSWLFLVAILKGVQCDIVMTQSHKFMSTSIGARVSITC
KASQDVRTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGS
GSGTDFTFTISSVQAEDLAVYYCQQHYGTPPWTFGGGTKLEI
KGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCEASR
FTFSSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRF
TISRDNAKNFLYLQMSSLRSEDTAMYYCARHYDGYLDYWGQG
TTLTVSSTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR
SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR
(B7-H3.CAR including 4-1BB co-stimulatory domain).
[0034] In particular embodiments, the chimeric antigen receptor
(CAR) of this invention has one, two, three, four, or more
components, and in some embodiments the one, two, three, four or
more components facilitate targeting and/or binding of the CAR to
the B7-H3 antigen-comprising cancer cell and/or CIC, although in
some cases one or more components can be useful to promote and/or
maintain growth and/or maturity of the cell comprising the CAR.
[0035] The present invention additionally provides a nucleic acid
molecule encoding the CAR of this invention. In some embodiments,
the nucleic acid molecule can comprise the nucleotide sequence of
SEQ ID NO:3, which encodes a B7-H3 CAR including a CD28
co-stimulatory domain. In some embodiments, the nucleic acid
molecule can comprise the nucleotide sequence of SEQ ID NO:4, which
encodes a B7-H3 CAR including a 4-1BB co-stimulatory domain.
[0036] Further provided herein is a vector comprising the nucleic
acid molecule of this invention.
[0037] In some embodiments, the present invention provides a cell
comprising the CAR of this invention and in some embodiments, the
present invention provides a cell comprising the nucleic acid
molecule of this invention.
[0038] Nonlimiting examples of a cell of this invention include a
.alpha..beta.T cell, a natural killer (NK) cell, a cytotoxic T
lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT)
cell, a Th17 cell, a .gamma..delta.T cell, and any combination
thereof.
[0039] In some embodiments, the present invention provides a
cytotoxic T lymphocyte comprising a CAR that recognizes and binds
B7-H3 antigen. The cytotoxic T lymphocyte can be transduced with a
viral vector or transfected with a plasmid or nucleic acid
construct comprising a nucleotide sequence encoding the CAR of this
invention and in some embodiments the nucleotide sequence can be
SEQ ID NO:3 and/or SEQ ID NO:4.
[0040] In certain embodiments, the present invention includes T
lymphocytes engineered to comprise a chimeric antigen receptor
having an antibody, antigen binding fragment and/or engineered
antibody specific for B7-H3, part or all of a cytoplasmic signaling
domain, and/or part or all of one or more costimulatory molecules,
for example endodomains of costimulatory molecules. In specific
embodiments, the antibody for B7-H3 is a single-chain variable
fragment (scFv), although in certain aspects the antibody can be
directed at other target antigens on the cell surface, such as HER2
or CD19, for example. In certain embodiments, a cytoplasmic
signaling domain, such as those derived from the T cell receptor
.zeta.-chain, can be included as at least part of the chimeric
antigen receptor in order to produce stimulatory signals for T
lymphocyte proliferation and effector function following engagement
of the chimeric antigen receptor with the target antigen. Examples
would include, but are not limited to, endodomains from
co-stimulatory molecules such as CD28, 4-1BB, and OX40 or the
signaling components of cytokine receptors such as interleukin 7
(IL7), interleukin 15 (IL15) and interleukin 12 (IL12). In
particular embodiments, costimulatory molecules are employed to
enhance the activation, proliferation and/or cytotoxicity of T
cells produced by the CAR after antigen engagement. In specific
embodiments, the costimulatory molecules can be CD28, OX40, and
4-1BB and cytokine receptors. Nonlimiting examples of cytokine
receptors of this invention include IL7 and IL15.
[0041] Genetic engineering of human T lymphocytes to express
tumor-directed chimeric antigen receptors (CAR) can produce
antitumor effector cells that bypass tumor immune escape mechanisms
that are due to abnormalities in protein-antigen processing and
presentation.
[0042] In certain embodiments, the present invention provides cells
specific for the B7-H3 antigen, wherein said cells have a chimeric
antigen receptor on the cell surface that is produced by joining an
extracellular antigen-binding domain derived from the
B7-H3-specific antibody 376.96 to a cytoplasmic signaling domain
derived from the T-cell receptor zeta-chain, and endodomains of the
costimulatory molecules CD28 and/or 4-1BB, as nonlimiting
examples.
[0043] In some embodiments, the CAR of this invention can comprise,
consist essentially of and/or consist of the effector domain of the
T cell receptor zeta chain or a related signal transduction
endodomain derived from a T cell receptor. In some embodiments the
chimeric antigen receptor is encoded by the nucleotide sequence of
SEQ ID NO:3 or SEQ ID NO:4. Thus, the present invention further
provides a vector (e.g., a viral vector) comprising the nucleotide
sequence of SEQ ID NO:3 and/or SEQ ID NO:4 and the T lymphocytes of
this invention can be transduced with a viral vector comprising the
nucleotide sequence of SEQ ID NO:3 and/or SEQ ID NO:4 under
conditions whereby the chimeric antigen receptor is produced in the
T lymphocyte.
[0044] As used herein, the term "co-stimulatory molecule" refers to
a molecular component that promotes activation, proliferation and
effector function of a T cell after engagement of an antigen
specific receptor. In some embodiments, the CAR of this invention
can comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)
co-stimulatory molecules and/or active fragments thereof,
nonlimiting examples of which include CD28, OX40, 4-1BB or any
other co-stimulatory molecule and/or active fragment thereof now
known or later identified, singly or in any combination.
[0045] In further embodiments, the chimeric antigen receptor (CAR)
of this invention can further comprise a detectable moiety as would
be known in the art and/or an effector molecule, nonlimiting
examples of which include a drug, a toxin, a small molecule, an
antibody, and/or an antibody fragment, singly or in any
combination.
[0046] As used herein, the term "cytoplasmic signaling domain"
refers to the component of a co-stimulatory molecule or cytokine
receptor that exists inside the cell and is responsible for
transducing the external signal received to the internal metabolic
processes of the cell, thereby altering its phenotype and
function.
[0047] In some embodiments of the present invention, the
overexpression of B7-H3 by cancer cells allows these cells to be
targeted in vitro and in vivo by B7-H3 CAR-expressing T cells, and
in some embodiments, incorporation of endodomains (e.g., from both
CD28 and OX40 molecules and/or from CD28 and/or from 4-1BB)
mediates co-stimulation of the T lymphocytes, inducing T cell
activation, proliferation, and/or cytotoxicity against
B7-H3-positive cancer and/or CIC cells.
[0048] In particular embodiments of the invention, there are
methods for killing cancer cells using genetically manipulated
T-cells that express a chimeric antigen receptor (CAR) directed
against the antigen B7-H3. In some embodiments, engagement (antigen
binding) of this CAR leads to activation of the linked T-cell
receptor C chain and the costimulatory molecules CD28 and
4-1BB.
[0049] In particular embodiments of the invention, the CAR receptor
comprises a single-chain variable fragment (scFv) that recognizes
B7-H3. The skilled artisan recognizes that scFv is a fusion protein
of the variable regions of the heavy (VH) and light chains (VL) of
immunoglobulins, connected with a short linker peptide of ten to
about 25 amino acids. The linker may be rich in glycine for
flexibility and/or it may have serine or threonine for solubility,
in certain cases. In a particular embodiment, the 376.96 scFv
antibody is used in the CAR of this invention. The scFv may be
generated by methods known in the art.
[0050] In certain aspects, one can use cytokine exodomains or other
ligand/receptor molecules as exodomains to provide targeting to the
tumor cells.
[0051] The skilled artisan recognizes that T cells utilize
co-stimulatory signals that are antigen non-specific to become
fully activated. In particular cases they are provided by the
interaction between co-stimulatory molecules expressed on the
membrane of an antigen presenting cell (APC) and the T cell. In
specific embodiments, the one or more costimulatory molecules in
the chimeric antigen receptor come from the B7/CD28 family, TNF
superfamily, or the signaling lymphocyte activation molecule (SLAM)
family. Exemplary costimulatory molecules include one or more of
the following in any combination: B7-1/CD80; CD28; B7-2/CD86;
CTLA-4; B7-H1/PD-L1; ICOS; B7-H2; PD-1; B7-H3; PD-L2; B7-H4; PDCD6;
BTLA; 4-1BB/TNFRSF9/CD137; CD40 Ligand/TNFSF5; 4-1BB Ligand/TNFSF9;
GITR/TNFRSF18; BAFF/BLyS/TNFSF13B; GITR Ligand/TNFSF18; BAFF
R/TNFRSF13C; HVEM/TNFRSF14; CD27/TNFRSF7; LIGHT/TNFSF14; CD27
Ligand/TNFSF7; OX40/TNFRSF4; CD30/TNFRSF8; OX40 Ligand/TNFSF4; CD30
Ligand/TNFSF8; TAC1/TNFRSF13B; CD40/TNFRSF5; 2B4/CD244/SLAMF4;
CD84/SLAMF5; BLAME/SLAMF8; CD229/SLAMF3; CD2 CRACC/SLAMF7;
CD2F-10/SLAMF9; NTB-A/SLAMF6; CD48/SLAMF2; SLAM/CD150; CD58/LFA-3;
CD2; Ikaros; CD53; Integrin alpha 4/CD49d; CD82/Kai-1; Integrin
alpha 4 beta 1; CD90/Thy1; Integrin alpha 4 beta 7/LPAM-1; CD96;
LAG-3; CD160; LMIR1/CD300A; CRTAM; TCL1A; DAP12; TIM-1/KIM-1/HAVCR;
Dectin-1/CLEC7A; TIM-4; DPPIV/CD26; TSLP; EphB6; TSLP R; and
HLA-DR.
[0052] The effector domain is a signaling domain that transduces
the event of receptor ligand binding to an intracellular signal
that partially activates the T lymphocyte. Absent appropriate
co-stimulatory signals, this event is insufficient for useful T
cell activation and proliferation. A nonlimiting example of an
effector domain of this invention is the effector domain of the T
cell receptor zeta chain.
[0053] The present invention additionally provides embodiments of
the amino acid sequences and nucleotide sequences of this invention
wherein the amino acid sequence and/or the nucleotide sequence has
at least 60% (e.g., 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or 100%) identity with the
amino acid sequence and/or nucleotide sequences described herein.
The present invention further encompasses all nucleotide sequences
that encode the amino acid sequences described herein.
[0054] In further embodiments, the present invention provides a
composition (e.g., a pharmaceutical composition) comprising,
consisting essentially of and/or consisting of the CAR of this
invention, the nucleic acid molecule of this invention, the vector
of this invention and/or the cell of this invention, in a
pharmaceutically acceptable carrier.
[0055] The present invention also provides methods employing the
CAR of this invention. Thus, in one embodiment, the present
invention provides a method of stimulating a T cell-mediated immune
response to a B7-H3 expressing target cell population and/or tissue
in a subject, comprising administering to the subject an effective
amount of the CAR of this invention, the nucleic acid molecule of
this invention, the vector of this invention, and/or the cell of
this invention, thereby stimulating a T cell-mediated immune
response to the B7-H3 expressing target cell population and/or
tissue in the subject.
[0056] In another embodiment, the present invention provides a
method of providing an anti-tumor immunity (e.g., an immune
response to tumor cells) in a subject, comprising administering to
the subject an effective amount of the CAR of this invention, the
nucleic acid molecule of this invention, the vector of this
invention, and/or the cell of this invention, thereby providing an
anti-tumor immunity in the subject.
[0057] In a further embodiment, the present invention provides a
method of treating a subject having a disease or disorder
associated with elevated expression of B7-H3 (CD276) by a cell of
the subject, comprising administering to the subject an effective
amount of the CAR of this invention, the nucleic acid molecule of
this invention, the vector of this invention, and/or the cell of
this invention, thereby treating the subject having the disease or
disorder associated with elevated expression of B7-H3 by the cell
of the subject.
