U.S. patent application number 17/013195 was filed with the patent office on 2021-02-25 for selection methods for genetically-modified t cells.
The applicant listed for this patent is BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Laurence J.N. COOPER, David RUSHWORTH.
Application Number | 20210054346 17/013195 |
Document ID | / |
Family ID | 1000005199019 |
Filed Date | 2021-02-25 |
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United States Patent
Application |
20210054346 |
Kind Code |
A1 |
RUSHWORTH; David ; et
al. |
February 25, 2021 |
SELECTION METHODS FOR GENETICALLY-MODIFIED T CELLS
Abstract
In some aspects, isolated transgenic cells (e.g., transgenic T
cells) are provided that comprise or express a transgene and
DHFR.sup.FS and/or TYMS.sup.SS. Methods for selecting transgeneic
cells are also provided.
Inventors: |
RUSHWORTH; David; (Houston,
TX) ; COOPER; Laurence J.N.; (Houston, TX) |
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Applicant: |
Name |
City |
State |
Country |
Type |
BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM |
AUSTIN |
TX |
US |
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Family ID: |
1000005199019 |
Appl. No.: |
17/013195 |
Filed: |
September 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15552821 |
Aug 23, 2017 |
10808230 |
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PCT/US2016/019288 |
Feb 24, 2016 |
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17013195 |
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62175794 |
Jun 15, 2015 |
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62120790 |
Feb 25, 2015 |
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62120329 |
Feb 24, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/04 20130101;
C12N 9/003 20130101; C12N 5/0637 20130101; C12N 9/48 20130101; C12N
2501/2312 20130101; C12N 9/1007 20130101; C12N 2510/00 20130101;
G01N 33/54326 20130101; C07K 14/575 20130101; C07K 14/7051
20130101; C12N 5/0638 20130101; C12Y 105/01003 20130101; C12Y
201/01045 20130101; C12N 2501/06 20130101; A61K 35/17 20130101;
C12N 2501/599 20130101; C12Y 304/22062 20130101; A61K 38/00
20130101; C07K 14/5434 20130101; C07K 14/5443 20130101 |
International
Class: |
C12N 9/06 20060101
C12N009/06; C12N 9/10 20060101 C12N009/10; A61K 35/17 20060101
A61K035/17; C07K 14/54 20060101 C07K014/54; C07K 14/575 20060101
C07K014/575; C07K 14/725 20060101 C07K014/725; C12N 5/0783 20060101
C12N005/0783; C12N 9/48 20060101 C12N009/48; G01N 33/543 20060101
G01N033/543 |
Claims
1-74. (canceled)
75. An isolated engineered mammalian T cell expressing a first
transgene and TYMS.sup.SS, wherein said T cell comprises (1) a
nucleotide sequence encoding the first transgene and (2) a
nucleotide sequence encoding TYMS.sup.SS.
76. The isolated engineered mammalian T cell of claim 75, wherein
the nucleotide sequence encoding the first transgene and the
nucleotide sequence encoding TYMS.sup.SS are operably linked.
77. The isolated engineered mammalian T cell of claim 75, wherein
the nucleotide sequence encoding the first transgene and the
nucleotide sequence encoding TYMS.sup.SS, upon expression, are
encoded on the same mRNA.
78. The isolated engineered mammalian T cell of claim 75, wherein
the nucleotide sequence encoding the first transgene and the
nucleotide sequence encoding TYMS.sup.SS are separated by an
internal ribosomal entry site (IRES) or a ribosomal slip
sequence.
79. The isolated engineered mammalian T cell of claim 75, wherein
nucleotide sequence encoding the first transgene is positioned 3'
relative to the nucleotide sequence encoding TYMS.sup.SS.
80. The isolated engineered mammalian T cell of claim 75, wherein
the first transgene is a chimeric antigen receptor (CAR) construct,
a polypeptide hormone, a suicide gene, a T-cell receptor (TCR), a
growth factor, or a cytokine.
81. The isolated engineered mammalian T cell of claim 80, wherein
the cytokine is IL-12 or IL-15.
82. The isolated engineered mammalian T cell of claim 75, further
comprising (3) a nucleotide sequence encoding DHFR.sup.FS.
83. The isolated engineered mammalian T cell of claim 82, wherein
the nucleotide sequence encoding DHFR.sup.FS is operably linked to
a nucleotide sequence encoding a second transgene.
84. The isolated engineered mammalian T cell of claim 83, wherein
the nucleotide sequence encoding the second transgene and the
nucleotide sequence encoding DHFR.sup.FS, upon expression, are
encoded on the same mRNA.
85. The isolated engineered mammalian T cell of claim 83, wherein
the nucleotide sequence encoding the second transgene and the
nucleotide sequence encoding DHFR.sup.FS are separated by an
internal ribosomal entry site (IRES) or a ribosomal slip
sequence.
86. The isolated engineered mammalian T cell of claim 83, wherein
the second transgene is a suicide gene, CAR, TCR, polypeptide
hormone, cytokine, chemokine, or transcription factor.
87. The isolated engineered mammalian T cell of claim 75, wherein
the isolated engineered mammalian T cell is a T helper cell (TH
cell), cytotoxic T cell (Tc cell or CTL), memory T cell (TCM cell),
effector T cell (TEM cell), regulatory T cell (Treg cell; also
known as suppressor T cell), natural killer T cell (NKT cell),
mucosal associated invariant T cell, alpha-beta T cell
(T.alpha..beta. cell), or gamma-delta T cell (T.gamma..delta.
cell).
88. A method of treating a patient with a cancer comprising to
administering to the patient a therapeutically effective amount of
the isolated engineered mammalian T cells of claim 75.
89. A method of enriching for regulatory T cells in a population of
T cells isolated from a mammal, the method comprising contacting
the population of T cells with a thymidine synthesis inhibitor
selected from the group consisting of methotrexate (MTX), 5-FU,
Raltitrexed, and Pemetrexed, or a combination thereof, to
selectively deplete effector T cells in the population.
90. The method of claim 89, wherein the population of T cells
isolated from a mammal is contacted with both MTX and 5-FU.
91. The method of claim 89, wherein the T cells express one or both
of DHFR.sup.FS and TYMS.sup.SS.
92. A method for selecting a T cell expressing a transgene of
interest comprising: a) applying a thymidine synthesis inhibitor to
a plurality of T cells that comprises a T cell expressing the
transgene of interest and TYMS.sup.SS; and b) selecting for one or
more T cells surviving after seven or more days of application of
the thymidine synthesis inhibitor, wherein the one or more
surviving T cell(s) expresses the transgene of interest and
TYMS.sup.SS.
93. The method of claim 92, wherein the T cell expressing the
transgene of interest and TYMS.sup.SS further expresses
DHFR.sup.FS.
94. The method of claim 92, wherein the thymidine synthesis
inhibitor is selected from the group consisting of methotrexate
(MTX), 5-FU, Raltitrexed, and Pemetrexed.
Description
[0001] This application is a division of U.S. application Ser. No.
15/552,821, filed Aug. 23, 2017, as a national phase application
under 35 U.S.C. .sctn. 371 of International Application No.
PCT/US2016/019288, filed Feb. 24, 2016, which claims the benefit of
United States Provisional Patent Application Nos. 62/120,329, filed
Feb. 24, 2015, 62/120,790, filed Feb. 25, 2015, and 62/175,794,
filed Jun. 15, 2015, the entirety of each of which is incorporated
herein by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing that is contained in the file named
"UTSCP1272USD1_ST25.txt", which is 13 KB (as measured in Microsoft
Windows.RTM.) and was created on Sep. 2, 2020, is filed herewith by
electronic submission and is incorporated by reference herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The disclosure relates to methods and compositions for
preparing transgenic T cells and enriching for regulatory T cells
in a population of T cells isolated from a mammal.
2. Description of Related Art
[0004] Targeting T cells to human disease has been in progress for
more than 25 years. See Yee C., Immunological reviews 2014,
257(1):250-263. The initial aim of clinical trials was to direct T
cells to target and kill diffuse cancers, for example metastatic
melanoma and leukemia. See Yee C., Immunological reviews 2014,
257(1):250-263 and Roddie C and Peggs K S, Expert opinion on
biological therapy 2011, 11(4):473-487.
[0005] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates, which
may need to be independently confirmed.
[0006] Antigens on cancers are often times overexpressed or mutated
versions of proteins found on non-cancerous cells. Although cancer
antigens ideally demarcate only the cancer, in many instances
cancer antigens are found on non-cancerous cells with the risk of
off-tumor toxicities that cause serious complications that many
times have led to morbidity and death. The powerful nature of T
cell therapies is one of the reasons that T cells continue to be
sought as a therapeutic, but have not yet reached FDA approval in
the United States for any form of disease.
[0007] While many of the T cell clinical trials are showing strong
benefit over standard of care, the cost of producing a T cell
therapy and risk to the patient continues to hamper development of
these technologies beyond a few specialized centers. Further
limitations exist due to the complex immunosuppressive environment
of the tumor, and difficulty of identifying appropriate tumor
antigens. See Corrigan-Curay J, Kiem H P et al., Molecular therapy:
the Journal of the American Society of Gene Therapy 2014,
22(9):1564-1574. It should be noted that T cell therapeutics in
cancer were initially developed for the treatment of melanoma and
leukemia, and in the intervening quarter century have not
significantly deviated from those cancer targets. Further
improvements in the technical aspects of T cell therapy as well as
continuing research and development of immune-modulatory drugs will
continue to promote T cell cancer therapies for cancer and
potentially broaden the applicability of these therapeutics.
[0008] Diseases of excessive inflammation are currently targeted by
immune-modulatory or immune-suppressive medications. These
therapies are often effective, but have untoward side effects as
discussed in the above section. Better targeted immunosuppression
may be possible using regulatory T cells (T.sub.regs). As
T.sub.regs are better understood and culturing techniques become
more advanced, cell therapies based on reconstituting T.sub.regs
will likely move toward clinical trials more rapidly. The use of
T.sub.regs in clinical trials has been limited to preventing GvHD
following hematopoetic stem cell transplantation (HSCT) for the
most part. It is likely that the number of uses for T.sub.reg will
expand as many other forms of inflammation have been targeted in
preclinical models. Technical challenges related to the isolation
and propagation of T.sub.reg is currently limiting the advance of
this T cell therapy. See Singer B D et al., Frontiers in immunology
2014, 5:46.
[0009] The development of MEC independent T cell propagation
methods has been a great technical advance for T cell therapies.
Growing T cells by antigen-specificity-independent selection (ASIS)
generates large numbers of T cells for reinfusion to a patient.
While it might seem counterintuitive to grow T cells without direct
selection for specificity, the large number of T cells can include
an activated and propagated subset of T cells that are specific to
the antigen targeted. Novel ASIS methods are sought to enhance the
selection of transgenic T cells and to select for therapeutically
useful T cell phenotypes. While in vitro ASIS using chimeric
cytokine receptors is a recently reported method of non-immunogenic
selection, it only utilizes the third signal in T cell
activation--cytokine signaling. See Wilkie S et al., The Journal of
biological chemistry 2010, 285(33):25538-25544. A strategy that can
utilize the first and second signals of T cell activation (CD3 and
costimulatory signaling) of human genes to activate and propagate T
cells independent of antigen specificity can be of further
benefit.
[0010] The adoptive transfer of antigen-specific T cells is a
rapidly developing field of cancer immunotherapy with various
approaches to their manufacture being tested and new antigens being
targeted. T cells can be genetically-modified for immunotherapy to
express chimeric antigen receptors (CAR) that recognize
tumor-associated antigens (TAAs) independent of HLA (HLA is the
human version of MEC) expression. Recent results from early-phase
clinical trials demonstrate that CAR.sup.+ T-cell (CART) therapies
can lead to partial and complete remissions of malignant diseases,
including in some recipients with advanced/relapsed B-cell tumors.
See Kalos M et al., Science translational medicine 2011,
3(95):95ra73 and Kochenderfer J N et al., Blood 2012,
119(12):2709-2720.
[0011] Therefore, notwithstanding what has previously been reported
in the literature, there exists a need for improved methods of
preparing transgenic T cells, propagating T cells for therapeutic
treatments and selecting for regulatory T cells. Additionally,
methods of making and using transgenic T cells and agents
regulating the propagation and selection of transgenic T cells will
greatly aid in the treatment of cancer, autoimmune diseases,
infectious diseases and any number of other medical conditions in
which the immune system plays a role.
SUMMARY OF THE INVENTION
[0012] In one aspect, an isolated transgenic mammalian T cell
comprising or expressing a transgene and one or more of DHFR.sup.FS
and TYMS.sup.SS is provided. In some embodiments, the isolated
transgenic mammalian T cell comprises or expresses a transgene,
DHFR.sup.FS and TYMS.sup.SS. In some embodiments, the transgene is
a suicide gene. In some embodiments, a suicide gene is further
included. In some embodiments, codon optimization is performed on
DHFR.sup.FS, TYMS.sup.SS, or both.
[0013] In another aspect is provided a method for inhibiting
anti-thymidylate (AThy) toxicity in a mammalian T cell comprising
expressing an anti-thymidylate resistance (AThyR) transgene in said
mammalian T cell. In some embodiments, the AThyR transgene is
DHFR.sup.FS. In some embodiments, the AThyR transgene is
TYMS.sup.SS. In some embodiments, the transgene is a suicide gene.
In some embodiments, a suicide gene is further included. In some
embodiments, codon optimization is performed on DHFR.sup.FS,
TYMS.sup.SS, or both.
[0014] In another aspect is provided a method for selecting a T
cell expressing a transgene of interest. The method comprises
applying a thymidine synthesis inhibitor to a plurality of T cells
that comprises a T cell expressing the transgene of interest and
DHFR.sup.FS and selecting for one or more T cells surviving after
seven or more days of application of the thymidine synthesis
inhibitor, wherein the one or more T cells expresses a vector
comprising the transgene of interest and DHFR.sup.FS. The thymidine
synthesis inhibitor may be selected from the group consisting of
methotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed. In some
embodiments, the transgene is a suicide gene. In some embodiments,
a suicide gene is further included. In some embodiments, codon
optimization is performed on DHFR.sup.FS, TYMS.sup.SS, or both.
[0015] Yet another aspect is a method for selectively propagating
peripheral blood mononuclear cells (PBMC) resistant to MTX and
5-FU. The method comprises transfecting peripheral PBMC with a
vector comprising an AThyR gene, treating the transfected PBMC with
a thymidine synthesis inhibitor and selecting for PBMC that express
the AThyR gene. In some embodiments of this aspect, the method
further comprises propagating a T cell population from the
transfected PBMC. In some embodiments, the thymidine synthesis
inhibitor may be selected from the group consisting of methotrexate
(MTX), 5-FU, Raltitrexed and Pemetrexed. In some embodiments, the
thymidine synthesis inhibitor is MTX. In some embodiments, the
AThyR gene is TYMS.sup.SS. In some embodiments, the AThyR gene is
DHFR.sup.FS. In some embodiments, codon optimization is performed
on DHFR.sup.FS, TYMS.sup.SS, or both.
[0016] Another aspect is an isolated transgenic mammalian T cell
comprising a nucleic acid sequence comprising a transgene of
interest and a nucleotide sequence encoding DHFR.sup.FS or
TYMS.sup.SS. In some embodiments, the isolated transgenic mammalian
T cell comprises a nucleic acid comprising a transgene of interest
and a nucleotide sequence encoding DHFR.sup.FS, wherein the
transgene of interest and the nucleotide sequence encoding
DHFR.sup.FS are operably linked. In some embodiments, the isolated
transgenic mammalian T cell comprises a nucleic acid comprising a
transgene of interest and a nucleotide sequence encoding
TYMS.sup.SS, wherein the transgene of interest and the nucleotide
sequence encoding TYMS.sup.SS are operably linked. In some
embodiments, the transgene is a suicide gene. In some embodiments,
a suicide gene is further included. In some embodiments, codon
optimization is performed on DHFR.sup.FS, TYMS.sup.SS, or both.
[0017] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and DHFR.sup.FS, wherein
the T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the DHFR.sup.FS. In some embodiments, the transgene is
a suicide gene. In some embodiments, a suicide gene is further
included. In some embodiments, codon optimization is performed on
DHFR.sup.FS.
[0018] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and TYMS.sup.SS, wherein
said T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the TYMS.sup.SS. In some embodiments, the transgene is
a suicide gene. In some embodiments, a suicide gene is further
included. In some embodiments, codon optimization is performed on
TYMS.sup.SS.
[0019] In yet another aspect is provided a method of treating a
patient with a cancer comprising administering to a patient a
therapeutically effective amount of a T cell of an isolated T cell
of any of the above embodiments.
[0020] In some embodiments, a combination therapy of AThyR.sup.+ T
cells with AThy therapies can be used to improve anti-tumor
immunity. An isolated T cell with a AThyR.sup.+ phenotype can be
administered with MTX, 5-FU, Raltitrexed and Pemetrexed, or any
other thymidine synthesis inhibitor.
[0021] In yet another aspect is provided a method for selecting for
a T cell expressing a transgene of interest, as shown in any of the
FIGS. or as described in the description.
[0022] In yet another aspect is provided a T cell, as shown in any
of the FIGS. or as described in the description.
[0023] In another aspect is a method for selectively propagating
human T cells resistant to one or more of MTX, 5-FU, Raltitrexed
and Pemetrexed, as shown in any of the FIGS. or as described in the
description. In some embodiments, the human T cells are primary
human T cells.
[0024] Another aspect is a method of enriching for regulatory T
cells in a population of T cells isolated from a mammal by
contacting said population with a thymidine synthesis inhibitor
selected from the group consisting of MTX, 5-FU, Raltitrexed and
Pemetrexed, or a combination thereof, to selectively deplete
effector T cells in the population. In some embodiments, the
population of T cells isolated from a mammal is contacted with both
MTX and 5-FU. In some embodiments, the T cells express one or more
of DHFR.sup.FS and TYMS.sup.SS. In some embodiments, the T cells
express both DHFR.sup.FS and TYMS.sup.SS. In some embodiments,
codon optimization is performed on DHFR.sup.FS, TYMS.sup.SS, or
both.
[0025] Another aspect is a method for depleting regulatory T cells
in a population of T cells isolated from a mammal by culturing said
population in the presence of one or more aminoglycosidases to
selectively deplete the regulatory T cells in said culture. In some
embodiments, the T cells express one or more of DHFR.sup.FS and
TYMS.sup.SS. In some embodiments, the T cells express both
DHFR.sup.FS and TYMS.sup.SS. In some embodiments, codon
optimization is performed on DHFR.sup.FS, TYMS.sup.SS, or both.
[0026] Another aspect is a method for selecting for a regulatory T
cell isolated from a mammal. The method comprises treating a
plurality of T cells expressing one or more of DHFR.sup.FS and
TYMS.sup.SS with a thymidine synthesis inhibitor and selecting a
regulatory T cell that expresses a marker for a regulatory T cell.
In some embodiments, the T cells express DHFR.sup.FS. In some
embodiments, the selecting step comprises cell isolating with
magnetic bead sorting using one or more of an anti-CD4 antibody, an
anti-CD25 antibody, an anti-CD3 antibody, an anti-CD8 antibody, an
anti-CD25 antibody, an anti-CD39 antibody, an anti-CD45 antibody,
an anti-CD152 antibody, an anti-KI-67 antibody, an anti-LAP
antibody and an anti-FoxP3 antibody. In some embodiments, the
thymidine synthesis inhibitor is selected from the group consisting
of methotrexate (MTX), 5-FU, Raltitrexed or Pemetrexed. In some
embodiments, the method further comprises treating the regulatory T
cell with one or more of folate, leucovarin and FU.
[0027] In another aspect is provided a composition comprising a
first plurality of T cells isolated from a mammal and a thymidine
synthesis inhibitor. The first plurality of T cells is enriched for
regulatory T cells as compared to a second plurality of T cells
isolated from a mammal that does not comprise a thymidine synthesis
inhibitor.
[0028] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
[0029] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and DHFR.sup.FS, wherein
the T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the DHFR.sup.FS. In some embodiments, codon
optimization is performed on DHFR.sup.FS and/or the sequence
encoding the transgene of interest. In some embodiments, the
transgene of interest and the nucleotide sequence encoding
DHFR.sup.FS, upon expression, are encoded on the same mRNA. In
further embodiments, the sequence encoding the transgene of
interest and the nucleotide sequence encoding DHFR.sup.FS are
separated by an internal ribosomal entry site (IRES) or a ribosomal
slip sequence. In certain embodiments, the transgene of interest
may encode a chimeric antigen receptor (CAR) construct, a T-cell
Receptor (TCR), a hormone (e.g., glucagon), a cytokine, a
chemokine, a suicide gene, a transcription factor or a cell surface
polypeptide, such as a receptor (e.g., an integrin, cytokine
receptor, chemokine receptor or hormone receptor).
[0030] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and TYMS.sup.SS, wherein
said T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the TYMS.sup.SS. In some embodiments, codon
optimization is performed on TYMS.sup.SS and/or the sequence
encoding the transgene of interest. In certain embodiments, the
transgene of interest and the nucleotide sequence encoding
TYMS.sup.SS, upon expression, are encoded on the same mRNA. In some
embodiments, the sequence encoding the transgene of interest and
nucleotide sequence encoding TYMS.sup.SS are separated by an IRES
or a ribosomal slip sequence. In specific embodiments, the isolated
transgenic mammalian T cell expressing a transgene and TYMS.sup.SS
further comprises a nucleotide sequence encoding DHFR.sup.FS
(optionally, the nucleotide sequence encoding DHFR.sup.FS is
operably linked to a second transgene of interest). In some
embodiments, the transgene of interest (e.g., operably linked to
TYMS.sup.SS) is a growth factor, a CAR construct, a TCR, a hormone
(e.g., glucagon), a cytokine, a chemokine, a suicide gene, a
transcription factor (e.g., FoxP3) or a cell surface polypeptide,
such as a receptor (e.g., an integrin, cytokine receptor, chemokine
receptor or hormone receptor). In particular embodiments, the
cytokine may be IL-12 or IL-15.