[0058] In addition, the present invention provides a method of
generating a population of genetically engineered T cells in a
subject (e.g., a subject diagnosed with cancer and/or otherwise in
need thereof), comprising administering to the subject a T cell
genetically engineered to express the CAR of this invention,
wherein the population of genetically engineered T cells persists
in the subject for a period of time (e.g., at least one week, one
month two months, three months, four months, five months, nine
months, one year, two years, five years, etc.) following
administration to the subject.
[0059] Additionally provided herein is a method of expanding a
population of genetically engineered cells in a subject (e.g., a
subject diagnosed with cancer and/or a subject in need thereof),
comprising administering to the subject a cell genetically
engineered to express the CAR of this invention, wherein the
administered genetically engineered cell produces a population of
progeny cells in the subject.
[0060] In additional embodiments of this invention, a method is
provided of treating cancer in a subject (e.g., a subject in need
thereof), comprising administering to the subject an effective
amount of the CAR of this invention, the nucleic acid molecule of
this invention, the vector of this invention, and/or the cell of
this invention, thereby treating cancer in the subject. In some
embodiments, the subject of this method has had and/or is having
therapy for cancer.
[0061] Thus, in an additional embodiment of this invention, the
present invention provides a method of treating cancer in a
subject, comprising administering to the subject cytotoxic T
lymphocytes having a chimeric antigen receptor that recognizes a
B7-H3 antigen on the surface of cancer cells and/or cancer
initiating cells (CICs).
[0062] In further embodiments of this invention, a method is
provided of preventing cancer in a subject (e.g., a subject in need
thereof), comprising administering to the subject an effective
amount of the CAR of this invention, the nucleic acid molecule of
this invention, the vector of this invention, and/or the cell of
this invention, thereby preventing cancer in the subject.
[0063] In one embodiment, the present invention provides a method
of targeting a cancer cell and/or a cancer initiating cell (CIC)
having a B7-H3 (CD276) antigen, comprising providing to the cancer
cell and/or the CIC or contacting the cancer cell and/or the CIC
with a cell comprising the CAR of this invention.
[0064] In some embodiments of this invention, the cell of this
invention (e.g., a .alpha..beta.T cell, a natural killer (NK) cell,
a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural
killer T (NKT) cell, a Th17 cell, a .gamma..delta.T cell) can be an
autologous cell from the subject to whom treatment is administered.
In some embodiments, the cell of this invention can be from a
different individual of the same species as the subject receiving
treatment or from an individual of a different species from the
subject receiving treatment.
[0065] In the methods of this invention, the cancer cell and/or CIC
can be in vitro, ex vivo, and/or in vivo. In some embodiments, the
cell can be in a subject. In some embodiments, the cell can be an
autologous cell. In some embodiments, the cell is not an autologous
cell. In some embodiments, the cell is of the same species of the
subject. In some embodiments, the cell is of a species that is
different than the species of the subject.
[0066] In further embodiments, the present invention provides a
method of detecting cancer cells and/or cancer initiating cells
(CICs) in a cell sample, comprising: a) contacting the cell sample
with the CAR of this invention under conditions whereby a binding
complex can form; and b) detecting formation of the binding
complex, wherein detection of the binding complex is indicative of
cancer cells and/or CICs in the cell sample.
[0067] In another embodiment, the present invention provides a
method of detecting cancer cells and/or cancer initiating cells
(CICs) in a subject, comprising: a) contacting a cell sample
obtained from the subject with the CAR of this invention under
conditions whereby a binding complex can form; and b) detecting
formation of the binding complex, wherein detection of the binding
complex is indicative of the presence of cancer cells and/or CICs
in the subject.
[0068] In methods of this invention, the cell can be an
.alpha..beta.T cell, a natural killer (NK) cell, a cytotoxic T
lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT)
cell, a Th17 cell, .alpha..delta.T cell and any combination
thereof. In some embodiments, the cell can be an autologous cell.
In some embodiments, the cell can be of the same species of the
subject and in some embodiments, the cell can be of a species that
is different than the species of the subject.
[0069] In some embodiments, the cancer of this invention can be a
cancer associated with increased expression or overexpression of
B7-H3 antigen and in some embodiments, cancer cells and CICs of
this invention can overexpress the B7-H3 antigen relative to a
noncancerous cell or a cancer cell of a cancer that is not
associated with increased expression or overexpression of B7-H3
antigen.
[0070] In some embodiments, the cancer cells and/or CICs of this
invention can be contacted with LDE225, an inhibitor of the sonic
hedgehog homolog (SHH) pathway, before, during and/or after
contacting with the CAR of this invention.
[0071] The term "cancer" as used herein is defined as 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,
[0072] Nonlimiting examples of a cancer that can be treated
according to the methods of this invention include B cell lymphoma,
T cell lymphoma, myeloma, leukemia, hematopoietic neoplasias,
thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins
lymphoma, Hodgkins lymphoma, skin cancer, uterine cancer, cervical
cancer, endometrial cancer, adenocarcinoma, breast cancer,
pancreatic cancer, colorectal cancer, anal cancer, lung cancer,
renal cancer, bladder cancer, liver cancer, prostate cancer,
ovarian cancer, primary or metastatic melanoma, squamous cell
carcinoma, basal cell carcinoma, brain cancer, angiosarcoma,
hemangiosarcoma, head and neck carcinoma, thyroid carcinoma, soft
tissue sarcoma, bone sarcoma, testicular cancer, gastrointestinal
cancer, and any other cancer now known or later identified (see,
e.g., Rosenberg (1996) Ann. Rev. Med. 47:481-491, the entire
contents of which are incorporated by reference herein).
[0073] The term "autoimmune disease" as used herein is defined as a
disorder that results from an autoimmune response. An autoimmune
disease is the result of an inappropriate and excessive response to
a self-antigen. Examples of autoimmune diseases include but are not
limited to, Addision's disease, alopecia greata, ankylosing
spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's
disease, diabetes (Type 1), dystrophic epidermolysis bullosa,
epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr
syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus
erythematosus, multiple sclerosis, myasthenia gravis, pemphigus
vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,
sarcoidosis, scleroderma, Sjogren's syndrome,
spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,
pernicious anemia, ulcerative colitis, among others.
[0074] In certain embodiments of the invention, methods of the
present invention for clinical aspects are combined with other
agents effective in the treatment of hyperproliferative disease,
such as anti-cancer agents. An "anti-cancer" agent is capable of
negatively affecting cancer in a subject, for example, by killing
cancer cells, inducing apoptosis in cancer cells, reducing the
growth rate of cancer cells, reducing the incidence or number of
metastases, reducing tumor size, inhibiting tumor growth, reducing
the blood supply to a tumor or cancer cells, promoting an immune
response against cancer cells or a tumor, preventing or inhibiting
the progression of cancer, and/or increasing the lifespan of a
subject with cancer. More generally, these other compositions would
be provided in a combined amount effective to kill or inhibit
proliferation of the cancer cell. This process may involve
contacting the cancer cells with the nucleic acid molecule, vector
and/or cell of this invention and the agent(s) or multiple
factor(s) at the same time. This may be achieved by contacting the
cell with a single composition or pharmacological formulation that
includes both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes the nucleic acid molecule, vector and/or cell
of the invention and the other composition includes the second
agent(s).
[0075] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir. In the context of the present invention, it is
contemplated that cell therapy could be used similarly in
conjunction with chemotherapeutic, radiotherapeutic, or
immunotherapeutic intervention, in addition to other pro-apoptotic
or cell cycle regulating agents.
[0076] Alternatively, the present inventive therapy may precede
and/or follow the other agent treatment(s) by intervals ranging
from minutes to weeks. In embodiments where the other agent and
present invention are applied separately to the individual, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with the multiple modalities within
about 12-24 h of each other and, more preferably, within about 6-12
h of each other. In some situations, it may be desirable to extend
the time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0077] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the inventive cell therapy.
[0078] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, abraxane,
altretamine, docetaxel, herceptin, methotrexate, novantrone,
zoladex, cisplatin (CDDP), carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the
foregoing.
[0079] In specific embodiments, chemotherapy for B7-H3 positive
cancer is employed in conjunction with the invention, for example
before, during and/or after administration of the invention.
[0080] Other factors that cause DNA damage and have been used
extensively include what are commonly known as gamma-rays, X-rays,
and/or the directed delivery of radioisotopes to tumor cells. Other
forms of DNA damaging factors are also contemplated such as
microwaves and UV-irradiation. It is most likely that all of these
factors affect a broad range of damage on DNA, on the precursors of
DNA, on the replication and repair of DNA, and on the assembly and
maintenance of chromosomes. Dosage ranges for X-rays range from
daily doses of 50 to 200 roentgens for prolonged periods of time (3
to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges
for radioisotopes vary widely, and depend on the half-life of the
isotope, the strength and type of radiation emitted, and the uptake
by the neoplastic cells.
[0081] The terms "contacted with," "provided to" and "exposed to,"
when applied to a cell, are used herein to describe the process by
which a therapeutic agent (e.g., a CAR) is delivered to a target
cell and/or are placed in direct juxtaposition with the target
cell, e.g., under conditions that facilitate binding of the CAR to
the target antigen in and/or on the target cell. In some
embodiments, chemotherapy and/or radiation therapy can also be
included before, after and/or during the contacting or exposing or
providing to step to achieve cell killing or stasis, wherein both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0082] Immunotherapeutics generally rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually affect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0083] Immunotherapy could thus be used as part of a combined
therapy, in conjunction with the present cell therapy. The general
approach for combined therapy is discussed herein. Generally, the
tumor cell must bear some marker that is amenable to targeting,
i.e., is not present on the majority of other cells. Many tumor
markers exist and any of these may be suitable for targeting in the
context of the present invention. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0084] Immunotherapy for a cancer of this invention may include
interleukin-2 (IL-2) or interferon (IFN), for example.
[0085] In yet another embodiment, the secondary treatment can be a
gene therapy in which a therapeutic polynucleotide is administered
before, after, and/or at the same time as the present invention
clinical embodiments. A variety of expression products are
encompassed within the invention, including inducers of cellular
proliferation, inhibitors of cellular proliferation, or regulators
of programmed cell death.
[0086] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0087] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0088] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0089] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abilities of the present invention by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increasing intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
Definitions
[0090] As used herein, "a," "an" and "the" can mean one or more
than one, depending on the context in which it is used. For
example, "a" cell can mean one cell or multiple cells.
[0091] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0092] Furthermore, the term "about," as used herein when referring
to a measurable value such as an amount of a compound or agent of
this invention, dose, time, temperature, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, .+-.0.5%,
or even .+-.0.1% of the specified amount.
[0093] As used herein, the transitional phrase "consisting
essentially of" means that the scope of a claim is to be
interpreted to encompass the specified materials or steps recited
in the claim, "and those that do not materially affect the basic
and novel characteristic(s)" of the claimed invention. See, In re
Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis
in the original); see also MPEP .sctn. 2111.03. Thus, the term
"consisting essentially of" when used in a claim of this invention
is not intended to be interpreted to be equivalent to
"comprising."
[0094] Also as used herein, "one or more" means one, two, three,
four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, etc.
[0095] Subjects that may be treated by the present invention
include both human subjects for medical and/or therapeutic purposes
and animal subjects for veterinary and drug screening and
development purposes. Other suitable animal subjects are, in
general, mammalian subjects such as primates, bovines, ovines,
caprines, porcines, equines, felines, canines, lagomorphs, rodents
(e.g., rats and mice), etc. Human subjects are the most preferred.
Human subjects include fetal, neonatal, infant, juvenile, adult and
geriatric subjects.
[0096] The term "anti-tumor effect" as used herein, refers to a
biological effect which can be manifested by a decrease in tumor
volume, a decrease in the number of tumor cells, a decrease in the
proliferation rate, a decrease in the number of metastases, an
increase in life expectancy, and/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 to
prevent and/or delay the occurrence of tumor in the first
place.
[0097] The term "auto-antigen" means, in accordance with the
present invention, any self-antigen which is mistakenly recognized
by the immune system as being foreign. Auto-antigens comprise, but
are not limited to, cellular proteins, phosphoproteins, cellular
surface proteins, cellular lipids, nucleic acids, and
glycoproteins, including cell surface receptors.