[0031] Yet a further aspect is a method for providing controlled
expression of a first transgene comprising providing a transgenic
mammalian cell comprising a nucleic acid comprising the first
transgene operably linked to a nucleotide sequence encoding
TYMS.sup.SS, said cell further comprising a nucleotide sequence
encoding DHFR.sup.FS. In some embodiments, the first transgene and
nucleotide sequence encoding TYMS.sup.SS, upon expression, are
encoded on the same mRNA. In further embodiments, the sequence
encoding the first transgene and the nucleotide sequence encoding
TYMS.sup.SS are separated by an IRES or a ribosomal slip sequence.
In certain embodiments, the first transgene of interest is a growth
factor, is a growth factor, a CAR construct, a TCR, a hormone
(e.g., glucagon), a cytokine, a chemokine, a suicide gene, a
transcription factor (e.g., FoxP3) or a cell surface polypeptide,
such as a receptor (e.g., an integrin, cytokine receptor, chemokine
receptor or hormone receptor). In particular embodiments, the
cytokine may be IL-12 or IL-15.
[0032] In further embodiments, the nucleotide sequence encoding
DHFR.sup.FS is operably linked to a second transgene. In some
embodiments, the second transgene and the nucleotide sequence
encoding DHFR.sup.FS, upon expression, are encoded on the same
mRNA. In other embodiments, the sequence encoding the second
transgene of interest and nucleotide sequence encoding DHFR.sup.FS
are separated by an IRES or a ribosomal slip sequence. In certain
embodiments, the second transgene is a suicide gene. In specific
embodiments, the suicide gene is an inducible suicide gene. In
particular embodiments, the suicide gene is an inducible Caspase 9.
In some embodiments, the mammalian cell is a T-cell.
[0033] In another aspect is provided a recombinant nucleic acid
molecule encoding TYMS.sup.SS and a first transgene coding
sequence. In some embodiments, the sequence encoding TYMS.sup.SS
and/or the sequence encoding the transgene of interest is codon
optimized. In certain embodiments, recombinant nucleic acid is a
DNA or a RNA (e.g., a mRNA). In some embodiments, the sequence
encoding the transgene of interest and nucleotide sequence encoding
TYMS.sup.SS are separated by an IRES or a ribosomal slip sequence.
In some embodiments, the transgene of interest is a growth factor,
is a growth factor, a CAR construct, a TCR, a hormone (e.g.,
glucagon), a cytokine, a chemokine, a suicide gene, a transcription
factor (e.g., FoxP3) or a cell surface polypeptide, such as a
receptor (e.g., an integrin, cytokine receptor, chemokine receptor
or hormone receptor). In particular embodiments, the cytokine may
be IL-12 or IL-15.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The drawings are exemplary only, and should not be construed
as limiting the invention.
[0035] FIG. 1A depicts a pathway showing the role of synthesis of
thymidine in DNA replication and cell survival.
[0036] FIG. 1B depicts the design of putative AThyR transgenes
resistant to AThy toxicity in order to confer resistance to T cells
that might be used in a combination therapeutic with AThy
chemotherapy. AThyRs were co-expressed with a fluorescent protein
to indicate that surviving cells contained the transgene. These
transgene utilized the Sleeping Beauty transposon/transposase
system to induce stable transgene expression in Jurkat. Human
muteins DHFR.sup.FS-resistant to MTX (left), human mutein
TYMS.sup.SS--resistant to 5-FU (center), and the gold-standard
Neomycin resistance gene (NeoR) drug resistance gene--resistance to
G418 (right) were used in this study. Codon optimized (CoOp)
versions of DHFR.sup.FS & TYMS.sup.SS replaced native codon
DHFR.sup.FS & TYMS.sup.SS to test whether known
post-transcriptional regulatory mechanisms were affecting AThyR
selection or survival.
[0037] FIG. 1C depicts three different panels showing the
percentage of eGFP+ viable Jurkat T cells following treatment with
MTX (left panel), 5-FU (center panel) and G418 (right panel) at
varying concentrations. The left panel relates to
DHFR.sup.FS-2A-GFP (DG), CoOp DG, and no DNA, that were
electroporated into Jurkat and subjected to MTX after 2 days. The
center panel relates to TYMS.sup.SS-2A-GFP (TSG), CoOp TSG, and No
DNA electroporated Jurkat that were treated on day 2 with 5-FU. The
right panel relates to NeoR-GFP and No DNA electroporated Jurkat
that were treated on day 2 with G418. For each experiment in C the
percentage of eGFP.sup.+ viable Jurkat is given after testing on
day 8-10 after the addition of drug.
[0038] FIG. 1D depicts the effect of MTX and Pemetrexid on the
survival of cells that expressed native DHFR and TYMS ("No DNA") or
expressed. DG and TYMS.sup.SS-2A-RFP (TSR) were co-electroporated
into Jurkat to determine whether combination DHFR.sup.FS &
TYMS.sup.SS confer enhanced survival to MTX (left) or Pemetrexid
(right).
[0039] FIG. 1E depicts that following 2 weeks of selection in 1
.mu.M MTX, [DHFR.sup.FS & TYMS.sup.SS].sup.+ Jurkat displayed a
uniform and repeatable pattern of correlated expression. Shown
here, four separate [DHFR.sup.FS & TYMS.sup.SS].sup.+ Jurkat
experiments are overlaid in different shades. Experiments were
independently repeated at least twice with 4-6 replicates.
*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.;
Dihydrofolate (DHF); DHF reductase (DHFR); deoxyuridine
monophosphate (dUMP); deoxythymidine monophosphate (dTMP); 5,
10-methylenetetrahydrofolate (5,10 CH.sub.2THF); nicotinamide
adenine dinucleotide phosphate (NADP).
[0040] FIG. 2A-I depicts experiments relating to viability of
Jurkat cells given for DHFR.sup.FS (left), TYMS.sup.SS (right), and
NeoR (center).
[0041] FIG. 2A-II depicts experiments relating to alternations of
mean fluorescent intensity (MFI) of eGFP given for DHFR.sup.FS
(left), TYMS.sup.SS (right), and NeoR (center).
[0042] FIG. 2B depicts a determination whether enhanced survival
occurs when Raltitrexed and DHFR.sup.FS & TYMS.sup.SS were
co-electroporated into Jurkat treated with Ral.
[0043] FIG. 2C depicts the correlation of expression of DHFR.sup.FS
and TYMS.sup.SS plasmids that were independently expressed.
Observations suggested that cells expressing DHFR.sup.FS &
TYMS.sup.SS as independent plasmids have correlated expression of
each plasmid. This could have implications in the co-regulation of
DHFR.sup.FS with TYMS.sup.SS. Hence, the MFI of eGFP and RFP were
correlated for treatments with multiple concentrations of MTX, Pem,
and Ral. The linear regression data is included in the FIG. Each
experiment was independently repeated at least twice with 4-6
replicates. *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001. There was observed improved expression over mock
electroporated Jurkat, and a weak survival improvement in 5 .mu.M
5-FU. Without wishing to be bound by theory, the lack of
significantly enhanced survival is likely due to an alternative
mechanism of 5-FU contributing to toxicity, which is likely the
known inhibition of mRNA and rRNA synthesis by 5-FU. See Longley D
B, et al., The Journal of biological chemistry 2010,
285(16):12416-12425.
[0044] FIG. 3A depicts a propagation schematic showing initial AaPC
stimulation. Two days after AaPC stimulation, the co-cultures
received 0.1 .mu.M MTX, 5 .mu.M 5-FU, or 1.4 mM G418 until day 14.
The co-cultures were re-stimulated with AaPC at a 1:1 ratio and
given 50 IU/mL IL-2 every 7 days from day 1 to 35. Phenotypic
changes in transgene expression were tracked during drug
administration for the first 14 days and for the 21 days after drug
administration had ended
[0045] FIG. 3B-i shows the tracking of T cells for expression of
AThyRs DHFR.sup.FS-DG, TYMS.sup.SS-TG, both [DG & TSR], and
NeoR-NRG in the presence (day 2-14) then absence (day 14-35) of
appropriate selection drug. All experiments contain 5-6 biological
replicates with each experiment independently repeated two times.
*=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.
[0046] FIG. 3B-ii shows the percentage of T cells shown in FIG.
4B-I that express co-receptor CD4.
[0047] FIG. 3C-i shows the tracking of T cells for expression of
Myc-ffLuc-2A-NeoR (NRF) combined with each AThyR transgene [DG
& NRF], [TSG & NRF], and [DG & TSR & NRF] in order
to improve selection for AThyRs selected by 5-FU. Selection
occurred under the same condition as FIG. 4B-I, with the exception
that 100 IU IL-2/mL was added to promote outgrowth of cells treated
with G418. All experiments contain 5-6 biological replicates with
each experiment independently repeated two times. *=p<0.05;
**=p<0.01; ***=p<0.001; ****=p<0.0001.
[0048] FIG. 3C-ii shows the percentage of T cells shown in FIG.
4C-I that express co-receptor CD4.
[0049] FIG. 3D-i shows that to elucidate the influence of 5-FU and
TYMS.sup.SS on the selection of DHFR.sup.FS, RFP or TYMS.sup.SS-RFP
(TSR) that were co-electroporated into T cells with DHFR.sup.FS.
All experiments contain 5-6 biological replicates with each
experiment independently repeated two times. *=p<0.05;
**=p<0.01; ***=p<0.001; ****=p<0.0001.
[0050] FIG. 3D-ii shows the percentage of T cells shown in FIG.
4D-I that express co-receptor CD4.
[0051] FIGS. 4A-4C show the propagation characteristics of AThyR+ T
cells in the presence or absence of MTX, 5-FU, and/or G418. FIG. 4A
shows AThyR and NeoR electroporated primary T cells were compared
on Day 21 to mock-electroporated T cells treated with the same
conditions. Each experiment was independently repeated at least
twice with 5-6 replicates. *=p<0.05, **=p<0.01. FIG. 4B,
panel I depicts the continued propagation of the experiment of FIG.
5A on day 35. Each experiment was independently repeated at least
twice with 5-6 replicates. *=p<0.05, **=p<0.01. FIG. 4B,
panel II depicts the day 35 changes in outgrowth potential for
primary T cells when NeoR is combined with DHFR.sup.FS and/or
TYMS.sup.SS. Each experiment was independently repeated at least
twice with 5-6 replicates. *=p<0.05, **=p<0.01. FIG. 4C shows
the influence of 5-FU on preserving outgrowth potential for primary
T cells on day 35. Each experiment was independently repeated at
least twice with 5-6 replicates. *=p<0.05, **=p<0.01.
[0052] FIGS. 5A-5H: Cis-transgenes downstream of DHFR.sup.FS
increase in the presence of MTX independent of mRNA sequence and
the increase is suppressed by restoration of thymidine synthesis.
FIG. 5A Jurkat cells were genetically-modified to express
FLAG-DHFR.sup.FS-2A-eGFP pSBSO (D.sup.FSG) with resistance to MTX
(n=4), codon optimized (CoOp) D.sup.FSG--with known mRNA binding
elements D.sup.FSG removed (n=5), and [D.sup.FSG &
FLAG-TYMS.sup.SS-2A-RFP pSBSO (TS.sup.SSR)]-- with enhanced
resistance to MTX beyond D.sup.FSG alone through the addition of
MTX resistant TYMS.sup.SS (n=7). Genetically-modified Jurkat cells
were selected for 2 weeks in 1 .mu.M MTX before culturing without
MTX for 3-5 weeks. The stable fluorescent protein expression, in
the absence of MTX, is depicted by mean fluorescence intensity
(MFI). FIG. 5B-I Jurkat cells were treated for 72 hours with 0.5
.mu.M MTX or no treatment. The MFI difference (.DELTA.=eGFP MFI MTX
treated--eGFP MFI untreated) is depicted. FIG. 5B-II a
representative histogram demonstrates the MTX induced change in
eGFP MFI for DHFR.sup.FS (left peak) and CoOp DHFR.sup.FS (right
peak) in Jurkat. FIGS. 5C-D show in primary T cells, transgenes
DHFR.sup.FS, TYMS.sup.SS, or the combination were selected for 2
weeks in the presence of cytotoxic drug and then propagated without
selection for 3 weeks (see examples). On day 35, T cells were
stimulated with anti-CD3, anti-CD28 antibodies, and 50 IU/mL IL-2
in the presence or absence of MTX. The fluorescent protein MFI of
untreated cells is shown in FIG. 5C, and FIG. 5D-I depicts the A
MFI after 72 hours of treatment with 0.5 .mu.M MTX in comparison to
no treatment. FIG. 5D-II, shows a representative histogram, which
demonstrates the observed shift in eGFP fluorescence for
DHFR.sup.FS+ T cells in the presence or absence of MTX (n=5). (No
DNA=far left peak; D.sup.FSG & NRF, No Trx=upper center peak;
D.sup.FSG & NRF, MTX=upper right peak; D.sup.FSG &
TS.sup.SSR, No Trx=lower center and lower right peak; D.sup.FSG
& TS.sup.SSR, MTX=lowest peak). FIG. 5E, a trans regulatory
pattern of DHFR and TYMS linked fluorescent proteins was observed.
A representative flow plot from the 1 .mu.M MTX selected Jurkat
left untreated in (5A) demonstrates that unselected
mock-electroporated (No DNA--lower left cluster) Jurkat and
D.sup.FSG+ Jurkat (lower right cluster) have a globular appearance
in the RFP channel, while co-expression of DHFR.sup.FS with
TYMS.sup.SS in [D.sup.FSG & TS.sup.SSR]+ Jurkat leads to a
linear clustering (upper right cluster). FIG. 5F T cells were
electroporated with DHFR.sup.FS and co-transformed with either RFP
control or FLAG-TYMS.sup.SS-2A-RFP pSBSO (TS.sup.SSR) before
propagation as before (in 5C) with selection in 0.1 .mu.M MTX from
days 2-14 before continued propagation in the absence of MTX. A
representative flow plot of primary human T cells from the same
donor where [D.sup.FSG & RFP (cluster on the far right)],
[D.sup.FSG & TS.sup.SSR (upper right cluster)], and
untransformed T cells (lower left quadrant) are shown on day 21. A
linear clustering of DHFR.sup.FS is again noted when co-expressed
with TYMS.sup.SS that is not noted with RFP alone. FIG. 5G further
studies to identify a trans pattern of linked expression between
DHFR.sup.FS and TYMS.sup.SS were identified in the selection of
[D.sup.FSG & TS.sup.SSR] electroporated Jurkat in anti-folates
MTX [0, 0.01, 0.1, 0.5, 1, 5 .mu.M], pemetrexed [0, 10, 50, 100
.mu.M], and raltitrexed [0, 1, 5, 10 .mu.M]. The MFI of D.sup.FSG
and TS.sup.SSR for each expression pattern was plotted after day
2-14 in selection. The values are plotted and a linear fitting was
performed with the R.sup.2 from the Pearson's correlation and the
slope of the linear regression provided on the graph. This data is
assembled from 4 technical replicates. FIG. 5H depicts a model of
post-transcriptional regulation of DHFR and TYMS. All experiments
other than that depicted in FIG. 5G were independently repeated
twice. Kruskall-Wallis test was used to determine significant
differences with multivariate analyses; *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001. TMP--thymidine monophosphate;
UMP--uridine monophosphate; DHF--dihydrofolate;
THF--tetrahydrofolate; 5, 10-methylenetetrahydrofolate (5, 10
CH2THF).
[0053] FIGS. 5I-5L, shows co-expression of DHFR.sup.FS with
TYMS.sup.SS leads to controlled expression of TYMS.sup.SS and cis
transgenes in the presence of MTX. FIGS. 5I-5J, T cells from the
experiment described in FIG. 5F were propagated to day 35. T cells
were stimulated for 72 hours with anti-CD3, anti-CD28 antibodies,
50 IU/mL IL-2, and varying concentrations of MTX. The MTX induced
change in eGFP MFI for DHFR.sup.FS is shown in (5I), while the
influence of MTX on RFP and RFP co-expressed with TYMS.sup.SS
(TS.sup.SSR) is shown in (5J) (n=6, repeated independently twice,
analyzed by Two-Way ANOVA with Sidak's multiple comparison test).
FIG. 5K, this regulatory pattern was applied to a clinically
relevant problem: The cytokine interleukin-12 (IL-12) is a strong
promoter of anti-tumor activity in T cells, but is highly toxic. A
construct expressing IL-12 following TYMS.sup.SS, called
TS.sup.SSIL-12, was used to modulate IL-12 expression in
conjunction with the construct D.sup.FSiC9. D.sup.FSiC9 is capable
of selecting T cells with DHFR.sup.FS or depleting T cells with
inducible caspase 9 (iC9). A representative flow diagram of the
same donor depicts intracellular expression of IL-12 and c-Myc-iC9
in [DFSiC9 & TS.sup.SSIL-12]-expressing T cells. These cells
are shown on day 21 after selection from day 2-14 in 0.1 .mu.M MTX
and subsequent treatment with 0.5 .mu.M MTX (right cluster) or no
treatment (left cluster) from days 14-21. Cellular excretion of
IL-12 was blocked for 6 hours before intracellular staining. Gating
is based on staining of untransformed, unselected T cells stained
in the same way. FIG. 5L, three donors were treated as in (K) and
the change in transgene expression noted after 7 days of treatment
with 0.5 .mu.M MTX is shown. Each measure was analyzed by t-tests.
ns=not significant; *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001.
[0054] FIGS. 6A-6C depict flow plots of transgene expression for
AThyR experiments on day 35. Flow plots of CD4 and GFP expression
depict day 35 of a series of experiments designed to characterize
the selection and maintenance of transgene expression in donor T
cells. T cells grown for 35 days with days 2-14 in the presence of
cytotoxic drugs MTX, 5-FU, G418, or a combination, as noted above
the flow plot. FIG. 6A depicts experimental conditions that
corresponds to the experiment described for FIG. 3B. FIG. 6B
depicts experimental conditions that corresponds to the experiment
described for FIG. 3C. FIG. 6C depicts experimental conditions that
corresponds to the experiment described for FIG. 3D.
[0055] FIG. 6D shows that the presence of ffLuc-2A-NeoR--NRF--on
day 35 for experiment noted in FIG. 6B is demonstrated using
D-luciferin to induce T cell chemiluminescence. Each experiment was
independently repeated at least twice with 6 replicates.
Representative flow plots are depicted. *=p<0.05, **=p<0.01,
***=p<0.001.
[0056] FIGS. 7A-7C depict AThyR rescue of AThyR.sup.+ and
AThyR.sup.neg T cells following 72 hours treatment in MTX. T cells
from the experiment described for FIG. 3D were stimulated on day 35
with anti-CD3, anti-CD28, and IL-2 along with varying doses of MTX
[0, 0.1, 0.5, 1 .mu.M] for 72 hours. FIG. 7A shows the gating
strategy and representative flow plots. FIG. 7B shows enhanced
viability of AThyR+ T cell cultures. FIG. 7C shows assessment of
Viable, CD3.sup.+, GFPneg, RFP.sup.neg T cells (AThyR.sup.neg) for
survival. Each experiment was independently repeated at least twice
with 6 biologic replicates total. Representative flow plots from
one are depicted; ns=no significance; *=p<0.05, **=p<0.01,
***=p<0.001; ****=p<0.0001.
[0057] FIGS. 8A-8E depict an example that AThyRs select for
transgenes of interest. Increased selection of DHFR.sup.FS is
desirable for difficult to isolate genes of interest, such as
suicide genes. FIG. 8A shows a construct in which the suicide gene
inducible caspase 9 (iC9) was designed to express with DHFR.sup.FS
in the plasmid DFSiC9 shown in (A). FIG. 8B shows the testing of
the construct depicted in FIG. 8A in PBMC of 3 healthy donors
stimulated with a 1:1 ratio of OKT3-loaded AaPC and treated with
MTX from day 2 until day 7 when survival is shown. FIG. 8C shows T
cells were electroporated with CD19-specific chimeric antigen
receptor (CAR), D.sup.FSiC9, and SB transposase and expanded on
CARL.sup.+ K562 in the presence of MTX for 21 days to select for
each transgene, with CARL an acryonym for ligand for CAR. The
expression of costimulatory T cell receptors CD4, CD8, and
transgenes CAR and DHFR.sup.FS are shown in 21 day CARL expanded
transgenic T cells in comparison to mock electroporated T cells
expanded on OKT3-loaded AaPC clone.4. Experiments were performed
with 4 normal donors and repeated twice. Significance for each
comparison was initially determined by Two-Way ANOVA followed by
Sidak's post-hoc analysis; *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001. FIG. 8D shows the effect of MTX
on cytotoxicity in DHFR.sup.FS+ CAR.sup.+ T cells was tested by
stimulating CAR.sup.+ T cells in the presence or absence of MTX for
7 days after stimulation on day 14. Cytotoxicity was assessed by
chromium release assay (CRA) on Day 21 using CD19 positive or CD19
negative murine lymphoma EL-4 cells. T cells were co-incubated with
EL-4 at a 1 target:5 effector ratio. Experiments were performed
with 4 normal donors and repeated twice. Significance for each
comparison was initially determined by Two-Way ANOVA followed by
Sidak's post-hoc analysis; *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001. FIG. 8E shows the assessment of
the functionality of iC9 on day 21 by resting T cells for 48 hours
in 10 nM AP20187. T cells had previously been stimulated for 7 days
in the presence or absence of MTX. Comparison of surviving
CAR.sup.+ T cells is made to matched, un-treated cells. Experiments
were performed with 4 normal donors and repeated twice.