[0098] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to whom it is later
to be re-introduced.
[0099] "Allogeneic" refers to a graft derived from a different
animal of the same species,
[0100] "Xenogeneic" refers to a graft derived from an animal of a
different species.
[0101] "Treat" or "treating" as used herein refers to any type of
treatment that imparts a benefit to a subject that has a disease or
disorder or is at risk of having or developing the disease or
disorder, including, for example, improvement in the condition of
the subject (e.g., in one or more symptoms) and/or slowing of the
progression of symptoms, etc.
[0102] As used herein, "prevent," "preventing" or "prevention"
includes prophylactic treatment of the subject to prevent the onset
or advancement of a disorder, as determined, e.g., by the absence
or delay in the manifestation of symptoms associated with the
disorder. As used herein, "prevent," "preventing" or "prevention"
is not necessarily meant to imply complete abolition of
symptoms.
[0103] "Treatment effective amount," "effective amount," "amount
effective to treat" or the like as used herein means an amount of
the antibody or fragment thereof or CAR or cell of this invention
sufficient to produce a desirable effect upon a patient that has a
disease, disorder and/or condition of this invention. This includes
improvement in the condition of the patient (e.g., in one or more
symptoms), delay in the progression of the disease, etc.
[0104] "Pharmaceutically acceptable" as used herein means that the
compound or composition is suitable for administration to a subject
to achieve the treatments described herein, without unduly
deleterious side effects in light of the severity of the disease
and necessity of the treatment.
[0105] "Antibody" or "antibodies" as used herein refers to all
types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
The term "immunoglobulin" includes the subtypes of these
immunoglobulins, such as IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, etc. The antibodies may be of any species of origin,
including (for example) mouse, rat, rabbit, horse, or human, or may
be chimeric or humanized antibodies. The term "antibody" as used
herein includes antibody fragments which retain the capability of
binding to a target antigen, for example, Fab, F(ab').sub.2, and Fv
fragments, and the corresponding fragments obtained from antibodies
other than IgG. Such fragments are also produced by known
techniques. In some embodiments antibodies may be coupled to or
conjugated to a detectable group or therapeutic group in accordance
with known techniques.
[0106] Furthermore, the term "antibody" as used herein, is intended
to refer to immunoglobulin molecules comprising four polypeptide
chains, two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. Each heavy chain comprises a
heavy chain variable region (abbreviated herein as HCVR or VH) and
a heavy chain constant region. The heavy chain constant region
comprises three domains, CH1, CH2 and CH3. Each light chain
comprises a light chain variable region (abbreviated herein as LCVR
or VL) and a light chain constant region. The light chain constant
region comprises one domain (CL1). The VH and VL regions can be
further subdivided into regions of hypervariability, termed
complementary determining regions (CDR), interspersed with regions
that are more conserved, termed framework regions (FR). In various
embodiments of the antibody or antigen binding fragment thereof of
the invention, the FRs may be identical to the human germline
sequences, or may be naturally or artificially modified. Each VH
and VL is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0107] In general, the antibodies and antigen binding fragments
thereof of the present invention possess very high affinities,
typically possessing K.sub.D values of from about 10.sup.-8 through
about 10.sup.-12 M or higher, for example, at least 10.sup.-8M, at
least 10.sup.-9 M, at least 10.sup.-10 M, at least 10.sup.-11 M, or
at least 10.sup.-12 M, when measured by binding to antigen
presented on cell surface.
[0108] The antibodies and antigen binding fragments thereof of the
present invention possess very high affinities, typically
possessing EC.sub.50 values of from about 10.sup.-8 through about
10.sup.-12 M or higher, for example, at least 10.sup.-8M, at least
10.sup.-9 M, at least 10.sup.-10 M, at least 10.sup.-11 M, or at
least 10.sup.-12 M, when measured by binding to antigen presented
on cell surface.
[0109] The term "antigen-binding portion" or "antigen-binding
fragment" of an antibody (or simply "antibody portion" or "antibody
fragment"), as used herein, refers to one or more fragments,
portions or domains of an antibody that retain the ability to
specifically bind to an antigen. It has been shown that fragments
of a full-length antibody can perform the antigen-binding function
of an antibody. Examples of binding fragments encompassed within
the term "antigen-binding portion" of an antibody include (i) an
Fab fragment, a monovalent fragment consisting of the VL, VH, CL1
and CH1 domains; (ii) an F(ab').sub.2 fragment, a bivalent fragment
comprising two F(ab)' fragments linked by a disulfide bridge at the
hinge region; (iii) an Fd fragment consisting of the VH and CH1
domains; (iv) an Fv fragment consisting of the VL and VH domains of
a single aim of an antibody; (v) a dAb fragment (Ward et al. (1989)
Nature 241:544-546), which consists of a VH domain; and (vi) an
isolated complementary determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single contiguous chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies arc also intended to be encompassed within the term
"antigen-binding portion" of an antibody. Other forms of single
chain antibodies, such as diabodies, are also encompassed (see
e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA
90:6444-6448).
[0110] The term "epitope" refers to an antigenic determinant that
interacts with a specific antigen binding site in the variable
region of an antibody molecule known as a paratope. A single
antigen may have more than one epitope. Epitopes may be either
conformational or linear. A conformational epitope is produced by
spatially juxtaposed amino acids from different segments of one (or
more) linear polypeptide chain(s). A linear epitope is an epitope
produced by adjacent amino acid residues in a polypeptide chain. In
certain embodiments, an epitope may include other moieties, such as
saccharides, phosphoryl groups, or sulfonyl groups on the
antigen.
[0111] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, scFv antibodies, and multispecific
antibodies formed from antibody fragments.
[0112] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations,
[0113] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations,
.kappa. and .lamda. light chains refer to the two major antibody
light chain isotypes.
[0114] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. 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 synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0115] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. 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 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. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a biological fluid.
[0116] Amino acid as used herein refers to a compound having a free
carboxyl group and a free unsubstituted amino group on the a
carbon, which may be joined by peptide bonds to form a peptide
active agent as described herein. Amino acids may be standard or
non-standard, natural or synthetic, with examples (and their
abbreviations) including but not limited to:
[0117] Asp=D=Aspartic Acid
[0118] Ala=A=Alanine
[0119] Arg=R=Arginine
[0120] Asn=N=Asparagine
[0121] Cys=C=Cysteine
[0122] Gly=G=Glycine
[0123] Glu=E=Glutamic Acid
[0124] Gln=Q=Glutamine
[0125] His=H=Histidine
[0126] Ile=I=Isoleucine
[0127] Leu=L=Leucine
[0128] Lys=K=Lysine
[0129] Met=M=Methionine
[0130] Phe=F=Phenylalanine
[0131] Pro=P=Proline
[0132] Ser=S=Serine
[0133] Thr=T=Threonine
[0134] Trp=W=Tryptophan
[0135] Tyr=Y=Tyrosine
[0136] Val=V=Valine
[0137] Orn=Ornithine
[0138] Nal=2-napthylalanine
[0139] Nva=Norvaline
[0140] Nle=Norleucine
[0141] Thi=2-thienylalanine
[0142] Pcp=4-chlorophenylalanine
[0143] Bth=3-benzothienyalanine
[0144] Bip=4,4'-biphenylalanine
[0145] Tic=tetrahydroisoquinoline-3-carboxylic acid
[0146] Aib=aminoisobutyric acid
[0147] Anb=.alpha.-aminonormalbutyric acid
[0148] Dip=2,2-diphenylalanine
[0149] Thz=4-Thiazolylalanine
[0150] All peptide sequences mentioned herein are written according
to the usual convention whereby the N-terminal amino acid is on the
left and the C-terminal amino acid is on the right. A short line
(or no line) between two amino acid residues indicates a peptide
bond.
[0151] Basic amino acid" refers to any amino acid that is
positively charged at a pH of 6.0, including but not limited to R,
K, and H.
[0152] Aromatic amino acid" refers to any amino acid that has an
aromatic group in the side-chain coupled to the alpha carbon,
including but not limited to F, Y, W, and H.
[0153] Hydrophobic amino acid" refers to any amino acid that has a
hydrophobic side chain coupled to the alpha carbon, including but
not limited to I, L, V, M, F, W and C, most preferably I, L, and
V.
[0154] Neutral amino acid" refers to a non-charged amino acid, such
as M, F, W, C and A.
[0155] As applied to polypeptides, the term "substantial
similarity" or "substantially similar" means that two peptide
sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, share at least 95% sequence
identity, even more preferably at least 98% or 99% sequence
identity. Preferably, residue positions, which are not identical,
differ by conservative amino acid substitutions. A "conservative
amino acid substitution" is one in which an amino acid residue is
substituted by another amino acid residue having a side chain (R
group) with similar chemical properties (e.g., charge or
hydrophobicity). In general, a conservative amino acid substitution
will not substantially change the functional properties of a
protein. In cases where two or more amino acid sequences differ
from each other by conservative substitutions, the percent or
degree of similarity may be adjusted upwards to correct for the
conservative nature of the substitution. Means for making this
adjustment are well-known to those of skill in the art. See, e.g.,
Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated
by reference. Examples of groups of amino acids that have side
chains with similar chemical properties include 1) aliphatic side
chains: glycine, alanine, valine, leucine and isoleucine; 2)
aliphatic-hydroxyl side chains: scrine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; 6) acidic side chains:
aspartate and glutamate, and 7) sulfur-containing side chains:
cysteine and methionine. Preferred conservative amino acids
substitution groups are: valine-leucine-1soleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine. Alternatively, a
conservative replacement is any change having a positive value in
the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992)
Science 256: 1443 45, herein incorporated by reference. A
"moderately conservative" replacement is any change having a
nonnegative value in the PAM250 log-likelihood matrix.
[0156] Sequence similarity for polypeptides is typically measured
using sequence analysis software. Protein analysis software matches
similar sequences using measures of similarity assigned to various
substitutions, deletions and other modifications, including
conservative amino acid substitutions. For instance, GCG software
contains programs such as GAP and BESTFIT which can be used with
default parameters to determine sequence homology or sequence
identity between closely related polypeptides, such as homologous
polypeptides from different species of organisms or between a wild
type protein and a mutein thereof. See, e.g., GCG Version 6.1.
Polypeptide sequences also can be compared using FASTA with default
or recommended parameters; a program in GCG Version 6.1. FASTA
(e.g., FASTA2 and FASTA3) provides alignments and percent sequence
identity of the regions of the best overlap between the query and
search sequences (Pearson (2000) supra). Another preferred
algorithm when comparing a sequence of the invention to a database
containing a large number of sequences from different organisms is
the computer program BLAST, especially BLASTP or TBLASTN, using
default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol.
215: 403 410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389
402, each of which is herein incorporated by reference in its
entirety.
[0157] "Therapeutic group" means any suitable therapeutic group,
including but not limited to radionuclides, chemotherapeutic agents
and cytotoxic agents.
[0158] "Radionuclide" as described herein may be any radionuclide
suitable for delivering a therapeutic dosage of radiation to a
tumor or cancer cell, including but not limited to .sup.227Ac,
.sup.211At, .sup.131Ba, .sup.77Br, .sup.109Cd, .sup.51Cr,
.sup.67Cu, .sup.165Dy, .sup.155Eu, .sup.153Gd, .sup.198Au,
.sup.166Ho, .sup.113mIn, .sup.115mIn, .sup.123I, .sup.125I,
.sup.131I, .sup.189Ir, .sup.191Ir, .sup.192Ir, .sup.194Ir,
.sup.52Fe, .sup.55Fe, .sup.59Fe, .sup.177Lu, .sup.109Pd, .sup.32P,
.sup.226Ra, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.46Se,
.sup.47Se, .sup.72Se, .sup.75Se, .sup.105Ag, .sup.89Sr, .sup.35S,
.sup.177Ta, .sup.117mSn, .sup.121Sn, .sup.166Yb, .sup.169Yb,
.sup.90Y, .sup.212Bi, .sup.119Sb, .sup.197H, .sup.97Ru, .sup.100Pd,
.sup.101mRh, and .sup.212Pb.
[0159] "Cytotoxic agent" as used herein includes but is not limited
to ricin (or more particularly the ricin A chain), aclacinomycin,
diphtheria toxin. Monensin, Verrucarin A, Abrin, Vinca alkaloids,
Tricothecenes, and Pseudomonas exotoxin A.
[0160] "Detectable group" as used herein includes any suitable
detectable group, such as radiolabels (e.g. .sup.35S, .sup.125I,
.sup.131I, etc.), enzyme labels (e.g., horseradish peroxidase,
alkaline phosphatase, etc.), fluorescence labels (e.g.,
fluorescein, green fluorescent protein, etc.), etc., as are well
known in the art and used in accordance with known techniques.
Formulations and Administration
[0161] For administration in the methods of use described below,
the active agent (e.g., the antibody or antigen-binding fragment
thereof, cell, nucleic acid molecule and/or vector of this
invention) will generally be mixed, prior to administration, with a
non-toxic, pharmaceutically acceptable carrier substance (e.g.,
normal saline or phosphate-buffered saline), and will be
administered using any medically appropriate procedure, e.g.,
parenteral administration (e.g., injection) such as by intravenous
or intra-arterial injection.
[0162] The active agents described above may be formulated for
administration in a pharmaceutical carrier in accordance with known
techniques. See, e.g., Remington, The Science And Practice of
Pharmacy (latest edition). In the manufacture of a pharmaceutical
formulation according to the invention, the active compound
(including the physiologically acceptable salts thereof) is
typically admixed with, inter alia, an acceptable carrier. The
carrier must, of course, be acceptable in the sense of being
compatible with any other ingredients in the formulation and must
not be deleterious to the subject. The carrier may be a liquid and
is preferably formulated with the compound as a unit-dose
formulation which may contain from 0.01 or 0.5% to 95% or 99% by
weight of the active compound. The carrier may be sterile or
otherwise free from contaminants that would be undesirable to
administer or deliver to a subject.
[0163] Formulations of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the active compound, which preparations are
preferably isotonic with the blood of the intended subject. These
preparations may contain anti-oxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended subject.
[0164] The active agents may be administered by any medically
appropriate procedure, e.g., normal intravenous or intra-arterial
administration. In certain cases, direct administration to a tumor
and/or a body cavity, orifice and/or tissue containing a tumor may
be desired.
[0165] Active agents may be provided in lyophylized form in a
sterile aseptic container or may be provided in a pharmaceutical
formulation in combination with a pharmaceutically acceptable
carrier, such as sterile pyrogen-free water or sterile pyrogen-free
physiological saline solution.
[0166] CAR-modified T cells of this invention may also serve as a
type of vaccine for ex vivo immunization and/or in vivo therapy in
a subject of this invention.
[0167] In some embodiments involving ex vivo immunization, at least
one of the following occurs in vitro prior to administering the
cell into a subject: i) expansion of the cells, ii) introducing a
nucleic acid encoding a CAR of this invention to the cells, and/or
iii) cryopreservation of the cells.
[0168] Ex vivo procedures are well known in the art and are
discussed more fully below. Briefly, cells are isolated from a
mammal (preferably a human) and genetically modified (i.e.,
transduced or transfected in vitro) with a vector expressing a CAR
of this invention. The resulting CAR-modified cell can be
administered to a subject of this invention to provide a
therapeutic benefit. In some embodiments, the subject can be a
human and the CAR-modified cell can be autologous with respect to
the subject who is the recipient of the CAR-modified cells.
Alternatively, the cells can be allogeneic, syngeneic or xenogeneic
with respect to the subject who is the recipient of the
CAR-modified cells.
[0169] In addition to using a cell-based vaccine for ex vivo
immunization, the present invention also provides compositions and
methods for in vivo immunization to elicit and/or enhance an immune
response directed against an antigen in a subject of this
invention.
[0170] Generally, the cells activated and expanded as described
herein can be used in the treatment and/or prevention of diseases
and/or disorders that arise in subjects; e.g., subjects who are
immunocompromised or at risking of becoming immunocompromised.
[0171] CAR-modified T 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
and/or other cytokines and/or cell populations. Briefly,
pharmaceutical compositions of the present invention may comprise a
target cell population 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, sterile
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
and/or glutathione; adjuvants (e.g., aluminum hydroxide) and/or
preservatives, singly or in any combination.
[0172] Pharmaceutical compositions of the present invention can be
administered in a manner appropriate to the disease to be treated
and/or prevented. The quantity and frequency of administration will
be determined by such factors as the condition of the subject, as
well as the type and severity of the subject's disease, although in
some embodiments, appropriate dosages may be determined by clinical
trials.
[0173] When "an immunologically effective amount," "an anti-tumor
effective amount," "a tumor-inhibiting effective amount," or a
"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). In some
embodiments, a pharmaceutical composition comprising cells of this
invention can be administered at a dosage of about 10.sup.3 to
about 10.sup.10 cells/kg body weight, and in some embodiments, the
dosage can be from about 10.sup.5 to about 10.sup.6 cells/kg body
weight, including all integer values (e.g., 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9) within those ranges.
[0174] The cell compositions of this invention can also be
administered multiple times (e.g., hourly, four times daily, three
times daily, two times daily, daily, twice weekly, three times
weekly, weekly, monthly, bi-monthly, semi-annually, annually, etc.)
at these dosages.
[0175] The cells of this invention 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)). The
optimal dosage and treatment regimen for a particular subject can
readily be determined by one skilled in the art of medicine by
monitoring the subject for signs of disease and adjusting the
treatment accordingly.
[0176] In some embodiments, it may be desirable to administer
activated T cells to a subject and then subsequently redraw blood
(or have an apheresis performed), activate T cells therefrom as
described herein, and reinfuse the subject with these activated and
expanded T cells. This process can be carried out multiple times,
e.g., weekly or every few weeks. In certain embodiments, T cells
can be activated from blood draws of from about 10 cc to about 400
cc. In certain embodiments, 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. Not to be bound by theory, using this multiple blood
draw/multiple reinfusion protocol may serve to select out certain
populations of T cells.
[0177] Administration of the compositions of this invention can be
carried out in any manner, including by aerosol inhalation,
injection, ingestion, transfusion, implantation and/or
transplantation. The compositions of this invention can be
administered to a patient subcutaneously, intradermally,
intratumorally, intranodally, intramedullary, intramuscularly, by
intravenous (i.v.) injection, and/or intraperitoneally. In some
embodiments, the T cell compositions of the present invention can
be administered to a subject by intradermal or subcutaneous
injection. In another embodiment, the T cell compositions of the
present invention can be administered by i.v. injection. In some
embodiments, the compositions of T cells can be injected directly
into a tumor, lymph node and/or site of infection.
[0178] In some embodiments of the present invention, cells
activated and expanded using the methods described herein, or other
methods known in the art where T cells are expanded to therapeutic
levels, can be administered to a subject in conjunction with (e.g.,
before, concurrently and/or following) any number of relevant
treatment modalities,
[0179] In some embodiments, the T cells of the invention may be
used in combination with chemotherapy, radiation, immunosuppressive
agents, such as cyclosporin, azathioprine, methotrexate,
mycophenolate, and FK506, antibodies, or other immunoablative
agents such as CAM PATH, anti-CD3 antibodies or other antibody
therapies, cytotoxin, fludaribine, cyclosporin, FK506, rapamycin,
mycophenolic acid, steroids, FR901228, cytokines, and/o
irradiation.
[0180] In some embodiments, the cell compositions of the present
invention can be administered to a patient in conjunction with
(e.g., before, concurrently and/or following) bone marrow
transplantation, T cell ablative therapy using either chemotherapy
agents such as fludarabine, external-beam radiation therapy (XRT),
cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another
embodiment, the cell compositions of the present invention can be
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 can receive an
infusion of the expanded immune cells of the present invention. In
an additional embodiment, expanded cells can be administered before
and/or following surgery.
[0181] In the treatment of cancers or tumors the CARs and/or
nucleic acid molecules encoding CARs of the present invention may
optionally be administered in conjunction with other, different,
cytotoxic agents such as chemotherapeutic or antineoplastic
compounds or radiation therapy useful in the treatment of the
disorders or conditions described herein (e.g., chemotherapeutics
or antineoplastic compounds). The other compounds may be
administered prior to, concurrently and/or after administration of
the antibodies or antigen binding fragments thereof of this
invention. As used herein, the word "concurrently" means
sufficiently close in time to produce a combined effect (that is,
concurrently may be simultaneously, or it may be two or more
administrations occurring before or after each other)
[0182] As used herein, the phrase "radiation therapy" includes, but
is not limited to, x-rays or gamma rays which are delivered from
either an externally applied source such as a beam or by
implantation of small radioactive sources.
[0183] Nonlimiting examples of suitable chemotherapeutic agents
which may be administered with the antibodies or antigen binding
fragments, cells, nucleic acid molecules and/or vectors as
described herein include daunomycin, cisplatin, verapamil, cytosine
arabinoside, aminopterin, democolcine, tamoxifen, Actinomycin D,
Alkylating agents (including, without limitation, nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas
and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide
(Cytoxan.RTM.), Ifosfamide, Melphalan, Chlorambucil,
[0184] Pipobroman, Triethylene-melamine,
Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,
Streptozocin, Dacarbazine, and Temozolomide; Antimetabolites
(including, without limitation, folic acid antagonists, pyrimidine
analogs, purine analogs and adenosine deaminase inhibitors):
Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine,
6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
Pentostatine, and Gemcitabine, Natural products and their
derivatives (for example, vinca alkaloids, antitumor antibiotics,
enzymes, lymphokines and epipodophyllotoxins): Vinblastine,
Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin,
Doxorubicin, Epirubicin, Idarubicin, Ara-C, paclitaxel (paclitaxel
is commercially available as Taxol.RTM.), Mithramycin,
Deoxyco-formycin, Mitomycin-C, L-Asparaginase, Interferons
(especially IFN-.alpha.), Etoposide, and Teniposide; Other
anti-proliferative cytotoxic agents are navelbene, CPT-11,
anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,
ifosamide, and droloxafine. Additional anti-proliferative cytotoxic
agents include, but are not limited to, melphalan, hexamethyl
melamine, thiotepa, cytarabin, idatrexate, trimetrexate,
dacarbazine, L-asparaginase, camptothecin, topotecan, bicalutamide,
flutamide, leuprolide, pyridobenzoindole derivatives, interferons,
and interleukins. Preferred classes of antiproliferative cytotoxic
agents are the EGFR inhibitors, Her-2 inhibitors, CDK inhibitors,
and Herceptin.RTM. (trastuzumab). (see, e.g., U.S. Pat. Nos.
6,537,988; 6,420,377). Such compounds may be given in accordance
with techniques currently known for the administration thereof.
[0185] Antibodies of the invention include antibodies that are
modified, i.e., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from specifically binding to its binding site. For
example, antibodies of the invention may be modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
or with other protecting/blocking groups, proteolytic cleavage,
linkage to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the antibodies may contain one or more non-classical
amino acids.
[0186] Monoclonal antibodies can be prepared using a wide variety
of techniques including the use of hybridoma, recombinant, and
phage display technologies, or a combination thereof. For example,
monoclonal antibodies can be produced using hybridoma techniques
including those taught, for example, in Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); and Hammerling et al., Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981). The term "monoclonal
antibody" as used herein is not limited to antibodies produced
through hybridoma technology. The term "monoclonal antibody" refers
to an antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced.
[0187] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and known. Briefly, mice are
immunized with an antigen or a cell expressing such antigen. Once
an immune response is detected, e.g., antibodies specific for the
antigen are detected in the mouse serum, the mouse spleen is
harvested and splenocytes isolated. The splenocytes are then fused
by known techniques to any suitable myeloma cells, for example
cells from cell line SP20 available from the ATCC. Hybridomas are
selected and cloned by limited dilution. The hybridoma clones are
then assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide or antigen of the
invention. Ascites fluid, which generally contains high levels of
antibodies, can be generated by immunizing mice with positive
hybridoma clones.