Significance for each comparison was initially determined by
Two-Way ANOVA followed by Sidak's post-hoc analysis; *=p<0.05,
**=p<0.01, ***=p<0.001, ****=p<0.0001. Co-expressing
DHFR.sup.FS with iC9 rather than CAR added the potential to ablate
T cells through the addition of iC9 chemical inducer of
dimerization AP20187. The addition of AP20187 significantly
depleted resting CAR.sup.+ T cells independent of MTX. This
demonstrates that D.sup.FSiC9 can select for iC9 expression and
deplete genetically-modified T cells as necessary. The use of
DHFR.sup.FS has the advantage of selecting transgene expression in
T cells independent of antigen-specificity and antigen expression,
making DHFR.sup.FS a more portable tool for use in a variety of T
cell studies.
[0058] FIG. 9 depicts that post-transcriptional regulation of
thymidine synthesis locks expression of DHFR to TYMS. MTX-induced
increases in DHFR expression were inhibited by restoration of
thymidine synthesis (TMP--thymidine monophosphate from UMP--uridine
monophosphate). Likewise, MTX-induced decreases in TYMS expression
were restored to normal levels by the restoration of DHFR activity
reducing DHF--dihydrofolate to THF--tetrahydrofolate.
[0059] FIGS. 10A-10D show that the drug selection of T.sub.CD4,
FoxP3 by MTX occurs in part through toxicity. The known selection
of T.sub.CD4, FoxP3 by MTX was analyzed by targeting enzymes that
contribute to the action of MTX. As T.sub.CD4, FoxP3 are a rare
component of PBMC, drug based inhibition was originally sought to
analyze the phenomenon. Multiple drugs with actions similar to MTX
were used to assay for the selection of T.sub.CD4, FoxP3. In this
case, .gamma.-irradiation, G418, and cisplatin (CDDP) were used for
controls as none of those treatments act on the known enzymatic
targets of MTX. FIG. 10A shows the association of each drug to the
enzyme targets of MTX. FIG. 10B, panel I shows PBMC stimulated with
anti-CD3/CD28 and soluble human IL-2 were given lethal doses of
each treatment and assayed after 7 days for viability. FIG. 10B,
panel II shows that these treatments resulted in variable selection
for T.sub.CD4, FoxP3 on day 7. The inability of folate analogs
targeting DHFR, TYMS, or GARFT to significantly select for
T.sub.CD4, FoxP3 suggested that inhibition of AICARtf/inosine
monophosphate (IMP) cyclohydrolase (ATIC) contributes to this
selection. A dose dependence study followed analyzing the
contribution of ATIC inhibitor in the selection of T.sub.CD4,
FoxP3. The study in B-II noted that G418 depleted T.sub.CD4, FoxP3,
thus, this was used as a negative control while the known selection
of T.sub.CD4, FoxP3 by rapamycin (Rapa) was a positive control. A
non-folate analog known to inhibit ATIC (iATIC) was used as a
specific inhibitor of ATIC. FIGS. 10C and 10D show the cytotoxicity
of G418 and MTX.
[0060] FIGS. 10E and 10F-i show the cytotoxicity of iATIC and
Rapa.
[0061] FIG. 10F-ii (four panels) shows the selection for TCD4,
FoxP3 for G418, MTX, iATIC, and Rapa.
[0062] FIG. 10G depicts flow plots for CD4 and FoxP3 expression.
FoxP3 expression was enhanced by iATIC similar to the action of
Rapa, suggesting that MTX selection relies in part on cytotoxicity
and in part by inhibition of ATIC to enhance selection of
T.sub.CD4, FoxP3. All assays used 4-7 donors independently repeated
2-3 times. Statistical significance was assessed using One-Way
ANOVA for viability and Kruskall-Wallis test for percentage of
T.sub.CD4, FoxP3; *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001.
[0063] FIGS. 11A-11B show correlative findings in the selection of
Tregs from primary T cells through resistance to the anti-DHFR and
anti-TYMS actions of MTX. FIG. 11A shows the selection of TCD4,
FoxP3 was assessed at day 21 in each experiment. Selection of TCD4,
FoxP3 was assessed at day 21 in each experiment. The selection of
T.sub.CD4, FoxP3 in the experiment corresponding to column I of
FIG. 2 is shown in A. It is notable for the rescue of T.sub.CD4,
FoxP3 with NeoR and early selection of T.sub.CD4, FoxP3 with MTX
selection of DHFR.sup.FS. FIG. 11B shows flow plots in which FoxP3
is co-expressed with IL-2 in the top row, LAP in the middle row or
CTLA-4 in the bottom row for the same experiment after stimulation
on Day 35. This experiment utilized 5 donors and was independently
repeated twice. Significance was assessed by Two-Way ANOVA and
Sidak's post-hoc; *=p<0.05, **=p<0.01.
[0064] FIGS. 12A-12D show that primary T cells resistant to the
anti-DHFR and anti-TYMS actions of MTX preferentially expand Tregs.
Primary T cells were electroporated with DHFR.sup.FS and
TYMS.sup.SS transgenes resistant to the anti-DHFR and anti-TYMS
actions of MTX, respectively, in order to assess the contribution
of each pathway to the selection of T.sub.CD4, FoxP3. T cells were
electroporated with plasmids expressing drug resistant transgenes
and stimulated with artificial antigen presenting cells (AaPCs)
weekly at a 1:1 ratio. T cells were selected for 2 weeks in the
combined with TYMSSS-2A-RFP (TSR) and selected using both MTX and
SFU, or control selection vector NeoR-2A-GFP (NRG) selected with
G418. Selection of TYMS.sub.SS by 5-FU was incomplete. Thus,
ffLuc-2A-NeoR (NRF) vector was included with the MTX resistant
transgenes DG, TSG, or [DG & TSR] to remove untransformed T
cells in the experiments shown in column II. Equivalent selection
for each transgene showed that MTX enhanced selected for T.sub.reg
in the presence of MTX resistant DHFR. It was still uncertain
whether the enzymatic activity of TYMS or 5-FU played a part in the
selection of T.sub.reg. Therefore, the experiment shown in column
III was performed to test the influence of TYMS inhibition in the
selection of T.sub.reg. Selection of T.sub.reg phenotype was found
to be associated with 5-FU, but independent of TYMS activity. The
Kruskall-Wallis test was used to assess differences between groups
for 5-6 biologic replicates and tests were independently repeated
twice; *=p<0.05, **=p<0.01.
[0065] FIG. 13 is a diagrammatic representation of biochemical and
protein interactions thought to influence selection of
T.sub.reg.
[0066] FIGS. 14A-14E show that ribosomal Inhibition by
aminoglycoside G418 selectively depletes replicating T.sub.CD4,
FoxP3. FIG. 14A shows that thawed PBMC were stimulated with
anti-CD3/CD28 and IL-2 in the presence of increasing concentrations
of G418, hygromycin, zeocin, or rapamycin for 7 days and the
selection for T.sub.CD4, FoxP3. FIG. 14B shows flow plots of FoxP3
and CD4 expression, which in turn show the representative trends
for one donor following the use of each drug. FIG. 14C, the top
panel shows the loss of T.sub.CD4, FoxP3 was tested in
un-stimulated, thawed PBMC over the course of 9 days with or
without G418 while the bottom panel shows the effects of G418 on
proliferating and non-proliferating T.sub.CD4, FoxP3 as indicated
by Ki-67. In FIG. 14D, representative flow plots for one donor
demonstrate the effect of G418 on CD4 and FoxP3 expression in the
top panel while FoxP3 and Ki-67 expression are shown in the bottom
panel. Gentamicin is an FDA approved aminoglycoside antibiotic and
was subsequently tested in comparison to G418 for depletion of
T.sub.CD4, FoxP3 over a 7 day period. All experiments were
performed with 6 normal donors and repeated independently twice.
FIG. 14E depicts the depletion of T.sub.CD4, FoxP3 in resting PBMC
after 7 days from gentamicin, an aminoglycosin, and demonstrates
the action of aminoglycosides in depleting T.sub.CD4, FoxP3. It was
next tested whether depletion of T.sub.CD4, FoxP3 corresponded with
a loss of T.sub.reg marker expression or selective T.sub.reg
toxicity.
[0067] FIGS. 15A-15D show the effects of MTX, 5-FU, and G418 in
sorted T.sub.reg. FIG. 15A diagrammatically shows the T.sub.reg and
T.sub.eff were treated with MTX, 5-FU, or G418 as before for 7 days
before stimulating without drug for the remaining 2 weeks of the
experiment. FIG. 15B shows an assessment of markers and activity of
T.sub.reg on Day 21 to determine the contribution of each drug to
selection or depletion of T.sub.reg, and the live T.sub.CD4, FoxP3
on Day 21 are shown in B. FIG. 15C shows that after stimulating
with soluble anti-CD3/CD28 and IL-2 for 48 hours T cells were
assessed for co-expression of FoxP3 with CD25 in C-I, FoxP3 with
CTLA-4 in C-II, and FoxP3 with LAP in C-IV. Six hours of
stimulation with PMA/ionomycin was used to assess loss of IL-2
secretion in FoxP3 expressing T cells, C-III. A 72 hour suppression
assay was performed by mixing treated T.sub.reg with untreated
T.sub.eff and looking at uptake of [.sup.3H] Thymidine at two
separate concentrations, shown in D. This experiment was performed
with 5 normal donors and repeated twice. All experiments were
assessed with Two-Way ANOVA and significance was determined by
Sidak's post-hoc analysis; *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001.
[0068] FIGS. 16A-E show that stimulation of T.sub.CD4, FoxP3
enhances adenosine monophosphate (AMP) Kinase (AMPK) activation and
leads to inhibition of translational elongation factor eEF2.
Differentiation of T.sub.CD4, FoxP3 from CD4+CD25.sub.neg T cells
was accomplished by gating in the stimulated and unstimulated
experiments. FIG. 16A depicts the mean fluorescence intensity (MFI)
of AMPK activated by phosphorylation at T172 after stimulation in
the top panel while the lower panel of FIG. 16A depicts the MFI of
activated S6 by phosphorylation at sites S235/S236. FIG. 16B
depicts a flow plot depicting the changes in phosphorylation for
T.sub.CD4, FoxP3 and CD4.sup.+ CD25.sub.neg T cells in the upper
panel for AMPK and in the lower panel for S6 with respect to FoxP3
expression in gated CD4.sup.+ cells. FIG. 16C is an image cytometry
gallery depicting fluorescent and morphologic changes in T.sub.CD4,
FoxP3 following stimulation. FIG. 16D shows an image cytometer was
used to analyze p-eEF2 T56 MFI and depicts an increase in
activation of T.sub.CD4, FoxP3. FIG. 16E shows the difference from
CD4.sup.+ FoxP3.sup.neg T cells in image cytometry gallery.
DEFINITIONS
[0069] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0070] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art. The
following terms are provided below.
[0071] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element. Thus, recitation of "a cell", for
example, includes a plurality of the cells of the same type.
[0072] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of +/-20% or +/-10%, more preferably +/-5%,
even more preferably +/-1%, and still more preferably +/-0.1% from
the specified value, as such variations are appropriate to perform
the disclosed methods.
[0073] By "animal" is meant any member of the animal kingdom
including vertebrates (e.g., frogs, salamanders, chickens, or
horses) and invertebrates (e.g., worms, etc.). "Animal" is also
meant to include "mammals." Preferred mammals include livestock
animals (e.g., ungulates, such as cattle, buffalo, horses, sheep,
pigs and goats), as well as rodents (e.g., mice, hamsters, rats and
guinea pigs), canines, felines, primates, lupine, camelid,
cervidae, rodent, avian and ichthyes.
[0074] As used herein, the term "antibody" is meant to refer to
complete, intact antibodies, and Fab fragments and F(ab).sub.2
fragments thereof. Complete, intact antibodies include monoclonal
antibodies such as murine monoclonal antibodies (mAb), chimeric
antibodies and humanized antibodies. The production of antibodies
and the protein structures of complete, intact antibodies, Fab
fragments and F(ab).sub.2 fragments and the organization of the
genetic sequences that encode such molecules are well known and are
described, for example, in Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1988) which is incorporated herein by reference.
[0075] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0076] An "effective amount" as used herein, means an amount which
provides a therapeutic or prophylactic benefit.
[0077] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0078] By "epitope" is meant a region on an antigen molecule to
which an antibody or an immunogenic fragment thereof binds
specifically. The epitope can be a three dimensional epitope formed
from residues on different regions of a protein antigen molecule,
which, in a native state, are closely apposed due to protein
folding. "Epitope" as used herein can also mean an epitope created
by a peptide or hapten portion of matriptase and not a three
dimensional epitope.
[0079] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter.
[0080] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses (e.g.,
lentiviruses, retroviruses, adenoviruses, and adeno-associated
viruses) that incorporate the recombinant polynucleotide.
[0081] As used herein, the term "fusion protein" or "fusion
polypeptide" is a polypeptide comprised of at least two
polypeptides and optionally a linking sequence, and that are
operatively linked into one continuous protein. The two
polypeptides linked in a fusion protein are typically derived from
two independent sources (i.e., not from the same parental
polypeptide), and therefore a fusion protein comprises two linked
polypeptides not normally found linked in nature. Typically, the
two polypeptides can be operably attached directly by a peptide
bond, or may be connected by a linking group, such as a spacer
domain. An example of a fusion polypeptide is a polypeptide that
functions as a receptor for an antigen, wherein an antigen binding
polypeptide forming an extracellular domain is fused to a different
polypeptide, forming a "chimeric antigen receptor".
[0082] By "knock-in" of a target gene means an alteration in a host
cell genome that results in altered expression (e.g., increased,
including ectopic) of the target gene, e.g., by introduction of an
additional copy of the target gene or by operatively inserting a
regulatory sequence that provides for enhanced expression of an
endogenous copy of the target gene. See U.S. Pat. No.
6,175,057.
[0083] By "knock-out" of a gene means an alteration in the sequence
of the gene that results in a decrease of function of the target
gene, preferably such that target gene expression is undetectable
or insignificant. See U.S. Pat. No. 6,175,057.
[0084] By "modulating" or "regulating" is meant the ability of an
agent to alter from the wild type level observed in the individual
organism the activity of a particular gene, protein, factor, or
other molecule.
[0085] By "mutant" with respect to a polypeptide or portion thereof
(such as a functional domain of a polypeptide) is meant a
polypeptide that differs in amino acid sequence from the
corresponding wild type polypeptide amino acid sequence by
deletion, substitution or insertion of at least one amino acid. A
"deletion" in an amino acid sequence or polypeptide is defined as a
change in amino acid sequence in which one or more amino acid
residues are absent as compared to the wild-type protein. As used
herein an "insertion" or "addition" in an amino acid sequence or
polypeptide is a change in an amino acid sequence that has resulted
in the addition of one or more amino acid residues as compared to
the wild-type protein.
[0086] As used herein "substitution" in an amino acid sequence or
polypeptide results from the replacement of one or more amino acids
by different amino acids, respectively, as compared to the
wild-type protein.
[0087] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0088] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, i.e., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, i.e., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, i.e., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (i.e., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0089] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used, "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0090] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0091] A "lentivirus" as used herein refers to a genus of the
Retroviridae family. Lentiviruses are unique among the retroviruses
in being able to infect non-dividing cells; they can deliver a
significant amount of genetic information into the DNA of the host
cell, so they are one of the most efficient methods of a gene
delivery vector. HIV, SIV, and FIV are all examples of
lentiviruses. Vectors derived from lentiviruses offer the means to
achieve significant levels of gene transfer in vivo.
[0092] The term "linker", also referred to as a "spacer" or "spacer
domain" as used herein, refers to a an amino acid or sequence of
amino acids that that is optionally located between two amino acid
sequences in a fusion protein.
[0093] The term "operably linked" (and also the term "under
transcriptional control") refers to functional linkage between a
regulatory sequence and a heterologous nucleic acid sequence
resulting in expression of the latter. For example, a first nucleic
acid sequence is operably linked with a second nucleic acid
sequence when the first nucleic acid sequence is placed in a
functional relationship with the second nucleic acid sequence. For
instance, a promoter is operably linked to a coding sequence if the
promoter affects the transcription or expression of the coding
sequence. Generally, operably linked DNA sequences are contiguous
and, where necessary to join two protein coding regions, in the
same reading frame.
[0094] "Parenteral" administration of an immunogenic composition
includes, e.g., subcutaneous (s.c.), intravenous (i.v.),
intramuscular (i.m.), or intrasternal injection, or infusion
techniques.
[0095] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to a human being.
[0096] The term "polynucleotide" is a chain of nucleotides, also
known as a "nucleic acid". As used herein polynucleotides include,
but are not limited to, all nucleic acid sequences which are
obtained by any means available in the art, and include both
naturally occurring and synthetic nucleic acids.
[0097] The terms "peptide," "polypeptide," and "protein" are used
interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide
must contain at least two amino acids, and no limitation is placed
on the maximum number of amino acids that can comprise a protein's
or peptide's sequence. Polypeptides include any peptide or protein
comprising two or more amino acids joined to each other by peptide
bonds. As used herein, the term refers to both short chains, which
also commonly are referred to in the art as peptides, oligopeptides
and oligomers, for example, and to longer chains, which generally
are referred to in the art as proteins, of which there are many
types. "Polypeptides" include, for example, biologically active
fragments, substantially homologous polypeptides, oligopeptides,
homodimers, heterodimers, variants of polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, among others.
The polypeptides include natural peptides, recombinant peptides,
synthetic peptides, or a combination thereof.
[0098] The term "promoter" means a DNA sequence recognized by the
synthetic machinery of the cell, or introduced synthetic machinery,
required to initiate the specific transcription of a polynucleotide
sequence.
[0099] By "somatic cell" is meant any cell of a multicellular
organism, preferably an animal, that does not become a gamete.
[0100] The term "therapeutically effective amount" shall mean that
amount of drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system or animal that
is being sought by a researcher or clinician.
[0101] The term "transfected" or "transformed" or "transduced means
to a process by which exogenous nucleic acid is transferred or
introduced into the host cell. A "transfected" or "transformed" or
"transduced" cell is one which has been transfected, transformed or
transduced with exogenous nucleic acid. The transduced cell
includes the primary subject cell and its progeny.
[0102] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0103] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Examples of vectors include
but are not limited to, linear polynucleotides, polynucleotides
associated with ionic or amphiphilic compounds, plasmids, and
viruses. Thus, the term "vector" includes an autonomously
replicating plasmid or a virus. The term is also construed to
include non-plasmid and non-viral compounds which facilitate
transfer of nucleic acid into cells, such as, for example,
polylysine compounds, liposomes, and the like. Examples of viral
vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0104] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
[0105] Where any amino acid sequence is specifically referred to by
a Swiss Prot. or GENBANK Accession number, the sequence is
incorporated herein by reference. Information associated with the
accession number, such as identification of signal peptide,
extracellular domain, transmembrane domain, promoter sequence and
translation start, is also incorporated herein in its entirety by
reference.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0106] In one aspect, an isolated transgenic mammalian T cell
comprising or expressing a transgene and one or more of DHFR.sup.FS
and TYMS.sup.SS is provided. In some embodiments, the isolated
transgenic mammalian T cell comprises or expresses a transgene,
DHFR.sup.FS and TYMS.sup.SS. Briefly, T cells can be obtained from
peripheral blood mononuclear cells, bone marrow, lymph node tissue,
cord blood, thymus tissue, tissue from a site of infection,
ascites, pleural effusion, spleen tissue, and tumors. T cell lines
available in the art may be used. Preferably, T cells are obtained
from a unit of blood collected from a subject using any number of
techniques known to those skilled in the art. Isolation of T cells
may proceed according to procedures known in the art, as described
in US2013/0287748 A1. The harvested T cells are then expanded using
methods well-known in the art, such as described in US2013/0287748
A1.
[0107] According to one embodiment, T-cells are harvested and
processed for lentiviral transduction as follows. Patient
peripheral blood mononuclear cells are purified and washed in
phosphate-buffered saline (PBS) with 1% human serum albumin.
Lymphocytes are enriched using magnetic bead depletion of
monocytes, according to known methods. Lymphocytes are cultured
according to Good Manufacturing Practice regulations as previously
described by Levine et al., (1998), J Hematother 7:437-448. The
cells are expanded ex vivo for 14 days in a serum-free
hematopoietic cell medium, e.g., X-VIVO 15 of Lonza Group Ltd. (a
chemically defined, serum-free hematopoietic cell medium)
supplemented with 10% Normal Human Antibody Serum, and then
processed for reinfusion on day 14 of culturing. The magnetic beads
are removed using a magnetic cell separation system. The cells are
harvested, washed and resuspended in a Plasmalyte A containing 1%
human serum albumin before being transduced with lentiviral
vectors.
[0108] As demonstrated herein, T cells are genetically modified to
express anti-thymidylate resistance (AThyR) transgenes, and other
transgenes. AThyRs are shown to rescue T cells from
anti-thymidylate (AThy) drug toxicity, such as AThy toxicity
mediated by 5-FU and anti-folates targeting DHFR and TYMS. Also, as
demonstrated herein DHFR muteins such as DHFR.sup.FS permits
methotrexate (MTX)-inducible increase in transgene expression that
is thymidine dependent, and TYMS muteins such as TYMS.sup.SS permit
MTX-inducible decrease in transgene expression that is
dihydrofolate dependent. As further demonstrated herein, AThyRs can
be used to positively select for transgenes of interest without the
use of immunogenic genes or magnetic selection.