[0188] Examples of techniques which can be used to produce
single-chain Fvs (scFv) and antibodies include those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al. Methods in
Enzymology 203:46-88 (1991); Shu et al. PNAS 90:7995-7999 (1993);
and Skerra et al. Science 240:1038-1040 (1988).
[0189] The term "humanized" as used herein refers to antibodies
from non-human species whose amino acid sequences have been
modified to increase their similarity to antibody variants produced
naturally in humans. Thus, humanized antibodies are antibody
molecules from a non-human species antibody that binds the desired
antigen, having one or more complementarity determining regions
(CDRs) from the non-human species and framework regions from a
human immunoglobulin molecule. Often, framework residues in the
human framework regions will be substituted with the corresponding
residue from the donor antibody to alter, preferably to improve,
antigen binding and/or reduce immunogenicity of the humanized
antibody in a subject. These framework substitutions are identified
by methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and/or
immunogenicity and sequence comparison to identify unusual
framework residues at particular positions. (See, e.g., Queen et
al. U.S. Pat. No. 5,585,089; Riechmann et al. Nature 332:323
(1988), which are incorporated herein by reference in their
entireties.) Antibodies can be humanized using a variety of
techniques known in the art including, for example, CDR-grafting
(see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089),
veneering or resurfacing (see, e.g., EP Patent No. 592,106; EP
Patent No. 519,596; Padlan, Molecular Immunology 28(4/5):489-498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);
Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S.
Pat. No. 5,565,332). A detailed description of the production and
characterization of the humanized monoclonal antibodies of the
present invention is provided in the Examples section herein.
[0190] Completely human antibodies are desirable for therapeutic
treatment, diagnosis, and/or detection of human subjects. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See, e.g.,
U.S. Pat. Nos. 4,444,887 and 4,716,111.
[0191] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318 and 5,939,598.
[0192] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0193] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-1diotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-ldiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-Idiotypes or
Fab fragments of such anti-Idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-1diotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0194] The invention further provides polynucleotides comprising a
nucleotide sequence encoding a chimeric antigen receptor of the
invention as described above. The polynucleotides may be obtained,
and the nucleotide sequence of the polynucleotides determined, by
any method known in the art. For example, if the nucleotide
sequence of the components of the chimeric antigen receptor are
known, a polynucleotide encoding the components may be assembled
from chemically synthesized oligonucleotides, which involves the
synthesis of overlapping oligonucleotides containing portions of
the sequence encoding the components of the chimeric antigen
receptor, annealing and ligation of those oligonucleotides, and
then amplification of the ligated oligonucleotides by polymerase
chain reaction (PCR). Alternatively, a polynucleotide encoding a
chimeric antigen receptor may be generated from nucleic acid from a
suitable source. Amplified nucleic acids generated by PCR may then
be cloned into replicable cloning vectors using any method well
known in the art.
[0195] The present invention is explained in greater detail in the
following non-limiting examples. The following examples are
presented in order to more fully illustrate the preferred
embodiments of the invention. They should in no way, however, be
construed as limiting the broad scope of the invention.
EXAMPLES
Example 1
[0196] B7-H3 (CD276) is a type I transmembrane protein and a member
of the B7 superfamily of ligands that has an inhibitory effect on
T-cells. B7-H3 is highly expressed in several human malignancies
and its expression correlates with poor survival. We have selected
B7-H3 as a target of chimeric antigen receptor (CAR)-redirected T
cells, since it is expressed in tumor cells, but has a restricted
distribution in normal tissues. Noteworthy, the mAb 376.96--from
which we have derived the B7-H3-specific CAR targets a defined
B7-H3 epitope that is not detectable in normal tissues, thus
further minimizing potential side effects due to "on target but off
tumor" recognition. Furthermore, this epitope is highly expressed
in cancer initiating cells and tumor-associated vasculature and
fibroblasts. We have generated a CAR from the single chain Fv
(scFv) obtained from the mAb 376.96. We found that B7-H3.CAR can be
stably expressed by human T lymphocytes upon gene transfer and that
B7-H3.CAR-modified T cells can specifically recognize and
efficiently eliminate B7-H3 positive cells, and cross-react with
both human and murine B7-H3. We have found that B7-H3 highly
express on pancreatic cancer (PDAC) cell lines, which can be
efficiently eliminated by B7-H3.CAR-Ts (using either CD28 or 4-1-BB
as co-stimulatory domains) in vitro. In vivo experiments showed
that B7-H3.CAR-T cells effectively target pancreatic tumor cells
both in systemic metastatic model and orthotopic pancreas model in
NSG mice. In view of the broad tumor expression of B7-H3, we
anticipate the applicability of the B7-H3.CAR derived from the mAb
376.96 for the treatment of many types of solid and liquid human
tumors.
[0197] Cell lines. Human pancreatic tumor cell lines Panc-1,
BxPC-3, Panc-10.05, Capan-1, Hpaf-II and AsPC-1 were purchased from
American Type Culture Collection (ATCC). 293T, Phenix Eco and
Capan-1 cell lines were cultured in IMDM (Gibco, Invitrogen)
supplemented with 10% FBS (Sigma), 2 mM GlutaMax (Gibco). BxPC-3,
Panc-10.05, KPC-4662 were cultured in RPMI1640 (Gibco) supplemented
with 10% FBS and 2 mM GlutaMax. AsPC-1 was cultured in RPMI1640
(Gibco) supplemented with 10% FBS, 2 mM GlutaMax and 1 mM Sodium
pyruvate (Gibco). Panc-1 cells were cultured in DMEM (GIBCO)
supplemented with 10% FBS and 2 mM GlutaMax. Hpaf-II was cultured
in MEM (Gibco) supplemented with 10% FBS and 2 mM GlutaMax.
Penicillin (100 unit/mL) (Gibco) and streptomycin (100 .mu.g/mL)
(Gibco) were added to all cell culture mediums. Cells were
maintained in a humidified atmosphere containing 5% CO.sub.2 at
37.degree. C. Panc-1, Panc-10.05, BxPC-3, Hpaf-II, Capan-1 and
Aspc-1 cells were transduced with a retroviral vector encoding the
eGFP, and Raji cell was transduced with retroviral vectors encoding
either human or murine B7-H3 edna. The murine pancreatic tumor cell
line KPC-4662 was transduced with a retroviral vector encoding the
murine B7-H3 edna. Panc-1 and BxPC-3 cells were also transduced
with a retroviral vector encoding the eGFP-Firefly-Luciferase
(eGFP-FFluc) gene. All lines were routinely checked over the course
of the experiments and always found mycoplasma free and routinely
validated by flow cytometry for surface markers and functional
readouts as needed.
[0198] Plasmid Construction and Retrovirus Production.
[0199] The full-length human 2Ig-B7-H3 (accession number
NM_001329628) and 4Ig-B7-H3 (accession number NM_001024736) genes
were amplified by PCR from cDNA generated from Panc-1 cell line,
and cloned into the retroviral vector SFG using NcoI and XhoI
restriction sites. Murine B7-H3 (accession number NM_133983) was
amplified by PCR from a plasmid obtained from InVivogene (San
Diego, Calif.) and cloned into the retroviral vector SFG using NcoI
and MluI restriction sites. The scFv.376.96 specific for human
B7-H3, was cloned into the retroviral vector SFG using NcoI and
MluI restriction sites, and the entire cassette of the B7-H3.CARs
are illustrated in FIG. 3A. The retroviral supernatant was prepared
as previously described. Briefly, 293T cells were cotransfected
with 3 plasmids (the retroviral construct, Peg-Pam-e encoding for
gag-pol, and RDF encoding for the RD114 envelop), using the
GeneJuice transfection reagent (Novagen), and supernatants were
collected at 48 and 72 hours later.
[0200] T Cell Transduction and Expansion.
[0201] Buffy coats from healthy donors were obtained through the
Gulf Coast Regional Blood Center, Houston, Tex. Peripheral blood
mononuclear cells (PBMCs) were isolated with Lymphoprep density
separation (Fresenius Kabi Norge), were activated using 1 .mu.g/mL
anti-CD3 (Miltenyi Biotec) and 1 .mu.g/mL anti-CD28 (BD
Biosciences) antibodies coated plates. On day 3, T lymphocytes were
transduced with retroviral supernatants using retronectin-coated
plates (Takara Bio Inc., Shiga, Japan). After removal from the
retronectin plates, T cells were expanded in complete medium (45%
RPMI-1640 and 45% Click's medium (Irvine Scientific), 10% FBS
(Hyclone), 2 mM GlutaMAX, 100 unit/mL of Penicillin and 100
.mu.g/mL of streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15
(5 ng/mL; PeproTech), changing medium every 2-3 days. On day 12-14,
cells were collected for in vitro or in vivo experiments. T cells
were cultured in IL-7/1L-15 depleted medium for two days prior to
functional assays.
[0202] Immunohistochemistry.
[0203] Frozen normal human tissue microarrays and normal murine
tissue microarrays were purchased from US Biomax. Frozen pancreatic
cancer samples were obtained from the Tissue Procurement Facility
at the UNC Lineberger Comprehensive Cancer Center. Tissues were
sectioned by the Histology Research core facility at University of
North Carolina. Slides were fixed in 4% PFA in PBS for 15 min,
dried for 30 min at room temperature and blocked with 1% BSA and
10% horse serum (Company) in PBS with 0.05% tween-20. Slides were
stained with the primary antibody specific for human B7-H3 (clone
376.96, 1:1000 dilution) at 4.degree. C. overnight, and probed with
HRP polymer conjugated goat anti-mouse secondary antibody (Dako,
code K4000, 1:8 dilution at 25.degree. C. for 1.5 h). Slides were
developed using DAB chromogen (Vector Labs), counterstained with
CAT hematoxylin (Biocare medical), dehydrated in ethanol, and
cleared in xylene (Fisher chemical). Cover slips were added using
histological mounting medium (Fisher, toluene solution). Stained
TMA slides were digitally imaged at 20.times. objective using the
Aperio ScanScope XT (Leica). Tissue microarray slides were
de-arrayed to visualize individual cores and each core was visually
inspected. Folded tissues were excluded from the analysis using a
negative pen, and all other artifacts were automatically excluded
with the Aperio Genie software. The B7-H3 staining was measured
using Aperio membrane v9 (cell quantification) algorithm.
Percentage of positive cells obtained with this algorithm at each
intensity level (negative, low, medium, high) were used to
calculate the H-Score using the formula: H-Score=(% at 1+)*1+(% at
2+)*2+(% at 3+)*3. The Aperio color deconvolution v9 algorithm with
the Genie classifier was also applied to calculate the area and
intensity of the positive stain and generate a Score (0-300).
[0204] ELISA
[0205] T cells (5.times.10.sup.5 or 1.times.10.sup.5) were
co-cultured with tumor cells (5.times.10.sup.5) in a 24 well plate
without exogenous cytokines. After 24 hours, supernatants were
collected and cytokines (interferon gamma (IFN.gamma.) and
interleukin 2 (IL2)) were measured by using ELISA kit (R&D
system) following manufacturer's instructions. Each supernatant was
measured in triplicate.
[0206] Flow Cytometry.
[0207] We performed flow cytometry using Abs specific to CD45,
CD56, CD8, CD4, CD3, CD45RA, CD45RO, CD62L, hB7-H3 (clone 7-517),
mB7-H3 (clone MIH32), mCD3, mCD4, mCD8, mCD11b, mCD11c, mLy6cG,
mCD19 (all from Becton Dickinson, San Jose, Calif.) and CCR7 (from
E&D) conjugated with BV421, AF488, FITC, PE, PE-cy7,
PerCP-cy5.5, APC, and APC-cy7 fluorochromes. Expression of B7-H3 in
tumor cell lines was assessed with anti-B7-H3 specific Abs (clone
7-517 from BD, and clone 376.96). The expression of B7-H3.CAR was
detected using Protein-L (Genscript) and Anti-Fab antibody (Jackson
ImmunoResearch Laboratories INC.). Samples were analyzed with BD
FACScanto II or BD FACSfortessa with the BD Diva software (BD
Biosciences), for each sample we acquired a minimum of 10,000
events, and data was analyzed using Flojo 10.
[0208] Long-Term In Vitro Cytotoxic Activity.
[0209] Tumor cells were seeded in 24-well plates at a concentration
of 5.times.10.sup.5/well. T cells were added to the culture at
different ratios (E:T of 1:1; 1:5, or 1:10) without the addition of
exogenous cytokines. Cells were analyzed by day 5-7 to measure
residual tumor cells and T cells by FACS. Dead cells were removed
by Zombie Aqua (Biolegend) staining, T cells and tumor cells were
identified by the expression of CD3, GFP (pancreatic cancer cell
lines and fibroblast cells), CD19 (Raji, Raji-2IgB7-H3,
Raji-4IgB7-H3 and Raji-mB7-H3) and mB7-H3 (KPC-4662 and
KPC-4662-mB7-H3).
[0210] Proliferation Assay.
[0211] T cells were labeled with 1.5 mM carboxyfluorescein
diacetate succinimidyl ester (CFSE; Invitrogen) and plated with
tumor cell targets at an E:T ratio of 1:1. CFSE dilution was
measured on CD3.sup.+ T cells by day 5 using flow cytometry.
[0212] Xenogenic Mouse Models.
[0213] NSG mice were used to assess the in vivo antitumor effect of
control and transduced T cells. All mouse experiments were approved
by the Institutional Animal Care and Use Committee of University of
North Carolina at Chapel Hill. For systemic metastatic model,
8-10-week-old male and female NSG mice (UNC animal facility) were
injected i.v. with FFluc transduced either Panc-1
(1.times.10.sup.6) or BxPC-3 (1.times.10.sup.6) tumor cells, 14
days after tumor cells inoculation, none-transduced T cell,
B7-H3.CAR-28 or B7-H3.CAR-BB T cells were injected i.v.
(1.times.10.sup.7 cells per mouse) (FIG. 6H). For pancreatic
orthotopic model, FFluc transduced Panc-1 (2.times.10.sup.5) or
BxPC-3 (1.times.10.sup.5) tumor cells were suspended in 25 .mu.L
DPBS and mixed with 25 .mu.L Matrigel (Corning), then surgically
implanted into pancreas of 8-10-week-old male mice using 28-gauge
needle. Briefly, an incision is performed in the left flank and
tumor cells mixed with Matrigel were injected using a 28-gauge
needle into a tail of the pancreas. The wound is closed in two
layers, with running 4-0 Vicryl, and wound clips or polypropylene
sutures for the skin. 12 days after tumor cells inoculation,
CD19.CAR-T (control) or B7-H3.CAR-T cells were injected i.v.
(1.times.10.sup.7 cells per mouse) (FIG. 6A). No randomization was
used. Investigators were not blinded, but mice were matched based
on the signal of tumor cells before assignment to control or
treatment groups. Tumor growth was monitored by bioluminescence
imaging weekly using IVIS lumina II in vivo imaging system
(PerkinElmer). Mice were euthanized when signs of discomfort were
detected by the investigator or as recommended by the veterinarian
who monitored the mice three times a week, or when luciferase
signal reached 2.times.10.sup.11 photons per second per cm.sup.2 to
investigate animal survival. Tumor specimen was collected and snap
froze for IHC staining to detect B7-H3 expression.
[0214] Statistical Analyses.
[0215] Unless otherwise noted, data were reported as mean.+-.s.d.
Student's t-test (two-sided) was used to determine statistically
significant differences between samples, with P<0.05 indicating
a significant difference. Graph generation and statistical analyses
were performed using Prism version 5.0d software (GraphPad, La
Jolla, Calif.).
[0216] B7-H3 is Highly Expressed on Pancreatic Cancer Tissues but
not Normal Human Tissues.
[0217] To evaluate the expression of B7-H3 on normal human tissues,
frozen normal human tissue microarray (TMA) slides were stained
with the 376.96 mAb. Frozen human pancreatic ductal adenocarcinoma
(PDAC) tissues were used as positive control. As shown in FIG. 1A,
PDAC tissues were positive for B7-H3, and the antigen is expressed
by both tumor cells and stroma fibroblasts, while normal pancreas
is B7-H3 negative. Similarly, six human PDAC tumor cell lines and
three primary pancreatic tumor cell lines from PDX models are B7-H3
positive (FIGS. 1B-C). Normal human tissues including heart, lung,
liver, kidney, spleen, muscle, cerebrum, cerebellum, spinal cord
and peripheral nerves were B7-H3 negative (FIG. 2). Weak positivity
was detected in adrenal gland, salivary gland, epithelia cells of
prostate and basal layer of the skin (FIG. 2).
[0218] B7-H3.CAR-Ts Specifically Target B7-H3 Positive Cells and
Cross-React with Both Human and Murine B7-H3.
[0219] We have generated a B7-H3.CAR using the 376.96 mAb. The scFv
sequence obtained from the hybridoma 376.96 was cloned into
previously validated CAR formats including the human CD8.alpha.
hinge and transmembrane domain, CD28 or 4-1-BB intracellular
costimulatory domains and CD31 intracellular signaling domain. The
B7-H3.CAR cassettes were cloned into the retroviral vector SFG and
are illustrated in FIG. 3A. The transduction efficiency of
B7-H3.CAR-Ts is 65%-85% (FIG. 3B). To verify the specificity of
B7-H3.CAR-Ts, the tumor cell line Raji that is B7-H3 negative was
genetically modified to express either the two isoforms of human
B7-H3 (4Ig-B7-H3 and 2Ig-B7-H3) or the corresponding mouse B7-H3
(mB7-H3). Single cell clones of these cells were obtained (FIG.
3C). Control and B7-H3-expressing Raji cells were then co-cultured
with either control or B7-H3.CAR-Ts. As shown in FIG. 3D,
B7-H3.CAR-Ts encoding either CD28 or 4-1-BB co-stimulatory domains
specifically targeted B7-H3-expressing Raji cells. B7-H3.CAR-Ts
also targeted Raji cells expressing mB7-H3 demonstrating that the
scFv derived from the 376.96 mAb targets both human and mouse B7-H3
molecule (FIG. 3D-E). The antitumor effect was also parallel by
IFN.gamma. and IL-2 release (FIG. 3F-G).
[0220] B7-H3.CAR-Ts Target PDAC Cell Lines In Vitro.
[0221] To assess the effects of B7-H3.CAR-Ts on tumor cell lines
naturally expressing B7-H3, we co-cultured six PDAC cell lines with
CD19.CAR-Ts (negative control) and B7-H3.CAR-Ts at different T cell
to tumor cell ratios. As shown in FIG. 4A, B7-H3.CAR-Ts effectively
eliminated PDAC cells, even at 1:10 T cell to tumor cell ratio
(FIG. 4B-C). For two PDAC tumor cell lines, BxPC-3 and Panc-10.05,
B7-H3.CAR-Ts encoding 4-1BB seemed more efficient than B7-H3.CAR-Ts
encoding CD28 (FIG. 4C). Cytolytic activity of B7-H3.CAR-Ts was
corroborated by cytokine release (IFN.gamma. and IL2) (FIG. 4D-E)
and proliferation (FIG. 4F). Similar results were obtained when
B7-H3.CAR-Ts were co-cultured with PDAC cell lines derived from PDX
(FIG. 5A-F).
[0222] B7-H3.CAR-Ts Show Antitumor Activity in Xenograft
Models.
[0223] To investigate the antitumor effects of B7-H3.CAR-Ts in
vivo, FFluc transduced Panc-1 and BxPC-3 tumor cells were implanted
into pancreas of NSG mice by surgery for pancreas orthotopic
models, and treated with control CD19.CAR-Ts and B7-H3.CAR-Ts (FIG.
6A). As shown in FIG. 6B-D, B7-H3.CAR-Ts effectively eliminated
Panc-1 tumor cells and mice remained tumor free up to day 80 after
treatment. In the BxPC-3 orthotopic model, B7-H3.CAR-Ts also
controlled tumor but in this model B7-H3.CAR-Ts encoding 4-1BB were
more effective than B7-H3.CAR-Ts encoding CD28 (FIG. 6E-G). In
metastatic model, FFluc transduced Panc-1 tumor cell was implanted
into NSG mice by i.v. injection (FIG. 6H). Mice were then treated
via tail vein injection with CD19.CAR-Ts and B7-H3.CAR-Ts. As shown
in FIG. 6I-J, B7-H3.CAR-Ts controlled Panc-1 tumor growth until day
70 post treatment when the experiment was terminated. In this model
both B7-H3.CAR-Ts encoding either CD28 or 4-1BB showed similar
activity.
[0224] All publications, patent applications, patents, patent
publications and other references cited herein are incorporated by
reference in their entireties for the teachings relevant to the
sentence and/or paragraph in which the reference is presented.