[0109] The use of AThyR transgenes DHFR.sup.FS and TYMS.sup.SS
alone or in combination, engineered into T cells expressing a
transgene of interest, provides a unique capacity to select for
transgene expression within the bulk population, can modulate the
expression of cis as well as trans transgenes of interest, and
promote survival in toxic concentrations of AThys. Thus, T cells
expressing transgenes of interest, such as T cells expressing
tumor-targeting chimeric antigen receptors (CARs), further
engineered to express AThyRs such as DHFR.sup.FS and/or
TYMS.sup.SS, find utility in treating cancers such as lung, colon,
breast, and pancreas that are in need of new therapeutic
options.
[0110] As demonstrated herein, combining AThyRs DHFR.sup.FS and
TYMS.sup.SS in T cells leads to significant survival advantages for
such cells treated with toxic concentrations of AThys: MTX, Pem, or
5-FU. These AThy drugs are regularly used to treat lung and colon
cancer among other common cancers. The findings described herein
indicate that AThyRs T cells can survive toxic AThy concentrations.
Combining the immunomodulatory effects of chemotherapy like 5-FU
with T cells resistant to the cytotoxic effects of 5-FU could
substantially improve the anti-cancer response of the patient
beyond that of either therapeutic used alone.
[0111] As described herein, for the purpose of selecting transgenes
of interest for T cell expression, AThyRs were compared to one of
the earliest drug resistance transgenes--NeoR. As described herein,
it was found that DHFR.sup.FS is superior to NeoR in promoting
survival, selection, and drug-dependent increases of expression of
a representative transgene (eGFP). Notably, DHFR.sup.FS and
TYMS.sup.SS have lower immunogenicity as human proteins, and MTX
can be used both in vitro and in vivo.sup.1 to improve transgene
selection, whereas G418 cannot. The findings described herein
demonstrate that DHFR.sup.FS can select for cells expressing
transgenes such as the suicide gene iC9. Thus, DHFR.sup.FS and
[DHFR.sup.FS & TYMS.sup.SS] are attractive alternatives to
alternative to magnetic beads for selecting T cells expressing one
or more transgenes of interest. In fact, the potential to select
for AThyR+ T cells in vivo using MTX indicates that transgene
selection could be performed within the patient rather than ex
vivo.
[0112] In another aspect is provided a method for inhibiting AThy
toxicity in a mammalian T cell comprising expressing an AThyR
transgene in said mammalian T cell. In some embodiments, the AThyR
transgene is DHFR.sup.FS. In some embodiments, the AThyR transgene
is TYMS.sup.SS.
[0113] In another aspect is provided a method for selecting a T
cell expressing a transgene of interest. The method comprises
applying a thymidine synthesis inhibitor to a plurality of T cells
that comprises a T cell expressing the transgene of interest and
DHFR.sup.FS and selecting for one or more T cells surviving after
seven or more days of application of the thymidine synthesis
inhibitor, wherein the one or more T cells expresses the vector
comprising the transgene of interest and DHFR.sup.FS. The thymidine
synthesis inhibitor may be selected from the group consisting of
methotrexate (MTX), 5-FU, Raltitrexed and Pemetrexed.
[0114] In some embodiments, a DNA sequence, including DNA sequences
from genes described herein, is inserted into the vector. Vectors
derived from retroviruses are preferred, as they provide long-term
gene transfer since and allow stable integration of a transgene and
its propagation in daughter cells. Expression of nucleic acids
encoding the AThyRs described herein may be achieved using
well-known molecular biology techniques by operably linking a
nucleic acid encoding the AThyRs to a promoter, and incorporating
the construct into a suitable expression vector. The vectors can be
suitable for replication and integration eukaryotes. Typical
cloning vectors contain transcription and translation terminators,
initiation sequences, and promoters useful for regulation of the
expression of the desired nucleic acid sequence.
[0115] In some embodiments, one or more DNA constructs encode the
transgene and one or more DNA constructs encoding one or more
AThyRs, DHFR.sup.FS and TYMS.sup.SS. In other embodiments, the
transgene and the one or more AThyRs, DHFR.sup.FS and TYMS.sup.SS
are operably linked. A chimeric construct encoding the various
nucleotide sequences encoding one or more transgenes and one or
more AThyRs, DHFR.sup.FS and TYMS.sup.SS may be prepared by
well-known molecular biology techniques, from naturally derived or
synthetically prepared nucleic acids encoding the components. The
chimeric constructs may be prepared using natural sequences. The
natural genes may be isolated and manipulated as appropriate so as
to allow for the proper joining of the various domains. Thus one
may prepare the truncated portion of the sequence by employing
polymerase chain reaction (PCR) using appropriate primers which
result in deletion of the undesired portions of the gene.
Alternatively, one may use primer repair where the sequence of
interest may be cloned in an appropriate host. In either case,
primers may be employed which result in termini which allow for
annealing of the sequences to result in the desired open reading
frame encoding the CAR protein. Thus, the sequences may be selected
to provide for restriction sites which are blunt-ended or have
complementary overlaps. Preferably, the constructs are prepared by
overlapping PCR.
[0116] As demonstrated herein, anti-thymidylates or thymidine
synthesis inhibitors, exemplified by MTX, can be used to regulate
transgene expression either to higher or lower expression levels
for a transgene expressed cis to DHFR.sup.FS or TYMS.sup.SS.
MTX-inducible positive or negative modulation of cis-transgenes is
believed clinically useful in situations where MTX is used to
modulate the spatial and temporal expression of dangerous but
necessary transgenes in T cells, such as transgenes expressing
certain chimeric antigen receptors (CAR) or cytokines. The
correlated expression of DHFR.sup.FS with trans expressed
TYMS.sup.SS is also useful in expressing proteins such as TCR
.alpha. and .beta. that need to be expressed at nearly equivalent
amounts and where the use of 2A mediated cleavage sites may
adversely affect protein structure and function.
[0117] Yet another aspect is a method for selectively propagating
peripheral blood mononuclear cells (PBMC) resistant to MTX and
5-FU. The method comprises transfecting peripheral PBMC with a
vector comprising an AThyR gene, treating the transfected PBMC with
a thymidine synthesis inhibitor and selecting for PBMC that express
the AThyR gene. In some embodiments of this aspect, the method
further comprises propagating a T cell population from the
transfected PBMC. In some embodiments, the thymidine synthesis
inhibitor may be selected from the group consisting of methotrexate
(MTX), 5-FU, Raltitrexed and Pemetrexed. In some embodiments, the
thymidine synthesis inhibitor is MTX. In some embodiments, the
AThyR gene is TYMS.sup.SS. In some embodiments, the AThyR gene is
DHFR.sup.FS.
[0118] Another aspect is an isolated transgenic mammalian T cell
comprising a nucleic acid sequence comprising a transgene of
interest and a nucleotide sequence encoding DHFR.sup.FS or
TYMS.sup.SS. In some embodiments, the isolated transgenic mammalian
T cell comprises a nucleic acid comprising a transgene of interest
and a nucleotide sequence encoding DHFR.sup.FS, wherein the
transgene of interest and the nucleotide sequence encoding
DHFR.sup.FS are operably linked. In some embodiments, the isolated
transgenic mammalian T cell comprises a nucleic acid comprising a
transgene of interest and a nucleotide sequence encoding
TYMS.sup.SS, wherein the transgene of interest and the nucleotide
sequence encoding TYMS.sup.SS are operably linked.
[0119] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and DHFR.sup.FS, wherein
the T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the DHFR.sup.FS.
[0120] In certain aspects, a sequence encoding DHFR.sup.FS encodes
a polypeptide at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 12. In some
embodiments, a sequence encoding DHFR.sup.FS encodes a polypeptide
having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid
deletions, insertions or substitutions relative to SEQ ID NO:
12.
[0121] In another aspect is provided an isolated transgenic
mammalian T cell expressing a transgene and TYMS.sup.SS, wherein
said T cell comprises (1) a polynucleotide comprising sequence that
encodes the transgene and (2) a polynucleotide comprising sequence
that encodes the TYMS.sup.SS.
[0122] In certain aspects, a sequence encoding TYMS.sup.SS encodes
a polypeptide at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 11. In some
embodiments, a sequence encoding TYMS.sup.SS encodes a polypeptide
having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid
deletions, insertions or substitutions relative to SEQ ID NO:
11.
[0123] In a further aspect, a composition is provided comprising a
plurality of human cells (e.g., T-cells), wherein the cells
comprise a sequence encoding TYMS.sup.SS and a first transgene,
said cells having been treated with MTX (e.g., in culture or in a
living organism), thereby changing expression of the transgene. In
certain embodiments, the transgene encodes a CAR, TCR, polypeptide
hormone (e.g., an endocrinological hormone, such as glucagon),
cytokine, a transcription factor or chemokine. In still further
aspects, a transgene of the embodiments encodes a cell surface
polypeptide, such as an integrin, cytokine receptor, chemokine
receptor or a receptor of a hormone (e.g., a neurological or
endocrine hormone).
[0124] In still a further aspect, a composition is provided
comprising a plurality of human cells (e.g., T-cells), wherein the
cells comprise a sequence encoding DHFR.sup.SS and a first
transgene, said cells having been treated with MTX (e.g., in
culture or in a living organism), thereby changing expression of
the transgene. In certain embodiments, the transgene encodes a CAR,
TCR, polypeptide hormone (e.g., an endocrinological hormone, such
as glucagon), cytokine, transcription factor or chemokine. In still
further aspects, a transgene of the embodiments encodes a cell
surface polypeptide, such as an integrin, cytokine receptor,
chemokine receptor or a receptor of a hormone (e.g., a neurological
or endocrine hormone).
[0125] In a further aspect, there is provided a composition
comprising a first plurality of T cells isolated from a mammal and
treated with a thymidine synthesis inhibitor, wherein the first
plurality of T cells is enriched for regulatory T cells as compared
to a second plurality of T cells isolated from a mammal that is
depleted by a thymidine synthesis inhibitor during stimulation with
a(n) antibody(ies) compromising any singular or combination use of
anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-41BB, anti-OX40,
phytohemagluttinin (PHA), ionomycin or peptide pulsed antigen
presenting cells (whether synthetic or biologic and of any cell
origin whether human or otherwise if utilized to stimulate T cells
in such a way that the T cells begin to replicate).
[0126] In yet another aspect is provided a method of treating a
patient with a cancer comprising to administering to a patient a
therapeutically effective amount of a T cell of an isolated T cell
of any of the above embodiments. While few cell therapies and no
cell-based gene therapies are currently approved by the FDA, any of
the transgenic techniques reported herein can be used to prepare a
composition to administer to a patient with cancer. Further,
CAR-mediated ex vivo expansion can be used to generate a
therapeutically effective amount of a T cell of an isolated T cell
of any of the above embodiments.
[0127] The processed T cells of the invention can be generated by
introducing a lentiviral vector containing any of the
above-described nucleic acid constructs into T cells, such as
autologous T cells of a patient to be treated for cancer or an
IgE-mediated allergic disease. A composition comprising autologous
T cells is collected from a patient in need of such treatment. The
cells are engineered into the processed T cells ex vivo, activated
and expanded using the methods described herein and known in the
art, and then infused back into the patient. The processed T cells
replicate in vivo resulting in persistent immunity against cancer
cells or other cells expressing mIgE.
[0128] Any of the above isolated T cells may be processed, with the
processed T cells then transduced with lentiviral vectors as
described above to generate processed T cells for administration.
Transduction is carried out according to known protocols.
[0129] The processed T cells are administered to a subject in need
of treatment for an IgE-mediated allergic disease. The processed T
cells are able to replicate in vivo, providing long-term
persistence that can lead to sustained allergic disease control.
The processed T cells may be administered either alone, or as a
pharmaceutical composition in combination with one or more
pharmaceutically acceptable carriers, diluents or excipients and/or
with other components, such as cytokines or other cell populations.
Such compositions may comprise buffers such as neutral buffered
saline, phosphate buffered saline and the like; carbohydrates such
as glucose, mannose, sucrose or dextrans, mannitol; proteins;
polypeptides or amino acids such as glycine; antioxidants;
chelating agents such as EDTA or glutathione; adjuvants (e.g.,
aluminum hydroxide); and preservatives. Compositions are preferably
formulated for intravenous administration. Preferably, the T cells
comprise autologous T cells that are removed from the subject and
engineered ex vivo to express the CAR and administered to the
subject.
[0130] The processed T cells or pharmaceutical composition thereof
may be administered by a route that results in the effective
delivery of an effective amount of cells to the patient for
pharmacological effect. Administration is typically parenteral.
Intravenous administration is the preferred route, using infusion
techniques that are commonly known in immunotherapy (see, e.g.,
Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The quantity
of CAR.sup.+ T cells and frequency of administration are determined
by such factors as the condition of the patient, and the type and
severity of the patient's disease, although appropriate dosages may
be determined by clinical trials. An "effective amount" is
determined by a physician with consideration of individual
differences in age, weight, disease state, and disease severity of
the patient. Generally, the amount of CAR.sup.+ T given in a single
dosage will range from about 10.sup.6 to 10.sup.9 cells/kg body
weight, including all integer values within those ranges. The
CAR.sup.+ T may be administered multiple times at these dosages.
The optimal dosage and treatment regime for a particular patient
can readily be determined by one skilled in the art of medicine by
monitoring the patient for signs of disease and adjusting the
treatment accordingly.
[0131] In yet another aspect is provided a method for selecting for
a T cell expressing a transgene of interest, as shown in any of the
FIGS. or as described in the description.
[0132] In yet another aspect is provided a T cell, as shown in any
of the FIGS. or as described in the description.
[0133] In another aspect is a method for selectively propagating
primary human T cells resistant to one or more of MTX, 5-FU,
Raltitrexed and Pemetrexed, as shown in any of the FIGS. or as
described in the description.
[0134] Another aspect is a method of enriching for regulatory T
cells in a population of T cells isolated from a mammal by
contacting said population with a thymidine synthesis inhibitor
selected from the group consisting of MTX, 5-FU, Raltitrexed and
Pemetrexed, or a combination thereof, to selectively deplete
effector T cells in the population. In some embodiments, the
population of T cells isolated from a mammal is contacted with both
MTX and 5-FU. In some embodiments, the T cells express one or more
of DHFR.sup.FS and TYMS.sup.SS. In some embodiments, the T cells
express both DHFR.sup.FS and TYMS.sup.SS.
[0135] Specific inhibition of 5-aminoimidazole-4-carboxamide
riboside (AICAR) synthesis has been shown herein to be neither
toxic to T cells nor selective for T.sub.CD4, FoxP3. FoxP3
expression in T.sub.CD4, FoxP3 has now been found to be enhanced by
the specific action of AICARtf inhibition, suggesting some action
of AMPK may improve T.sub.reg phenotype. Without wishing to be
bound by theory, isolated T.sub.reg studies described herein show
that the action of MTX is twofold: 1) Selection of T.sub.reg is
dependent on the depletion of T.sub.eff, as removal of T.sub.eff
prevents the selective increase of T.sub.reg following MTX
treatment. 2) The action of MTX does enhance T.sub.reg functional
activity in some regard as latency associated peptide (LAP)
expression and suppression of T.sub.eff proliferation were
increased above untreated T.sub.reg. The activation of AMPK in the
absence of folate depletion by MTX was achieved in the transgenic T
cell experiments and increased the percent of T cells with a
functional T.sub.reg phenotype. Thus, MTX depletes T.sub.eff and
promotes an immunosuppressive T.sub.reg phenotype.
[0136] Another aspect is a method for depleting regulatory T cells
in a population of T cells isolated from a mammal by culturing said
population in the presence of one or more aminoglycosidases to
selectively deplete the regulatory T cells in said culture. In some
embodiments, the T cells express one or more of DHFR.sup.FS and
TYMS.sup.SS. In some embodiments, the T cells express both
DHFR.sup.FS and TYMS.sup.SS. In some embodiments, Treg can be
rescuded from G418-mediated depletion when Neomycin resistance
gene, which prevents G418 toxicity, was present. The aminoglycoside
depletion may be specifically limited to regulatory T cells. While
aminoglycosides have been in use for several decades the capacity
of this drug to deplete Treg has not been described. Without
wishing to be bound by theory, the most likely explanation is that
the drug is used at much lower doses in vivo than those used to
deplete Treg in vitro, and is often discontinued for toxicity to
multiple tissues.
[0137] In some embodiments, aminoglycosides can be administered to
a patient with a tumor in order to enhance anti-tumor activity.
Aminoglycosides can be administered by pretreatment in a therapy,
for example.
[0138] Another aspect is a method for selecting for a regulatory T
cell isolated from a mammal. The method comprises treating a
plurality of T cells expressing one or more of DHFR.sup.FS and
TYMS.sup.SS with a thymidine synthesis inhibitor and selecting a
regulatory T cell that expresses a marker for a regulatory T cell.
In some embodiments, the T cells express DHFR.sup.FS. In some
embodiments, the selecting step comprises cell isolating with
magnetic bead sorting using one or more of an anti-CD4 antibody, an
anti-CD25 antibody, an anti-CD3 antibody, an anti-CD8 antibody, an
anti-CD25 antibody, an anti-CD39 antibody, an anti-CD45 antibody,
an anti-CD152 antibody, an anti-KI-67 antibody, and an anti-FoxP3
antibody. In some embodiments, the thymidine synthesis inhibitor is
selected from the group consisting of methotrexate (MTX), 5-FU,
Raltitrexed or Pemetrexed. In some embodiments, the method further
comprises treating the regulatory T cell with one or more of
folate, leucovarin and FU.
[0139] As further demonstrated herein, AThyRs protect AThyRs T
cells from anti-folate toxicity from MTX or Pem. Results described
herein establish that MTX is more toxic to T cells than Pem and
that MTX susceptibility to <1 .mu.M MTX could be completely
abrogated by the codon optimization of DHFR.sup.FS or by the
addition of TYMS.sup.SS to DHFR.sup.FS in T cells. Concentrations
of up to 1 .mu.M MTX are achieved during the treatment of
rheumatoid arthritis. Higher doses of MTX are achieved in cancer
chemotherapy (>1 mM MTX) with the use of leucovorin. Leucovorin
rescues thymidine synthesis through the same pathway as combination
DHFR.sup.FS and TYMS.sup.SS. Thus, it is believed that [DHFR.sup.FS
& TYMS.sup.SS].sup.+ T cells will likely resist cytotoxicity
induced by the range of MTX experienced for both immune suppression
and cancer treatment.
[0140] In another aspect is provided a composition comprising a
first plurality of T cells isolated from a mammal and a thymidine
synthesis inhibitor. The first plurality of T cells is enriched for
regulatory T cells as compared to a second plurality of T cells
isolated from a mammal that does not comprise a thymidine synthesis
inhibitor.
[0141] In various embodiments of any of the above aspects and
embodiments, T cells (T lymphocytes) as used herein may comprise or
consist of any naturally occurring or artificially (e.g.,
synthetically, genetically, recombinantly) engineered immune cells
expressing naturally occurring or made to express or present on the
cell surface artificially (e.g., synthetically, genetically,
recombinantly) engineered T cell receptors or portions thereof,
including, for example but not limited to, chimeric, humanized,
heterologous, xenogenic, allogenic, and autologous T cell
receptors.
[0142] In various embodiments of any of the above aspects and
embodiments, "T cells" as used herein include all forms of T cells,
for example, but not limited to T helper cells (T.sub.H cells),
cytotoxic T cells (T.sub.c cells or CTLs), memory T cells (T.sub.CM
cells), effector T cells (TEM cells), regulatory T cells (Treg
cells; also known as suppressor T cells), natural killer T cells
(NKT cells), mucosal associated invariant T cells, alpha-beta T
cells (T.alpha..beta. cells), and gamma-delta T cells
(T.gamma..delta. cells).
[0143] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only, and are not restrictive of the invention as
claimed. The accompanying drawings, which are incorporated herein
by reference, and which constitute a part of this specification,
illustrate certain embodiments of the invention and together with
the detailed description, serve to explain the principles of the
present invention.
[0144] All cited patents and publications referred to in this
application are herein incorporated by reference in their
entirety.
Example 1
Materials and Methods
Cells and Culture Conditions:
[0145] Cells: Peripheral blood mononuclear cells (PBMC) derived
from healthy donors at the Gulf Coast Regional Blood Bank or MDACC
Blood Bank, both in Houston, Tex., was subjected to density
gradient centrifugation using Ficoll-Paque Plus (GE Healthcare
Biosciences, Piscataway Township, N.J.; Cat No. 17-1440-02). PBMC
were washed once in CliniMACS Plus PBS/EDTA buffer (Miltenyi
Biotec, Gladbach, Germany, Cat. No. 130-070-525) and twice in
Dulbecco's PBS (D-PBS) (Sigma-Aldrich, St. Louis, Mo., Cat. No.
D8537) before resting in complete media (CM) made of RPMI 1640
(Thermo Scientific Hyclone, Bridgewater, N.J.; Cat. No.
SH30096.01), 10% heat-inactivated fetal bovine serum (FBS--Thermo
Scientific Hyclone, Cat. No. SH30070.03), and 2 mM GlutaMAX
supplement (Life Technologies, Grand Island, N.Y.; Cat. No.