TABLE-US-00005 scFV sequence of B7-H3-CAR (SEQ ID NO: 5)
GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCA
ATTGGAGCCAGGGTCAGCATCACCTGCAAGGCCAGTCAGGAT
GTGAGAACTGCTGTAGCCTGGTATCAACAGAAACCAGGCCAG
TCTCCTAAACTACTAATTTACTCGGCATCCTACCGGTACACT
GGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGAT
TTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCA
GTTTATTACTGTCAGCAACATTATGGTACTCCTCCGTGGACG
TTCGGTGGAGGCACCAAGCTGGAAATCAAAGGCGGCGGAGGA
TCTGGCGGAGGCGGAAGTGGCGGAGGGGGCTCTGAAGTGCAG
CTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCC
CTGAAACTCTCCTGTGAAGCCTCTAGATTCACTTTCAGTAGC
TATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTG
GAGTGGGTCGCAGCCATTAGTGGAGGTGGTAGGTACACCTAC
TATCCAGACAGTATGAAGGGTCGATTCACCATCTCCAGAGAC
AATGCCAAGAATTTCCTGTACCTGCAAATGAGCAGTCTGAGG
TCTGAGGACACGGCCATGTATTACTGTGCAAGACACTATGAT
GGTTATCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTC TCCTCA
Sequence CWU 1
1
51483PRTArtificialchimeric antigen receptor CD28 1Met Glu Phe Gly
Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr 20 25 30Ser Ile
Gly Ala Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val 35 40 45Arg
Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 50 55
60Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg65
70 75 80Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser 85 90 95Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His
Tyr Gly 100 105 110Thr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val 130 135 140Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser Leu145 150 155 160Lys Leu Ser Cys Glu
Ala Ser Arg Phe Thr Phe Ser Ser Tyr Ala Met 165 170 175Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ala 180 185 190Ile
Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Met Lys Gly 195 200
205Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Phe Leu Tyr Leu Gln
210 215 220Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Ala Arg225 230 235 240His Tyr Asp Gly Tyr Leu Asp Tyr Trp Gly Gln
Gly Thr Thr Leu Thr 245 250 255Val Ser Ser Thr Arg Thr Thr Thr Pro
Ala Pro Arg Pro Pro Thr Pro 260 265 270Ala Pro Thr Ile Ala Ser Gln
Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285Arg Pro Ala Ala Gly
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300Cys Asp Ile
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu305 310 315
320Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg
325 330 335Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
Gly Pro 340 345 350Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
Asp Phe Ala Ala 355 360 365Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
Ala Asp Ala Pro Ala Tyr 370 375 380Gln 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 Arg2484PRTArtificialchimeric antigen
receptor 4-1-BB 2Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala
Ile Leu Lys Gly1 5 10 15Val Gln Cys Asp Ile Val Met Thr Gln Ser His
Lys Phe Met Ser Thr 20 25 30Ser Ile Gly Ala Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val 35 40 45Arg Thr Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys 50 55 60Leu Leu Ile Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Asp Arg65 70 75 80Phe Thr Gly Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95Val Gln Ala Glu Asp
Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Gly 100 105 110Thr Pro Pro
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 115 120 125Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 130 135
140Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser
Leu145 150 155 160Lys Leu Ser Cys Glu Ala Ser Arg Phe Thr Phe Ser
Ser Tyr Ala Met 165 170 175Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val Ala Ala 180 185 190Ile Ser Gly Gly Gly Arg Tyr Thr
Tyr Tyr Pro Asp Ser Met Lys Gly 195 200 205Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Phe Leu Tyr Leu Gln 210 215 220Met Ser Ser Leu
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg225 230 235 240His
Tyr Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 245 250
255Val Ser Ser Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
260 265 270Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
Ala Cys 275 280 285Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
Leu Asp Phe Ala 290 295 300Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
Gly Thr Cys Gly Val Leu305 310 315 320Leu Leu Ser Leu Val Ile Thr
Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335Leu Leu Tyr Ile Phe
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350Gln Glu Glu
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365Gly
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375
380Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg385 390 395 400Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu 405 410 415Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu Gly Leu Tyr Asn 420 425 430Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu Ile Gly Met 435 440 445Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 450 455 460Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala465 470 475 480Leu
Pro Pro Arg37811DNAArtificialplasmid CD28 3aagctttgct cttaggagtt
tcctaataca tcccaaactc aaatatataa agcatttgac 60ttgttctatg ccctaggggg
cggggggaag ctaagccagc tttttttaac atttaaaatg 120ttaattccat
tttaaatgca cagatgtttt tatttcataa gggtttcaat gtgcatgaat
180gctgcaatat tcctgttacc aaagctagta taaataaaaa tagataaacg
tggaaattac 240ttagagtttc tgtcattaac gtttccttcc tcagttgaca
acataaatgc gctgctgagc 300aagccagttt gcatctgtca ggatcaattt
cccattatgc cagtcatatt aattactagt 360caattagttg atttttattt
ttgacatata catgtgaatg aaagacccca cctgtaggtt 420tggcaagcta
gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat
480agaaaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
caaacaggat 540atctgtggta agcagttcct gccccggctc agggccaaga
acagatggaa cagctgaata 600tgggccaaac aggatatctg tggtaagcag
ttcctgcccc ggctcagggc caagaacaga 660tggtccccag atgcggtcca
gccctcagca gtttctagag aaccatcaga tgtttccagg 720gtgccccaag
gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
780tcgcttctgt tcgcgcgctt atgctccccg agctcaataa aagagcccac
aacccctcac 840tcggggcgcc agtcctccga ttgactgagt cgcccgggta
cccgtgtatc caataaaccc 900tcttgcagtt gcatccgact tgtggtctcg
ctgttccttg ggagggtctc ctctgagtga 960ttgactaccc gtcagcgggg
gtctttcatt tgggggctcg tccgggatcg ggagacccct 1020gcccagggac
caccgaccca ccaccgggag gtaagctggc cagcaactta tctgtgtctg
1080tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
gttagctaac 1140tagctctgta tctggcggac ccgtggtgga actgacgagt
tcggaacacc cggccgcaac 1200cctgggagac gtcccaggga cttcgggggc
cgtttttgtg gcccgacctg agtcctaaaa 1260tcccgatcgt ttaggactct
ttggtgcacc ccccttagag gagggatatg tggttctggt 1320aggagacgag
aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggga
1380ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
tctgtctgac 1440tgtgtttctg tatttgtctg aaaatatggg cccgggctag
cctgttacca ctcccttaag 1500tttgacctta ggtcactgga aagatgtcga
gcggatcgct cacaaccagt cggtagatgt 1560caagaagaga cgttgggtta
ccttctgctc tgcagaatgg ccaaccttta acgtcggatg 1620gccgcgagac
ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
1680acctggcccg catggacacc cagaccaggt ggggtacatc gtgacctggg
aagccttggc 1740ttttgacccc cctccctggg tcaagccctt tgtacaccct
aagcctccgc ctcctcttcc 1800tccatccgcc ccgtctctcc cccttgaacc
tcctcgttcg accccgcctc gatcctccct 1860ttatccagcc ctcactcctt
ctctaggcgc ccccatatgg ccatatgaga tcttatatgg 1920ggcacccccg
ccccttgtaa acttccctga ccctgacatg acaagagtta ctaacagccc
1980ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct
ggagacctct 2040ggcggcagcc taccaagaac aactggaccg accggtggta
cctcaccctt accgagtcgg 2100cgacacagtg tgggtccgcc gacaccagac
taagaaccta gaacctcgct ggaaaggacc 2160ttacacagtc ctgctgacca
cccccaccgc cctcaaagta gacggcatcg cagcttggat 2220acacgccgcc
cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta gactgccatg
2280gaattcggcc tgagctggct gttcctggtg gccatcctga agggcgtgca
gtgcgacatt 2340gtgatgaccc agtctcacaa attcatgtcc acatcaattg
gagccagggt cagcatcacc 2400tgcaaggcca gtcaggatgt gagaactgct
gtagcctggt atcaacagaa accaggccag 2460tctcctaaac tactaattta
ctcggcatcc taccggtaca ctggagtccc tgatcgcttc 2520actggcagtg
gatctgggac ggatttcact ttcaccatca gcagtgtgca ggctgaagac
2580ctggcagttt attactgtca gcaacattat ggtactcctc cgtggacgtt
cggtggaggc 2640accaagctgg aaatcaaagg cggcggagga tctggcggag
gcggaagtgg cggagggggc 2700tctgaagtgc agctggtgga gtctggggga
ggcttagtga agcctggagg gtccctgaaa 2760ctctcctgtg aagcctctag
attcactttc agtagctatg ccatgtcttg ggttcgccag 2820actccggaga
agaggctgga gtgggtcgca gccattagtg gaggtggtag gtacacctac
2880tatccagaca gtatgaaggg tcgattcacc atctccagag acaatgccaa
gaatttcctg 2940tacctgcaaa tgagcagtct gaggtctgag gacacggcca
tgtattactg tgcaagacac 3000tatgatggtt atcttgacta ctggggccaa
ggcaccactc tcacagtctc ctcaacgcgt 3060accacgacgc cagcgccgcg
accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 3120tccctgcgcc
cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
3180gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg
ggtccttctc 3240ctgtcactgg ttatcaccct ttactgcagg agtaagagga
gcaggctcct gcacagtgac 3300tacatgaaca tgactccccg ccgccccggg
cccacccgca agcattacca gccctatgcc 3360ccaccacgcg acttcgcagc
ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc 3420cccgcgtacc
agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag
3480gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg
aaagccgaga 3540aggaagaacc ctcaggaagg cctgtacaat gaactgcaga
aagataagat ggcggaggcc 3600tacagtgaga ttgggatgaa aggcgagcgc
cggaggggca aggggcacga tggcctttac 3660cagggtctca gtacagccac
caaggacacc tacgacgccc ttcacatgca ggccctgccc 3720cctcgctaag
catgcacctc gagatcgatc cggattagtc caatttgtta aagacaggat
3780atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc
ctatagagta 3840cgagccatag ataaaataaa agattttatt tagtctccag
aaaaaggggg gaatgaaaga 3900ccccacctgt aggtttggca agctagctta
agtaacgcca ttttgcaagg catggaaaaa 3960tacataactg agaatagaga
agttcagatc aaggtcagga acagatggaa cagctgaata 4020tgggccaaac
aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga
4080tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
gccccggctc 4140agggccaaga acagatggtc cccagatgcg gtccagccct
cagcagtttc tagagaacca 4200tcagatgttt ccagggtgcc ccaaggacct
gaaatgaccc tgtgccttat ttgaactaac 4260caatcagttc gcttctcgct
tctgttcgcg cgcttctgct ccccgagctc aataaaagag 4320cccacaaccc
ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt
4380gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
ccttgggagg 4440gtctcctctg agtgattgac tacccgtcag cgggggtctt
tcacacatgc agcatgtatc 4500aaaattaatt tggttttttt tcttaagtat
ttacattaaa tggccatagt acttaaagtt 4560acattggctt ccttgaaata
aacatggagt attcagaatg tgtcataaat atttctaatt 4620ttaagatagt
atctccattg gctttctact ttttctttta tttttttttg tcctctgtct
4680tccatttgtt gttgttgttg tttgtttgtt tgtttgttgg ttggttggtt
aatttttttt 4740taaagatcct acactatagt tcaagctaga ctattagcta
ctctgtaacc cagggtgacc 4800ttgaagtcat gggtagcctg ctgttttagc
cttcccacat ctaagattac aggtatgagc 4860tatcattttt ggtatattga
ttgattgatt gattgatgtg tgtgtgtgtg attgtgtttg 4920tgtgtgtgac
tgtgaaaatg tgtgtatggg tgtgtgtgaa tgtgtgtatg tatgtgtgtg
4980tgtgagtgtg tgtgtgtgtg tgtgcatgtg tgtgtgtgtg actgtgtcta
tgtgtatgac 5040tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
tgtgtgttgt gaaaaaatat 5100tctatggtag tgagagccaa cgctccggct
caggtgtcag gttggttttt gagacagagt 5160ctttcactta gcttggaatt
cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 5220tggcgttacc
caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
5280cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
gcgaatggcg 5340cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
tcacaccgca tatggtgcac 5400tctcagtaca atctgctctg atgccgcata
gttaagccag ccccgacacc cgccaacacc 5460cgctgacgcg ccctgacggg
cttgtctgct cccggcatcc gcttacagac aagctgtgac 5520cgtctccggg
agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgatgac
5580gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
atggtttctt 5640agacgtcagg tggcactttt cggggaaatg tgcgcggaac
ccctatttgt ttatttttct 5700aaatacattc aaatatgtat ccgctcatga
gacaataacc ctgataaatg cttcaataat 5760attgaaaaag gaagagtatg
agtattcaac atttccgtgt cgcccttatt cccttttttg 5820cggcattttg
ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
5880aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
ggtaagatcc 5940ttgagagttt tcgccccgaa gaacgttttc caatgatgag
cacttttaaa gttctgctat 6000gtggcgcggt attatcccgt attgacgccg
ggcaagagca actcggtcgc cgcatacact 6060attctcagaa tgacttggtt
gagtactcac cagtcacaga aaagcatctt acggatggca 6120tgacagtaag
agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
6180tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
aacatggggg 6240atcatgtaac tcgccttgat cgttgggaac cggagctgaa
tgaagccata ccaaacgacg 6300agcgtgacac cacgatgcct gtagcaatgg
caacaacgtt gcgcaaacta ttaactggcg 6360aactacttac tctagcttcc
cggcaacaat taatagactg gatggaggcg gataaagttg 6420caggaccact
tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
6480ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
aagccctccc 6540gtatcgtagt tatctacacg acggggagtc aggcaactat
ggatgaacga aatagacaga 6600tcgctgagat aggtgcctca ctgattaagc
attggtaact gtcagaccaa gtttactcat 6660atatacttta gattgattta
aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6720tttttgataa
tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
6780accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
gtaatctgct 6840gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
tttgccggat caagagctac 6900caactctttt tccgaaggta actggcttca
gcagagcgca gataccaaat actgtccttc 6960tagtgtagcc gtagttaggc
caccacttca agaactctgt agcaccgcct acatacctcg 7020ctctgctaat
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
7080tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
gggggttcgt 7140gcacacagcc cagcttggag cgaacgacct acaccgaact
gagataccta cagcgtgagc 7200attgagaaag cgccacgctt cccgaaggga
gaaaggcgga caggtatccg gtaagcggca 7260gggtcggaac aggagagcgc
acgagggagc ttccaggggg aaacgcctgg tatctttata 7320gtcctgtcgg
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
7380ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
gccttttgct 7440ggccttttgc tcacatgttc tttcctgcgt tatcccctga
ttctgtggat aaccgtatta 7500ccgcctttga gtgagctgat accgctcgcc
gcagccgaac gaccgagcgc agcgagtcag 7560tgagcgagga agcggaagag
cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 7620ttcattaatg
cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
7680caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
atgcttccgg 7740ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
cacaggaaac agctatgacc 7800atgattacgc c
781147814DNAArtificialplasmid 4-1-BB 4aagctttgct cttaggagtt
tcctaataca tcccaaactc aaatatataa agcatttgac 60ttgttctatg ccctaggggg
cggggggaag ctaagccagc tttttttaac atttaaaatg 120ttaattccat
tttaaatgca cagatgtttt tatttcataa gggtttcaat gtgcatgaat
180gctgcaatat tcctgttacc aaagctagta taaataaaaa tagataaacg
tggaaattac 240ttagagtttc tgtcattaac gtttccttcc tcagttgaca
acataaatgc gctgctgagc 300aagccagttt gcatctgtca ggatcaattt
cccattatgc cagtcatatt aattactagt 360caattagttg atttttattt
ttgacatata catgtgaatg aaagacccca cctgtaggtt 420tggcaagcta
gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat
480agaaaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc
caaacaggat 540atctgtggta agcagttcct gccccggctc agggccaaga
acagatggaa cagctgaata 600tgggccaaac aggatatctg tggtaagcag
ttcctgcccc ggctcagggc caagaacaga 660tggtccccag atgcggtcca
gccctcagca gtttctagag aaccatcaga tgtttccagg 720gtgccccaag
gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
780tcgcttctgt tcgcgcgctt atgctccccg agctcaataa aagagcccac
aacccctcac 840tcggggcgcc agtcctccga ttgactgagt cgcccgggta
cccgtgtatc caataaaccc 900tcttgcagtt gcatccgact tgtggtctcg
ctgttccttg ggagggtctc ctctgagtga 960ttgactaccc gtcagcgggg
gtctttcatt tgggggctcg tccgggatcg ggagacccct 1020gcccagggac
caccgaccca ccaccgggag gtaagctggc cagcaactta tctgtgtctg
1080tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
gttagctaac 1140tagctctgta tctggcggac ccgtggtgga actgacgagt
tcggaacacc cggccgcaac
1200cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
agtcctaaaa 1260tcccgatcgt ttaggactct ttggtgcacc ccccttagag
gagggatatg tggttctggt 1320aggagacgag aacctaaaac agttcccgcc
tccgtctgaa tttttgcttt cggtttggga 1380ccgaagccgc gccgcgcgtc
ttgtctgctg cagcatcgtt ctgtgttgtc tctgtctgac 1440tgtgtttctg
tatttgtctg aaaatatggg cccgggctag cctgttacca ctcccttaag
1500tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt
cggtagatgt 1560caagaagaga cgttgggtta ccttctgctc tgcagaatgg
ccaaccttta acgtcggatg 1620gccgcgagac ggcaccttta accgagacct
catcacccag gttaagatca aggtcttttc 1680acctggcccg catggacacc
cagaccaggt ggggtacatc gtgacctggg aagccttggc 1740ttttgacccc
cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
1800tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc
gatcctccct 1860ttatccagcc ctcactcctt ctctaggcgc ccccatatgg
ccatatgaga tcttatatgg 1920ggcacccccg ccccttgtaa acttccctga
ccctgacatg acaagagtta ctaacagccc 1980ctctctccaa gctcacttac
aggctctcta cttagtccag cacgaagtct ggagacctct 2040ggcggcagcc
taccaagaac aactggaccg accggtggta cctcaccctt accgagtcgg
2100cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct
ggaaaggacc 2160ttacacagtc ctgctgacca cccccaccgc cctcaaagta
gacggcatcg cagcttggat 2220acacgccgcc cacgtgaagg ctgccgaccc
cgggggtgga ccatcctcta gactgccatg 2280gaattcggcc tgagctggct
gttcctggtg gccatcctga agggcgtgca gtgcgacatt 2340gtgatgaccc
agtctcacaa attcatgtcc acatcaattg gagccagggt cagcatcacc
2400tgcaaggcca gtcaggatgt gagaactgct gtagcctggt atcaacagaa
accaggccag 2460tctcctaaac tactaattta ctcggcatcc taccggtaca
ctggagtccc tgatcgcttc 2520actggcagtg gatctgggac ggatttcact
ttcaccatca gcagtgtgca ggctgaagac 2580ctggcagttt attactgtca
gcaacattat ggtactcctc cgtggacgtt cggtggaggc 2640accaagctgg
aaatcaaagg cggcggagga tctggcggag gcggaagtgg cggagggggc
2700tctgaagtgc agctggtgga gtctggggga ggcttagtga agcctggagg
gtccctgaaa 2760ctctcctgtg aagcctctag attcactttc agtagctatg
ccatgtcttg ggttcgccag 2820actccggaga agaggctgga gtgggtcgca
gccattagtg gaggtggtag gtacacctac 2880tatccagaca gtatgaaggg
tcgattcacc atctccagag acaatgccaa gaatttcctg 2940tacctgcaaa
tgagcagtct gaggtctgag gacacggcca tgtattactg tgcaagacac
3000tatgatggtt atcttgacta ctggggccaa ggcaccactc tcacagtctc
ctcaacgcgt 3060accacgacgc cagcgccgcg accaccaaca ccggcgccca
ccatcgcgtc gcagcccctg 3120tccctgcgcc cagaggcgtg ccggccagcg
gcggggggcg cagtgcacac gagggggctg 3180gacttcgcct gtgatatcta
catctgggcg cccttggccg ggacttgtgg ggtccttctc 3240ctgtcactgg
ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc
3300aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg
tagctgccga 3360tttccagaag aagaagaagg aggatgtgaa ctgagagtga
agttcagcag gagcgcagac 3420gcccccgcgt accagcaggg ccagaaccag
ctctataacg agctcaatct aggacgaaga 3480gaggagtacg atgttttgga
caagagacgt ggccgggacc ctgagatggg gggaaagccg 3540agaaggaaga
accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag
3600gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca
cgatggcctt 3660taccagggtc tcagtacagc caccaaggac acctacgacg
cccttcacat gcaggccctg 3720ccccctcgct aagcatgcac ctcgagatcg
atccggatta gtccaatttg ttaaagacag 3780gatatcagtg gtccaggctc
tagttttgac tcaacaatat caccagctga agcctataga 3840gtacgagcca
tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
3900agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca
aggcatggaa 3960aaatacataa ctgagaatag agaagttcag atcaaggtca
ggaacagatg gaacagctga 4020atatgggcca aacaggatat ctgtggtaag
cagttcctgc cccggctcag ggccaagaac 4080agatggaaca gctgaatatg
ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 4140ctcagggcca
agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa
4200ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
tatttgaact 4260aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
gctccccgag ctcaataaaa 4320gagcccacaa cccctcactc ggggcgccag
tcctccgatt gactgagtcg cccgggtacc 4380cgtgtatcca ataaaccctc
ttgcagttgc atccgacttg tggtctcgct gttccttggg 4440agggtctcct
ctgagtgatt gactacccgt cagcgggggt ctttcacaca tgcagcatgt
4500atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat
agtacttaaa 4560gttacattgg cttccttgaa ataaacatgg agtattcaga
atgtgtcata aatatttcta 4620attttaagat agtatctcca ttggctttct
actttttctt ttattttttt ttgtcctctg 4680tcttccattt gttgttgttg
ttgtttgttt gtttgtttgt tggttggttg gttaattttt 4740ttttaaagat
cctacactat agttcaagct agactattag ctactctgta acccagggtg
4800accttgaagt catgggtagc ctgctgtttt agccttccca catctaagat
tacaggtatg 4860agctatcatt tttggtatat tgattgattg attgattgat
gtgtgtgtgt gtgattgtgt 4920ttgtgtgtgt gactgtgaaa atgtgtgtat
gggtgtgtgt gaatgtgtgt atgtatgtgt 4980gtgtgtgagt gtgtgtgtgt
gtgtgtgcat gtgtgtgtgt gtgactgtgt ctatgtgtat 5040gactgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tgtgaaaaaa
5100tattctatgg tagtgagagc caacgctccg gctcaggtgt caggttggtt
tttgagacag 5160agtctttcac ttagcttgga attcactggc cgtcgtttta
caacgtcgtg actgggaaaa 5220ccctggcgtt acccaactta atcgccttgc
agcacatccc cctttcgcca gctggcgtaa 5280tagcgaagag gcccgcaccg
atcgcccttc ccaacagttg cgcagcctga atggcgaatg 5340gcgcctgatg
cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
5400cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
acccgccaac 5460acccgctgac gcgccctgac gggcttgtct gctcccggca
tccgcttaca gacaagctgt 5520gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg tcatcaccga aacgcgcgat 5580gacgaaaggg cctcgtgata
cgcctatttt tataggttaa tgtcatgata ataatggttt 5640cttagacgtc
aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
5700tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
atgcttcaat 5760aatattgaaa aaggaagagt atgagtattc aacatttccg
tgtcgccctt attccctttt 5820ttgcggcatt ttgccttcct gtttttgctc
acccagaaac gctggtgaaa gtaaaagatg 5880ctgaagatca gttgggtgca
cgagtgggtt acatcgaact ggatctcaac agcggtaaga 5940tccttgagag
ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
6000tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
cgccgcatac 6060actattctca gaatgacttg gttgagtact caccagtcac
agaaaagcat cttacggatg 6120gcatgacagt aagagaatta tgcagtgctg
ccataaccat gagtgataac actgcggcca 6180acttacttct gacaacgatc
ggaggaccga aggagctaac cgcttttttg cacaacatgg 6240gggatcatgt
aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
6300acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
ctattaactg 6360gcgaactact tactctagct tcccggcaac aattaataga
ctggatggag gcggataaag 6420ttgcaggacc acttctgcgc tcggcccttc
cggctggctg gtttattgct gataaatctg 6480gagccggtga gcgtgggtct
cgcggtatca ttgcagcact ggggccagat ggtaagccct 6540cccgtatcgt
agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
6600agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
caagtttact 6660catatatact ttagattgat ttaaaacttc atttttaatt
taaaaggatc taggtgaaga 6720tcctttttga taatctcatg accaaaatcc
cttaacgtga gttttcgttc cactgagcgt 6780cagaccccgt agaaaagatc
aaaggatctt cttgagatcc tttttttctg cgcgtaatct 6840gctgcttgca
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
6900taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
aatactgtcc 6960ttctagtgta gccgtagtta ggccaccact tcaagaactc
tgtagcaccg cctacatacc 7020tcgctctgct aatcctgtta ccagtggctg
ctgccagtgg cgataagtcg tgtcttaccg 7080ggttggactc aagacgatag
ttaccggata aggcgcagcg gtcgggctga acggggggtt 7140cgtgcacaca
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
7200agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
ccggtaagcg 7260gcagggtcgg aacaggagag cgcacgaggg agcttccagg
gggaaacgcc tggtatcttt 7320atagtcctgt cgggtttcgc cacctctgac
ttgagcgtcg atttttgtga tgctcgtcag 7380gggggcggag cctatggaaa
aacgccagca acgcggcctt tttacggttc ctggcctttt 7440gctggccttt
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
7500ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
cgcagcgagt 7560cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
gcctctcccc gcgcgttggc 7620cgattcatta atgcagctgg cacgacaggt
ttcccgactg gaaagcgggc agtgagcgca 7680acgcaattaa tgtgagttag
ctcactcatt aggcacccca ggctttacac tttatgcttc 7740cggctcgtat
gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
7800accatgatta cgcc 78145720DNAArtificialchimeric antigen receptor
5gacattgtga tgacccagtc tcacaaattc atgtccacat caattggagc cagggtcagc
60atcacctgca aggccagtca ggatgtgaga actgctgtag cctggtatca acagaaacca
120ggccagtctc ctaaactact aatttactcg gcatcctacc ggtacactgg
agtccctgat 180cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 240gaagacctgg cagtttatta ctgtcagcaa
cattatggta ctcctccgtg gacgttcggt 300ggaggcacca agctggaaat
caaaggcggc ggaggatctg gcggaggcgg aagtggcgga 360gggggctctg
aagtgcagct ggtggagtct gggggaggct tagtgaagcc tggagggtcc
420ctgaaactct cctgtgaagc ctctagattc actttcagta gctatgccat
gtcttgggtt 480cgccagactc cggagaagag gctggagtgg gtcgcagcca
ttagtggagg tggtaggtac 540acctactatc cagacagtat gaagggtcga
ttcaccatct ccagagacaa tgccaagaat 600ttcctgtacc tgcaaatgag
cagtctgagg tctgaggaca cggccatgta ttactgtgca 660agacactatg
atggttatct tgactactgg ggccaaggca ccactctcac agtctcctca 720
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