35050061). Alternatively, PBMC were frozen using a prepared mixture
of 50% heat-inactivated FBS, 40% RPMI 1640, and 10% DMSO
(Sigma-Aldrich, PA; Cat. No. D2650)--freeze media (FM) at
4.times.107 cells/mL. The use of rested or frozen PBMC is outlined
in each experiment, below. The Jurkat cell line, a human T cell
acute lymphoblastic leukemia (American Type Culture Collection,
Manassas, Va., Cat. No. TIB-152) was used and maintained in CM. The
identity of this cell line was assured by short tandem repeat DNA
fingerprinting performed by MDACC Cancer Center Support Grant
Characterized Cell Line Core. Activating and propagating cells
(AaPC) were used to stimulate T cells. The AaPC cell line K562
clone.4, expressing CD86, CD137, CD64, along with membrane bound
IL-15, was modified to present OKT3 antibody for the polyclonal
stimulation of T cells, as previously described (Singh et al.,
Journal of immunotherapy 2014, 37(4):204-213). For the propagation
of chimeric antigen receptor (CAR)+ T cells, the AaPC CARL+K562
(Rushworth et al., Journal of immunotherapy 2014, 37(4):204-213)
was utilized.
[0146] All AaPC were rapidly thawed in a 37.degree. C. water bath
and washed twice before stimulation of T cells (Singh et al.,
supra). Jurkat and AaPC were tested for the presence of mycoplasma
before use. Cell counting was accomplished in a mixture of 0.1%
Trypan Blue (Sigma-Aldrich, T8154) with the Cellometer K2 Image
Cyotmeter (Nexcelom, Lawrence, Mass.).
Chemical and Biological Agents:
[0147] Stimulation via CD3 and CD28 was achieved by the addition of
30 ng/mL OKT3 antibody (eBioscience, San Diego, Calif., Cat. No.
16-0037-85), 100 ng/mL anti-CD28 antibody (EMD Millipore, Temecula,
Calif., Cat. No. CBL517). T cell stimulation included recombinant
human IL-2 (Proleukin, Prometheus Labs, San Diego, Calif.). When
indicated, the following drugs were used: 5-FU, MTX, pemetrexed,
raltitrexed, G418, and AP20187. Further information regarding each
drug is given in Table 1.
DNA Expression Plasmids:
[0148] DNA plasmids for testing anti-thymidylate resistance (AThyR)
transgenes were generated using the previously described DNA
plasmid G4CAR as a backbone (Rushworth et al., supra). Commercially
synthesized FLAG-DHFR.sup.FS, codon optimized (CoOp) DHFR.sup.FS,
FLAG-TYMS.sup.SS, and CoOp TYMS.sup.SS DNA (Life Technologies, Gene
Art), and neomycin resistance gene (NeoR) DNA product were cleaved
by NheI and ApaI. Reporter genes mCherry with N-terminus SV40
nuclear localization sequence (RFP), inducible suicide gene CoOp
iC9 (both produced by GeneArt), and enhanced green fluorescent
protein.
TABLE-US-00001 TABLE 1 Chemical Agents Agent Manufacturer ID No.
5-fluorouracil APP Pharmaceuticals, Schaumburg, IL NDC 63323-117-10
Methotrexate Hospira, Lake Forest IL NDC 61703-350-38 Pemetrexed
Lilly, Indianapolis, IN NDC 0002-7610-01 Raltitrexed Abcam
Biochemicals, Cambridge, MA AB142974 G418 Invivogen, San Diego, CA
Ant-gn-1 AP20187 Clontech, Mountain View, CA 635060
[0149] (eGFP) DNA were digested by ApaI and KpnI. The G4CAR
backbone was restriction enzyme digested by NheI and KpnI. The
G4CAR backbone was ligated with NheI and ApaI digested fragments
and ApaI and KpnI digested fragments in a three component ligation.
Enzyme digestion locations of NheI, KpnI, and ApaI are shown in
FIG. 1B. The drug resistant component (DHFR.sup.FS, TYMS.sup.SS, or
NeoR) was permutated with the transgenes (RFP, CoOp iC9, and GFP)
to make the following DNA plasmids: FLAG-DHFR.sup.FS-2A-eGFP pSBSO
(DG), FLAG-CoOp DHFR.sup.FS-2A-eGFP pSBSO (CoOp DG);
FLAG-TYMS.sup.SS-2A-GFP pSBSO (TSG); FLAG-CoOp TYMS.sup.SS-2A-GFP
pSBSO (CoOp TSG); FLAG-TYMS.sup.SS-2A-RFP pSBSO (TSR); NeoR-2A-GFP
pSBSO (NRG); and FLAG-DHFR.sup.FS-2A-iC9 pSBSO (DFSiC9). The
construct FLAG-TYMS.sup.SS-2A-IL-12p35-2A-IL-12p40 pSBSO
(TS.sup.SSIL-12) was synthesized from codon optimized (GeneArt,
Life Technologies) IL-12 p35 and IL-12 p40 transgenes and digested
within the 2A regions to ligate IL-12 p35 and IL-12 p40 with a
TYMS.sup.SS fragment also digested within the 2A region. TS.sup.SSG
backbone digestion points 5' to the start site of TYMS.sup.SS and
3' to the IL-12p40 stop site ligated the three components into the
TS.sup.SSG backbone in a four part ligation. A construct is also
provided, which encodes Myc-DHFR.sup.FS-2A (the polypeptide
sequence corresponding to Myc-DHFR.sup.FS-2A is provided as SEQ ID
NO: 10). The polypeptide sequence for TYMS.sup.SS is provided as
SEQ ID NO: 11. The polypeptide sequence for DHFR.sup.FS is provided
as SEQ ID NO: 12. Codon optimization of DHFR.sup.FS and TYMS.sup.SS
DNA was performed to avoid the mRNA transcript from being bound by
DHFR and TYMS proteins, respectively. Known RNA binding motifs of
DHFR and TYMS mRNA are recognized by DHFR (Tai et al., The
Biochemical journal 2004, 378(Pt 3):999-1006) and TYMS (Lin et al.,
Nucleic acids research 2000, 28(6):1381-1389), respectively. Codons
of DHFR.sup.FS and TYMS.sup.SS were altered as much as possible
while maintaining the amino acid sequence of each protein in order
to avoid protein binding of the mRNA transcript. Previously
described CD19-specific chimeric antigen receptor (CAR) (Rushworth
et al., supra) was utilized without modification.
[0150] Myc-ffLuc-NeoR pSBSO (NRF) was constructed using the
backbone of CD19-2A-Neo pSBSO (Rushworth et al., supra) isolated
after restriction digestion with NheI and SpeI. NheI and SpeI
digested Myc-firefly Luciferase (ffLuc) insert was ligated to
CD19-2A-Neo backbone followed by digestion of the ligation product
with SpeI and EcoRV. SpeI and EcoRV digested NeoR fragments were
then ligated to the digested backbone to yield NRF. All constructs
contain Sleeping Beauty (SB) indirect/direct repeat (IR/DR) sites
to induce genomic integration in the presence of SB transposase.
Each transgene is promoted using elongation factor 1 alpha
(EF1-.alpha.) promoter. Cartoon representations of constructs can
be seen in FIG. 1 B and FIG. 8A. Select DNA and protein sequences
can be found in Table 2.
TABLE-US-00002 TABLE 2 Synthetic DNA/protein sequences FLAG-
Atggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaagacgacga-
tgata dmDHFR
aggacgtcgttggttcgctaaactgcatcgtcgctgtgteccagaacatgggcatcggcaagaacgg-
ggacttcc
cctggccaccgctcaggaatgaatccagatatttccagagaatgaccacaacctettcagtagaaggtaaaca-
ga
atctggtgattatgggtaagaagacctggttctccattectgagaagaatcgacctttaaagggtagaattaa-
tttagt
tctcagcagagaactcaaggaacctccacaaggagctcattttctttccagaagtctagatgatgccttaaaa-
cttac
tgaacaaccagaattagcaaataaagtagacatggtctggatagttggtggcagttctgtttataaggaagcc-
atga
atcacccaggccatcttaaactatttgtgacaaggatcatgcaagactttgaaagtgacacgifitttccaga-
aattga
tttggagaaatataaacttctgccagaatacccaggtgttctctctgatgtccaggaggagaaaggcattaag-
taca aatttgaagtatatgagaagaatgat (SEQ ID NO: 1) FLAG-CoOp
Atggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaggacg-
atgatgaca dmDHFR
aagacgtcgtgggcagcctgaactgcatcgtggccgtgtcccagaacatgggcatcggcaagaacgg-
cgactt
cccctggccccctctgcggaacgagagccggtacttccagcggatgaccaccaccagcagcgtggaaggcaa
gcagaacctcgtgatcatgggcaagaaaacctggttcagcatccccgagaagaaccggcccctgaagggccgg
atcaacctggtgctgagcagagagctgaaagagccccctcagggcgcccacttcctgagcagatctctggacg-
a
cgccctgaagctgaccgagcagccagagctggccaacaaggtggacatggtgtggatcgtgggcggcagctc
cgtgtacaaagaagccatgaaccaccctggccacctgaaactgttcgttacccgtataatgcaggatttcgag-
agc
gataccttettccccgagatcgacctggaaaagtacaagctgcttcccgagtaccccggcgtgctgtccgatg-
tgc aggaagagaagggcatcaagtacaagttcgaggtgtacgagaagaatgac (SEQ ID NO:
2) FLAG-
Atgtatccgtacgacgtaccagactacgcatatccgtacgacgtaccagactacgcagacgtccctgt-
ggccgg dmTYMS
ctcggagctgccgcgccggcccttgccccccgccgcacaggagcgggacgccgagccgcgtccgccg-
cacg
gggagctgcagtacctggggcagatccaacacatcctccgctgcggcgtcaggaaggacgaccgctcgagca
ccggcaccctgtcggtattcggcatgcaggcgcgctacagcctgagagatgaattccctctgctgacaaccaa-
ac
gtgtgttctggaagggtgttttggaggagttgctgtggtttatcaagggatccacaaatgctaaagagctgtc-
ttcca
agggagtgaaaatctgggatgccaatggatcccgagactttttggacagcctgggattctccaccagagaaga-
ag
gggacttgggaccagtttatggcttccagtggaggcattttggggcagaatacagagatatggaatcagatta-
ttca
ggacagggagttgaccaactgcaaagagtgattgacaccatcaaaaccaaccctgacgacagaagaatcatca-
t
gtgcgcttggaatccaagagatcttcctctgatggcgctgcctccatgccatgccctctgccagttctatgtg-
gtgaa
cagtgagctgtcctgccagctgtaccagagatcgggagacatgggcctcggtgtgcctttcaacatcgccagc-
ta
cgccctgctcacgtacatgattgcgcacatcacgggcctgaagccaggtgactttatacacactttgggagat-
gca
catatttacctgaatcacatcgagccactgaaaattcagcttcagcgagaacccagacctttcccaaagctca-
ggat
tcttcgaaaagttgagaaaattgatgacttcaaagctgaagactttcagattgaagggtacaatccgcatcca-
actat taaaatggaaatggctgtt (SEQ ID NO: 3) FLAG-CoOp-
Atggactacaaggacgacgacgacaaggattacaaggatgatgatgataaggactataaggac-
gatgatgaca dmTYMS
aagacgtccccgtggccggcagcgagctgcctagaaggcctctgcctcctgccgctcaggaaaggga-
cgccg
aacctagacctcctcacggcgagctgcagtacctgggccagatccagcacatcctgagatgcggcgtgcggaa
ggacgacagaagcagcacaggcaccctgagcgtgttcggaatgcaggccagatacagcctgcgggacgagtt
ccctctgctgaccaccaagegggtgttctggaagggcgtgctggaagaactgctgtggttcatcaagggcagc-
a
ccaacgccaaagagctgagcagcaagggcgtgaagatctgggacgccaacggcagcagagacttcctggaca
gcctgggcttcagcaccagagaggaaggcgatctgggtcccgtgtacgggtttcaatggcggcacttcggcgc-
c
gagtatcgggacatggagagcgactacagcggccagggcgtggaccagctgcagagagtgatcgacaccatc
aagaccaaccccgacgaccggcggatcatcatgtgcgcctggaaccccagagatctgcccctgatggccctgc
ctccatgtcacgccctgtgccagttctacgtcgtgaactccgagctgagctgccagctgtaccagcggagcgg-
cg
atatgggactgggcgtgcccttcaatatcgccagctacgccctgctgacctacatgatcgcccacatcaccgg-
cct
gaagcccggcgactttatccacaccctgggcgacgcccatatctacctgaaccacatcgagcccctgaagatt-
ca
gctgcagcgcgagcccagacccttcccaaagctgcggatcctgcggaaggtggaaaagatcgacgacttcaag
gccgaggacttccagatcgagggctacaacccccaccccacaatcaagatggaaatggccgtg
(SEQ ID NO: 4) eGFP forward 5'
cccgggcccggcgccatgccacctcctcgcctcctcttc 3' (SEQ ID NO: 5) eGFP
reverse 5' ggtacccttgtacagctcgtccatgccgagagtgatcccggcggcggtcac 3'
(SEQ ID NO: 6) NeoR forward 5'
gctagcacatgtgccaccatgattgaacaagatggattgcacgcaggttctccggccgcttgg 3'
(SEQ ID NO: 7) Neo R reverse 5'
aagcttccgcggccctctccgctaccgaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatc
3' (SEQ ID NO: 8) NLS MAPKKKRKVGIHRGVP (SEQ ID NO: 9)
Genetic Modification and Propagation of Cells:
[0151] The Amaxa Nucleofector.RTM. II (Lonza, Allendale, N.J.) was
used to electroporate both Jurkat and human PBMC. Electroporation
of Jurkat cells utilized a modified buffer (Chicaybam et al.,
Proceedings of the National Academy of Sciences of the United
States of America 2002, 99(6):3400-3405) containing 5 mM KCl, 15 mM
MgCl.sub.2, 120 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH 7.2, and
50 mM DMSO, where 10.sup.6 Jurkat cells per cuvette were
electroporated using program T-14 before immediate transfer to CM.
The addition of drug occurred 48 hours after electroporation and
cell culture remained undisturbed until sampling for gene
expression on days 10-12 post electroporation. Human PBMC
electroporation followed a previously described protocol (Rushworth
et al., supra). Briefly, 1 to 2.times.10.sup.7 thawed PBMC per
cuvette were electroporated in Amaxa T cell Nucleofector solution
(Lonza Biosciences; Cat No. VPA-1002) using program U14. On the
following day, PBMC were stimulated in fresh CM with AaPC at a
ratio of 1:1 including 50 IU/mL IL-2, unless otherwise noted. The
cellular co-culture concentration of 10.sup.6 cells/mL was
maintained at each stimulation, and PBMC derived T cells were
re-stimulated every 7 days using the same concentrations. IL-2 was
added when media was changed between stimulations. Drug treatment
initiated 48 hours after co-culture began and continued until day
14. Drug was only added with fresh CM.
Western Blot:
[0152] 10.sup.6 T cells were centrifuged from culture, supernatant
aspirated, and the pellet rapidly frozen in liquid nitrogen.
Whole-cell extracts were harvested using 50 mM Tris, 150 mM NaCl, 1
mM EDTA, 1% NP-40, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl
fluoride, 150 mM p-nitrophenyl phosphate and 0.3 .mu.M Aprotinin,
pH 7.4. Proteins were separated by SDS-PAGE in reducing conditions
and analyzed using specific primary antibodies indicated in Table
3. Detection was performed using an enhanced chemiluminescence
detection system.
Flow Cytometry:
[0153] Cultured cells were resuspended, and washed once in FACS
staining solution (Rushworth et al., supra). If transgene
expression alone was sought, the specimen was then analyzed on a
flow cytometer. The BD LSRFortessa (BD Biosciences) was used to
analyze RFP expression; otherwise, BD FACSCalibur (BD Biosciences)
was used. Surface antibody staining was performed in FACS staining
solution with fluorochrome-conjugated antibodies at 4.degree. C.
for at least 30 minutes. Antibody targets, concentrations, and
manufacturers are listed in Table 4. Analysis of flow cytometry
data utilized FlowJo v 10.0.5 (Tree Star Inc., Ashland, Oreg.).
Luciferase Assay:
[0154] Cultured T cells were tested for the persistence of ffLuc
transgene by the cleavage of D-luciferin (Perkin Elmer, Waltham,
Mass., Cat. No. 122796). Resuspended cells were plated and washed
once in D-PBS before testing in a D-PBS solution of D-luciferin at
0.14 mg/mL. After incubation at 37.degree. C. for 10 min, the plate
was analyzed on a TopCount NXT Luminescence Counter (Perkin
Elmer).
TABLE-US-00003 TABLE 3 Western Blot Antibodies Antibody
Manufacturer Cat. No. Dilution Actin Sigma A2228 1:10000 Hsp-70
Santa Cruz Biotechnology, SC-24 1:5000 Dallas, TX DHFR Santa Cruz
Biotechnology SC-377091 1:1000 TYMS Millipore MAB4130 1:1000 Myc
Tag CST 2276S 1:1000 DYKDDDDK Tag Pierce MA1-91876 1:1000
TABLE-US-00004 TABLE 4 Flow Cytometry Antibodies Antibody
Manufacturer Cat. No. Dilution CD3-APC BD Pharmingen 340661 1:33
CD3-PerCP-Cy5.5 BD Pharmingen 340949 1:33 CD4 FITC BD Pharmingen
340133 1:33 CD4-PE BD Pharmingen 347327 1:33 CD4-PerCP-Cy5.5 BD
Pharmingen 341645 1:33 CD8-APC BD Pharmingen 340359 1:33 Annexin
V-PE BD Pharmingen 556422 1:20 7-AAD BD Pharmingen 559925 1:20
Propidium Iodide BD Pharmingen 556463 Human Fc-PE Invitrogen H10104
1:40 Myc- AF488 MBL M047-A48 1:33 FLAG-AF647 Cell Signaling 3916S
1:33
Chromium Release Assay:
[0155] Antigen specific cytotoxicity was assessed by CRA. This
assay was previously described (Rushworth et al., supra). Briefly,
antigen positive CD19+EL-4 were compared to antigen negative
CD19.sup.neg EL-4 after each cell line was loaded with .sup.51Cr
for 3 hours and subsequently incubated with CD19-specific CAR+ T
cells at a 1 target:5 effector cell ratio for 6 hours. Release of
.sup.51Cr from cell lysis was assessed by the TopCount NXT
scintillation counter.
Statistical Analysis:
[0156] Statistical analysis and graphical representation of data
was achieved using Prism v6.0 (Graph Pad Software Inc., La Jolla,
Ca). Experiments of more than one variable were analyzed by
multivariate analysis: Two-Way ANOVA was used when appropriate with
Sidak's multiple comparison test, One-Way ANOVA was used when
appropriate with Tukey's or Dunnett's multiple comparison tests as
applicable, non-Gaussian distributions were assessed by the
Kruskall-Wallis test followed by Dunn's multiple comparison test.
Single variable tests (experimental vs. control) were made using
the Mann-Whitney test. Statistical significance was designated as
.alpha.<0.05.
Results
A. Testing AThyR Transgene Selection in Jurkats
[0157] DHFR.sup.FS were used to determine whether T cells can be
genetically-modified to resist toxic doses of AThys used in the
initial treatment of malignancy. DHFR.sup.FS+ T cells resistant to
MTX are described by Jonnalagadda et al., PloS one 2013,
8(6):e65519, and Jonnalagadda et al., Gene therapy 2013,
20(8):853-860. 5-FU resistant TYMS muteins previously identified
within a bacterial culture system (Landis et al., Cancer research
2001, 61(2):666-672) were tested in human cells (data not shown)
and TYMS.sup.SS was chosen for further study.
[0158] To test the enhanced survival of each AThyR, constructs
individually expressing DHFR.sup.FS, TYMS.sup.SS, and NeoR were
ligated into the same backbone containing Sleeping Beauty (SB)
transposable elements upstream of eGFP (FIG. 1B). eGFP was used to
track the predominance of surviving genetically-modified T cells.
Jurkat cells were co-electroporated with each construct and SB11
transposase (Singhet et al., Cancer Research 2008,
68(8):2961-2971), which mediated genomic integration of each
construct. Cytotoxic drugs were added two days after
electroporation. Jurkat were assessed for eGFP expression in viable
cells by propidium iodide (PI) exclusion on day 10-12 (FIG. 1C).
Increased percentage expression of eGFP was sought as a measure for
transgene selection in the presence of drug. Overall survival and
mean fluorescence intensity (MFI) of eGFP are also given in FIGS.
2AI and AII, respectively. Overall, the data demonstrate that
DHFR.sup.FS has much better selection than the traditional
drug-resistance transgene NeoR. The data also demonstrate that
TYMS.sup.SS has no independent capacity to enhance Jurkat
survival.
[0159] More specifically, it was found that DHFR.sup.FS confers
resistance to MTX at concentrations range of 0.01-0.5 .mu.M, and
codon optimization of DHFR.sup.FS enhanced the drug resistance
range of CoOp DHFR.sup.FS to 0.01-1 .mu.M (FIG. 1C). Codon
optimization removed potential endogenous DHFR binding to the
DHFR.sup.FS mRNA as well as possible micro RNA binding domains.
Notably, gating on eGFP.sup.+ cells demonstrated that DHFR.sup.FS
constructs lead to a MTX dependent increase in eGFP MFI. Hence,
eGFP expression within a single cell increased based on the
addition of MTX. This finding occurred independent of mRNA
regulation until 5 .mu.M MTX where endogenous codon DHFR.sup.FS
expression significantly decreased compared to CoOp DHFR.sup.FS
(p<0.0001) (FIG. 2A-II). Drug inducible transgene expression is
a rare phenomenon. This phenomenon, although rare, is not novel.
While the capacity of DHFR to increase cis-expressed eGFP in an MTX
dependent manner was previously described for native DHFR, the
phenomenon was attributed to MTX binding DHFR, DHFR releasing DHFR
mRNA, and free DHFR mRNA leading to increased translation of DHFR
protein (Meyer et al., Proceedings of the National Academy of
Sciences of the United States of America 2002, 99(6):3400-3405).
Here it is noted that the phenomenon also occurs with MTX resistant
DHFR.sup.FS, and with DHFR.sup.FS occurs independent of mRNA
regulation from 0.01-1 .mu.M MTX. Hence, without wishing to be
bound by any theory, it is believed that the regulation of DHFR
expression occurs partially through a heretofore unknown mRNA
independent mechanism.
[0160] There was no drug selective advantage for TYMS.sup.SS
expressing Jurkat when tested with 5-FU (FIG. 1C). Native codon
TYMS.sup.SS had no expression advantage over No DNA Jurkat at any
concentration of 5-FU. Further analysis of eGFP.sup.+ cells for
eGFP MFI revealed that TYMS.sup.SS expressed at a lower eGFP MFI
compared to CoOp TYMS.sup.SS (FIG. 2A). It is concluded that lower
expression of TYMS.sup.FS due to mRNA based suppression contributed
to the lack of TYMS.sup.SS survival advantage. When mRNA regulatory
mechanisms are ablated by codon optimization, TYMS.sup.SS has a
significant expression advantage over mock electroporated Jurkat,
and a weak survival advantage in 5 .mu.M 5-FU. Without wishing to
be bound by any theory, the lack of significantly enhanced survival
is likely due to an alternative mechanism of 5-FU contributing to
toxicity.
[0161] NeoR was used to select for enhanced survival of Jurkat in
the presence of G418. This was intended to serve as a standard to
gauge the utility of DHFR.sup.FS and TYMS.sup.SS. Electroporation
of NeoR into Jurkat improved survival in the presence of G418 at
0.72-1.1 mM G418 (FIG. 1C). The survival advantage of NeoR over No
DNA was not significant due to variability (FIG. 2A), but a G418
dependent increase in GFP MFI was noted. The GFP MFI significantly
increased above No DNA Jurkat at 1.4 mM G418 (FIG. 2A-II). These
results reinforce that DHFR.sup.FS and NeoR are capable of
providing dose-dependent transgene selection advantage in surviving
Jurkat. However, only DHFR.sup.FS conferred reliable survival
advantages to Jurkat in this experiment (FIG. 2A-II).
[0162] The next experiment combined DHFR.sup.FS and TYMS.sup.SS by
co-electroporating each plasmid into Jurkat. The capacity of the
combined transgenes to resist commonly used anti-folate AThys: MTX,
Pem, and Raltitrexed (Ral), were tested. As before, drug was added
on day 2 and cells were tested on day 10-12. There was clear
selection for [DHFR.sup.FS & TYMS.sup.SS] expressing Jurkat in
0.1-1 .mu.M MTX when compared to similarly treated No DNA or
untreated [DHFR.sup.FS & TYMS.sup.SS].sup.+ Jurkat (FIG. 1D).
It should be noted that endogenous codon DHFR.sup.FS was used in
these experiments and the resistance to MTX was enhanced from 0.5
(FIG. 1C) to 1 .mu.M MTX (FIG. 1D) by the addition of TYMS.sup.SS
with no other changes to the experimental conditions. Selection was
also noted for 50-100 .mu.M Pem (FIG. 1D). Moderate selection was
also noted with 10 .mu.M Ral when compared to untreated
[DHFR.sup.FS & TYMS.sup.SS].sup.+ Jurkat (FIG. 2B). Ral
primarily targets TYMS, whereas MTX and Pem target both DHFR and
TYMS (Walling, Investigational new drugs 2006, 24(1):37-77), hence
the improved selection for MTX and Pem over Ral in [DHFR.sup.FS
& TYMS.sup.SS].sup.+ Jurkat. After 2 weeks within 1 .mu.M MTX,
surviving [DHFR.sup.FS & TYMS.sup.SS].sup.+ Jurkat were
refreshed in untreated media and grown for 3-5 weeks. Subsequently,
the stability of transgene expression of [DHFR.sup.FS &
TYMS.sup.SS]+ Jurkat was tested by flow cytometry with the
co-expression of eGFP representing DHFR.sup.FS expression and RFP
representing TYMS.sup.SS expression as seen in FIG. 1E. Each color
represents a separate experiment and is overlaid to represent the
trend that DHFR.sup.FS and TYMS.sup.SS co-express in a correlated
fashion. In fact, analysis of GFP MFI representing DHFR.sup.FS
expression and RFP MFIs representing TYMS.sup.SS expression over
multiple anti-folate drugs, at multiple concentrations demonstrated
that DHFR.sup.FS & TYMS.sup.SS co-express with a strong
Pearson's correlation (R.sup.2=0.9) (FIG. 2C). Without wishing to
be bound by theory, this finding suggests that expression of
DHFR.sup.FS is somehow regulated by the expression of TYMS.sup.SS,
or vice versa.
B. Selective Propagation of Primary Human T Cells Resistant to MTX
and/or 5-FU.
[0163] As demonstrated, TYMS.sup.SS enhances the ability of Jurkat
expressing DHFR.sup.FS to survive in the presence of MTX and Pem,
which both target endogenous DHFR and TYMS to prevent thymidine
synthesis. Given the more robust survival to toxic MTX
concentrations conferred by DHFR.sup.FS and TYMS.sup.SS,
experiments with MTX were undertaken to demonstrate anti-folate and
AThy resistance. TYMS.sup.SS with DHFR.sup.FS were tested in human
cells by electroporation into human PBMC. The day following
electroporation, cells were stimulated with an OKT3-loaded AaPC
capable of polyclonal T cell propagation. The propagation schematic
is shown in FIG. 3A. Two days after AaPC stimulation, the
co-cultures received 0.1 .mu.M MTX, 5 .mu.M 5-FU, or 1.4 mM G418
until day 14, as designated in FIG. 3. The co-cultures were
re-stimulated with AaPC at a 1:1 ratio and given 50 IU/mL IL-2
every 7 days from day 1 to 35. Phenotypic changes in transgene
expression were tracked during drug administration for the first 14
days and for the 21 days after drug administration had ended. The
weekly changes in transgene expression can be noted in FIG. 3B-I,
C-I, D-I.
[0164] Initial testing of DHFR.sup.FS, TYMS.sup.SS, and NeoR
co-expressed with fluorescent proteins demonstrated rapid and
persistent selection to nearly complete selection for expression of
DHFR.sup.FS with MTX and NeoR with G418 (FIG. 3B-I). Survival and
propagation of AThyR+ T cells (TAThyR) compared to No DNA T cells
on day 21 showed that the presence of AThyR or NeoR transgene plays
a role in T cell survival and growth (FIG. 4A). On day 35, total
inferred cell count for T cells expressing AThyR and NeoR
transgenes were compared to untreated No DNA T cells, and
NeoR.sup.+ T cells were the only T cells with significantly
inferior growth at Day35 (FIG. 4B-I). In opposition to experiments
in Jurkat, TYMS.sup.SS demonstrated selection within the population
of surviving T cells on Day 21 in the presence of 5-FU. However,
the selected TYMS.sup.SS expressing T cells did not persist to Day
35, and the lack of persistence was also noted when [DHFR.sup.FS
& TYMS.sup.SS] were selected using MTX and 5-FU. Without
wishing to be bound by any theory, thymidine synthesis may be
restored by TYMS.sup.SS and thymidine transporters then make
thymine available to un-transformed cells. Without wishing to be
bound by any theory, this is likely mediated by an equilibrative
nucleoside transporter as the same transporter that permits 5-FU
entry also mediates equilibrative transport of thymine. As
TYMS.sup.SS restores thymidine synthesis in the presence of
methotrexate, DHFR.sup.FS is no longer able to select for T cells
expressing DHFR.sup.FS & TYMS.sup.SS as noted in FIG. 3B-I.
[0165] In order to achieve complete selection of TYMS.sup.SS for
possible use in combination therapies, NeoR was co-electroporated
into primary T cells with DHFR.sup.FS, TYMS.sup.SS, and
[DHFR.sup.FS & TYMS.sup.SS]. The only change made to the
propagation method was the addition of 100 IU/mL IL-2 rather than
50 IU/mL from days 14-35 to supplement the poor outgrowth already
noted in G418 selected T cells. The higher doses of IL-2 were
insufficient to rescue poor outgrowth when G418 and 5-FU were
combined for T cell selection (FIG. 4B-II). With the
co-transfection of NeoR into DHFR.sup.FS and/or TYMS.sup.SS
expressing T cells, nearly 100% transgenes selection was observed
with the same transgene selection kinetics among all groups (FIG.
3C-I).
[0166] The influence of TYMS.sup.SS on DHFR.sup.FS selection in T
cells subjected to MTX was tested. Plasmids expressing DHFR.sup.FS
were co-electroporated into T cells along with either TYMS.sup.SS
co-expressing RFP or a vector expressing RFP alone. This experiment
followed the same strategy as described for FIG. 3B. Due to
technical limitations, the total amount of DHFR.sup.FS expressing
plasmid DNA electroporated into the same number of T cells was
decreased. Consequently, fewer T cells initially expressed
DHFR.sup.FS at the beginning of the experiment and DHFR.sup.FS was
incompletely selected by the addition of MTX within a 14 day time
period (FIG. 3D-I). The progressive loss of DHFR.sup.FS after day
14 is reminiscent of TYMS.sup.SS expression in FIG. 3B-I. This
demonstrates that AThyR transgenes must select for a large portion
of the T cell population to maintain stable expression within the
population. With regards to the influence of TYMS.sup.SS on the
selection of DHFR.sup.FS, it appears that TYMS.sup.SS blunts
DHFR.sup.FS selection in T cells as selection of [DHFR.sup.FS &
RFP] expressing T cells was more robust than selection of
[DHFR.sup.FS & TYMS.sup.SS] expressing T cells. This is
attributed to the restoration of thymidine synthesis in the
presence of TYMS.sup.SS (FIG. 3D-I). The presence of 5-FU prevents
selection of DHFR.sup.FS with or without TYMS.sup.SS, and this is
attributed to the TYMS.sup.SS independent inhibition of mRNA and
rRNA.
[0167] It was also noted that transgenic selection tended to
increase the population of CD4.sup.+ T cells by day 35 in all T
cell experiments, which was not seen with un-modified T cell
cultures. This was noted in any experiment involving one or more
transgenes selected in the presence of cytotoxic drug (FIG. 3B-II,
3C-II, 3D-II, respective flow plots seen in FIGS. 6A, 6B, and 6C).
The experiment in FIG. 3D-II demonstrates that it is not caused by
cytotoxic drug, rather, the presence of transgene in combinations
with cytotoxic drug leads to CD4.sup.+ T cell predominance by day
35. The selection towards CD4.sup.+ T cell predominance was not
noted 7 days after initial drug selection for AThyR+ T cells (FIG.
4C), which is consistent with previously published findings using
DHFR.sup.FS T cells (Jonnalagadda et al., Gene therapy 2013,
20(8):853-860). The longer period of follow-up than prior
experiments demonstrated a previously unknown phenomenon that
CD8.sup.+ T cells are unable to persist for long periods of time
following cytotoxic insult, or are selectively outgrown by
CD4.sup.+ T cells.
C. MTX Increases Cis-Transgene Expression in DHFR.sup.FS+ T
Cells
[0168] MTX mediated changes in transgene expression are useful for
in vivo control of transgene expression in both animals and humans.
Thus, according to the present invention, MTX, a clinically
available drug, is used to mediate transgene expression either up
or down in T cells. To investigate the persistence of this
regulation, DHFR.sup.FS, CoOp DHFR.sup.FS, and [DHFR.sup.FS &
TYMS.sup.SS] expressed in Jurkat were selected in 1 .mu.M MTX for 2
weeks and rested for 3-5 weeks before testing MTX mediated
regulation of DHFR.sup.FS expression. The expression of DHFR.sup.FS
and codon optimization (CoOp) DHFR.sup.FS selected for uniform
expression in Jurkat I cell line is shown in FIG. 5A. CoOp
DHFR.sup.FS did not contribute to a significantly higher expression
of DHFR.sup.FS as indicated by a cis-expressed eGFP, nor did it
prevent MTX induced increases in transgene expression as noted in
FIG. 5B. This was unexpected. However, the loss of MTX induced
increase in DHFR.sup.FS expression was noted when TYMS.sup.SS was
co-expressed with DHFR.sup.FS as seen in FIG. 5A and FIG. 5B. The
addition of TYMS.sup.SS led to an insignificant reduction in the
expression of native DHFR.sup.FS in the absence of MIX, The
addition of MTX was unable to induce the same increase in
DHFR.sup.FS expression seen during the sole expression of either
DHFR.sup.FS version. Thus, TYMS.sup.SS is playing a role in the MTX
inducible increase of DHFR.sup.FS. In certain experiments, the
co-expression of TYMS.sup.SS with DHFR.sup.FS in Jurkat blunts the
MTX induced increase in eGFP MFI (FIG. 3B-I). Thus, DHFR.sup.FS
maintains MTX-inducible expression of cis-transgenes which is
dependent on MTX mediated inhibition of TYMS.
[0169] Expression of these transgenes in primary cells was next
attempted to recapitulate the findings of MTX inducible increases
in DHFR.sup.FS expression that were prevented by TYMS.sup.SS.
Expression of DHFR.sup.FS, TYMS.sup.SS, or [DHFR.sup.FS &
TYMS.sup.SS] was achieved with stability and purity by selecting
from days 2-14 of propagation with the respective drugs MTX,
5-fluorouracil (5-FU), and G418 when the selection vector
containing neomycin resistance was included. The expression of
DHFR.sup.FS linked eGFP and TYMS.sup.SS linked RFP can be noted in
FIG. 5C for DHFR.sup.FS, TYMS.sup.SS, or [DHFR.sup.FS &
TYMS.sup.SS]. Again it is noted that DHFR.sup.FS expression is
increased in the presence of MTX (FIG. 5D), but this increase is
blunted and no longer significant when TYMS.sup.SS is co-expressed
with DHFR.sup.FS, as in Jurkat. Of note, expression of TYMS.sup.SS
without DHFR.sup.FS was successfully achieved in primary T cells by
selection with 5-FU and a trans neomycin resistance gene selected
by G418. When TYMS.sup.SS was tested for inducible changes in the
presence of high doses of MTX (FIG. 5D), it was found that
TYMS.sup.SS linked RFP decreased significantly. The presence of
DHFR.sup.FS along with TYMS.sup.SS in the same treatment conditions
prevented this decrease. MTX induced a reduction in the expression
of TYMS.sup.SS that MTX resistant DHFR.sup.FS restored. These
findings could indicate that TYMS.sup.SS is being repressed by a
lack of 5, 10-methylenetetrahydrofolate (5, 10 CH.sub.2THF).
Without being limited by any particular mechanism, it is proposed
that MTX, which leads to a drop in 5, 10 CH.sub.2THF, is causing
TYMS protein to bind TYMS and TYMS.sup.SS mRNA preventing
expression. It should be noted that TYMS.sup.SS is equivalent to
the native sequence with the exception of the point mutations.
[0170] Based on findings in FIG. 5A-B, it is proposed that
DHFR.sup.FS expression is also regulated by the synthesis of
thymidine. Likewise, based on findings in FIGS. 5C & D, it is
proposed that TYMS.sup.SS expression is regulated by the synthesis
of tetrahydrofolate (THF). As a derivative of THF is used to make
thymidine, a logical conclusion was made that DHFR.sup.FS regulates
the expression of TYMS.sup.SS and TYMS.sup.SS regulates the
expression of DHFR.sup.FS. Therefore, a correlated expression of
DHFR.sup.FS and TYMS.sup.SS should be noted within individual
cells. When a correlated expression of DHFR.sup.FS and TYMS.sup.SS
was tested by observing flow plots of Jurkat in FIG. 5E and primary
T cells in FIG. 5F, it was noted. A control RFP vector co-expressed
with DHFR.sup.FS, but not modulated by cis expression with
TYMS.sup.SS, did not appear to have the same co-expression pattern
(FIG. 5F). To quantify this observation, Jurkat expressing
[DHFR.sup.FS & TYMS.sup.SS] were treated with antifolates MTX,
pemetrexed, and raltitrexed at varying concentrations for 2 weeks.
DHFR.sup.FS linked eGFP MFI and TYMS.sup.SS linked RFP MFI for each
separate well were then plotted and correlated. The linked
expression between DHFR.sup.FS and TYMS.sup.SS was significant and
fit a linear regression (FIG. 5G). These findings support a general
mechanism for regulation of DHFR and TYMS, which leads to a linear
co-expression of DHFR.sup.FS and TYMS.sup.SS. This model is shown
in FIG. 511.
[0171] Based on the above model in FIG. 511, it appears that
TYMS.sup.SS expression will be stabilized by DHFR.sup.FS from
strong expression changes in the presence of MTX. This was tested
in FIG. 5I with primary T cells expressing DHFR.sup.FS along with
either RFP or TYMS.sup.SS linked to RFP by applying increasing
doses of MTX. As expected, DHFR.sup.FS linked eGFP was increased by
increasing concentrations of MTX, and this increase was blunted by
the presence of TYMS.sup.SS (FIG. 5I-J). This conserves the model
in FIG. 511 where restoration of thymidine synthesis prevents the
MTX induced increase in DHFR.sup.FS. Further conserving the model,
RFP linked to TYMS.sup.SS did not significantly increase over any
concentration of MTX used (FIG. 5I-J). When DHFR.sup.FS linked eGFP
increased so too did the control RFP, and an increase in the
expression of RFP alone was not expected. A possible explanation is
that this increase was noted above 0.5 .mu.M MTX, and DHFR.sup.FS
alone is only resistant to 0.5 .mu.M MTX..sup.9 This suggests that
higher doses of MTX begin to select for cells with higher gene
content of DHFR.sup.FS and associated transgenes. Notably,
DHFR.sup.FS co-expressed with TYMS.sup.SS is resistant to
concentrations of up to 1 .mu.M MTX. This further supports the use
of TYMS.sup.SS to modulate transgene expression and prevent
unwanted selection towards higher gene expression levels of genes
expressed in cis or trans with DHFR.sup.FS.
[0172] Next, a construct of DHFR.sup.FS cis expressing an inducible
suicide gene--inducible caspase 9 (iC9) was employed. This
construct, called D.sup.FSiC9, selects for T cells expressing
D.sup.FS iC9 in the presence of MTX and ablates D.sup.FSiC9.sup.+ T
cells in the presence of drug that activates iC9 to induce
apoptosis. Based on the above findings, the DHFR.sup.FS in
D.sup.FSiC9 could be used to modulate and potentially ablate the
expression of a transgene of interest which is otherwise too toxic
to express without regulation. Interleukin-12 (IL-12) is such a
transgene. IL-12 is a cytokine capable of inducing a strong immune
response against tumor from tumor specific T cells. However,
systemic IL-12 is highly toxic and of low efficacy. Presented here
is an alternative approach where IL-12 is expressed cis to
TYMS.sup.SS in order to decrease and stabilize the expression level
of IL-12. In FIG. 5K, a flow plot demonstrates the expression of
IL-12 cis expressed with TYMS.sup.SS and iC9 cis expressed with
DHFR.sup.FS expression. The donor cells were either left untreated
or treated with high doses of MTX for 7 days. This expression
pattern appears to indicate that IL-12 can be stably expressed even
in prolonged toxic doses of MTX. A further analysis of similarly
manipulated donors (FIG. 5L) demonstrates the potential of
TYMS.sup.SS when co-expressed with DHFR.sup.FS to stabilize the
expression of the potentially toxic transgenes of
interest--IL-12.
[0173] T cells from the experiment shown in FIG. 3D were also
subjected to varying concentrations of MTX. On day 35, T cells
received anti-CD3/CD28 stimulation and were subjected to a range of
MTX from 0 to 1 .mu.M for 72 hours. On day 35, no T cell group
significantly expressed DHFR.sup.FS, as indicated by co-expressed
eGFP, above background (FIG. 3D-I). However, DHFR.sup.FS+ T cells
selected with MTX alone persisted enough to significantly improve
survival when MTX was re-introduced at concentrations up to 0.5
.mu.M MTX (FIG. 7B). Flow plots in FIG. 7A demonstrate
MTX-dependent increases in transgene expression and improved
survival for transgene expressing T cells for one donor. It should
be noted that the addition of TYMS.sup.SS in [DHFR.sup.FS+ &
TYMS.sup.SS].sup.+ T cells permitted the survival of transgene
negative cells at 1 .mu.M MTX, which was not seen in TYMS.sup.SS
neg T cells subjected to MTX (FIG. 7C).
D. AThyR Permits Independent Selection for Transgenes of
Interest
[0174] AThyRs are human proteins and therefore have lower
immunogenicity in humans than NeoR or similar drug resistance
transgenes, typically originating from bacteria. Thus, using AThyRs
to select transgenes of interest is desirable due to lower
immunogenicity, and ease of use in vitro. As a demonstration, the
suicide gene inducible caspase 9 (iC9) was selected by
co-expressing iC9 with DHFR.sup.FS in a construct designated
D.sup.FS iC9 (FIG. 8A). Current methods to select iC9 utilize
surface-expressed antigen and isolation by magnetic beads. However,
this method of selection is more labor intensive than adding drug
and does not add the functionality of AThy resistance. The
D.sup.FSiC9 plasmid significantly selected for survival in T cells
after 7 days of AaPC based stimulation including days 2-7 days in
0.1 .mu.M MTX (FIG. 8B). Next, D.sup.FSiC9 was co-electroporated
with CAR to express in T cells. The CAR was specifically selected
by a CAR exodomain binding ligand (CARL).sup.+ K562 AaPC (Rushworth
et al., supra) while D.sup.FSiC9 was selected using 0.1 .mu.M MTX.
After days 2-14 in 0.1 .mu.M MTX, CAR+D.sup.FSiC9+ T cells were
rested from MTX or selected for another 7 days in 0.1 .mu.M MTX. T
cells selected in 0.1 .mu.M MTX from day 2-21 are shown in FIG. 8C
compared to mock-electroporated T cells. As before, there is no
selection towards CD4.sup.+ T cell predominance following MTX
selection by day 21.
[0175] These cells also demonstrated cytotoxicity at the levels
expected for the given 5:1 target to effector ratio (FIG. 8D).
Co-expressing DHFR.sup.FS with iC9 rather than CAR added the
potential to ablate T cells through the addition of iC9 chemical
inducer of dimerization AP20187 (FIG. 8E). The addition of AP20187
significantly depleted resting CAR.sup.+ T cells independent of
MTX. This demonstrates that D.sup.FSiC9 can effectively select for
iC9 expression and deplete genetically-modified T cells as
necessary. The use of DHFR.sup.FS has the advantage of selecting
transgene expression in T cells independent of antigen-specificity
and antigen expression.
Example 2
Materials and Methods
[0176] Healthy donor derived peripheral blood from MDACC Blood
Bank, Houston, Tex., was subjected to density gradient
centrifugation to isolate mononuclear cells which were either
rested in complete media (CM) or frozen as previously outlined. The
use of rested or frozen peripheral blood derived mononuclear cells
(PBMC) is outlined in each experiment. T cells from PBMC were
stimulated using thawed OKT3 antibody-loaded K562 clone #4, an
activating and propagating cell (AaPC). See Singh H, et al, PloS
one 2013, 8(5). The presence of mycoplasma was tested in AaPC
before stimulation of T cells. Cell counting was accomplished by
0.1% Trypan Blue (Sigma-Aldrich, T8154) exclusion using automated
cell counting (Nexcelcom, Lawrence, Mass.). Cell Isolation was
accomplished using magnetic bead based sorting with the CD4+, CD25+
Regulatory T Cell Isolation Kit following the manufacturer's
instructions (Miltenyi Biotec, San Diego, Calif., 130-091-301).
Briefly, CD4.sup.+ T cells were negatively selected before sorting
one time with anti-CD25 beads was used to differentiate between
effector T cells (CD25.sup.neg) and T.sup.reg (CD25.sup.pos).
[0177] Culture Conditions: Acellular stimulation was accomplished
as previously described using soluble anti-CD3--30 ng/mL,
anti-CD28--100 ng/mL, and human IL-2--50 IU/mL, as previously
described. When indicated, the following drugs were used: 5-FU,
MTX, cisplatin (CDDP), pemetrexed, raltitrexed, G418, hygromycin B,
zeocin, rapamycin, metformin, AICARtf/inosine monophosphate (IMP)
cyclohydrolase (ATIC) dimerization inhibitor (iATIC) (Table 5).
Acellular stimulation experiments received addition of toxic drug
or treatment on the same day as stimulation.
TABLE-US-00005 TABLE 5 Chemical Agents Agent Manufacturer ID No.
5-fluorouracil APP Pharmaceuticals, Schaumburg, IL NDC 63323-117-10
Methotrexate Hospira, Lake Forest, IL NDC 61703-350-38 CDDP Pfizer,
New York, NY NDC 0069-0084-07 Pemetrexed Lilly, Indianapolis, IN
NDC 0002-7640-01 Raltitrexed Abcam Biochemicals, Cambridge, MA
Ab142974 iATIC EMD Millipore 118490 G418 Invivogen, San Diego, CA
Ant-gn-1 Hygromycin Invivogen Ant-hg-1 Zeocin Invivogen Anti-zn-1
Rapamycin Wyeth, Philadelphia, PA NDC 0008-1030-04
DNA Expression Plasmids:
[0178] Selection vectors: FLAG-DHFR.sup.FS-2A-eGFP pSBSO (noted as
DHFR.sup.FS-GFP (DG)), FLAG-TYMS.sup.SS-2A-eGFP pSBSO (noted as
TYMS.sup.SS-GFP (TSG)), NLS-mCherry pSBSO (RFP),
FLAG-TYMS.sup.SS-2A-NLS-mCherry pSBSO (noted as TYMS.sup.SS-RFP
(TRG)), Neomycin Resistance (NeoR)-2A-eGFP pSBSO (noted as NeoR-GFP
(NRG)), and Myc-ffLuc-NeoR pSBSO (NRF), were designed constructed
and utilized as previously described. Sleeping Beauty (SB)
indirect/direct repeat (IR/DR) sites were present in each construct
to induce genomic integration with SB transposase. Each transgene
was expressed by elongation factor 1 alpha (EF1.alpha.)
promoter.
Genetic Transformation and Propagation of Cells:
[0179] The Amaxa Nucleofector.RTM. II was utilized to transform
human PBMC, where 1-2*10.sup.7 thawed PBMC were electroporated in
Amaxa T cell Nucleofector solution using program U14, as previously
described. The next day, PBMC were stimulated with CM with AaPC at
a ratio of 1:1 including 50 IU/mL IL-2. The co-culture of T cells
and AaPC was maintained at 1*10.sup.6 cells/mL with each subsequent
stimulation. Outgrowth of T cells was promoted by re-stimulated of
co-cultures every 7 days with IL-2 and AaPC at the concentrations
noted. Fresh IL-2 was added when media was changed between
stimulations. During transgenic experiments, drugs were added 48
hours after co-culture initiation and maintained at the given
concentration until day 14. After day 14, no drugs were added to T
cell cultures.
Western Blot:
[0180] When noted, T cells were removed from cultures for western
blot by centrifugation of 1*10.sup.6 T cells, and rapid freezing of
the cell pellet in liquid nitrogen. T cell pellets were lysed and
prepared with 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5%
deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 150 mM
p-nitrophenyl phosphate and 0.3 .mu.M Aprotinin, pH 7.4. SDS-PAGE
separated proteins and primary antibodies noted in Table 6 were
used to detect the presence of protein via chemiluminescence.
TABLE-US-00006 TABLE 6 Western Blot Antibodies Antibody
Manufacturer Cat. No. Dilution AMPK.alpha. Cell Signaling
Technology (CST), 2603S 1:1000 Danvers, MA p-AMPK.alpha. CST 2535S
1:1000 (T172) S6 CST 2317S 1:1000 p-S6 CST 3945S 1:1500 (S235/236)
Actin Sigma A2228 1:10000 Hsp-70 Santa Cruz Biotechnology, Dallas,
SC-24 1:5000 TX eEF2 LifeSpan Biosciences, Seattle, WA LS-B8940
p-eEF2 (T56) LifeSpan Biosciences LS-C198899
Flow Cytometry:
[0181] Cultured T cells were washed in FACS staining solution[95]
before surface antibody staining was performed in FACS staining
solution with fluorochrome-conjugated antibodies at 4.degree. C.
for at least 30 minutes. Intracellular transcription factor and
cytokine staining utilized the FoxP3/transcription factor staining
buffer set manufacturer's protocol (eBioscience, 00-5523-00), and
was performed following surface staining. The BD FACSCalibur (BD
Biosciences) analyzed most samples expressing FoxP3. Antibody
targets, concentrations, and manufacturers are listed in Table 7.
Flow cytometry data analysis utilized FlowJo v 10.0.5 (Tree Star
Inc., Ashland, Oreg.). Flow cytometric imaging of cells stained for
phosphorylated antigens was accomplished using the ImageStreamX
Mark II (Amnis, Seattle, Wash.) with the following protocol; after
surface staining, samples were fixed in 100% methanol (Sigma) for 1
hour at 4.degree. C. before washing and staining in
FoxP3/transcription factor staining buffer set wash buffer as
outlined by the manufacturer's protocol. Analysis of image
cytometry data utilized Amnis IDEAS v 6.0.
TABLE-US-00007 TABLE 7 Flow Cytometry Antibodies Antibody
Manufacturer Cat. No. Dilution CD3-APC BD Pharmingen 340661 1:33
CD3-PerCP-Cy5.5 BD Pharmingen 340949 1:33 CD4 FITC BD Pharmingen
340133 1:33 CD4-PE BD Pharmingen 347327 1:33 CD4-PerCP-Cy5.5 BD
Pharmingen 341645 1:33 CD8-APC BD Pharmingen 340659 1:33 CD25-APC
BD Pharmingen 555434 1:33 CD39-APC BD Pharmingen 560239 1:33
CD45RO-APC BD Pharmingen 559865 1:33 CD152-APC BD Pharmingen 555855
1:33 KI-67-AF647 BD Pharmingen 561126 1:50 Annexin V BD Pharmingen
556422 1:20 7-AAD BD Pharmingen 559925 1:20 Propidium Iodide BD
Pharmingen 556463 FoxP3-PE eBiosciences 12-4777-42 1:20 Helios-APC
Biolegend 137222 1:05 LAP-APC Biolegend 349608 1:20 IFN-g-APC
Biolegend 502516 1:20 IL-2-APC Biolegend 500315 1:20 p-eEF2 (T56)
LifeSpan LS-C198899 1:20 Biosciences p-AMPK.alpha. (T172) AbCam
Ab133448 1:20 CD4-Pacific Blue BD Pharmingen 558116 1:33 p-S6
(S244) - BD Pharmingen 560465 1:20 AF647 Goat anti-Rabbit - Life
Technologies A-11034 1:100 AF488
Thymidine Incorporation Assay:
[0182] A thymidine incorporation assay was performed with
anti-CD3/CD28 and IL-2 used to stimulate each well containing
2*10.sup.5 viable cells. Varying ratios of effector T cells
(T.sub.eff) to T.sub.reg were combined in each well and all wells
were run in triplicate in U-bottom 96 well plates. At 48 hours 1
.mu.Ci [.sup.3H] Thymidine (Perkin-Elmer, Waltham, Mass.) was added
to each well, and 24 hours later the cells were assessed for
radioactivity on a Top Count NXT (Perkin-Elmer). T.sub.reg mediated
suppression of growth was determined by the following equation: (No
Treatment T.sub.eff [cpm]-(T.sub.reg & No Treatment T.sub.eff
[cpm]))/No Treatment T.sub.eff [cpm].
Statistical Analysis:
[0183] Graphical representation and statistical analysis of data
was performed with Prism v6.0 (Graph Pad Software Inc., La Jolla,
Ca). One-Way ANOVA was used when appropriate with Tukey's or
Dunnett's multiple comparison tests as applicable, non-Gaussian
distributions were assessed by the Kruskall-Wallis test followed by
Dunn's multiple comparison test. Total cell counts and expression
data involving T.sub.CD4, FoxP3 tended to be non-Gaussian in
distribution. Single variable tests (experimental vs. control) were
made using the Mann-Whitney test. Statistical significance was
designated as .alpha.<0.05.
Results
[0184] Drug selection of TCD4, FoxP3 by MTX occurs in part through
toxicity. In order to determine how MTX contributes to the
selection of T.sub.CD4, FoxP3, freshly derived PBMC were stimulated
with anti-CD3/CD28 antibodies and IL-2 in the presence of cytotoxic
drugs or lethal .gamma.-irradiation. After 7 days there was a
significant difference in survival markers Annexin V and 7-AAD in
stimulated T cells receiving any cytotoxic insult with stimulation
(FIG. 1B-I). The selection of T.sub.CD4, FoxP3 was not as
consistent as cytotoxicity. Following 7 days of stimulation, 2 Grey
.gamma.-irradiation significantly increased the amount of
T.sub.CD4, FoxP3 in the surviving population (FIG. 1B-II). This
lethal treatment did not target a common pathway being considered,
nor did cisplatin, yet both increased T.sub.CD4, FoxP3. However,
the T.sub.CD4, FoxP3 increase induced by cisplatin is
insignificant. Significant increases were derived from 5-FU and
MTX. With the exception of ribosomal elongation inhibitor G418,
each cytotoxic treatment appeared to increase the percentage of
surviving T.sub.CD4, FoxP3. See Bar-Nun S. et al., Biochimica et
biophysica acta 1983, 741(1):123-127. This pattern of increasing
T.sub.CD4, FoxP3 percentage in the face of varied cytotoxic insult
suggests a common pathway that can be enhanced by certain drugs.
Without wishing to be bound by theory, this pathway is likely
related to the reduced proliferation rate of T.sub.reg, and appears
to be ribosomally mediated as G418 can inhibit this general trend
of increasing T.sub.CD4, FoxP3 percentage. See Cao M. et al.,
International journal of radiation biology 2011, 87(1):71-80.
[0185] The findings of T.sub.reg depletion with G418 and T.sub.reg
selection by MTX were further evaluated for dose dependence by
stimulating thawed PBMC with anti-CD3/CD28+IL-2 for 7 days, as
before. G418 was significantly cytotoxic at all doses tested, but
significantly depleted T.sub.CD4, FoxP3 at two moderate drug doses
(FIG. 10C). MTX was also cytotoxic at all doses tested, but had
significant elevation of T.sub.CD4, FoxP3 at lower doses (FIG.
10D). Rapamycin (Rapa) was used as a T.sub.reg selection
control.sub.[138] and showed similar T.sub.CD4, FoxP3 selection at
a moderate drug concentration independent of cytotoxicity, which
only occurred at the highest doses (FIG. 10F). The selection for or
against T.sub.reg at moderate drug doses rather than higher doses
suggests that T.sub.reg have a narrow therapeutic window for drug
induced selection or depletion. A specific inhibitor of
ATIC.sub.[142] was used to test whether MTX mediates selection of
T.sub.CD4, FoxP3 through inhibition of ATIC. Without wishing to be
bound by theory, inhibition of AICARtf or the heterodimeric complex
ATIC, in which AICARtf is found, increases AICAR. FIG. 10E
demonstrates that ATIC inhibition alone was neither cytotoxic nor
selective for T.sub.CD4, FoxP3. Further analysis of flow plots
represented by the same donor in FIG. 10G show expression of CD4
and FoxP3 for several of the drugs used. Use of iATIC
characteristically mediated increased expression of FoxP3 in CD4+ T
cells similar to that of Rapa, but did not inhibit proliferation of
FoxP3.sub.neg T cells as MTX, G418, or Rapa. Thus, iATIC enhanced
FoxP3 expression in CD4+ T cells but diluted these cells by
permitting proliferation of FoxP3.sub.neg T cells. It appears that
MTX mediated selection of T.sub.CD4, FoxP3 occurs by depletion of
rapidly proliferating effector T cells and enhancement of FoxP3
expression via a pathway similar to Rapa that includes ribosomal
inhibition. The increased susceptibility of T.sub.regs to ribosomal
inhibitor G418 solidifies this relationship between enhanced FoxP3
expression and increased susceptibility to ribosomal
inhibition.
[0186] T.sub.regs are preferentially expanded in primary T cells
resistant to the anti-folate and anti-thymidine actions of MTX. It
was hypothesized that regulatory T cells were inhibiting CD8+ T
cells proliferation following drug selection. To test this
hypothesis, drug resistant T cells were derived by transformation
with DHFR.sup.FS, TYMS.sup.SS, NeoR, or a combination, and
numerically expanded as previously described. Briefly, transformed
T cells were selected in the presence of 0.1 .mu.M MTX, 5 .mu.M
5-FU, or 1.6 mM G418 as designated from day 2 to 14 while
stimulation with OKT3-loaded AaPC and 50 IU/mL IL-2 occurred every
7 days until day 35. See Singh H. et al., PloS one 2013, 8(5).
Initial testing for T.sub.regs by elevated expression of FoxP3 in
the CD4.sup.+ T cell population demonstrated there was a
significant T.sub.CD4, FoxP3 percentage increase in DHFR.sup.FS
expressing T cells. Selection using MTX in comparison to
mock-electroporated (No DNA) T cells on Day 21 showed this increase
(FIG. 11), and this increase persisted to Day 35 when 5-FU was
combined with MTX during selection (FIG. 12A). The transgenic T
cells were almost entirely CD4.sup.+ in each experimental
population after selection, but the predominance of T.sub.regs
appeared to often exceed the 5-10% typically found in the
un-manipulated CD4.sup.+ T cell compartment. Markers of T.sub.reg
function were also assessed. Low IL-2 expression.sub.[ ] is a known
trait of T.sub.regs and is assessed with FoxP3 expression. The
percentage of the T cell population with a FoxP3.sub.pos,
IL-2.sub.neg expression pattern is shown in FIG. 12B. Expression of
latency associated peptide (LAP)--a part of the TGF-.beta. complex
and strongly associated with activated T.sub.reg, and is seen in
FIG. 12C.
[0187] The transgenes DHFR.sup.FS and TYMS.sup.SS were compared
individually and in combination to the control selection vector
NeoR and un-treated No DNA T cells. Selection towards T.sub.reg in
this experiment may be noted in FIG. 12A, B, C-I. This experiment
demonstrated that [DHFR.sup.FS-GFP (DG) & TYMS.sup.SS-RFP
(TSR)].sup.+ T cells selected in MTX+5-FU had an increased
population of cells characteristic of T.sub.reg when compared to
mock-transformed T cells. To further elucidate the contribution of
DHFR.sup.FS and TYMS.sup.SS to T.sub.reg selection, NeoR was
co-electroporated with DHFR.sup.FS, TYMS.sup.SS, or the
combination. The addition of NeoR permitted equivalent selection of
DHFR.sup.FS, TYMS.sup.SS, and the combination in all T cell
populations. With un-transformed T cells removed, it became clear
that DHFR.sup.FS alone, but not TYMS.sup.SS alone could select for
cells characteristic of T.sub.regs (FIGS. 12A, B, and C-II). [DG
& TSR].sup.+ T cells continued to select for cells with
T.sub.reg features. Finally, the contribution of TYMS.sup.SS to the
selection of T.sub.reg by DHFR.sup.FS was assessed by
co-electroporation of TSR or a control vector--RFP. The
characteristics of T.sub.regs from this experiment are shown in
FIGS. 12A, B, and C-III. This experiment demonstrates that
selection of DHFR.sup.FS with MTX can enhance outgrowth of
T.sub.reg and that 5-FU enhances this selection independent of
TYMS.sup.SS. Selection of T.sub.reg benefits from folate rescue by
DHFR.sup.FS. This is expected as folate is known to play a role in
T.sub.reg survival. See Kunisawa J. et al., PloS one 2012,
7(2):e32094. Surprisingly, selection of T.sub.reg did not require
de novo thymidine synthesis as TYMS.sup.SS, which alleviates MTX
and 5-FU inhibition of TYMS, was dispensable.
[0188] Previous findings showed survival and toxicity of 5-FU in
PBMC is mediated by TYMS and an alternative mechanism. See
Eisenthal A et al., Anticancer research 2009, 29(10):3925-3930.
Combining the known mechanisms of T.sub.reg selecting drugs MTX,
5-FU, and rapamycin yielded the diagram in FIG. 13, which details
how each drug interacts with ribosomal function. It was noted in an
experiment depicted in Supplemental FIG. 1A that Neomycin
resistance gene rescued T.sub.CD4, FoxP3 from the treatment of
G418. This finding suggests that a specific action of G418 is
responsible for T.sub.CD4, FoxP3 depletion, and this phenomenon was
further explored.
[0189] Ribosomal Inhibition by aminoglycoside G418 selectively
depletes replicating T.sub.CD4, FoxP3. Thawed PBMC were activated
with anti-CD3/CD28+IL-2 for 7 days in the presence of alternative
doses of G418, Hygromycin B--a different aminoglycoside, Zeocin--a
DNA targeting antibiotic, and Rapa to assess the dose dependent
selection or depletion of T.sub.CD4, FoxP3 by aminoglycosides (FIG.
14A). Depletion of T.sub.CD4, FoxP3 is again noted in the presence
of aminoglycoside G418. The alternative
aminoglycoside--hygromycin--developed an insignificant increase in
T.sub.CD4, FoxP3 at 0.2 mM hygromycin. This increase significantly
decreased with higher doses of hygromycin--1.5 and 2.3 mM.
Hygromycin showed no significant depletion of T.sub.CD4, FoxP3 from
untreated control.
[0190] Ribosomal Inhibition by aminoglycoside G418 selectively
depletes replicating T.sub.CD4, FoxP3. Thawed PBMC were activated
with anti-CD3/CD28+IL-2 for 7 days in the presence of alternative
doses of G418, Hygromycin B--a different aminoglycoside,.sub.[146]
Zeocin--a DNA targeting antibiotic, and Rapa to assess the dose
dependent selection or depletion of T.sub.CD4, FoxP3 by
aminoglycosides (FIG. 14A). Depletion of T.sub.CD4, FoxP3 is again
noted in the presence of aminoglycoside G418. The alternative
aminoglycoside--hygromycin--developed an insignificant increase in
T.sub.CD4, FoxP3 at 0.2 mM hygromycin. This increase significantly
decreased with higher doses of hygromycin--1.5 and 2.3 mM.
Hygromycin showed no significant depletion of T.sub.CD4, FoxP3 from
untreated control.
[0191] This dose dependent depletion of T.sub.CD4, FoxP3 is
consistent with that seen for G418, and was not noted with
increasing doses Zeocin or Rapa. An increase of T.sub.CD4, FoxP3
was noted with increasing doses of Zeocin, yet this was
insignificant, similar to that seen for other cytotoxic drugs in
FIG. 10B-II. A representative flow plot of CD4 and FoxP3 expression
from the same donor can be seen in FIG. 14B. Here, the trends can
be visualized.
[0192] It was considered that polyclonal stimulation may play some
part in the G418 depletion of T.sub.CD4, FoxP3. To test this, PBMC
were rested in CM for 9 days after thawing +/-G418 and tested for
the presence of T.sub.CD4, FoxP3. Significant depletion of
T.sub.CD4, FoxP3 by G418 persisted under resting conditions (FIG.
14C--left panel). This was replication dependent as
CD4+,FoxP3+,Ki-67+ cells showed significant G418 mediated depletion
while CD4+,FoxP3+,Ki-67neg cells were not significantly depleted by
the same post-Hoc measure (FIG. 14C--right panel). Representative
flow diagrams of resting PBMC in FIG. 14D--upper panel show the
loss in expression of FoxP3 for CD4+ T cells after treatment with
G418. An alternative view of Ki-67 and FoxP3 expression in FIG.
14D--lower panel demonstrates that FoxP3neg T cells continue to
proliferate in the presence of G418, further supporting the
selective targeting of G418 to T.sub.CD4, FoxP3 at this
concentration. Thus, proliferating T.sub.CD4, FoxP3 are depleted
following treatment with aminoglycoside G418.
[0193] As G418 and hygromycin are considered toxic to live animals,
gentamicin, an aminoglycoside well known for its use in humans and
animal models, was tested for selective TCD4, FoxP3 depletion. See
Lopez-Novoa J M. et al., Kidney international 2011, 79(1):33-45.
FIG. 3E depicts this depletion of T.sub.CD4, FoxP3 in resting PBMC
after 7 days and demonstrates the consistent action of
aminoglycosides in depleting TCD4, FoxP3. It was next tested
whether depletion of T.sub.CD4, FoxP3 corresponded with a loss of
T.sub.reg marker expression or selective T.sub.reg toxicity.
[0194] Sorted Treg differentiate the effects of MTX, 5-FU, and G418
on selection in bulk PBMC. Magnetic sorting for CD4 and CD25
expressing PBMC yielded a CD4+CD25+ population that is widely
considered to contain T.sub.reg, and a CD25.sub.neg population of
effector T cells (T.sub.eff). See Miyara M. et al., Immunity 2009,
30(6):899-911. These populations were treated with the same
concentrations of MTX, 5-FU, G418, or no treatment, as above, for
the first 7 days of co-culture with AaPC. After this period of
time, co-culture continued without drug by stimulating with AaPC
every 7 days until Day 21. Cells were assayed at this time for
expression of CD25, CTLA-4, LAP, and IL-2, as before. The
experimental outline can be seen in FIG. 15A. A [.sup.3H] thymidine
incorporation assay was also performed to determine the effect of
each drug on the functionality of propagated T.sub.reg.
[0195] When the surviving CD4.sup.+ cells were assayed on day 21 it
was found that no drug significantly selected for T.sub.CD4, FoxP3
in the T.sub.eff compartment, nor did MTX and 5-FU improve
selection for T.sub.CD4, FoxP3 in the T.sub.reg compartment (FIG.
15B). The most consistent finding was that G418 persistently
decreased surviving T.sub.reg following drug treatment. This was
demonstrated by loss of surviving T.sub.CD4, FoxP3 (FIG. 15B).
T.sub.reg markers such as CD25 (FIG. 15C-I), CTLA-4 (FIG. 15C-II),
decreased IL-2 expression (FIG. 4C-III), or LAP (FIG. 15C-IV), in
combination with FoxP3 expression was also decreased following
stimulation on day 21. Thus, T.sub.reg are lost, likely due to
toxicity of G418, rather than inhibited as 2 weeks of growth
promoting co-culture conditions could not sufficiently restore
T.sub.regs following G418 treatment.
[0196] The T.sub.reg promoting properties of MTX and 5-FU appeared
to depend in part upon the presence of T.sub.eff, as the enhanced
selection of T.sub.CD4, FoxP3 was no longer noticeable after
T.sub.eff were removed from the culture system (FIG. 15B). The
improved selection towards T.sub.reg phenotypes may have been
accomplished by depletion of T.sub.eff which are known to
contaminate T.sub.reg sorting..sub.[113] It is likely that the
ability of T.sub.reg to survive the cytotoxic insult of MTX or 5-FU
in comparison to T.sub.eff was a primary component of the enhanced
selection. Although there was a trend towards improved selection of
T.sub.reg phenotypes (FIG. 15C-I, II, III) when MTX or 5-FU was
used, there was no significant difference for expression of CD25,
CTLA-4, or loss of IL-2. However, the T.sub.reg-specific marker LAP
was significantly increased by early treatment with MTX or 5-FU
(FIG. 15C-IV). As LAP was the only increased marker of those
assayed, it is likely that LAP and the associated expression of
TGF-.beta..sub.[143] was the probable cause for improved
suppression of MTX and 5-FU treated T.sub.reg above untreated
T.sub.reg (FIG. 15D). Thus, MTX and 5-FU appear to have two
components in enhancing selection of T.sub.reg: 1) T.sub.eff are
selectively depleted by MTX and 5-FU, and 2) MTX and 5-FU increase
the expression of LAP weeks after treatment.
[0197] Stimulation of T.sub.CD4, FoxP3 enhances AMPK activation and
leads to inhibition of eEF2--a factor that plays a role in
translational elongation. AMPK is hypothesized to play a role in
selection of T.sub.CD4, FoxP3, as noted above (FIG. 13).
Furthermore, enhanced activation of AMPK may lead to inhibition of
eEF2 in T.sub.CD4, FoxP3. See Browne G J. et al., The Journal of
biological chemistry 2004, 279(13):12220-12231. Preferential
inhibition of translational elongation could explain selection for
T.sub.CD4, FoxP3 in the presence of many cytotoxic drugs and
depletion of T.sub.CD4, FoxP3 in the presence of inhibitors of
translational elongation. This was tested by assessing
phosphorylation of AMPK 24 hours after activation of PBMC using
flow cytometry (FIGS. 16A & B) and imaging cytometry (FIG.
16C). The phosphorylation of AMPK on T172 indicates activation and
was enhanced in stimulated over unstimulated T.sub.CD4, FoxP3. See
Hardie D G et al., Diabetes 2013, 62(7):2164-2172. This enhanced
activation of AMPK was increased in CD4.sup.+, FoxP3.sub.neg T
cells (FIG. 16A--upper panel) as well, but the significant increase
(p=0.03 by t-test) did not persist following post-hoc analysis.
Likewise, flow plots of activated AMPK with FoxP3 show this
enhancement of AMPK activation is much more noticeable in the
FoxP3-expressing subset (FIG. 16B--upper panel). See MacIver N J et
al., Journal of immunology 2011, 187(8):4187-4198. A marker of
translational initiation--S6--is susceptible to mTOR regulation,
and is phosphorylated when active. See Mahoney S J et al., Progress
in molecular biology and translational science 2009, 90:53-107.
Phosphorylation of S6 (p-S6) was significantly enhanced in
T.sub.CD4, FoxP3 following stimulation (FIG. 16A--lower panel),
which was previously shown by Cabone et al. See Carbone F. et al.,
Nature medicine 2014, 20(1):69-74. While p-S6 increased in the
FoxP3.sub.neg T cells (p=0.01 by t-test), this increase was not
significant following post-hoc analysis. The enhancement of p-S6 is
observable in the representative flow plot for FIG. 16B--lower
panel. The activation of metabolic regulators AMPK and S6 was
enhanced in both FoxP3.sup.+ and FoxP3.sup.neg CD4.sup.+ T cells
following activation, but the increase was only significant in
T.sub.CD4, FoxP3 in a Two-Way ANOVA with post-hoc Sidak's test. The
increased activation of AMPK and S6 following activation of
T.sub.CD4, FoxP3 can be seen with image cytometry profiles shown in
FIG. 16D before--top panel--and after stimulation with
anti-CD3/CD28 and IL-2--bottom panel. The same compensation and
visualization were applied to each panel making the top and bottom
panels comparable.
[0198] Without wishing to be bound by theory, enhanced activation
of AMPK in T.sub.CD4, FoxP3 suggests translational elongation may
be inhibited by phosphorylation of eEF2 and could account for the
increased survival of T.sub.CD4, FoxP3 in the presence of cytotoxic
drugs and susceptibility to inhibitors of translational elongation,
like aminoglycosides. The same experiment as in FIG. 16 A-C was
performed to assess the inactivation of eEF2 by phosphorylation at
T56.[135] Image cytometry was used to quantify and visualize all
events. FIG. 16D demonstrates a significant increase in
phosphorylation of eEF2 in the same subset of T cells--T.sub.CD4,
FoxP3--following stimulation. Also, inhibitory phosphorylation of
eEF2 was significantly increased above stimulated FoxP3.sub.neg T
cells, which was not noted with AMPK or S6 phosphorylation. The
increased phosphorylation of eEF2 only in stimulated T.sub.CD4,
FoxP3 suggests that T.sub.CD4, FoxP3 would have decreased
replicative capacity upon stimulation, as shown by Cao et al.
Decreased levels of active eEF2, which inhibit progression through
the cell cycle, suggest that increased phosphorylation of eEF2 may
account for the survival of T.sub.CD4, FoxP3 in cytotoxic
environments, which was noted in FIG. 10. Similarly, decreased
translational capacity would make T.sub.CD4, FoxP3 increasingly
susceptible to inhibitors of translational elongation, as was shown
with aminoglycosides in FIG. 14. Therefore, the activity of eEF2
may be the primary factor influencing both selection and depletion
of T.sub.reg in these studies.
[0199] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
[0200] All cited patents and publications referred to in this
application are herein incorporated by reference in their entirety.
Sequence CWU 1
1
121639DNAArtificial sequenceSynthetic oligonucleotide 1atggactaca
aggacgacga cgacaaggat tacaaggatg atgatgataa ggactataaa 60gacgacgatg
ataaggacgt cgttggttcg ctaaactgca tcgtcgctgt gtcccagaac
120atgggcatcg gcaagaacgg ggacttcccc tggccaccgc tcaggaatga
atccagatat 180ttccagagaa tgaccacaac ctcttcagta gaaggtaaac
agaatctggt gattatgggt 240aagaagacct ggttctccat tcctgagaag
aatcgacctt taaagggtag aattaattta 300gttctcagca gagaactcaa
ggaacctcca caaggagctc attttctttc cagaagtcta 360gatgatgcct
taaaacttac tgaacaacca gaattagcaa ataaagtaga catggtctgg
420atagttggtg gcagttctgt ttataaggaa gccatgaatc acccaggcca
tcttaaacta 480tttgtgacaa ggatcatgca agactttgaa agtgacacgt
tttttccaga aattgatttg 540gagaaatata aacttctgcc agaataccca
ggtgttctct ctgatgtcca ggaggagaaa 600ggcattaagt acaaatttga
agtatatgag aagaatgat 6392639DNAArtificial sequenceSynthetic
oligonucleotide 2atggactaca aggacgacga cgacaaggat tacaaggatg
atgatgataa ggactataag 60gacgatgatg acaaagacgt cgtgggcagc ctgaactgca
tcgtggccgt gtcccagaac 120atgggcatcg gcaagaacgg cgacttcccc
tggccccctc tgcggaacga gagccggtac 180ttccagcgga tgaccaccac
cagcagcgtg gaaggcaagc agaacctcgt gatcatgggc 240aagaaaacct
ggttcagcat ccccgagaag aaccggcccc tgaagggccg gatcaacctg
300gtgctgagca gagagctgaa agagccccct cagggcgccc acttcctgag
cagatctctg 360gacgacgccc tgaagctgac cgagcagcca gagctggcca
acaaggtgga catggtgtgg 420atcgtgggcg gcagctccgt gtacaaagaa
gccatgaacc accctggcca cctgaaactg 480ttcgttaccc gtataatgca
ggatttcgag agcgatacct tcttccccga gatcgacctg 540gaaaagtaca
agctgcttcc cgagtacccc ggcgtgctgt ccgatgtgca ggaagagaag
600ggcatcaagt acaagttcga ggtgtacgag aagaatgac 6393999DNAArtificial
sequenceSynthetic oligonucleotide 3atgtatccgt acgacgtacc agactacgca
tatccgtacg acgtaccaga ctacgcagac 60gtccctgtgg ccggctcgga gctgccgcgc
cggcccttgc cccccgccgc acaggagcgg 120gacgccgagc cgcgtccgcc
gcacggggag ctgcagtacc tggggcagat ccaacacatc 180ctccgctgcg
gcgtcaggaa ggacgaccgc tcgagcaccg gcaccctgtc ggtattcggc
240atgcaggcgc gctacagcct gagagatgaa ttccctctgc tgacaaccaa
acgtgtgttc 300tggaagggtg ttttggagga gttgctgtgg tttatcaagg
gatccacaaa tgctaaagag 360ctgtcttcca agggagtgaa aatctgggat
gccaatggat cccgagactt tttggacagc 420ctgggattct ccaccagaga
agaaggggac ttgggaccag tttatggctt ccagtggagg 480cattttgggg
cagaatacag agatatggaa tcagattatt caggacaggg agttgaccaa
540ctgcaaagag tgattgacac catcaaaacc aaccctgacg acagaagaat
catcatgtgc 600gcttggaatc caagagatct tcctctgatg gcgctgcctc
catgccatgc cctctgccag 660ttctatgtgg tgaacagtga gctgtcctgc
cagctgtacc agagatcggg agacatgggc 720ctcggtgtgc ctttcaacat
cgccagctac gccctgctca cgtacatgat tgcgcacatc 780acgggcctga
agccaggtga ctttatacac actttgggag atgcacatat ttacctgaat
840cacatcgagc cactgaaaat tcagcttcag cgagaaccca gacctttccc
aaagctcagg 900attcttcgaa aagttgagaa aattgatgac ttcaaagctg
aagactttca gattgaaggg 960tacaatccgc atccaactat taaaatggaa atggctgtt
99941017DNAArtificial sequenceSynthetic oligonucleotide 4atggactaca
aggacgacga cgacaaggat tacaaggatg atgatgataa ggactataag 60gacgatgatg
acaaagacgt ccccgtggcc ggcagcgagc tgcctagaag gcctctgcct
120cctgccgctc aggaaaggga cgccgaacct agacctcctc acggcgagct
gcagtacctg 180ggccagatcc agcacatcct gagatgcggc gtgcggaagg
acgacagaag cagcacaggc 240accctgagcg tgttcggaat gcaggccaga
tacagcctgc gggacgagtt ccctctgctg 300accaccaagc gggtgttctg
gaagggcgtg ctggaagaac tgctgtggtt catcaagggc 360agcaccaacg
ccaaagagct gagcagcaag ggcgtgaaga tctgggacgc caacggcagc
420agagacttcc tggacagcct gggcttcagc accagagagg aaggcgatct
gggtcccgtg 480tacgggtttc aatggcggca cttcggcgcc gagtatcggg
acatggagag cgactacagc 540ggccagggcg tggaccagct gcagagagtg
atcgacacca tcaagaccaa ccccgacgac 600cggcggatca tcatgtgcgc
ctggaacccc agagatctgc ccctgatggc cctgcctcca 660tgtcacgccc
tgtgccagtt ctacgtcgtg aactccgagc tgagctgcca gctgtaccag
720cggagcggcg atatgggact gggcgtgccc ttcaatatcg ccagctacgc
cctgctgacc 780tacatgatcg cccacatcac cggcctgaag cccggcgact
ttatccacac cctgggcgac 840gcccatatct acctgaacca catcgagccc
ctgaagattc agctgcagcg cgagcccaga 900cccttcccaa agctgcggat
cctgcggaag gtggaaaaga tcgacgactt caaggccgag 960gacttccaga
tcgagggcta caacccccac cccacaatca agatggaaat ggccgtg
1017539DNAArtificial sequenceSynthetic oligonucleotide 5cccgggcccg
gcgccatgcc acctcctcgc ctcctcttc 39651DNAArtificial
sequenceSynthetic oligonucleotide 6ggtacccttg tacagctcgt ccatgccgag
agtgatcccg gcggcggtca c 51763DNAArtificial sequenceSynthetic
oligonucleotide 7gctagcacat gtgccaccat gattgaacaa gatggattgc
acgcaggttc tccggccgct 60tgg 63871DNAArtificial sequenceSynthetic
oligonucleotide 8aagcttccgc ggccctctcc gctaccgaag aactcgtcaa
gaaggcgata gaaggcgatg 60cgctgcgaat c 71916PRTArtificial
sequenceSynthetic polypeptide 9Met Ala Pro Lys Lys Lys Arg Lys Val
Gly Ile His Arg Gly Val Pro1 5 10 1510251PRTArtificial
sequenceSynthetic polypeptide 10Met Glu Gln Lys Leu Ile Ser Glu Glu
Asp Leu Glu Gln Lys Leu Ile1 5 10 15Ser Glu Glu Asp Leu Glu Gln Lys
Leu Ile Ser Glu Glu Asp Val Val 20 25 30Gly Ser Leu Asn Cys Ile Val
Ala Val Ser Gln Asn Met Gly Ile Gly 35 40 45Lys Asn Gly Asp Phe Pro
Trp Pro Pro Leu Arg Asn Glu Ser Arg Tyr 50 55 60Phe Gln Arg Met Thr
Thr Thr Ser Ser Val Glu Gly Lys Gln Asn Leu65 70 75 80Val Ile Met
Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys Asn Arg 85 90 95Pro Leu
Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu Lys Glu 100 105
110Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp Ala Leu
115 120 125Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met
Val Trp 130 135 140Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met
Asn His Pro Gly145 150 155 160His Leu Lys Leu Phe Val Thr Arg Ile
Met Gln Asp Phe Glu Ser Asp 165 170 175Thr Phe Phe Pro Glu Ile Asp
Leu Glu Lys Tyr Lys Leu Leu Pro Glu 180 185 190Tyr Pro Gly Val Leu
Ser Asp Val Gln Glu Glu Lys Gly Ile Lys Tyr 195 200 205Lys Phe Glu
Val Tyr Glu Lys Asn Asp Gly Thr Gly Glu Gly Arg Gly 210 215 220Ser
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Leu Gly225 230
235 240Leu Met Gly Leu Pro Phe Thr Ala Arg Phe Pro 245
25011316PRTArtificial sequenceSynthetic polypeptide 11Asp Val Pro
Val Ala Gly Ser Glu Leu Pro Arg Arg Pro Leu Pro Pro1 5 10 15Ala Ala
Gln Glu Arg Asp Ala Glu Pro Arg Pro Pro His Gly Glu Leu 20 25 30Gln
Tyr Leu Gly Gln Ile Gln His Ile Leu Arg Cys Gly Val Arg Lys 35 40
45Asp Asp Arg Ser Ser Thr Gly Thr Leu Ser Val Phe Gly Met Gln Ala
50 55 60Arg Tyr Ser Leu Arg Asp Glu Phe Pro Leu Leu Thr Thr Lys Arg
Val65 70 75 80Phe Trp Lys Gly Val Leu Glu Glu Leu Leu Trp Phe Ile
Lys Gly Ser 85 90 95Thr Asn Ala Lys Glu Leu Ser Ser Lys Gly Val Lys
Ile Trp Asp Ala 100 105 110Asn Gly Ser Arg Asp Phe Leu Asp Ser Leu
Gly Phe Ser Thr Arg Glu 115 120 125Glu Gly Asp Leu Gly Pro Val Tyr
Gly Phe Gln Trp Arg His Phe Gly 130 135 140Ala Glu Tyr Arg Asp Met
Glu Ser Asp Tyr Ser Gly Gln Gly Val Asp145 150 155 160Gln Leu Gln
Arg Val Ile Asp Thr Ile Lys Thr Asn Pro Asp Asp Arg 165 170 175Arg
Ile Ile Met Cys Ala Trp Asn Pro Arg Asp Leu Pro Leu Met Ala 180 185
190Leu Pro Pro Cys His Ala Leu Cys Gln Phe Tyr Val Val Asn Ser Glu
195 200 205Leu Ser Cys Gln Leu Tyr Gln Arg Ser Gly Asp Met Gly Leu
Gly Val 210 215 220Pro Phe Asn Ile Ala Ser Tyr Ala Leu Leu Thr Tyr
Met Ile Ala His225 230 235 240Ile Thr Gly Leu Lys Pro Gly Asp Phe
Ile His Thr Leu Gly Asp Ala 245 250 255His Ile Tyr Leu Asn His Ile
Glu Pro Leu Lys Ile Gln Leu Gln Arg 260 265 270Glu Pro Arg Pro Phe
Pro Lys Leu Arg Ile Leu Arg Lys Val Glu Lys 275 280 285Ile Asp Asp
Phe Lys Ala Glu Asp Phe Gln Ile Glu Gly Tyr Asn Pro 290 295 300His
Pro Thr Ile Lys Met Glu Met Ala Val Gly Thr305 310
31512186PRTArtificial sequenceSynthetic polypeptide 12Val Gly Ser
Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly Ile1 5 10 15Gly Lys
Asn Gly Asp Phe Pro Trp Pro Pro Leu Arg Asn Glu Ser Arg 20 25 30Tyr
Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln Asn 35 40
45Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys Asn
50 55 60Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu
Lys65 70 75 80Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu
Asp Asp Ala 85 90 95Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys
Val Asp Met Val 100 105 110Trp Ile Val Gly Gly Ser Ser Val Tyr Lys
Glu Ala Met Asn His Pro 115 120 125Gly His Leu Lys Leu Phe Val Thr
Arg Ile Met Gln Asp Phe Glu Ser 130 135 140Asp Thr Phe Phe Pro Glu
Ile Asp Leu Glu Lys Tyr Lys Leu Leu Pro145 150 155 160Glu Tyr Pro
Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly Ile Lys 165 170 175Tyr
Lys Phe Glu Val Tyr Glu Lys Asn Asp 180 185
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